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UBC Theses and Dissertations

Choline metabolism in Nb 2 rat node lymphoma cells Ko, Kerry Woon Sing 1985

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CHOLINE METABOLISM IN Nb 2 RAT NODE LYMPHOMA CELLS by KERRY WOON SING KO B . S c , The U n i v e r s i t y o f B r i t i s h C o l i u m b i a , 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f B i o c h e m i s t r y ) 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 t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September 1985 © K e r r y Woon S i n g Ko, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of $1° die^i rjr> The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) ABSTRACT P h o s p h a t i d y l c h o l i n e m e t a b o l i s m was i n v e s t i g a t e d i n Nb 2 n o d e r a t l y m p h o m a , a c e l l l i n e w h i c h i s d e p e n d a n t on p r o l a c t i n f o r g r o w t h i n c u l t u r e . T r e a t m e n t o f s t a t i o n a r y c u l t u r e s w i t h p r o l a c t i n s t i m u l a t e d t h e i n c o r p o r a t i o n o f [Me-^ H ] c h o l i n e i n t o p h o s p h a t i d y l c h o l i n e ( 1 . 7 - f o l d a f t e r 4 h) and i t ' s aqueous p r e c u r s o r s , m a i n l y p h o s p h o c h o l i n e ( 2 - f o l d a f t e r 4 h a n d 3 - f o l d a f t e r 10 h ) . T h e s e e f f e c t s w e r e b l o c k e d by c y c l o h e x i m i d e . P u l s e - c h a s e s t u d i e s d e m o n s t r a t e d t h a t t h e r e a c t i o n c a t a l y z e d by C T P : p h o s p h o c h o l i n e c y t i d y l y l t r a n s f e r a s e (EC 2.7.7.15) was r a t e - l i m i t i n g f o r p h o s p h a t i d y l c h o l i n e s y n t h e s i s i n Nb 2 c e l l s . The s p e c i f i c a c t i v i t y o f c h o l i n e k i n a s e ( E C 2.7.1.32) i n c r e a s e d 1 . 9 - f o l d a f t e r 4 h o f p r o l a c t i n t r e a t m e n t , i n c o r r e s p o n d e n c e w i t h t h e i n c r e a s e i n c h o l i n e i n c o r p o r a t i o n m e n t i o n e d a b o v e , and t h i s was a l s o b l o c k e d by c y c l o h e x i m i d e . The a c t i v i t i e s o f t h e o t h e r e n z y m e s o f p h o s p h a t i d y l c h o l i n e s y n t h e s i s w e r e u n c h a n g e d . These r e s u l t s s u g g e s t e d t h a t p h o s p h a t i d y l c h o l i n e b i o s y n t h e s i s was n o t a l t e r e d i n Nb 2 c e l l s a f t e r p r o l a c t i n t r e a t m e n t . H o w e v e r , p h o s p h a t i d y l c h o l i n e l e v e l s i n c r e a s e d i n p r o l a c t i n t r e a t e d - c e l l s ( 1 . 4 - f o l d a f t e r 16 h ) . T u r n o v e r o f l a b e l e d p h o s p h a t i d y l c h o l i n e was r e d u c e d 3 . 4 - f o l d i n p r o l a c t i n t r e a t e d c e l l s . C a l c u l a t e d t u r n o v e r r a t e s f o r p h o s p h a t i d y l c h o l i n e a v e r a g e d 3 . 2 - f o l d l o w e r i n p r o l a c t i n t r e a t e d c e l l s w h e r e a s t h e s y n t h e t i c r a t e s w e r e s i m i l a r i n p r o l a c t i n t r e a t e d and s t a t i o n a r y c e l l s . T h u s , Nb 2 c e l l s u t i l i z e a n o v e l m echanism, i n h i b i t i o n o f t u r n o v e r , t o r e g u l a t e t h e c e l l u l a r l e v e l s o f p h o s p h a t i d y l c h o l i n e d u r i n g g r o w t h . i TABLE OF CONTENTS Page ABSTRACT i . L I S T OF TABLES v i . L I S T OF FIGURES v i i . L I S T OF ABBREVIATIONS v i i i . ACKNOWLEDGEMENTS i x . INTRODUCTION 1. 1. P r o p e r t i e s o f P h o s p h a t i d y l c h o l i n e 1. 2. B i o l o g i c a l R o l e s o f P h o s p h a t i d y l c h o l i n e 3. 3. B i o s y n t h e s i s o f P h o s p h a t i d y l c h o l i n e 4. (a) C D P - c h o l i n e P athway 5. (b) O t h e r P a t h w a y s f o r P h o s p h a t i d y l c h o l i n e B i o s y n t h e s i s 7. ( i ) S t e p w i s e M e t h y l a t i o n o f P h o s p h a t i d y l e t h a n o l a m i n e 7. ( i i ) B a s e E x c h a n g e 7. ( i i i ) A c y l a t i o n o f L y s o p h o s p h a t i d y l c h o l i n e 8. ( i v ) T r a n s a c y l a t i o n o f P h o s p h a t i d y l c h o l i n e 8. 4. C a t a b o l i s m o f P h o s p h a t i d y l c h o l i n e 8. (a) P h o s p h o l i p a s e A 8. (b) L y s o p h o s p h o l i p a s e s and P h o s p h o l i p a s e B 11. (c) G l y c e r o p h o s p h o c h o l i n e P h o s p h o d i e s t e r a s e . . 11. (d) P h o s p h o l i p a s e C 12. (e) P o s p h o l i p a s e D 12. i i Page 5. The T h e s i s I n v e s t i g a t i o n s 13. MATERIALS AND METHODS 15. 1. C h e m i c a l s , Enzymes, and I s o t o p e s 15. 2. C e l l s 17. (a) S o u r c e 17. (b) M a i n t e n a n c e o f C u l t u r e s 17. (c) P r e p a r a t i o n o f S t a t i o n a r y C u l t u r e s 18. 3. P r e p a r a t i v e P r o c e d u r e s 18. (a) C e n t r i f u g a t i o n s 18. (b) H a r v e s t i n g o f C e l l s . 18. (c) S u b c e l l u l a r F r a c t i o n a t i o n 19. (d) Medium 20. (e) P r o l a c t i n S o l u t i o n 20. (f ) [Me- 3H] P h o s p h o c h o l i n e 21. (g) T o t a l Rat L i v e r P h o s p h o l i p i d 22. 4. A n a l y t i c a l P r o c e d u r e s 23. (a) T h i n - l a y e r C h r o m a t o g r a p h y 23. ( i ) S e p a r a t i o n o f Aqueous C h o l i n e M e t a b o l i t e s (System I) 23. ( i i ) S e p a r a t i o n o f P h o s p h o l i p i d s (System II) 24. (b) L i q u i d S c i n t i l l a t i o n C o u n t i n g 24. (c) P r o t e i n A s s a y ... 25. (d) L i p i d P h o s p h o r u s A s s a y 25. 5. C e l l L a b e l i n g S t u d i e s 26. (a) P u l s e - l a b e l i n g E x p e r i m e n t s 26. (b) P u l s e - c h a s e E x p e r i m e n t s . . . . . 26. (c) P h o s p h a t i d y l c h o l i n e T u r n o v e r 27. i i i Page 6. P o o l S i z e Measurements 27. (a) P h o s p h o c h o l i n e 27. (b) P h o s p h a t i d y l c h o l i n e 28. 7. E s t i m a t i o n o f M e t a b o l i c R a t e s 28. (a) P h o s p h a t i d y l c h o l i n e T u r n o v e r 28. (b) P h o s p h a t i d y l c h o l i n e S y n t h e s i s 29. 8. Enzyme A s s a y s 29. (a) C h o l i n e K i n a s e 29. (b) C T P : P h o s p h o c h o l i n e C y t i d y l y l t r a n s f e r a s e . . 30. (c) C D P - c h o l i n e : D i a c y l g l y c e r o l C h o i i n e p h o s p h o t r a n s f e r a s e 31. RESULTS 1. E f f e c t of P r o l a c t i n on t h e C D P - c h o l i n e Pathway. 32. (a) [ M e - 3 H ] C h o l i n e Uptake 32. (b) P u l s e - c h a s e S t u d i e s 37. (c) P h o s p h o c h o l i n e P o o l S i z e 40. 2. E f f e c t o f P r o l a c t i n on P h o s p h a t i d y l c h o l i n e T u r n o v e r 43. (a) P h o s p h a t i d y l c h o l i n e P o o l S i z e 43. (b) T u r n o v e r o f P h o s p h a t i d y l c h o l i n e 44. (c) E s t i m a t i o n o f P h o s p h a t i d y l c h o l i n e M e t a b o l i c R a t e s 48. 3. E f f e c t o f P r o l a c t i n on t h e Enzymes o f t h e C D P - c h o l i n e Pathway 50. (a) C h o l i n e K i n a s e 52. (b) C y t i d y l y l t r a n s f e r a s e 55. (c) C h o l i n e p h o s p h o t r a n s f e r a s e 59. DISCUSSION 1. I n t e r a c t i o n o f P r o l a c t i n w i t h Nb 2 C e l l s 60. i v Pag 2. E f f e c t o f P r o l a c t i n on P h o s p h a t i d y l c h o l i n e B i o s y n t h e s i s 61 (a) C h o l i n e K i n a s e 62 (b) C y t i d y l y l t r a n s f e r a s e 64 3. E f f e c t o f P r o l a c t i n on P h o s p h a t i d y l c h o l i n e C a t a b o l i s m 65 (a) P h o s p h o l i p a s e D o r C 67 (b) D e a c y l a t i o n Pathways 68 4. C o n c l u d i n g Remarks 70 v LIST OF TABLES T a b l e Page 1 T y p i c a l p r o t e i n p r o f i l e o f Nb 2 c e l l s a f t e r s u b c e l l u l a r f r a c t i o n a t i o n 21. 2 C o m p a r i s o n o f c h a n g e s i n s p e c i f i c r a d i o -a c t i v i t y o f p h o s p h o c h o l i n e and l a b e l i n g o f p h o s p h a t i d y l c h o l i n e i n Nb 2 c e l l s + p r o l a c t i n . 42. 3 P h o s p h o l i p i d c o m p o s i t i o n o f Nb 2 c e l l s 46. 4 C a l c u l a t e d p h o s p h a t i d y l c h o l i n e t u r n o v e r and s y n t h e s i s r a t e s 51. 5 In. v i t r o a c t i v i t i e s o f enzymes of C D P - c h o l i n e pathway from Nb 2 c e l l s 53. 6 M i c r o s o m a l r e c o v e r y 56. 7 R e c o v e r y o f c y t i d y l y l t r a n s f e r a s e a f t e r s u b -c e l l u l a r f r a c t i o n a t i o n o f Nb 2 c e l l s 58. v i LIST OF FIGURES F i g u r e Pag 1 S t r u c t u r e and s p a c e - f i l l i n g model o f p h o s p h a t i d y l c h o l i n e 2 2 The C D P - c h o l i n e pathway f o r p h o s p h a t i d y l -c h o l i n e b i o s y n t h e s i s 6 3 The s i t e s o f a t t a c k o f v a r i o u s p h o s p h o l i p a s e s on p h o s p h a t i d y l c h o l i n e 9 4 C o n t i n u o u s p u l s e o f Nb 2 c e l l s w i t h [ M e - 3 H ] c h o l i n e 33 5 E f f e c t o f p r o l a c t i n on [Me- H ] c h o l i n e u p t a k e i n t o Nb 2 c e l l s 35 6 E f f e c t o f c y c l o h e x i m i d e on p r o l a c t i n - m e d i a t e d [Me- 3H] c h o l i n e u p t a k e i n t o Nb 2 c e l l s 36 7 E f f e c t o f p r o l a c t i n on [ M e - 3 H ] c h o l i n e p u l s e -c h a s e i n Nb 2 c e l l s 38 8 E f f e c t o f p r o l a c t i n on p h o s p h o c h o l i n e p o o l s i n Nb 2 c e l l s 41 9 E f f e c t o f p r o l a c t i n on p h o s p h a t i d y l c h o l i n e l e v e l s i n Nb 2 c e l l s 45 10 E f f e c t o f p r o l a c t i n on t h e t u r n o v e r o f l a b e l e d p h o s p h a t i d y l c h o l i n e 47 11 A n a l y s i s o f medium f o r r a d i o a c t i v i t y i n aqueous c h o l i n e m e t a b o l i t e s 49 v i i LIST OF ABBREVIATIONS ACS aqueous c o u n t i n g s c i n t i l l a n t ATP a d e n o s i n e t r i p h o s p h a t e C C e l s i u s cAMP a d e n o s i n e 3':5'-monophosphate CDP- c y t i d i n e d i p h o s p h o r y l -CHO Chinese hamster o v a r y C i C u r i e CoA coenzyme A CTP c y t i d i n e t r i p h o s p h a t e DNA d e o x y r i b o n u c l e i c a c i d dpm d i s i n t e g r a t i o n s p e r m i n u t e EDTA e t h y l e n e d i a m i n e - t e t r a a c e t a t e EGF e p i d e r m a l growth f a c t o r EGTA e t h y l e n e g l y c o l - b i s - ( b e t a - a m i n o e t h y l e t h e r ) N , N ' -t e t r a a c e t a t e F i g . F i g u r e g gram £ g r a v i t y h hour HDL h i g h d e n s i t y l i p o p r o t e i n HPLC h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y Km M i c h a e l i s - M e n t e n c o n s t a n t 1 l i t e r LDL low d e n s i t y l i p o p r o t e i n m meter M mo1a r Me- m e t h y l -min m i n u t e moL mole N normal n= number o f samples PC p h o s p h a t i d y l c h o l i n e PE p h o s p h a t i d y l e t h a n o l a m i n e PI p h o s p h a t i d y l i n o s i t o l PS p h o s p h a t i d y l s e r i n e rpm r e v o l u t i o n s p e r mi n u t e S.D. s t a n d a r d d e v i a t i o n SM s p h i n g o m y e l i n U u n i t s U.V. u l t r a v i o l e t v volume VLDL v e r y low d e n s i t y l i p o p r o t e i n Vmax maximum v e l o c i t y w w e i g h t S t a n d a r d p r e f i x e s a r e : n (nano) - 10 ; u ( m i c r o ) - 10 m ( m i l l i ) - 1 0 ~ 3 ; c ( c e n t i ) - 10~ 2 v i i i ACKNOWLEDGEMENTS I am g r a t e f u l t o my s u p e r v i s o r , Dr. D e n n i s E. Vance, f o r h i s p a t i e n t g u i d a n c e . The c o u n s e l i n g o f Dr. H a r o l d W. Cook d u r i n g t h e e a r l y s t a g e s o f t h i s work i s g r e a t l y a p p r e c i a t e d . D r s . C h a r l e s B e e r and P e t e r G o u t a r e t h a n k e d f o r t h e i r a s s i s t a n c e on c u l t u r i n g t h e Nb 2 c e l l s . I am a l s o t h a n k f u l t o t h e o t h e r members o f t h i s l a b g r o u p , D r s . J e a n V a n c e , R o s e m a r y C o r n e l l , and H o w a r d M u e l l e r , my f e l l o w g r a d u a t e s t u d e n t s J.L., J.S., N.R., Z.Y. and my many o t h e r a c q u a i n t -a n c e s t h e i r f r i e n d s h i p and a d v i c e a l o n g t h e way. i x INTRODUCTION 1. P r o p e r t i e s o f P h o s p h a t i d y l c h o l i n e P h o s p h a t i d y l c h o l i n e (PC) i s t h e m a j o r p h o s p h o l i p i d i n h i g h e r o r g a n i s m s , a l t h o u g h i t i s r a r e i n p r o k a r y o t e s . I n e u k a r y o t e s , PC i s an i m p o r t a n t s t r u c t u r a l c o m p o n e n t and r e p r e s e n t s a b o u t h a l f t h e membrane p h o s p h o l i p i d (1). The s t r u c t u r e o f PC ( F i g . 1) c o n s i s t s o f a g l y c e r o l b a c k b o n e w i t h f a t t y a c i d s e s t e r i f i e d t o t h e C I and C2 p o s i t i o n s and a c h o l i n e r e s i d u e a t t a c h e d t o t h e C3 p o s i t i o n b y a p h o s p h a t e e s t e r l i n k a g e . T he f a t t y a c i d a t t h e C I p o s i t i o n i s u s u a l l y s a t u r a t e d w h e r e a s a t C2 i t i s u s u a l l y u n s a t u r a t e d . V a r i a t i o n s i n t h e c h a i n l e n g t h and d e g r e e o f u n s a t u r a t i o n o f t h e f a t t y a c i d s r e s u l t s i n m o l e c u l a r s p e c i e s w i t h i n t h i s p h o s p h o l i p i d c l a s s . PC i s an a m p h i p a t h i c m o l e c u l e , owing t o i t ' s h y d r o p h o b i c h y d r o c a r b o n t a i l and i t ' s h y d r o p h i l i c p o l a r head group. The t e n d e n c y o f PC and o t h e r p h o s p h o l i p i d s t o a d o p t a b i l a y e r o r g a n i z a t i o n upon h y d r a t i o n i s i n a m a j o r p a r t due t o t h e i r a m p h i p a t h i c n a t u r e . T h i s b i l a y e r o r g a n i z a t i o n o f phospho-l i p i d s f o r m s t h e b a s i s o f a l l b i o l o g i c a l membranes a c c o r d i n g t o t h e f l u i d m o s a i c m o d e l o f S i n g e r and N i c h o l s o n ( l a ) and p r o v i d e s a m a t r i x f o r m e m b r a n e p r o t e i n s . B i o l o g i c a l membranes c o n t a i n l i p i d s o t h e r t h a n PC and t h e s e may s e r v e t o m o d u l a t e membrane f l u i d i t y and p e r m e a b i l i t y o r c o n t r i b u t e t o 1 0 0 II CH - 0 - C - R 1 R ?-C -0»»C-H Q + CH o - 0 - P - 0 - C H o C H oN(CH oL 2 i 2 2 3 3 0 _ F i g u r e 1. S t r u c t u r e and s p a c e - f i l l i n g model o f p h o s p h a t i d y l -c h o l i n e , and R2 a r e f a t t y a c y l s u s t i t u e n t s . 2 l i p i d p o l y m o r p h i s m s (2). 2. B i o l o g i c a l R o l e s o f P h o s p h a t i d y l c h o l i n e I n a n i m a l s , PC i s a c o n s t i t u e n t o f b i l e , p l a s m a l i p o p r o t e i n s and l u n g s u r f a c t a n t . B i l i a r y PC a s s i s t s i n the s o l u b i l i z a t i o n o f t r i g l y c e r i d e s d u r i n g d i g e s t i o n i n t h e s m a l l i n t e s t i n e s . The m a j o r p h o s p h o l i p i d component o f HDL, LDL and VLDL i s PC ( 2 a ) . Why PC p r e d o m i n a t e s i n l i p o p r o t e i n s i s u n c l e a r a l t h o u g h i t does s e r v e t o c o a t t h e s u r f a c e and t h u s r e n d e r t h e c o r e o f t r i a c y l g l y c e r o l and c h o l e s t e r o l e s t e r s w a t e r - s o l u b l e . PC i s a l s o a s u b s t r a t e f o r t h e l e c i t h i n r c h o l -e s t e r o l a c y l - C o A t r a n s f e r a s e r e a c t i o n v i t a l f o r c h o l e s t e r o l e f f l u x f r o m n o n - h e p a t i c c e l l s t o p l a s m a l i p o p r o t e i n s ( 2 a ) . Lung s u r f a c t a n t c o n t a i n s a p p r o x i m a t e l y 70% PC by w e i g h t (3) and l i n e s t h e a l v e o l i t o l o w e r t h e s u r f a c e t e n s i o n and p r e v e n t a l v e o l a r c o l l a p s e d u r i n g e x p i r a t i o n . The i m p o r t a n c e o f t h e s e s e c r e t i o n s has l e a d t o c o n s i d e r a b l e i n t e r e s t i n t h e pathways r e s p o n s i b l e f o r t h e s y n t h e s i s o f t h e PC component, as w e l l as t h e s e c r e t o r y p r o c e s s e s . PC i s t h e d o n o r o f f a t t y a c y l m o i e t y i n t h e s y n t h e s e s o f p r o s t a g l a n d i n s and r e l a t e d compounds (4). A p a r t i c u l a r s p e c -i e s o f PC ( l - a l k y l - 2 - a c e t y l PC) h a s b e e n i d e n t i f i e d a s a p o t e n t b i o l o g i c a l a g e n t c a u s i n g p l a t e l e t a g g r e g a t i o n ( 5 ) . P h o s p h o l i p i d s a r e r e q u i r e d f o r t h e c a t a l y t i c a c t i o n s o f v a r i o u s membrane e n z y m e s a n d p l a y i m p o r t a n t r o l e s i n many c e l l u l a r f u n c t i o n s (1,6,6a). D - b e t a - h y d r o x y b u t y r a t e d e h y d r o -g e n a s e (7) and a c y l - C o A s y n t h e t a s e (7a) a r e e x a m p l e s o f 3 enzymes w h i c h r e q u i r e PC f o r a c t i v i t y . However, t h e c e n t r a l b i o l o g i c a l r o l e o f PC i s as a main s t r u c t u r a l c o m p o n e n t o f c e l l m e m b r a n e s . The l i p i d b i l a y e r s a t i s f i e s t h e c e l l ' s r e q u i r e m e n t f o r a p e r m e a b i l i t y b a r r i e r and a l s o s e r v e s as a m a t r i x f o r membrane p r o t e i n s , p r e s u m a b l y p r o v i d i n g an a p p r o p r i a t e e n v i r o n m e n t f o r t h e m a i n t e n a n c e o f a f u n c t i o n a l p r o t e i n c o n f o r m a t i o n . A l t h o u g h a l o t o f membrane f u n c t i o n s a r e m e d i a t e d by p r o t e i n s , p h o s p h o l i p i d s have been i m p l i c a t e d i n a number o f c e l l e v e n t s s u c h as membrane f u s i o n (8) and e n d o c y t o s i s ( 9 ) . I t was once b e l i e v e d t h a t t h e s t r u c t u r a l p h o s p h o l i p i d s were m e t a b o l i c a l l y i n e r t . A f t e r t h e i n t r o d u c t i o n o f i s o t o p e s i n t o b i o l o g i c a l r e s e a r c h i n 1923 by Hevesy (10), t h r e e g r o u p s d e m o n s t r a t e d t h a t r a d i o a c t i v e p h o s p h o r u s was r a p i d l y i n c o r p o r a t e d i n t o t i s s u e p h o s p h o l i p i d s ( 1 1 - 1 3 ) . As w e l l , i n c o r p o r a t e d l a b e l r a p i d l y d i s a p p e a r s f r o m t h e p h o s p h o l i p i d f r a c t i o n . T h i s d i s p e l l e d t h e myth t h a t p h o s p h o l i p i d s w e r e i n a c t i v e s t r u c t u r a l e l e m e n t s and t h e y w e r e r e c o g n i z e d as d y n a m i c c o m p o n e n t s c o n t i n u a l l y b e i n g s y n t h e s i z e d a n d d e g r a d e d . As w e l l as t h i s c o n t i n u a l b a l a n c e , t h e r e needed t o be n e t s y n t h e s i s o f p h o s p h o l i p i d d u r i n g g r o w t h o r s e c r e t o r y a c t i v i t y and n e t l o s s d u r i n g s t a r v a t i o n o r d i s e a s e . 3. B i o s y n t h e s i s o f P h o s p h a t i d y l c h o l i n e I n mammals, a l l c e l l s e x c e p t e r y t h r o c y t e s a r e a b l e t o s y n t h e s i z e p h o s h o l i p i d s f r o m f u n d a m e n t a l c o m p o u n d s (e.g. p h o s p h a t e , c h o l i n e ) . T h e r e a r e f i v e p a t h w a y s f o r t h e 4 s y n t h e s i s o f PC: ( i ) t h e C D P - c h o l i n e p a t h w a y ; ( i i ) s t e p w i s e m e t h y l a t i o n o f p h o s p h a t i d y l e t h a n o l a m i n e ( P E ) ; ( i i i ) b a s e e x c h a n g e ; ( i v ) a c y l a t i o n o f l y s o p h o s p h a t i d y l c h o l i n e ( l y s o -PC); (v) t r a n s a c y l a t i o n o f l y s o - P C . (a) C D P - c h o l i n e Pathway T h i s p a t h w a y was e l u c i d a t e d l a r g e l y by K e n n e d y and c o w o r k e r s ( 1 4 ) . I t i n v o l v e s t h e s e q u e n t i a l c o n v e r s i o n o. c h o l i n e t o p h o s p h o c h o l i n e , C D P - c h o l i n e and PC ( F i g . 2) and i s t h e m a j o r b i o s y n t h e t i c pathway. C h o l i n e k i n a s e (EC 2.7.1.32) c a t a l y z e s t h e i n i t i a l and +2 c o m m i t t e d s t e p and i s a c y t o s o l i c Mg -ATP dep e n d a n t enzyme p r e s e n t i n t h e c y t o s o l o f mammalian c e l l s . The enzyme i n r a t t i s s u e s e x i s t s as m u l t i p l e f o r m s w h i c h a l s o have e t h a n o l a m i n e k i n a s e a c t i v i t y (15). The enzyme has been p u r i f i e d f r o m r a t k i d n e y (16) and i s a d i m e r o f m o l e c u l a r w e i g h t 75,000-80,000. A v a r i e t y o f t r e a t m e n t s o f c e l l s ( o r a n i m a l s ) h a v e l e a d t o s t i m u l a t i o n o f c h o l i n e k i n a s e , p r o b a b l y by enzyme s y n t h e s i s (17-20). P o l y a m i n e a c c u m u l a t i o n i n c e l l s can a l s o i n f l u e n c e c h o l i n e k i n a s e a c t i v i t y (21). C T P : p h o s p h o c h o l i n e c y t i d y l y l t r a n s f e r a s e (EC 2.7.7.15) i s Mg - d e p e n d a n t enzyme and i t i s a m b i q u i t o u s i n t h a t i t i s r e c o v e r e d i n b o t h c y t o s o l i c and m i c r o s o m a l c e l l f r a c t i o n s . P o o l s i z e and p u l s e c h a s e d a t a p r o v i d e s t r o n g e v i d e n c e t h a t t h i s e n zyme c a t a l y z e s t h e r a t e - 1 i m i t i n g s t e p i n t h e CDP-c h o l i n e pathway (22) a l t h o u g h some i n v e s t i g a t o r s a r e o f t h e o p i n i o n t h a t c h o l i n e k i n a s e i s a l s o r a t e - 1 i m i t i n g ( 2 3 , 2 3 a , 2 4 ) . C y t i d y 1 y 1 1 r a n s f e r a s e i s a c t i v a t e d ix\ v i t r o by 5 H , C \ « H H H , C — N — C — C — O H / H H H , C CHOLINE ATP ADP-H , C CHOLINE KINASE \ » H H n HjC—N—C—C—O—P—O 0 / H H I H , C O e PHOSPHOCHOLINE CTP-PPi CYTIDYLYLTRA NSFERASE „ > . H H ? ? O ^ N ' H 3C-N-C-C—O-P—O-P-O—CH, / H H H3C CDP-CHOLINE OH OH DIGLYCERIDE CMP-CHOLINEPHOSPHOTRANSFERASE O H,C—O—C—R • II ? l R'—C—O—CH 0 CH, I II H H «>/ H,C—O—P—O—C—C—N—CH, I H H \ CH;, PHOSPHATIDYLCHOLINE F i g u r e 2. The C D P - c h o l i n e pathway f o r p h o s p h a t i d y l c h o l i n e b i o s y n t h e s i s 6 p h o s p h o l i p i d s (22) and _in v i v o r e g u l a t i o n i s b e l i e v e d t o i n v o l v e t r a n s l o c a t i o n o f t h e enzyme f r o m c y t o s o l t o e n d o p l a s -m i c r e t i c u l u m ( 4 ) . T h i s p r o c e s s a p p e a r s t o be m e d i a t e d by p r o t e i n p h o s p h o r y l a t i o n (25) o r l o n g c h a i n f a t t y a c i d s ( 4 ) . S u b s t r a t e l e v e l s may a l s o i n f l u e n c e _i n v h o a c t i v i t y ( 2 6 , 2 7 , 2 7 a ) . C D P - c h o l i n e : 1 , 2 - d i a c y l g l y c e r o l c h o l i n e p h o s p h o t r a n s f e r a s e (EC 2.7.8.2) i s an i n t r i n s i c p r o t e i n l o c a t e d on t h e c y t o -p l a s m i c f a c e o f t h e e n d o p l a s m i c r e t i c u l u m (28). The enzyme shows p r e f e r e n c e f o r l - p a l m i t o y l - 2 - a c y l g l y c e r o l s where the 2-a c y l g r o u p i s an u n s a t u r a t e d f a t t y a c i d (29). (b) O t h e r Pathways f o r P h o s p h a t i d y l c h o l i n e B i o s y n t h e s i s ( i ) S t e p w i s e M e t h y l a t i o n o f P h o s p h a t i d y l e t h a n o l a m i n e T h i s pathway i n v o l v e s t h e t r a n s f e r o f m e t h y l g r o u p s from S - a d e n o s y l m e t h i o n i n e t o PE and i s c a t a l y z e d b y one o r more m e t h y l t r a n s f e r a s e s . T h e s e enzymes a r e l o c a t e d on m i c r o s o m a l membranes and t h e r e a r e s u g g e s t i o n s t h a t t h e f i r s t m e t h y l a -t i o n o c c u r s on t h e l u m i n a l s i d e o f t h e e n d o p l a s m i c r e t i c u l u m and t h e o t h e r two on t h e c y t o p l a s m i c s i d e (30). The pathway i s s i g n i f i c a n t o n l y i n l i v e r where i t can a c c o u n t f o r 20-40% o f PC b i o s y n t h e s i s (30). ( i i ) Base Exchange T h i s r e a c t i o n i n v o l v e s t h e e n e r g y - i n d e p e n d a n t exchange o f c h o l i n e w i t h t h e h e a d g r o u p o f an e x i s t i n g p h o s p h o l i p i d and t h i s a c t i v i t y i s l o c a t e d on m i c r o s o m e s . Base exchange p l a y s 7 a m i n o r r o l e i n PC b i o s y n t h e s i s i n l i v e r (31) and b r a i n (32) and i t ' s p h y s i o l o g i c a l s i g n i f i c a n c e i s unknown. ( i i i ) A c y l a t i o n o f L y s o p h o s p h a t i d y l c h o l i n e S p e c i f i c a c y l t r a n s f e r a s e s t r a n s f e r f a t t y a c i d s f r o m a c y l - C o A t o t h e 1 o r 2 p o s i t i o n o f l y s o - P C . S a t u r a t e d f a t t y a c i d s a r e p r e f e r e n t i a l l y t r a n s f e r r e d t o t h e 1 - p o s i t i o n and u n s a t u r a t e d f a t t y a c i d s , e s p e c i a l l y a r a c h i d o n a t e a r e r e a d i l y t r a n s f e r r e d i f t h e 2 - p o s i t i o n i s a v a i l a b l e ( 3 0 ) . T h e s e r e a c t i o n s a r e c r i t i c a l f o r t h e s y n t h e s i s o f m o l e c u l a r s p e c i e s o f PC. ( i v ) T r a n s a c y l a t i o n o f L y s o p h o s p h a t i d y l c h o l i n e T h i s r e a c t i o n t a k e s two m o l e c u l e s o f l y s o - P C w i t h t h e OH-group a t t h e 2 - p o s i t i o n and y i e l d s PC p l u s g l y c e r o p h o s p h o -c h o l i n e . T h i s r e a c t i o n may be i m p o r t a n t i n l u n g (30). 4. C a t a b o l i s m o f P h o s p h a t i d y l c h o l i n e I n p r i n c i p l e , a n y e s t e r l i n k a g e i n p h o s p h o l i p i d s i s s u s c e p t i b l e t o e n z y m a t i c h y d r o l y s i s and t h e enzymes a r e named a c c o r d i n g t o t h e i r s i t e s o f a t t a c k ( F i g . 3). (a) P h o s p h o l i p a s e s A P h o s p h o l i p a s e A a c t i v i t y i s u b i q u i t o u s i n n a t u r e and has b e e n f o u n d i n a l m o s t e v e r y c e l l w h e r e i t h a s b e e n s o u g h t ( 3 3 ) . I n a d d i t i o n , t h e i r s u b c e l l u l a r l o c a t i o n i s n o t r e s t r i c t e d t o any s i n g l e s i t e . In r a t l i v e r , p h o s p h o l i p a s e A^ 8 P H O S P H O L I P A S E B (EC. 3.1.1.5. ) P H O S P H O L I P A S E A 2 ( EC. 3.1.1.4.) P H O S P H O L I P A S E A-, (EC. 3.1.1.32) O II H 2 C - 0 - C - R -O ^ C - O - P - O - C H 2 - C H2-N(CH3). P H O S P H O L I P A S E C ( EC. 3.1.4.3.) i s 3 P H O S P H O L I P A S E D (EC. 3.1.4.4.) F i g u r e 3. The s i t e s o f a t t a c k o f v a r i o u s p h o s p h o 1 i p a s e s on p h o s p h a t i d y l c h o l i n e (Reproduced from Ref. 34) 9 and A 2 a c t i v i t i e s have been f o u n d on p l a s m a membrane, m i c r o -somes, g o l g i , m i t o c h o n d r i a , i n c y t o s o l and w i t h i n l y s o s o m e s (33) . < The s o l u b l e and membrane bound p h o s p h o l i p a s e A-^  enzymes 2 + i n r a t l i v e r d i f f e r i n pH o p t i m a a n d a c t i v a t i o n b y Ca , s u g g e s t i n g t h a t t h e y a r e d i f f e r e n t p r o t e i n s (34). P h o s p h o l i -p a s e A^ p u r i f i e d f r o m v a r i o u s s o u r c e s v a r i e s w i d e l y i n m o l e c u l a r w e i g h t (33). P h o s p h o l i p a s e A 2 i s p r o b a b l y t h e m o s t t h o r o u g h l y i n v e s t i g a t e d p h o s p h o l i p a s e and i n mammals, o c c u r s i n h i g h e s t a m o u n t s i n p a n c r e a t i c s e c r e t i o n s . I n r a t l i v e r , p h o s p h o -l i p a s e A 2 i s e a s i l y d e m o n s t r a t e d i n m i t o c h o n d r i a , s i n c e t h i s o r g a n e l l e d o e s n o t c o n t a i n p h o s p h o l i p a s e A-^  a c t i v i t y ( 3 4 ) . In o t h e r s u b c e l l u l a r f r a c t i o n s , p h o s p h o l i p a s e A 2 a c t i v i t y i s a l w a y s f o u n d i n c o n j u n c t i o n w i t h A-^  a c t i v i t y , s o t h e p o s s i b i l i t y o f a p h o s p h o l i p a s e B c a t a l y z i n g b o t h a c t i v i t i e s c a n n o t be d i s c o u n t e d ( 3 4 ) . H e p a t i c p h o s p h o l i p a s e A 2 a c t i v i t i e s f r o m v a r i o u s s u b c e l l u l a r f r a c t i o n s had a a l k a l i n e O J . pH o p t i m u m and w e r e s t i m u l a t e d b y Ca , e x c e p t l y s o s o m a l a c t i v i t y w h i c h had a b a s i c pH o p t i m u m and was i n h i b i t e d b y Ca (34). Many p u r i f i e d p h o s p h o l i p a s e s A 2 have m o l e c u l a r w e i g h t s o f 12,000-15,000, w h i c h i s c o m p a r a b l e t o p a n c r e a t i c p h o s p h o l i p a s e A 2 (33). P h o s p h o l i p a s e A 2 a c t i v i t y i s i m p l i c a t e d i n t h e r e l e a s e o f a r a c h i d o n a t e f o r e n d o p e r o x i d e and p r o s t a g l a n d i n f o r m a t i o n and f o r t h i s r e a s o n , much a t t e n t i o n has been f o c u s s e d on t h e r e g u l a t i o n o f t h i s e nzyme. S e v e r a l m o d e l s h a v e b e e n p u t f o r w a r d f o r che r e g u l a t i o n o f membrane-bound p h o s p h o l i p a s e A 2 10 i n c l u d i n g : zymogen a c t i v a t i o n , a v a i l a b i l i t y o f Ca , i n t e r -a c t i o n w i t h r e g u l a t o r y p r o t e i n s , i n f l u e n c e o f cAMP o r hormones, and changes i n membrane s t r u c t u r e (33,34). (b) L y s o p h o s p h o l i p a s e s and P h o s p h o l i p a s e B C r u d e p h o s p h o l i p a s e A p r e p a r a t i o n s u s u a l l y c o n t a i n l y s o p h o s p h o l i p a s e a c t i v i t y w h i c h c o n v e r t i n i t i a l l y p r o d u c e d l y s o p h o s p h o l i p i d s i n t o f u l l y d e a c y l a t e d p r o d u c t s . I n t h e a s s a y i n g o f p h o s p h o l i p a s e A, d e t e r g e n t s a r e u s e d t o i n h i b i t l y s o p h o s p h o l i p a s e a c t i v i t y . L y s o p h o s p h o l i p a s e s a r e d i s t i n c t f r o m p h o s p h o l i p a s e B s i n c e l y s o p h o s p h o l i p a s e o a r e n o t a c t i v e a g a i n s t i n t a c t p h o s p h o l i p i d s . The p r o d u c t s o f p h o s p h o l i p a s e A a c t i v i t y a r e l y t i c and t h u s need t o be r a p i d l y m e t a b o l i z e d . The l y s o p h o s p h o l i p a s e s a p p a r e n t l y f i l l t h i s r o l e and t h e y a r e u s u a l l y p r e s e n t i n c r u d e p r e p a r a t i o n s a t h i g h e r l e v e l s t h an t h e p h o s p h o l i p a s e A t h e m s e l v e s . More t h a n one l y s o p h o s p h o -l i p a s e e x i s t s i n t i s s u e s (35) and i n l i v e r , t h i s a c t i v i t y i s m i c r o s o m a l (36) and c y t o s o l i c (33). (c) G l y c e r o p h o s h o c h o l i n e P h o s p h o d i e s t e r a s e T h i s a c t i v i t y h y d r o l y z e s g l yc e r o - 3 - p h o s p h o c h o l i n e , t h e p r o d u c t a f t e r p h o s p h o l i p a s e B o r c o m b i n e d p h o s p h o l i p a s e A and l y s o p h o s p h o l i p a s e a c t i v i t y , i n t o g 1 y c e r o 1 - 3 - p h o s p h a t e and c h o l i n e . I t i s p r o b a b l y a l s o a c t i v e a g a i n s t g l y c e r o - 3 - p h o s -p h o e t h a n o l a m i n e ( 3 7 ) . The e nzyme h a s w i d e d i s t r i b u t i o n i n r a t t i s s u e s (37a) and f r o m r a t l i v e r , i t i s r e c o v e r e d f r o m s o l u b l e and p a r t i c u l a t e s u b c e l l u l a r f r a c t i o n s ( 3 7 b ) . The p a r t i c u l a t e a c t i v i t y i s a s s o c i a t e d w i t h n u c l e i , m i c r o s o m e s , 11 and p l a s m a membrane (37b). The p r e s e n c e o f g l y c e r o p h o s p h o -c h o l i n e i n c e l l s s u g g e s t s t h a t s t e p w i s e d e a c y l a t i o n i s a m a j o r pathway f o r t h e c a t a b o l i s m o f t h e c h o l i n e (and e t h a n o l -amine) c o n t a i n i n g p h o s p h o l i p i d s , and a c c o u n t s i n p a r t f o r t h e a c t i v i t y o f p h o s p h o l i p a s e A and l y s o p h o s p h o l i p a s e . (d) P h o s p h o l i p a s e C P h o s p h o l i p a s e C i s n o t a w i d e l y d i s t r i b u t e d o r m a j o r p h o s p h o l i p a s e i n m a m m a l i a n t i s s u e . The b e s t d o c u m e n t e d e x a m p l e i s t h e p h o s p h a t i d y l i n o s i t o l (PI) - s p e c i f i c e nzyme. The m a j o r p a t h w a y o f PI h y d r o l y s i s i s v i a p h o s p h o l i p a s e C, l i b e r a t i n g d i a c y l g y c e r o l and i n o s i t o l p h o s p h a t e ( 3 8 ) . The enzyme i s m a i n l y c y t o s o l i c a l t h o u g h some a c t i v i t y i s f o u n d i n l y s o s o m e s ( 3 8 ) . A s p e c i a l r o l e f o r t h i s e n z y m e i s i n t h e r e c e p t o r - m e d i a t e d breakdown o f PI 4 , 5 - b i s p h o s p h a t e , y i e l d i n g d i a c y l g l y c e r o l w h i c h c a n a c t i v a t e p r o t e i n k i n a s e C, and i n o s i t o l 1 , 4 , 5 - t r i p h o s p h a t e , w h i c h c a n m o b i l i z e i n t r a c e l l u l a r Ca s t o r e s i n a v a r i e t y o f t i s s u e s ( 3 8 - 4 0 ) . M a t s u z a w a an" 1 H o s t e t l e r h a v e d e s c r i b e d a l y s o s o m a l p h o s p h o l i p a s e C w h i c i . was a c t i v e a g a i n s t PC ( 4 1 ) . T h e r e i s e v i d e n c e t h a t t h i s a c t i v i t y i s w i d e l y d i s t r i b u t e d i n r a t t i s s u e s (42). R e c e n t -l y , W o l f and G r o s s (43) i d e n t i f i e d a c y t o s o l i c p h o s p h o l i p a s e C a c t i v e a g a i n s t PC a n d t h e p r e s e n c e o f a n e n d o g e n o u s i n h i b i t o r . T h i s d i s c o v e r y s u g g e s t s t h a t PC may p l a y a r o l e i n t h e f o r m a t i o n o f t h e s e c o n d m e s s e n g e r d i a c y l g l y c e r o l and p r o t e i n k i n a s e C a c t i v a t i o n . (e) P h o s p h o l i p a s e D Few p h o s p h o l i p a s e D's have been d e s c r i b e d i n mammalian 12 t i s s u e s . Wykle and c o - w o r k e r s (44) d e s c r i b e d a s p e c i a l i z e d p h o s p h o l i p a s e D i n b r a i n s p e c i f i c f o r 1 - e t h e r - l y s o - P C or -PE. K a n f e r and a s s o c i a t e s h a v e s e p a r a t e d p h o s p h o l i p a s e D f r o m t r a n s p h o s p h a t i d y 1 a t i n g b a s e e x c h a n g e a c t i v i t i e s i n b r a i n (30,45) . 5. The T h e s i s I n v e s t i g a t i o n s A l t h o u g h t h e m a j o r p a t h w a y f o r PC s y n t h e s i s , v i a CDP-c h o l i n e , was d e s c r i b e d o v e r 20 y e a r s ago, o u r k n owledge about i t ' s r e g u l a t i o n i s s t i l l n ot c o m p r e h e n s i v e . Much i n s i g h t has b e e n g a i n e d by s t u d y i n g t h e e f f e c t s o f a l t e r e d n u t r i t i o n a l , h o r m o n a l , and p h a r m a c o l o g i c a l s t a t e s on a c e l l s a b i l i t y t o s y n t h e s i z e PC ( 4 6 ) . We know t h a t u n d e r m o s t c i r c u m s t a n c e s , t h e r a t e - l i m i t i n g s t e p i s c a t a l y z e d by c y t i d y l y l t r a n s f e r a s e and s e v e r a l m o d e l s f o r t h e r e g u l a t i o n o f t h i s e nzyme h a v e b e e n p u t f o r w a r d ; p u r i f i c a t i o n o f t h e e n z y m e i s a w a i t e d so t h e s e h y p o t h e s e s can be t e s t e d d i r e c t l y . M e a n w h i l e , new e x p e r i m e n t a l models were s o u g h t t o s t u d y t h e r e g u l a t i o n o f PC b i o s y n t h e s i s , l e a d i n g t o t h e Nb 2 node r a t lymphoma s y s t e m . T h i s c e l l l i n e i s s t r i c t l y d e pendant on p r o l a c t i n f o r g r o w t h i n c u l t u r e (47) and upon h o r m o n a l s t i m u l a t i o n , p r o c e e d s t h r o u g h the c e l l c y c l e i n a s y n c h r o n o u s manner. A v i r t u e o f t h i s s y s t e m i s t h a t m a n i p u l a t i o n o f c e l l g r o w t h i s s i m p l e and t h i s s y s t e m s e e m s s u i t e d f o r s t u d y i n g f u n d a m e n t a l e v e n t s r e l a t e d t o c e l l g r o w t h . F o r i n s t a n c e , w e l l d e f i n e d c h a n g es i n DNA s y n t h e s i s and l e v e l s o f p o l y a m i n e b i o s y n t h e t i c e n z y m e s d u r i n g t h e c e l l c y c l e h a v e b e e n 13 d e s c r i b e d u s i n g t h i s s y s t e m (48). Membrane s y n t h e s i s s h o u l d be c o n s i d e r e d a k e y p r o c e s s d u r i n g c e l l g r o w t h so we s e t f o r t h t o examine how Nb 2 c e l l s w o u l d meet t h e i n c r e a s e d demand f o r PC f o l l o w i n g h o r m o n a l s t i m u l a t i o n . I n i t i a l l y , t h e a c t i v i t y o f t h e C D P - c h o l i n e p a t h w a y was i n v e s t i g a t e d . T h e n , c h a n g e s i n c e l l u l a r PC l e v e l s a f t e r p r o l a c t i n - t r e a t m e n t w e r e m e a s u r e d and t h e mechanism f o r t h e s e changes was s t u d i e d . A l s o , t h e enzymes o f t h e C D P - c h o l i n e p a t h w a y w e r e a s s a y e d i n an a t t e m p t t o c o r r e l a t e in. v i v o changes i n t h e pathway t o _in v i t r o c hanges i n e n zyme a c t i v i t y . The r o l e s o f t h e b i o s y n t h e t i c and c a t a b o l i c p a t h w a y s i n t h e c o n t r o l o f PC l e v e l s i n Nb 2 c e l l s d u r i n g growth a r e d i s c u s s e d . 14 MATERIALS AND METHODS 1. C h e m i c a l s , Enzymes, and I s o t o p e s The c h e m i c a l s , e n z y m e s , i s o t o p e s , and o t h e r m a t e r i a l s u s e d i n t h i s s t u d y a r e l i s t e d b e l o w u n d e r t h e i r s u p p l y company. A l l o t h e r c h e m i c a l s used were r e a g e n t g r a d e . Grand I s l a n d B i o l o g i c a l Company, Grand I s l a n d , NY F i s c h e r ' s medium (powdered), p e n i c i l l i n - s t r e p t o m y c i n , f e t a l c a l f serum Sigma C h e m i c a l Company, S t . L o u i s , MO 2 - M e r c a p t o e t h a n o l , p r o l a c t i n ( o v i n e , " h i g h l y p u r i f i e d by e x c l u s i o n c h r o m a t o g r a p h y " , N I H - P - S 1 3 ) , c y c 1 o h e x i m i d e , p h o s p h o c h o l i n e , t r i z m a b a s e , i n o r g a n i c p y r o p h o s p h a t a s e , p h e n y l m e t h y 1 s u l f o n y l f l u o r i d e , s o d i u m f l u o r i d e , a d e n o s i n e t r i p h o s p h a t e , 1 , 2 - d i o l e i n , EGTA, b o v i n e a l b u m i n , c h o l i n e k i n a s e MA B i o p r o d u c t s , W a l k e r s v i l l e , MD, c/o J o h n s S c i e n t i f i c ,  T o r o n t o , Ont. Horse serum F a l c o n P l a s t i c s , Oxnard, CA 75 cm T i s s u e c u l t u r e f l a s k s BDH C h e m i c a l s Canada L t d . , V a n c o u v e r , B.C. C h o l i n e c h l o r i d e , s o d i u m s u c c i n a t e , ammonium m o l y b d a t e , 70% p e r c h l o r i c a c i d , 15 E. Merck L a b o r a t o r i e s , c/o BDH C h e m i c a l s (see above) P l a s t i c - and g l a s s - b a c k e d s i l i c a g e l 60 t h i n - l a y e r c h r o m a t o -g r a p h y p l a t e s , 0.2 mm l a y e r t h i c k n e s s Amersham C o r p o r a t i o n , O a k v i l l e , O n t a r i o •a A q u e o u s c o u n t i n g s c i n t i l l a n t ( A C S ) , [Me_- H] c h o l i n e , [ 5 -•3 H ] c y t i d i n e t r i p h o s p h a t e New E n g l a n d N u c l e a r P r o d u c t s , c/o Dupont Canada, Inc., L a c h i n e , Que. 14 [Me- C ] C y t i d i n e d i p h o s p h o c h o l i n e F i s h e r S c i e n t i f i c L t d . , V a n c o u v e r , B.C. 5 ml and 20 ml p l a s t i c s c i n t i l l a t i o n c o u n t i n g v i a l s , magnesium a c e t a t e , EDTA, s o d i u m p h o s p h a t e , s o d i u m c i t r a t e , p h e n o l r e a g e n t s o l u t i o n 2N, Darco G-60 a c t i v a t e d c h a r c o a l , o l e i c a c i d H auser S c i e n t i f i c , B l u e B e l l , PA Hemacytometer S e r d a r y R e s e a r c h L a b o r a t o r i e s , London, Ont. C y t i d i n e t r i p h o s p h a t e , c y t i d i n e d i p h o s p h o c h o l i n e , p h o s p h a t i -d y l c h o l i n e , l y s o p h o s p h a t i d y l c h o l i n e , s p h i n g o m y e l i n , p h o s -p h a t i d y l s e r i n e , p h o s p h a t i d y l i n o s i t o l , p h o s p h a t i d y l e t h a n o l -amine MCB R e a g e n t s , C i n c i n n a t i , OH Ammonium a c e t a t e Whatman L t d . , E n g l a n d , c/o VWR S c i e n t i f i c , S e a t t l e , WA Whatman 4 c h r o m a t o g r a p h y pap e r C a n a d i a n S c i e n t i f i c P r o d u c t s L t d . , London, Ont. Enzyme g r a d e s u c r o s e 16 J . T. B a k e r C h e m i c a l Company, P h i l l i p s b u r g , NJ Magnesium c h l o r i d e , Tween 20 M a l l i n c k d r o d t Inc., S t . L o u i s , MO A s c o r b i c a c i d A s s o c i a t e d C o n c e n t r a t e s , Woodside, NY A s o l e c t i n E astman Kodak Company, R o c h e s t e r , NY 2 ' , 7 ' - d i c h l o r o f l u o r e s c e i n 2. C e l l s (a) S o u r c e Nb 2 c e l l c u l t u r e s w e r e p r o v i d e d by Dr. C h a r l e s T. B e e r o f t h e C a n c e r C o n t r o l A g e n c y o f B r i t i s h C o l u m b i a . The c u l t u r e s w e r e e s t a b l i s h e d by P. W. G o u t o f t h e B e e r l a b o r a -t o r y f r o m an Nb '2 n o d e 1 r a t l y m p h o m a s u p p l i e d b y D r . R. L. N o b l e , as p r e v i o u s l y d e s c r i b e d (49). (b) M a i n t e n a n c e o f C u l t u r e s E x p o n e n t i a l l y g r o w i n g c u l t u r e s were m a i n t a i n e d by s u b -c u l t u r i n g t h e c e l l s s u c h t h a t t h e y w o u l d r e a c h a d e n s i t y o f 1.0x10^ c e l l s / m l e v e r y 72 o r 96 h. I n i t i a l c o n c e n t r a t i o n s o f new c u l t u r e s was d e t e r m i n e d u s i n g t h e e q u a t i o n n/nQ = 2^~^*~ (n, n Q : f i n a l , i n i t i a l c e l l d e n s i t y ; t , d t : t i m e , d o u b l i n g t i m e ) . C e l l d e n s i t y was m o n i t o r e d w i t h a h e m a c y t o m e t e r and t h e d o u b l i n g t i m e o f e x p o n e n t i a l l y g r o w i n g c u l t u r e s was c_a. 15 h. The m a i n t e n a n c e m edium was F i s c h e r ' s m e dium, s u p p l e m e n t e d w i t h 10% h o r s e s e r u m , 10% f e t a l c a l f s e r u m , 0.1 mM 2-17 m e r c a p t o e t h a n o l , 50 U/ml p e n i c i l l i n , and 50 ug/ml s t r e p t o m y -c i n . F i f t y ml c u l t u r e s i n 75 cm f l a s k s w e r e i n c u b a t e d a t 3 7 ° i n a F o r m a S c i e n t i f i c m o d e l 3029 i n c u b a t o r and t h e f l a s k s w e r e v e n t e d f o r 5 m i n u n d e r 5% C 0 2 / 9 5 % a i r i n s i d e t h e i n c u b a -t o r , and t h e n s e a l e d f o r t h e r e m a i n d e r o f t h e i n c u b a t i o n p e r i o d . (c) P r e p a r a t i o n o f S t a t i o n a r y C u l t u r e s C e l l g r o w t h was a r r e s t e d by s e d i m e n t i n g g r o w i n g c e l l s by c e n t r i f u g a t i o n (300 x g_, 3 m i n ) , f o l l o w e d by w a s h i n g t h e c e l l s t w i c e and t h e n r e s u s p e n d i n g them i n ' s t a t i o n a r y medium' and i n c u b a t i o n o f t h e c u l t u r e s f o r 15-18 h. S t a t i o n a r y medium was F i s c h e r ' s medium s u p p l e m e n t e d w i t h 10% h o r s e serum, and 2 - m e r c a p t o e t h a n o l p l u s a n t i b i o t i c s a t same c o n c e n t r a t i o n s as m a i n t e n a n c e medium. 3. P r e p a r a t i v e P r o c e d u r e s (a) C e n t r i f u g a t i o n s Low speed c e n t r i f u g a t i o n s were done i n a W e s t e r n H-103N b e n c h t o p c e n t r i f u g e . Medium speed c e n t r i f u g a t i o n s were done u s i n g a SS 34 r o t o r i n a S o r v a l l RC-5 S u p e r s p e e d C e n t r i f u g e . H i g h s p e e d c e n t r i f u g a t i o n s were p e r f o r m e d u s i n g a T i 75 r o t o r i n a B e c k m a n L8-70 U l t r a c e n t r i f u g e . M e d i u m and h i g h s p e e d c e n t r i f u g a t i o n s were done at 4° C. (b) H a r v e s t i n g o f C e l l s C e l l i n c u b a t i o n s i n t h e l a b e l i n g s t u d i e s and p o o l s i z e 18 m e a s u r e m e n t s d e s c r i b e d l a t e r w e r e r o u t i n e l y t e r m i n a t e d by e x t r a c t i o n a c c o r d i n g t o t h e method o f F o l c h e t al (50). F i v e m i s a l i q u o t s o f c e l l s were c o l l e c t e d by c e n t r i f u g a t i o n (300 x g, 3 min) and washed t w i c e w i t h i c e - c o l d p h o s p h a t e - b u f f e r e d s a l i n e ( 5 1 ) , t h e n 1.6 ml 0.1 M KC1 and 6.0 ml C H C l 3 / M e O H (2:1; v/v) w e r e a d d e d , w i t h v o r t e x i n g a f t e r e a c h a d d i t i o n . The m i x t u r e was c e n t r i f u g e d (3500 rpm, 10 m i n ) , r e s u l t i n g i n two p h a s e s s e p a r a t e d by a b a n d o f p r o t e i n a t t h e i n t e r f a c e . The u p p e r CH^OH/H^O p h a s e was r e m o v e d and t h e l o w e r C H C l ^ p h a s e was w a s h e d t w i c e w i t h 2.0 ml t h e o r e t i c a l u p p e r p h a s e c o n s i s t i n g o f H 20/CH 30H/CHC1 3 (47:48:3, v / v / v ) . Samples were s t o r e d a t - 2 0 ° C. (c) S u b c e l l u l a r F r a c t i o n a t i o n C e l l c u l t u r e s (250 ml) were c o l l e c t e d by c e n t r i f u g a t i o n (300 x g, 3 min) a f t e r t h e d e s c r i b e d t r e a t m e n t s and w a s h e d t w i c e w i t h p h o s p h a t e b u f f e r e d s a l i n e . The c e l l s w e r e r e s u s p e n d e d i n 2.5 m l i c e - c o l d h o m o g e n i z a t i o n b u f f e r c o n t a i n i n g 10 mM T r i s - H C l , pH 7.3, 0.1 mM ph e n y l m e t h y l -s u l f o n y l f l u o r i d e , 10 mM NaF, and 1 mM EDTA and d i s r u p t e d by 35 s t r o k e s i n a P o t t e r E l v e h j e m h o m o g e n i z e r . The homogenate was c e n t r i f u g e d (10, 000 x g_, 10 m i n ) , t h e s u p e r n a t a n t was removed and t h e p e l l e t was r e h o m o g e n i z e d i n a f u r t h e r 2.5 ml o f h o m o g e n i z a t i o n b u f f e r . A n o t h e r c e n t r i f u g a t i o n g a v e a p e l l e t c o n t a i n i n g c e l l d e b r i s and u n b r o k e n c e l l s , and t h e s e c o n d s u p e r n a t a n t was c o m b i n e d w i t h t h e f i r s t and s p u n (130,000 x g, 1 h ) . The r e s u l t i n g s u p e r n a t a n t , o r c y t o s o l i c f r a c t i o n , was r e m o v e d and s t o r e d a t - 7 0 ° C. The p e l l e t , o r 19 m i c r o s o m a l f r a c t i o n , was r e s u s p e n d e d i n 1 ml h o m o g e n i z a t i o n b u f f e r p l u s 0.25 M s u c r o s e and t h e n s t o r e d a t - 7 0 ° C. A t y p i c a l p r o t e i n p r o f i l e o f t h e f r a c t i o n a t i o n i s shown ( T a b l e 1) • (d) Medium Powdered F i s c h e r ' s medium (52.6 g) was d i s s o l v e d i n 5 L d i s t i l l e d H 2 0 a l o n g w i t h 4.745 g NaHCOj. The s o l u t i o n was a d j u s t e d t o pH 7.2 w i t h HC1 and t h e n s t e r i l i z e d b y membrane f i l t r a t i o n . T he a n t i b i o t i c s w e r e d i s s o l v e d w i t h f r e s h l y s t e r i l i z e d medium t o a c o n c e n t r a t i o n o f 10,000 U/ml p e n i c i l -l i n and 10,000 u g / m l s t r e p t o m y c i n and 3.2 ml was a d d e d p e r 500 ml o f m e d i urn. A t t h e t i m e o f u s e , t h e medium was s u p p l e m e n t e d w i t h 2-m e r c a p t o e t h a n o l . A 5 mM s o l u t i o n of 2 - m e r c a p t o e t h a n o l was p r e p a r e d by c o m b i n i n g : 100 ml d i s t i l l e d H 20 0.9 g N a C l 35 u l 2 - m e r c a p t o e t h a n o l The s o l u t i o n was f i l t e r s t e r i l i z e d and s t o r e d f r o z e n i n s m a l l a l i q u o t s . A t t i m e o f s u b c u l t u r i n g , e t c . , 1 ml was added p e r 50 ml medium ( i n c l u d i n g s e r a ) . (e) P r o l a c t i n S o l u t i o n O v i n e p r o l a c t i n (1 mg) was d i s s o l v e d i n 2.0 ml 0.01 N NaOH. The s o l u t i o n was n e u t r a l i z e d w i t h 300 u l 0.05 M K H 2 P 0 4 a n d F i s c h e r ' s medium s u p p l e m e n t e d w i t h 10% h o r s e s e r u m was a d d e d t o g i v e a s o l u t i o n o f l l x l O 4 ng p r o l a c t i n / m l . The s o l u t i o n was f u r t h e r d i l u t e d w i t h medium t o g i v e a s o l u t i o n 20 T a b l e 1. T y p i c a l p r o t e i n p r o f i l e o f Nb 2 c e l l s a f t e r s u b c e l l u l a r f r a c t i o n a t i o n . P r o t e i n F r a c t i o n T o t a l % o f F r a c t i o n C o n c e n t r a t i o n Volume P r o t e i n Homo-(mg/ml) (ml) (mg) g e n a t e Homogenate 8.8 10,000 x g 6.5 p e l l e t C y t o s o l 2.2 M i c r o s o m e s 5.6 2.5 22.0 100 1.0 6.5 29.5 4.5 9.9 45.0 1.0 5.6 25.5 Data a r e means o f two s e p a r a t e p r e p a r a t i o n s 21 of 1000 ng/ml and t h i s was s t o r e d at -20° C i n s m a l l aliquots. When c e l l s were to be treated with p r o l a c t i n , 1 ml of solution was added per 50 ml c e l l s . (f) [Me- H]Phosphocholine This substrate f o r the c y t i d y l y l t r a n s f e r a s e assay was 3 e n z y m a t i c a l l y synthesized from [Me- H]choline, ATP and choline kinase e s s e n t i a l l y as previously described (52). The reaction mixture contained: The reaction was incubated and the [iMe- H] phosphocholine was isolated as previously described (52). The [Me- H]phospho-choline was eluted from the s i l i c a gel by several washes with d i s t i l l e d H20, to a f i n a l volume of 11 ml. The r a d i o a c t i v i t y was assayed and the s o l u t i o n was adjusted to 15 mM, 10 uCi/umol by the a d d i t i o n of cold phosphocholine and H 20, i f necessary. The f i n a l product was stored at -20° C in small aliquots. (g) Total Rat Liver Phospholipid Exogenous phospholipid for the c y t i d y l y l t r a n s f e r a s e assay was prepared by B l i g h and Dyer e x t r a c t i o n (53) of the p e l l e t obtained f o l l o w i n g c e n t r i f u g a t i o n (170,000 x g, 1 h) of a rat l i v e r homogenate. The p e l l e t was resuspended in 80 ml H90, followed by 100 ml CHC1-. and 200 ml CH-OH. Then the 2 mCi [Me- H]choline (dried down under N2) 1 M T r i s - H C l (pH 8.0) 0.1 M MgCl 2 0.1 M ATP dialyzed choline kinase (0.25 units) 25 ul 25 ul 25 ul 175 ul 22 m i x t u r e was s t i r r e d f o r 1 h a t room t e m p e r a t u r e and c e n t r i -f u g e d (8000 rpm, 20 min) and t h e s u p e r n a t a n t was f i l t e r e d t h r o u g h g l a s s w o o l . A d d i t i o n o f 80 ml H 2 0 a " d 100 ml C H C 1 3 b r o u g h t t h e p r o p o r t i o n s o f C H C 1 3 / C H 3 O H / H 2 0 t o (1:1:0.8; v / v / v ) . The m i x t u r e was c e n t r i f u g e d as above and t h e o r g a n i c l o w e r p h a s e was t r a n s f e r r e d t o a p r e w e i g h e d r o u n d - b o t t o m f l a s k . The C H C 1 3 was r e m o v e d by r o t a r y e v a p o r a t i o n and t h e f l a s k was c o o l e d t o - 2 0 ° C and r i n s e d w i t h i c e c o l d a c e t o n e t o r e m o v e n e u t r a l l i p i d s . R e s i d u a l a c e t o n e was e v a p o r a t e d u n d e r N 2 and t h e p h o s p h o l i p i d was d i s s o l v e d i n C H C 1 3 a t a f i n a l c o n c e n t r a t i o n o f 20 mg/ml and s t o r e d a t -20° C. 4. A n a l y t i c a l P r o c e d u r e s (a) T h i n - l a y e r C h r o m a t o g r a p h y ( i ) S e p a r a t i o n o f Aqueous C h o l i n e M e t a b o l i t e s (System I) CH 3OH/0.6% NaCl/NH 40H (10:10:1; v/v/v) was used f o r t h e s e p a r a t i o n o f w a t e r - s o l u b l e c h o l i n e m e t a b o l i t e s . P l a s t i c b a c k e d s i l i c a g e l 60 p l a t e s (10 cm x 20 cm) w e r e u s e d . S a m p l e s and s t a n d a r d s (0.25 mg c h o l i n e , 0.80 mg p h o s p h o -c h o l i n e , 0.15mg C D P - c h o l i n e a n d / o r 0.30 mg b e t a i n e ) w e r e s p o t t e d 2 cm above t h e b o t t o m o f t h e p l a t e . R e l a t i v e m o b i l i -t i e s f o r c h o l i n e and p h o s p h o c h o 1 i n e w e r e 0.11 and 0.51 r e s -p e c t i v e l y and 0.76 f o r C D P - c h o l i n e and b e t a i n e w h i c h were not r e s o l v e d . C D P - c h o l i n e was v i s u a l i z e d under U.V. l i g h t a f t e r s p r a y i n g t h e p l a t e w i t h 2 •, 7 ' - d i c h l o r o f 1 u o r e s c e i n i n 0.01% MeOH. The o t h e r compounds were d e t e c t e d by i o d i n e s t a i n i n g . 23 T h i s s y s t e m was u s e d t o a n a l y z e i n c o r p o r a t i o n o f r a d i o -a c t i v i t y i n t o a q u e o u s c h o l i n e m e t a b o l i t e s i n u p p e r p h a s e s a m p l e s o f c e l l e x t r a c t s and i n medium s a m p l e s . I t was a l s o used t o s e p a r a t e t h e r a d i o l a b e l e d r e a c t a n t s and p r o d u c t s o f t h e c h o l i n e k i n a s e and t h e c y t i d y l y l t r a n s f e r a s e a s s a y s and d u r i n g t h e p r e p a r a t i o n o f [Me- H] phosphocho^. i n e . ( i i ) S e p a r a t i o n o f P h o s p h o l i p i d s (System II) CHCl 3/CH 3OH/CH 3COOH/H 20 (50:30:8:3; v/v/v/v) was used t o s e p a r a t e t h e m a j o r p h o s p h o l i p i d c l a s s e s . G l a s s b a c k e d s i l i c a g e l 60 c h r o m a t o g r a p h y p l a t e s (20 cm x 20cm) w e r e u s e d . S a m p l e s o r s t a n d a r d s w e r e s p o t t e d 2 cm a b o v e t h e b o t t o m o f t h e p l a t e and a f t e r d e v e l o p m e n t , t h e p h o s p h o l i p i d s were v i s u a l i z e d by i o d i n e s t a i n i n g . The r e l a t i v e m o b i l i t i e s were l y s o p h o s p h a t i d y l c h o l i n e 0.06, s p h i n g o m y e l i n 0.18, p h o s p h a t i -d y l c h o l i n e 0.35, p h o s p h a t i d y l s e r i n e / p h o s p h a t i d y l i n o s i t o l 0.52, a n d p h o s p h a t i d y l e t h a n o l a m i n e 0.87. T h i s s y s t e m was u s e d t o d e t e r m i n e t h e i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n t o p h o s p h o l i p i d s c o n t a i n e d i n l o w e r p h a s e s a m p l e s o f c e l l e x t r a c t s and a l s o t o s e p a r a t e p h o s p h a t i d y l c h o l i n e f o r p o o l s i z e d e t e r m i n a t i o n . (b) L i q u i d S c i n t i l l a t i o n C o u n t i n g Aqueous s a m p l e s were c o u n t e d i n 5 ml ACS, as were l i p i d s a m p l e s w h i c h w e r e f i r s t d r i e d u n d e r N 2 t o r e m o v e a n y c h l o r o f o r m . S i l i c a g e l s r a p i n g s were d e a c t i v a t e d w i t h 0.5 ml H 2 0 and t h e n c o u n t e d i n 4.5 ml ACS. C o u n t i n g was d o n e i n a C h i c a g o - N u c l e a r I s o c a p / 3 0 0 c o u n t e r . C o u n t i n g e f f i c i e n c i e s 24 w e r e d e t e r m i n e d by e i t h e r e x t e r n a l s t a n d a r d s r a t i o ( l o w , - o u n t s , i . e . l e s s t h a n 1000 cpm) o r c h a n n e l s r a t i o ( h i g h c o u n t s ) o f c h l o r o f o r m q u e n c h e d s t a n d a r d s c o n t a i n i n g e i t h e r [ 3H] h e x a d e c a n e o r [ ^ C ] t o l u e n e . (c) P r o t e i n A s s a y P r o t e i n was d e t e r m i n e d by t h e m e t h o d o f L o w r y e t a l . (54). The f o l l o w i n g s o l u t i o n s were p r e p a r e d : A. 0.1 N NaOH i n 2% (w/v) N a 2 C 0 3 B. 0.5% (w/v) CuSO^, i n 1% s o d i u m c i t r a t e C. A m i x t u r e o f A/B (50:1; v / v ) S a m p l e s w e r e made up t o 200 u l w i t h H 2 0 and 1 ml o f C was a d d e d . A f t e r 10 m i n , 100 u l o f IN p h e n o l r e a g e n t was a d d e d . A f t e r 10 min, t h e a b s o r b a n c e was r e a d a t 500 nm i n a Beckman DU Q u a r t z S p e c t r o p h o t o m e t e r . A s t a n d a r d c u r v e p r e p a r e d w i t h b o v i n e a l b u m i n was l i n e a r f r o m 0-40 ug w i t h an a b s o r b a n c e o f a p p r o x i m a t e l y 0.008/ug p r o t e i n . (d) L i p i d P h o s p h o r u s A s s a y L i p i d p h o s p h o r u s was d e t e r m i n e d by t h e method o f R o u s e r e t a l . (55). The f o l l o w i n g s t o c k s o l u t i o n s were p r e p a r e d : A. 2.5% (w/v) ammonium m o l y b d a t e B. 10% (w/v) a s c o r b i c a c i d S t a n d a r d s ( s o d i u m p h o s p h a t e s o l u t i o n ) and s a m p l e s , e i t h e r s i l i c a g e l s c r a p i n g s o r d i s s o l v e d i n C H C l ^ , w h i c h was t h e n e v a p o r a t e d , were combined w i t h 450 u l o f 70% p e r c h l o r i c a c i d i n c l e a n t e s t t u b e s . The t u b e s were c o v e r e d w i t h m a r b l e s and h e a t e d a t 1 8 0 ° C f o r 45 m i n . The t u b e s w e r e c o o l e d and 2.5 ml H~0 was a d d e d , f o l l o w e d by 0.5 ml A and 0.5 ml B, w i t h 25 m i x i n g b e t w e e n e a c h a d d i t i o n . The t u b e s were t h e n h e a t e d i n a h o t w a t e r b a t h f o r 15 m i n and i f s i l i c a g e l was p r e s e n t , i t was s p u n down, t h e n t h e a b s o r b a n c e was r e a d a t 820 nm. The s t a n d a r d c u r v e g e n e r a t e d w i t h s o d i u m p h o s p h a t e was l i n e a r f r o m 0-150 nmol and t h e a b s o r b a n c e was t y p i c a l l y 0.006/nmol P. 5. C e l l L a b e l i n g S t u d i e s (a) P u l s e - l a b e l i n g E x p e r i m e n t s S t a t i o n a r y c u l t u r e s were d i v i d e d i n t o 2 o r more s e t s , one u n t r e a t e d and a n o t h e r t r e a t e d w i t h p r o l a c t i n (20 n g / m l ) . A t t h e i n d i c a t e d t i m e s , 5 ml a l i q u o t s o f c e l l s w e r e s e d i m e n t e d and t h e medium was r e p l a c e d w i t h f r e s h s t a t i o n a r y medium c o n t a i n i n g [Me- H ] c h o l i n e (9.4 uM, 2-4 u C i / m l ) . The c e l l s w e r e i n c u b a t e d a t 3 7 ° C f o r 2 h, u n l e s s s p e c i f i e d o t h e r w i s e , and t h e n h a r v e s t e d as p r e v i o u s l y d e s c r i b e d ( S e c t i o n 3 b ) . (b) P u l s e - c h a s e E x p e r i m e n t s •3 S t a t i o n a r y c e l l s w e r e p u l s e d w i t h [ M e - J H ] c h o l i n e (2-3 u C i / m l ) added t o t h e c u l t u r e medium f o r 1 h. Then t h e c e l l s w e r e w a s h e d t w i c e w i t h warm s t a t i o n a r y medium and d i v i d e d i n t o 2 o r more s e t s , one u n t r e a t e d and a n o t h e r t r e a t e d w i t h p r o l a c t i n (20 n g / m l ) . A t t h e i n d i c a t e d t i m e s , 5 ml a l i q u o t s were h a r v e s t e d . 26 (c) P h o s p h a t i d y l c h o l i n e T u r n o v e r The p r o c e d u r e s were as f o r t h e p u l s e - c h a s e e x p e r i m e n t s d e s c r i b e d p r e v i o u s l y , e x c e p t t h a t t h e c e l l s were p u l s e d f o r 3 h and t h e c h a s e p e r i o J was much l o n g e r . 6. P o o l S i z e M e a s u r e m e n t s (a) P h o s p h o c h o l i n e P h o s p h o c h o l i n e p o o l s i z e s w e r e d e t e r m i n e d b y a n a d a p t a t i o n o f t h e m e t h o d o f Choy e t a_l. ( 5 6 ) . S t a t i o n a r y u n t r e a t e d and p r o l a c t i n t r e a t e d c e l l s were h a r v e s t e d a t t h e i n d i c a t e d t i m e s . Upper phase s a m p l e s f r o m c e l l e x t r a c t s were t r e a t e d w i t h c h a r c o a l (50 mg/ml) a t 0° C f o r 30 m i n w i t h o c c a s i o n a l s h a k i n g t o a b s o r b n u c l e o t i d e s w h i c h w o u l d o t h e r -w i s e i n t e r f e r e w i t h t h e a s s a y . Then, t h e p h o s p h o c h o l i n e was d e t e r m i n e d i n a l i q u o t s ( 300-600 u l ) o f t h i s t r e a t e d u p p e r p h a s e . F i r s t l y , 30 u l o f r a t l i v e r p h o s p h o l i p i d was d r i e d under N 2 i n s i d e e a c h t u b e , f o l l o w e d by t h e upper p h a s e s w h i c h were a l s o e v a p o r a t e d u n d e r N 2 , and t h e n 20 u l o f E^O was a d d e d t o e a c h t u b e . P h o s p h o c h o l i n e s t a n d a r d s i n 20 u l w e r e p r e p a r e d s i m u l t a n e o u s l y . Then, 50 u l o f p a r t i a l l y p u r i f i e d r a t l i v e r c y t i d y l y l t r a n s f e r a s e was added and t h e m i x t u r e was v i g o r o u s l y v o r t e x e d . T h i s was f o l l o w e d by 30 u l o f r e a c t i o n c o c k t a i l c o n t a i n i n g : 7.5 u l o f 1 M T r i s - s u c c i n a t e , pH 6.5 and 100 mM magnesium a c e t a t e 10 u l o f [ 3 H ] C T P (20 mM, 5 u C i / u m o l ) 27 12.5 u l o f i n o r g a n i c p y r o p h o s p h a t a s e (0.008 umol/min) The m i x t u r e was i n c u b a t e d a L 3 7 ° C f o r 1 h and t h e r e a c t i o n was s t o p p e d by b o i l i n g f o r 2 min. The C D P - c h o l i n e f o r m e d d u r i n g t h e r e a c t i o n was i s o l a t e d b y p a p e r c h r o m a t o g r a p h y i n a s o l v e n t s y s t e m o f e t h a n o l / 1 M ammonium a c e t a t e , pH 7 (7:3; v / v ) . F i f t e e n u l o f r e a c t i o n m i x t u r e was s p o t t e d o n t o c h r o m a t o g r a p h y p a p e r a l o n g w i t h 0.30 mg C D P - c h o l i n e c a r r i e r and f o l l o w i n g d e v e l o p m e n t , t h e CDP-c h o l i n e s p o t s w e r e d e t e c t e d u n d e r U.V. l i g h t a f t e r s p r a y i n g w i t h 2 ' , 7 ' - d i c h l o r o f 1 u o r e s c e i n and c o u n t e d f o r r a d i o a c t i v i t y i n 1 m l H 2 0 p l u s 9 m l ACS. T h e r e was s t o i c h i o m e t r i c c o n v e r s i o n o f s t a n d a r d p h o s p h o c h o l i n e t o r a d i o l a b e l e d CDP-c h o l i n e f r o m 0-7.5 nmol. (b) P h o s p h a t i d y l c h o l i n e The l o w e r p h a s e s a m p l e s o b t a i n e d d u r i n g t h e PC l a b e l t u r n o v e r s t u d y ( S e c t i o n 5c) w e r e u s e d t o d e t e r m i n e t h e c e l l u l a r PC c o n t e n t . T h r e e ml l o w e r p h a s e was s p o t t e d o n t o g l a s s s i l i c a - 6 0 p l a t e s a l o n g w i t h p h o s p h o l i p i d s t a n d a r d s i n an empty l a n e and t h e p l a t e was d e v e l o p e d i n System I I . The PC b a n d was s c r a p e d and s u b j e c t e d t o t h e l i p i d p h o s p h o r u s a s s a y p r e v i o u s l y d e s c r i b e d ( S e c t i o n 4d). 7. E s t i m a t i o n o f M e t a b o l i c R a t e s (a) P h o s p h a t i d y l c h o l i n e T u r n o v e r I n t h e PC t u r n o v e r s t u d i e s , t h e r e was l o s s o f c h o l i n e 28 l a b e l f r o m c e l l a s s o c i a t e d PC t o t h e c e l l medium. S i n c e t h e p o o l s i z e o f PC was d e t e r m i n e d , t h e s p e c i f i c r a d i o a c t i v i t y c o u l d be c a l c u l a t e d . The d e c r e a s e i n l a b e l i n g o f PC a t each i n t e r v a l o f t h e t u r n o v e r s t u d y c o u l d be used t o e s t i m a t e the mass o f PC l o s t and t h e r a t e o f t u r n o v e r c o u l d be c a l c u l a t e d a c c o r d i n g t o t h e e q u a t i o n : T u r n o v e r r a t e = d e c r e a s e i n P C l a b e l i n g mean s p e c i f i c r a d i o a c t i v i t y x t i m e (b) P h o s p h a t i d y l c h o l i n e S y n t h e s i s The r a t e o f PC s y n t h e s i s was e s t i m a t e d as t h a t w h i c h was n e c e s s a r y t o compensate f o r t u r n o v e r ( d e s c r i b e d above) p l u s any change i n p o o l s i z e . The e q u a t i o n d e c r i b i n g t h i s i s : S y n t h e s i s r a t e = t u r n o v e r r a t e + change i n p o o l s i z e t i m e (an i n c r e a s e i n p o o l s i z e i s p o s i t i v e , a d e c r e a s e n e g a t i v e ) 8. Enzyme A s s a y s (a) C h o l i n e K i n a s e C h o l i n e k i n a s e was a s s a y e d a c c o r d i n g t o t h e m e t h o d o f P e l e c h e_t ajk (57). The r e a c t i o n m i x t u r e c o n s i s t e d o f : 20 u l 500 mM T r i s - H C l ( p H 8 . 5 ) , c o n t a i n i n g : 50 mM MgCl-, and 50 mM ATP 10 u l 10 mM [iMe- 3H] c h o l i n e c h l o r i d e (2 u C i / u m o l ) 70 u l c y t o s o l i c p r o t e i n The r e a c t i o n was i n c u b a t e d a t 37° C and t e r m i n a t e d by b o i l i n g 29 f o r 2 min. The p r o t e i n p r e c i p i t a t e was p e l l e t e d by c e n t r i f u -g a t i o n (5000 rpm, 10 min) and t h e n 30 u l was s p o t t e d o n t o p l a s t i c - b a c k e d s i l i c a - 6 0 p l a t e s (2.5 x 10 cm l a n e s ) a l o n g w i t h 0.80 mg p h o s p h o c h o l i n e c a r r i e r . The p l a t e was d e v e l o p e d i n S y s t e m I , and t h e n t h e p h o s p h o c h o l i n e b a n d was s c r a p e d , and c o u n t e d f o r r a d i o a c t i v i t y . The a s s a y was l i n e a r f o r a t l e a s t 60 min and 150 mg c y t o s o l i c p r o t e i n . (b) C T P : P h o s p h o c h o l i n e C y t i d y l y l t r a n s f e r a s e C y t i d y l y l t r a n s f e r a s e was a s s a y e d a c c o r d i n g t o t h e method o f P e l e c h e t a_l. (58) w i t h s l i g h t m o d i f i c a t i o n s . Each 100 u l r e a c t i o n m i x t u r e c o n t a i n e d : 30 u l 0.25 M T r i s - s u c c i n a t e (pH 6.5) r e a c t i o n c o c k t a i l 25 mM magnesium a c e t a t e c o n t a i n i n g : 3.33 mM CTP 5 mM [Me- H ] p h o s p h o c h o l i n e (10 u C i / u m o l ) 10 u l 1 mM o l e a t e 60 u l c y t o s o l i c or m i c r o s o m a l p r o t e i n When c y t i d y l y l t r a n s f e r a s e was d e t e r m i n e d i n t h e p r e s e n c e o f e x o g e n o u s p h o s p h o l i p i d s , 0.3 mg r a t l i v e r p h o s p h o l i p i d , was d r i e d down i n t o e a c h t u b e u n d e r N 2- The e n z y m e and o l e a t e w e r e e m u l s i f i e d w i t h t h e p h o s p h o l i p i d u s i n g a v o r t e x m i x e r a n d t h e n p r e i n c u b a t e d a t 3 7 ° C f o r 3 m i n . The r e a c t i o n was s t a r t e d by a d d i t i o n o f t h e r e a c t i o n c o c k t a i l a n d s t o p p e d by b o i l i n g f o r 2 m i n . The p r o t e i n p r e c i p i t a t e was p e l l e t e d by c e n t r i f u g a t i o n (5000 rpm, 10 min) and 40 u l was s p o t t e d o n t o p l a s t i c - b a c k e d s i l i c a - 6 0 p l a t e s (2.5 x 10 cm) a l o n g w i t h 0.8 mg p h o s p h o c h o l i n e and 0.15 mg C D P - c h o l i n e c a r r i e r s . The 30 p l a t e was d e v e l o p e d i n s y s t e m I and t h e C D P - c h o l i n e r e g i o n was s c r a p e d and c o u n t e d f o r r a d i o a c t i v i t y . The s e p a r a t i o n o f p h o s p h o c h o l i n e and C D P - c h o l i n e was c h e c k e d by v i s u a l i z i n g t h e p h o s p h o c h o l i n e under I 2 s t a i n i n g . F o r c y t o s o l i c p r o t e i n , t h e a s s a y was l i n e a r f o r a t l e a s t 100 ug p r o t e i n and 60 min. (c) C D P - c h o l i n e : D i a c y l g l y c e r o l C h o l i n e p h o s p h o t r a n s f e r a s e C h o i i n e p h o s p h o t r a n s f e r a s e was a s s a y e d a c c o r d i n g t o t h e m e t h o d o f C o r n e l l and G o l d f i n e (59) w i t h s l i g h t m o d i f i c a -t i o n s . Each r e a c t i o n m i x t u r e c o n t a i n e d : 10 u l 500 mM T r i s - H C l (pH 7.4), and 5 mM EGTA 10 u l 4 mM [ M e - 1 4 C ] C D P - c h o l i n e (2 uCi/umol) 15 u l 2.4 mM 1 , 2 - d i o l e i n 65 u l m i c r o s o m a l p r o t e i n The d i o l e i n was made i n t o a s u s p e n s i o n by c o m b i n i n g 5 mg w i t h 12.5 u l Tween 20 (20 mg/ml). T h i s was f o l l o w e d b y 0.25 ml 10% a s o l e c t i n and 0.25 ml H 2 0 and t h e m i x t u r e was v o r t e x e d t o h o m o g e n e i t y . The r e a g e n t s minus C D P - c h o l i n e were combined an d p r e i n c u b a t e d a t 3 7 ° C f o r 5 m i n . T h e r e a c t i o n was s t a r t e d by a d d i t i o n o f C D P - c h o l i n e and s t o p p e d by a d d i t i o n o f 1.5 ml C H C 1 3 / C H 3 0 H (1:2; v/v) , f o l l o w e d by 0.3 ml H 2 0 , 0.5 ml C H C 1 3 , and a n o t h e r 0.5 ml H 20. The f i n a l p r o p o r t i o n s w e r e e q u i v a l e n t t o a B l i g h and D y e r (53) e x t r a c t i o n . T he u p p e r phase was d i s c a r d e d and t h e l o w e r phase was washed t w i c e w i t h 0.75 ml t h e o r e t i c a l u p p e r p h a s e and t h e n d r i e d u n d e r N 2 and c o u n t e d f o r r a d i o a c t i v i t y i n 10 ml ACS. The a s s a y was l i n e a r f o r up t o 60 ug m i c r o s o m a l p r o t e i n and f o r a t l e a s t 30 m i n . 31 RESULTS 1. E f f e c t o f P r o l a c t i n on t h e C D P - c h o l i n e Pathway (a) [ M e - 3 H ] C h o l i n e Uptake The e f f e c t o f h o r m o n a l s t i m u l a t i o n on t h e a c t i v i t y o f t h e C D P - c h o l i n e p a t h w a y was i n i t i a l l y s t u d i e d by a p u l s e -l a b e l i n g e x p e r i m e n t . T h i s i n v o l v e d p u l s i n g c e l l s w i t h [Me-3 H] c h o l i n e f o r a c o n s t a n t t i m e p e r i o d a t v a r i o u s i n t e r v a l s a f t e r p r o l a c t i n - t r e a t m e n t and l o o k i n g f o r c h a n g e s i n l a b e l u p t a k e i n t o PC and i t s p r e c u r s o r s . A c o n t r o l e x p e r i m e n t was p e r f o r m e d t o s t u d y t h e k i n e t i c s 3 o f [Me- H ] c h o l i n e u p t a k e . T o t a l u p t a k e o f c h o l i n e l a b e l i n t o s t a t i o n a r y c o n t r o l o r p r o l a c t i n - t r e a t e d (4 h) c e l l s i n c r e a s e d l i n e a r l y w i t h t i m e f o r a t l e a s t 2 h ( F i g . 4 ) . The r a t e o f t o t a l u p t a k e was a l s o s t i m u l a t e d 2 . 2 - f o l d by p r o l a c t i n -t r e a t m e n t f o r 4 h. T h u s , a t l e a s t one o f t h e s t e p s f r o m e x t r a c e l l u l a r c h o l i n e t o p h o s p h a t i d y l c h o l i n e was s t i m u l a t e d by p r o l a c t i n - t r e a t m e n t . The u p t a k e o f c h o l i n e i n l o aqueous m e t a b o l i t e s c o n t a i n e d i n t h e upper phase o f t h e c e l l e x t r a c t s was s t i m u l a t e d 2 - f o l d and i n t o l o w e r phase o r g a n i c compounds t h e s t i m u l a t i o n was 3 - f o l d ( F i g . 4 ) . T h e s e p r o p o r t i o n s emerged a f t e r 1 h o f p u l s i n g and were p r a c t i c a l l y unchanged a f t e r 2h, so a 2 h p u l s e was a d o p t e d f o r s t u d y i n g t h e e f f e c t 3 o f p r o l a c t i n on [_Me-H] c h o l i n e u p t a k e as a f u n c t i o n o f t i m e a f t e r h o r m o n e t r e a t m e n t . 32 0. 30 60 90 120 P U L S E P E R I O D (MIN) F i g u r e 4 . C o n t i n u o u s p u l s e o f N b 2 c e l l s w i t h [ H e -H] c h o l i n e . S t a t i o n a r y c o n t r o l a n d p r o l a c t i n - t r e a t e d (4 h) c e l l s w e r e p u l s e d w i t h [ Me- H] c h o l i ne i n ' s t a t i o n a r y m e d i u m 1 (9.4 uM, 3.9 u C i / m l ) . A t t h e i n d i c a t e d t i m e s , t h e c e l l s w e r e h a r v e s t e d a s d e s c r i b e d i n " M a t e r i a l s a n d M e t h o d s " a n d t h e t o t a l c o u n t s ( O / • ) a n d i n c o r p o r a t i o n i n t o a q u e o u s (•,.,>) a n d o r g a n i c ( A , A ) m e t a b o l i t e s a r e d i s p l a y e d . O p e n s y m b o l s = c o n t r o l s , c l o s e d s y m b o l s = p r o l a c t i n - t r e a t e d . E a c h p o i n t i s t h e mean o f d u p l i c a t e s a m p l e s . 33 P r o l a c t i n - t r e a t m e n t l e a d t o m a r k e d and w e l l - d e f i n e d 3 c h a n g e s i n [f4e- H] c h o l i n e u p t a k e i n t o upper and l o w e r phase m e t a b o l i t e s whereas t h e c o n t r o l c e l l s m a i n t a i n e d a b a s a l r a t e o f u p t a k e ( F i g . 5). Uptake i n t o upper phase m e t a b o l i t e s was s t i m u l a t e d 2 - f o l d a f t e r 4 h and 3 - f o l d a f t e r 10 h, p l a t e a u i n g t h e r e a f t e r ( F i g . 5A). A n a l y s i s o f t h e upper p h a s e s r e v e a l e d t h a t m a j o r i t y o f t h e a q u e o u s c o u n t s w e r e a s s o c i a t e d w i t h p h o s p h o c h o l i n e i n b o t h c o n t r o l and p r o l a c t i n - t r e a t e d c e l l s (89% i n c o n t r o l s , 95% i n p r o l a c t i n - t r e a t e d c e l l s ) . Thus, t h e 3 b u l k o f t h e i n c r e a s e s i n [ M e - H ] c h o l i n e u p t a k e i n t o p r o l a c t i n - t r e a t e d c e l l s was due t o i n c r e a s e d l a b e l i n g o f p h o s p h o c h o l i n e , s u g g e s t i n g t h a t t h e r e was s t i m u l a t i o n o f one o f t h e s t e p s l e a d i n g t o p h o s p h o c h o l i n e , i . e . c h o l i n e t r a n s -p o r t o r c h o l i n e k i n a s e . B oth t h e s e a c t i v i t e s a r e m e d i a t e d by p r o t e i n s so t h e i n v o l v e m e n t o f p r o t e i n s y n t h e s i s i n t h e c h a n g e s i n l a b e l i n g o f p h o s p h o c h o l i n e was s t u d i e d . C y c l o h e x -i m i d e (10 u g / m l ) a b o l i s h e d t h e e f f e c t o f p r o l a c t i n on [Me-3 H ] c h o l i n e u p t a k e ( F i g . 6 ) . T h u s , p r o t e i n s y n t h e s i s was r e q u i r e d f o r t h e i n d u c t i o n o f c h o l i n e t r a n s p o r t o r c h o l i n e k i n a s e a f t e r p r o l a c t i n - t r e a t m e n t . 3 [Me- H J C h o l i n e u p t a k e i n t o l o w e r phase m e t a b o l i t e s was r a p i d l y s t i m u l a t e d f o l l o w i n g p r o l a c t i n - t r e a t m e n t , r e a c h i n g 1 . 7 - f o l d a f t e r 4 h, b u t g r a d u a l l y r e t u r n e d to c o n t r o l l e v e l s t h e r e a f t e r ( F i g . 5B). A n a l y s i s o f t h e l o w e r p h a s e s r e v e a l e d t h a t t h e m a j o r i t y (90%) o f t h e c o u n t s were a s s o c i a t e d w i t h PC i n b o t h c o n t r o l a n d p r o l a c t i n - t r e a t e d c e l l s . T h i s demon-s t r a t e s t h e p r e c u r s o r - p r o d u c t r e l a t i o n s h i p b e t w e e n e x t r a -c e l l u l a r c h o l i n e and PC. The e a r l i e r s t u d y r e v e a l e d t h a t 34 4 0 i CD X 0 5f-A _i L I ... I I i ' i ' i I i ' B —i i i _ Q 6 8 10 12 14 16 18 T I M E (HOURS ) F i g u r e 5. E f f e c t o f P r o l a c t i n o n [ M e - H ] c h o l i n e u p t a k e i n t o Nb 2 c e l l s . P r o l a c t i n ( 2 0 n g / m l ) w a s a d d e d t o s t a t i o n a r y c u l t u r e s a t 0 h a n d a t t h e i n d i c a t e d t i m e s , c e l l s w e r e p u l s e d w i t h [M_e- H] c h o l i n e i n ' s t a t i o n a r y m e d i u m 1 (9.4 uM, 2.8 u C i / m l ) f o r 2 h , t h e n h a r v e s t e d a s d e s c r i b e d i n " M a t e r i a l s a n d M e t h o d s " . I n c o r p o r a t i o n i n t o A) a q u e o u s a n d B) o r g a n i c m e t a b o l i t e s i s d i s p l a y e d . O p e n s y m b o l s = c o n t r o l s , c l o s e d s y m b o l s = p r o l a c t i n - t r e a t e d . E a c h p o i n t i s t h e m e a n o f d u p l i c a t e s a m p l e s a n d t h e e x p e r i m e n t was r e p e a t e d t w i c e with s i m i l a r r e s u l t s . T h e m a j o r i t y o f t h e a q u e o u s c o u n t s w e r e i n p h o s p h o c h o l i n e ( 8 9 % i n c o n t r o l s , 9 5 % i n p r o l a c t i n - t r e a t e d ) a n d t h e m a j o r i t y o f t h e o r g a n i c c o u n t s ( 9 0 % ) w e r e i n PC. 35 F i g u r e 6. E f f e c t o f c y c l o h e x i m i d e o n p r o l a c t i n - m e d i a t e d [ M e -3 H ] c h o l i n e u p t a k e i n t o Nb 2 c e l l s . P r o c e d u r e s w e r e a s d e s c r i b e d i n F i g . 5 e x c e p t t h a t t h e r e w e r e t w o a d d i t i o n a l s e t s o f c e l l s , o n e t r e a t e d w i t h c y c l o h e x i m i d e (10 u g / m l ) a n d o n e w i t h b o t h c y c l o h e x i m i d e a n d p r o l a c t i n . T h e s p e c i f i c r a d i o a c t i v i t y o f t h e m e d i u m c h o l i n e w a s 9.4 u M , 3.5 u C i / m l . S h o w n a r e i n c o r p o r a t i o n i n t o A) a q u e o u s a n d B) o r g a n i c m e t a -b o l i t e s o f c o n t r o l {O) , p r o l a c t i n ( • ) , c y c l o h e x i m i d e ( A ) , a n d b o t h p r o l a c t i n a n d c y c l o h e x i m i d e (<^>) t r e a t e d c e l l s . E a c h p o i n t i s t h e m e a n d u p l i c a t e s a m p l e s . 36 l a b e l u p t a k e i n t o u p p e r p h a s e m e t a b o l i t e s p r e c e d e d u p t a k e i n t o l o w e r phase compounds ( F i g . 4 ) , s h o w i n g t h a t t h e pathway f o r u t i l i z a t i o n o f e x t r a c e l l u l a r c h o l i n e p r o c e e d s f r o m a q u e o u s c h o l i n e m e t a b o l i t e s t o PC. The i n c r e a s e d l a b e l u p t a k e i n t o PC a f t e r p r o l a c t i n -t r e a t m e n t may have been due t o an i n c r e a s e i n PC b i o s y n t h e s i s b u t a n o t h e r p o s s i b i l i t y i s a c h a n g e i n t h e s p e c i f i c r a d i o -a c t i v i t y o f one o f t h e p r e c u r s o r p o o l s . T h i s i s d i s c u s s e d i n g r e a t e r d e t a i l i n a l a t e r s e c t i o n d e s c r i b i n g c h a n g e s i n p h o s p h o c h o l i n e p o o l s i z e a f t e r p r o l a c t i n - t r e a t m e n t . (b) P u l s e - c h a s e S t u d i e s P u l s e - c h a s e s t u d i e s were u n d e r t a k e n f o r two r e a s o n s : (i) t h e y have been i m p o r t a n t i n t h e argument o f t h e r a t e - l i m i t i n g s t e p i n PC b i o s y n t h e s i s ( 2 2 ) , a n d ( i i ) t h e y h a v e b e e n e f f e c t i v e f o r d e t e c t i n g c h a n g e s i n t h e r a t e o f PC b i o s y n t h e s i s ( 6 0 , 6 1 ) . P r o l a c t i n h a d no e f f e c t on t h e r a t e o f c h o l i n e l a b e l t r a n s f e r f r o m aqueous m e t a b o l i t e s to PC ( F i g . 7). T h r o u g h o u t t h e d u r a t i o n o f t h e c h a s e , v e r y l i t t l e l a b e l a c c u m u l a t e d i n c h o l i n e o r C D P - c h o l i n e ( F i g . 7). The low l a b e l i n g o f c h o l i n e i m p l i e s t h a t o n c e i t e n t e r s t h e c e l l , i t i s r a p i d l y p h o s p h o r y l a t e d . The l a c k o f a c c u m u l a t i o n o f l a b e l i n CDP-c h o l i n e s u g g e s t s t h a t upon f o r m a t i o n , i t i s r a p i d l y c o n v e r t e d t o PC. T h i s p r o v i d e s e v i d e n c e t h a t t h e s y n t h e s i s o f CDP-c h o l i n e , c a t a l y z e d b y C T P : p h o s p h o c h o 1 i n e c y t i d y l y l -t r a n s f e r a s e , i s t h e s l o w s t e p and t h u s r a t e - l i m i t i n g f o r PC b i o s y n t h e s i s i n Nb 2 c e l l s . 37 0 1 2 3 4 5 C H A S E I ME 1 2 3 4 ( H O U R S ) F i g u r e 7. E f f e c t o f p r o l a c t i n on [Me- H ] c h o l i n e p u l s e c h a s e i n Nb 2 c e l l s . S t a t i o n a r y c e l l s w e r e p u l s e d w i t h [Me-3 H ] c h o l i n e (9.4 uM, 3 u C i / m l ) as d e s c r i b e d i n " M a t e r i a l s and M e t h o d s " . P r o l a c t i n w a s a d d e d a t 0 h a n d c e l l s w e r e h a r v e s t e d a t t h e i n d i c a t e d t i m e s , f o l l o w e d by a n a l y s i s o f t h e aqu e o u s and o r g a n i c m e t a b o l i t e s by t h i n - l a y e r c h r o m a t o g r a p h y a s d e s c r i b e d i n " M a t e r i a l s a n d M e t h o d s " . A) a q u e o u s c o u n t s i n c h o l i n e ( O / • ) »• p h o s p h o c h o l i n e ( A , A ) , C D P - c h o l i n e (• ,B ) a n d B) medium c o u n t s (<0>'^ ) an& o r g a n i c c o u n t s i n PC ( 0 / # ) a r e d i s p l a y e d . Open s y m b o l s = c o n t r o l s , c l o s e d ' s y m b o l s = p r o l a c t i n - t r e a t e d . E a c h p o i n t i s t h e mean o f d u p l i c a t e s a m p l e s and t h e e x p e r i m e n t was r e p e a t e d t w i c e w i t h s i m i l a r r e s u l t s . 38 Since the prolactin-treated c e l l s were only exposed to pro l a c t i n during the chase, the s p e c i f i c r a d i o a c t i v i t i e s of the precursor pools were i d e n t i c a l in the c o n t r o l and prolactin-treated c e l l s at the beginning of the chase. Thus, any rapid changes in the rate of l a b e l accumulation in PC would be i n d i c a t i v e of a change i n the r a t e of PC biosynthesis. The pulse-chase s t u d i e s suggested that there was no change i n the r a t e of PC b i o s y n t h e s i s up to 4 h a f t e r prolactin-treatment. This interval was also when much of the increased l a b e l i n g of PC was observed during the pulse-l a b e l i n g s t u d i e s (Fig. 5B). However, t h i s study did not reveal whether the rate of PC b i o s y n t h e s i s was a f f e c t e d at times l a t e r than 4 h after prolactin-treatment. To examine t h i s by pulse-chase would require p u l s i n g c e l l s in the presence and absence of p r o l a c t i n and measuring the pool sizes of phosphocholine so that the s p e c i f i c r a d i o a c t i v i t y of phosphocholine can be determined. Since CDP-choline i s a very s h o r t - l i v e d intermediate in the pathway, the s p e c i f i c r a d i o a c t i v i t y of newly formed PC should be the same as the s p e c i f i c r a d i o a c t i v i t y of phosphocholine. Then from the pulse-chase curves and the s p e c i f i c r a d i o a c t i v t y data, the mass of PC newly formed during the chase can be c a l c u l a t e d , as w e l l as the rate of formation. A p r e l i m i n a r y t r i a l of t h i s method suggested that the rate of PC b i o s y n t h e s i s was unchanged from 4 to 7 h after prolactin-treatment. 39 (c) P h o s p h o c h o l i n e P o o l S i z e T h e r e was i n c r e a s e d l a b e l i n g o f p h o s p h o c h o l i n e i n t h e p r o l a c t i n - t r e a t e d c e l l s d u r i n g t h e p u l s e - l a b e l i n g s t u d y ( F i g . 5A), so t h e e f f e c t o f p r o l a c t i n on p h o s p h o c h o l i n e p o o l s i z e s was d e t e r m i n e d . P r o l a c t i n - t r e a t m e n t l e a d t o e n l a r g e m e n t o f t h e p h o s p h o c h o l i n e p o o l ( F i g . 8). The i n c r e a s e s i n p o o l s i z e w e r e n o t p r o p o r t i o n a l t o t h e i n c r e a s e s i n l a b e l i n g o f p h o s p h o c h o l i n e d u r i n g t h e p u l s e - l a b e l i n g s t u d y . T h u s , t h e s p e c i f i c r a d i o a c t i v i t y o f p h o s p h o c h o l i n e was g r e a t e r i n t h e p r o l a c t i n - t r e a t e d c e l l s and t h i s c o u l d a c c o u n t l a r g e l y f o r t h e i n c r e a s e i n l a b e l i n g o f PC a f t e r 4h p r o l a c t i n - t r e a t m e n t ( T a b l e 2). I f t h i s a rgument can be e x t e n d e d , PC b i o s y n t h e s i s may e v e n be d e p r e s s e d i n t h e p r o l a c t i n - t r e a t e d c e l l s a t t h e l a t e r t i m e p o i n t s . F o r i n s t a n c e , a f t e r 10 h, l a b e l i n g o f PC was s i m i l a r i n c o n t r o l and p r o l a c t i n - t r e a t e d c e l l s w h e r e a s t h e s p e c i f i c r a d i o a c t i v i t y o f p h o s p h o c h o l i n e was g r e a t e r i n t h e p r o l a c t i n - t r e a t e d c e l l s ( T a b l e 2). In t h e c o n t r o l c e l l s , t h e p h o s p h o c h o l i n e p o o l s i z e r e m a i n e d c o n s t a n t , i n d i c a t i n g t h a t t h e r e was a b a l a n c e b e t w e e n r e a c t i o n s i n v o l v e d i n t h e f o r m a t i o n o f p h o s p h o c h o l i n e , s u c h a s c h o l i n e k i n a s e , a n d r e a c t i o n s i n v o l v e d i n i t ' s u t i l i z a t i o n , s u c h as c y t i d y l y l t r a n s f e r a s e . O t h e r r e a c t i o n s w h i c h c o u l d i n f l u e n c e t h e p o o l s i z e a r e p h o s p h o c h o l i n e p h o s p h a t a s e o r c a t a b o l i c r e a c t i o n s r e s u l t i n g i n t h e r e l e a s e o f p h o s p h o c h o l i n e f r o m PC. In t h e p r o l a c t i n -t r e a t e d c e l l s , s t i m u l a t i o n o f c h o l i n e k i n a s e may e x p l a i n t h e i n c r e a s e i n p h o s p h o c h o l i n e p o o l s i z e i n p a r t b u t c h a n g e s i n t h e o t h e r r e a c t i o n s may a l s o have o c c u r r e d . 40 I I I 0 2 4 6 8 10 12 14 16 T I M E ( H O U R S ) F i g u r e 8. E f f e c t o f p r o l a c t i n o n p h o s p h o c h o l i n e p o o l s i n Nb 2 c e l l s . P h o s p h o c h o l i n e p o o l s i n c o n t r o l (O) a n d p r o l a c t i n -t r e a t e d ( # ) c e l l s w e r e d e t e r m i n e d a s d e s c r i b e d i n " M a t e r i a l s a n d M e t h o d s " . E a c h p o i n t i s t h e m e a n + S.D. o f 3 m e a s u r e -m e n t s . T h e w e t w e i g h t o f t h e c e l l s w a s 0.030 g / 1 0 7 c e l l s , t h u s t h e c o n c e n t r a t i o n o f p h o s p h o c h o l i n e i n c o n t r o l c e l l s " w a s 0.62 uM a s s u m i n g 1 g c e l l s = 1 m l . 41 T a b l e 2. C o m p a r i s o n o f changes i n s p e c i f i c r a d i o a c t i v i t y o f p h o s p h o c h o l i n e and l a b e l i n g o f p h o s p h a t i d y l c h o l i n e i n Nb 2 c e l l s + p r o l a c t i n . +/- P h o s p h o c h o l i n e S p e c i f i c R a t i o F o l d i n c T ime P r o - M a s s 1 L a b e l R a d i o a c t i v i t y +/- i n PC l a c t i n l a b e l i n g ^ 4 h - 1.8 7.5 4.2 + 1.9 17.5 9.2 2.2 1.7 10 h 1.7 2.8 9.0 27. 3 5. 3 9.6 1.8 1.1 nmol/10 c e l l s , from F i g . 8 dpm x 1 0 / 1 0 c e l l s , from F i g . 5A c o r r e c t e d f o r p h o s p h o c h o l i n e dpm x 1 0 - 4 / n m o l from F i g . 5B 42 In t h e p r o l a c t i n - t r e a t e d c e l l s , t h e p h o s p h o c h o l i n e p o o l s i z e i n c r e a s e d 1 . 8 - f o l d a f t e r 16 h, w h e r e a s t h e l a b e l i n g o f p h o s p h o c h o l i n e was s t i m u l a t e d 3 - f o l d a f t e r 10 h i n t h e p u l s e -l a b e l i n g s t u d y . One e x p l a n a t i o n f o r t h i s l a c k o f p a r a l l e l i s m b e t w e e n p o o l s i z e s and c h o l i n e l a b e l u p t a k e may be t h a t i n p u l s e - l a b e l i n g s t u d y , t h e p h o s p h o c h o l i n e p o o l s w e r e n o t l a b e l e d t o c o n s t a n t s p e c i f i c r a d i o a c t i v i t y so l a b e l i n g o f t h e p o o l s do n o t r e p r e s e n t r e l a t i v e p o o l s i z e s . O t h e r w i s e , t h e r e may j u s t be a d e l a y b e t w e e n i n d u c t i o n o f c h o l i n e k i n a s e and i t ' s e f f e c t on i n t r a c e l l u l a r p h o s p h o c h o l i n e l e v e l s . A n o t h e r p o s s i b i l i t y i s t h a t a c a t a b o l i c r e a c t i o n c o n t r i b u t i n g t o t h e p h o s p h o c h o l i n e p o o l may be i n a c t i v a t e d i n t h e p r o l a c t i n -t r e a t e d c e l l s . 2. E f f e c t o f P r o l a c t i n on P h o s p h a t i d y l c h o l i n e T u r n o v e r (a) P h o s p h a t i d y l c h o l i n e P o o l S i z e The p u l s e - c h a s e r e s u l t s s u g g e s t e d t h a t t h e r a t e o f PC b i o s y n t h e s i s was n o t i n f l u e n c e d by p r o l a c t i n - t r e a t m e n t f o r a t l e a s t up t o 4 h. The p h o s p h o c h o l i n e p o o l s i z e d e t e r m i n a t i o n s s u g g e s t e d t h a t t h e i n c r e a s e s i n l a b e l i n g o f PC i n t h e [Me-3 H ] c h o l i n e u p t a k e s t u d y c o u l d be l a r g e l y a c c o u n t e d f o r by an i n c r e a s e i n t h e s p e c i f i c r a d i o a c t i v i t y o f t h e p h o s p h o c h o l i n e p r e c u r s o r p o o l . These s e p a r a t e l i n e s o f e v i d e n c e s u g g e s t e d t h a t t h e a c t i v i t y o f t h e C D P - c h o l i n e pathway i s n o t a f f e c t e d g r e a t l y a f t e r t h e c e l l s w e r e s t i m u l a t e d t o g r o w w i t h p r o l a c t i n . T h i s r e s u l t was somewhat s u r p r i s i n g so c e l l u l a r l e v e l s o f PC w e r e m e a s u r e d i n o r d e r t o c h e c k o u r i n i t i a l 43 p r e m i s e t h a t c e l l g r o w t h s h o u l d be a s s o c i a t e d w i t h i n c r e a s e d membrane s y n t h e s i s and PC l e v e l s . C e l l u l a r PC l e v e l s i n c r e a s e d g r a d u a l l y i n t h e p r o l a c t i n -t r e a t e d c e l l s , r e a c h i n g 1 . 4 - f o l d a f t e r 16 h, whereas c o n t r o l c e l l s m a i n t a i n e d a c o n s t a n t l e v e l ( F i g . 9). Thus, p r o l a c t i n -c r e a t m e n t was l e a d i n g t o a n e t a c c u m u l a t i o n o f PC a l t h o u g h t h e m e c h a n i s m d i d n o t a p p e a r t o be an i n c r e a s e i n t h e r a t e o f b i o s y n t h e s i s . PC was a l s o f o u n d t o be t h e m a j o r p h o s p h o l i p i d i n Nb 2 c e l l s ( T a b l e 3) and p r e l i m i n a r y r e s u l t s s u g g e s t t h a t t h e r a t i o o f PC t o t o t a l p h o s p h o l i p i d r e m a i n s c o n s t a n t i n t h e p r o l a c t i n - t r e a t e d c e l l s . (b) T u r n o v e r o f P h o s p h a t i d y l c h o l i n e C a t a b o l i s m o f PC was i n v e s t i g a t e d a s a p o s s i b l e m e c h a n i s m f o r r e g u l a t i n g c e l l u l a r PC l e v e l s i n Nb 2 c e l l s . T h i s was done by l a b e l i n g c e l l u l a r p o o l s w i t h [Me- H ] c h o l i n e a n d c h a s i n g i t i n t o PC, t h e n o b s e r v i n g d i f f e r e n c e s i n t h e t u r n o v e r o f t h e l a b e l e d PC b e t w e e n c o n t r o l and p r o l a c t i n -t r e a t e d c e l l s . F o l l o w i n g a 3 h p u l s e , t h e m a j o r i t y (70%) o f t h e c h o l i n e c o u n t s c o u l d be c h a s e d f r o m t h e a q u e o u s m e t a b o l i t e p o o l w i t h i n 4 h and l a b e l i n g o f PC r e a c h e d a maximum ( F i g . 1 0 ) . A f t e r 4 h, t h e r e was t u r n o v e r o f c h o l i n e l a b e l f r o m PC w i t h t h e a p p e a r a n c e o f c o r r e s p o n d i n g a m o u n t s o f l a b e l i n t h e medium. R e m a r k a b l y , t h e c o n t r o l c e l l s l o s t l a b e l a t a 3.4-f o l d g r e a t e r r a t e t h a n t h e p r o l a c t i n - t r e a t e d c e l l s ( F i g . 10). T h u s , a l o w e r e d r a t e o f PC t u r n o v e r i n p r o l a c t i n - t r e a t e d 44 1 h • 2 4 6 8 10 12 14 16 TIME (HOURS) F i g u r e 9. E f f e c t o f p r o l a c t i n on p h o s p h a t i d y l c h o l i n e l e v e l s i n Nb 2 c e l l s . PC p o o l s i n c o n t r o l (O) and p r o l a c t i n -t r e a t e d ( f t ) c e l l s were d e t e r m i n e d as d e s c r i b e d i n " M a t e r i a l s and M e t h o d s " . D a t a a r e means + S.D. o f 4 o r 5 d e t e r m i n a -t i o n s . 45 T a b l e 3. P h o s p h o l i p i d c o m p o s i t i o n o f Nb 2 c e l l s . P h o s p h o l i p i d % PC 42 PE 28 PI/PS 16 SM 11 l y s o - P C 3 E x p o n e n t i a l l y g r o w i n g Nb 2 c e l l s w e r e h a r v e s t e d , p h o s p h o -l i p i d s w e r e s e p a r a t e d and s u b j e c t e d t o p h o s p h o r u s d e t e r -m i n a t i o n as d e s c r i b e d i n " M a t e r i a l s and Methods". 46 in CJ to a i a x Q_ a 2 4 6 8 10 12 14 16 C H A S E T I M E ( H O U R S ) F i g u r e 10. E f f e c t o f p r o l a c t i n o n t u r n o v e r o f l a b e l e d p h o s p h a t i d y l c h o l i n e . S t a t i o n a r y c e l l s w e r e p u l s e d w i t h [ M e -R] c h o l i n e (9.4 u M , 4.4 u C i / m l ) a s d e s c r i b e d i n " M a t e r i a l s a n d M e t h o d s " . P r o l a c t i n w a s a d d e d a t 0 h a n d c e l l s w e r e h a r v e s t e d a t t h e i n d i c a t e d a n d M e t h o d s " . S h o w n a r e ( A , A ) , m e d i u m ( < > ' • ) ' = c o n t r o l s , c l o s e d s y m b o l s i s t h e m e a n o f d u p l i c a t e t i m e s , a s d e s c r i b e d i n " M a t e r i a l s c o u n t s i n a q u e o u s m e t a b o l i t e s a n d P C ( O f # ) - O p e n s y m b o l s = p r o l a c t i n - t r e a t e d . E a c h p o i n t s a m p l e s a n d t h e e x p e r i m e n t w a s r e p e a t e d 3 t i m e s w i t h s i m i l a r r e s u l t s . 47 c e l l s a p p e a r e d t o be an e x p l a n a t i o n f o r how t h e y i n c r e a s e d t h e i r c e l l u l a r l e v e l s o f PC w i t h o u t a l t e r i n g t h e i r r a t e o f b i o s y n t h e s i s . A n o t h e r p o s s i b i l i t y was t h a t d i l u t i o n o f t h e PC p o o l i n t h e p r o l a c t i n - t r e a t e d c e l l s due t o t h e p o o l s i z e i n c r e a s e was l e a d i n g t o a d e c r e a s e i n t h e r a t e o f l a b e l t u r n o v e r . H o w e v e r , t h i s s e e m e d u n l i k e l y b e c a u s e t h e p o o l s i z e i n c r e a s e d o n l y 1 . 4 - f o l d a f t e r 16 h whereas t h e 3 . 4 - f o l d d i f f e r e n c e i n l a b e l t u r n o v e r was a p p a r e n t a f t e r 4 h. The medium was a n a l y z e d f o r aqueous c h o l i n e m e t a b o l i t e s and t h e i n c r e a s e i n m e d ium c o u n t s f o r t h e c o n t r o l c e l l s was l a r g e l y a c c o u n t e d f o r b y an i n c r e a s e i n c h o l i n e l a b e l a p p e a r i n g i n t h e m e d ium ( F i g . 1 1 ) . T h e r e was some l a b e l i n p h o s p h o c h o l i n e b u t i t d i d n o t a c c u m u l a t e w i t h t i m e i n e i t h e r c o n t r o l o r p r o l a c t i n - t r e a t e d c e l l s ( F i g . 11). T h u s , i n c o n t r o l c e l l s , t h e r e i s s t e a d y r e l e a s e o f c h o l i n e i n t o t h e medium w i t h a c o r r e s p o n d i n g l o s s f r o m the PC f r a c t i o n o f t h e c e l l s . I n t h e p r o l a c t i n - t r e a t e d c e l l s , t h i s r e l e a s e o f c h o l i n e a p p e a r e d t o be c o m p l e t e y a b o l i s h e d . H o w e v e r , t h i s does n o t n e c e s s a r i l y mean t h a t a l l c a t a b o l i c r e a c t i o n s c e a s e i n t h e p r o l a c t i n - t r e a t e d c e l l s . R e m o d e l i n g o f PC o r r e c y c l i n g may be o c c u r r i n g , j u s t v e r y l i t t l e c h o l i n e i s b e i n g l o s t f r o m t h e c e l l s . (c) E s t i m a t i o n o f P h o s p h a t i d y l c h o l i n e M e t a b o l i c R a t e s The PC l a b e l t u r n o v e r s t u d i e s and t h e PC p o o l s i z e m easurements were c o m b i n e d t o e s t i m a t e t h e s p e c i f i c r a d i o -a c t i v i t y o f PC d u r i n g t h e t u r n o v e r s t u d y and f r o m t h i s , t h e mass o f t h e PC b e i n g t u r n e d o v e r and t h u s t h e r a t e o f PC 48 CO b 1 X — I X 0 Q_ • LU 2 5d • I X V a u CONTROL T PROLACTIN 0 T 1 1 4 8 12 16 T I M E ( H O U R S ) F i g u r e 11. A n a l y s i s o f m e d i u m f o r r a d i o a c t i v i t y i n a q u e o u s c h o l i n e m e t a b o l i t e s . T w e n t y u l a l i q u o t s o f m e d i u m ' f r o m e x p e r i m e n t d e s c r i b e d i n F i g . 10 w e r e c h r o m a t o g r a p h e d aV d e s c r i b e d i n " M a t e r i a l s a n d M e t h o d s " . S h o w n a r e t h e m e a n + r a n g e o f t w o s u c h e x p e r i m e n t s . 49 t u r n o v e r was c a l c u l a t e d a s d e s c r i b e d i n " M a t e r i a l s and Methods". The d e f i n i t i o n o f t u r n o v e r i n t h i s c a s e means n e t d e g r a d a t i o n o f PC w i t h t h e l o s s o f t h e c h o l i n e h e a d g r o u p . T h i s c o u l d a r i s e t h r o u g h p h o s p h o l i p a s e C o r D a c t i v i t y o r t h r o u g h s u b s e q u e n t d e g r a d a t i o n f o l l o w i n g p h o s p h o l i p a s e A a c t i v i t y . The f a t e o f t h e r e m a i n d e r o f t h e m o l e c u l e m i g h t be a s c e r t a i n e d by l a b e l i n g a d i f f e r e n t p o r t i o n o f t h e PC m o l e -c u l e . The r a t e o f PC b i o s y n t h e s i s was c a l c u l a t e d as t h a t r e q u i r e d t o o f f s e t PC t u r n o v e r p l u s any c h a n g e i n t h e p o o l s i z e , as d e s c r i b e d i n " M a t e r i a l s and Methods". T h i s s y n t h e -s i s r a t e i n c l u d e s a l l s o u r c e s , a l t h o u g h t h e C D P - c h o l i n e pathway i s c o n s i d e r e d t h e m a j o r r o u t e o f s y n t h e s i s . When the PC t u r n o v e r and s y n t h e s i s r a t e s were d e t e r m i n e d f r o m 4 t o 16 h a f t e r p r o l a c t i n t r e a t m e n t , on t h e a v e r a g e , PC t u r n o v e r r a t e s were 3 . 2 - f o l d l o w e r i n t h e p r o l a c t i n - t r e a t e d c e l l s whereas t h e s y n t h e s i s r a t e s were s i m i l a r i n t h e c o n t r o l a n d p r o l a c t i n - t r e a t e d c e l l s ( T a b l e 4 ) . T h e s e r e s u l t s a r e f u r t h e r s u g g e s t i o n t h a t i n r e s p o n s e t o p r o l a c t i n - t r e a t m e n t , Nb 2 c e l l s r e g u l a t e c e l l u l a r PC l e v e l s n o t by a l t e r i n g t h e r a t e o f s y n t h e s i s but t h e r a t e o f t u r n o v e r . 3. E f f e c t o f P r o l a c t i n on t h e E n z y m e s o f t h e C D P - c h o l i n e Pathway The e n z y m e s o f t h e C D P - c h o l i n e p a t h w a y w e r e a s s a y e d u n d e r o p t i m u m c o n d i t i o n s i n o r d e r t o s e e i f t h e e f f e c t s o f p r o l a c t i n on t h e pathway as s u g g e s t e d by t h e p r e v i o u s s t u d i e s 50 T a b l e 4. C a l c u l a t e d p h o s p h a t i d y l c h o l i n e t u r n o v e r a n d s y n t h e s i s r a t e s . CONTROL: R a d 1 (dpm x I O - 4 / 1 0 6 c e l l s ) PC (nmol/ 1 0 6 c e l l s ) PC t u r n o v e r ( n m o l / h / 1 0 6 c e l l s ) PC nmol/ h / 1 0 6 c e l l s ) PC s y n t h e s i s (nmol/ h / 1 0 6 c e l l s ) PROLACTIN: TIME (HOUR) 4 8 10 12 14 16 15.1 13.2 12.3 11.3 10.2 9.0 4.07 4.03 4.00 3.96 3.90 3.82 .136 .142 . 168 .201 . 240 -.010 -.015 -. 020 -.020 -.040 .126 .127 .148 .181 . 200 R a d 1 16.1 15.7 15.3 14.9 14.6 14. 2 P C 2 4.35 4.85 5.15 5. 38 5. 55 5. 72 PC t u r n o v e r .029 .065 .070 .056 . 078 PC . 125 . 150 . 100 . 085 . 085 PC s y n t h e s i s .154 .215 .185 .141 . 163 RATIO (CONTROL/PROLACTIN): Avg . PC t u r n o v e r 4.86 2.18 2.40 3.59 3. 08 3. 22 PC s y n t h e s i s . 82 1. 69 .87 1. 28 1. 23 1. 18 • ' • r a d i o a c t i v i t y i n PC, from F i g . 10 2 P C p o o l s i z e , f r o m F i g . 8 51 would be r e f l e c t e d by c h a n g es i n in v i t r o enzyme a c t i v i t i e s . The enzymes were a s s a y e d i n c o n t r o l and p r o l a c t i n - t r e a t e d (4 h) c e l l s . C e l l s were a l s o t r e a t e d w i t h c y c l o h e x i m i d e t o see i f any o f t h e changes o b t a i n e d m i g h t r e q u i r e p r o t e i n s y n t h e -s i s . The e n z y m e s w e r e a s s a y e d i n t h e a p p r o p r i a t e c e l l f r a c t i o n and t h e r e s u l t s a r e o u t l i n e d ( T a b l e 5). (a) C h o l i n e K i n a s e P r o l a c t i n t r e a t m e n t f o r 4 h l e a d t o a 1 . 9 - f o l d s t i m u l a t i o n o f c h o l i n e k i n a s e a c t i v i t y and t h i s e f f e c t was a b o l i s h e d by c y c l o h e x i m i d e ( T a b l e 5). The i n c r e a s e i n c h o l i n e k i n a s e a c t i v i t y c o r r e l a t e s w e l l w i t h t h e 2 - f o l d i n c r e a s e i n l a b e l u p t a k e i n t o p h o s p h o c h o l i n e a f t e r 4 h d u r i n g t h e [Me_-H ] c h o l i n e u p t a k e s t u d i e s , w h i c h was a l s o b l o c k e d b y c y c l o h e x i m i d e ( F i g . 6 ) . T h u s , t h e m e c h a n i s m o f i n d u c t i o n r e q u i r e d p r o t e i n s y n t h e s i s a n d w a s s e n s i t i v e t o c y c l o h e x i m i d e . The i n d u c t i o n o f c h o l i n e k i n a s e has been d e s c r i b e d i n a number o f o t h e r s y s t e m s . T r e a t m e n t o f c u l t u r e d l i v e r c e l l s w i t h i n s u l i n ( 1 7 ) , r o o s t e r l i v e r w i t h d i e t h y l s t i l b e s t r o 1 ( 1 8 ) , a n d r a t l i v e r w i t h c y c l i c a r o m a t i c h y d r o c a r b o n c a r c i n o g e n s (19) o r C C 1 4 (20) h a v e r e s u l t e d i n e l e v a t i o n o f c h o l i n e k i n a s e , m o s t l i k e l y b y s y n t h e s i s o f new e n zyme. P o l y a m i n e s a r e a c t i v a t o r s o f c h o l i n e k i n a s e (62) a n d a c c u m u l a t i o n o f t h e s e compounds i n mammary g l a n d a f t e r g l u c o -c o r t i c o i d s t i m u l a t i o n c a n a l s o r e g u l a t e c h o l i n e k i n a s e a c t i v i t y (21). D r a m a t i c i n c r e a s e s i n t h e l e v e l s o f p o l y a m i n e b i o s y n t h e t i c e n z y m e s h a v e a l s o b e e n d e s c r i b e d i n Nb 2 c e l l s 52 r a b l e 5. I_n v i t r o a c t i v i t i e s o f e n z y m e s o f C D P - c h o l i n e pathway from Nb 2 c e l l s . •CK S p e c i f i c A c t i v i t y (nmol/min/mg) *r*T CT ^ c y t o s o l microsome CPT C o n t r o l 0.26+0.03 (n=3) 1.61+0.33 (n = 5) 0.0 5+0.0 3 (n=3) 3.98+0.10 (n=2) P r o l a c t i n (4 h) 0.49+0.02 (n=3) 1.52+0.30 (n=5) 0.05+0.03 (n=3) 3.83+0.04 (n=2) C y c l o h e x i m i d e (4 h) P r o l a c t i n p l u s c y c l o -h e x i m i d e 0.23+0.02 (n=2) 0.20+0.05 (n=3) 1.43 (n=l) 1.42+0.02 (n=2) n.d n.d, n.d. n.d. v a l u e s a r e mean + S.D. n.d. n o t d e t e r m i n e d ^ c h o l i n e k i n a s e 2 c y t i d y l y l t r a n s f e r a s e c h o l i n e p h o s p h o t r a n s f e r a s e 53 f o l l o w i n g p r o l a c t i n - t r e a t m e n t ( 4 8 ) , w h i c h m i g h t a c t i v a t e c h o l i n e k i n a s e by an i n c r e a s e i n p o l y a m i n e s . Thus, the mechanism o f c h o l i n e k i n a s e i n d u c t i o n i n Nb 2 c e l l i s u n c l e a r . I m m u n o t i t r a t i o n o f c h o l i n e k i n a s e may r e v e a l i f e nzyme s y n t h e s i s was i n v o l v e d . A way t o t e s t w h e t h e r t h e r e i s a n a c t i v a t o r i n t h e c y t o s o l o f t h e p r o l a c t i n - t r e a t e d c e l l s w ould be t o b o i l t h e c y t o s o l and t h e n t e s t i t f o r a b i l i t y t o s t i m u l a t e c h o l i n e k i n a s e a c t i v i t y i n c o n t r o l c y t o s o l . A v a r i a t i o n o f t h i s w o u l d be t o c o m b i n e c o n t r o l and p r o l a c t i n - t r e a t e d c y t o s o l s and m e a s u r e t h e c h o l i n e k i n a s e a c t i v i t y . The p r e s e n c e o f an a c t i v a t o r s h o u l d r e s u l t i n g r e a t e r t o t a l a c t i v i t y t h a n t h e sum o f t h e s e p a r a t e a c t i v i t i e s , whereas i f i n d u c t i o n was by enzyme s y n t h e s i s , t h e t o t a l a c t i v i t y s h o u l d e q u a l t h e sum. A l t e r n a t i v e l y , t h e p o l y a m i n e p o o l s i z e s can be measured by HPLC. C h o l i n e k i n a s e e x i s t s as m u l t i p l e f o r m s i n r a t t i s s u e s (15) and i n d u c t i o n by h y d r o c a r b o n c a r c i n o g e n s o r C C l ^ a p p e a r s t o l e a d t o s y n t h e s i s o f f o r m s o f c h o l i n e k i n a s e w h i c h a r e n o t c r o s s - r e a c t i v e w i t h a n t i b o d i e s g e n e r a t e d a g a i n s t t h e non-i n d u c e d f o r m (16). However, t h e d i e t h y l s t i l b e s t r o l - i n d u c e d f o r m o f c h o l i n e k i n a s e i n r o o s t e r l i v e r i s i m m u n o l o g i c a l l y s i m i l a r t o t h e n o n - i n d u c e d f o r m ( 1 8 ) . T h u s , i n d u c t i o n o f c h o l i n e k i n a s e s y n t h e s i s f o l l o w i n g hormone or c a r c i n o g e n (or C C l ^ ) t r e a t m e n t may i n v o l v e d i f f e r e n t m o l e c u l a r mechanisms. I f e nzyme s y n t h e s i s was r e s p o n s i b l e f o r c h o l i n e k i n a s e i n d u c t i o n i n Nb 2 c e l l s , i t w o u l d be i n t e r e s t i n g t o f i n d o u t w h i c h mechanism i t r e s e m b l e s . 54 (b) C y t i d y l y l t r a n s f e r a s e C y t i d y l y t r a n s f e r a s e a c t i v i t y i n Nb 2 c e l l s was n o t a f f e c t e d by p r o l a c t i n - t r e a t m e n t f o r 4 h ( T a b l e 5 ) . T h i s s u p p o r t s t h e o t h e r s t u d i e s i n s u g g e s t i n g t h a t t h e r a t e o f PC b i o s y n t h e s i s i n Nb 2 c e l l s i s n o t i n f l u e n c e d by p r o l a c t i n -t r e a t m e n t . A p u z z l i n g a s p e c t a b o u t c y t i d y l y l t r a n s f e r a s e was t h e low amount o f a c t i v i t y a s s o c i a t e d w i t h t h e m i c r o s o m a l f r a c t i o n ( T a b l e 5 ) . C y t i d y l y l t r a n s f e r a s e i s t h o u g h t t o be r e g u l a t e d by a t r a n s l o c a t i o n mechanism (4) and t h e m i c r o s o m a l a c t i v i t y i s s u p p o s e d t o r e f l e c t t h e r a t e o f t h e p a t h w a y . A l t h o u g h c y t i d y l y l t r a n s f e r a s e a c t i v i t y was n o t a f f e c t e d by p r o l a c t i n , t h e p u l s e - c h a s e and o t h e r s t u d i e s s u g g e s t t h a t b o t h c o n t r o l and p r o l a c t i n - t r e a t e d c e l l s a r e a c t i v e i n t h e s y n t h e s i s o f PC v i a t h e C D P - c h o l i n e p a t h w a y . The m i c r o s o m a l r e c o v e r y was a s s e s s e d by d e t e r m i n i n g th e d i s t r i b u t i o n o f c h o l i n e p h o s p h o -t r a n s f e r a s e a c t i v i t y as a m i c r o s o m a l m a r k e r . M i c r o s o m a l r e c o v e r y was 31% ( T a b l e 6), and a s s u m i n g p r o t e i n c o n t e n t s as i n T a b l e 1, t h e d i s t r i b u t i o n o f c y t i d y l y l t r a n s f e r a s e a c t i v i t y i n Nb 2 c e l l s i s 9 5 % c y t o s o l i c a n d 5% m i c r o s o m a l . I n c o n t r a s t , m i c r o s o m a l a c t i v i t y i n r a t l i v e r i s 24% (60) and i n HeLa c e l l s i s 35% ( 6 3 ) . A p o s s i b l e e x p l a n a t i o n f o r t h e low m i c r o s o m a l a c t i v i t y may be i n t h e p r o c e d u r e f o r s u b c e l l u l a r f r a c t i o n a t i o n . C y t i d y l y l t r a n s f e r a s e i s a m b i q u i t o u s and t h e d i s t r i b u t i o n i s i n f l u e n c e d b y t h e m e t h o d o f c e n t r i f u g a t i o n and t h e i o n i c s t r e n g t h o f t h e h o m o g e n i z a t i o n b u f f e r , w i t h d i s t i l l e d t^O g i v i n g m a x i m a l r e c o v e r y (85%) i n t h e p a r t i c u l a t e f r a c t i o n and 55 i a b l e 6. M i c r o s o m a l r e c o v e r y . C h o i i n e p h o s p h o t r a n s f e r a s e d i s t r i b u t i o n a f t e r s u b c e l l u l a r f r a c t i o n a t i o n S p e c i f i c T o t a l T o t a l % o f F r a c t i o n A c t i v i t y P r o t e i n A c t i v i t y Homo-(nmol/min/mg) (mg) (nmol/min) g e n a t e Homogenate C 2. .77 22. . 5 62 .3 100 P 2. , 44 21. .0 51 .2 10,000 x g C 2. , 98 12. , 4 37 .0 63.7 p e l l e t P 3. . 17 11. . 2 35 . 5 C y t o s o l C 0. , 01 12. .6 0. 14 0.3 P 0. .01 14. .3 0. 18 Mi c r o s o m e C 3. ,79 4. .6 17 .4 31. 3 P 3. .87 4. .7 18 . 2 F r a c t i o n s f r o m c o n t r o l (C) and p r o l a c t i n - t r e a t e d (P) c e l l s . T h i s e x p e r i m e n t was r e p e a t e d w i t h s i m i l a r r e s u l t s . 56 i s o t o n i c s a l i n e g i v i n g 99% a c t i v i t y i n t h e s u p e r n a t a n t o f a r a t l i v e r homogenate (64). The h o m o g e n i z a t i o n b u f f e r used i n t h i s s t u d y was l o w i o n i c s t r e n g t h ( M a t e r i a l s and M e t h o d s , S e c t i o n 3 c ) . A n o t h e r p o s s i b i l i t y i s t h a t t h e membrane p r e p a r a t i o n i s somehow i n h i b i t o r y t o c y t i d y l y l t r a n s f e r a s e , p e r h a p s a s a r e s u l t o f t h e h o m o g e n i z a t i o n p r o c e d u r e . In t h e m i c r o s o m e s , t h e c y t i d y l y l t r a n s f e r a s e i s l o c a t e d on t h e c y t o s o l i c s i d e o f s e a l e d f r a g m e n t s o f e n d o p l a s m i c r e t i c u l u m (28). A p r o f i l e o f r e c o v e r y o f c y t i d y l y l t r a n s f e r a s e a c t i v i t y f r o m Nb 2 c e l l s shows t h a t a f t e r s u b c e l l u l a r f r a c t i o n a t i o n , t h e m a j o r i t y o f t h e c y t i d y l y l t r a n s f e r a s e a c t i v i t y r e s i d e s i n t h e c y t o s o l i c f r a c t i o n when a s s a y e d u n d e r o p t i m u m c o n d i t i o n s ( T a b l e 7 ) . The c y t o s o l i c a c t i v i t y f a r e x c e e d s t h e homogenate a c t i v i t y , s u g g e s t i n g t h a t t h e homogenate i s i n h i b i t o r y ( o t h e r w i s e , t h e 10,000 x g p e l l e t , c y t o s o l i c , a n d m i c r o s o m a l a c t i v i t i e s s h o u l d add up t o t h e h o m o g e n a t e a c t i v i t y ) . The h o m o g e n a t e , 10,000 x g p e l l e t , and m i c r o s o m a l f r a c t i o n s w e r e a l l f a i r l y v i s c o u s and t h i s m i g h t h a v e h i n d e r e d d i s p e r s i o n o f t h e membranes d u r i n g t h e a s s a y . P e r h a p s t h e h o m o g e n i z a t i o n was too h a r s h and d i s r u p t e d t h e c y t i d y l y l t r a n s f e r a s e i n t e r a c t i o n w i t h t h e membrane o r a f f e c t e d t h e membrane s t r u c t u r e i n a i n h i b i t o r y way. A way t o t e s t w h e t h e r t h e Nb 2 membrane p r e p a r a t i o n s a r e i n h i b i t o r y t o c y t i d y l y l t r a n s f e r a s e m i g h t be t o s e e i f Nb 2 c y t o s o l i c c y t i d y 1 y 1 t r a n s f e r a s e w i l l t r a n s l o c a t e t o r a t l i v e r m i c r o s o m e s . A p r e l i m i n a r y s t u d y u s i n g f a t t y a c i d t o s t i m u l a t e t r a n s l o c a t i o n i n Nb 2 c e l l s r e s u l t e d i n d e c r e a s e d c y t o s o l i c a c t i v i t y w i t h o u t i n c r e a s e d 57 T a b l e . 7. R e c o v e r y o f c y t i d y l y l t r a n s f e r a s e a f t e r s u b c e l l u l a r f r a c t i o n a t i o n o f Nb 2 c e l l s S p e c i f i c T o t a l T o t a l F r a c t i o n A c t i v i t y P r o t e i n A c t i v i t y (nmol/min/mg) (mg) (nmol/min) Homogenate C 0. 069 23.6 1.63 P 0.059 24.2 1.43 10,000 x £ C 0. 057 3.9 0. 22 p e l l e t P 0. 060 3.9 0.23 C y t o s o l C 1. 46 9.2 13.4 P 1.58 9.0 14. 2 Microsome C 0. 098 4.4 0.43 P 0.087 4.3 0.37 F r a c t i o n s f r o m c o n t r o l (C) and p r o l a c t i n - t r e a t e d (P) c e l l s . 58 m i c r o s o m a l a c t i v i t y , and t h i s may be e x p l a i n e d by t h e i n h i b i t i o n h y p o t h e s i s . As an a d d e d n o t e , t h e m i c r o s o m a l a c t i v i t i e s r e p o r t e d i n T a b l e 5 a r e p r o b a b l y m i n i m a l v a l u e s , s i n c e t h e a c t i v i t y was t o o low t o o b t a i n r e a c t i o n c u r v e s f o r l i n e a r i t y w i t h t i m e o r p r o t e i n . (c) C h o i i n e p h o s p h o t r a n s f e r a s e C h o i i n e p h o s p h o t r a n s f e r a s e a c t i v i t y i n Nb 2 c e l l s was n o t a f f e c t e d b y p r o l a c t i n - t r e a t m e n t f o r 4 h ( T a b l e 5 ) . C h o l i n e p h o s p h o t r a n s f e r a s e i s n o t r a t e - l i m i t i n g f o r PC b i o s y n t h e s i s (23a) and i n m o s t s y s t e m s , t h e r e i s a l a c k o f c o r r e l a t i o n b etween a l t e r a t i o n s i n PC b i o s y n t h e s i s and in. v i t r o c h o l i n e -p h o s p h o t r a n s f e r a s e a c t i v i t y (65). 59 DISCUSSION 1. I n t e r a c t i o n o f P r o l a c t i n w i t h Nb 2 C e l l s P r o l a c t i n i s a v e r s a t i l e hormone w h i c h a f f e c t s a v a r i e t y o f p h y s i o l o g i c a l p r o c e s s e s i n a number o f t a r g e t t i s s u e s ( 4 8 ) . F o r Nb 2 c e l l s , p r o l a c t i n and o t h e r l a c t o g e n i c h o r -mones a r e p o t e n t m i t o g e n s . The r e a s o n f o r t h i s h o r m o n e d e p e n d a n c e i s unknown and i t may be due t o m a l i g n a n t t r a n s -f o r m a t i o n o r t h e c e l l s may h a v e a r i s e n f r o m a t h y m o c y t e ' f r o z e n ' a t some i n t e r m e d i a t e s t a g e o f d i f f e r e n t i a t i o n (66). The p r o l a c t i n r e c e p t o r s o f Nb 2 c e l l s h a v e 20-30 t i m e s g r e a t e r a f f i n i t y f o r t h e hormone t h a n t h o s e o f n o r m a l t a r g e t t i s s u e s ( 6 7 ) , so t h e y may be u n i q u e . The i n t e r a c t i o n o f p r o l a c t i n w i t h Nb 2 c e l l s i n v o l v e s b i n d i n g t o r e c e p t o r s , i n t e r n a l i z a t i o n , a n d d e g r a d a t i o n o f t h e h o r m o n e ( 6 7 ) , f o l l o w e d by m e t a b o l i c a l t e r a t i o n s c u l m i n a t i n g w i t h c e l l d i v i -s i o n . T h i s s e q u e n c e o f e v e n t s i s s i m i l a r t o t h e a c t i o n o f a n o t h e r w e l l c h a r a c t e r i z e d m i t o g e n , e p i d e r m a l g r o w t h f a c t o r (EGF) ( 6 8 ) . S t u d y o f t h e s i g n a l l i n k i n g r e c e p t o r b i n d i n g t o m e t a -b o l i c c h a n g e s may p r o v i d e i n s i g h t on t h e r e g u l a t i o n o f m e t a b o l i c p a t h w a y s and c e l l p r o l i f e r a t i o n . F o r t h e EGF r e c e p t o r , t h e s i g n a l a p p e a r s t o be p h o s p h o r y l a t i o n o f e n d o -genous p r o t e i n s (68). P h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n r e a c t i o n s a r e known t o p a r t i c i p a t e i n t h e r e g u l a t i o n o f many 60 m e t a b o l i c p a t h w a y s and s t u d i e s on Nb 2 c e l l s i n d i c a t e t h a t p r o t e i n p h o s p h o r y l a t i o n i s a l s o a n e a r l y e v e n t a f t e r p r o l a c t i n - t r e a t m e n t ( 6 7 ) . O r n i t h i n e d e c a r b o x y l a s e a c t i v i t y i s s t i m u l a t e d m a r k e d l y i n Nb 2 c e l l s 5 h a f t e r p r o l a c t i n -t r e a t m e n t (48). T h i s enzyme has t h e s h o r t e s t h a l f l i f e (8-30 min) y e t r e p o r t e d i n e u k a r y o t e s (69) and p r o p o s e d r e g u l a t o r y m e c h a n i s m s i n c l u d e a n t i z y m e p r o t e i n , p h o s p h o r y l a t i o n , and t r a n s a m i d a t i o n (70). Thus, f o l l o w i n g p r o l a c t i n - t r e a t m e n t o f Nb 2 c e l l s , many mechanisms may come i n t o p l a y i n p r o d u c i n g a l t e r a t i o n s i n m e t a b o l i c pathways. We were i n t e r e s t e d i n the e f f e c t o f m i t o g e n i c s t i m u l a t i o n on t h e a c t i v i t y o f t h e CDP-c h o l i n e p a t h w a y s i n c e g r o w i n g Nb 2 c e l l s s h o u l d h a v e an i n c r e a s e d demand f o r PC f o r membrane s y n t h e s i s . We hoped t o d e m o n s t r a t e s t i m u l a t i o n o f t h i s p a t h w a y and t h e n s t u d y t h e iuechanism (s) l e a d i n g t o t h e s e changes. 2. E f f e c t o f P r o l a c t i n on P h o s p h a t i d y l c h o l i n e B i o s y n t h e s i s The C D P - c h o l i n e pathway, assumed t o be t h e m a j o r r o u t e f o r PC b i o s y n t h e s i s i n Nb 2 c e l l s , was i n v e s t i g a t e d by r a d i o -l a b e l i n g e x p e r i m e n t s , p o o l s i z e m e a s u r e m e n t s and e n z y m e a s s a y s i n o r d e r t o d e t e r m i n e i f i t ' s a c t i v i t y was a l t e r e d a f t e r p r o l a c t i n t r e a t m e n t . P r o l a c t i n t r e a t m e n t l e a d t o i n c r e a s e d u p t a k e and p h o s p h o r y l a t i o n o f [Me_- H] c h o l i n e . I t was c o n c l u d e d t h a t t h i s may h a v e b e e n due t o i n c r e a s e d c h o l i n e t r a n s p o r t o r c h o l i n e k i n a s e . A t t e m p t s t o measure t h e p a r a m e t e r s o f c h o l i n e t r a n s p o r t i n Nb 2 c e l l s h a v e b e e n u n s u c e s s f u l t h u s f a r due m a i n l y t o 61 t h e l a c k o f a w o r k i n g , r a p i d t r a n s p o r t a s s a y . C h o l i n e t r a n s -p o r t has been shown t o be i n d u c i b l e i n a p o l y a m i n e a u x o t r o p h o f S a c c h a r o m y c e s c e r e v i s i a e where i t i n v o l v e s a c t i v e t r a n s -p o r t m e d i a t e d by a s p e c i f i c c a r r i e r ( 7 1 ) . I n c o n t r a s t , i n r a t h e p a t o c y t e s (72) and N o v i k o f f h e p a t o m a ( 7 3 ) , s a t u r a b l e a n d n o n - s a t u r a b l e c o m p o n e n t s f o r c h o l i n e t r a n s p o r t e x i s t . C h o l i n e u p t a k e a t l o w c o n c e n t r a t i o n s ( i . e . b e l o w 20 uM) f o l l o w e d M i c h a e l i s - M e n t o n k i n e t i c s , s u g g e s t i n g f a c i l i t a t e d t r a n s p o r t , b u t a t h i g h c o n c e n t r a t i o n s , u p t a k e r e s e m b l e d p a s s i v e d i f f u s i o n . The n o r m a l c o n c e n t r a t i o n o f c h o l i n e i n r a t p l a s m a i s 10-20 uM (74) and a t t h e s e c o n c e n t r a t i o n s , t h e f a c i l i t a t e d mechanisms would be n e a r s a t u r a t i o n so a q u e s t i o n a r i s e s w h e t h e r c h o l i n e t r a n s p o r t can l i m i t PC b i o s y n t h e s i s . P l a g e m a n n and R o t h h a v e s u g g e s t e d t h a t c h o l i n e t r a n s p o r t l i m i t s i n c o r p o r a t i o n i n t o PC s i n c e t h e a p p a r e n t Km's and Vmax's f o r c h o l i n e p h o s p h o r y l a t i o n were a t l e a s t one o r d e r o f m a g n i t u d e h i g h e r t h a n t h o s e f o r e n t r y o f c h o l i n e i n t o N o v i k o f f c e l l ( 7 5 ) . H o w e v e r , t h e r e i s t h e p o s s i b i l i t y t h a t c h o l i n e k i n a s e c o u l d d r i v e u p t a k e by p a s s i v e d i f f u s i o n . The q u e s t i o n o f w h e t h e r p r o l a c t i n - t r e a t m e n t i n f l u e n c e s c h o l i n e t r a n s p o r t i n Nb 2 c e l l s r e m a i n s open. (a) C h o l i n e k i n a s e C h o l i n e k i n a s e a c t i v i t y was s t i m u l a t e d i n Nb 2 c e l l s to a s i m i l a r d e g r e e as t h e i n c r e a s e i n p h o s p h o r y l a t i o n o f [Me-3 H ] c h o l i n e d u r i n g t h e p u l s e - l a b e l i n g e x p e r i m e n t . Thus, the i n c r e a s e d p h o s p h o r y l a t i o n was e x p l a i n e d and p o s s i b l y , c h o l i n e k i n a s e d r i v e s c h o l i n e u p t a k e i n Nb 2 c e l l s . The m e c h a n i s m 62 f o r i n d u c t i o n r e m a i n s u n c l e a r and t h e p o s s i b i l i t i e s i n c l u d e enzyme s y n t h e s i s o r a c t i v a t i o n by p o l y a m i n e s . The p h y s i o l o g i c a l r o l e o f c h o l i n e k i n a s e i n d u c t i o n i s u n c l e a r . I n mouse mammary g l a n d f o l l o w i n g g l u c o c o r t i c o i d t r e a t m e n t , i t was s u g g e s t e d t h a t s t i m u l a t i o n o f c h o l i n e k i n a s e m i g h t be i m p o r t a n t i n r o u g h e n d o p l a s m i c r e t i c u l u m a c c u m u l a t i o n f o r t h e f o r m a t i o n o f s e c r e t o r y p r o t e i n s (21) but t h e r e l a t i o n b etween c h o l i n e k i n a s e i n d u c t i o n and t h e r a t e o f PC b i o s y n t h e s i s was not examined. The i n d u c t i o n o f h e p a t i c c h o l i n e k i n a s e i n r a t s a d m i n i s t e r e d p o l y c y c l i c a r o m a t i c h y d r o c a r b o n c a r c i n o g e n s o r C C l ^ i s p r o p o s e d t o be i n v o l v e d i n a p r o t e c t i o n m e c h a n i s m a g a i n s t t h e s e h i g h l y c y t o t o x i c i n v a d e r s and a p p e a r s t o be a s p e c i f i c f u n c t i o n o f t h e l i v e r s i n c e e x t r a h e p a t i c t i s s u e s were n o t a f f e c t e d by t h e c a r c i n o -gens (15). P o l y c y c l i c a r o m a t i c compounds had c o u n t e r a c t i v e e f f e c t s on PC b i o s y n t h e s i s i n r a t l i v e r , s t i m u l a t i n g c h o l i n e k i n a s e b u t i n h i b i t i n g c h o l i n e p h o s p h o t r a n s f e r a s e a c t i v i t y (76). In r o o s t e r l i v e r , t h e c o n c e n t r a t i o n o f p h o s p h o c h o l i n e i s n o r m a l l y n e a r t h e Km o f c y t i d y l y l t r a n s f e r a s e (0.17 mM) and s t i m u l a t i o n o f c h o l i n e k i n a s e l e a d s t o an i n c r e a s e i n p h o s p h o c h o l i n e c o n c e n t r a t i o n and c o n c o m i n a n t 1 y i n PC b i o -s y n t h e s i s ( 2 7 ) . P h o s p h o c h o l i n e i s an i n h i b i t o r o f o r n i t h i n e d e c a r b o x y l a s e and g l u t a m i c a c i d d e c a r b o x y l a s e (77) and s t i m u -l a t i o n o f c h o l i n e k i n a s e w i t h a t t e n d a n t i n c r e a s e s i n i n t r a -c e l l u l a r p h o s p h o c h o l i n e l e v e l s may s e r v e t o r e g u l a t e t h e s e c a r b o x y l a s e s in v i v o . C e r t a i n i n v e s t i g a t o r s a r g u e t h a t c h o l i n e k i n a s e i s r e g u l a t o r y f o r PC b i o s y n t h e s i s (23,24). I n f a n t e and K i n s e l l a 63 a r g u e t h a t c h o l i n e k i n a s e i s most r a t e - l i m i t i n g f o r PC b i o -s y n t h e s i s and i n e s s e n t i a l - f a t t y a c i d - d e f i c i e n t r a t s , t h e y f o u n d an i n c r e a s e i n t h e in v i t r o a c t i v i t y o f c h o l i n e k i n a s e c o u p l e d t o i n c r e a s e d f l u x t h r o u g h t h e pathway (23). However, t h e y a l s o s a i d t h a t t h e f l u x i n c r e a s e may have been t r a n s m i t -t e d by i n c r e a s e d p h o s p h o c h o l i n e c o n c e n t r a t i o n s a c t i n g on c y t i d y l y l t r a n s f e r a s e , w h i c h i s a l s o r a t e - l i m i t i n g ( 2 3 ) . N i s h i j i m a and c o - w o r k e r s i s o l a t e d a m u t a n t CHO c e l l d e f e c t i v e i n c h o l i n e k i n a s e w h i c h was a l s o d e f e c t i v e i n PC b i o s y n t h e s i s ( 2 4 ) . T h u s , i n t h e s e c a s e s a n d i n r o o s t e r l i v e r u p o n d i e t h y l s t i l b e s t r o l t r e a t m e n t , c h o l i n e k i n a s e i s a p p a r e n t l y a b l e t o e x e r t a r e g u l a t o r y r o l e on PC b i o s y n t h e s i s . However, c h o l i n e k i n a s e a c t i v i t y has been shown t o f l u c t u a t e w i t h c e l l c y c l e i n N o v i k o f f r a t h e p a t o m a a l t h o u g h t h e r a t e o f PC b i o -s y n t h e s i s a p p e a r e d t o r e m a i n c o n s t a n t (78). (b) C y t i d y l y l t r a n s f e r a s e The c y t i d y l y l t r a n s f e r a s e r e a c t i o n was i d e n t i f i e d a s b e i n g r a t e - 1 i m i t i n g f o r PC b i o s y n t h e s i s i n Nb 2 c e l l s by p u l s e - c h a s e s t u d i e s . I n Nb 2 c e l l s , an i n c r e a s e i n c h o l i n e k i n a s e a c t i v i t y was n o t a c c o m p a n i e d by i n c r e a s e d f l u x t h r o u g h t h e p a t h w a y f o r a t l e a s t 4 and p o s s i b l y 7 h. T h u s , c h o l i n e k i n a s e d i d n o t a p p e a r t o e x e r t a r e g u l a t o r y e f f e c t on PC b i o s y n t h e s i s i n Nb 2 c e l l s . H o w e v e r , p h o s p h o c h o l i n e p o o l s i z e i n c r e a s e s l a g g e d c o n s i d e r a b l y b e h i n d t h e i n d u c t i o n o f c h o l i n e k i n a s e a f t e r p r o l a c t i n - t r e a t m e n t , t h u s i f c y t i d y l y l t r a n s f e r a s e a c t i v i t y i n Nb 2 c e l l s was r e g u l a t e d by c h a n g e s i n t h e c o n c e n t r a t i o n o f t h i s s u b s t r a t e , i t would n o t 64 a p p e a r u n t i l a f t e r a t l e a s t 6 h ( F i g . 8). The p h o s p h o c h o l i n e c o n c e n t r a t i o n i n s t a t i o n a r y Nb 2 c e l l s i s 0.62 uM and i t i s n o t known w h e t h e r t h i s l i m i t s t h e c y t i d l y l t r a n s f e r a s e r e a c t i o n i n t h e s e c e l l s . No d i f f e r e n c e i n t h e _in v i t r o a c t i v i t y o f c y t i d y l y l -t r a n s f e r a s e was d e t e c t e d a f t e r 4 h p r o l a c t i n - t r e a t m e n t . The e n z y m e was a s s a y e d u n d e r o p t i m u m ( s a t u r a t i n g ) c o n d i t i o n s . S u c h an a s s a y d o e s n o t r e f l e c t i n v i v o s u b s t r a t e c o n c e n t r a -t i o n s b u t i t may d e t e c t c h a n g e s i n e n z y m e a c t i v i t y due t o c o v a l e n t m o d i f i c a t i o n o r a l l o s t e r i c i n t e r a c t i o n s , as w e l l as c hanges i n s u b c e l l u l a r l o c a l i z a t i o n . However, s e v e r a l l i n e s o f e v i d e n c e ( p u l s e - l a b e l i n g , p u l s e - c h a s e s t u d i e s , p h o s p h o -c h o l i n e s p e c i f i c r a d i o a c t i v i t y ) s u g g e s t t h a t t h e in_ v i v o f l u x t h r o u g h t h e r a t e - 1 i m i t i n g s t e p , c a t a l y z e d b y c y t i d y l y l -t r a n s f e r a s e , was n o t a f f e c t e d g r e a t l y by p r o l a c t i n - t r e a t m e n t . In a d d i t i o n , i n c r e a s e s i n c e l l u l a r PC l e v e l s a f t e r p r o l a c t i n -t r e a t m e n t w e r e a c c o u n t e d f o r by c h a n g e s i n t h e r a t e o f PC t u r n o v e r a n d when t h e s e w e r e c a l c u l a t e d a l o n g w i t h c o r r e s -p o n d i n g s y n t h e t i c r a t e s , t h e s y n t h e t i c r a t e s i n c o n t r o l and p r o l a c t i n - t r e a t e d c e l l s were f o u n d n o t t o d i f f e r g r e a t l y . 3. E f f e c t o f P r o l a c t i n on P h o s p h a t i d y l c h o l i n e C a t a b o l i s m Dynamic t u r n o v e r o f p h o s p h o l i p i d s was r e c o g n i z e d a f t e r t h e e x p e r i m e n t s o f H e v e s y and o t h e r s ( 1 1 - 1 3 ) . H o w e v e r , t h e r o l e o f p h o s p h o l i p i d t u r n o v e r r e m a i n s o b s c u r e . The m o s t l i k e l y r e a s o n w o u l d be t o m a i n t a i n membrane i n t e g r i t y and f u n c t i o n b u t s u c h an c o n c l u s i o n r e m a i n s vague u n t i l i t c a n be 65 e x p l a i n e d i n m o l e c u l a r t e r m s . C o n t i n u a l s y n t h e s i s o f p h o s -p h o l i p i d i s n o t r e q u i r e d f o r m a i n t e n a n c e o f membrane i n t e -g r i t y a s e r y t h r o c y t e s c a n r e m a i n a c t i v e f o r many m o n t h s (6) (however, e r y t h r o c y t e s can exchange p h o s p h o l i p i d s w i t h p l a s m a l i p o p r o t e i n s and r e a c y l a t e l y s o p h o s p h o l i p i d s ) . M o r e o v e r , t u r n o v e r o f p h o s p h o l i p i d s i n m y e l i n s h e a t h i s v e r y s l o w (6), so t u r n o v e r i s a l s o not e s s e n t i a l f o r membrane f u n c t i o n . D i f f e r e n t c l a s s e s o f p h o s p h o l i p i d t u r n o v e r i n d e p e n d a n t l y and a t d i f f e r e n t r a t e s (33). A l s o , t h e t u r n o v e r r a t e v a r i e s d e p e n d i n g on t h e r a d i o a c t i v e p r e c u r s o r u s e d t o l a b e l t h e p h o s p h o l i p i d , s u g g e s t i n g t h a t c e r t a i n p o r t i o n s o f t h e m o l e c u l e t u r n o v e r f a s t e r t h a n o t h e r s i . e . a c y l c h a i n s a r e more e x t e n s i v e l y r e u t i l i z e d t h a n t h e g l y c e r o l - p h o s p h a t e - b a s e b a c k b o n e ( 3 3 ) . The r e l a t i o n s h i p b e t w e e n p h o s p h o l i p i d t u r n o v e r and c e l l g r o w t h i s a l s o u n c l e a r . W a r r e n and G l i c n . s u g g e s t t h a t membrane t u r n o v e r i s an i n d i c a t i o n o f c e l l u l a r i n a c t i v i t y ( 7 9 ) . H o w e v e r , i n P815Y n e o p l a s t i c m a s t c e l l s , p h o s p h o l i p i d t u r n o v e r i s i n d e p e n d a n t o f c e l l g r o w t h ( 8 0 ) , w h e r e a s i n c u l t u r e d l y m p h o c y t e s , s t i m u l a t i o n o f g r o w t h i s p r e c e d e d by an i n c r e a s e i n p h o s p h o l i p i d t u r n o v e r (81). T h e r e i s a l s o e v i d e n c e t h a t p h o s p h o l i p i d s y n t h e s i s and c a t a b o l i s m a r e c o u p l e d . W i t h mouse f i b r o b l a s t s , m e t a b o l i c i n h i b i t o r s w h i c h s u p p r e s s e d p h o s p h o l i p i d s y n t h e s i s a l s o l e a d t o an e q u i v a l e n t d e c l i n e i n t h e d e g r a d a t i o n r a t e ( 7 9 ) . S l e i g h t and K e n t h a v e shown t h a t d e g r a d a t i o n o f PC i n CHO c e l l s by e x o g e n o u s l y added p h o s p h o l i p a s e C l e a d s t o s t i m u l a -t i o n o f PC b i o s y n t h e s i s ( 8 2 ) . R e c e n t l y , a CHO m u t a n t d e f e c t i v e i n PC b i o s y n t h e s i s a l s o h a d a r e d u c t i o n i n t u r n -66 o v e r , l e a d i n g t o a n o r m a l PC c o n t e n t ( 2 4 ) . I n Nb 2 c e l l s , PC l a b e l e d a t t h e c h o l i n e h e a d g r o u p t u r n e d o v e r a t d i f f e r e n t r a t e s i n c o n t r o l a n d p r o l a c t i n -t r e a t e d c e l l s . The t u r n o v e r o f PC was a c c o m p a n i e d by a p p e a r -a n c e o f c o r r e s p o n d i n g amounts o f r a d i o a c t i v e c h o l i n e i n t h e medium, and t u r n o v e r was r e d u c e d i n p r o l a c t i n - t r e a t e d c e l l s , w h i c h c o u l d a c c o u n t f o r a n e t i n c r e a s e i n PC l e v e l s i n t h e s e c e l l s e v e n i n t h e a b s e n c e o f a n y c h a n g e i n t h e b i o s y n t h e t i c r a t e . T h i s i m p l i e s a r e g u l a t o r y r o l e f o r t u r n o v e r i n c o n t r o l -l i n g PC l e v e l s i n Nb 2 c e l l s u n d e r d i f f e r e n t g r o w t h c o n d i -t i o n s . PC c a t a b o l i s m c a n f o l l o w many d i f f e r e n t r o u t e s (see I n t r o d u c t i o n ) a n d t h u s t h e r e a r e many p o s s i b l e s i t e s f o r r e g u l a t i o n t o o c c u r . (a) P h o s p h o l i p a s e D o r C I f t h e c h o l i n e r e l e a s e d i n t o t h e medium was an i m m e d i a t e b r e a k d o w n p r o d u c t , p h o s p h o l i p a s e D w o u l d be i m p l i c a t e d as t h e c a t a b o l i c a c t i v i t y r e g u l a t e d by p r o l a c t i n - t r e a t m e n t . I f s u c h a m e c h a n i s m was i n v o l v e d , t h e most p r o b a b l e l o c a t i o n w o u l d be o n t h e e x t e r n a l s i d e o f t h e c e l l m e m b r a n e s i n c e i f t h e a c t i v i t y was i n t r a c e l l u l a r , t h e f r e e c h o l i n e s h o u l d be r a p i d -l y p h o s p h o r y l a t e d by c h o l i n e k i n a s e . P h o s p h o l i p a s e D f o u n d i n r a t b r a i n m i c r o s o m e s i s a c t i v a t e d by f r e e f a t t y a c i d s and d e t e r g e n t s ( 8 3 ) , s u g g e s t i n g p o s s i b l e r e g u l a t o r y m e c h a n i s m s . T h e m e d i u m c h o l i n e c o u l d a l s o h a v e r e s u l t e d f r o m c o m b i n e d p h o s p h o l i p a s e C and p h o s p h o c h o l i n e ( o r a l k a l i n e ) p h o s p h a t a s e a c t i v i t i e s . Guy and M u r r a y h a v e p r o p o s e d s u c h a m e c h a n i s m f o r t h e s t i m u l a t i o n o f PC t u r n o v e r i n HeLa c e l l s 67 f o l l o w i n g t u m o r p r o m o t e r s t i m u l a t i o n (84) and t h i s i s s u p p o r t e d by t h e e x i s t e n c e o f a l k a l i n e p h o s p h a t a s e a c t i v i t y on HeLa c e l l m e m b r a n e s ( 8 5 ) . The r e c e n t i s o l a t i o n o f a mammalian p h o s p h o l i p a s e C a c t i v e a g a i n s t PC and an endogenous i n h i b i t o r (43) a r e f u r t h e r s u g g e s t i o n o f a p o s s i b l e r o l e f o r t h i s enzyme i n PC c a t a b o l i s m . However, t h e q u a n t i t a t i v e s i g n i f i c a n c e o f p h o s p h o l i p a s e C or D a c t i v i t y i n PC t u r n o v e r has y e t t o be d e t e r m i n e d . The m a m m a l i a n PI s p e c i f i c p h o s p h o l i p a s e C a p p e a r s t o a c t t r a n s i e n t l y f o l l o w i n g c e l l s t i m u l a t i o n i n t h e p r o d u c t i o n o f s e c o n d m e s s e n g e r s d i a c y l g l y c e r o l and i n o s i t o l t r i p h o s p h a t e and t h e mechanism o f r e g u l a t i o n i s u n c l e a r . I t i s not known w h e t h e r a s i m i l a r f u n c t i o n c a n be a s c r i b e d t o t h e p h o s p h o -l i p a s e C a c t i v e a g a i n s t PC o r w h e t h e r i t i s i n v o l v e d i n a c o n t i n u a l c a t a b o l i c p r o c e s s . A l s o , p r o l a c t i n - t r e a t m e n t l e a d t o a l m o s t c o m p l e t e c e s s a t i o n o f t u r n o v e r o f PC l a b e l e d i n t h e h e a d g r o u p ( F i g . 1 0 ) . I f p h o s p h o l i p a s e C o r D w e r e t h e m a j o r c a t a b o l i c a c t i v i t i e s i n v o l v e d , i t w o u l d s u g g e s t t h a t PC t u r n o v e r was an a l l "on" o r a l l " o f f " ' p r o c e s s i n Nb 2 c e l l s d e p e n d i n g on t h e h o r m o n a l c o n d i t i o n , u n l e s s t h e r e was d i f f e r -e n t i a l r e u t i 1 i z a t i o n o f t h e d e g r a d a t i o n p r o d u c t s ( p h o s p h o -c h o l i n e o r c h o l i n e ) under the two c o n d i t i o n s . Such a p r o c e s s i s u n l i k e l y b e c a u s e t h e c o n c e p t o f c o n t i n u a l t u r n o v e r o f membrane p h o s p h o l i p i d s would n o t a p p l y t o p r o l a c t i n - t r e a t e d Nb 2 c e l l s s i n c e t h e y would not a p pear t o c a t a b o l i z e PC. (B) D e a c y l a t i o n p a t hways I t i s g e n e r a l l y t h o u g h t t h e m a j o r i t y o f t h e c a t a b o l i c 68 aspect of phospholipid turnover is catalyzed by the ubiquit-ous phospholipases A 1 and A 2. The a v a i l a b l e data i n d i c a t e that the a c t i v i t y of membrane-associated phospholipases A and lysophospholipases i s s u f f i c i e n t to account for the h a l f -l i v e s of the major membrane phospholipids (33). Acting on PC to liberate lyso-PC, phospholipase A plays r o l e s i n the r e l e a s e of p r o s t a g l a n d i n p r e c u r s o r s and synthesis of molecular species of PC, as well as PC turnover. The lyso-PC r e s u l t i n g from phospholipase A a c t i v i t y can be transacy 1ated or re a c y l a t e d or degraded to glycerophospho-choline and subsequently to choline and glycerol -3-phosphate. These d i f f e r e n t f a t e s provide i n t e r e s t i n g p o s s i b i l i t i e s regarding turnover in Nb 2 c e l l s after prolactin-treatment. Perhaps regulation i s a matter of changing the t o t a l a c t i v i t y of the c a t a b o l i c pathway. Another p o s s i b i l i t y i s that in p r o l a c t i n - t r e a t e d c e l l s , lyso-PC generated a f t e r phospho-l i p a s e A a c t i v i t y i s shunted towards the r e a c y l a t i o n (or t r a n s a c y l a t i o n ) pathway, r e s u l t i n g i n r e t e n t i o n of the ch o l i n e l a b e l e d PC by the c e l l . In c o n t r o l c e l l s , lyso-PC may be p r e f e r e n t i a l l y degraded by lysophospholipase and other enzymes, r e s u l t i n g in the l i b e r a t i o n of ch o l i n e into the medium. This might involve a c t i v a t i o n of a membrane bound phospholipase A and/or lysophopholipase. The r e s u l t i n g glycerophosphocholine would would then need to be degraded, presumably by a membrane bound g 1ycerophosphocho 1ine phosphodiesterase. Another p o s s i b i l i t y i s that there i s "background" PC turnover via deacylation mechanisms going on continually with 69 r e t e n t i o n o f t h e c h o l i n e h e a d g r o u p i n b o t h c o n t r o l and p r o l a c t i n - t r e a t e d c e l l s and t h e d i f f e r e n c e between t h e two i s i n t h e a c t i v i t y o f t h e p h o s p h o l i p a s e C o r D m e c h a n i s m s . I n l i g h t o f t h e u n c l e a r r e l a t i o n s h i p between p h o s p h o l i p i d t u r n -o v e r and c e l l g r o w t h , a n o t h e r q u e s t i o n i s w h e t h e r PC c a t a -b o l i s m i s i n h i b i t e d i n p r o l a c t i n - t r e a t e d c e l l s o r a c t i v a t e d i n p r o l a c t i n - d e f i c i e n t c e l l s . 4 . C o n c l u d i n g Remarks Our i n v e s t i g a t i o n o f PC m e t a b o l i s m i n Nb 2 c e l l s f o l l o w -i n g m i t o g e n i c s t i m u l a t i o n l e a d t o some i n t e r e s t i n g f i n d i n g s b u t a l s o t o some new q u e s t i o n s . A l t h o u g h PC l e v e l s i n c r e a s e d i n Nb 2 c e l l s a f t e r p r o l a c t i n - t r e a t m e n t , t h i s d i d n o t a p p e a r t o be due t o s t i m u l a t i o n o f t h e b i o s y n t h e t i c p a t h w a y . An i n d u c t i o n o f c h o l i n e k i n a s e was n o t e d b u t t h e i n d u c t i o n mechanism and p h y s i o l o g i c a l s i g n i f i c a n c e a r e u n c l e a r . The mechanism f o r t h e i n c r e a s e i n PC l e v e l s a p p e a r e d t o be r e d u c -t i o n i n t h e r a t e o f t u r n o v e r . T h i s may s h e d new m e a n i n g t o t h e dogma t h a t t h e r a t e o f PC r a r e l y u n d e r g o e s d r a m a t i c changes (22). The p h o s p h o l i p a s e s and a s s o c i a t e d d e g r a d a t i v e enzymes seem s u i t e d as mechanisms f o r r e g u l a t i o n o f c e l l u l a r PC l e v e l s . T h e i r w i d e s u b c e l l u l a r d i s t r i b u t i o n o f f e r s t h e p o t e n t i a l f o r l o c a l i z a t i o n o f t u r n o v e r t o s p e c i f i c o r g a n e l l e s o r m e m branes and t h e r e g u l a t i o n o f t h e p h o s p h o l i p a s e s A 2 a p p e a r s v e r y c o m p l e x , w h i l e more a t t e n t i o n n e e d s t o be f o c u s s e d on t h e r e g u l a t i o n o f t h e o t h e r p h o s p h o l i p a s e s . T h e c o n t r i b u t i o n s o f t h e v a r i o u s p a t h w a y s f o r PC 70 catabolism in growing and non-growing Nb 2 c e l l s needs to be determined and then d e t a i l s such as the subcellular l o c a l i z a -t i o n of the enzymes and the mechanisms of r e g u l a t i o n can be approached. 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