Chapter 19
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Insect Synaptosomes in Superfusion A Technique To Investigate the Actions of Ion Channel Directed Neurotoxicants by Monitoring Their Effects on Transmitter Release Russell A. Nicholson, Peter Baines, and Philip S. Robinson Wellcome Research Laboratories, Berkhamsted, Hertfordshire HP4 2DY, England
A study of the interaction of selected neurotoxicants with ion channels in the synaptosomal membrane as measured by a superfusion technique is described. The alkaloid neurotoxin veratridine(10 M)stimulated release of radiolabel from [ H]-choline loaded synaptosomes in superfusion. Hemicholinium-3 (10-4 M) was inactive. Veratridine-induced release, although unaffected by picrotoxinin (10 M) was strongly inhibited by tetrodotoxin (IC : 5x10 ). Deltamethrin produced a potentiation of veratridine-stimulated transmitter release, this effect being more pronounced upon preincubation with the pyrethroid. Ivermectin and certain milbemycins induce release of label from insect synaptosomes. Picrotoxinin, dieldrin and γ-hexachlorocyclohexane are potent inhibitors of ivermectin-dependent release whereas t-butylbicyclophosphorothionate (10 M) and tetrodotoxin (10 Μ) have no antagonist activity. These observations support the concept that insect central nerve terminals contain functional sodium and chloride channels. -4
3
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C e r t a i n n e u r o t o x i c a n t s a c t by d i r e c t l y i n f l u e n c i n g t h e e l e c t r i c a l a c t i v i t y o f t h e i n s e c t nerve membrane t h r o u g h a s p e c i f i c a c t i o n at i o n - s e l e c t i v e channels. These phenomena can be s t u d i e d u s i n g many o f the n e u r o b i o c h e m i c a l t e c h n i q u e s t h a t have s u c c e s s f u l l y been a p p l i e d t o v e r t e b r a t e nervous s y s t e m s . In t h i s s t u d y we use the t e c h n i q u e o f i n s e c t synaptosomes i n s u p e r f u s i o n t o examine the i n t e r a c t i o n o f a range o f i n s e c t i c i d a l a g e n t s w i t h i o n channels i n t h e nerve t e r m i n a l by m o n i t o r i n g e f f e c t s on t r a n s m i t t e r r e l e a s e . Methods Head and t h o r a c i c g a n g l i a from e i g h t y c o c k r o a c h e s iP_. americana) were homogenized i n i c e - c o l d 0.25M s u c r o s e ( b u f f e r e d t o pH 7.2 w i t h T r i s . H C l ) and f r a c t i o n a t e d as f a r as t h e crude synaptosomal
0097-6156/87/0356-0262$06.00/0 © 1987 American Chemical Society Hollingworth and Green; Sites of Action for Neurotoxic Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
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19. NICHOLSON ET AL.
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( P 2 ) s t a g e a c c o r d i n g to p r o c e d u r e s p u b l i s h e d f o r l o c u s t nervous tissue M ). In a p r e v i o u s s t r u c t u r a l a n a l y s i s o f c o c k r o a c h ?^ f r a c t i o n we had c o n f i r m e d t h e p r e s e n c e o f c o n s i d e r a b l e numbers o f i n t a c t synaptosomal p r o f i l e s which r e t a i n e d the c h a r a c t e r i s t i c morphology o f t e r m i n a l s w i t h i n the i n t a c t c o c k r o a c h nervous system [2). C o n t a m i n a t i n g fragments ( m i t o c h o n d r i a , l a r g e empty v e s i c l e s , e l o n g a t e d membranous p r o f i l e s and o t h e r c e l l u l a r d e b r i s ) a r e a l s o present. The f r a c t i o n was g e n t l y resuspended i n b u f f e r e d s u c r o s e (0.2ml) and then s l o w l y i n t r o d u c e d i n t o oxygenated i n s e c t s a l i n e (1.8ml) o f the f o l l o w i n g c o m p o s i t i o n : NaCl 1 2 . 5 g / l ; KC1 0 . 2 3 g / l C a C l . 6 H 0 0 . 4 5 g / l j N a ^ H P O .12hhG 0 . 8 5 g / l ; NaH^PG-^hUD 0 . 0 3 g / l ; MgCn.BH^O 1 . 3 g / l g l u c o s e 3gA. Synaptosomes suspended i n s a l i n e were i n c u b a t e d w i t h me t h y l p H ] - c h o l i n e f o r 15 minutes a t 30 C and d i s t r i b u t e d between twenty s u p e r f u s i o n u n i t s c o n t a i n i n g Whatman G F / B f i l t e r s . [ H ] - C h o l i n e l o a d e d synaptosomes were s u p e r f u s e d w i t h oxygenated s a l i n e t o wash out most o f the e x t r a s y n a p t o somal r a d i o a c t i v i t y . S a l i n e (20ml) was then added which c o n t a i n e d the n e u r o t o x i c a n t ( s ) . S u p e r f u s i o n r a t e s were m a i n t a i n e d a t 0 . 8 8 m l / m i n u t e u s i n g a p e r i s t a l t i c pump. Depending on the e x p e r i m e n t , 1 3 - 2 0 f r a c t i o n s were c o l l e c t e d . When the e f f e c t o f p r e - i n c u b a t i o n w i t h d e l t a m e t h r i n was examined, synaptosomes were exposed t o the p y r e t h r o i d i n s a l i n e 34 minutes p r i o r t o a c h a l l e n g e w i t h v e r a t r i d i n e p l u s p y r e t h r o i d . L i p o p h i l i c n e u r o t o x i c a n t s were added t o the s u p e r f u s i o n s a l i n e d i s s o l v e d i n a c e t o n e ( f i n a l c a r r i e r c o n c e n t r a t i o n 0.1%). R a d i o a c t i v i t y i n s u p e r f u s a t e samples was determined using 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 . 5
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Synaptosomes i s o l a t e d from the c e n t r a l nervous system o f the c o c k r o a c h and m a i n t a i n e d on f i l t e r p l a t f o r m s , as o u t l i n e d , show no d e t e r i o r a t i o n i n response t o the n e u r o t o x i c a n t s examined f o r a t l e a s t one hour a f t e r the s t a r t o f s u p e r f u s i o n . V a r i a b i l i t y within an experiment was l e s s than 8%, and t h e r e was good agreement between experiments c a r r i e d out on d i f f e r e n t d a y s . R e s u l t s and D i s c u s s i o n I s o l a t e d nerve endings (synaptosomes) i n s u p e r f u s i o n r e p r e s e n t an i d e a l p r e p a r a t i o n f o r the s t u d y o f n e u r o t o x i c a n t a c t i o n . Indeed the use o f such an approach t o examine t h e i n t e r a c t i o n o f pharmacol o g i c a l agents w i t h i o n c h a n n e l s i n v e r t e b r a t e synaptosomal p r e p a r a t i o n s i s w e l l e s t a b l i s h e d Î3), (4). It i s only r e l a t i v e l y r e c e n t l y t h a t f r a c t i o n a t i o n p r o c e d u r e s f o r i n v e r t e b r a t e synaptosomes have been o p t i m i z e d and s u p e r f u s i o n t e c h n i q u e s a p p l i e d t o t h e s t u d y o f drug and t o x i n a c t i o n i n i n s e c t s iî), [5) . In the p r e s e n t communication we have attempted t o c l a r i f y the mode o f a c t i o n o f c e r t a i n i n s e c t i c i d a l n e u r o t o x i c a n t s by d e t e r m i n i n g t h e i r e f f e c t s on t r a n s m i t t e r r e l e a s e i n the p r e s e n c e and absence o f c h a n n e l s p e c i f i c modulators. The e f f e c t s o f v e r a t r i d i n e on s u p e r f u s e d synaptosomes p r e l o a d e d w i t h p H ] - c h o l i n e a r e summarized i n F i g u r e s 1 and 2. Upon exposure o f the synaptosomal bed t o the a l k a l o i d t h e r e f o l l o w s a r a p i d i n c r e a s e i n the r a t e o f r e l e a s e o f l a b e l which peaks a f t e r ten minutes then d e c l i n e s r a p i d l y . V e r a t r i d i n e i s o f moderate p o t e n c y , p r o d u c i n g t h r e s h o l d e f f e c t s a t 1 0 ~ ° N and a maximal response a t 2 . 5 x 1 0 " M , the l a t t e r c o n c e n t r a t i o n a p p r o a c h i n g t h e 4
Hollingworth and Green; Sites of Action for Neurotoxic Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
264
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Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 25, 2018 | https://pubs.acs.org Publication Date: November 9, 1987 | doi: 10.1021/bk-1987-0356.ch019
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F i g u r e 1. The e f f e c t o f v e r a t r i d i n e (added a t arrow) on r e l e a s e o f l a b e l from s u p e r f u s e d i n s e c t synaptosomes l o a d e d w i t h [ HJ-choline. 3
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F i g u r e 2. The r e l a t i o n s h i p between v e r a t r i d i n e c o n c e n t r a t i o n and r e l e a s e o f n e u r o t r a n s m i t t e r . Reproduced w i t h p e r m i s s i o n R e f . 2. C o p y r i g h t 1985, S o c i e t y o f C h e m i c a l I n d u s t r y .
Hollingworth and Green; Sites of Action for Neurotoxic Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
19. NICHOLSON ET AL.
Insect Synaptosomes in Superfusion
265
l i m i t of i t s s o l u b i l i t y in saline. The v e r a t r i d i n e - s t i m u l a t e d e f f l u x o f l a b e l from t h e s y n a p t o s o m a l p r e p a r a t i o n c o u l d a r i s e from i t s c l a s s i c a l n e u r o t r a n s m i t t e r - r e l e a s i n g a c t i o n t h r o u g h sodium c h a n n e l a c t i v a t i o n , as d e s c r i b e d f o r mairmalian n e r v e t e r m i n a l s ( 4 ) . A l t e r n a t i v e l y i t c o u l d a c t by i n h i b i t i n g r e u p t a k e i n t o t h e t e r m i n a l s o f a n y [ H ] - c h o l i n e t h a t might d i f f u s e i n t o t h e e x t r a t e r m i n a l spaces o f the synaptosomal bed. C l e a r l y an i m p o r t a n t r e q u i r e m e n t i n any r e l e a s e s t u d y i s t h a t r e u p t a k e o f r e l e a s e d r a d i o l a b e l e d material i s prevented. I f t h i s i s not a c h i e v e d , i n h i b i t i o n o f r e u p t a k e may be m i s i n t e r p r e t e d as a r e l e a s i n g e f f e c t . Evidence from v e r t e b r a t e s u p e r f u s i o n e x p e r i m e n t s demonstrates t h a t r e u p t a k e o f r e l e a s e d n e u r o t r a n s m i t t e r s can be overcome i f synaptosomes a r e s u p e r f u s e d as a t h i n l a y e r a t a p p r o p r i a t e f l o w r a t e s (J5). In t h e p r e s e n t s t u d y i n h i b i t i o n o f r e u p t a k e o f ' d i f f u s i b l e ' c h o l i n e can be d i s c o u n t e d because h e m i c h o l i n i u m - 3 , w h i c h has been shown t o be an e f f e c t i v e i n h i b i t o r of [ H ] - c h o l i n e accumulation i n l o c u s t c e n t r a l n e r v e t e r m i n a l s [7), f a i l e d t o s t i m u l a t e r e l e a s e o f l a b e l a t 10"^M (Figure 3). F u r t h e r m o r e , t h e r e s u l t s show c l e a r l y t h a t t e t r o d o t o x i n , a s e l e c t i v e b l o c k e r o f sodium c h a n n e l s (j3), i s a v e r y p o t e n t i n h i b i t o r o f v e r a t r i d i n e - i n d u c e d r e l e a s e ( F i g u r e 4 ) , w i t h 90% s u p p r e s s i o n o c c u r r i n g a t an i n h i b i t o r c o n c e n t r a t i o n o f 10"^M. The h i g h l y s p e c i f i c n a t u r e o f t h e s e i n t e r a c t i o n s a r e f u r t h e r emphasized by t h e o b s e r v a t i o n t h a t p i c r o t o x i n i n which a c t s by b l o c k i n g c h l o r i d e c h a n n e l s (9) f a i l s t o i n f l u e n c e v e r a t r i d i n e - d e p e n d e n t r e l e a s e , even at high concentrations (Figure 5). In marrmalian s y n a p t o s o m a l p r e p a r a t i o n s low c o n c e n t r a t i o n s o f p y r e t h r o i d s have been shown t o s t i m u l a t e , i n a t e t r o d o t o x i n ^ s e n s i t i v e f a s h i o n , both sodium e n t r y (10) and r e l e a s e o f t h e n e u r o t r a n s m i t t e r GABA (JJ_). In view o f t h e s e e x p e r i m e n t s and t h e s t r o n g e l e c t r o p h y s i o l o g i c a l e v i d e n c e t h a t e x i s t s f o r the occurrence o f nerve t e r m i n a l d e p o l a r i z a t i o n i n i n s e c t s poisoned w i t h p y r e t h r o i d s (12), the a c t i o n o f d e l t a m e t h r i n was i n v e s t i g a t e d u s i n g t h e s u p e r f u s i o n t e c h n i q u e . Insect synaptosomes must be exposed t o d e l t a m e t h r i n a t h i g h c o n c e n t r a t i o n s (10"^M) t o o b s e r v e i m m é d i a t e e f f e c t s o r f o r much l o n g e r p e r i o d s a t l o w e r c o n c e n t r a t i o n s (10" N t o 10~ M) t o o b s e r v e s i g n i f i c a n t changes i n the r a t e o f t r a n s m i t t e r r e l e a s e i n t h i s system. In b o t h c a s e s the s t i m u l a t i o n s o b s e r v e d a r e a g r e a t d e a l weaker (
