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UBC Theses and Dissertations

Regulation of urea synthesis in skate hepatocytes Nener, Jennifer C. 1986

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REGULATION OF UREA SYNTHESIS IN SKATE HEPATOCYTES by JENNIFER C. NENER B . S c , D a l h o u s i e U n i v e r s i t y , H a l i f a x , 1984 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 a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September 1986 © J e n n i f e r C. Nener, 1986 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f ^OOfo, The U n i v e r s i t y o f B r i t i s h Columbia 1 9 5 6 Main Mall Vancouver, Canada V 6 T 1 Y 3 Date Qdt. % , )C[%b i i ABSTRACT Urea s y n t h e s i s and a l a n i n e o x i d a t i o n were measured i n s k a t e ( R a j a e r i n a c e a ) h e p a t o c y t e s i n c u b a t e d w i t h p h a r m a c o l o g i c a l doses of f o u r hormones: i n s u l i n , g l u c a g o n , la-OH, and 1,2-dehydro-A. P e p t i d e hormones were used o n l y f o r s h o r t (2.25 h o u r s ) i n c u b a t i o n s , w h i l e i n c u b a t i o n s w i t h s t e r o i d s were 2.25, 24, or 48 h o u r s . None of the hormones e x e r t e d any e f f e c t s on urea s y n t h e s i s or a l a n i n e o x i d a t i o n d u r i n g the s h o r t e s t i n c u b a t i o n p e r i o d , however a f t e r 24 hours 1a-0H caused a s i g n i f i c a n t (p<.05) e l e v a t i o n i n a l a n i n e o x i d a t i o n , which may be an a d a p t i v e response of s k a t e s t o d i l u t e s e a w a t e r . Long-term i n c u b a t i o n w i t h s t e r o i d s had no e f f e c t on urea s y n t h e s i s i n s k a t e h e p a t o c y t e s . Urea s y n t h e s i s , a l a n i n e o x i d a t i o n , and g l u c o n e o g e n e s i s were measured i n s k a t e h e p a t o c y t e s i n c u b a t e d i n a wide range of o s m o l a r i t i e s (50%, 75%, 100%, 112.5%, and 125% of normal plasma o s m o l a r i t y ) . C o n c e n t r a t i o n of the i n c u b a t i o n medium was a l t e r e d by v a r y i n g amounts of u r e a , N a C l , TMAO, MgSO„, M g C l 2 , and KC1, w h i l e NaHC0 3, NaH 2PO a, C a C l 2 , Hepes, and BSA were m a i n t a i n e d a t c o n s t a n t l e v e l s . Urea s y n t h e s i s was s i g n i f i c a n t y d e p r e s s e d (P<.05) a t 50% o s m o l a r i t y , but d i d not v a r y s i g n i f i c a n t l y a t h i g h e r s o l u t e c o n c e n t r a t i o n s . G l u c o n e o g e n e s i s was s i g n i f i c a n t l y e l e v a t e d (P<,05) a t 50% and 75% of normal o s m o l a r i t y , as was a l a n i n e o x i d a t i o n a t 50% o s m o l a r i t y . Rates of g l u c o n e o g e n e s i s and a l a n i n e o x i d a t i o n d i d not v a r y s i g n i f i c a n t l y a t h i g h e r s o l u t e c o n c e n t r a t i o n s . In o t h e r e x p e r i m e n t s o s m o l a r i t y was m a i n t a i n e d a t 87-113% w h i l e one of the v a r i a b l e s o l u t e s was reduced t o 50% of s t a n d a r d c o n c e n t r a t i o n . I n a l l c a s e s r e d u c t i o n of c o n c e n t r a t i o n of one s o l u t e f a i l e d t o cause a d e p r e s s i o n of u r e a s y n t h e s i s . I t was c o n c l u d e d t h a t a s h a r p d e c r e a s e i n o s m o l a r i t y i n d i c e s s p e c i f i c a d a p t i v e m e t a b o l i c changes i n R. E r i n a c e a h e p a t o c y t e s . i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS v i i i O s m o r e g u l a t o r y Mechanisms i n M a r i n e Elasmobranchs 1 Urea 4 T r i m e t h y l a m i n e - N - o x i d e 7 Amino A c i d s 10 Responses of R a j a e r i n a c e a t o Osmotic S t r e s s 12 M a t e r i a l s and Methods 14 A n i m a l s 14 C h e m i c a l s 14 H e p a t o c y t e P r e p a r a t i o n 15 C e l l I n t e g r i t y 16 A l a n i n e O x i d a t i o n and G l u c o n e o g e n e s i s 17 Urea S y n t h e s i s 19 C a l c u l a t i o n s 21 S t a t i s t i c a l A n a l y s i s 21 E f f e c t s of I n s u l i n , G l ucagon, 1a-OH, and 1,2-dehydro-A on Urea S y n t h e s i s and A l a n i n e O x i d a t i o n 24 I n t r o d u c t i o n 24 Hormones and Urea S y n t h e s i s 25 Hormones and O s m o r e g u l a t i o n 26 E x p e r i m e n t a l D e sign 30 S h o r t Term I n c u b a t i o n s 30 Long Term Hormone I n c u b a t i o n s 31 R e s u l t s and D i s c u s s i o n 33 C e l l I n t e g r i t y 33 Short-Term I n c u b a t i o n s 33 Long-Term I n c u b a t i o n s 43 Summary 53 E f f e c t s of A l t e r i n g O s m o l a r i t y and S p e c i f i c S o l u t e C o n c e n t r a t i o n s on M e t a b o l i c Pathways 54 I n t r o d u c t i o n 54 E x p e r i m e n t a l D e s i g n 56 O s m o l a r i t y 57 Minor S o l u t e s 57 Major S o l u t e s 58 R e s u l t s and D i s c u s s i o n 61 O s m o l a r i t y E x p e r i m e n t s 61 Minor S o l u t e s 71 Major S o l u t e s 77 Summary 84 C o n c l u s i o n s 87 R e f e r e n c e s 91 v i LIST OF TABLES T a b l e 1. S o l u t i o n s f o r I s o l a t i o n and I n c u b a t i o n of Skate H e p a t o c y t e s 22 T a b l e 2. E f f e c t s of S h o r t Term Hormone Treatments on Urea S y n t h e s i s and A l a n i n e O x i d a t i o n 39 T a b l e 3. Mean Rates of Urea S y n t h e s i s and A l a n i n e O x i d a t i o n F o l l o w i n g I n c u b a t i o n w i t h S t e r o i d Hormones f o r 24 and 48 Hours 49 T a b l e 4. C o n c e n t r a t i o n s of Major S o l u t e s i n I n c u b a t i o n Media 59 T a b l e 5. E f f e c t s of O s m o l a r i t y on Urea S y n t h e s i s , A l a n i n e O x i d a t i o n , and G l u c o n e o g e n e s i s i n Skate H e p a t o c y t e s .... 66 T a b l e 6. E f f e c t s of V a r y i n g C o n c e n t r a t i o n s of M g + + and K + on Urea S y n t h e s i s i n Sk a t e H e p a t o c y t e s 74 T a b l e 7. E f f e c t s of Reduced C o n c e n t r a t i o n s of Urea, N a C l , and TMAO on Urea S y n t h e s i s i n Skate H e p a t o c y t e s a t Co n s t a n t O s m o l a r i t y 82 LIST OF FIGURES F i g u r e 1 . The O r n i t h i n e Urea C y c l e 8 F i g u r e 2. E f f e c t s of Short-Term Hormone I n c u b a t i o n s on Urea S y n t h e s i s i n Skate H e p a t o c y t e s 35 F i g u r e 3. E f f e c t s of Short-Term Hormone I n c u b a t i o n s on A l a n i n e O x i d a t i o n i n Skate H e p a t o c y t e s 37 F i g u r e 4. E f f e c t s of Long-Term S t e r o i d I n c u b a t i o n on Urea S y n t h e s i s i n Skate H e p a t o c y t e s 45 F i g u r e 5. E f f e c t s of Long-Term S t e r o i d I n c u b a t i o n on A l a n i n e O x i d a t i o n i n Skate H e p a t o c y t e s 47 F i g u r e 6. E f f e c t s of O s m o l a r i t y on Rates of Urea S y n t h e s i s and A l a n i n e O x i d a t i o n i n S k a t e H e p a t o c y t e s 62 F i g u r e 7. E f f e c t s of O s m o l a r i t y on Rates of Urea S y n t h e s i s and G l u c o n e o g e n e s i s i n S k a t e H e p a t o c y t e s 64 F i g u r e 8. E f f e c t s of A l t e r i n g C o n c e n t r a t i o n s of M g + + and K + on Urea S y n t h e s i s 72 F i g u r e 9. E f f e c t s of Reduced C o n c e n t r a t i o n s of Urea and TMAO on Urea S y n t h e s i s i n S k a t e H e p a t o c y t e s 78 F i g u r e 10. E f f e c t s of Reduced C o n c e n t r a t i o n s of NaCl and H i g h Urea L e v e l s on Urea S y n t h e s i s i n Skate H e p a t o c y t e s 80 v i i i ACKNOWLEDGEMENTS The p e o p l e whom I would l i k e t o thank f o r h e l p i n g me d u r i n g the c o u r s e of t h i s work a r e numerous. I w i l l b e g i n w i t h my s u p e r v i s o r , Dr. T.P. Mommsen, who i n t r o d u c e d me t o t h i s f i e l d of b i o l o g y . He c o n t i n u o u s l y c h a l l e n g e d me, p a t i e n t l y t a u g h t me numerous s k i l l s , and showed me how much fun s c i e n c e can be. I would a l s o l i k e t o thank Dr. P.W. Hochachka f o r i n c l u d i n g me as one of h i s l a b members; h i s i n f e c t i o u s e n t h u s i a s m f o r s c i e n c e i s always i n s p i r i n g . Dr. R.K. Suarez always p r o v i d e d good c o n v e r s a t i o n and w i l l i n g l y o f f e r e d h e l p and a d v i c e d u r i n g my time a t U.B.C.. Dr. T.W. Moon g e n e r o u s l y a l l o w e d me t o work f o r many months i n h i s l a b i n New B r u n s w i c k , and p r o v i d e d i n t e r e s t i n g d i s c u s s i o n s . The s t a f f a t Huntsman M a r i n e L a b o r a t o r y i n S t . Andrews, New B r u n s w i c k , d i d e v e r y t h i n g they c o u l d t o h e l p t h i n g s run smoothly d u r i n g the c o u r s e of my r e s e a r c h . Dr. M. D a d s w e l l , of the S t . Andrews B i o l o g i c a l S t a t i o n , p r o v i d e d me w i t h a l a r g e number of e x p e r i m e n t a l a n i m a l s . Ms. B e r y l l . T r u s c o t t g e n e r o u s l y donated the h i g h l y p u r i f i e d elasmobranch hormones which were used i n t h i s s t u d y . I would a l s o l i k e t o thank A l i s t a i r B l a c h f o r d , Susan E r t i s , and S h i r l e y G r a y s t o n e , of the B i o s c i e n c e s Data C e n t e r , f o r t e a c h i n g me a l l about computers, and p a t i e n t l y a n s w e r i n g many q u e s t i o n s . I am i n d e b t e d t o a number of s p e c i a l f r i e n d s , G a y l e Brown, Maggie C a s t e l l i n i , P e t e r C l i f f o r d , E r i k D e B r u i j n , Joanne Green, Heather McClean, and Tom P e t e r s e n , f o r l o t s of encouragement and fun which they o f f e r e d a l o n g the way. 1 OSMOREGULATORY MECHANISMS IN MARINE ELASMOBRANCHS Elasmobranchs a r e an a n c i e n t group of f i s h e s which have numerous p e c u l i a r p h y s i o l o g i c a l c h a r a c t e r i s t i c s . They a r e the l a r g e s t group of v e r t e b r a t e osmoconformers, and p o s s e s s a c o m b i n a t i o n of unique a d a p t i o n s which e n a b l e them t o s u c c e s s f u l l y r e g u l a t e t h e i r plasma and i n t r a c e l l u l a r o s m o t ic c o m p o s i t i o n . Such a d a p t a t i o n s i n c l u d e the a b i l i t y t o s y n t h e s i z e a number of o s m o l y t e s such as u r e a , t r i m e t h y l a m i n e - N - o x i d e (TMAO) and s p e c i f i c amino a c i d s , which a r e m e t a b o l i c a l l y c o m p a t i b l e . I n a d d i t i o n elasmobranchs have t h e a b i l i t y t o r e a b s o r b t h e s e o s m o l y t e s w i t h h i g h e f f i c i e n c y from t h e i r g l o m e r u l a r f i l t r a t e . Elasmobranchs a l s o have m o d i f i e d g i l l s and s k i n which render them e s s e n t i a l l y impermeable t o s e l e c t e d o s m o l y t e s , and t h e r e b y reduces the l o s s of m e t a b o l i c a l l y c o s t l y compounds t o the envir o n m e n t . O s m o r e g u l a t i o n i n e u r y h a l i n e elasmobranchs i s more complex, as such a n i m a l s must be a b l e t o a l t e r t h e i r s o l u t e c o m p o s i t i o n i n response t o changes i n e n v i r o n m e n t a l s a l i n i t y . L i t t l e i s known about the b i o c h e m i s t r y of elasmobranchs, and the mechanisms wh i c h e n a b l e t h e s e a n i m a l s t o adapt t o d i l u t e seawater a r e p o o r l y u n d e r s t o o d . The p r e s e n t s t u d y a d d r e s s e s the s p e c i f i c q u e s t i o n of how s y n t h e s i s of one major o s m o l y t e , u r e a , i s r e g u l a t e d i n a e u r y h a l i n e elasmobranch, R a j a e r i n a c e a . Important g o a l s of t h e p r e s e n t study were as f o l l o w s : 2 1 . t o demonstrate urea s y n t h e s i s i n an ir\ v i t r o i s o l a t e d m e t a b o l i c system which a d e q u a t e l y r e f l e c t s osmoconforming a b i l i t i e s ; 2 . t o i d e n t i f y s p e c i f i c r e g u l a t o r s of urea s y n t h e s i s i n t h i s p h y s i o l o g i c a l system. The p o w e r f u l t o o l of i s o l a t e d h e p a t o c y t e s was used as the m e t a b o l i c system i n t h i s s t u d y . P o s s i b l e r e g u l a t o r s of urea s y n t h e s i s which were s t u d i e d i n c l u d e d f o u r hormones of i n t e r e s t , and a l t e r i n g t h e c o m p o s i t i o n of the e x t r a c e l l u l a r f l u i d . A l l o rganisms must be c a p a b l e of r e g u l a t i n g t h e f l u x of s a l t s and water i n t o and out of the body i n o r d e r t o m a i n t a i n a m e t a b o l i c a l l y c o m p a t i b l e i n t e r n a l m i l i e u . M a r i n e and f r e s h w a t e r a n i m a l s a r e c o n t i n u o u s l y immersed i n a medium wh i c h i s a t l e a s t somewhat i n c o m p a t i b l e w i t h t h e i r p h y s i o l o g i c a l r e q u i r e m e n t s . Some marine a n i m a l s have t o co n t e n d w i t h the problem of d e h y d r a t i o n ; they must o b t a i n enough water t o meet t h e i r p h y s i o l o g i c a l r e q u i r e m e n t s and expend energy t o r i d themselves of e x c e s s i n o r g a n i c i o n s . Among marine f i s h e s two main s t r a t e g i e s have e v o l v e d f o r d e a l i n g w i t h t h e s e c o n s t r a i n t s . M a r i n e t e l e o s t s a r e o s m o r e g u l a t o r s which a r e h y p o t o n i c t o t h e i r e n v i r o n m e n t . They d r i n k c o p i o u s amounts of seawater and expend energy pumping e x c e s s i o n s out of t h e body a c r o s s the g i l l s . The a l t e r n a t e s t r a t e g y i s found i n p r i m i t i v e groups of f i s h e s , i n c l u d i n g elasmobranchs ( S e l a c h i i ) , h a g f i s h ( M y x i n i d a e ) , h o l o c e p h a l a ( H o l o c e p h a l i ) , and the c o e l o c a n t h ( C r o s s o p t e r y g i i ) . These a n i m a l s a r e osmoconformers; they have a plasma o s m o l a r i t y which i s n o r m a l l y j u s t s l i g h t l y h y p e r t o n i c t o t h a t of the 3 e x t e r n a l medium, and are t h e r e f o r e a s s u r e d of a c o n t i n u o u s i n f l u x of water a c r o s s permeable s u r f a c e s such as the g i l l s . These a n i m a l s do not d r i n k s e a w a t e r , as marine t e l e o s t o s m o r e g u l a t o r s do, and t h e r e f o r e have a r e l a t i v e l y low r a t e of i n t a k e of s a l t s . C o n s e q u e n t l y , elasmobranchs expend c o m p a r a t i v e l y l i t t l e energy e x c r e t i n g i n o r g a n i c i o n s . E x c e s s i n o r g a n i c i o n s must be e x c r e t e d as t hey d i s r u p t the s t r u c t u r e of m a c r o m o l e c u l e s , i n c l u d i n g enzymes, and t h e r e f o r e i n t e r f e r e w i t h m e t a b o l i s m . I n o r g a n i c i o n s a r e known t o a f f e c t b o t h the c a t a l y t i c r a t e and M i c h a e l i s c o n s t a n t (Km) of enzymes w h i l e e q u i m o l a r amounts of " c o m p a t i b l e " s o l u t e s , as r e t a i n e d by e l a s m o b r a n c h s , have no such d e t r i m e n t a l e f f e c t s (Yancey e t a l . , 1982). C o m p a t i b l e o s m o l y t e s a r e t y p i c a l l y low m o l e c u l a r weight m e t a b o l i c p r o d u c t s i n c l u d i n g urea and methylamine d e r i v a t i v e s such as t r i m e t h y l a m i n e - N - o x i d e (TMAO) and b e t a i n e . S p e c i f i c f r e e amino a c i d s and amino a c i d d e r i v a t i v e s , e x e m p l i f i e d by t a u r i n e , s a r c o s i n e , and /3-alanine, a r e a l s o r e t a i n e d w i t h i n c e l l s , where t h e y c omprise up t o 20% of the o s m o t i c a l l y a c t i v e s o l u t e s ( G o l d s t e i n , 1981). C o n c e n t r a t i o n s of a l l t h e s e o s m o l y t e s a r e r e g u l a t e d by the a n i m a l and a r e s e l e c t i v e l y r educed i n r e s p o n s e t o d i l u t i o n of the e n v i r o n m e n t a l medium, i n o r d e r t o d e c r e a s e the a n i m a l - t o - e n v i r o n m e n t o s m o t i c g r a d i e n t ( G o l d s t e i n and F o r s t e r , 1971; K i n g et a l . , 1980; Leech and G o l d s t e i n , 1983). The major c h a r a c t e r i s t i c s h a r e d by chosen s o l u t e s i s t h a t t h ey a r e e s s e n t i a l l y m e t a b o l i c a l l y i n e r t ; t h ey a r e not i n v o l v e d 4 i n s y n t h e s i s of any o t h e r major c e l l p r o d u c t s , thus l a r g e f l u c t u a t i o n s i n t h e i r c o n c e n t r a t i o n , which o c c u r i n response t o changes i n e n v i r o n m e n t a l s a l i n i t y , do not d i s r u p t c e l l f u n c t i o n ( G o l d s t e i n , 1981 ). These o r g a n i c o s m o l y t e s may be s e p a r a t e d i n t o two f u n c t i o n a l c a t e g o r i e s : 1) t h o s e which a r e o s m o t i c a l l y a c t i v e between f i s h and environment, such as u r e a ; t h e y a r e d i s t r i b u t e d homogeneously t h r o u g h o u t the body due t o t h e i r d i f f u s a b i l i t y ( K i n g and G o l d s t e i n , 1983), and 2) t h o s e which a r e o s m o t i c a l l y a c t i v e between c e l l s and plasma, such as amino a c i d s ; they a r e p r e s e n t a t h i g h c o n c e n t r a t i o n s i n c e l l s of some t i s s u e s and do not d i f f u s e f r e e l y a c r o s s membranes. Osmolytes which f a l l i n t o the l a t t e r c a t e g o r y a r e i m p o r t a n t f o r c e l l volume r e g u l a t i o n , as opposed t o w h o l e - a n i m a l o s m o r e g u l a t i o n (Boyd e t a l . , 1977). Urea M a r i n e e l a s m o b r a n c h s , h o l o c e p h a l a n s , and t h e c o e l o c a n t h a r e the o n l y m u l t i c e l l u l a r a n i m a l s which s y n t h e s i z e urea p u r e l y f o r o s m o r e g u l a t o r y p u r p o s e s . S e m i - t e r r e s t r i a l and t e r r e s t r i a l a n i m a l s s y n t h e s i z e urea as a mechanism f o r d e t o x i f y i n g ammonia, a p o t e n t i a l l y h i g h l y t o x i c p r o d u c t of p r o t e i n and amino a c i d m e t a b o l i s m . Amino a c i d s i n e x c e s s of t h o s e r e q u i r e d f o r p r o t e i n s y n t h e s i s a r e m e t a b o l i z e d . The a-amino group i s removed and must be d i s p o s e d o f , w h i l e the r e s u l t i n g c a r b o n s k e l e t o n i s c o n v e r t e d t o a major m e t a b o l i c i n t e r m e d i a t e such as a c e t y l CoA, p y r u v a t e , or c i t r i c a c i d c y c l e i n t e r m e d i a t e s . E x c r e t i o n of waste n i t r o g e n as ammonia (NH 3) or ammonium 5 (NH U +) r e q u i r e s l a r g e amounts of water, as ammonium i s t o x i c a t a c o n c e n t r a t i o n of 70 uM or h i g h e r i n mammals (Kashiwagura e t a l . , 1984), and thus must not be a l l o w e d t o accumulate t o h i g h l e v e l s w i t h i n t i s s u e s . T e r r e s t r i a l a n i m a l s o f t e n have a l i m i t e d s u p p l y of water and c i r c u m v e n t problems by d e t o x i f y i n g waste n i t r o g e n ; t h e y i n c o r p o r a t e i t i n t o e i t h e r u r ea or p u r i n e d e r i v a t i v e s ( u r i c a c i d , guanine) which a r e r e l a t i v e l y i n n o c u o u s , and may t h e r e f o r e accumulate t o c o m p a r a t i v e l y h i g h l e v e l s . The u s u a l f a t e of t h e s e n i t r o g e n o u s end p r o d u c t s i s e x c r e t i o n i n the u r i n e i n a h i g h l y c o n c e n t r a t e d form. Plasma urea c o n c e n t r a t i o n s i n mammals a r e t y p i c a l l y i n the range of 6 mM. Thus, the a b i l i t y t o s y n t h e s i z e urea d e c r e a s e s mammalian demands f o r water by a f a c t o r of about 170. The v a s t m a j o r i t y of a q u a t i c a n i m a l s d i s p o s e of waste n i t r o g e n i n the form of ammonia or ammonium by e x c r e t i o n a c r o s s the g i l l s (Kormanik and Cameron, 1981). T h i s i s the l e a s t m e t a b o l i c a l l y e x p e n s i v e mechanism f o r waste n i t r o g e n d i s p o s a l , p r o v i d e d t h a t adequate water i s a v a i l a b l e . D e t o x i f i c a t i o n of ammonia by i n c o r p o r a t i o n i n t o urea c o s t s 2 ATP per atom of n i t r o g e n f i x e d . I t must be emphasised t h a t a l t h o u g h elasmobranchs l o s e some urea t o the environment they do not produce u r e a as a waste p r o d u c t . S u p e r f i c i a l l y i t appears t h a t urea i s a n i t r o g e n o u s end p r o d u c t t o e l a s m o b r a n c h s , as 80% of n i t r o g e n w h i c h they l o s e t o the environment i s i n the form of u r e a . A l t h o u g h elasmobranch g i l l s a r e r e l a t i v e l y impermeable t o urea t h e r e i s s t i l l a p p r e c i a b l e l o s s of t h i s o s m olyte d u r i n g the c o u r s e of a day due t o p a s s i v e d i f f u s i o n down i t s c o n c e n t r a t i o n 6 g r a d i e n t ( G o l d s t e i n and F o r s t e r , 1971). U n f o r t u n a t e l y , c o n c l u s i v e s t u d i e s have not been done t o answer the i n t e r e s t i n g q u e s t i o n of how ammonia e x c r e t i o n and r a t e s of urea s y n t h e s i s and l o s s v a r y w i t h changes i n d i e t a r y p r o t e i n s u p p l y i n el a s m b r a n c h s . H i g h l e v e l s of u r e a i n marine elasmobranchs were f i r s t n o t e d by S t a d e l e r and F r e r i c h s i n 1858 ( S m i t h , 1936). In f a c t , t h e s e a n i m a l s m a i n t a i n plasma urea c o n c e n t r a t i o n s i n the range of 350-400 mM. Such h i g h l e v e l s of u r e a a r e a t t a i n e d t h r o u g h a c o m b i n a t i o n of f a c t o r s . Elasmobranchs have a l a r g e c a p a c i t y f o r h e p a t i c u r e a s y n t h e s i s ; u rea i s made v i a a 5-step pathway which i s v i r t u a l l y i d e n t i c a l t o t h a t of mammals ( F i g u r e 1 ) . Urea i s l o s t t o the environment o n l y a t low r a t e s . Both s k i n and g i l l s of elasmobranchs a r e e x t r e m e l y impermeable t o u r e a . C e l l membranes of elasmobranch g i l l s and s k i n a r e s p e c i f i c a l l y a d apted t o p r e v e n t l o s s of urea t o t h e environment; urea p a s s e s f r e e l y a c r o s s membranes of a l l o t h e r c e l l t y p e s (Fenstermacher et a l . , 1972). The p e r m e a b i l i t y c o e f f i c i e n t of R. e r i n a c e a g i l l f o r u r e a i s 1.0X10"J c m / s e c , a v a l u e e q u a l t o o n e - t e n t h of t h a t d e t e r m i n e d f o r t o a d b l a d d e r (Payan e t a l . , 1973). F u r t h e r , the p e r m e a b i l i t y of R. e r i n a c e a g i l l t o u r e a i s not s e n s i t i v e t o o s m o l a r i t y of the e x t e r n a l medium. Payan e t a_l. (1973) r e p o r t t h a t b r a n c h i a l urea e x c r e t i o n d e c r e a s e s from 57 Mmoles/100 g/hour t o 20 jumoles/100 g/ hour when a n i m a l s a r e t r a n s f e r e d from 100% t o 50% se a w a t e r , i n d i r e c t p r o p o r t i o n t o d e c r e a s e i n plasma urea c o n c e n t r a t i o n (see b e l o w ) . Elasmobranchs a r e h i g h l y e f f i c i e n t a t a b s o r b i n g urea a c r o s s the 7 r e n a l t u b u l e s . A p p r o x i m a t e l y 95% of a l l urea which i s f i l t e r e d out of the plasma a t the g l o m e r u l u s i s r e a b s o r b e d a c r o s s the r e n a l t u b u l e s a g a i n s t a c h e m i c a l g r a d i e n t . R e n a l l o s s e s of urea amount t o a p p r o x i m a t e l y 2 /_mol/l00 g/hour i n R. e r i n a c e a i n 100% seawater and i n c r e a s e t o 9 jumol/100 g/hour when a n i m a l s a r e a c c l i m a t e d t o 50% seawater (Payan e t a l . , 1973). G o l d s t e i n and F o r s t e r (1971) d i s c o v e r e d t h a t a d a p t i n g R. e r i n a c e a t o 50% seawater caused plasma urea c o n c e n t r a t i o n t o d e c r e a s e by 45%, and t h a t t o t a l urea e x c r e t i o n was reduced. The a u t h o r s t h e r e f o r e c o n c l u d e d t h a t r e d u c t i o n i n plasma urea c o n c e n t r a t i o n i n reponse t o e n v i r o n m e n t a l d i l u t i o n was a consequence of i n c r e a s e d r e n a l c l e a r a n c e and a d e c r e a s e i n b i o s y n t h e s i s . T r i m e t h y l a m i n e - N - o x i d e The p r e s e n c e of t r i m e t h y l a m i n e - N - o x i d e (TMAO) was f i r s t d e m o n s t r a t e d i n elasmobranchs i n 1909 ( S m i t h , 1936). Plasma l e v e l s of t h i s compound i n elasmobranchs a r e t y p i c a l l y i n the range of 80 mM (Cohen e t a l . , 1958; G o l d s t e i n and P a l a t t , 1974). The o r i g i n of t h i s TMAO v a r i e s among s p e c i e s . Some el a s m o b r a n c h s , i n c l u d i n g t he nu r s e shark (Ginqlymostoma  c i r r a t u m ) and lemon shark ( N e q a p r i o n b r e v i r o s t r i s ) , s y n t h e s i z e TMAO from t e r t i a r y amines such as c h o l i n e and t r i m e t h y l a m i n e . Other s p e c i e s , such as the s p i n y d o g f i s h ( S q u a l u s a c a n t h i a s ) and l i t t l e s k a t e ( R a j a e r i n a c e a ) , l a c k the enzymes n e c e s s a r y f o r TMAO s y n t h e s i s and a r e thought t o r e l y upon d i e t a r y s o u r c e s ( G o l d s t e i n and D e w i t t - H a r l e y , 1973; G o l d s t e i n and Funkhouser, 1972). As i s the case f o r u r e a , 95-99% of TMAO f i l t e r e d a t the e 1 . The O r n i t h i n e Urea C y c l e . T h i s f i g u r e i l l u s t r a t e s the enzyme compartmentation found i n elasmobranchs; i n mammals a r g i n a s e i s l o c a t e d i n the c y t o s o l . Enzymes c o r r e s p o n d i n g t o numbers a r e as f o l l o w s : 1 . Carbamyl phosphate s y n t h e t a s e 2 . O r n i t h i n e t r a n s c a r b a m y l a s e 3 . A r g i n i n o s u c c i n a t e s y n t h e t a s e 4 . A r g i n i n o s u c c i n a t e l y a s e 5 . A r g i n a s e M I T O C H O N D R I O N G L U T A M I N E + H C O , ~ + H _ 0 A S P A R T A T E C Y T O S O L 10 g l o m e r u l u s i s a c t i v e l y r e a b s o r b e d by the r e n a l t u b u l e (Cohen e t a l . , 1958; G o l d s t e i n and P a l a t t , 1974). Only minute amounts of TMAO a r e l o s t a c r o s s the g i l l s ( G o l d s t e i n , 1982). R e t e n t i o n of TMAO i s p e c u l i a r t o elasmobranchs and perhaps o t h e r osmoconformers; when t h i s c h e m i c a l i s a d m i n i s t e r e d t o o t h e r v e r t e b r a t e s i n c l u d i n g t e l e o s t s , i t i s pr o m p t l y e x c r e t e d i n the u r i n e ( N o r r i s e t a l ., 1945; De La Huerga e t a l . , 1951). I n c o n t r a s t w i t h u r e a , TMAO c o n c e n t r a t i o n s a r e not f u l l y e q u i l i b r a t e d t h r o u g h o u t a l l body compartments, and the e x a c t r o l e of TMAO i n c e l l volume r e g u l a t i o n i s unknown ( K i n g and G o l d s t e i n , 1983). G o l d s t e i n and F o r s t e r (1971) r e p o r t t h a t plasma TMAO l e v e l s d e c r e a s e by 27% when R. e r i n a c e a i s adapted t o 50% seawater. I t s h o u l d be no t e d , however, t h a t i n t r a c e l l u l a r TMAO c o n c e n t r a t i o n s were found t o change v e r y l i t t l e i n marine elasmobranchs upon a d a p t a t i o n t o a d i l u t e environment ( F o r s t e r and G o l d s t e i n , 1976). Amino Ac i d s Among a q u a t i c i n v e r t e b r a t e s ( S c h o f f e n i e l s , 1976; G i l l e s , 1979) and v e r t e b r a t e s (Yancey e_t ajL. , 1982) c e r t a i n amino a c i d s and amino a c i d d e r i v a t i v e s , w h i c h v a r y from s p e c i e s t o s p e c i e s and among t i s s u e s , a r e r e t a i n e d a t h i g h c o n c e n t r a t i o n s w i t h i n c e l l s . Most n o n - o s m o t i c a l l y a c t i v e amino a c i d s a r e p r e s e n t i n the plasma a t l e v e l s not e x c e e d i n g 1 mM (Boyd e t a _ l . , 1977). I n t r a c e l l u l a r c o n c e n t r a t i o n s of 0 - a l a n i n e and s a r c o s i n e , two o s m o t i c a l l y a c t i v e amino a c i d s i n s k a t e wing muscle, a r e 40.7 mM, and 44 mM, r e s p e c t i v e l y ; t a u r i n e and /3-alanine a r e the 11 predominant amino a c i d s i n R. er i n a c e a e r y t h r o c y t e s , where t h e y o c c u r a t l e v e l s of 75.6 mM and 50.6 mM r e s p e c t i v e l y . T o t a l f r e e amino a c i d s c o m p r i s e more than 20% of o s m o t i c a l l y a c t i v e s o l u t e s i n s k a t e muscle ( F o r s t e r and G o l d s t e i n , 1976). In c o n t r a s t , plasma l e v e l s of t h e s e amino a c i d s do not exceed 0.5 mM (Boyd e t a l . , 1977). T i s s u e s which m a i n t a i n h i g h c o n c e n t r a t i o n s of s p e c i f i c amino a c i d s a p p a r e n t l y l a c k the c a p a c i t y t o e i t h e r s y n t h e s i z e o r o x i d i z e them ( G o l d s t e i n , 1981). 0 - a l a n i n e i s s y n t h e s i z e d i n the l i v e r , r e l e a s e d i n t o the b l o o d , and s u b s e q u e n t l y t a k e n up by o t h e r t i s s u e s f o r c e l l volume r e g u l a t i o n ( S h u t t l e w o r t h and G o l d s t e i n , 1984) and t h e same i s a p p a r e n t l y t r u e f o r s a r c o s i n e . T a u r i n e appears t o be o b t a i n e d o n l y from d i e t a r y s o u r c e s ( K i n g e t a l . , 1980; G o l d s t e i n , 1981). D u r i n g e n v i r o n m e n t a l d i l u t i o n c o n c e n t r a t i o n s of o s m o t i c a l l y a c t i v e amino a c i d s and amino a c i d d e r i v a t i v e s d e c r e a s e s i g n i f i c a n t l y (Boyd e_t a l . , 1977; G o l d s t e i n , 1981). E x c r e t i o n of amino a c i d s would i n v o l v e a s u b s t a n t i a l l o s s of p o t e n t i a l m e t a b o l i c energy and the a b i l i t y t o h a r v e s t t h i s energy would p r o v i d e a s e l e c t i v e advantage. In R. e r i n a c e a s a r c o s i n e and 0 - a l a n i n e a r e r e l e a s e d from c e l l s d u r i n g hypoosmotic s t r e s s and t a k e n up by the l i v e r where t h e y a r e o x i d i z e d ( K i n g e t a l . , 1980; B a l l a n t y n e e t a l . , 1986). Elasmobranchs appear unable t o m e t a b o l i z e t a u r i n e , and e x c r e t e i t i n the u r i n e ( G o l d s t e i n , 1981). 1 2 Responses of R a j a e r i n a c e a t o Osmotic S t r e s s The r e s p o n s e s of R. e r i n a c e a t o hypoosmotic s t r e s s have been w e l l documented by G o l d s t e i n and F o r s t e r (1971). They found t h a t a d a p t a t i o n of s k a t e s t o 50% seawater caused plasma c o n c e n t r a t i o n s of u r e a , c h l o r i d e , and TMAO t o d e c l i n e by 45, 30, and 27% r e s p e c t i v e l y . U r i n e f l o w was e l e v a t e d s i x f o l d , and g l o m e r u l a r f i l t r a t i o n r a t e was i n c r e a s e d f o u r - f o l d , as de t e r m i n e d w i t h i n u l i n c l e a r a n c e measurements. R e n a l c l e a r a n c e s of u r e a , c h l o r i d e and TMAO i n c r e a s e d 22, 6, and 13 t i m e s r e s p e c t i v e l y f o l l o w i n g e n v i r o n m e n t a l d i l u t i o n . The p e r c e n t f i l t e r e d u r e a e x c r e t e d by t h e k i d n e y s i n c r e a s e d s i x - f o l d i n s k a t e s m a i n t a i n e d i n 50% seawater w h i l e p e r c e n t a g e s of f i l t e r e d c h l o r i d e and TMAO e x c r e t e d remain e s s e n t i a l l y unchanged ( G o l d s t e i n and F o r s t e r , 1971). Rate of b r a n c h i a l urea l o s s d e c r e a s e d p r o p o r t i o n a t e l y w i t h plasma urea c o n c e n t r a t i o n s (Payan e t a l . , 1973). T o t a l body c l e a r a n c e of urea was e s s e n t i a l l y u n a f f e c t e d by hypoosmotic s t r e s s . Ammonia p r o d u c t i o n was found t o be not s i g n i f i c a n t l y a f f e c t e d by e n v i r o n m e n t a l d i l u t i o n ( G o l d s t e i n and F o r s t e r , 1971), however r e s u l t s were somewhat ambiguous and a d e c r e a s e i n n i t r o g e n m e t a b o l i s m c o u l d not be r u l e d out as an e x p l a n a t i o n of t h e i r o b s e r v e d d e c r e a s e i n r a t e of urea s y n t h e s i s . Thus, d e c r e a s e d plasma urea c o n c e n t r a t i o n s i n a n i m a l s a c c l i m a t e d t o 50% seawater a r e a t t r i b u t e d t o a de c r e a s e d r a t e of urea b i o s y n t h e s i s , p o s s i b l y due t o a d e c r e a s e i n p r o t e i n and amino a c i d m e t a b o l i s m , combined w i t h a m a i n t a i n e d r a t e of ur e a l o s s . The p r e s e n t s t u d y was u n d e r t a k e n i n an e f f o r t answer the 13 i n t r i g u i n g q u e s t i o n of how e u r y h a l i n e elasmobranchs r e g u l a t e the r a t e of h e p a t i c urea s y n t h e s i s i n response t o o s m o t i c s t r e s s . I s o l a t e d s k a t e h e p a t o c y t e s p r o v i d e d an i d e a l m e t a b o l i c system, and were t h e s u b j e c t of a l l e x p e r i m e n t s . Two p o s s i b l e p h y s i o l o g i c a l mechanisms were i n v e s t i g a t e d . I t was h y p o t h e s i z e d t h a t hormonal r e g u l a t i o n might be i n v o l v e d , t h e r e f o r e the e f f e c t s of f o u r s e l e c t e d hormones ( i n s u l i n , g l u c a g o n , 1a~OH, and 1,2-dehydro-A), on urea s y n t h e s i s were s t u d i e d . A second h y p o t h e s i s e x p l o r e d was the p o s s i b l e r e g u l a t o r y r o l e on urea s y n t h e s i s of a l t e r i n g the c o m p o s i t i o n of the e x t r a c e l l u l a r f l u i d . I n many c a s e s t r e a t m e n t e f f e c t s on a l a n i n e o x i d a t i o n and g l u c o n e o g e n e s i s were measured s i m u l t a n e o u s l y w i t h urea s y n t h e s i s f o r c o m p a r a t i v e p u r p o s e s . The g i v e n r e g u l a t o r y mechanisms a r e d e a l t w i t h i n s e p a r a t e c h a p t e r s . E x p e r i m e n t a l t r e a t m e n t s and t h e r a t i o n a l e b e h i n d them a r e f u l l y e x p l a i n e d i n the i n t r o d u c t o r y s e c t i o n of a p p r o p r i a t e c h a p t e r s . 1 4 MATERIALS AND METHODS An i m a l s S k a t e s ( R a j a e r i n a c e a ) were o b t a i n e d by o t t e r t r a w l i n Passamaquoddy Bay, near S t . Andrews, New B r u n s w i c k . A n i m a l s were h e l d w i t h o u t f e e d i n g i n f l o w i n g seawater f o r s e v e r a l days t o a p p r o x i m a t e l y two weeks p r i o r t o use i n the summer, and f o r up t o t s e v e r a l months i n the w i n t e r when i t was d i f f i c u l t t o c o l l e c t f r e s h specimens. S a l i n i t y was 29 ppt and te m p e r a t u r e v a r i e d from 6°C t o 12°C between w i n t e r and summer. Ch e m i c a l s A l l amino a c i d s , enzymes, and d e f a t t e d b o v i n e serum a l b u m i n (BSA) were o b t a i n e d from Sigma Ch e m i c a l Company, S t . L o u i s , MO. S a l t s used i n i s o l a t i o n and i n c u b a t i o n media were of the h i g h e s t a v a i l a b l e p u r i t y and were o b t a i n e d from e i t h e r F i s h e r S c i e n t i f i c or BDH. Hyamine h y d r o x i d e , U - 1 f C - a l a n i n e and NaH'fC0 3 were a l l p u r c h a s e d from New E n g l a n d N u c l e a r , L a c h i n e , Quebec. U- 1|C-a l a n i n e had a s p e c i f i c a c t i v i t y of 170 Ci/mole and was d i s s o l v e d i n 0.01 M H C l . 250 juCi of NaH'fCOa was pu r c h a s e d i n powder form and d i s s o l v e d i n 5.0 ml of NaOH, pH 11.0, y i e l d i n g a s p e c i f i c a c t i v i t y of 7.7 C i / m o l . 15 H e p a t o c y t e P r e p a r a t i o n H e p a t o c y t e s were p r e p a r e d a c c o r d i n g t o F r e n c h e t a l . (1981) w i t h a number of m o d i f i c a t i o n s . B r i e f l y , a n i m a l s were p i t h e d and l i v e r s were i m m e d i a t e l y e x c i s e d and p l a c e d i n a watch g l a s s . The r i g h t and l e f t h e p a t i c p o r t a l v e i n s were c a n n u l a t e d w i t h PE50 t u b i n g , which was s e c u r e d i n the v e s s e l s w i t h s i l k t h r e a d . A two c h a n n e l G i l s o n M i n i p u l s p e r i s t a l t i c pump, w i t h f l o w r a t e a d j u s t e d t o 2.5 m l / m i n / l i n e , was used t o p e r f u s e the l i v e r w i t h a p p r o p r i a t e s o l u t i o n s . I c e - c o l d s k a t e s a l i n e ( S o l u t i o n A, T a b l e 1) was used i n the i n i t i a l 3~10 minutes of p e r f u s i o n , d u r i n g which time most b l o o d was f l u s h e d from the l i v e r . Once the l i v e r was c l e a r e d of b l o o d , c o l l a g e n a s e (Sigma type IV , C l o s t r i d i u m sp. ) was added t o the p e r f u s i o n medium (20 mg/50 m l ) . P e r f u s i o n w i t h r e c i r c u l a t e d c o l l a g e n a s e s o l u t i o n was c o n t i n u e d f o r 20-60 minutes u n t i l the l i v e r showed s i g n s of d i s i n t e g r a t i o n ( p u f f i n e s s , b l i s t e r i n g ) . P e r f u s i o n was then stopped and the g a l l b l a d d e r c a r e f u l l y removed from t h e l i v e r . T i s s u e was t r a n s f e r r e d i n t o a p e t r i d i s h w i t h i c e - c o l d S o l u t i o n A. P o o r l y p e r f u s e d a r e a s were removed and r e m a i n i n g t i s s u e was f i n e l y minced w i t h a r a z o r b l a d e and poured t h r o u g h two s u c c e s s i v e s c r e e n s of p l a n k t o n n e t t i n g (253 and 73 (xm r e s p e c t i v e l y ) . M assaging of t i s s u e t h r o u g h the c o a r s e r s c r e e n and washing t i s s u e p i e c e s w i t h c o l d S o l u t i o n A g r e a t l y i n c r e a s e d h e p a t o c y t e y i e l d . C e l l s were c o l l e c t e d by low speed c e n t r i f u g a t i o n (approx. 70 g) f o r two minutes a t 4°C. The r e s u l t i n g s u p e r n a t a n t and f a t pad were d e c a n t e d and c e l l s r esuspended and washed once i n each of S o l u t i o n s B and C ( T a b l e 1 6 1) . C e l l s were f i n a l l y resuspended i n S o l u t i o n C a t a c o n c e n t r a t i o n of 30-100 mg/ml. C e l l s were e i t h e r used i m m e d i a t e l y or e l s e p l a c e d i n p l a s t i c s c i n t i l l a t i o n v i a l s w i t h a 99% 0 2 / l % C 0 2 environment and r e f r i g e r a t e d u n t i l use. In the l a t t e r case h e p a t o c y t e s were p e r i o d i c a l l y r esuspended by g e n t l e s w i r l i n g and washed once more w i t h S o l u t i o n C i m m e d i a t e l y p r i o r t o use. H e p a t o c y t e s were always used w i t h i n 48 hours of p r e p a r a t i o n , d u r i n g > w h i c h time t h e i r p h y s i o l o g i c a l c o n d i t i o n d i d not d e t e r i o r a t e (see r e s u l t s , hormone c h a p t e r ) . C e l l I n t e g r i t y Y i e l d of h e p a t o c y t e s from t h e i s o l a t i o n p r o c e d u r e v a r i e d w i d e l y depending upon the l i p i d c o n t e n t of the l i v e r . Some a n i m a l s , p a r t i c u l a r l y d u r i n g the summer, had h i g h i n t r a c e l l u l a r h e p a t i c l i p i d c o n t e n t , which made c e l l s p o s i t i v e l y buoyant and i m p o s s i b l e t o c o l l e c t by c e n t r i f u g a t i o n . C e l l s were r o u t i n e l y examined m i c r o s c o p i c a l l y t o a s s e s s c o n t a m i n a t i o n by r e d b l o o d c e l l s and m e l a n o c y t e s , which d i d not exceed 2% i n e i t h e r c a s e . C e l l v i a b i l i t y was e s t a b l i s h e d by the a b i l i t y of h e p a t o c y t e s t o e x c l u d e 0.02% Trypan b l u e (>98%). R. er i n a c e a h e p a t o c y t e s m a i n t a i n i n t e g r i t y over a wide range of o s m o l a r i t i e s as judged by v e r y low l e a k a g e of mala t e dehydrogenase from c e l l s ( B a l l a n t y n e e t a l . , 1986). Performance of m e t a b o l i c p r o c e s s e s which r e q u i r e f u n c t i o n a l l y d i f f e r e n t c e l l u l a r compartments (urea s y n t h e s i s , g l u c o n e o g e n e s i s ) a l s o e s t a b l i s h e d c e l l v i a b i l i t y (Moon e t a l . , 1985; C o r n e l l , 1983). P r e l i m i n a r y e x p e r i m e n t s 1 7 were done t o ensure t h a t r a t e s of u r e a s y n t h e s i s , a l a n i n e o x i d a t i o n , and g l u c o n e o g e n e s i s were l i n e a r o v er the two hour i n c u b a t i o n p e r i o d used. . A l a n i n e O x i d a t i o n and G l u c o n e o g e n e s i s A l a n i n e o x i d a t i o n was measured by c o l l e c t i n g and c o u n t i n g 1 f C 0 2 e v o l v e d when c e l l s were i n c u b a t e d i n the presence of u n i f o r m l y l a b e l l e d 1 | C ~ a l a n i n e . Rates of g l u c o n e o g e n e s i s were measured i n the same samples by d e t e r m i n i n g t h e amount of 1 f C -g l u c o s e which was s y n t h e s i s e d d u r i n g t h e i n c u b a t i o n p e r i o d ( F r e n c h e t a l . , 1981). H e p a t o c y t e s were suspended i n a f i n a l volume of 1.4 ml, and i n c u b a t e d i n 20 ml g l a s s s c i n t i l l a t i o n v i a l s i n a s h a k i n g w a t e r b a t h a t 10°C. I n c u b a t i o n v i a l s were s e t up as f o l l o w s : 1. 700 ul of an a p p r o p r i a t e 0 2 s a t u r a t e d i n c u b a t i o n medium c o n t a i n i n g u n l a b e l l e d a l a n i n e ( f i n a l c o n c e n t r a t i o n 1 mm) was p i p e t t e d i n t o each i n c u b a t i o n v i a l . 2. V i a l s were capped w i t h rubber w e l l c a p s , each of which c o n t a i n e d a 2.4 cm Whatman GFA m i c r o f i b e r f i l t e r . ^ 3. V i a l s were t e m p e r a t u r e - e q u i l i b r a t e d i n t h e w a t e r b a t h f o r a few m i n u t e s . 4. Caps were removed and 500 M1 of c e l l s u s p e n s i o n c o n t a i n i n g a known amount of c e l l s was p i p e t t e d i n t o each f l a s k . 18 5. Caps were r e p l a c e d and c e l l s were p r e i n c u b a t e d a t 10°C f o r 15 m i n u t e s . 6. 200 M1 of S o l u t i o n C c o n t a i n i n g 0.1 uCi of u n i f o r m l y 1|C-a l a n i n e ( f i n a l s p e c i f i c a c t i v i t y = a p p r o x i m a t e l y 180,000 DPM/Mmol) was i n j e c t e d i n t o each f l a s k t h r o u g h the rubber c a p s . 7. C o n t r o l f l a s k s c o n t a i n e d 1200 ul of S o l u t i o n C and 200 ul of l a b e l l e d S o l u t i o n C. A f t e r a 2 hour i n c u b a t i o n a t 10°C e x p e r i m e n t s were t e r m i n a t e d by i n j e c t i o n t h r o u g h the cap of 100 ul of 70% p e r c h l o r i c a c i d (PCA). Hyamine h y d r o x i d e (1M i n methanol) (150 M D was then i n j e c t e d onto GFA f i l t e r s and f l a s k s were shaken a t room t e m p e r a t u r e f o r 90 minutes f o r c o l l e c t i o n of C 0 2 . F i l t e r s were then removed and coun t e d i n 5 ml of a t o l u e n e - b a s e d s c i n t i l l a t i o n f l u i d (2.4 1 t o l u e n e , 0.6 1 e t h a n o l , 6.0 g PPO ( 2 , 5 - d i p h e n y l o x a z o l e ) , 0.3 g POPOP ( 1 , 4 - b i s [ 2 - ( 5 -p h e n y l o x a z o l y l ) ] - b e n z e n e ) ) f o r 10 m i n u t e s . A Rackbeta 1211 s c i n t i l l a t i o n c o u n t e r w i t h an e x t e r n a l s t a n d a r d r a t i o (ESR) f o r quench c o r r e c t i o n was used. F o l l o w i n g c o l l e c t i o n of C 0 2 a l l f l a s k s were p a r t i a l l y n e u t r a l i z e d w i t h 2M K 2 C 0 3 and r e f r i g e r a t e d f o r no l o n g e r than 5 days p r i o r t o a s s a y i n g f o r ' f C - g l u c o s e . Immediately b e f o r e use 1 f C - g l u c o s e samples were f u l l y n e u t r a l i z e d (pH 6.5-7.5) w i t h 2 M K 2 C 0 3 . For each sample 2 g of A m b e r l i t e MB-3 i o n exchange r e s i n was p l a c e d i n a g l a s s s c i n t i l l a t i o n v i a l and c o v e r e d w i t h 3.6 ml of a 1 M g l u c o s e s o l u t i o n . 400 j i l of n e u t r a l i z e d sample (from above) was the n added, y i e l d i n g a f i n a l volume of 4.0 ml. V i a l s were c o v e r e d w i t h P a r a f i l m and shaken v i g o r o u s l y a t room 19 t e m p e r a t u r e f o r 2 h o u r s . A 1.2 ml a l i q u o t of g l u c o s e s o l u t i o n was then removed from each v i a l and c e n t r i f u g e d a t h i g h speed f o r 2 m i n u t e s t o remove s m a l l and broken r e s i n p a r t i c l e s . 1.0 ml of t h e s u p e r n a t a n t was removed and c o u n t e d i n 8.0 ml of S c i n t i v e r s e 1 ( F i s h e r S c i e n t i f i c ) , an aqueous s c i n t i l l a t i o n f l u i d , f o r 10 m i n u t e s . T o t a l r a d i o a c t i v i t y added was d e t e r m i n e d by c o u n t i n g 100 ul of n e u t r a l i z e d sample from 2 s e p a r a t e i n c u b a t i o n v i a l s chosen a t random. I t s h o u l d be noted t h a t t h i s a s s a y i s not e n t i r e l y s p e c i f i c f o r g l u c o s e . S t e p s have been t a k e n , however, t o m i n i m i z e p o s s i b l e c o n t a m i n a t i o n from o t h e r n e u t r a l 1 f C compounds which may have been s y n t h e s i s e d d u r i n g the i n c u b a t i o n p e r i o d from 1 f C a l a n i n e , such as g l y c e r o l and u r e a . U s i n g the above p r o c e d u r e Mommsen and Moon ( u n p u b l i s h e d ) have found t h a t c o n t a m i n a t i o n caused by o t h e r n e u t r a l m e t a b o l i c p r o d u c t s i s i n s i g n i f i c a n t . T h i s method has been shown t o remove more than 99.9 % of e x c e s s c h a r g e d l a b e l l e d s u b s t r a t e s (Walton and Cowey, 1979) and use of a p p r o p r i a t e c o n t r o l s a c c o u n t s f o r the r e m a i n d e r . Urea S y n t h e s i s Urea s y n t h e s i s was measured by d e t e r m i n i n g r a t e s of f i x a t i o n of 1 f C - b i c a r b o n a t e i n t o u r e a . A h i g h l y s p e c i f i c two-s t e p assay u s i n g u r e a s e was employed. I n c u b a t i o n v i a l s were s e t up e x a c t l y as d e s c r i b e d f o r a l a n i n e o x i d a t i o n / g l u c o n e o g e n e s i s e x p e r i m e n t s , however N a H 1 f C 0 3 was d i s s o l v e d i n S o l u t i o n C and i n j e c t e d i n t o i n c u b a t i o n f l a s k s (0.2 M C i / f l a s k ) i n s t e a d of l a b e l l e d a l a n i n e . A g a i n , c e l l s were 20 i n c u b a t e d f o r 2 hours a t 10°C i n a s h a k i n g water b a t h . E x p e r i m e n t s were t e r m i n a t e d and C 0 2 was c o l l e c t e d and counted as d e s c r i b e d above. C o l l e c t i o n of a l l 1 f C r a d i o a c t i v i t y made i t p o s s i b l e t o a c c u r a t e l y d e t e r m i n e the s p e c i f i c a c t i v i t y of each i n d i v i d u a l f l a s k . Samples were r e f r i g e r a t e d f o r a maximum of 5 days p r i o r t o c o m p l e t i o n of the a s s a y . For the second s t e p of t h e a s s a y samples were n e u t r a l i s e d w i t h 2M KOH i n 0.5 M i m i d a z o l e . For each a s s a y 500 ul of the r e s u l t i n g s u p e r n a t a n t was p i p e t t e d i n t o a f r e s h s c i n t i l l a t i o n v i a l which was then s e a l e d w i t h a rubber w e l l cap ( h i g h background l e v e l s of urea p r e s e n t i n the i n c u b a t i o n medium (350 mM), combined w i t h the l i m i t e d c a p a c i t y of Hyamine h y d r o x i d e f o r a b s o r b t i o n of C 0 2 , made i t n e c e s s a r y t o reduce the volume used i n the urease i n c u b a t i o n from 1.5 m l ) . A g a i n , each w e l l c o n t a i n e d a Whatman GFA f i l t e r . 100 ul of a u r e a s e s o l u t i o n (40 mg of Sigma Type IX u r e a s e / m l of 50 mM i m i d a z o l e , pH 7.4) was i n j e c t e d t h r o u g h the rubber cap onto the 500 ul of n e u t r a l i z e d sample t o h y d r o l y z e the u r e a c o n t a i n e d i n the o r i g i n a l medium. Only newly s y n t h e s i z e d u r e a c o u l d be l a b e l l e d w i t h carbon-14. F o l l o w i n g a d d i t i o n of u r e a s e , f l a s k s were shaken v i g o r o u s l y f o r 90 minutes a t room t e m p e r a t u r e and then a c i d i f i e d by i n j e c t i o n of 100 ul of 36% PCA. Once a g a i n C 0 2 was c o l l e c t e d and c o u n t e d as d e s c r i b e d above. T h i s l a t t e r s t e p e n s u r e s t h a t a l l c o u n t s o b t a i n e d a r e d e r i v e d from 1|C which was i n c o r p o r a t e d i n t o u r e a . 21 C a l c u l a t i o n s R a t es of s y n t h e s i s were c a l c u l a t e d f o r 60 minute i n c u b a t i o n s and f o r 1 g of packed h e p a t o c y t e s , u s i n g the s p e c i f i c a c t i v i t y d e t e r m i n e d f o r i n d i v i d u a l e x p e r i m e n t s . Rates of s u b s t r a t e o x i d a t i o n a r e g i v e n i n Mmoles of C0 2 e v o l v e d per gram of c e l l s per hour. T h e o r e t i c a l l y , d i v i s i o n by the number of c a r b o n s i n t h e compound (3 i n t h e case of a l a n i n e ) w i l l y i e l d t he r a t e of s u b s t r a t e u t i l i z a t i o n . R a t e s of g l u c o n e o g e n e s i s a r e g i v e n i n jumoles of g l u c o s e measured. I f the as s u m p t i o n s v a l i d a t e d f o r t e l e o s t s (Mommsen, 1986) a r e a l s o l e g i t i m a t e f o r elasmo b r a n c h s , r a t e s of a l a n i n e u t i l i z a t i o n as a g l u c o n e o g e n i c s u b s t r a t e can be c a l c u l a t e d by m u l t i p l y i n g by two the computed r a t e of g l u c o n e o g e n e s i s . E x p e r i m e n t s were always done i n d u p l i c a t e w i t h a complete b l o c k d e s i g n ; numerous t r e a t m e n t s were performed i n p a r a l l e l w i t h a l i q u o t s of c e l l s d e r i v e d from one a n i m a l . A maximum of 60 f l a s k s (15 t r e a t m e n t s ) c o u l d be p r o c e s s e d a t one t i m e . S t a t i s t i c a l A n a l y s i s B a r t l e t t ' s c h i - s q u a r e t e s t was used t o e s t a b l i s h h o m o s c e d a s t i c i t y of d a t a . Two-way a n a l y s i s of v a r i a n c e was a p p l i e d t o a l l complete b l o c k d e s i g n d a t a s e t s , f o l l o w e d by Tukey's t e s t f o r m u l t i p l e c o m p a r i s o n s ( Z a r , 1974) u s i n g the G e n l i n s t a t i s t i c a l package a v a i l a b l e on UNIX. 1 . S o l u t i o n s f o r I s o l a t i o n and I n c u b a t i o n of Skate H e p a t o c y t e s . S o l u t i o n A. ( P e r f u s i o n medium, m o d i f i e d from K i n g e t a l . , 1980). 281.0 mM NaCl 6.0 mM KC1 0.5 mM MgSO, 2.5 mM M g C l 2 1.0 mM NaH 2PO« 350.0 mM Urea 5.0 mM Hepes 8.0 mM NaHCOa S o l u t i o n B. (Washing of c e l l s ) S o l u t i o n A t o which i s added 1.0 mM C a C l 2 2% b o v i n e serum a l b u m i n , f a t t y a c i d f r e e 0.13 mM T a u r i n e 0.06 mM A s p a r a g i n e 0.01 mM S a r c o s i n e 0.03 mM /3-Alanine 0.10 mM A s p a r t a t e * 0.80 mM O r n i t h i n e * 0.10 mM G l u t a m i n e * * added a t 10 t i m e s p h y s i o l o g i c a l s k a t e plasma c o n c e n t r a t i o n as r e p o r t e d by Boyd et a l . , (1977). A l l o t h e r amino a c i d s added were at p h y s i o l o g i c a l plasma c o n c e n t r a t i o n s . S o l u t i o n C. ( I n c u b a t i o n of c e l l s ) . S o l u t i o n B w i t h the f o l l o w i n g m o d i f i c a t i o n s : 241.0 mM NaCl 80.0 mM TMAO 24 EFFECTS OF INSULIN, GLUCAGON, 1a-OH, AND 1,2-DEHYDRO-A ON UREA SYNTHESIS AND ALANINE OXIDATION I n t r o d u c t i o n A number of f a c t o r s may a f f e c t urea s y n t h e s i s i n a n i m a l s w h i c h make t h i s compound p u r e l y as a waste p r o d u c t . The r e l a t i v e importance of t h e s e r e g u l a t o r s i s d i f f i c u l t t o q u a n t i f y i n view of both the c o m p l e x i t y of i n t e r a c t i o n s between them, and the wide range of p h y s i o l o g i c a l s t a t e s e x p e r i e n c e d by most a n i m a l s . For example, some r e g u l a t o r s may predominate d u r i n g s t a r v a t i o n , w h i l e o t h e r s may be of importance when d i f f e r e n t t y p e s of food a r e p l e n t i f u l . A number of g e n e r a l mechanisms which a r e i n v o l v e d i n r e g u l a t i o n of u r e a s y n t h e s i s i n t e r r e s t r i a l a n i m a l s a r e as f o l l o w s : 1. D i r e c t e f f e c t s of n u t r i t i o n a l s t a t e of t h e a n i m a l ; h i g h l e v e l s of p r o t e i n i n t a k e i n t r o d u c e l a r g e amounts of n i t r o g e n w h i c h must be e l i m i n a t e d (Schimke, 1962). 2. Hormones a s s o c i a t e d w i t h r e g u l a t i o n of plasma m e t a b o l i t e l e v e l s such as i n s u l i n and g l u c a g o n . 3. A c i d - b a s e r e g u l a t i o n and removal of HC0 3~; t h i s i s a somewhat c o n t r o v e r s i a l c o n c e p t put f o r t h by A t k i n s o n and co w o r k e r s (Bean and A t k i n s o n , 1984; A t k i n s o n and Bourke, 1984) and d e a l t w i t h i n p a r t by H a l p e r i n and Jungas (1983), and Walser ( 1 9 8 6 ) . C o m p a r a t i v e l y l i t t l e i s known about the e n d o c r i n o l o g y of o s m o r e g u l a t i o n i n el a s m o b r a n c h s , and no p r e v i o u s s t u d i e s have 25 been done e x p l o r i n g the r o l e of hormones i n r e g u l a t i n g urea s y n t h e s i s i n the s e a n i m a l s . V a r i o u s p e p t i d e and s t e r o i d hormones have r e g u l a t o r y e f f e c t s on urea s y n t h e s i s i n mammals, and o t h e r s a r e i n v o l v e d w i t h o s m o r e g u l a t i o n i n t e l e o s t s . Hormones and Urea S y n t h e s i s C o r t i s o l and gl u c a g o n a r e known t o i n f l u e n c e u rea s y n t h e s i s i n mature mammals, and i n s u l i n a l l e g e d l y e x e r t s some i n d i r e c t e f f e c t s . I n j e c t i o n of p h y s i o l o g i c a l t o p h a r m a c o l o g i c a l doses of C o r t i s o l i n t o r a t s r a i s e d l e v e l s of urea c y c l e enzymes and thus i n c r e a s e d the f l u x c a p a c i t y of the pathway ( C h r i s t o w i t z e t a l . , 1981). I t s h o u l d be n o t e d t h a t r a t s s e c r e t e m a i n l y c o r t i c o s t e r o n e , r a t h e r than C o r t i s o l ( B e n t l e y , 1982), thu s t h e r e p o r t e d response may not be of p h y s i o l o g i c a l importance t o the a n i m a l s . I n f u s i o n of p h y s i o l o g i c a l c o n c e n t r a t i o n s of gl u c a g o n i n t o r a t s c auses c o o r d i n a t e d i n d u c t i o n of urea c y c l e enzymes (Snodgrass e t a l . , 1978) and s i m i l a r r e s u l t s were o b t a i n e d i n e x p e r i m e n t s where g l u c a g o n was a p p l i e d t o r a t h e p a t o c y t e s . A d d i t i o n a l l y , t h e r e i s a p o s i t i v e c o r r e l a t i o n between r a t e s of g l u c o n e o g e n e s i s and urea s y n t h e s i s i n mammals, w i t h u rea s y n t h e s i s b e i n g the dependant v a r i a b l e ( Kraus-Friedmann, 1984; M e i j e r e t al. , 1978); as g l u c a g o n i n c r e a s e s the r a t e of g l u c o n e o g e n e s i s i n b o t h mammals ( A q u i l a r - P a r a d a e t a _ l . , 1 969) and f i s h ( P e t e r s e n , 1987) i t may a l s o have some e f f e c t s , d i r e c t or i n d i r e c t , on urea s y n t h e s i s i n elasmobranchs. I n s u l i n has been found t o s t i m u l a t e uptake of amino a c i d s and t h e i r i n c o r p o r a t i o n i n t o p r o t e i n s i n both mammals and f i s h 26 ( P l i s e t s k a y a e_t a l . , 1986). A l s o , i n one s e t of e x p e r i m e n t s , i n f u s i o n of mammalian i n s u l i n i n t o unfed s p i n y d o g f i s h r e s u l t e d i n p r o l o n g e d d e p r e s s i o n of plasma a l a n i n e l e v e l s (DeRoos e t a l . , 1985), p o s s i b l y by s t i m u l a t i n g g l u c o n e o g e n e s i s and i n c o r p o r a t i n g a l a n i n e from the plasma i n t o g l u c o s e . A l a n i n e i s s u p p o s e d l y the o n l y amino a c i d r e l e a s e d i n s i g n i f i c a n t q u a n t i t i e s from d o g f i s h muscle d u r i n g s t a r v a t i o n (Leech e t a l . , 1979) and thus may be an i m p o r t a n t g l u c o n e o g e n i c s u b s t r a t e i n elas m o b r a n c h s . Because i n s u l i n a f f e c t s amino a c i d and t h e r e f o r e n i t r o g e n m e t a b o l i s m , I h y p o t h e s i z e d t h a t i t may e x e r t a t l e a s t an i n d i r e c t r o l e i n r e g u l a t i n g u rea s y n t h e s i s . Hormones and O s m o r e g u l a t i o n I n t e g r a t i o n of o s m o r e g u l a t o r y mechanisms r e l i e s l a r g e l y upon hormones, which c o o r d i n a t e the f u n c t i o n s of a l l o s m o r e g u l a t o r y o r g a n s . O s m o r e g u l a t i o n i n v e r t e b r a t e s depends upon k i d n e y s , u r i n a r y b l a d d e r , and the g u t , i n a d d i t i o n t o g i l l s and s a l t - s e c r e t i n g g l a n d s i f p r e s e n t . In elasmobranchs the l i v e r may a l s o be i n c l u d e d as i t i s the s i t e of s y n t h e s i s of o s m o t i c a l l y i m p o r t a n t amino a c i d s and u r e a , and i s a l s o the organ i n which o s m o t i c a l l y a c t i v e amino a c i d s a r e o x i d i s e d ( G o l d s t e i n , 1981). Hormones of o s m o r e g u l a t o r y importance t o t e l e o s t s i n c l u d e n e u r o h y p o p h y s i a l hormones ( v a s o t o c i n , v a s o p r e s s i n ) , a d r e n o c o r t i c o s t e r o i d s , p r o l a c t i n , and c a t e c h o l a m i n e s . W i t h the e x c e p t i o n of c o r t i c o s t e r o i d s t h e s e hormones were not u t i l i s e d i n the p r e s e n t s t u d y as they f u n c t i o n p r i m a r i l y i n r e g u l a t i n g f l u x 27 of s a l t s i n t o and out of the body, r a t h e r than i n r e g u l a t i n g r a t e of f l u x t h r o u g h m e t a b o l i c pathways. For a r e v i e w of the mechanisms of a c t i o n of t h e s e hormones i n t e l e o s t o s m o r e g u l a t i o n p l e a s e r e f e r t o B e n t l e y (1982). C o r t i c o s t e r o i d s have a w e l l - e s t a b l i s h e d r o l e i n t e l e o s t o s m o r e g u l a t i o n . Maintenance of adequate amounts of Na +/K +-ATPase i n t e l e o s t g u t , g i l l s ( c h l o r i d e c e l l s ) , and kidneys depends upon s e c r e t i o n of C o r t i s o l i n a p p r o p r i a t e amounts ( B e n t l e y , 1982). Na +/K +-ATPase f u n c t i o n s i n m a i n t a i n i n g i n t r a c e l l u l a r i o n c o m p o s i t i o n , and i n t r a n s p o r t i n g i o n s i n t o and out of the body. L i t t l e i s known about t h e o s m o r e g u l a t o r y e f f e c t s of c o r t i c o s t e r o i d s i n e l a s m o b r a n c h s , which have e x t r e m e l y low t o u n d e t e c t a b l e l e v e l s of C o r t i s o l and c o r t i c o s t e r o n e compared with nanomolar c o n c e n t r a t i o n s found i n t e l e o s t s (Hazon and Henderson, 1984). I n s t e a d , t h e s e a n i m a l s p o s s e s s a unique dominant c o r t i c o s t e r o i d which i s s y n t h e s i s e d i n the i n t e r r e n a l g l a n d , i d e n t i f i e d as 1 a - h y d r o x y c o r t i c o s t e r o n e (la-OH) by I d l e r and c o -workers (1967). R e c e p t o r s f o r 1a-OH a r e l o c a l i s e d i n the l i v e r , g i l l , k i d n e y , and r e c t a l g l a n d (Moon and I d l e r , 1974; I d l e r and Kane, 1980) w h i c h a r e t h e p r i n c i p l e o s m o r e g u l a t o r y o r g a n s . The f a c t t h a t t h i s hormone b i n d s p r e d o m i n a n t l y t o o s m o r e g u l a t o r y t i s s u e s s u g g e s t s t h a t i t may be i n v o l v e d w i t h o s m o r e g u l a t i o n i n elasmobranchs. In f a c t , 1a-OH i s known t o f u n c t i o n i n m a i n t a i n i n g the m e t a b o l i c i n t e g r i t y of the r e c t a l g l a n d ( H o l t and I d l e r , 1975), w h i c h p l a y s a r o l e i n e x c r e t i o n of d i v a l e n t i o n s . Hazon and Henderson (1984) found t h a t i n d o g f i s h h e l d i n 28 a wide range of s a l i n i t i e s (50%-140% seawater) plasma c o n c e n t r a t i o n s of 1a-OH were i n v e r s e l y r e l a t e d t o e n v i r o n m e n t a l s a l i n i t y and c o n s e q u e n t l y a l s o t o plasma s o l u t e c o n c e n t r a t i o n s . R e s u l t s of t h e i r s t u d y l e a d them t o suggest t h a t h o m e o s t a s i s of plasma c o m p o s i t i o n , w i t h p a r t i c u l a r r e s p e c t t o u r e a , may i n p a r t r e g u l a t e d by 1a-OH. R e c e n t l y , Ms. B. T r u s c o t t , of Dr. I d l e r s l a b o r a t o r y , has i s o l a t e d and p u r i f i e d a d e r i v a t i v e of 1a-OH, known as 1,2-dehydro-A. T h i s d e r i v a t i v e i s more p o l a r than 1a-OH, and T r u s c o t t e t a_l. (1978 ) suggest t h a t i t may be the m e t a b o l i c a l l y a c t i v e form of the hormone. To d a t e , however, no s t u d i e s have been p u b l i s h e d on the b i o l o g i c a l e f f e c t s of 1 ,2-dehydro-A. The purpose of t h e f o l l o w i n g s t u d y was t o examine the e f f e c t s of f o u r hormones ( i n s u l i n , g l u c a g o n , 1a-OH and 1,2-dehydro-A), on urea s y n t h e s i s and a l a n i n e o x i d a t i o n i n i s o l a t e d s k a t e l i v e r c e l l s . These hormones were s e l e c t e d f o r use i n the p r e s e n t s t u d y as i n f o r m a t i o n a v a i l a b l e i n r e c e n t l i t e r a t u r e s u g g e s t s a p o s s i b l e r o l e f o r each i n o s m o r e g u l a t i o n and/or r e g u l a t i o n of p r o t e i n m e t a b o l i s m , and t h e r e f o r e p o s s i b l y i n r e g u l a t i o n of u rea s y n t h e s i s . I n s u l i n and g l u c a g o n a r e p e p t i d e hormones, b o t h of which have been i d e n t i f i e d i n v i r t u a l l y a l l v e r t e b r a t e s ( B e n t l e y , 1 9 8 2 ) , i n c l u d i n g elasmobranchs (Conlon e t a l . , 1 9 8 6 ) . Responses of c e l l s t o t h e s e hormones a r e i n i t i a t e d when th e y b i n d t o plasma membranes, and a r e m e d i a t e d t h r o u g h a c t i v a t i o n or d e a c t i v a t i o n of enzymes by p h o s p h o r y l a t i o n . (For a d e t a i l e d review of mechanisms i n v o l v e d p l e a s e r e f e r t o Cohen, 1 9 8 5 ) . 29 R e g u l a t o r y e f f e c t s of p e p t i d e hormones a r e t y p i c a l l y i n s t a n t a n e o u s , t h u s o n l y s h o r t - t e r m i n c u b a t i o n s of h e p a t o c y t e s w i t h t h e s e hormones were performed. C o r t i c o s t e r o i d s a r e l a r g e , 1 i p i d - s o l u a b l e m o l e c u l e s which have two mechanisms of a c t i o n upon c e l l s , c a t e g o r i z e d as d i r e c t and p e r m i s s i v e e f f e c t s . D i r e c t a c t i o n of s t e r o i d hormones u s u a l l y i n v o l v e s , n u c l e a r e v e n t s i n which a hormone-receptor complex i n t e r r a c t s w i t h t a r g e t c e l l c h r o m a t i n i n such a way as t o a l t e r t r a n s l a t i o n of DNA ( K i n g and Green, 1984; Welshons, 1984). RNA s y n t h e s i s i s t h e r e b y e a l t e r e d , and u l t i m a t e l y a f f e c t s s y n t h e s i s of p r o t e i n s ( u s u a l l y enzymes or i n h i b i t o r y p r o t e i n s ) . There may be a c o n s i d e r a b l e time l a g between r e l e a s e of s t e r o i d hormones and a c c u m u l a t i o n of s p e c i f i c p r o t e i n s t o l e v e l s a t which they a l t e r m e t a b o l i s m . For t h i s reason h e p a t o c y t e s were i n c u b a t e d w i t h 1a-OH and 1,2-dehydro-A f o r p e r i o d s of 24 and 48 h o u r s . Mechanisms of a c t i o n of p e r m i s s i v e e f f e c t s a r e p o o r l y u n d e r s t o o d . I t was n o t e d by I n g l e (1952) t h a t some hormones, p a r t i c u l a r l y s t e r o i d s , can " p e r m i t " a second s t i m u l u s such as gl u c a g o n t o e x e r t an e f f e c t which would not o t h e r w i s e be p o s s i b l e . P e r m i s s i v e e f f e c t s o c c u r i n s t a n t l y ; t h e r e i s no l a g p e r i o d a s s o c i a t e d w i t h p r o t e i n s y n t h e s i s as o c c u r s w i t h d i r e c t e f f e c t s ( K r a u s - F r i e d m a n n , 1984). Thus, a f f e c t s of 1a-OH and 1,2-dehydro-A when combined w i t h i n s u l i n and g l u c a g o n were i n v e s t i g a t e d o n l y o ver a s h o r t i n c u b a t i o n p e r i o d . 3 0 E x p e r i m e n t a l D e s i g n S h o r t Term I n c u b a t i o n s H e p a t o c y t e s were i n c u b a t e d w i t h s c o m b r o i d i n s u l i n , b ovine g l u c a g o n , and elasmobranch 1a-OH and 1,2-dehydro-A, t o determine e f f e c t s of s h o r t term exposure on urea s y n t h e s i s and a l a n i n e o x i d a t i o n . Measurements of g l u c o n e o g e n i c r a t e c o u l d not be o b t a i n e d as a f a u l t y b a t c h of ion-exchange r e s i n was r e c e i v e d . I n s u l i n was o b t a i n e d from Dr. P.J. Clemens of Connaught L a b o r a t o r i e s , T o r o n t o , O n t a r i o , and g l u c a g o n was s u p p l i e d by C a l b i o c h e m , San D i e g o , CA. Ms. B. T r u s c o t t , M a r i n e S c i e n c e s R e s e a r c h L a b o r a t o r y , Memorial U n i v e r s i t y , N f l d . , g e n e r o u s l y donated the h i g h l y p u r i f i e d elasmobranch s t e r o i d hormones. I n s u l i n and g l u c a g o n were d i s s o l v e d i n M HCl a t a c o n c e n t r a t i o n of 1 mg per ml. F i n a l hormone c o n c e n t r a t i o n s i n c e l l i n c u b a t i o n s were 1X10'] M i n each case (Plasma c o n c e n t r a t i o n s of b o t h hormones a r e t y p i c a l l y i n t h e range of 1 t o 3X10"— M (E.M. P l i s e t s k a y a , p e r s o n a l c o m m u n i c a t i o n ) . S i n c e o n l y minute volumes of a c i d c o n t a i n i n g p e p t i d e hormones were added t o c e l l s , pH of i n c u b a t i o n media was u n a f f e c t e d . S t e r o i d hormones were d i s s o l v e d a t 40,000 t i m e s p h y s i o l o g i c a l c o n c e n t r a t i o n i n p r o p y l e n e g l y c o l ; o n l y s m a l l volumes (0.5 jul per ml of f i n a l i n c u b a t i o n volume) were added t o c e l l s . The f i n a l c o n c e n t r a t i o n of each s t e r o i d used i n e x p e r i m e n t s was 5X10"f M, a p p r o x i m a t e l y 10 t i m e s the p h y s i o l o g i c a l plasma c o n c e n t r a t i o n of 1a-OH r e p o r t e d f o r R. e r i n a c e a ( I d l e r and T r u s c o t t , 1969) . 31 Urea s y n t h e s i s and a l a n i n e o x i d a t i o n were measured as p r e v i o u s l y d e s c r i b e d , w i t h the f o l l o w i n g changes: hormones were added t o volumes of S o l u t i o n C a t t w i c e the d e s i r e d f i n a l c o n c e n t r a t i o n . 700 ju.1 of s o l u t i o n C c o n t a i n i n g hormone was then p i p e t t e d i n t o i n c u b a t i o n f l a s k s f o r g i v e n t r e a t m e n t s . Subsequent a d d i t i o n of 500 jul of c e l l s u s p e n s i o n and 200 jul of r a d i o a c t i v e l y l a b e l l e d s u b s t r a t e t o each f l a s k y i e l d e d the d e s i r e d f i n a l c o n c e n t r a t i o n of hormone i n a t o t a l volume of 1.4 ml. Treatments were as f o l l o w s : 1. C o n t r o l s ( p r o p y l e n e g l y c o l o n l y ) 2. I n s u l i n + p r o p y l e n e g l y c o l 3. I n s u l i n + 1a-OH 4. Glucagon + p r o p y l e n e g l y c o l 5. Glucagon + 1a-OH 6. 1a-OH 7. 1 ,2-dehydro-A E x p e r i m e n t s were done u s i n g a complete b l o c k d e s i g n , and d u p l i c a t e s f o r each t r e a t m e n t were done w i t h a l l b a t c h e s of c e l l s . Long Term Hormone I n c u b a t i o n s P l a s t i c s c i n t i l l a t i o n v i a l s were s e t up c o n t a i n i n g 1.0 ml of S o l u t i o n C and 5 ul of hormone s o l u t i o n ( p r o p y l e n e g l y c o l f o r c o n t r o l s ) . 9.0 ml of h e p a t o c y t e s u s p e n s i o n c o n t a i n i n g 500 mg of c e l l s was t h e n added t o each f l a s k . H e p a t o c y t e s were gassed b r i e f l y w i t h 99% 0 2 - 1 % C 0 2 , capped, and kept i n a r e f r i g e r a t o r a t 32 4°C f o r 24 h o u r s . C e l l s were p e r i o d i c a l l y resuspended d u r i n g t h i s time by g e n t l e s w i r l i n g . A f t e r 24 hou r s 5.0 ml of c e l l s u s p e n s i o n was removed, c e n t r i f u g e d , and resuspended i n 4.5 ml of S o l u t i o n C c o n t a i n i n g 5X10" f M hormone or 0.5 jul per ml of p r o p y l e n e g l y c o l i n the case of c o n t r o l s . C e l l s were then i n c u b a t e d i n g l a s s s c i n t i l l a t i o n v i a l s and r a t e s of urea s y n t h e s i s and a l a n i n e o x i d a t i o n were measured as p r e v i o u s l y d e s c r i b e d w i t h the f o l l o w i n g a d d i t i o n s . S o l u t i o n C added t o i n c u b a t i o n v i a l s c o n t a i n e d 5.85X10"f M hormone i n o r d e r t o compensate f o r d i l u t i o n of hormone which o c c u r s when r a d i o a c t i v e l a b e l s a r e added t o i n c u b a t i o n v i a l s . F o r reasons s t a t e d p r e v i o u s l y , measurements of g l u c o n e o g e n i c r a t e s c o u l d not be o b t a i n e d . The r e m a i n i n g 5.0 ml of c e l l s u s p e n s i o n f o r each t r e a t m e n t were g i v e n 1.0 ml of S o l u t i o n C c o n t a i n i n g a d d i t i o n a l f r e s h hormone (or 3 M1 of p r o p y l e n e g l y c o l f o r c o n t r o l t r e a t m e n t s ) . C e l l s were then gassed and r e f r i g e r a t e d as above f o r a n o t h e r 24 h o u r s . F o l l o w i n g a t o t a l i n c u b a t i o n p e r i o d of 48 hou r s r e m a i n i n g c e l l s were washed and resuspended as d e s c r i b e d , and urea s y n t h e s i s and a l a n i n e o x i d a t i o n were a s s e s s e d . 3 3 R e s u l t s and D i s c u s s i o n C e l l I n t e g r i t y M e t a b o l i c a l l y i n t a c t h e p a t o c y t e s were s u c c e s s f u l l y i s o l a t e d from s k a t e l i v e r . C e l l p r e p a r a t i o n s were homogeneous, and n e i t h e r r e d b l o o d c e l l s nor m e l a n o c y t e s c o m p r i s e d more than 2% of c e l l s u s p e n s i o n s . C e l l s e x c l u d e d the v i t a l s t a i n , Trypan B l u e (>98%), and c l u m p i n g of h e p a t o c y t e s d i d not o c c u r . F l u x r a t e s t h r o u g h urea s y n t h e s i s and a l a n i n e o x i d a t i o n (and g l u c o n e o g e n e s i s i n o t h e r e x p e r i m e n t s ) , were l i n e a r over the 2-hour e x p e r i m e n t a l i n c u b a t i o n p e r i o d s , and c e l l s were found t o be m e t a b o l i c a l l y a c t i v e even 48 hours a f t e r i s o l a t i o n . In c o n t r a s t w i t h h e p a t o c y t e s from o t h e r s p e c i e s , s k a t e h e p a t o c y t e s d i d not have t o be e s t a b l i s h e d i n p r i m a r y c u l t u r e s f o r r e l a t i v e l y l o n g -term s t u d i e s . The use of i s o l a t e d h e p a t o c y t e s t o s t u d y u r e a s y n t h e s i s i n elasmobranchs i s a n o v e l a p p r o a c h ; h i g h c e l l y i e l d s p e r m i t t e d t h e use of b l o c k - d e s i g n e x p e r i m e n t s , which a l l o w e d f o r p a i r e d c o m p a r i s o n s of t r e a t m e n t e f f e c t s on c e l l s o b t a i n e d from the same a n i m a l . Such a system was h i g h l y s e n s i t i v e as i n d i v i d u a l v a r i a t i o n i n b a s e l i n e r a t e s of f l u x t h r o u g h m e t a b o l i c pathways d i d not mask t r e a t m e n t e f f e c t s . Short-Term I n c u b a t i o n s I n s u l i n , g l u c a g o n , 1a~0H, and 1,2-dehydro-A d i d not induce c o n s i s t e n t r e s p o n s e s i n r a t e s of urea s y n t h e s i s or a l a n i n e o x i d a t i o n i n h e p a t o c y t e s d u r i n g 2.25 hours of exposure t o the 34 hormones. F u r t h e r m o r e , no p e r m i s s i v e e f f e c t s on e i t h e r of the two pathways i n v e s t i g a t e d were e v i d e n t when c e l l s were i n c u b a t e d w i t h 1a-OH combined w i t h i n s u l i n o r g l u c a g o n ( F i g u r e s 2 & 3, T a b l e 2 ) . re 2 . E f f e c t s of Short-Term Hormone I n c u b a t i o n s on Urea S y n t h e s i s i n S k a t e H e p a t o c y t e s . P e r c e n t r a t e of urea s y n t h e s i s r e l a t i v e t o c o n t r o l s i s c a l c u l a t e d from the r a t e d e t e r m i n e d w i t h a g i v e n t r e a t m e n t d i v i d e d by t h e r a t e under s t a n d a r d c o n d i t i o n s f o r h e p a t o c y t e s from the same a n i m a l . The mean p e r c e n t r a t e i s o b t a i n e d by c a l c u l a t i n g t h e sum of the p e r c e n t r a t e s f o r a l l a n i m a l s w i t h t h e g i v e n t r e a t m e n t and d i v i d i n g by N. 3 7 F i g u r e 3 . E f f e c t s of Short-Term Hormone I n c u b a t i o n s on A l a n i n e O x i d a t i o n i n S k a t e H e p a t o c y t e s . P e r c e n t r a t e of a l a n i n e o x i d a t i o n r e l a t i v e t o c o n t r o l s i s c a l c u l a t e d from t h e r a t e measured f o r a g i v e n t r e a t m e n t d i v i d e d by t h e r a t e under s t a n d a r d c o n d i t i o n s f o r h e p a t o c y t e s from t h e same a n i m a l , m u l t i p l i e d by one hundred. The mean p e r c e n t r a t e i s o b t a i n e d by c a l c u l a t i n g the sum of the p e r c e n t r a t e s f o r a l l a n i m a l s w i t h the g i v e n t r e a t m e n t and d i v i d i n g by N. 3 9 T a b l e 2 . E f f e c t s of S h o r t Term Hormone Treatments on Urea S y n t h e s i s and A l a n i n e O x i d a t i o n . Rate of urea s y n t h e s i s and a l a n i n e o x i d a t i o n a r e e x p r e s s e d as /imoles of p r o d u c t (urea and C0 2 r e s p e c t i v e l y ) e v o l v e d per gram of h e p a t o c y t e s per hour. Treatment Mean Rate of Urea Syn. N ± S t d . E r r o r (nmol/g/hour) Mean Rate of A l a . O x i d . ± S t d . E r r o r (mnol/g/hour) C o n t r o l 6 0.858±0.026 0.219±0.006 I n s u l i n 6 0.896±0.025 0.245±0.006 Ins.+1a-0H 6 0.674±0.025 0.239±0.006 Glucagon 6 0.879±0.025 0.240±0.006 Glue.+1a-OH 6 0.942±0.025 0.253±0.006 1a-OH 6 0.886±0.025 0.231±0.006 1,2-dehydro-A 6 0.877±0.028 0.239±0.006 41 E f f e c t s of i n s u l i n and g l u c a g o n show marked s e a s o n a l v a r i a t i o n i n f i s h e s (Moon, Mommsen, and P l i s e t s k a y a , p e r s o n a l c o m m u n i c a t i o n s ) . Hormones y i e l d maximal response d u r i n g s p r i n g and summer, and may have g r e a t l y reduced m e t a b o l i c e f f e c t s d u r i n g w i n t e r months. The p r e s e n t s t u d y was c o n d u c t e d d u r i n g t h e month of F e b r u a r y , however l a c k of a u r e a g e n i c response t o i n s u l i n and glucagon because of s e a s o n a l f a c t o r s can be r u l e d o u t ; Mommsen and Moon (1987) a p p l i e d g l u c agon and i n s u l i n t o R. e r i n a c e a h e p a t o c y t e s i s o l a t e d from summer f i s h and found no b i o l o g i c a l l y s i g n i f i c a n t r e s p o n s e . Both hormones caused a minor (11%) i n c r e a s e i n r a t e of urea s y n t h e s i s . I t has y e t t o be d e t e r m i n e d whether or not i n s u l i n s t i m u l a t e s uptake of amino a c i d s and t h e i r i n c o r p o r a t i o n i n t o p r o t e i n s i n elasmobranchs as i t does i n t e l e o s t s and mammals ( P l i s e t s k a y a e t a _ l . , 1986), however, r e s u l t s of the p r e s e n t s t u d y , and the s t u d y by Mommsen and Moon (1987), i n d i c a t e t h a t even i f i t does, t h e r e a r e no e f f e c t s , d i r e c t or o t h e r w i s e , on u r e a s y n t h e s i s . F u r t h e r m o r e , i f i n s u l i n does s t i m u l a t e uptake of amino a c i d s , i t was not r e f l e c t e d i n i n c r e a s e d r a t e s of a l a n i n e o x i d a t i o n . The l a c k of p e r m i s s i v e e f f e c t s of 1a-OH w i t h i n s u l i n and g l u c a g o n on urea s y n t h e s i s and a l a n i n e o x i d a t i o n cannot be c o n s i d e r e d c o n c l u s i v e due t o s e a s o n a l c o n s i d e r a t i o n s . I t would be a c c e p t a b l e i n terms of the b i o l o g y of R. e r i n a c e a i f t h e s e a n i m a l s d i d not r e g u l a t e urea s y n t h e s i s a c c o r d i n g t o e n v i r o n m e n t a l s a l i n i t y d u r i n g w i n t e r . T h i s s p e c i e s moves o f f -s h o r e i n the f a l l and o v e r - w i n t e r s i n the mud, where s a l i n i t y i s 42 l i k e l y t o be s t a b l e (M. D a d s w e l l , p e r s o n a l c o m m u n i c a t i o n ) . Only d u r i n g the summer months does R. e r i n a c e a move near t h e c o a s t , where s a l i n i t y v a r i e s c o n s i d e r a b l y , t o f e e d . I n f u s i o n of p h y s i o l o g i c a l c o n c e n t r a t i o n s of g l u c a g o n i n t o r a t s i s r e p o r t e d t o cause a c o o r d i n a t e d i n d u c t i o n of u r e a c y c l e enzymes (Snodgrass e t a l . , 1978). I n t e r e s t i n g l y , the mechanism of t h i s r esponse i s somewhat a t y p i c a l f o r g lucagon and resembles t h a t of a c o r t i c o s t e r o i d . A s s o c i a t e d w i t h an i n c r e a s e d a c t i v i t y of u r e a c y c l e enzymes, which o c c u r r e d between 24 and 72 h o u r s a f t e r i n i t i a t i o n of hormone t r e a t m e n t , was an i n c r e a s e i n i m m u n o p r e c i p i t a b l e urea c y c l e enzyme. The a u t h o r s found f a i r l y s t r o n g e v i d e n c e s u g g e s t i n g t h a t RNA s y n t h e s i s was r e q u i r e d f o r the o b s e r v e d i n d u c t i o n of u r e a c y c l e enzymes. I t i s p o s s i b l e t h a t p r o l o n g e d i n c u b a t i o n of s k a t e h e p a t o c y t e s w i t h g l u c a g o n might induce a u r e a g e n i c r e s p o n s e . A p p l i c a t i o n of elasmobranch i n s u l i n and g l u c a g o n t o s k a t e h e p a t o c y t e s might y i e l d r e s u l t s d i f f e r e n t from t h o s e o b t a i n e d w i t h b o v i n e hormones. Recent s t u d i e s , however, have shown a h i g h degree of amino a c i d homology between f i s h ( s a l m o n i d ) and mammalian i n s u l i n and g l u c a g o n , e s p e c i a l l y between r e s i d u e s which a r e i n v o l v e d w i t h • hormone-receptor i n t e r a c t i o n ( P l i s e t s k a y a a t a l . , 1985; P l i s e t s k a y a e t a l . , 1986). Muggeo e t a l . (1979) r e p o r t t h a t the i n s u l i n r e c e p t o r i s e v o l u t i o n a r i l y more c o n s e r v e d than the hormone m o l e c u l e i t s e l f , and d i s p l a y s no s p e c i e s s p e c i f i c i t y w i t h r e s p e c t t o hormone b i n d i n g . T h i s i n f o r m a t i o n s h o u l d be kept i n mind, however, the p o s s i b i l i t y of elasmobranch i n s u l i n and g l u c a g o n h a v i n g r e g u l a t o r y r o l e s i n 43 urea s y n t h e s i s cannot be r u l e d o u t . As p r e v i o u s l y s t a t e d , the r e c e p t o r s f o r b o t h i n s u l i n and gl u c a g o n a r e l o c a t e d on c e l l membranes. Thus, i t i s p o s s i b l e t h a t l a c k of response t o t h e s e hormones w i t h b o t h urea s y n t h e s i s and a l a n i n e o x i d a t i o n was due t o damage or l o s s of r e c e p t o r s d u r i n g h e p a t o c y t e i s o l a t i o n . T h i s i s h i g h l y u n l i k e l y as t h e r e was no e v i d e n c e t o suggest t h a t c e l l s were o v e r - d i g e s t e d . T e l e o s t h e p a t o c y t e s i s o l a t e d under i d e n t i c a l c o n d i t i o n s responded t o p e p t i d e hormones (Mommsen and S u a r e z , 1984). A d d i t i o n a l l y , B h a t t a c h a r y a e_t a l . (1985) r e p o r t t h a t c e l l s r e s y n t h e s i z e r e c e p t o r s w i t h i n 2-4 hours of i s o l a t i o n g i v e n a p p r o p r i a t e c o n d i t i o n s . C e l l s used i n the p r e s e n t s t u d y a l w a y s had a r e c o v e r y p e r i o d between i s o l a t i o n and use i n e x p e r i m e n t s of a p p r o x i m a t e l y 24 h o u r s . No d i r e c t e f f e c t s of 1a-OH and 1,2-dehydro-A on urea s y n t h e s i s or a l a n i n e o x i d a t i o n were o b s e r v e d d u r i n g s h o r t - t e r m i n c u b a t i o n s . T h i s l a c k of response i s not unexpected i n view of the mechanism of a c t i o n of t h e s e hormones. T h i s t r e a t m e n t was i n c l u d e d p r i m a r i l y t o s e r v e as a c o n t r o l f o r s t e r o i d - p e p t i d e i n c u b a t i o n s . Long-Term I n c u b a t i o n s I n t e r e s t i n g l y , f o r a l l t r e a t m e n t s , r a t e s of b o t h u r e a s y n t h e s i s and a l a n i n e o x i d a t i o n were s i g n i f i c a n t l y h i g h e r (37% and 40% r e s p e c t i v e l y ) (P<0.05) f o l l o w i n g 48 hours of i n c u b a t i o n i n comparison w i t h r a t e s measured i n the same b a t c h e s of c e l l s a t 24 hours ( T a b l e 3 ) . T h i s s u g g e s t s t h a t c e l l s may a c t u a l l y 44 have been r e c o v e r i n g from the i s o l a t i o n p r o c e d u r e , and were d e f i n i t e l y not l o s i n g t h e i r m e t a b o l i c i n t e g r i t y d u r i n g the c o u r s e of t h e e x p e r i m e n t . Long term i n c u b a t i o n (24 and 48 hours) of s k a t e h e p a t o c y t e s w i t h 1a-OH and 1,2-dehydro-A y i e l d e d no e f f e c t s on urea s y n t h e s i s ( F i g u r e 4, T a b l e 3 ) , however f o l l o w i n g 24 hours of i n c u b a t i o n w i t h the same hormones, h e p a t o c y t e s d i s p l a y e d s i g n i f i c a n t l y (P<0.05) e l e v a t e d r a t e s of a l a n i n e o x i d a t i o n ( F i g u r e 5, T a b l e 3 ) . T h i s t r e n d was s t i l l o b s e r v a b l e a f t e r a t o t a l i n c u b a t i o n p e r i o d of 48 h o u r s . 45 F i g u r e 4. E f f e c t s of Long-Term S t e r o i d I n c u b a t i o n on Urea S y n t h e s i s i n S k a t e H e p a t o c y t e s . Mean p e r c e n t r a t e s r e l a t i v e t o c o n t r o l s a r e c a l c u l a t e d as d e s c r i b e d f o r s h o r t - t e r m hormone i n c u b a t i o n s . 4 7 F i g u r e 5. E f f e c t s of Long-Term S t e r o i d I n c u b a t i o n on A l a n i n e O x i d a t i o n i n Sk a t e H e p a t o c y t e s . Mean p e r c e n t r a t e s r e l a t i v e t o c o n t r o l s a r e c a l c u l a t e d as d e s c r i b e d f o r s h o r t - t e r m hormone i n c u b a t i o n s . * S i g n i f i c a n t l y d i f f e r e n t (P<0.05) from c o n t r o l r a t e as d e t e r m i n e d w i t h Tukey's M u l t i p l e Range T e s t . 87 49 T a b l e 3. Mean R a t e s of Urea S y n t h e s i s and A l a n i n e O x i d a t i o n F o l l o w i n g I n c u b a t i o n w i t h S t e r o i d Hormones f o r 24 and 48 Hours. * S i g n i f i c a n t l y d i f f e r e n t (P<0.05) from c o n t r o l r a t e . ** S i g n i f i c a n t l y d i f f e r e n t (P<0.05) from the same t r e a t m e n t a t 24 hours as d e t e r m i n e d w i t h Tukey's M u l t i p l e Range T e s t . Rate of u r e a s y n t h e s i s and a l a n i n e o x i d a t i o n a r e e x p r e s s e d as jumoles of p r o d u c t (urea and C 0 2 r e s p e c t i v e l y ) e v o l v e d per gram of h e p a t o c y t e s per hour. Treatment Time N Rate of Urea Rate of A l a . S y n t h e s i s O x i d . ± S t d . E r r o r ± S t d . E r r o r (Hours) (/zmol/g/hour) (umol/g/hour) C o n t r o l 24 4 1 .088±0. 056 0. 159±0. 007 1 a-OH 24 4 1 .165±0. 056 0. 195±0. 007 * 1,2-dehydro-A 24 4 1 .102±0. 056 0. 175±0. 007 * C o n t r o l 48 5 1 .307±0. 063 ** 0. 241±0. 01 1 ** 1 a-OH 48 5 1 .315±0. 063 ** 0. 245±0. 011 ** 1,2-dehydro-A 48 5 1 .511±0. 063 ** 0. 251±0. 011 ** 51 A d a p t a t i o n of d o g f i s h ( S c y l i o r h i n u s c a n i c u l a ) t o a wide range of o s m o l a r i t i e s (50-140% seawater) has been shown t o a f f e c t b o t h p r o d u c t i o n r a t e s and plasma l e v e l s of 1a-OH (Hazon and Henderson, 1984). Both of the s e parameters were found t o be i n v e r s l e y r e l a t e d t o o s m o l a r i t y of the e x t e r n a l medium. F u r t h e r m o r e , Hazon and Henderson (1984) deduced t h a t a t low o s m o l a r i t i e s r a t e of urea p r o d u c t i o n was d e c r e a s e d and t h a t u rea l o s s was i n c r e a s e d p r e f e r e n t i a l l y over l o s s e s of sodium and TMAO, i n agreement w i t h f i n d i n g s of G o l d s t e i n and F o r s t e r (1971) f o r R. e r i n a c e a . Hazon and Henderson thus s u g g e s t e d t h a t 1a-OH may have a r o l e i n r e g u l a t i n g plasma u r e a c o n c e n t r a t i o n s . Data o b t a i n e d i n t h e p r e s e n t s t u d y i n d i c a t e t h a t i f 1a-OH i s i n v o l v e d w i t h r e g u l a t i o n of plasma urea l e v e l s i t i s not t h r o u g h any d i r e c t e f f e c t s on h e p a t i c u rea s y n t h e s i s . In mammals t h e r e i s a p o s i t i v e c o r r e l a t i o n between r a t e s of g l u c o n e o g e n e s i s and urea s y n t h e s i s ( K r a u s - F r i e d m a n n , 1984). T h i s r e l a t i o n s h i p between t h e two major h e p a t i c m e t a b o l i c pathways has been a t t r i b u t e d t o malate b e i n g r e l e a s e d from the urea c y c l e ( F i g u r e 1) s e r v i n g as a g l u c o s e p r e c u r s o r ( M e i j e r e t a l . , 1978). A d d i t i o n a l l y , i n mammals on a h i g h p r o t e i n d i e t the two pathways a r e i n s e p a r a b l e . E x c e s s p r o t e i n s and amino a c i d s cannot be s t o r e d as such. P r o t e i n s a r e m e t a b o l i s e d and amino a c i d s a r e deaminated. R e s u l t i n g carbon c h a i n s a r e i n c o r p o r a t e d i n t o g l u c o s e , and may be s t o r e d i n t i s s u e s i n t h e form of g l y c o g e n . Amino groups r e s u l t i n g from d e a m i n a t i o n of amino a c i d s must then be d e t o x i f i e d and e l i m i n a t e d , t h u s r a t e s of urea s y n t h e s i s and g l u c o n e o g e n e s i s a r e s i m u l t a n e o u s l y e l e v a t e d . 5 2 The g l y c o g e n i c a c t i v i t y of 1a-OH i n elasmobranchs has y e t t o be e s t a b l i s h e d . 1a-OH n e g l e c t e d t o promote d e p o s i t i o n of l i v e r g l y c o g e n i n a s t a n d a r d mouse b i o a s s a y ( I d l e r e_t a l . , 1 9 6 7 ) . C o n v e r s l e y , Hartmann et a l . ( 1 9 4 4 ) r e p o r t t h a t i n t e r r e n a l e c t o m i s e d s k a t e s c o n s i s t e n t l y had h e p a t i c g l y c o g e n c o n t e n t s which were s u b s t a n t i a l l y lower than i n sham-operated a n i m a l s , s u g g e s t i n g t h a t 1a-OH may i n f a c t have a r e g u l a t o r y r o l e i n elasmobranch g l u c o n e o g e n e s i s or g l y c o g e n o l y s i s . I t would be i n t e r e s t i n g t o d e t e r m i n e whether or not u r e a s y n t h e s i s and g l u c o n e o g e n e s i s a r e l i n k e d i n elasmobranchs as they a r e i n mammals, c o n s i d e r i n g the r e l a t i v e unimportance of g l u c o s e t o elasmobranchs r e p o r t e d by DeRoos e t a l . , ( 1 9 8 5 ) and Mommsen and Moon ( 1 9 8 7 ) . The i n c r e a s e d r a t e s of a l a n i n e o x i d a t i o n i n h e p a t o c y t e s exposed t o t o s t e r o i d hormones f o r 2 4 hours may be of o s m o r e g u l a t o r y s i g n i f i c a n c e . T h i s response may be a s s o c i a t e d w i t h i n c r e a s e d energy demands d u r i n g exposure t o hypoosmotic s t r e s s and might a l s o i n d i c a t e a r o l e f o r 1a-OH i n c e l l - v o l u m e r e g u l a t i o n . A l o g i c a l next s t e p would be t o i n v e s t i g a t e e f f e c t s of 1a-OH on o x i d a t i o n of o s m o t i c a l l y a c t i v e amino a c i d s such as / 3 - a l a n i n e and s a r c o s i n e . W i t h r e s p e c t t o e f f e c t s on a l a n i n e o x i d a t i o n , the t i m e -c o u r s e of the p r e s e n t s t u d y c o u l d not d i f f e r e n t i a t e whether 1 a -OH or 1 , 2 - d e h y d r o - A i s the b i o l o g i c a l l y a c t i v e form of the s t e r o i d . The i n a c t i v e form of the hormone s h o u l d have a l o n g e r l a g phase between a d m i n i s t r a t i o n t o c e l l s and m a n i f e s t a t i o n of a response than the a c t i v e form would, as the former must be 5 3 m e t a b o l i s e d i n t o t he l a t t e r form b e f o r e i t can e x e r t i t s e f f e c t s . T r u s c o t t e t a l . (1978 ) i s o l a t e d 1 ,2-dehydro-A from s k a t e b i l e , and b e l i e v e t h a t i t i s a p r o d u c t of h e p a t i c m e t a b o l i s m of 1 a - 0 H . Summary I n c u b a t i o n of s k a t e h e p a t o c y t e s w i t h i n s u l i n , g l u c a g o n , 1a-OH, and 1 ,2-dehydro-A d i d not have any e f f e c t s on ur e a s y n t h e s i s or a l a n i n e o x i d a t i o n , nor were t h e r e any p e r m i s s i v e e f f e c t s of 1a-OH when combined w i t h the two p e p t i d e hormones. The s t e r o i d hormones i n d u c e d an i n c r e a s e d r a t e of a l a n i n e o x i d a t i o n i n h e p a t o c y t e s a f t e r 24 h o u r s , which d i m i n i s h e d over the next 24 h o u r s . T h i s response may be an a d a p t a t i o n t o hypoosmotic s t r e s s . Urea s y n t h e s i s d i d not respond t o 1a-OH or 1 , 2-dehydro-A a f t e r 24 or 48 hours of i n c u b a t i o n , t h u s c o n t r a r y t o s u g g e s t i o n s made by Hazon and Henderson ( 1 9 8 4 ) , 1a-OH does not appear t o be i n v o l v e d w i t h r e g u l a t i n g u rea s y n t h e s i s . 54 EFFECTS OF ALTERING OSMOLARITY AND SPECIFIC SOLUTE CONCENTRATIONS ON METABOLIC PATHWAYS I n t r o d u c t i o n Upon e n t e r i n g a r e l a t i v e l y hypoosmotic environment osmoconformers i n i t i a l l y e x p e r i e n c e an i n f l u x of w a t e r , and t h e r e f o r e a d i l u t i o n of plasma s o l u t e s . D u r i n g p e r i o d s of hypoosmotic s t r e s s elasmobranchs a c t i v e l y r i d th e m s e l v e s of plasma s o l u t e s ( u r e a , TMAO, and NaCl) ( G o l d s t e i n and F o r s t e r , 1971; Payan e t a_l. , 1973), and water s u b s e q u e n t l y d i f f u s e s down i t s c o n c e n t r a t i o n g r a d i e n t and r e - e q u i l i b r a t e s between the a n i m a l and en v i r o n m e n t . Thus, g i v e n time t o adapt t o a hypoosmotic e n v i r o n m e n t , t h e s e a n i m a l s w i l l r e c o v e r a normal w a t e r - s o l u t e b a l a n c e . A s i m i l a r r esponse i s seen a t the c e l l u l a r l e v e l i n elasmobranchs and o t h e r osmoconformers ( F i s h e r and S p r i n g , 1984; Boyd e t §_1., 1977; K i n g and G o l d s t e i n , 1983). F o l l o w i n g exposure t o hypoosmotic media an i n i t i a l phase of o s m o t i c s w e l l i n g o c c u r s , succeeded by a more l e n g t h y phase of c e l l s h r i n k a g e , or volume r e g u l a t o r y - d e c r e a s e (Hoffmann, 1983). D u r i n g t h i s l a t t e r phase, c e l l volume g r a d u a l l y approaches a new s t e a d y - s t a t e l e v e l which t r a n s i e n t l y remains s l i g h t l y above the o r i g i n a l ( H e n d i l and Hoffmann, 1974). In f i s h , c e l l volume r e g u l a t i o n i s a c c o m p l i s h e d by d e c r e a s i n g i n t r a c e l l u l a r c o n c e n t r a t i o n s of b o t h i n o r g a n i c i o n s ( F u g e l l i and R o h r s , 1980; L a u f , 1982) and s p e c i f i c o s m o t i c a l l y a c t i v e amino a c i d s (Boyd e t a l . , 1977; G o l d s t e i n , 1981). The 5 5 r o l e of i n o r g a n i c i o n s i n c e l l volume r e g u l a t i o n i s l i m i t e d , however, by t h e need t o m a i n t a i n a c o n s t a n t i n t r a c e l l u l a r i o n i c c o m p o s i t i o n , i n o r d e r t o p r o v i d e a c o m p a t i b l e m i l i e u f o r m e t a b o l i c p r o c e s s e s and e l e c t r i c a l p r o p e r t i e s of the c e l l s . I n t r a c e l l u l a r i o n c o n t e n t i s a d j u s t e d by changes i n membrane p e r m e a b i l i t y t o s p e c i f i c i o n s , and by a c t i v e pumping of i o n s a c r o s s the plasma membrane (Hoffmann, 1983). When osmoconformers a r e a d a p t e d t o a d i l u t e e nvironment, i n t r a c e l l u l a r p o o l s of amino a c i d s a r e reduced by an amount e q u a l t o or g r e a t e r than the p e r c e n t d e c r e a s e i n o s m o l a r i t y of the e x t r a c e l l u l a r f l u i d (Boyd ejt a l . , 1977). In R. er i n a c e a , a c c u m u l a t i o n of /3-alanine and t a u r i n e , and perhaps s a r c o s i n e , i n v a r i o u s t i s s u e s o c c u r s by an Na +-dependent t r a n s p o r t system which i s l o c a t e d i n the c e l l membrane ( F o r s t e r e_t a l . . , 1978; G o l d s t e i n and Boyd, 1978). D u r i n g hypoosmotic s t r e s s a net o u t f l u x of s p e c i f i c amino a c i d s from c e l l s o c c u r s , perhaps i n p a r t due t o a d e c r e a s e i n Na + c o n c e n t r a t i o n of t h e e x t r a c e l l u l a r f l u i d . Thus, i n t r a c e l l u l a r c o n c e n t r a t i o n s of amino a c i d s d e c r e a s e , w i t h a c o n c o m i t a n t i n c r e a s e i n plasma l e v e l s . |3-a l a n i n e and s a r c o s i n e i n plasma a r e d e l i v e r e d t o t h e l i v e r where th e y a r e m e t a b o l i s e d . /3-alanine o x i d a t i o n i s thought t o be r e g u l a t e d by s u b s t r a t e a v a i l a b i l i t y ( S h u t t l e w o r t h and G o l d s t e i n , 1984). I n t e r e s t i n g l y , r a t e of s a r c o s i n e o x i d a t i o n i n b oth h e p a t o c y t e s and h e p a t i c m i t o c h o n d r i a i s o l a t e d from R. e r i n a c e a a ppears t o be i n v e r s l e y r e l a t e d t o o s m o l a r i t y of the i n c u b a t i o n medium ( B a l l a n t y n e e t a l . , 1986). These a u t h o r s p o s t u l a t e t h a t t h e r e may be s t i m u l a t i o n or i n h i b i t i o n of amino a c i d o x i d a t i o n 56 by d i r e c t o s m o t i c e f f e c t s on the m i t o c h o n d r i a . From the f i n d i n g s d i s c u s s e d above i t appears t h a t a l t e r i n g o s m o l a r i t y of e x t r a c e l l u l a r f l u i d may a f f e c t m e t a b o l i s m t h r o u g h a number of mechanisms, such as t r a n s p o r t of s u b s t r a t e s a c r o s s membranes, d i l u t i o n of enzymes and s u b s t r a t e s w i t h i n c e l l s , and by o t h e r mechanisms not y e t f u l l y u n d e r s t o o d . The p r e s e n t s t u d y was u n d e r t a k e n t o i n v e s t i g a t e e f f e c t s of hypoosmotic s t r e s s on urea s y n t h e s i s i n s k a t e h e p a t o c y t e s ; a l a n i n e o x i d a t i o n and g l u c o n e o g e n e s i s were examined s i m u l t a n e o u s l y f o r comparison w i t h f l u x t h r o u g h the urea c y c l e . As a l t e r i n g s o l u t e c o n c e n t r a t i o n of the h e p a t o c y t e i n c u b a t i o n medium was found t o cause s p e c i f i c i n h i b i t i o n of urea s y n t h e s i s , subsequent e x p e r i m e n t s were done to d e t e r m i n e whether or not one s p e c i f i c s o l u t e was r e s p o n s i b l e f o r t h i s e f f e c t . E x p e r i m e n t a l D e s ign Urea s y n t h e s i s , a l a n i n e o x i d a t i o n , and g l u c o n e o g e n e s i s were measured i n s k a t e (R. e r i n a c e a ) h e p a t o c y t e s i n c u b a t e d i n a wide range of o s m o l a r i t i e s (50%, 75%, 100%, 112.5%, and 125% of normal plasma l e v e l s as r e p o r t e d by Boyd e t a_l. , 1977). Subsequent e x p e r i m e n t s were done t o d e t e r m i n e e f f e c t s of r e d u c i n g t h e c o n c e n t r a t i o n of one s p e c i f i c s o l u t e on urea s y n t h e s i s . For t h e s e s e t s of e x p e r i m e n t s a l a n i n e o x i d a t i o n and g l u c o n e o g e n e s i s were measured o n l y w i t h s t a n d a r d i n c u b a t i o n medium f o r assessment of c e l l i n t e g r i t y . F l u x r a t e s t h r o u g h m e t a b o l i c pathways were measured as d e s c r i b e d i n the main M a t e r i a l s and Methods s e c t i o n of t h i s t h e s i s . 57 O s m o l a r i t y O s m o l a r i t y of the i n c u b a t i o n medium was v a r i e d by a l t e r i n g p r o p o r t i o n a t e l y c o n c e n t r a t i o n s of u r e a , N a C l , TMAO, MgSO„, M g C l 2 , and KC1, w h i l e NaH 2PO„, C a C l 2 , Hepes, BSA, and amino a c i d s were m a i n t a i n e d a t c o n s t a n t l e v e l s f o r v a r i o u s r e a s o n s . B l o c k d e s i g n e x p e r i m e n t s were used f o r comparison of r a t e s of urea s y n t h e s i s , a l a n i n e o x i d a t i o n and g l u c o n e o g e n e s i s a t 50%, 75%, and 100% of normal o s m o l a r i t y . At 112.5% and 125% of normal plasma o s m o l a r i t y , r a t e s of f l u x t h r o u g h the 3 m e t a b o l i c pathways were measured o n l y i n t h e f i r s t 3 or 4 e x p e r i m e n t s t o c o n s e r v e l a b e l l e d s u b s t r a t e , as t h e s e t r e a t m e n t s had no a p p a r e n t e f f e c t s on any of the pathways of i n t e r e s t . Minor S o l u t e s H e p a t o c y t e s were i n c u b a t e d i n s o l u t i o n s i n which the f i n a l c o n c e n t r a t i o n of e i t h e r M g + + s a l t s , o r KC1, was h a l f of t h e l e v e l s p r e s e n t i n s t a n d a r d i n c u b a t i o n medium. These s a l t s c o n t r i b u t e d a n e g l i g i b l e amount t o t h e o s m o l a r i t y of t h e i n c u b a t i o n s o l u t i o n , t h u s o t h e r s o l u t e s were not used t o compensate f o r the s l i g h t y r educed o s m o l a r i t y . Complete b l o c k d e s i g n e x p e r i m e n t s were done i n which urea s y n t h e s i s was measured a t 50% and 100% of normal plasma o s m o l a r i t y , s i m u l t a n e o u s l y w i t h reduced i o n c o n c e n t r a t i o n t r e a t m e n t s . 58 M a j or S o l u t e s E x p e r i m e n t s were done i n which c e l l s were i n c u b a t e d i n media w i t h 50% of the s t a n d a r d c o n c e n t r a t i o n of one major s o l u t e ( u r e a , N a C l , or TMAO). As a l l of t h e s e s o l u t e s 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 the o s m o l a r i t y of the i n c u b a t i o n medium, o t h e r s o l u t e s were added i n " e x c e s s " t o r e t u r n o s m o l a r i t y t o a l e v e l of a t l e a s t 85% of normal plasma o s m o l a r i t y . C o n c e n t r a t i o n s of major s o l u t e s used i n t h e s e t r e a t m e n t s a r e shown i n T a b l e 4. Urea s y n t h e s i s was measured a t 50% and 100% of normal o s m o l a r i t y s i m u l t a n e o u s l y w i t h each of the o t h e r t r e a t m e n t s , w i t h the e x c e p t i o n of Treatment 4 (40 mM TMAO, 260 mM N a C l ) . In t h i s c a s e th e 50% o s m o l a r i t y t r e a t m e n t was o m i t t e d as a n i m a l s were becoming s c a r c e a t the time of the e x p e r i m e n t s , and the e f f e c t s on u r e a s y n t h e s i s of r e d u c i n g o s m o l a r i t y t o 50% of normal were a l r e a d y w e l l e s t a b l i s h e d . 59 T a b l e 4. C o n c e n t r a t i o n s of Major S o l u t e s i n I n c u b a t i o n M e d i a . S o l u t i o n s were made up w i t h 50% of the normal c o n c e n t r a t i o n of one s o l u t e , and a n o t h e r major s o l u t e was added i n e x c e s s i n o r d e r t o b r i n g o s m o l a r i t y up t o a p p r o x i m a t e l y 100%. Urea s y n t h e s i s d i d not d i f f e r s i g n i f i c a n t l y from c o n t r o l r a t e s when h e p a t o c y t e s were i n c u b a t e d i n media between 75% and 125% o s m o l a r i t y , and f i n a l o s m o l a r i t y was always w i t h i n t h i s r ange. When urea and TMAO were reduced i n c o n c e n t r a t i o n , NaCl was added t o compensate f o r o s m o l a r i t y ; NaCl y i e l d s two o s m o t i c a l l y a c t i v e p a r t i c l e s f o r each m o l e c u l e added, thus the amount of NaCl which was added was h a l f the number of moles by which the o t h e r s o l u t e was d e c r e a s e d . T r e a t -ment O s m o l a r i t y (% of s t a n d a r d l e v e l (952 mOsm) Urea (mM) NaCl (mM) TMAO (mM) 1 50 175 120 40 2 100 350 240 80 3 100 175 326 80 4 1 02 350 259 40 5 87 470 120 80 6 . 113 470 240 80 61 R e s u l t s and D i s c u s s i o n O s m o l a r i t y E x p e r i m e n t s I n c u b a t i o n of h e p a t o c y t e s i n d i l u t e media r e s u l t e d i n a marked d e p r e s s i o n of urea s y n t h e s i s , which was s t a t i s t i c a l l y s i g n i f i c a n t (P<0.05) a t 50% of normal o s m o l a r i t y , but the t r e n d was a l s o apparent a t 75% of s t a n d a r d s o l u t e c o n c e n t r a t i o n s ( T a b l e 5, F i g u r e s 6 & 7 ) . H i g h o s m o l a r i t i e s (112% and 125% of normal l e v e l s ) had no n o t a b l e e f f e c t on urea s y n t h e s i s . In c o n t r a s t w i t h urea s y n t h e s i s , s u r p r i s i n g l y b o t h a l a n i n e o x i d a t i o n and g l u c o n e o g e n e s i s were e l e v a t e d a t low o s m o l a r i t i e s . In the case of a l a n i n e o x i d a t i o n t h i s i n c r e a s e d r a t e was s t a t i s t i c a l l y s i g n i f i c a n t (P<0.05) a t 50% o s m o l a r i t y , w h i l e g l u c o n e o g e n e s i s was s i g n i f i c a n t l y e l e v a t e d (P<0.05) a t both 50% and 75% of normal s o l u t e c o n c e n t r a t i o n s (Table 5, F i g u r e s 6 & 7 ) . 6 2 F i g u r e 6. E f f e c t s , of O s m o l a r i t y on Rates of Urea S y n t h e s i s and A l a n i n e O x i d a t i o n i n Sk a t e H e p a t o c y t e s . Mean p e r c e n t r a t e s r e l a t i v e t o c o n t r o l s a r e c a l c u l a t e d as d e s c r i b e d f o r s h o r t - t e r m hormone i n c u b a t i o n s . * r a t e i s s i g n i f i c a n t l y d i f f e r e n t from r a t e measured under s t a n d a r d c o n d i t i o n s (P<0.05) f as d e t e r m i n e d w i t h Tukey's M u l t i p l e Range Te s t on b l o c k d e s i g n d a t a . Mean X (±SEM) of Standard Rate (100% Osmolarity) of Urea S y n t h e s i s and A l a n i n e O x i d a t i o n 20 40 60 80 100 120 140 160 130 o I I I I 1 1 1 1 n co rf _ (0 M - CD M- O U> 3 _ O e9 6 4 F i g u r e 7. E f f e c t s of O s m o l a r i t y on Rates of Urea S y n t h e s i s and G l u c o n e o g e n e s i s i n Skate H e p a t o c y t e s . Mean p e r c e n t r a t e s r e l a t i v e t o c o n t r o l s a r e c a l c u l a t e d as d e s c r i b e d f o r s h o r t - t e r m hormone i n c u b a t i o n s . * r a t e i s s i g n i f i c a n t l y d i f f e r e n t from r a t e measured under s t a n d a r d c o n d i t i o n s (P<0.05), as d e t e r m i n e d w i t h Tukey's M u l t i p l e Range Te s t on b l o c k d e s i g n d a t a . 59 6 6 T a b l e 5. E f f e c t s of O s m o l a r i t y on Urea S y n t h e s i s , A l a n i n e O x i d a t i o n , and G l u c o n e o g e n e s i s i n Sk a t e H e p a t o c y t e s . * r a t e i s s i g n i f i c a n t l y d i f f e r e n t from r a t e measured under s t a n d a r d c o n d i t i o n s (P<0.05), as d e t e r m i n e d w i t h Tukey's M u l t i p l e Range T e s t on b l o c k d e s i g n d a t a . R a t e s a r e e x p r e s s e d i n terms of Mmoles of p r o d u c t ( u r e a , C 0 2 , and g l u c o s e ) e v o l v e d per gram of h e p a t o c y t e s per hour. 67 O s m o l a r i t y (% of c o n t r o l l e v e l ) N Urea S y n t h e s i s UM/g/hr.) N A l a n i n e O x i d a t i o n UM/g/hr.) N Gluconeo-g e n e s i s UM/g/hr.) 50% 7 .559±.157 * 7 .4651.068 * 5 .4721.102 * 75% 7 .8351.180 7 .4311.069 4 .4331.039 * 1 00% 7 .9141.196 7 .3911.082 4 .2751.037 1 12% 4 .490+.133 3 .3251.069 3 .2811.065 125% 4 .5661.122 4 .2941.089 3 .2301.029 68 D e p r e s s i o n of urea s y n t h e s i s i n h e p a t o c y t e s i n c u b a t e d a t low o s m o l a r i t i e s r e f l e c t s t h e i n - v i v o response found i n elasmobranchs m a i n t a i n e d i n d i l u t e e n v i r o n m e n t s . G o l d s t e i n and F o r s t e r (1971) ada p t e d R. e r i n a c e a t o d i l u t e seawater and found a c o r r e s p o n d i n g d r o p i n the r a t e of urea b i o s y n t h e s i s . U s i n g a s i m i l a r e x p e r i m e n t a l p r o t o c o l Hazon and Henderson (1984) o b t a i n e d comparable f i n d i n g s f o r the d o g f i s h , S c y l i o r h i n u s  c a n i c u l a . The mechanisms o p e r a t i n g t o f a c i l i t a t e a d e c r e a s e i n ur e a s y n t h e s i s i n t h e p r e s e n t s t u d y may a l s o a c t t o i n d u c e t h e a d a p t i v e response o b s e r v e d i n i n t a c t a n i m a l s e x p e r i e n c i n g hypoosmotic s t r e s s , and t h e r e f o r e a d i l u t i o n of e x t r a c e l l u l a r f l u i d s . As u rea s y n t h e s i s was d e p r e s s e d a t low o s m o l a r i t i e s and bo t h a l a n i n e o x i d a t i o n and g l u c o n e o g e n e s i s were e l e v a t e d under the same c o n d i t i o n s , i t can be t e n t a t i v e l y c o n c l u d e d t h a t exposure of s k a t e h e p a t o c y t e s t o low o s m o l a r i t i e s c a u s e s a s p e c i f i c i n h i b i t i o n of u r e a s y n t h e s i s . Reduced r a t e s of u r e a s y n t h e s i s under such c o n d i t i o n s cannot be a t t r i b u t e d t o d i l u t i o n of enzymes and s u b s t r a t e s w i t h i n h e p a t o c y t e s , a p o s s i b l e consequence of c e l l s s w e l l i n g i n hypoosmotic media, because t h e same response was not o b s e r v e d i n the o t h e r two m u l t i -compartment m e t a b o l i c pathways s t u d i e d . R e p o r t s of e l e v a t e d l e v e l s of amino a c i d o x i d a t i o n i n i s o l a t e d c e l l s d u r i n g hypoosmotic s t r e s s a r e numerous. Hoffmann (1984) found i n c r e a s e d r a t e s of o x i d a t i v e c a t a b o l i s m of b o t h a l a n i n e and g l y c i n e i n E h r l i c h c e l l s under hypoosmotic c o n d i t i o n s . Lambert and Hoffmann (1982) r e p o r t e d t h a t changes 69 i n r a t e of p r o t e i n t u r n o v e r were not f u e l i n g the i n c r e a s e d o x i d a t i v e r a t e s . B a l l a n t y n e e t a l . (1986) o b s e r v e d i n c r e a s e d r a t e s of s a r c o s i n e o x i d a t i o n i n i s o l a t e d R. er i n a c e a h e p a t o c y t e s and m i t o c h o n d r i a i n c u b a t e d i n d i l u t e media. I n c r e a s e d r a t e s of amino a c i d o x i d a t i o n d u r i n g hypoosmotic s t r e s s may have two a d a p t i v e f u n c t i o n s i n el a s m o b r a n c h s . One p o s s i b l e f u n c t i o n i s t o complete the p r o c e s s of c e l l volume r e g u l a t i o n ; amino a c i d s a r e r e l e a s e d from c e l l s i n t o the plasma d u r i n g hypoosmotic s t r e s s , and must then be e i t h e r m e t a b o l i s e d or e x c r e t e d as they cannot be p e r m i t t e d t o accumulate t o e x c e s s i v e l e v e l s i n t h e b l o o d . The second f u n c t i o n may be t o p r o v i d e a n i m a l s w i t h energy t o meet the m e t a b o l i c demands of p r o c e s s e s a c t i v a t e d or a m p l i f i e d by the imposed s t r e s s . I t has been s u g g e s t e d t h a t the o s m o t i c optimum of some m e t a b o l i c pathways i s " d e l i b e r a t e l y " s e t lower than t y p i c a l i n t r a c e l l u l a r o s m o l a r i t y ( B a l l a n t y n e and S t o r e y , 1984; B a l l a n t y n e and Moon, 1985) t o f a c i l i t a t e e l i m i n a t i o n of amino a c i d s d u r i n g hypoosmotic s t r e s s . One q u e s t i o n of i n t e r e s t i s whether a d e c r e a s e i n the s o l u t e c o n c e n t r a t i o n of t h e e x t r a c e l l u l a r f l u i d enhances o x i d a t i o n of a l l amino a c i d s i n elasmobranchs, o r i f such a response i s l i m i t e d t o a few s e l e c t e d amino a c i d s . A l a n i n e i s not an i m p o r t a n t o s molyte i n t h e l i t t l e s k a t e as s a r c o s i n e i s , s u g g e s t i n g t h a t the o b s e r v e d e f f e c t s may w e l l a p p l y t o a l l amino a c i d s . I f m e t a b o l i s m of a l l amino a c i d s i s enhanced d u r i n g hypoosmotic s t r e s s when u r e a s y n t h e s i s i s d e p r e s s e d , one would expect t o f i n d a s i m u l t a n e o u s i n c r e a s e i n ammonia e x c r e t i o n . 70 U n f o r t u n a t e l y no c o n c l u s i v e d a t a a r e a v a i l a b l e t o c o n f i r m whether or not such i s the c a s e . G o l d s t e i n and F o r s t e r (1971) a t t e m p t e d t o measure ammonia e x c r e t i o n i n s k a t e s d u r i n g a d a p t a t i o n t o d i l u t e seawater and a f t e r r e t u r n i n g t o f u l l -s t r e n g t h s e a w a t e r , however r e s u l t s of t h e i r s t u d y were ambiguous. Mechanisms a c c o u n t i n g f o r the i n c r e a s e d r a t e s of amino a c i d o x i d a t i o n d u r i n g d i l u t i o n of e x t r a c e l l u l a r f l u i d s a r e open t o s p e c u l a t i o n . Hoffmann (1978) and Hoffmann and Lambert (1983) obse r v e d t h a t i n c r e a s e s i n c e l l volume of E h r l i c h c e l l s , due t o hypoosmotic s t r e s s , a r e accompanied by an i n c r e a s e i n p a s s i v e p e r m e a b i l i t y t o p a r t i c u l a r amino a c i d s . In the p r e s e n t e x p e r i m e n t s the i n i t i a l c o n c e n t r a t i o n of a l a n i n e i n t h e i n c u b a t i o n medium was t h r e e t i m e s t h e l e v e l r e p o r t e d f o r s k a t e l i v e r , thus hypoosmotic s t r e s s may have i n c r e a s e d the l e a k i n e s s of membranes t o a l a n i n e , a l l o w i n g i t t o move down a c o n c e n t r a t i o n g r a d i e n t i n t o h e p a t o c y t e s , thus f a c i l i t a t i n g an i n c r e a s e i n o x i d a t i v e r a t e a t low o s m o l a r i t i e s . R e s u l t s of the p r e s e n t s t u d y show t h a t i n c o n t r a s t w i t h mammalian systems t h e r e may be no c o r r e l a t i o n between r a t e s of f l u x t h r o u g h u r e a g e n i c and g l u c o n e o g e n i c pathways i n elasmobranch h e p a t o c y t e s . In mammalian systems t h e s e pathways were f i r s t shown t o be l i n k e d i n 1893 ( M i n k o w s k i , 1893). M e i j e r e t a l . (1978) propose t h a t an i n c r e a s e d r a t e of urea s y n t h e s i s would make more mala t e a v a i l a b l e f o r g l u c o n e o g e n e s i s and t h u s s t i m u l a t e t h e l a t t e r b i o s y n t h e t i c pathway. The i m p l i c i t a ssumption of t h i s h y p o t h e s i s i s t h a t g l u c o n e o g e n i c s u b s t r a t e s 71 a r e n o r m a l l y r a t e - l i m i t i n g f o r t h i s pathway. As p r e v i o u s l y s u g g e s t e d , hypoosmotic c o n d i t i o n s may enhance movement of amino a c i d s i n t o c e l l s w h i c h have the c a p a c i t y t o o x i d i z e them, thus the e l e v a t e d g l u c o n e o g e n i c r a t e s o b s e r v e d i n the p r e s e n t s t u d y a t low o s m o l a r i t i e s may be a consequence of i n c r e a s e d i n t r a c e l l u l a r s u b s t r a t e a v a i l a b i l i t y . A g a i n , t h i s c o u l d be a n o t h e r a d a p t i v e " s t r a t e g y " of elasmobranchs t o maximize e f f i c i e n c y of t h e i r energy r e s e r v e s . Amino a c i d s cannot be s t o r e d a such and t h o s e not r e q u i r e d i m m e d i a t e l y as s u b s t r a t e s f o r o t h e r m e t a b o l i c pathways must be deaminated; carbon s k e l e t o n s l i b e r a t e d a r e i n c o r p o r a t e d i n t o o t h e r compounds f o r l o n g - t e r m s t o r a g e . Enhanced h e p a t i c g l u c o n e o g e n i c r a t e s d u r i n g hypoosmotic s t r e s s , when amino a c i d s a r e r e l e a s e d from c e l l s i n t o plasma, e n a b l e s s t o r a g e of energy and r e d u c t i o n of o s m o l a r i t y a t the same t i m e . Carbon s k e l e t o n s d e r i v e d from amino a c i d s can t h u s be i n c o r p o r a t e d i n t o g l u c o s e , and s u b s e q u e n t l y i n t o g l y c o g e n , which i s o s m o t i c a l l y i n a c t i v e , f o r l o n g - t e r m s t o r a g e . Minor S o l u t e s Reducing c o n c e n t r a t i o n s of M g + + s a l t s and KC1 t o h a l f of the normal l e v e l s had no d e p r e s s i n g e f f e c t s on urea s y n t h e s i s . In f a c t , t h e s e t r e a t m e n t s caused a s l i g h t e l e v a t i o n of urea s y n t h e s i s , which was not s i g n i f i c a n t l y d i f f e r e n t (P<0.05) from l e v e l s measured under s t a n d a r d c o n d i t i o n s ( F i g u r e 8, T a b l e 6 ) . 7 2 F i g u r e 8. E f f e c t s of A l t e r i n g C o n c e n t r a t i o n s of M g + + and K + on Urea S y n t h e s i s . Mean p e r c e n t r a t e s r e l a t i v e t o c o n t r o l s a r e c a l c u l a t e d as d e s c r i b e d f o r s h o r t - t e r m hormone i n c u b a t i o n s . * r a t e i s s i g n i f i c a n t l y d i f f e r e n t from r a t e measured under s t a n d a r d c o n d i t i o n s (P<0.05), as d e t e r m i n e d w i t h Tukey's M u l t i p l e Range T e s t on b l o c k d e s i g n d a t a . 74 T a b l e 6. E f f e c t s of V a r y i n g C o n c e n t r a t i o n s of M g + + and K + on Urea S y n t h e s i s i n Skate H e p a t o c y t e s . * r a t e i s s i g n i f i c a n t l y d i f f e r e n t from r a t e measured under s t a n d a r d c o n d i t i o n s (P<0.05), as d e t e r m i n e d w i t h Tukey's M u l t i p l e Range Test on b l o c k d e s i g n d a t a . Rate of urea s y n t h e s i s i s e x p r e s s e d as Mmoles of urea s y n t h e s i z e d per gram of h e p a t o c y t e s per hour. Treatment Urea S y n t h e s i s N (MM/g/hr.) ± SEM C o n t r o l 1. 100% o s m o l a r i t y 5 0.785 ± 0.089 C o n t r o l 2. 50% o s m o l a r i t y 5 0.569 ± 0.044 * 100% o s m o l a r i t y , 50% Mg 5 0.863 + 0.087 100% o s m o l a r i t y , 50% K 5 0.837 + 0.102 7 6 Both Mg + + and K + p o s s e s s v a r i o u s , i m p o r t a n t r o l e s i n m e t a b o l i c systems, and i t was h y p o t h e s i s e d t h a t r e d u c t i o n of the c o n c e n t r a t i o n of one of t h e s e i o n s may have been r e s p o n s i b l e f o r the reduced urea b i o s y n t h e t i c r a t e s o b s e r v e d a t 50% of normal s o l u t e c o n c e n t r a t i o n s . Magnesium i s a c o f a c t o r i n numerous e n z y m a t i c r e a c t i o n s , i n c l u d i n g the f i r s t s t e p of u r e a s y n t h e s i s i n which c a r b a m y l phosphate i s made. Organisms expend l a r g e amounts of energy pumping p o t a s s i u m i n t o c e l l s a g a i n s t a c o n c e n t r a t i o n g r a d i e n t i n o r d e r t o m a i n t a i n the h i g h i n t r a c e l l u l a r c o n c e n t r a t i o n s n e c e s s a r y f o r m e t a b o l i c i n t e g r i t y . 77 Major S o l u t e s None of t h e major s o l u t e s ( u r e a , N a C l , and TMAO) was found t o a c t as a d i r e c t e f f e c t o r of the r e d u c t i o n of urea b i o s y n t h e s i s which o c c u r s when s k a t e h e p a t o c y t e s a r e i n c u b a t e d i n d i l u t e media. D e c r e a s i n g the l e v e l of urea or TMAO i n the i n c u b a t i o n medium, w h i l e s i m u l t a n e o u s l y compensating f o r o s m o l a r i t y w i t h i n c r e a s e d c o n c e n t r a t i o n s of NaCl (136% and 108% of s t a n d a r d c o n c e n t r a t i o n s r e s p e c t i v e l y ) (Treatments 3 & 4) had no e f f e c t on u r e a s y n t h e s i s ( T a b l e 7, F i g u r e s 9 & 10). When the c o n c e n t r a t i o n of NaCl was d e c r e a s e d by 50%, and urea was used t o compensate f o r o s m o l a r i t y (136% of the s t a n d a r d c o n c e n t r a t i o n was used) (Treatment 5 ) , a marked d e p r e s s i o n of urea s y n t h e s i s o c c u r e d . In f a c t , w i t h t h i s t r e a t m e n t , urea s y n t h e s i s was s i g n i f i c a n t l y l o wer (P<0.05) than r a t e s measured s i m u l t a n e o u s l y i n c e l l s i n c u b a t e d i n media w i t h 50% or 100% of normal o s m o l a r i t y . A t t h i s p o i n t a n o t h e r t r e a t m e n t (Treatment 6) was added t o the e x p e r i m e n t a l p r o t o c o l t o d e t e r m i n e whether low l e v e l s of NaCl o r h i g h c o n c e n t r a t i o n s of ur e a were c a u s i n g t h e i n h i b i t i o n of u r e a s y n t h e s i s . H e p a t o c y t e s were i n c u b a t e d i n media c o n t a i n i n g s t a n d a r d amounts of a l l s o l u t e s e xcept u r e a , which was p r e s e n t a t 136% of the normal c o n c e n t r a t i o n . A g a i n , as i n the low N a C l - h i g h urea t r e a t m e n t , a d e c r e a s e i n urea s y n t h e s i s o c c u r r e d which was more pronounced than t h a t which o c c u r e d a t 50% o s m o l a r i t y . 7 8 F i g u r e 9. E f f e c t s , of Reduced C o n c e n t r a t i o n s of Urea and TMAO on Urea S y n t h e s i s i n S k a t e H e p a t o c y t e s . * r a t e i s s i g n i f i c a n t l y d i f f e r e n t from r a t e measured under s t a n d a r d c o n d i t i o n s (P<0.05), as d e t e r m i n e d w i t h Tukey's M u l t i p l e Range T e s t on b l o c k d e s i g n d a t a . 8 0 F i g u r e 10. E f f e c t s of Reduced C o n c e n t r a t i o n s of NaCl and H i g h Urea L e v e l s on Urea S y n t h e s i s i n Skate H e p a t o c y t e s . * r a t e i s s i g n i f i c a n t l y d i f f e r e n t from r a t e measured under s t a n d a r d c o n d i t i o n s (P<0.05), as d e t e r m i n e d w i t h Tukey's M u l t i p l e Range T e s t on b l o c k d e s i g n d a t a . ** r a t e i s s i g n i f i c a n t l y d i f f e r e n t from r a t e s measured a t b oth 50% and 100% o s m o l a r i t y , as d e t e r m i n e d w i t h Tukey's t e s t as above. 18 8 2 T a b l e 7. E f f e c t s of Reduced C o n c e n t r a t i o n s of U r e a , N a C l , and TMAO on Urea S y n t h e s i s i n Skate H e p a t o c y t e s a t C o n s t a n t O s m o l a r i t y . * r a t e i s s i g n i f i c a n t l y d i f f e r e n t from r a t e measured under s t a n d a r d c o n d i t i o n s (P<0.05), as d e t e r m i n e d w i t h Tukey's M u l t i p l e Range T e s t on b l o c k d e s i g n d a t a . ** r a t e i s s i g n i f i c a n t l y d i f f e r e n t from r a t e s measured a t b o t h 50% and 100% o s m o l a r i t y , as d e t e r m i n e d w i t h Tukey's t e s t as above. Rate of urea s y n t h e s i s i s e x p r e s s e d as /imoles of u r e a s y n t h e s i z e d per gram of h e p a t o c y t e s per hour. Treatment N Rate of Urea S y n t h e s i s (timols/g/hour) ± SEM. 100% O s m o l a r i t y 7 1.047 ± 0.089 50% O s m o l a r i t y 7 0.768 ± 0.095 * 50% Urea/330 mM NaCl 7 0.985 ± 0.094 100% O s m o l a r i t y 4 1.205 ± 0.205 50% TMAO/280 mM NaCl 4 1.206 ± 0.255 100% O s m o l a r i t y 11 1.031 ± 0.216 50% O s m o l a r i t y 11 0.803 ± 0.123 * 50% NaCl/470 mM Urea 11 0.553 ± 0.122 ** 100% O s m o l a r i t y 5 1.214 ± 0.185 50% NaCl/470 mM Urea 5 0.876 ± 0.169 * 100% Osmolarity+470 mM Urea 5 0.486 ± 0.127 * 84 A l t e r i n g c o n c e n t r a t i o n s of the major s o l u t e s ( u r e a , N a C l , and TMAO) was not r e s p o n s i b l e f o r c a u s i n g t h e d e c r e a s e i n urea s y n t h e s i s which was o b s e r v e d when h e p a t o c y t e s were i n c u b a t e d i n media w i t h low o s m o l a r i t y . From r e s u l t s o b t a i n e d w i t h Treatment 6 i t was c o n c l u d e d t h a t h i g h l e v e l s of u r e a , as opposed t o low l e v e l s of N a C l , were c a u s i n g a d e p r e s s i o n of ur e a s y n t h e s i s i n Treatment 5. I t i s u n c e r t a i n whether t h i s i s a n e g a t i v e feedback e f f e c t on urea s y n t h e s i s , or whether h i g h urea c o n c e n t r a t i o n s were c a u s i n g a g e n e r a l m e t a b o l i c d e p r e s s i o n i n h e p a t o c y t e s . I n Treatment 6, o s m o l a r i t y of t h e i n c u b a t i o n was s l i g h t l y g r e a t e r than s t a n d a r d l e v e l s (113% of n o r m a l ) . T h i s s l i g h t l y e l e v a t e d o s m o l a r i t y does not acco u n t f o r t h e d e c r e a s e d r a t e of urea s y n t h e s i s as i n c u b a t i o n of h e p a t o c y t e s i n media w i t h 125% of normal plasma o s m o l a r i t y had no e f f e c t on urea s y n t h e s i s ( F i g u r e 6, T a b l e 5 ) . Summary I n c u b a t i o n of s k a t e h e p a t o c y t e s w i t h low s o l u t e c o n c e n t r a t i o n s c a u s e s a s p e c i f i c i n h i b i t i o n of urea s y n t h e s i s . No i n d i v i d u a l s o l u t e was i d e n t i f i e d as an e f f e c t o r of t h i s r e s p o n s e . T h e r e f o r e , the q u e s t i o n s t i l l remains as t o what p r e c i s e l y i s the mechanism i n v o l v e d which i n d u c e s a d e c r e a s e i n urea s y n t h e s i s i n h e p a t o c y t e s a t low o s m o l a r i t i e s . B a l l a n t y n e e t a l . (1986) s t a t e , w i t h r e g a r d t o e f f e c t s of o s m o l a r i t y on m i t o c h o n d r i a l o x i d a t i o n of s a r c o s i n e , " . . . i t i s apparent t h a t o s m o t i c e f f e c t s ... a r e s u f f i c i e n t t o i n i t i a t e t he r e g u l a t o r y r e s p o n s e " , and the same appears t o be t r u e f o r ur e a s y n t h e s i s i n 8 5 s k a t e h e p a t o c y t e s . Many m e t a b o l i c pathways, i n c l u d i n g u r e a s y n t h e s i s and g l u c o n e o g e n e s i s i n mammalian systems, a r e s e n s i t i v e t o changes i n pH. One p o s s i b l e e x p l a n a t i o n f o r the r e s u l t s o b t a i n e d i n t h e l o w - o s m o l a r i t y e x p e r i m e n t s i s t h a t a l t e r i n g the s o l u t e c o n c e n t r a t i o n of the e x t r a c e l l u l a r f l u i d r e s u l t e d i n changes i n pH w i t h i n h e p a t o c y t e s , which i n t u r n a f f e c t e d urea s y n t h e s i s , a l a n i n e o x i d a t i o n , and g l u c o n e o g e n e s i s . I t s h o u l d be n o t e d , however, t h a t when i n t r a c e l l u l a r pH i s a l t e r e d i n r a t h e p a t o c y t e s r a t e s of f l u x t h r o u g h urea and g l u c o s e b i o s y n t h e t i c pathways change i n the same d i r e c t i o n (Kashiwagura e t a l . , 1984). Thus, i f changes i n i n t r a c e l l u l a r pH a r e r e s p o n s i b l e f o r changes i n the r a t e of f l u x t h r o u g h m e t a b o l i c pathways as seen i n t h i s s t u d y a t low o s m o l a r i t i e s , u rea s y n t h e s i s and g l u c o n e o g e n e s i s show c o m p l e t e l y d i f f e r e n t pH s e n s i t i v i t i e s i n elasmobranchs as compared w i t h mammals. Many m e t a b o l i c r e a c t i o n s r e q u i r e the p r e s e n c e of s p e c i f i c i n o r g a n i c i o n s t o a c t as enzyme c o f a c t o r s . Hypoosmotic s t r e s s , w hich i n i t i a l l y c auses c e l l s t o s w e l l , may r e s u l t i n e f f l u x of s p e c i f i c i o n s . I t i s a w e l l - e s t a b l i s h e d f a c t t h a t f o l l o w i n g o s m o t i c s w e l l i n g , d u r i n g v o l u m e - r e g u l a t o r y s h r i n k a g e , membranes become more permeable t o K +, which r e s u l t s i n a net e f f l u x of p o t a s s i u m i o n s from c e l l s (Kregenow, 1971; H e n d i l and Hoffman, 1974). A n o t h e r f e a s i b l e e x p l a n a t i o n f o r t h e d e p r e s s i o n of urea s y n t h e s i s w h i c h i s obser v e d a t low o s m o l a r i t i e s i s t h a t an e f f l u x of one s p e c i f i c i o n i n v o l v e d w i t h u rea s y n t h e s i s i s o c c u r r i n g f o l l o w i n g hypoosmotic shock, and i s t h u s l i m i t i n g the 86 r e a c t i o n r a t e ( J . P h i l l i p s , p e r s o n a l c o m m u n i c a t i o n ) . T h i s h y p o t h e s i s may be t e s t e d by m e a s u r i n g urea s y n t h e s i s i n h e p a t o c y t e s i n c u b a t e d w i t h d i f f e r e n t i o n o p h o r e s , each of which s h o u l d cause an e f f l u x of one s p e c i f i c i o n . A d e c r e a s e i n urea s y n t h e s i s when a g i v e n ionophore i s a p p l i e d t o h e p a t o c y t e s would p r o v i d e c i r c u m s t a n t i a l e v i d e n c e r e g a r d i n g the i d e n t i t y of the e f f e c t o r of the a d a p t i v e response i n u r e a s y n t h e s i s which o c c u r s a t low o s m o l a r i t i e s . 8 7 CONCLUSIONS R a t e s of a l a n i n e o x i d a t i o n and g l u c o n e o g e n e s i s from a l a n i n e d e t e r m i n e d i n the p r e s e n t s t u d y a r e s i m i l a r t o r a t e s measured i n the sea raven ( H e m i t r i p t e r u s americanus) (Walsh e t a l . , 1985) and i n s a l m o n i d s (Mommsen e t a l . , 1985). Rates of f l u x t h r o u g h t h e s e pathways were measured p r i m a r i l y t o e n a b l e assessment of the o v e r a l l m e t a b o l i c s t a t e of h e p a t o c y t e s w i t h d i f f e r e n t t r e a t m e n t s , and f o r comparison w i t h r e s p o n s e s seen i n urea s y n t h e s i s . A l t h o u g h the p r e s e n t s t u d y was not f o c u s s e d on t h e s e pathways some i n t r i n s i c a l l y i n t e r e s t i n g f i n d i n g s , worthy of f u r t h e r c o n s i d e r a t i o n , were o b t a i n e d . I n c u b a t i o n of h e p a t o c y t e s w i t h i n s u l i n and g l u c a g o n f o r 2.25 h o u r s had no a f f e c t on a l a n i n e o x i d a t i o n , p o s s i b l y due t o s e a s o n a l f a c t o r s (T. Mommsen, E. P l i s e t s k a y a , and T. Moon, p e r s o n a l c o m m u n i c a t i o n s ) , as e x p e r i m e n t s were done i n the w i n t e r when some f i s h a r e known t o be u n r e s p o n s i v e t o t h e s e hormones. 1a-OH and 1,2-dehydro-B caused a s i g n i f i c a n t e l e v a t i o n (30% and 25% r e s p e c t i v e l y ) i n a l a n i n e o x i d a t i o n i n h e p a t o c y t e s a f t e r a 24 hour i n c u b a t i o n p e r i o d . A g a i n , t h i s may r e p r e s e n t an a d a p t i v e r e s p o n s e of e u r y h a l i n e s k a t e s t o e n v i r o n m e n t a l d i l u t i o n . P r o d u c t i o n r a t e s and plasma c o n c e n t r a t i o n s of 1a-OH a r e known t o i n c r e a s e i n elasmobranchs d u r i n g hypoosmotic s t r e s s (Hazon and Henderson, 1984); a l l the r e c e p t o r s f o r t h i s hormone a r e l o c a t e d i n o s m o r e g u l a t o r y o r g a n s , i n c l u d i n g the l i v e r (Moon and I d l e r , 1974), s u g g e s t i n g t h a t the o b s e r v e d response i s more than c o i n c i d e n t a l . In o r d e r t o o b t a i n a more c o n c l u s i v e answer r e g a r d i n g the n a t u r e of t h e o s m o r e g u l a t o r y importance of 1a-OH, 88 f u t u r e s t u d i e s s h o u l d be done t o d e t e r m i n e e f f e c t s of t h i s hormone on o x i d a t i o n r a t e s of o s m o t i c a l l y a c t i v e amino a c i d s . Measurements of h e p a t i c u r e a s y n t h e s i s f o r R. e r i n a c e a o b t a i n e d i n the p r e s e n t s t u d y and i n a p a r a l l e l s t u d y by Mommsen and Moon (1987) ( a p p r o x i m a t e l y 1 /mole per g per hour) a r e comparable w i t h r a t e s measured i n i n d o g f i s h ( S q u a l u s a c a n t h i a s ) l i v e r s l i c e s (3 /mole per g per h o u r ) , as d e t e r m i n e d by i n c o r p o r a t i o n of 1 f C - b i c a r b o n a t e ( S c h o o l e r e t a l . , 1966). A c c o r d i n g t o T r u s c o t t e t a l . ( 1 9 7 6 ) , the l i v e r may a c c o u n t f o r as much as 10% of body weight i n s k a t e s . Thus, a 1 kg a n i m a l c o u l d s y n t h e s i z e 100 /xmoles of u rea per hour. Payan e t a l . ( 1973) r e p o r t r a t e s of b r a n c h i a l u r e a l o s s of 570 jumoles per kg per hour, and r e n a l l o s s e s of 110 jumoles per kg per hour i n R. e r i n a c e a , a t o t a l r a t e of u rea l o s s e q u a l t o 671 umoles per kg per hour under c o n d i t i o n s of normal s a l i n i t y . Thus, h e p a t i c u r ea s y n t h e s i s appears t o be i n a d e q u a t e t o b a l a n c e l o s s e s t o t h e e n vironment. I t i s p o s s i b l e t h a t measurements o b t a i n e d i n c e l l and t i s s u e - s l i c e s t u d i e s u n d e r e s t i m a t e urea p r o d u c t i o n , or t h a t measured r a t e s of l o s s a r e t o o h i g h , however o t h e r f a c t o r s s h o u l d be t a k e n i n t o c o n s i d e r a t i o n . P r o l o n g e d s t a r v a t i o n has been found t o cause a d e c r e a s e i n plasma urea c o n c e n t r a t i o n s i n pyjama s h a r k s (Poroderma a f r i c a n u m ) , a s t e n o h a l i n e elasmobranch (Haywood, 1973), and plasma urea l e v e l s were found t o i n c r e a s e a l m o s t i m m e d i a t e l y f o l l o w i n g f e e d i n g . Haywood c o n c l u d e d t h a t m e t a b o l i s m of i n g e s t e d p r o t e i n s p r o v i d e s ammonia f o r urea s y n t h e s i s . A more d i r e c t r e l a t i o n s h i p between d i e t a r y p r o t e i n i n t a k e and plasma urea l e v e l s may e x i s t . L i k e many 89 e l a s m o b r a n c h s , R. e r i n a c e a i s c a r n i v o r o u s ( p e r s o n a l o b s e r v a t i o n ) , and f e eds p r i m a r i l y on marine worms, sh r i m p , and s m a l l f i s h . A r g i n i n e t y p i c a l l y a c c o u n t s f o r 20% of the amino a c i d c o n t e n t of muscle p r o t e i n (Mommsen e t a l . , 1980); t h u s , t h e enzyme a r g i n a s e may produce s u b s t a n t i a l amounts of urea s i m p l y by h y d r o l y s i n g d i e t a r y a r g i n i n e ( F i g u r e 1 ) . T h i s c o u l d save a n i m a l s the m e t a b o l i c expense of b u i l d i n g u rea m o l e c u l e s v i a t h e o r n i t h i n e - u r e a c y c l e , a t f o u r ATPs per m o l e c u l e . U n f o r t u n a t e l y no q u a n t i t a t i v e f e e d i n g s t u d i e s have been done on R. e r i n a c e a , t h u s i t i s unknown whether or not d i e t a r y a r g i n i n e can a c c o u n t f o r t h e apparent urea d e f i c i t . The e u r y h a l i n e s k a t e , R. e r i n a c e a , i s known t o d e c r e a s e t h e r a t e of urea b i o s y n t h e s i s upon exposure t o d i l u t e seawater ( G o l d s t e i n and F o r s t e r , 1971). The p r e s e n t s t u d y i n v o l v e d a s e a r c h f o r the r e g u l a t o r y mechanisms which f u n c t i o n t o b r i n g about t h i s r e s p o n s e . None of the hormone t r e a t m e n t s a p p l i e d t o h e p a t o c y t e s i n t h i s s tudy had any a f f e c t on u r e a s y n t h e s i s . Hypoosmotic s t r e s s c a u s e d a s i g n i f i c a n t d e c r e a s e ( a p p r o x i m a t e l y 50% i n 50% o s m o l a r i t y i n c u b a t i o n media) i n r a t e of u rea s y n t h e s i s , i n marked c o n t r a s t w i t h r e s p o n s e s seen f o r a l a n i n e o x i d a t i o n and g l u c o n e o g e n e s i s . No one s o l u t e was i d e n t i f i e d as an e f f e c t o r of t h i s r e s p o n s e . I t has been h y p o t h e s i s e d t h a t an e f f l u x of one s p e c i f i c i o n , which then becomes r a t e - l i m i t i n g t o u r e a s y n t h e s i s , o c c u r s when c e l l s s w e l l i n t h e i r i n i t i a l r esponse t o hypoosmotic s t r e s s . F u t u r e s t u d i e s w i l l i n v o l v e i n c u b a t i o n of s k a t e h e p a t o c y t e s i n s o l u t i o n s c o n t a i n i n g s p e c i f i c i o n o p h o r e s , 90 i n an attempt t o i d e n t i f y the r e g u l a t i n g f a c t o r i n v o l v e d w i t h r e d u c t i o n of u r e a s y n t h e s i s a t low o s m o l a r i t i e s . < o 91 REFERENCES A q u i l a r - P a r a d a , E., A.M. E i s e n t r a u t , and R.H. Unger ( 1 9 6 9 ) . E f f e c t s of s t a r v a t i o n on plasma p a n c r e a t i c g l u c a g o n i n normal man. D i a b e t e s 18:717-723. A t k i n s o n , D.E. and E Bourke (1984). The r o l e of u r e a g e n e s i s i n pH h o m e o s t a s i s . TIBS, J u l y 1984, 297-300 B a l l a n t y n e , J.S. and T.W. Moon (1985). Hepatopancreas m i t o c h o n d r i a from M y t i l u s e d u l i s : s u b s t r a t e p r e f e r e n c e and e f f e c t s of pH and o s m o l a r i t y . Mar. B i o l . , 87:239-244. B a l l a n t y n e , J.S. and K.B. S t o r e y (1984). M i t o c h o n d r i a from t h e hep a t o p a n c r e a s of the marine clam M e r c e n a r i a m e r c e n a r i a : S u b s t r a t e p r e f e r e n c e s and s a l t and pH e f f e c t s on t h e o x i d a t i o n of p a l m i t o y l - L - c a r n i t i n e and s u c c i n a t e . J . Exp. Z o o l . , 230:165-174. B a l l a n t y n e , J.S., C. Moyes, and T.W. Moon (1986). O s m o l a r i t y a f f e c t s o x i d a t i o n of s a r c o s i n e by i s o l a t e d h e p a t o c y t e s and m i t o c h o n d r i a from a e u r y h a l i n e elasmobranch. J . Exp. Z o o l . , 238:267-271. B h a t t a c h a r y a , S., E. P l i s e t s k a y a , W.W. D i c k o f f , and A. Gorbman (1985). The e f f e c t s of e s t r a d i o l and t r i i o d o t h r y o n i n e on p r o t e i n s y n t h e s i s by h e p a t o c y t e s of j u v e n i l e coho salmon (Onchorhynchus k i s u t c h ) . Gen. & Comp. Endocr. 57:103-109. Bean, E.S. and D.E. A t k i n s o n (1984). R e g u l a t i o n of the r a t e of urea s y n t h e s i s i n l i v e r by e x t r a c e l l u l a r pH. A major f a c t o r i n pH h o m e o s t a s i s i n mammals. J . B i o l . Chem. 259: 1552-1559. B e n t l e y , P . J . (1982). Comparative V e r t e b r a t e E n d o c r i n o l o g y . Cambridge U n i v e r s i t y P r e s s , New York. Boyd, T.A., C.J. Cha, R.P. F o r s t e r , and L. G o l d s t e i n ( 1 9 7 7 ) . F r e e amino a c i d s i n t i s s u e s of the s k a t e R a j a e r i n a c e a and the s t i n g r a y D a s y a t i s s a b i n a : E f f e c t s of e n v i r o n m e n t a l d i l u t i o n . J . Exp. Z o o l . , 199:435-442. 92 C h r i s t o w i t z , D., F . J . M a t t h e y s e , and J.B. B a l i n s k y (1981). D i e t a r y and hormonal r e g u l a t i o n of urea c y c l e enzymes i n r a t l i v e r . Enzyme, 26:113-121. Cohen, J . J . , M.A. Krupp, and C.A. C h i d s e y (1958). R e n a l c o n s e r v a t i o n of t r i e t h y l a m i n e o x i d e by the s p i n y d o g f i s h , S q u a l u s a c a n t h i a s . Am. J . P h y s i o l . 194(2):229-235. Cohen, P.P. (1985). The r o l e of p r o t e i n p h o s p h o r y l a t i o n i n n e u r a l and hormonal c o n t r o l of c e l l u l a r a c t i v i t y . N a t u r e 296:613-620. C o r n e l l , N.W. (1983). E v a l u a t i o n of h e p a t o c y t e q u a l i t y : c e l l i n t e g r i t y and m e t a b o l i c r a t e s . IN I s o l a t i o n , C h a r a c t e r i z a t i o n , and Use of H e p a t o c y t e s . Ed. R.A. H a r r i s and N.W. C o r n e l l . E l s e v i e r B i o m e d i c a l , New Yo r k : , pp. 11-20. C o n l o n , J.M. and L. Thim (1986). P r i m a r y s t r u c t u r e of g l u c a g o n from an e l a s m o b r a n c h i a n f i s h , Torpedo marmorata. Gen. Comp. Endocr. 60:406-413. Cowey, C.B., K.W. D a i s l e y , and G. P a r r y ( 1 9 6 2 ) . Study of amino a c i d s , f r e e or as components of p r o t e i n , and of some B v i t a m i n s i n the t i s s u e s of the A t l a n t i c salmon, Salmo  s a l a r , d u r i n g spawning m i g r a t i o n . Comp. Biochem. P h y s i o l . 7:29-38. De La Huerga, J . , H. Popper, and F. Steigmann (1958). U r i n a r y e x c r e t i o n of c h o l i n e and t r i m e t h y l a m i n e s a f t e r i n t r a v e n o u s a d m i n i s t r a t i o n of c h o l i n e i n l i v e r d i s e a s e s . J . Lab. and C l i n . Med. 38:904-910. DeRoos, R., C C . DeRoos, C.S. Werner, and H.Werner (1985). Plasma l e v e l s of g l u c o s e , a l a n i n e , l a c t a t e , and (S-h y d r o x y b u t y r a t e i n the unfed s p i n y d o g f i s h shark ( S q u a l u s  a c a n t h i a s ) a f t e r s u r g e r y and f o l l o w i n g mammalian i n s u l i n i n f u s i o n . Gen. Comp. Endocr. 58:28-43. F e n s t e r m a c h e r , J . , F. S h e l d o n , J . R a t n e r , and A. Roomet (1972). The b l o o d t o t i s s u e d i s t r i b u t i o n of v a r i o u s p o l a r m a t e r i a l s i n t h e d o g f i s h , Squalus a c a n t h i a s . Comp. Biochem. P h y s i o l . , 42A:195-204. 93 F i s h e r , R.S. and K.R. S p r i n g (1984). I n t r a c e l l u l a r a c t i v i t i e s d u r i n g volume r e g u l a t i o n by N e c t u r u s g a l l b l a d d e r . J . Membrane B i o l . , 78;187-199. F o r s t e r , R.P., and L. G o l d s t e i n (1976). I n t r a c e l l u l a r o s m o r e g u l a t o r y r o l e of amino a c i d s and urea i n marine e l a s m o b r a n c h s . Am. J . P h y s i o l . , 230(4):925-931 . F r e n c h , C . J . , P.W. Hochachka, and T.P. Mommsen (1 9 8 3 ) . M e t a b o l i c o r g a n i z a t i o n of l i v e r d u r i n g spawning m i g r a t i o n of sockeye salmon. Am. J . P h y s i o l . 245:R827-R830. F u g e l l i , K. and H. Rohrs (1980). The e f f e c t of Na + and o s m o l a l i t y on the i n f l u x and st e a d y s t a t e d i s t r i b u t i o n of t a u r i n e and gamma-aminobutyric a c i d i n f l o u n d e r ( P l a t i c h t h y s f l e s u s ) e r y t h r o c y t e s . Comp. Biochem. P h y s i o l . 67A:545-551. G i l l e s , R. (1979). I n t r a c e l l u l a r o r g a n i c o s m o t i c e f f e c t o r s . In Mechanisms of O s m o r e g u l a t i o n on A n i m a l s , ed R. G i l l e s . pp. 111-153. New York: W i l e y . G o l d s t e i n , L. (1981). F r e e amino a c i d s and c e l l volume r e g u l a t i o n i n the elasmobranch, R a j a e r i n a c e a . J . Exp. Z o o l . , 215:371-377. G o l d s t e i n , L. (1982). G i l l n i t r o g e n e x c r e t i o n . In G i l l s , S o c i e t y f o r E x p e r i m e n t a l B i o l o g y Seminar S e r i e s _1_6:1 93-206. . Cambridge U n i v e r s i t y P r e s s , New York, 1982. G o l d s t e i n , L. and T.A. Boyd (1978). R e g u l a t i o n of /3-alanine t r a n s p o r t i n s k a t e ( R a j a e r i n a c e a ) e r y t h r o c y t e s . Comp. Biochem. P h y s i o l . 60:319-325. G o l d s t e i n , L. and S. D e w i t t - H a r l e y (1973). T r i m e t h y l a m i n e o x i d a s e of nurse shark l i v e r a n d i t s r e l a t i o n t o mammalian f i x e d f u n c t i o n amine o x i d a s e . Comp. Biochem. P h y s i o l . 45B: 895-903. G o l d s t e i n , L., and R.P. F o r s t e r ( 1 9 71). O s m o r e g u l a t i o n and urea m e t a b o l i s m i n the l i t t l e s k a t e R a j a er i n a c e a . Am. J . P h y s i o l . , 220(3)-.742-746. 94 G o l d s t e i n , L. and D. Funkhouser (1972). B i o s y n t h e s i s of t r i m e t h y l a m i n e o x i d e i n the nurse s h a r k , Ginqlymostoma  c i r r a t u m . Comp. Biochem. P h y s i o l . , 42A;51-52 . G o l d s t e i n , L. and P . J . P a l a t t (1974). T r i m e t h y l a m i n e o x i d e e x c r e t i o n r a t e s i n elasmobranchs. Am. J . P h y s i o l . 2 2 7 ( 6 ) : 1268-1272. H a l p e r i n , M.L. and R.L. Jungas (1983). M e t a b o l i c p r o d u c t i o n and r e n a l d i s p o s a l of hydrogen i o n s . K i d n e y I n t e r n a t i o n a l , 24(6):709-713. Hartman, F.A., L.A. L e w i s , K.A. B r o w n e l l , C.A. A n g e r e r , and F.F. Sheldon ( 1 9 4 4 ) . E f f e c t s of i n t e r r e n a l e c t o m y on some b l o o d c o n s t i t u e n t s i n the s k a t e . P h y s i o l . Z o o l . 17;228-238. Haywood, G.P. ( 1 9 7 3 ) . Hypo-osmotic r e g u l a t i o n c o u p l e d w i t h reduced m e t a b o l i c urea i n the d o g f i s h Poroderma a f r i c a n u m : an a n a l y s i s of serum o s m o l a r i t y , c h l o r i d e , and u r e a . Mar. B i o l . 23:121-127. Hazon, N. and I,W. Henderson (1984). S e c r e t o r y dynamics of 1a-h y d r o x y c o r t i c o s t e r o n e i n the elasmobranch f i s h , S c y l i o r h i n u s c a n i c u l a . J . Endocr. 103:205-211. H e n d i l , K.B. and E.K. Hoffmann (1974). C e l l volume r e g u l a t i o n i n E h r l i c h a s c i t e s tumor c e l l s . J . Gen. P h y s i o l . , 84:115-125. Hoffmann, E.K. ( 1 9 7 8 ) . R e g u l a t i o n of c e l l volume by s e l e c t i v e changes i n t h e l e a k p e r m e a b i l i t i e s of E h r l i c h a s c i t e s tumor c e l l s . I n Osmotic and Volume R e g u l a t i o n , A l f r e d Benson Symposium X I , ed, C.B. J o r g e n s e n & E. Skadhauge, pp. 397-417. Coopenhagen: Munksgaard. Hoffmann, E.K. ( 1 9 8 4 ) . Volume r e g u l a t i o n by a n i m a l c e l l s . I n C e l l u l a r A c c l i m a t i s a t i o n t o E n v i r o n m e n t a l Change, S o c i e t y f o r E x p e r i m e n t a l B i o l o g y Seminar S e r i e s , j_7:55-80. . Cambridge U n i v e r s i t y P r e s s , New York. Hoffmann, E.K. and I . Lambert (1983). Amino a c i d t r a n s p o r t and c e l l volume r e g u l a t i o n i n E h r l i c h a s c i t e s tumour c e l l s . J . P h y s i o l . 338:613-625. 9 5 H o l t , W.F. and D.R. I d l e r (1975). I n f l u e n c e of t h e i n t e r r e n a l g l a n d on the r e c t a l g l a n d of a s k a t e . Comp. Biochem. P h y s i o l . , 50C:111-119. I d l e r , D.R. and K.M. Kane (1980). C y t o s o l r e c e p t o r g l y c o p r o t e i n f o r 1 a - h y d r o x y c o r t i c o s t e r o n e i n t i s s u e s of an elasmobranch f i s h ( R a j a o c e l l a t a ) . Gen. & Comp. Endocr. 42;259-266. I d l e r , D.R., H.C. Freeman, and B. T r u s c o t t ( 1 9 6 7 ) . A p r e l i m i n a r y communication on the b i o l o g i c a l a c t i v i t y of 1a-h y d r o x y c o r t i c o s t e r o n e i s o l a t e d from c a r t i l a g i n o u s f i s h . J . F i s h . Res. Bd. Canada, 2 4 ( 0 :205-206. I d l e r , D.R. and B. T r u s c o t t (1969). P r o d u c t i o n of 1a-h y d r o x y c o r t i c o s t e r o n e in_ v i v o and j j i v i t r o by ela s m o b r a n c h s . Gen. Comp. Endocr. Supp. 2:325-330. I n g l e , D.J. (1952). The r o l e of the a d r e n a l c o r t e x i n h o m e o s t a s i s . J . E n d o c r i n o l . 8:23-27. Kashi w a g u r a , T., C.J. De u t s c h , J . T a y l o r , . M . E r e c i n s k a , and D.F. W i l s o n (1984). Dependence of g l u c o n e o g e n e s i s , urea s y n t h e s i s , and energy m e t a b o l i s m of h e p a t o c y t e s on i n t r a c e l l u l a r pH. J . B i o l . Chem. 2 5 9 ( 0 : 2 3 7 - 2 4 3 . K i n g , P.A. and L. G o l d s t e i n (1983). O r g a n i c o s m o l y t e s and c e l l volume r e g u l a t i o n i n f i s h . M o l e c . P h y s i o l . 4:53-66. K i n g , P.A., C . J . Cha, and L. G o l d s t e i n (1980). Amino a c i d m e t a b o l i s m and c e l l volume r e g u l a t i o n i n the l i t t l e s k a t e , R a j a e r i n a c e a . 11. S y n t h e s i s . J . Exp. Z o o l . , 212:79-86. K i n g , W.J. and G.L. Green (1984). M o n o c l o n a l a n t i b o d i e s l o c a l i z e e s t r o g e n r e c e p t o r i n t h e n u c l e i of t a r g e t c e l l s . N ature 307:745-747. Kormanik, G.A. and J.N. Cameron (1981). Ammonia e x c r e t i o n i n a n i m a l s t h a t b r e a t h e w a t e r : a r e v i e w . Mar. B i o . L e t t . 2: 11-23. 96 K r a u s - F r i e d m a n n , N. (1984). Hormonal r e g u l a t i o n of h e p a t i c g l u c o n e o g e n e s i s . P h y s i o l . Rev. 64(1):170-259. Lambert, I . , and E.K. Hoffmann (1982). Amino a c i d m e t a b o l i s m and p r o t e i n t u r n o v e r under d i f f e r e n t o s m o t i c c o n d i t i o n s i n E h r l i c h a s c i t e s tumor c e l l s . J . Molec. P h y s i o l . , 2:273-286. L a u f , P.K. (1 9 8 2 ) . E v i d e n c e f o r c h l o r i d e dependent p o t a s s i u m and water t r a n s p o r t induced by hypoosmotic s t r e s s i n e r y t h r o c y t e s of the marine t e l e o s t , Opsanus t a u . J . Comp. P h y s i o l . , 146:9-16. Leech, A.R. and L. G o l d s t e i n ( 1 983). 0 - a l a n i n e o x i d a t i o n i n the l i v e r of the l i t t l e s k a t e , R a j a er i n a c e a . J . Exp. Z o o l . 225:9-14. Lee c h , A.R., L. G o l d s t e i n , C.J. Cha, and J.M. G o l d s t e i n (1979). A l a n i n e b i o s y n t h e s i s d u r i n g s t a r v a t i o n i n s k e l e t a l muscle of t h e s p i n y d o g f i s h , Squalus a c a n t h i a s . J . Exp. Z o o l . , 207:73-80. L o v e l l - S m i t h , C . J . and P. Garcia-Webb (1986). G l u c o c o r t i c o i d s and t h e i s o l a t e d r a t h e p a t o c y t e . Biochem. B i o p h y s . Res. Comm. 135(1):160-165. M e i j e r , A . J . , J.A. G i m p e l , G. Deleeuw, M.E. T i s c h l e r , J.M. Tager, and J.R. W i l l i a m s o n (1978). I n t e r r e l a t i o n s h i p s between g l u c o n e o g e n e s i s and u r e o g e n e s i s i n i s o l a t e d h e p a t o c y t e s . J . B i o l . Chem. 2_53( 7) : 2308-2320. M i n k o w s k i , O., (1893) i n Kraus-Friedmann, 1984. Mommsen, T.P. (1986). Comparative g l u c o n e o g e n e s i s i n h e p a t o c y t e s from s a l m o n i d f i s h e s . Can. J . Z o o l . , 64:1110-1115. Mommsen, T.P., C.J. F r e n c h , and P.W. Hochachka (1980). S i t e s and p a t t e r n s of p r o t e i n and amino a c i d u t i l i z a t i o n d u r i n g the spawning m i g r a t i o n of salmon. Can. J . Z o o l . 58:1785-1799. 9 7 Mommsen, T.P. and T.W. Moon (1987). The m e t a b o l i c p o t e n t i a l of h e p a t o c y t e s and k i n e y i n the l i t t l e s k a t e , Raja e r i n a c e a . ( S u b m i t t e d ) Mommsen, T.P., and R.K. Suarez (1984). C o n t r o l of g l u c o n e o g e n e s i s i n rainbow t r o u t h e p a t o c y t e s : r o l e of p y r u v a t e b r a n c h p o i n t and p h o s p h e n o l p y r u v a t e p y r u v a t e c y c l e . M o l e c . P h y s i o l . 6:9-18. Mommsen, T.P., P . J . Walsh, and T.W. Moon (1985). G l u c o n e o g e n e s i s i n h e p a t o c y t e s and k i d n e y of a t l a n t i c salmon. Molec. Phys. 8:89-100. Moon, T.W. and D.R. I d l e r (1974). The b i n d i n g of 1a-h y d r o x y c o r t i c o s t e r o n e t o t i s s u e s o l u b l e p r o t e i n s i n the s k a t e , R a j a o c e l l a t a . Comp. Biochem. P h y s i o l . 48B:499-506. Moon, T.W., P . J . Walsh, and T.P. Mommsen (1986). F i s h h e p a t o c y t e s : a model m e t a b o l i c system. Can." J . F i s h . Aquat. S c i . 42:1772-1782. Muggeo, M. , B.H. G i n s b e r g , J . Roth, D.M. N e v i l l e J r . , P. Demeytes, C.R. Kahn (1979). The i n s u l i n r e c e p t o r i n v e r t e b r a t e s i s f u n c t i o n a l l y more c o n s e r v e d d u r i n g e v o l u t i o n than i n s u l i n i t s e l f . E n d o c r i n o l o g y , 104:1393-1402. N o r r i s , E.R. and G.J. B e n o i t J r . (1945). S t u d i e s on t r i m e t h y l a m i n e o x i d e 111. T r i m e t h y l a m i n e o x i d e e x c r e t i o n by the r a t . J . B i o l . Chem. 158:443-448. Payan, P., L. G o l d s t e i n , and R.P. F o r s t e r ( 1 9 73). G i l l s and k i d n e y s i n u r e o s m o t i c r e g u l a t i o n i n e u r y h a l i n e s k a t e s . Am. J . P h y s i o l . , 224(2):367-372. P e t e r s e n , T.D.P. (1987). Acute hormonal r e g u l a t i o n of g l u c o n e o g e n e s i s i n rainbow t r o u t h e p a t o c y t e s . S u b m i t t e d . P l i s e t s k a y a , E., H.G. P o l l o c k , J.B. Rouse, J.W. H a m i l t o n , J.R. Kimmel, and A. Gorbman (1985). C h a r a c t e r i z a t i o n of coho salmon (Onchorynchus k i s u t c h ) i n s u l i n . R e g u l a t o r y P e p t i d e s 11:105-116. 9 8 P l i s e t s k a y a , E., W.W. D i c k h o f f , T.L. P a q u e t t e , and A. Gorbman (1 9 8 6 ) . The assay of salmon i n s u l i n by homologous r a d i o i m m i n o a s s a y . F i s h P h y s i o l , and Biochem. J_:37-43. Schimke, R.T. (1962). S t u d i e s on f a c t o r s a f f e c t i n g the l e v e l s of u r e a c y c l e enzymes i n r a t l i v e r . J . B i o l . Chem. 2 3 8 ( 3 ) : 1012-1018. S c h o f f e n i e l s , E. (1976). A d a p t a t i o n s w i t h r e s p e c t t o s a l i n i t y . Biochem. Soc. Symp. 41:179-204. S h u t t l e w o r t h , T .J. and L. G o l d s t e i n (1984). 0 - a l a n i n e t r a n s p o r t i n t he i s o l a t e d h e p a t o c y t e s of the elasmobranch R a j a  e r i n a c e a . J . Exp. Z o o l . 231:39-44. S m i t h , H.W. (1936). The r e t e n t i o n and p h y s i o l o g i c a l r o l e of urea i n t h e e l a s m o b r a n c h i i . • B i o l . Review. 11:49-82. S n o d g r a s s , P . J . , R.C. L i n , W.A. M u l l e r , and T.T. A o k i (1978). I n d u c t i o n of urea c y c l e enzymes of r a t l i v e r by g l u c a g o n . J . B i o l . Chem., 253_(8) : 2748-2753. S t a d e l e r , G. and F r . Th. F r e r i c h s (1858) i n S m i t h , 1936. T r u s c o t t , B., K.M. Kane, and D.R. I d l e r (1976). D i s t r i b u t i o n and me t a b o l i s m of 1 a - h y d r o x y c o r t i c o s t e r o n e i n the s k a t e , R a j a  r a d i a t a . Comp. Biochem. P h y s i o l . 56B:341-345. T r u s c o t t , B., K.M. Kane, and D.R. I d l e r (1978). 21-h y d r o x y p r e g n a - 1 , 4 ~ d i e n e - 3 , 1 1 , 2 0 - t r i o n e : a b i l i a r y m e t a b o l i t e of a c a r t i l a g i n o u s f i s h , R a j a sp. S t e r o i d s , 3 J _ ( 4 ) : 2269-2270. W a l s e r , M. (1986). R o l e of ur e a p r o d u c t i o n , ammonium e x c r e t i o n , and amino a c i d o x i d a t i o n i n a c i d - b a s e b a l a n c e . Am. J . P h y s i o l . 250(2):F181-F188. Walsh, P . J . , T.W. Moon, and T.P. Mommsen (1985). I n t e r a c t i v e e f f e c t s of a c u t e changes i n te m p e r a t u r e and pH on m e t a b o l i s m i n h e p a t o c y t e s from the sea raven H e m i t r i p t e r u s  a m e r i c a n u s . P h y s i o l . Z o o l . 58(6):727-735. Welshons, W.V., M.E. Lieberman, and J . G o r s k i (1984). N u c l e a r l o c a l i z a t i o n of u n o c c u p i e d o e s t r o g e n r e c e p t o r s . N a t u r e 307:747-749. Yancey, P.H., M.E. C l a r k , S.C. Hand, R.D. Bowlus, and G.N. Somero (1982). L i v i n g w i t h water s t r e s s : e v o l u t i o n of os m o l y t e systems. S c i e n c e , 217:1214-1222. Zar, J.H. (1984). B i o s t a t i s t i c a l a n a l y s i s , 2nd e d i t i o n . P r e n t i c e - H a l l , Englewood C l i f f s , N.J. 

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