UBC Theses and Dissertations

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UBC Theses and Dissertations

Cyclic AMP and ionic efflux in Dictyostelium discoideum Chi, Yea Yuan 1969

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C Y C L I C A M P A N D I O N I C E F F L U X I N D I C T Y O S T E L I U M D I S C O I D E U M by Y E A Y U A N C H I B.Sc., T a i w a n N o r m o n a l U n i v e r s i t y A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E M A S T E R O F S C I E N C E i n t h e D e p a r t m e n t o f Z O O L O G Y W e a c c e p t , t h i s t h e s i s a s 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 T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A A U G U S T , 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a 1 fu 1 f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s thes. is f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada Department A B S T R A C T T h e e f f e c t o f c y c l i c a d e n o s i n e 3',5' m o n o p h o s p h a t e ( c y c l i c A M P ) - o n i o n i c m o v e m e n t a c r o s s t h e c e l l - m e m b r a n e o f t h e c e l l u l a r s l i m e m o l d D i c t y o s t e l i u m d i s c o i d e u m , s t r a i n s V-12 a n d N C U , h a s b e e n s t u d i e d b y 22 k 5 u s i n g r a d i o i s o t o p e s , N a a n d C a . D . d i s c o i d e u m w a s g r o w n o n w a t e r a g a r a s s o c i a t e d w i t h a u t o c l a v e d r a d i o a c t i v e E . c o l i 2 8 l . T h e e x p e r i -m e n t a l a m o e b a e w e r e c e n t r i f u g e d t o f r e e t h e m f r o m b a c t e r i a . C y c l i c A M P w a s a d d e d t o t h e e x p e r i m e n t a l c u l t u r e e v e r y 10 o r 20 m i n u t e s . T h e r e s u l t s s h o w e d t h a t c y c l i c A M P h a s a s i g n i f i c a n t e f f e c t o n c a l c i u m i o n m o v e m e n t a c r o s s t h e m e m b r a n e b o t h i n t h e p r e - a g g r e g a t i v e a m o e b o i d s t a g e a n d d u r i n g t h e a g g r e g a t i n g p h a s e . T h e m o v e m e n t o f s o d i u m i o n w a s c h a n g e d o n l y d u r i n g a g g r e g a t i o n . T h e a d d i t i o n o f p h o s p h o d i e s t e r a s e t h e e n z y m e - w h i c h i n a c t i v e s c y c l i c A M P b y c o n v e r t i n g i t t o 5' A M P , s h o w e d n o e f f e c t o n i o n t r a n s p o r t e i t h e r i n t h e p r e - a g g r e g a t i v e s t a g e o r t h e a g g r e g a t i n g s t a g e . T h e i o n i c c o n t e n t s o f s o d i u m , p o t a s s i u m , a n d c a l c i u m , w e r e m e a s u r e d a t t h e s l u g s t a g e . T h e r e l a t i o n s h i p o f c a l c i u m i o n a n d c y c l i c A M P t o a m o e b o i d m o v e m e n t w a s c o m p a r e d w i t h t h e r o l e o f c y c l i c A M P i n m u s c u l a r c o n t r a c t i o n a n d o t h e r f u n c t i o n . - i i -TABLE OF CONTENTS PAGE INTRODUCTION 1 MATERIALS AND METHODS k I . E f f e c t o f c y c l i c a d e n o s i n e 3 ' , 5 ' monophosphate h I I . I o n c o n t e n t measurement 5 RESULTS I . The e f f e c t s o f c y c l i c AMP on i o n f l u x e s i n D. d i s c o i d e u m 8 A. E f f e c t s on c a l c i u m i o n s a t v a r i o u s s t a g e s o f t h e l i f e c y c l e • . 8 1. P r e - a g g r e g a t i v e st.age 8 2. A g g r a g a t i n g s t a g e 8 B. E f f e c t s on sodium i o n s a t v a r i o u s s t a g e s o f t h e l i f e c y c l e 9 1. P r e - a g g r e g a t i v e s t a g e 9 2. A g g r a g a t i n g s t a g e 9 I I . The e f f e c t s o f p h o s p h o d i e s t e r a s e on c a l s i u m i o n e f f l u x a t v a r i o u s s t a g e s i n t h e l i f e c y c l e 9 A. P r e - a g g r a g a t i v e s t a g e 9 B. A g g r e g a t i n g s t a g e 10 I I I . I o n c o n t e n t s i n amoeboid c e l l s a t t h e s l u g s t a g e 10 DISCUSSION 21". I . The r o l e o f c y c l i c AMP on i o n e f f l u x i n D. d i s c o i d e u m 21 I I . The r o l e o f p h o s p h o d i e s t e r a s e i n D. d i s c o i d e u m 23 I I I . F u n c t i o n s o f c y c l i c AMP i n o t h e r c e l l s 2k A. C o n t r a c t i l e mechanism-: 2k B. O t h e r f u n c t i o n s - 25 SUMMARY . 2 7 BIBLIOGRAPHY 28 — i i i — L I S T OF F I G U R E S F I G U R E 1. P e t r i d i s h w i t h g l a s s h e a d s w h i c h u s e d i n t h e e x p e r i m e n t s . 7 F I G U R E 2. T h e a m o e b a e o n t h e f i l t e r p a p e r s a t u r a t e d w i t h 3.5 c c B o n n e r ' s s o l u t i o n i n a d i s h w i t h g l a s s b e a d s . 7 F I G U R E 3. T h e e f f e c t o f c y c l i c A M P o n c a l c i u m i o n e f f l u x i n p r e -a g g r e g a t i v e s t a g e o f L \ _ d i s c o i d e u m . 1.1 F I G U R E h. T h e e f f e c t o f a s i n g l e a d d i t i o n o f c y c l i c A M P t o C a ^ t r e a t e d c u l t u r e s , a t p r e - a g g r e g a t i v e s t a g e . 12 F I G U R E 5. T h e e f f e c t o f c y c l i c A M P o n c a l c i u m i o n e f f l u x i n p r e -a g g r e g a t i v e s t a g e o f D_. d i s c o i d e u m ; t h e a d d i t i o n o f c y c l i c A M P a t 10 m i n u t e i n t e r v a l s . 13 F I G U R E 6 . T h e e f f e c t o f c y c l i c A M P o n c a l c i u m i o n e f f l u x d u r i n g a g g r e g a t i n g s t a g e o f d i s c o i d e u m ; t h e a d d i t i o n o f c y c l i c A M P a t 20 m i n u t e i n t e r v a l s . lh F I G U R E 7. T h e e f f e c t o f a s i n g l e a d d i t i o n o f c y c l i c A M P t o a C a t r e a t e d c u l t u r e d u r i n g a g g r e g a t i v e s t a g e o f p_. d i s c o i d e u m . 1 5 F I G U R E 8. T h e e f f e c t o f c y c l i c A M P o n s o d i u m i o n e f f l u x i n p r e -a g g r e g a t i v e s t a g e o f D_. d i s c o i d e u m ; t h e a d d i t i o n o f c y c l i c A M P a t 20 m i n u t e i n t e r v a l s . 16 F I G U R E 9. T h e e f f e c t o f c y c l i c A M P o n s o d i u m i o n e f f l u x d u r i n g a g g r e g a t i n g s t a g e o f p_. d i s c o i d e u m ; t h e a d d i t i o n o f c y c l i c A M P a t 20 m i n u t e i n t e r v a l s . 1? 5^ F I G U R E 10. T h e e f f e c t o f s i n g l e a d d i t i o n o f p h o s p h o d i e s t e r a s e t o C a t r e a t e d c u l t u r e s a t p r e - a g g r e g a t i v e s t a g e . l c k5 F I G U R E 11. T h e e f f e c t o f s i n g l e a d d i t i o n o f p h o s p h o d i e s t e r a s e t o C a t r e a t e d c u l t u r e s d u r i n g a g g r e g a t i n g s t a g e . I S - i v -LIST OF TABLE PAGE TKBLE 1. Ion contents i n amoebae at slug stage. 20 - V -' ACKNOWLEDGEMENT I w i s h t o e x p r e s s my h e a r t f e l t t h a n k s t o Dr. D. F r a n c i s f o r h i s i n s t r u c t i o n , d i s c u s s i o n , encouragement and s u p p o r t g i v e n t o me t h r o u g h o u t t h i s s t u d y . I w o u l d a l s o l i k e t o t h a n k Dr. J . P h i l l i p s and Dr. T. P e r k s f o r t h e i r c r i t i c a l r e a d i n g o f t h e m a n u s c r i p t . T h i s r e s e a r c h was s u p p o r t e d i n p a r t by f u n d s f r o m t h e N a t i o n a l R e s e a r c h C o u n c i l o f Canada. INTRODUCTION The most i m p o r t a n t event i n t h e morphogenesis o f c e l l u l a r s l i m e molds i s t h e a g g r e g a t i o n o f t h e amoebae i n t o a mass o f c e l l s w h i c h s u b s e q u e n t l y d i f f e r e n t i a t e s as a u n i t i n t o a m i g r a t i n g s l u g and f i n a l l y a f r u i t i n g body. I t has been known f o r many y e a r s t h a t t h e amoebae a r e a t t r a c t e d t o t h e c e n t e r by c h e m o t a x i s . The a g g r e g a t i o n o f c e l l s i s m e d i a t e d by a c h e m i c a l messenger w h i c h a t t r a c t s t h e c e l l s o v e r a d i s t a n c e . Bonner (19^7) c a l l e d t h i s a t t r a c t i n g s u b s t a n c e a c r a s i n . S h a f f e r (1953) was a b l e t o i s o l a t e t h e s u b s t a n c e in_ v i t r o . r e i n t r o d u c e i t , and o b t a i n t h e o r i e n t a t i o n o f s e n s i t i v e amoebae. I n r e c e n t y e a r s , much a t t e n t i o n has been g i v e n t o t h e p r o b l e m o f a g g r e g a t i o n and t h e c h e m i c a l i d e n t i t y o f a c r a s i n . K o n i j n et_ a l _ (1967) f o u n d t h a t c y c l i c a d e n o s i n e 3 ' , 5 ' monophosphate ( c y c l i c AMP) a t a c o n c e n t r a t i o n o f l :_x ' 1 0 ^ g / l has t h e a b i l i t y t o a t t r a c t s e n s i t i v e amoebae o f D i c t y o s t e l i u m d i s c o i d e u m . They were a b l e t o i d e n t i f y t h e a t t r a c t a n t f r o m e x t r a c t o f E. c o l i as c y c l i c AMP, .H.pd t o show t h a t P o l y s p h o n d y l i u m p a l l i d u m c a n s y n t h e s i z e c y c l i c AMP ( K o n i j n et_ a l , 1967, 1968) . A l t h o u g h D. d i s c o i d e u m s y n t h e s i z e s an a t t r a c t a n t , t h e r e were d i f f i c u l t i e s a t f i r s t i n o b t a i n i n g l a r g e enough q u e n t i t i e s o f t h i s s u b s t a n c e f o r i d e n t i f i c a t i o n , b ecause t h e a t t r a c t a n t o f t e n d i s a p p e a r e d more r a p i d l y t h a n i t was s y n t h e s i z e d ( K o n i j n et_ a l _ , 1968) . Chang (1968) d i s c o v e r e d l a t e r t h a t t h e amoebae o f D. d i s c o i d e u m s e c r e t e d q u a n t i t i e s o f c y c l i c AMP enzymes, p h o s p h o d i e s t e r a s e , i n t o t h e aqueous medium a r o u n d them. P h o s p h o d i e s t e r a s e , she f o u n d , showed many s i m i -l a r i t i e s t o t h e mammalian p h o s p h o d i e s t e r a s e o r i g i n a l l y d e s c r i b e d by B u t c h e r and S u t h e r l a n d ( 1962) . I t was s u g g e s t e d t h a t t h i s enzyme c o u l d be t h e a c r a s i n d e s t r o y i n g enzyme, a c r a s i n a s e , as o r i g i n a l l y assumed by S h a f f e r (1956). Q u i t e r e c e n t l y , Bonner (1969) and Bonner e t a l (1969) by s e p a r a t i n g t h e d e s t r u c t i v e enzyme, p h o s p h o d i e s t e r a s e , f r o m t h e a t t r a c t a n t were a b l e t o d e m o n s t r a t e t h a t p_. d i s c o i d e u m does s y n t h e s i z e c y c l i c AMP. They a l s o f o u n d t h a t s e c r e t i o n i n c r e a s e s a t l e a s t a h u n d r e d - f o l d d u r i n g t h e a g g r e g a t i o n phase. S i m u l t a n e o u s l y t h e r e i s a h u n d r e d - f o l d i n c r e a s e i n t h e amoebae's s e n s i t i v i t y t o c y c l i c AMP. The mechanism o f a g g r e g a t i o n i s s t i l l unknown. Though c y c l i c AMP i n c r e a s e s t h e a g g r e g a t i n g a c t i v i t y o f s l i m e m o l d s , no one knows t h e mode o f a c t i o n . A c c o r d i n g t o t h e work o f S u t h e r l a n d and h i s c o l l a b o r a t o r s (1966), c y c l i c AMP i s a second messenger i n mammalian hormone r e a c t i o n s . The hormone, o r f i r s t m essenger, s e c r e t e d by t h e e n d o c r i n e o r g a n , i n t e r a c t s w i t h a d e n y l c y c l a s e t o p r o d u c e a change i n t h e c o n c e n t r a t i o n o f c y c l i c AMP, t h e second messenger. T h i s t h e n p r o d u c e s c e l l u l a r changes w h i c h c o n s t i t u t e t h e c e l l ' s r e s p o n s e t o t h e hormone. By com p a r i n g c e l l u l a r s l i m e molds w i t h mammals, i t has been s u g g e s t e d t h a t c y c l i c AMP s e r v e s as a p r i m a r y messenger i n t h e s l i m e m o l d s , and c a n c a r r y messages a c r o s s l a r g e i n t e r c e l l u l a r s p a ces ( K o n i j n e t a l , 1968). There i s some i n f o r m a t i o n c o n c e r n i n g t h e f u n c t i o n o f c y c l i c AMP i n o t h e r o r g a n i s m s : i t has been shown t h a t t h e a d d i t i o n o f c y c l i c AMP' t o i . t h e b a s a l s'ucface o f t h e t o a d b l a d d e r membrane i n c r e a s e s b o t h t h e s h o r t c i r c u i t c u r r e n t a c r o s s i s o l a t e d t o a d b l a d d e r and t h e permea-b i l i t y t o w a t e r ( O r l o f f & H a n d l e r , 196l ; Grantham & B r u g , 1966), 2k Baba e_t a l _ (1967), by u s i n g Na , i n d i c a t e d t h a t n o t o n l y t h e s h o r t c i r c u i t c u r r e n t g e n e r a t e d by t h e i s o l a t e d f r o g s k i n i n c r e a s e d , b u t a l s o sodium t r a n s p o r t a c r o s s t h e e p i t h e l i a l membrane i n c r e a s e d . I t i s known t h a t t h e i n t r a c e l l u l a r d i s t r i b u t i o n and c o n c e n t r a t i o n s " - 3 - ' o f c a l c i u m i o n i n m u s c l e c e l l s f a c i l i t a t e e i t h e r c o n t r a c t i o n o r r e l a x a t i o n . The mechanism o f how c a l c i u m a c t i v a t e s c o n t r a c t i o n i s n o t known, b u t many s t u d i e s have shown t h a t i t i s t h e c o n c e n t r a t i o n o f c a l c i u m w h i c h d e t e r m i n e s m u s c l e c o n t r a c t i o n . The m u s c l e e x c i t a t i o n o c c u r s when t h e membrane d e p o l a r i z e s i n v o l v i n g a change i n t h e s e l e c t i v e p e r m e a b i l i t y o f t h e e x c i t e d c e l l membrane t o c e r t a i n o f t h e i o n s , sodium i o n , p o t a s s i u m i o n and c a l c i u m i o n . W i n i f r e d and N a y l e r (1967) showed t h a t c a l c i u m i o n t r a n s p o r t o f m u s c l e was changed by t h e a d d i t i o n o f c y c l i c AMP. I t i s t h e p u r p o s e o f t h i s s t u d y t o d e t e r m i n e whether o r n o t c y c l i c AMP' changes t h e membrane p e r m e a b i l i t y o f c e l l u l a r s l i m e mold c e l l s t o i o n s , i . e . sodium i o n and c a l c i u m i o n . -k-MATERIALS AND METHODS I . E f f e c t o f c y c l i c a d e n o s i n e 3 ' , 5 ' monophosphate ( c y c l i c AMP ): D i c t y o s t e l i u m d i s c o i d e u m , s t r a i n s V -12 and NCh, were u s e d f o r t h e s e e x p e r i m e n t s . S t o c k c u l t u r e s o f t h i s s l i m e mold were grown on n o n - n u t r i e n t w a t e r a g a r p l a t e s w i t h l i v i n g b a c t e r i a E s c h e r i c h i a c o l i 2 8 l . They were k e p t a t room t e m p e r a t u r e . The 2% w a t e r a g a r (20 g. D i f c o a g a r t o 1000 m l . d i s t . w a t e r ) was a u t o c l a v e d a t 15 l b s p r e s s u r e and 121°C f o r 20 m i n u t e s . The e x p e r i m e n t a l E. c o l i was grown on kO cc l i g u i d , r a d i o a c t i v e 22 ^5 (Na o r Ca ) NTGY n u t r i e n t media ( T r y p t o s e 5 g.» y e a s t e x t r a c t 5 g. , D e x t r o s e 1 g. , K H P 0 ^ ' 3 H 2 0 1 g., d i s t . w a t e r 1000ml . , pH 7 . ) f o r about 35 h o u r s a t room t e m p e r a t u r e i n a s h a k e r . The r a d i o a c t i v i t y u s e d was 5oo C.P.M. p e r .'. /• cc NTGY medium. They were h a r v e s t e d by s u s p e n d i n g i n Bonner's s a l t s o l u t i o n ( N a C l 0 . 6 0 g., K C 1 0 . 7 5 g . s CaClg, 0.3 g., d i s t . w a t e r 1000ml ) and washed by c e n t r i f u g a t i o n t h r e e t i m e s . F i n s . l l y t h e y were suspended i n t h e same s o l u t i o n a t a con c e n - :•• t r a t i o n g i v i n g an o p t i c a l d e n s i t y o f 0.85 a t wave l e n g t h 695 mu when d i l u t e d a t 6 t i m e s . The b a c t e r i a l s u s p e n s i o n was t h e n a u t o c l a v e d f o r 20 m i n u t e s a t 121°C. F r u i t i n g b o d i e s o f t h e s t o c k D. d i s c o i d e u m were i n n o c u l a t e d i n t o n o n - n u t r i e n t w a t e r agar p l a t e s w i t h 1 cc l a b e l l e d a u t o c l a v e d E.' c o l i 2 8 l s u s p e n s i o n and t h e p l a t e s were l e f t f o r two days a t room t e m p e r a t u r e . The amoebae were t h e n h a r v e s t e d by c e n t r i -f u g a t i o n a t 1000 x g r a v i t y f o r 5 m i n u t e s , and washed by c e n t r i - . -f u g a t i o n t h r e e t i m e s w i t h Bonner's s a l t s o l u t i o n i n o r d e r t o remove t h e r e m a i n i n g b a c t e r i a and e x t r a c e l l u l a r r a d i o i s o t o p e . The amoebae were p l a c e d on a s u b s t r a t u m , w h i c h was 5-5 cm d i s c s o f Whatman No. 50 f i l t e r p a p e r r e s t i n g on g l a s s b e a d s , s a t u r a t e d w i t h 3.5 cc Bonner's s o l u t i o n , i n s i d e a 5-6 cm g l a s s p e t r i d i s h ( F i g . 1 and F i g . 2). The d i s h e s were t h e n k e p t a t 22°C i n t h e d a r k f o r 3 t o h h o u r s , f o l l o w e d b y 12 o r more h o u r s a t 5°C. When t h e amoebae were b r o u g h t back t o room t e m p e r a t u r e , f u r t h e r m o r p h o g e n e s i s t o o k p l a c e . The r e s p o n s e a p p e a r e d t o be s y n c h r o n i z e d f o r . a l l t h e amoebae. T h i s p r o c e d u r e f o l l o w s K o n i j n et_ a l _ (1968) and Sussman and Sussman (1969). The f i l t e r p aper was t h e n t r a n s f e r r e d t o a new d i s h w i t h 3-5 cc f r e s h Bonner's s o l u t i o n . T h i s p r o a e d u r e was r e p e a t e d t w i c e a t 10 m i n u t e s t o r i n s e t h e amoebae f r e e o f any r e m a i n i n g r a d i o -a c t i v e f l u i d . The f i l t e r p a p e r was n e x t p l a c e d i n a d i s h w i t h 3-5 cc f r e s h Bonner's s a l t s o l u t i o n f o r t h e c o n t r o l , w h i l e t h e e x p e r i m e n t a l f i l t e r p a p e r was p l a c e d i n 1.5 cc o f f r e s h Bonner's s a l t s o l u t i o n and 2cc o f c y c l i c AMP, c o n c e n t r a t i o n o f 0.2 g / l , added t o t h e t r e a t e d amoebae e v e r y t e n o r t w e n t y m i n u t e s , d e p e n d i n g on t h e e x p e r i m e n t . The f i n a l c o n c e n --k t r a t i o n o f c y c l i c AMP u s e d was 3 x 10 M.. S u b s e q u e n t a l y , 2cc samples were t a k e n e v e r y t e n m i n u t e s f r o m b o t h t h e c o n t r o l and t h e c y c l i c AMP t r e a t e d amoebae. The e x p e r i m e n t s .were done m o s t l y a t t h e p r e - a g g r e g a t i v e : s t a g e b u t sometimes a t t h e a g g r e g a t i n g s t a g e . The r a d i o a c t i v i t y o f each sample was c o u n t e d w i t h a p l a n c h e t c o u n t e r , made by N u c l e a r C h i c a g o , • f o r t e n m i n u t e s . F i v e u n i t s o f p h o s p h o d i e s t e r a s e p e r 3.5 c c of:B3nner's. s o l u t i o n was u s e d i n s t e a d o f c y c l i c AMP i n some e x p e r i m e n t s . I I . I o n c o n t e n t measurement: D. d i s c o i d e u m was grown on w a t e r a g a r w i t h l i v i n g b a c t e r i a E. c o l i 2 8 l 0 s p r e a d on a s t r a i g h t l i n e o n . t h e w a t e r a g a r . A lamp was p l a c e d i n f r o n t o f t h e c u l t u r e d i s h e s i n o r d e r t o a t t r a c t t h e m i g r a t i n g s l u g s . - 6 -The s l u g s w o u l d t h e n move away f r o m t h e b a c t e r i a . A t l e a s t f i f t e e n s l u g s were p i c k e d up f r o m t h e w a t e r a g a r p l a t e s , homogenized i n 2 cc g l a s s d i s t i l l e d w a t e r and.the i o n i c c o n t e n t s (sodium i o n , p o t a s s i u m i o n and c a l c i u m i o n ) o f t h e homogenized s l u g s d e t e r m i n e d by f l a m e s p e c t r o m e t r y u s i n g t h e f l a m e s p e c t r o p h o t o m e t e r by Evans E l e c t r o S e l e n i u m L t d . , E n g l a n d , f o r p o t a s s i u m i o n and sodium i o n and sp. 900 Flame S p e c t r o p h o t o m e t e r by Unicam I n s t r u m e n t s L t d . f o r c a l c i u m i o n . C o l l i n s and Polkinhorne(l952) have d e m o n s t r a t e d t h a t t h e r e i s no i n t e r f e r e n c e f r o m o t h e r i o n s i n m e a s u r i n g p o t a s s i u m i D n and sodium i o n c o n c e n t r a t i o n s u s i n g t h e EEL f l a m e s p e c t r o p h o t o m e t e r . No r e f e r e n c e s t o p r e v i o u s measurement o f t h e i o n c o n t e n t s o f s l i m e mold amoebae were f o u n d . -7-F i g . 1. P e t r i dish with glass beads which were used.in the experiments. F i g . 2 . The amoebae on the f i l t e r paper saturated with 3.5 cc Bonner's s o l u t i o n i n a dish with glass beads. -8-RESULTS I . The e f f e c t s o f c y c l i c AMP on i o n f l u x e s i n D. "discoideum: A. E f f e c t s on c a l c i u m i o n s a t v a r i o u s s t a g e s o f t h e l i f e c y c l e : 1. P r e - a g g r e g a t i v e ' s t a g e : An i n c r e a s e o f c a l c i u m i o n e f f l u x was o b s e r v e d a f t e r t h e a d d i t i o n o f c y c l i c AMP, w h i l e a g r a d u a l decrea.se was a p p a r e n t i n t h e u n t r e a t e d c o n t r o l . T h i s e f f e c t was o b s e r v e d when c y c l i c AMP was added e v e r y 20 m i n u t e s ( F i g . 3) o r o n l y added once a t t=10 m i n u t e s a f t e r r e a c h i n g room t e m p e r a t u r e ( F i g . k). A n a l y s i s o f t h e s e d a t a by S t u d e n t ' s " t " t e s t showed t h a t t h e i n c r e a s e i n p e r c e n t a g e o u t p u t o f c a l c i u m i o n a f t e r t h e a d d i t i o n o f c y c l i c AMP was s i g n i f i c a n t l y d i f f e r e n t f r o m t h e n o r m a l d e c r e a s e o f o u t p u t i n t h e c o n t r o l ( F i g . 3 and F i g . U). There was no o b s e r v e d i n c r e a s e i n c a l c i u m e f f l u x when c y c l i c AMP was added e v e r y 10 m i n u t e s b u t t h e r e s u l t s a r e d i f f e r e n t i n e x p e r i m e n t a l and c o n t r o l samples ( F i g . 5). When f r e s h c y c l i c .AMP was added e v e r y 10 m i n u t e s t o a d i s h o f amoebae, a g g r e g a t i o n s t o p p e d . T h i s phenomena w i l l be d i s c u s s e d l a t e r . One can see t h a t t h e e f f l u x r a t e d e c r e a s e s w i t h t i m e i n t h e c o n t r o l s o f a l l e x p e r i m e n t s . T h i s i s p r o b a b l y due t o a f a l l i n t h e 1+5 i n t e r n a l c o n c e n t r a t i o n o f Ca as i t i s l o s t f r o m t h e c e l l s . 2. A g g r e g a t i n g s t a g e : There i s t h e same e f f e c t as i n t h e p r e - a g g r e g a t e s t a g e . An i n c r e a s e d . e f f l u x o f c a l c i u m a p p e a r e d a f t e r t h e a d d i t i o n o f c y c l i c AMP d u r i n g t h e a g g r e g a t i n g s t a g e when c y c l i c AMP was added e v e r y 20 m i n u t e s o r when i t was added a s i n g l e t i m e a t 10 m i n u t e s ( F i g . 6 and F i g . 7). T h i s i n c r e a s e d c a l c i u m i o n o u t p u t was s i g n i f i c a n t l y d i f f e r e n t f r o m t h e d e c r e a s e i n t h e c o n t r o l c u r v e . There i s a l a r g e i n c r e a s e d e f f l u x a f t e r the. a d d i t i o n a t f i f t y m i n u t e s . A l s o , an i n c r e a s e o f c a l c i u m o u t p u t r e s u l t e d i n t h e c o n t r o l c u l t u r e between 20 and kO m i n u t e s ( F i g . 6 ) . B. E f f e c t s on sodium i o n s a t v a r i o u s s t a g e s o f t h e l i f e c y c l e : 1. P r e - a g g r e g a t i v e - s t a g e : When c y c l i c AMP was added e v e r y 20 m i n u t e s , t h e r e was no s i g n i f i -c a n t d i f f e r e n c e between e x p e r i m e n t a l and c o n t r o l c u r v e s i n sodium i o n e f f l u x ( F i g . 8 ) . The p e r c e n t a g e o f sodium o u t p u t g r a d u a l l y d e c r e a s e d i n b o t h c u r v e s . A p p a r e n t l y about h a l f o f t h e sodium i o n came o u t o f t h e c e l l s i n t h e f i r s t t e n m i n u t e s whereas 28% o f t h e c a l c i u m i o n came fr o m t h e c e l l s i n t h e f i r s t t e n m i n u t e s , s u g g e s t i n g t h a t sodium i s l o s t f r o m t h e c e l l s f a s t e r t h a n c a l c i u m . 2. A g g r e g a t i n g s t a g e : An i n c r e a s e o f sodium e f f l u x wa,s a p p a r e n t a f t e r t h e f i r s t a d d i t i o n o f c y c l i c AMP and l i t l l e i n c r e a s e . a f t e r t h e l a t e r a d d i t i o n ( F i g . 9). An i n c r e a s e o f sodium e f f l u x a t 30 - 50 m i n u t e s f o l l o w i n g an i n i t i a l d e c r e a s e i n e f f l u x o c c u r r e d i n t h e c o n t r o l c u l t u r e . The i n i t i a l i n c r e a s e i n t h e e x p e r i m e n t a l c u r v e and t h e l a t e r i n c r e a s e i n t h e c o n t r o l were b o t h examined by S t u d e n t ' s " t " t e s t and f o u n d t o be s i g n i f i c a n t a t t h e 95% c o n f i d e n c e l e v e l . II. The e f f e c t s o f p h o s p h o d i e s t e r a s e on c a l c i u m i o n e f f l u x a t v a r i o u s s t a g e s i n t h e l i f e c y c l e A. P r e - a g g r e g a t i v e ' s t a g e : C y c l i c AMP enzyme, p h o s p h o d i e s t e r a s e , w h i c h c o n v e r t s c y c l i c AMP t o 5'AMP, was added once a t t=10 m i n u t e s a f t e r room t e m p e r a t u r e was r e a c h e d , i n s t e a d o f c y c l i c AMP. There was no o b s e r v e d e f f e c t s on c a l c i u m e f f l u x ( F i g . 1 0 ) . - 1 0 -2. A g g r e g a t i n g s t a g e : There was no change i n t h e c a l c i u m e f f l u x a t t h e a g g r e g a t i n g s t a g e a f t e r t h e a d d i t i o n o f p h o s p h o d i e s t e r a s e ( F i g . 11). There i s an i n c r e a s e o f c a l c i u m i o n e f f l u x l a t e r i n t h e e x p e r i m e n t a l amoebae, w h i c h i s s i g n i -f i c a n t . I I I . I o n c o n t e n t s i n amoeboid c e l l s a t t h e s l u g s t a g e : The c o n c e n t r a t i o n s o f sodium i o n , p o t a s s i u m i o n and c a l c i u m i c n were measured at the slug stage (Table i ) instead of the amoebae stage. I t i s i m p o s s i b l e t o c o m p l e t e l y remove t h e b a c t e r i a f r o m t h e amoebae. The l e n g t h and d i a m e t e r o f t h e s l u g were r e c o r d e d b e f o r e homo-g e n i z a t i o n . F r a n c i s (1962) has shown t h e e q u a t i o n f o r t h e volume o f 2 a s l u g t o be i r r 1 , because o f t h e p a r t i c u l a r shape o f t h e s l u g . 1.7k The w i d e s t d i a m e t e r was measured. A c c o r d i n g t o F r a n c i s (19^2), t h e ave r a g e d i a m e t e r o f t h e amoebae i s 10 u. The volume o f t h e amoebae was c a l c u l a t e d by t h e f o r m u l a f o r t h e Volume o f a s p h e r e , k_ ir r . The 3 6 i o n c o n c e n t r a t i o n c o u l d t h e n be e x p r e s s e d as m i l l i e q u i v a l e n t p e r 10 c e l l s o r p e r m i c r o l i t e r ( T a b l e I ) . P r e s u m a b l y t h e i o n c o n t e n t a t t h i s s t a g e i s t h e same as d u r i n g t h e e a r l i e r s t a g e s when e f f l u x e x p e r i m e n t s were p e r f o r m e d . The r e s u l t s showed t h a t p o t a s s i u m i o n c o n c e n t r a t i o n was o n l y about one t h i r d t h a t o f t h e sodium i o n . C a l c i u m i o n c o n c e n t r a t i o n was t h e h i g h e s t . -11-F i g . 3. The e f f e c t o f c y c l i c AMP on c a l c i u m i o n f l u x e s i n p r e - a g g r e g a t i v e s t a g e o f D. d i s c o i d e u m ; t h e a d d i t i o n o f c y c l i c AMP a t 20 m i n u t e i n t e r v a l s . R a d i o a c t i v i t y o f each sample i n c o u n t s p e r 10 m i n u t e s was e x p r e s s e d as a p e r c e n t a g e o f t h e t o t a l r a d i o a c t i v i t y r e l e a s e d o v e r t h e e n t i r e p e r i o d shown. The t=0 i s i m m e d i a t e l y a f t e r ' -t h r e e r i n s e s o f t h e f i l t e r p aper i n Bonner's s a l t s o l u t i o n (see t e x t ) . ( A v e r a g e o f seven e x p e r i m e n t s ) j . i n d i c a t e s t h e t i m e when c y c l i c AMP was 1 added. c-AMP TIMECMI NUTES) - 1 2 -F i g . h. The e f f e c t o f a s i n g l e a d d i t i o n o f c y c l i c AMP t o Ca t r e a t e d c u l t u r e s , a t p r e - a g g r e g a t i v e s t a g e . ( A v e rage o f two e x p e r i m e n t s ) O r d i n a t e s as i n . F i g u r e 3 . O C O N T R O L PRE-AGGREGATE ..' S T A G E • <B E X P E R I M E N T A L 10 2 0 3 0 4 0 5 0 T I M E ( M I N U T E S ) - 1 3 -F i g . 5- The e f f e c t o f c y c l i c AMP on c a l c i u m i o n f l u x e s i n p r e - a g g r e g a t i v e s t a g e o f D. d i s c o i d e u m ; t h e a d d i t i o n o f c y c l i c AMP a t 10 m i n u t e i n t e r v a l s . ( A verage o f f o u r e x p e r i m e n t s ) O r d i n a t e s as i n F i g u r e 3. C-AMP TIME (MINUTES) - I k -F i g . 6. The e f f e c t o f c y c l i c AMP on c a l c i u m i o n f l u x e s d u r i n g a g g r e g a t i v e s t a g e o f D. d i s c o i d e u m ; t h e a d d i t i o n o f c y c l i c AMP a t 20 m i n u t e i n t e r -v a l s . T=0 was when a g g r e g a t i o n s were f i r s t f o u n d d u r i n g t h e a g g r e g a t i o n p e r i o d . (Average o f f o u r e x p e r i m e n t s ) O r d i n a t e s as i n F i g u r e 3. A G G R E G A T I N G S T A G E O C O N T R O L E X P E R I M E N T A L - * 1 1 1~ 1 H » 10 2 0 3 0 4 0 5 0 6 0 7 0 TIME (Ml N U T E S ) -15-F i g . 7. The e f f e c t o f a s i n g l e a d d i t i o n o f c y c l i c AMP t o Ca t r e a t e d c u l t u r e d u r i n g a g g r e g a t i v e s t a g e o f IK d i s c o i d e u m . ( A v e rage o f 2 e x p e r i m e n t s ) O r d i n a t e s as i n F i g u r e 6. IQ 2 0 30 AO T I M E ( M I N U T E S ) - 1 6 -F i g . 8 . The e f f e c t o f c y c l i c AMP on sodium i o n f l u x e s i n p r e - a g g r e g a t i v e s t a g e o f D_. d i s c o i d e u m ; t h e a d d i t i o n o f c y c l i c AMP a t 20 m i n u t e i n t e r v a l s . ( Average o f s i x e x p e r i m e n t s ) O r d i n a t e s as f o r F i g u r e 3 . -17-F i g . 9. The e f f e c t o f c y c l i c AMP on sodium i o n f l u x e s . d u r i n g a g g r e g a t i n g s t a g e o f D. d i s c o i d e u m ; t h e a d d i t i o n o f c y c l i c AMP a t 20 m i n u t e i n t e r v a l s . (Average o f t h r e e e x p e r i m e n t s ) O r d i n a t e s as i n F i g u r e 6. T I M E ( M I N U T E S ) -.18-F i g . 10. T h e e f f e c t o f s i n g l e a d d i t i o n o f p h o s p h o d i e s t e r a s e t o C a t r e a t e d c u l t u r e s a t p r e - a g g r e g a t i v e s t a g e . ( A v e r a g e o f t w o e x p e r i m e n t s ' ) O r d i n a t e s a s i n F i g u r e 3. P R E - A G G R E G A T E S T A G E _| 1 (. 1 ! 1 0 2 0 3 0 4 0 5 0 T I M E C M I N U T E S ) -19 -F i g . 11. T h e e f f e c t o f s i n g l e a d d i t i o n o f p h o s p h o d i e s t e r a s e t o C a t r e a t e d c u l t u r e s d u r i n g a g g r e g a t i n g s t a g e . ( A v e r a g e o f t w o e x p e r i m e n t s ) O r d i n a t e s a s f o r F i g u r e 6 . 10 20 30 4 0 50 T I M E ( M I N U T E S ) -20-T a b l e I . I o n c o n t e n t s i n amoebae a t s l u g s t a g e . TABLE I mE/10^cells mE/ul c e l l m a t e r i a l Number of slugs Sodium ion (l.k + 0.28) x 10"^ (3.26 + 0.6h) x 10~^ 6 l Potassium i on (0.5 + O.lh) x 10~^ ( l . l 6 +0.75) x 10"^ 69 Calcium ion (6.5 + 0.93) x 10~^ (15 + 2.1 ) x 10 - 1 + 6h -21-DISCUSSION The c o n c e n t r a t i o n o f c y c l i c AMP u s e d i n t h i s s e t o f e x p e r i m e n t s -k was 3 x 10 M., w h i c h i s f a r h i g h e r t h a n t h e n a t u r a l c o n c e n t r a i t o n o f c y c l i c AMP. A c c o r d i n g t o t h e s t u d i e s o f Bonner e t _ a l _ (1969), t h e -12 n a t u r a l s e c r e t i o n o f c y c l i c AMP by amoebae r a n g e s f r o m 10 M. t o 10 M. I n K o n i j n ' s t e s t s , a g g r e g a t i n g amoebae were s e n s i t i v e t o c o n c e n t r a t i o n s o f c y c l i c AMP as l o w as 10 ^ M., a l t h o u g h v e g e t a t i v e amoebae were o n l y s e n s i t i v e t o c o n c e n t r a t i o n s o f c y c l i c AMP as l o w as -1+ 1 x 10 M. However, K o n i j n ' s r e s u l t s were o n l y a p p r o x i m a t e . The r e a s o n why amoebae a r e s e n s i t i v e t o c o n c e n t r a t i o n s o f c y c l i c AMP above t h e n a t u r a l s e c r e t i o n l e v e l s i s unknown. No s i g n i f i c a n t e f f e c t s r e s u l t e d i n u s i n g c y c l i c AMP c o n c e n t r a t i o n s o f 1 x 10 ^ g / l a t t h e b e g i n n i n g o f t h i s r e s e a r c h . I . The r o l e o f c y c l i c AMP on i o n e f f l u x i n D i c t y o s t e l i u m A c c o r d i n g t o F i g . h, i n w h i c h c y c l i c AMP was added o n l y once a t t=10, an i n c r e a s e o f c a l c i u m i o n o u t p u t a f t e r t h e a d d i t i o n o f c y c l i c AMP f o l l o w e d a g r a d u a l d e c r e a s e o f ca.lcium i o n e f f l u x w h i l e an i n c r e a s e d c a l c i u m i o n e f f l u x a p p e a r e d a g a i n i f c y c l i c AMP was added l a t e r a t t h e p r e - a g g r e g a t i v e s t a g e ( F i g . 3 ) . The same phenomenon was o b s e r v e d d u r i n g t h e a g g r e g a t i n g s t a g e ( F i g . 6 and F i g . 7 ) . One mi g h t c o n c l u d e t h a t c y c l i c AMP d e p o l a r i z e s o r h y p e r p o l a r i z e s t h e c e l l membrane and ca u s e s c a l c i u m i o n movement a c r o s s t h e membrane b o t h i n t h e p r e - a g g r e g a t i v e and a g g r e g a t i n g s t a g e s . The a g g r e g a t i n g amoebae may p r o d u c e enough o f t h e i r own c y c l i c AMP (Bonne r , 1969) t o a f f e c t a change i n membrane p e r m e a b i l i t y w i t h a consequent e f f l u x o f c a l c i u m . Bonner (1969) r e p o r t e d t h a t t h e r e i s a hundred f o l d i n c r e a s e i n c y c l i c AMP c o n c e n t r a t i o n between t h e b e g i n n i n g -22-o f a g g r e g a t i o n and l a t e a g g r e g a t i o n . The s e n s i t i v i t y t o c y c l i c AMP a l s o i n c r e a s e s hundred f o l d d u r i n g t h e a g g r e g a t i o n s t a g e . The g r a d u a l i n c r e a s e i n Ca o u t p u t i n t h e c o n t r o l c u r v e d u r i n g t h e a g g r e g a t i n g s t a g e ( F i g . 6) w o u l d t h e n be e x p l a i n e d . The samples t a k e n d u r i n g t h e a g g r e g a t i n g s t a g e showed t h a t sodium i o n e f f l u x changed d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t s ( F i g . 9 ) . The i n c r e a s e i n sodium i o n o u t p u t was m i m i c k e d by t h e c y c l i c AMP t r e a t e d c u l t u r e s i n t h e f i r s t sample. The sodium i o n e f f l u x f r o m t h e c o n t r o l i s h i g h e r t h a n t h a t f r o m t h e e x p e r i m e n t a l c u l t u r e d u r i n g t h e p e r i o d t=30 t o 60 m i n u t e s . T h i s p r o b a b l y r e s u l t e d from t h e d e p l e t i o n o f t h e i n t r a c e l l u a l r sodium i n t h e e x p e r i m e n t a l d i s h a f t e r t h e f i r s t a d d i t i o n o f c y c l i c AMP. P o s s i b l y t h e membrane p e r m e a b i l i t y t o sodium i o n changed f o r s i m i l i a r r e a s o n s i n b o t h i n s t a n c e s . T h i s . p e r m e a b i l i t y change may be i m p o r t a n t f o r a g g r e g a t i o n . The r e a s o n f o r t h e change o f sodium i o n e f f l u x d u r i n g t h e e x p e r i m e n t a l c o u r s e m i g h t be e x p l a i n e d by t h e i n c r e a s e o f s e n s i t i v i t y t o t h e a t t r a c t a n t ( B o n n e r , 1969) o r t h e r e m i ght be some o t h e r a t t r a c t i n g c h e m i c a l p r e s e n t w h i c h w o u l d change sodium e f f l u x d u r i n g t h e a g g r e g a t i n g s t a g e , s i n c e c y c l i c - A M P i s o n l y one k i n d o f a t t r a c t a n t . As Bonner (1969) r e p o r t e d , c y c l i c AMP a p p e a r s t o be t h e main hormone r e s p o n s i b l e f o r t h e amoebae's s o c i a l e x i s t a n c e i n D i c t y o s t e l i u m . L i k e mammalian hormones, c y c l i c AMP i s e x t r a c e l l u l a r and p r o v i d e s c o m m u n i c a t i o n between c e l l s t h a t a r e s e p a r a t e d f r o m one a n o t h e r i n s p a c e , b u t i n mammals, c y c l i c AMP i s t r i g g e r e d by t h e e x t r a c e l l u l a r hormones and a c t s as a "second messenger" ( S u t h e r l a n d e t a l , 1966) i n s i d e t h e c e l l s . As e x p e c t e d , t h e c h a i n o f c h e m i c a l e v e n t s i n v o l v i n g c y c l i c AMP i n mammals i s l o n g e r and more complex t h a n i t i s i n t h e l e s s c o m p l i c a t e d s o c i a l amoebae. -23-I I . The r o l e of phosphodiesterase i n Dicyostelium discoideum The ad d i t i o n of phosphodiesterase did not a f f e c t the calcium ion e f f l u x e i t h e r i n pre-aggregative stage or aggregating stage ( F i g . 10 and F i g . l l ) . The reason f o r t h i s might he that the temperature used, 22°C, was not the optimal temperature f o r the phosphodiesterase, around 35°C. I t might also have been that the amount, 5 u n i t s per 3 .5 cc, of phosphodiesterase used was not high enough to cause a change i n membrane permeability. Although phosphodiesterase" d i d not a f f e c t the e f f l u x of clacium ion ( F i g . 1 1 ) , we would l i k e to propose that t h i s c y c l i c AMP enzyme i s also playing a r o l e i n aggregation. The c o n t r o l amoebae formed an aggregate, while aggregation did not occur i n the treated amoebae where c y c l i c AMP was added every 10 minutes. The addition of c y c l i c AMP every 10 minutes would f l o o d the c e l l s with c y c l i c AMP, more than could be eliminated by the phosphodiesterase, and' no gradient i n c y c l i c AMP would then e x i s t . As Shaffer (1956b) pointed out, the destruction of acr a s i n by acrasinase would produce a gradient and increase the p o s s i b i l i t y of o r i e n t a t i o n of aggregating amoebae. Bonner et a l (1969) were able to show that the amoebae produce the enzyme, phosphodiesterase, that destroys the a t t r a c t a n t , c y c l i c AMP, i n a l l the stages of development, but they were not able to show the quantities of phosphodiesterase produced. This mechanism of simult-aneous production and destruction evidently serves an improtant purpose i n the amoeba's l i f e cycle (Eonner, 1969) . C y c l i c AMP must be produced and broken down to 5 ' AMP by phosphodiesterase during the aggregating stage. The highest enzyme concentration i s found i n the aggregate, destroying any at t r a c t a n t i n the v i c i n i t y of the amoebae. -2k-S i n c e t h e amoebae a r e a t t r a c t e d by t h e c y c l i c AMP g r a d i e n t ( S h a f f e r , 1957) and f e e d on b a c t e r i a , w h i c h a l s o s e c r e t e c y c l i c AMP, pe r h a p s t h e amoebae l o c a t e t h e b a c t e r i a v i a c y c l i c AMP ( B o n n e r , 1969); i n o r d e r t o t r a c k new p r e y , c y c l i c AMP must be b r o k e n down, o t h e r w i s e t h e amoebae w o u l d become d i s o r i e n t e d . Bonner (1969) assumed t h a t s o c i a l . amoebae a g g r e g a t e by t h e same mechanism o f c h e m i c a l a t t r a c t i o n t h a t - - a s s i s t s t h e i r q u e s t f o r f o o d . The o n l y r e q u i r e d change wo u l d be t o _ __' i n c r e a s e t h e s e n s i t i v i t y o f t h e mechanism e n o r m o u s l y , so t h a t i n d i -" v i d u a l s " w o u l d i g n o r e t h e i r s u r r o u d i n g s , s t o p h u n t i n g , and swarm t o -g e t h e r . P h o s p h o d i e s t e r a s e , t h e r e f o r e , i s most l i k e l y q u i t e importe.nt i n a g g r e g a t i o n . I I I . F u n c t i o n s o f c y c l i c AMP i n o t h e r c e l l s A. C o n t r a c t i l e mechanisms: I n m u s c u l a r c o n t r a c t i o n s , c y c l i c AMP changes t h e c a l c i u m i o n t r a n s p o r t a c r o s s t h e membrane ( N a y l e r , 1967) . The c o n t r a c t i o n depends • on t h e i n t r a c e l l u l a r c o n c e n t r a t i o n o f c a l c i u m i o n s ( N a y l e r , 1967 T a i s i j a e t a l , 1965; E b a s h i , 1 9 6 l ; Weber & H e r z , 1 9 6 l ; and Weber & W i n i c u r , 1 9 6 l ) . C a l c i u m ' s h y p e r p o l a r i z i n g e f f e c t upon t h e r e s t i n g c e l l membrane w i t h t h e consequent change i n t h e a c t i o n p o t e n t i a l , can be seen i n v a r i o u s e x c i t a b l e c e l l s . C y c l i c AMP i n c r e a s e s c a l c i u m permea-b i l i t y and m u s c u l a r c o n t r a c t i o n o f t h e m u s c l e c e l l s . M u s c u l a r c o n t r a c -t i o n i s assumed t o be t r i g g e r e d by an i n c r e a s e i n a v a i l a b i l i t y o f c a l c i u m i o n t o t h e c o n t r a c t i l e mechanism ( H u x l e y & H u x l e y , 196*+; P a u l , D a n i e l , Kay, & Monckton, 1965) . I t has a l s o been s u g g e s t e d t h a t t h e i n t e r a c t i o n between a c t i n and m y o s i n must u l t i m a t e l y be a f f e c t e d , b u t a d i r e c t e f f e c t o f c y c l i c AMP on t h i s s y s t e m has n o t been e s t a b l i s h e d . J a h n and Bovee (1966) s u g g e s t e d t h a t t h e c o n t r a c t i l e p r o t e i n o f ^25-t h e p r o t o z o a n c e l l i s v e r y s i m i l i a r t o a c t o m y o s i n o f r a b b i t s t r i a t e d m u s c l e . Hatana and Oosawa i n 1966 were a b l e t o e x t r a c t a p r o t e i n w i t h many o f t h e p r o p e r t i e s o f r a b b i t s t r i a t e d m u s c l e a c t i n f r o m t h e p l a s m o d i a o f a myxomycete, Physarum p o l y c e p h a l u m . B i o -C h e m i c a l r e s e a r c h a l s o has shown t h e n e c e s s i t y o f c a l c i u m i o n s i n t h e r e a c t i o n s o f c o n t r a c t i l e p r o t e i n ( G r e b e c k i , 1965). --The s t r o n g e v i d e n c e -for c y c l i c AMP e f f e c t on t h e c a l c i u m i o n p e r m e a b i l i t y o f t h e D. d i s c o i d e u m c e l l membrane ( F i g . 3 and F i g . k) and t h e i n c r e a s e i n c a l c i u m p e r m e a b i l i t y d u r i n g a g g r e g a t i o n ( F i g . 6) w o u l d s u g g e s t t h a t t h e mechanism o f amoeboid movement i s s i m i l a r t o t h a t o f m u s c l e c o n t r a c t i o n . Though changes i n c a l c i u m i o n c o n c e n t r a t i o n m i g h t be r e s p o n s i b l e f o r amoeboid movement, o r i e n t a t i o n o f amoeboid movement d u r i n g a g g r e g a t i o n i s d i r e c t e d by a c y c l i c AMP g r a d i e n t . B. Other F u n c t i o n s : I t has been s u g g e s t e d t h a t c e l l u l a r a d h e s i o n r e s u l t s f r o m b i n d i n g a c r o s s i n t e r c e l l u l a r c a l c i u m i o n ' b r i d g e s ' (Dehaan, 1959)• The l i t e r a t u r e i n d i c a t e s t h a t one o f t h e c h a r a c t e r i s t i c s o f membrane-l i n k e d phenomena w h i c h seems common t o many metazoan c e l l s i s t h e r e s p o n s e t o t h e r e m o v a l o f c a l c i u m (Dehaan, 1958). Removal o f c a l c i u m i o n c a u s e s t h e d i s f o r m a t i o n o f c e l l l a y e r s . D u r i n g m o r p h o g e n e s i s , i n t r a c e l l u l a r c a l c i u m i s n e c e s s a r y f o r t h e m a i n t a i n a n c e 'of t h e m i g r a t i n g c e l l l a y e r s (Dehaan, 1959). A c c o r d i n g t o S h a f f e r (1957), t h e s u r f a c e c h a r a c t e r i s t i c s o f t h e amoebae a l t e r so t h a t t h e amoebae become " s t i c k y " , f o r m i n g p s e u d o p o d i a l a t t a c h m e n t s t o one a n o t h e r d u r i n g t h e a g g r e g a t i v e s t a g e . C h e l a t i n g c a l c i u m i o n c a u s e s d i s i n t e g r a t i o n o f pseudoplasmodium i n t o a d i s o r g a n i z e d mass o f amoebae (Dehaan, 1959)--26-Hence, c a l c i u m i o n a p p e a r s t o have a p r i m e r o l e i n t h e p r e s e r v a t i o n o f m u l t i c e l l u l a r i t y . T h i s c o u l d he r e l a t e d t o t h e change o f c a l c i u m a c t i v i t y d u r i n g a g g r e g a t i o n and t h e d i s c o v e r y o f h i g h e s t c a l c i u m c o n c e n t r a t i o n a t t h i s p o i n t . L i t e r a t u r e r e v i e w s i n d i c a t e t h a t an a s s o c i a t i o n o f c y c l i c AMP and c a l c i u m i o n i s a p p a r e n t i n a v a r i e t y o f o t h e r f u n c t i o n s ( O r l o f f e t a l , 1962; N o v a l e s & D a v i s , 1967; Szego & D a v i s , 1967; P e r o n , G u e r r a , & M c C a r t h y , 1965; R i z a c k , I96U; Mansour & Mansour, 1962; R a i l & West, 1963). The main e f f e c t o f c y c l i c AMP and c a l c i u m i o n a r e i n t h e changes o f membrane s t r u c t u r e and f u n c t i o n . I t i s t o o e a r l y t o d e f i n e t h i s r e l a t i o n s h i p b u t g e n e r a l e v i d e n c e p o i n t s t o w a r d s t h e a l t e r a t i o n s i n t h e l e v e l o f f r e e c a l c i u m i o n i n t h e c y t o s o l o f t h e s e c e l l s . T h i s i s c o n s i s t a n t w i t h t h e r e s u l t s o f t h i s s t u d y . The r e l e a s e o f c a l c i u m i o n s p l a y s a r o l e i n t h e amoeboid movement and a d h e s i o n . SUMMARY (1) T h e e f f e c t s o f c y c l i c A M P o n t h e e f f l u x o f i o n s a c r o s s t h e c e l l m e m b r a n e s o f c e l l u l a r s l i m e m o l d s , s t r a i n s NCH a n d V-12, v e r e s t u d i e d 22 1+5 b y u s i n g r a d i o - i s o t o p e s , N a a n d C a (2) C y c l i c A M P w a s a d d e d a t t h e p r e - a g g r e g a t e s t a g e o r d u r i n g a c t u a l a g g r e g a t i o n , e i t h e r t e n o r t w e n t y m i n u t e s a f t e r r e a c h i n g r o o m t e m p e r a t u r e , d e p e n d i n g o n t h e e x p e r i m e n t . (3) C y c l i c A M P c a u s e s c h a n g e s i n c a l c i u m e f f l u x e i t h e r a t t h e a m o e b o i d s t a g e o r d u r i n g a g g r e g a t i o n . C h a n g e s i n s o d i u m e f f l u x w e r e f o u n d o n l y d u r i n g t h e a g g r e g a t i n g s t a g e . (1+) P h o s p h o d i e s t e r a s e , t h e e n z y m e w h i c h i n a c t i v a t e s c y c l i c A M P t o t h e i n a c t i v e f o r m 5 ' A M P , h a d n o e f f e c t o n m e m b r a n e p e r m e a b i l i t y w h e n t e s t e d . (5) T h e i o n c o n t e n t s , i . e . s o d i u m i o n , p o t a s s i u m i o n , a n d c a l c i u m i o n , w e r e m e a s u r e d i n t h e m i g r a t i n g s l u g o r . p s e u d o p l a s m o d i u m . I t w a s f o u n d -1+ 6 t h a t t h e c o n c e n t r a t i o n s o f p o t a s s i u m i o n , 5.3 x 10 mE/10 c e l l s i s o n l y a b o u t o n e t h i r d t h e c o n t e n t o f t h e c o n c e n t r a t i o n o f s o d i u m i o n , -1+ 6 l.k x 10 mE/10 c e l l s . - 2 8 -BIBLIOGRAPHY Argy, W.P. J r . , J.S. Handler, and J . O r l o f f , 1967. Ca + +and M g + + e f f e c t s on toad bladder response to c y c l i c AMP, theophylline, and ADH analogues. American J . of Phys. 213:803-808. Baba, W.I., A.J. Smith, and M.Townsend, 1967. The e f f e c t s of vasopressin, theophylline and c y c l i c 3 5 ' - a d e n o s i n e monophosphate ' ( c y c l i c AMP) on sodium transport across the frog skin. Quarterly Journal of Experimental Physiology 52:^16-1*21. Bonner, J.T., 19^7. Evidence for the formation of c e l l aggregates by chemotaxis i n the development of the slime mold Dictyostelium discoideum. Journal of Experi. Zool. 106:1-26. Bonner, J.T., I967. The C e l l u l a r Slime Molds. 2nd E d i t i o n , Princeton U n i v e r s i t y Press, Princeton, New Jersey. Bonner, J.T . , 1 9 6 9 . Hormones i n s o c i a l amoebae and mammals. S c i e n t i f i c American June:78-91. Bonner, J.T., D.S. Barkley, E.M. H a l l , T.M.Konijn, J.W. Mason, G.O'Keefe, ' ". I l l , and P.B. Wolfe. 1969. Acrasin, acrasinase, and the s e n s i t i v i t y to acrasin i n Dictyostelium discoideum.Develop. B i o l . 20:72-87• Breckenridge, B., J.H. Burn, and F.M. Matschinsky, 1967. 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