Myotropic Neuropeptides - ACS Symposium Series (ACS Publications)

Chapter 6 Myotropic Neuropeptides Physiological and Pharmacological Actions 1

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 25, 2018 | https://pubs.acs.org Publication Date: January 25, 1991 | doi: 10.1021/bk-1991-0453.ch006

Benjamin J. Cook and Renée M. Wagner

Veterinary Toxicology and Entomology Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, TX 77840

Proctolin, the first peptide identified from insects, has a broad distribution among members of the class and does not appear to function as a conventional neurotransmitter. More recently, eight peptides of quite similar structure were isolated from head extracts of the cockroach Leucophaea maderae. Experiments with these peptides (Leucokinins I-VIII) on various types of visceral muscle showed a selective activation of the hindgut. Subnanomolar amounts of the leucokinins (3 x 10 M) caused an increase in the frequency and amplitude of hindgut contractions. The muscles of the foregut and oviduct were 100 to 1000 fold less sensitive to these peptides. The heart by comparison was either unresponsive or inconsistent. Another myotropic peptide leucopyrokinin showed a similar response profile with the same visceral muscles, but it caused a more consistent stimulation of the heart. A peptide (leucomyosuppressin) that inhibits spontaneous visceral muscle activity was also isolated from head extracts of L. maderae. This peptide suppresses muscle activity of the foregut and hindgut in the 5 to 9 x10 M)range. The other visceral organs were less responsive. -10

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I t has l o n g been r e c o g n i z e d t h a t muscle c e l l s p r o v i d e t h e i n s t r u m e n t a l means f o r body movement i n a n i m a l s . The c e l l u l a r b a s i s f o r t h i s a c t i v i t y e x i s t s i n the unique o r g a n e l l e s c a l l e d m y o f i b r i l s , w h i c h c o n s i s t o f a l t e r n a t i n g s t r a n d s o f two d i f f e r e n t p r o t e i n m o l e c u l e s known as a c t i n and myosin. These m y o f i b r i l s a r e o r i e n t e d p a r a l l e l t o t h e l o n g i t u d i n a l a x i s o f t h e e l o n g a t e muscle c e l l and shorten i n the process o f c o n t r a c t i o n to generate a mechanical Current address: Agricultural Research Service, U.S. Department of Agriculture, Beltsville, M D 20705 This chapter not subject to U.S. copyright Published 1991 American Chemical Society

Menn et al.; Insect Neuropeptides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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force. The s t i m u l i t h a t i n i t i a t e t h e s e c o n t r a c t i o n s c a n o c c u r i n t h r e e ways: 1) by n e r v e i m p u l s e s t r a n s m i t t e d t o t h e s u b s y n a p t i c membrane on t h e s u r f a c e o f t h e muscle f i b e r ; 2) by s u b s t a n c e s t h a t c i r c u l a t e i n t h e b l o o d and i n t e r a c t w i t h t h e s u r f a c e membrane; and 3) by spontaneous changes i n t h e l e v e l o f e x c i t a b i l i t y w i t h i n t h e muscle i t s e l f . I n each o f t h e s e c a s e s , a c h e m i c a l messenger i s r e q u i r e d a t some p o i n t d u r i n g t h e p r o c e s s o f s t i m u l a t i o n . F o r example, when an e l e c t r i c a l impulse r e a c h e s t h e n e r v e t e r m i n a l i n v e r t e b r a t e muscle a c e t y l c h o l i n e i s r e l e a s e d i n t o t h e s y n a p t i c gap. Once t h i s messenger a c t i v a t e s r e c e p t o r p r o t e i n s i n t h e membrane o f the muscle f i b e r , t h e f o l l o w i n g sequence o f e v e n t s t a k e s p l a c e : 1) s p e c i f i c i o n c h a n n e l s a r e opened; 2) an e l e c t r i c a l s i g n a l i s g e n e r a t e d which p r o p a g a t e s a l o n g the s u r f a c e o f t h e f i b e r ; and 3) the r e l e a s e o f i n t r a c e l l u l a r c a l c i u m c a u s e s a s h o r t e n i n g o f t h e myofilaments. I n r e c e n t y e a r s , a whole a r r a y o f c h e m i c a l messengers w h i c h c o n t r o l o r r e g u l a t e muscle f u n c t i o n have been i d e n t i f i e d and d e s c r i b e d i n i n s e c t s . These s u b s t a n c e s have been v a r i o u s l y d e s c r i b e d as n e u r o t r a n s m i t t e r s , n e u r o m o d u l a t o r s , and neurohormones i n t h e c o n t e x t o f some p a r t i c u l a r n e u r o p h y s i o l o g i c a l p r e p a r a t i o n under i n v e s t i g a t i o n . However, i t i s now c l e a r t h a t a s p e c i f i c c h e m i c a l e f f e c t o r may f u n c t i o n as a n e u r o t r a n s m i t t e r a t one n e r v e - m u s c l e j u n c t i o n and as a neuromodulator a t a n o t h e r . Thus i t has become c o n v e n t i o n a l t o s i m p l y r e f e r t o such n e u r o c h e m i c a l s as t r a n s m i t t e r s u b s t a n c e s , and t h e n e u r o p e p t i d e s have q u i c k l y become the l a r g e s t group o f such c h e m i c a l s ( 1 ) . Moreover, t h e s t u d y o f t h e a c t i o n s o f t h e s e p e p t i d e s has opened a new d i m e n s i o n t o t h e e v e r w i d e n i n g spectrum o f c h e m i c a l communication t h a t seems t o r e g u l a t e insect behavior. The f o l l o w i n g p r o p e r t i e s o u t l i n e t h e d i s t i n c t i v e f u n c t i o n a l a s p e c t s o f t h i s group o f c h e m i c a l messengers ( 2 , 3 ) : 1) the e v e n t s a c t i v a t e d by p e p t i d e s g e n e r a l l y have a t h r e s h o l d i n t h e nanomolar ( r a t h e r t h a n t h e m i c r o m o l a r ) range; 2) n e u r o p e p t i d e s o f t e n i n i t i a t e a s u s t a i n e d r e g u l a t i o n o f some type o f complex a c t i v i t y o r cause a l o n g - l a s t i n g i n c r e a s e i n e x c i t a b i l i t y ; 3) many p e p t i d e s a r e r e l e a s e d from neurohemal r e g i o n s a l o n g axons o r a t n e u r o s e c r e t o m o t o r e n d i n g s , r a t h e r t h a n a t s y n a p t i c j u n c t i o n s ; and 4) p e p t i d e s c a n a c t i v a t e r e c e p t o r s on c e l l s q u i t e remote from t h e p o i n t o f t h e i r release. A l t h o u g h many o f t h e e a r l i e r s t u d i e s on a n i m a l e t h o l o g y and n e u r o p h y s i o l o g y gave an i n a d e q u a t e a c c o u n t o f t h e r o l e t h a t muscles have i n b e h a v i o r , i t i s now r e c o g n i z e d t h a t t h e s e organs a r e t h e p r i n c i p l e avenues f o r the e x p r e s s i o n o f n e a r l y a l l v a r i a t i o n s i n a n i m a l b e h a v i o r ( 4 ) . Moreover, r e c e n t s t u d i e s have shown t h a t even a s i n g l e muscle c a n be the c h a n n e l f o r e x p r e s s i n g d i f f e r e n t p a t t e r n s of behavior. These d i f f e r e n c e s a r e r e f l e c t e d n o t so much i n t h e geometry o f i n n e r v a t i o n , as i n t h e c a p a c i t y o f n e r v e t e r m i n a l s t o r e l e a s e more t h a n a s i n g l e c h e m i c a l e f f e c t o r . Each t r a n s m i t t e r s u b s t a n c e seems t o evoke i t s own u n i q u e p a t t e r n o f c o n t r a c t i l e events. Such a f a c t i s p a r t i c u l a r l y w e l l i l l u s t r a t e d i n t h e e x t e n s o r - t i b i a e o f t h e h i n d l e g o f t h e l o c u s t ( 5 ) . T h i s muscle i s i n n e r v a t e d by t h r e e i d e n t i f i e d motor neurons and one i d e n t i f i e d m o d u l a t o r y neuron. A s m a l l group o f f i b e r s a t t h e p r o x i m a l end o f the muscle e x h i b i t s a spontaneous and r h y t h m i c p a t t e r n o f c o n t r a c t i o n and r e l a x a t i o n . A p p a r e n t l y t h i s myogenic rhythm h e l p s t o pump b l o o d and a i r i n t o t h e l a r g e jumping l e g o f t h e l o c u s t .


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C O O K AND WAGNER

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Although these rhythmic c o n t r a c t i o n s o f the e x t e n s o r - t i b i a e a r e i n i t i a t e d by spontaneous d e p o l a r i z a t i o n s i n c e r t a i n f i b e r s w i t h i n the muscle, t h e y a r e r e g u l a t e d by a s p e c i f i c c e l l on t h e d o r s a l s u r f a c e o f the metathoracic g a n g l i o n . T h i s c e l l i s found i n a c l u s t e r o f n e u r o s e c r e t o r y c e l l s c a l l e d t h e d o r s a l u n p a i r e d median (DUM) neurons. Neuromuscular t r a n s m i s s i o n and m u s c u l a r c o n t r a c t i o n i n t h e e x t e n s o r - t i b i a e muscle i s modulated b y t h e b i o g e n i c amine octopamine and by t h e p e p t i d e s , p r o c t o l i n , and FMRFamide. Octopamine and p r o c t o l i n a r e c o n t a i n e d w i t h i n neurons w h i c h i n n e r v a t e t h e muscle, w h i l e FMRFamide- and s m a l l c a r d i o a c t i v e p e p t i d e ( S C P ) - l i k e p e p t i d e s appear t o be r e l e a s e d i n t o t h e l o c u s t hemolymph as neurohormones. P r o c t o l i n speeds up t h e myogenic rhythm and g e n e r a t e s an i n c r e a s e i n b a s a l t e n s i o n , w h i l e octopamine r e d u c e s the f r e q u e n c y o f t h e rhythm. I n a d d i t i o n , t h e rhythm i s s t i m u l a t e d by such p e p t i d e s as t h e a d i p o k i n e t i c hormone ( A K H ) - r e l a t e d p e p t i d e s M and M and b y t h e s m a l l c a r d i o a c t i v e p e p t i d e (SCP ) . The f u l l 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 o f such a d i v e r g e n t s e t o f p e p t i d e s t h a t c a n a c t i v a t e t h e myogenic rhythm o f t h i s muscle i s d i f f i c u l t t o a s s e s s , o t h e r t h a n t o s a y t h a t they must i n some way f i n e - t u n e b e h a v i o r a l commands and r e s p o n s e s i n t h e l i v i n g s t a t e . The m a j o r i t y o f i n s e c t n e u r o p e p t i d e s d i s c o v e r e d thus f a r seem to have t h e p r o p e r t y o f r e g u l a t i n g t h e c o n t r a c t i l e a c t i v i t y o f e i t h e r s k e l e t a l and/or v i s c e r a l muscles. Whether t h e s e a r e t h e p r i n c i p a l o r a u x i l i a r y modes o f a c t i o n f o r a g i v e n p e p t i d e must a w a i t a more comprehensive assessment o f t h e i r i n d i v i d u a l p r o f i l e s o f a c t i v i t y i n a v a r i e t y o f p h y s i o l o g i c a l systems. What f o l l o w s i s a summary o f o u r c u r r e n t u n d e r s t a n d i n g o f t h e p h y s i o l o g i c a l and pharmacological p r o p e r t i e s o f the i n s e c t neuropeptides that r e g u l a t e muscle a c t i v i t y .


B

x

2

B

Proctolin The d i s c o v e r y o f t h e n e u r o p e p t i d e p r o c t o l i n was t h e f r u i t f u l consequence o f B r i a n Brown's s u s t a i n e d i n t e r e s t i n p h a r m a c o l o g i c a l l y a c t i v e agents t h a t a f f e c t v i s c e r a l muscles o f t h e c o c k r o a c h P e r i p l a n e t a americana. I n 1967, he r e p o r t e d (6) on t h e p r e s e n c e o f an u n i d e n t i f i e d s u b s t a n c e i n e x t r a c t s o f t h e h i n d g u t w h i c h c a u s e d a slow type graded c o n t r a c t i o n o f t h e l o n g i t u d i n a l muscles o f t h a t organ. N e u r a l l y evoked c o n t r a c t i o n s were q u i t e s i m i l a r and t h i s " g u t - f a c t o r " was d e p l e t e d from t h e h i n d g u t a f t e r s u r g i c a l s e c t i o n o f the p r o c t o d e a l n e r v e . Moreover, i t s s p e c i f i c a c t i v i t y ( b i o a c t i v e u n i t s p e r m i l l i g r a m p r o t e i n ) was a p p r o x i m a t e l y 25 t i m e s h i g h e r i n the p r o c t o d e a l and stomodeal n e r v e s t h a n i n t h e v i s c e r a t h a t t h e y innervate. A l t h o u g h t h e s u b s t a n c e was h e a t s t a b l e and d i a l y z a b l e , i t was n o t i n a c t i v a t e d by c h y m o t r y p s i n . In an e f f o r t t o o b t a i n more i n f o r m a t i o n on t h e n a t u r e o f t h i s s u b s t a n c e , Holman and Cook (7) attempted t o i s o l a t e and c h a r a c t e r i z e the a c t i v e agent from t h e c o c k r o a c h Leucophaea maderae. Three s u b s t a n c e s were found i n t h e s e e x t r a c t s t h a t s t i m u l a t e t h e i s o l a t e d hindgut. Two o f t h e compounds were i d e n t i f i e d as L - g l u t a m i c a c i d and L - a s p a r t i c a c i d . When t h e h i n d g u t was exposed t o t h e s e amino a c i d s , a s i n g l e slow c o n t r a c t i o n r e s u l t e d which was i n d i s t i n g u i s h a b l e from a n e u r a l l y evoked r e s p o n s e . The t h i r d s u b s t a n c e , however, c a u s e d a complex s e r i e s o f changes i n t h e



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ACTION

f r e q u e n c y and a m p l i t u d e o f r h y t h m i c c o n t r a c t i o n s and the c h a r a c t e r o f t h i s r e s p o n s e was r e m a r k a b l y s i m i l a r t o t h a t r e p o r t e d by Davey (8) and Brown (9) f o r e x t r a c t s o f the c o r p u s c a r d i a c u m (CC). F u r t h e r s t u d y o f t h i s t h i r d m a t e r i a l by Holman and Cook (10) showed t h a t i t s b i o l o g i c a l a c t i v i t y was d e s t r o y e d by p r o n a s e , b u t n o t by chymotrypsin. Moreover, t h i s p e p t i d e was found i n e x t r a c t s o f h i n d g u t s , t e r m i n a l g a n g l i a , p r o c t o d e a l n e r v e s , and heads o f L. maderae. Once Brown and S t a r r a t t (11,12,13) i s o l a t e d and i d e n t i f i e d p r o c t o l i n ( A r g - T y r - L e u - P r o - T h r ) from the c o c k r o a c h P. a m e r i c a n a . i t was p o s s i b l e f o r n e u r o b i o l o g i s t s t o commence s t u d i e s on the p h y s i o l o g i c a l and p h a r m a c o l o g i c a l a c t i o n s o f p r o c t o l i n i n many insects. I t was q u i c k l y r e c o g n i z e d t h a t n o t o n l y v i s c e r a l m u s c l e s , b u t a l s o s k e l e t a l muscles, were s t i m u l a t e d by p r o c t o l i n . O'Shea and Adams (14) have r e c e n t l y p r o v i d e d an e x c e l l e n t r e v i e w o f the d e t a i l s o f t h i s p e p t i d e ' s a c t i v a t i o n o f s k e l e t a l muscle, a l o n g w i t h many other important h i g h l i g h t s . The commercial a v a i l a b i l i t y o f s y n t h e t i c p r o c t o l i n a l s o p r o v i d e d the o c c a s i o n t o compare the 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 i c (HPLC) p r o f i l e o f p r o c t o l i n w i t h p r e v i o u s l y r e p o r t e d p e p t i d e s o f undetermined s t r u c t u r e . Holman and Cook (15) f o u n d by HPLC f r a c t i o n a t i o n and q u a n t i t a t i v e b i o a s s a y t h a t the p r o c t o l i n - l i k e a c t i v i t y r e p o r t e d e a r l i e r i n e x t r a c t s o f the f o r e g u t and h i n d g u t o f L. maderae was the r e s u l t o f t h a t p e p t i d e . However, the h i n d g u t s t i m u l a t i n g neurohormone (HSN) r e p o r t e d i n head e x t r a c t s o f the same c o c k r o a c h c o u l d n o t be a c c o u n t e d f o r as proctolin. Thus a n o t h e r m y o t r o p i c n e u r o p e p t i d e must have been r e s p o n s i b l e f o r the i n v i t r o s y n t h e s i s (16) and r e l e a s e (17) o f HSN a c t i v i t y from the b r a i n - C C complex o f L. maderae. Indeed, the development o f a r e v e r s e - p h a s e HPLC system e v e n t u a l l y p r o v i d e d a c l e a r s e p a r a t i o n o f f i v e a c t i v e f r a c t i o n s from t h e s e head e x t r a c t s (18). T h i s f i n d i n g r e s u l t e d i n the p u r i f i c a t i o n and p r i m a r y s t r u c t u r e d e t e r m i n a t i o n o f 11 m y o t r o p i c p e p t i d e s (19-25), and o f one that suppresses c o n t r a c t i l e a c t i v i t y (26). E v i d e n c e o f the i n v o l v e m e n t o f p r o c t o l i n i n the r e g u l a t o r y mechanisms o f i n s e c t nerve-muscle systems has c o n t i n u e d t o grow and the remainder o f t h i s s e c t i o n d i s c u s s e s t h e s e more r e c e n t developments and t h e i r s i g n i f i c a n c e . Proctolin-like immunoreactive ( P L I ) neurons have been d e t e c t e d i n the b l o w f l y , C a l l i p h o r a e r v t h r o c e p h a l a ( 2 7 ) ; the C o l o r a d o p o t a t o b e e t l e , L e p t i n o t a r s a d e c e m l i n e a t a ( 2 8 ) ; and the f r u i t f l y l a r v a e , D r o s o p h i l a m e l a n o g a s t e r (29). I n the blow f l y , 80-90 neurons i n the b r a i n - s u b o e s o p h a g e a l complex were f o u n d t o be P L I . The t h o r a c i c g a n g l i a , by comparison, c o n t a i n e d 100-130 PLI neurons, w h i l e the abdominal s e c t i o n had o n l y 60. These neurons were o f d i f f e r e n t t y p e s : i n t e r n e u r o n s , motor neurons, and n e u r o s e c r e t o r y c e l l s . A small, stereotyped population o f PLI neurons was found i n the D r o s o p h i l a l a r v a l c e n t r a l nervous system (CNS). I n the p e r i p h e r y , p r o c t o l i n - i m m u n o r e a c t i v e n e u r o m u s c u l a r e n d i n g s were i d e n t i f i e d on b o t h v i s c e r a l and s k e l e t a l muscle f i b e r s . On the h i n d g u t , the n e u r o p e p t i d e i s a s s o c i a t e d w i t h e n d i n g s on i n t r i n s i c c i r c u l a r muscle f i b e r s . I n t h i s s t u d y , the p r e s e n c e o f p r o c t o l i n was v e r i f i e d i n the CNS, h i n d g u t , and segmental body w a l l by t i s s u e e x t r a c t i o n f o l l o w e d by r e v e r s e - p h a s e HPLC and q u a n t i t a t i v e b i o a s s a y . E v i d e n c e f o r a p r o c t o l i n - l i k e s u b s t a n c e has a l s o been f o u n d i n the a d u l t C o l o r a d o p o t a t o b e e t l e



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COOK AND WAGNER


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(28). Numerous PLI neurons o c c u r i n a l l v e n t r a l g a n g l i a and i n t h e f r o n t a l ganglion. Two groups o f n e u r o s e c r e t o r y c e l l s i n t h e s u b o e s o p h a g e a l g a n g l i o n c o n t a i n a PLI s u b s t a n c e . I n these c e l l s , t h i s s u b s t a n c e i s c o - l o c a l i z e d w i t h FMRF-amide-like m a t e r i a l . Nerve t e r m i n a l s on t h e m u s c u l a t u r e o f the f o r e - and h i n d - g u t and o f t h e vas d e f e r e n s , and on c e r t a i n segmental muscles show e v i d e n c e o f PLI. Upon f r a c t i o n a t i o n o f e x t r a c t s o f t h e n e r v o u s system o f L e p t i n o t a r s a d e c e m l i n e a t a by HPLC, most o f t h e p r o c t o l i n - l i k e b i o a c t i v e m a t e r i a l co-migrated with authentic p r o c t o l i n . Obviously such d e t a i l e d mapping and/or l o c a l i z a t i o n o f neurons b y immunocytochemical t e c h n i q u e s c a n be o f g r e a t a s s i s t a n c e t o t h e n e u r o b i o l o g i s t who i s a n a l y z i n g nerve-muscle systems t h a t i n v o l v e p r o c t o l i n e r g i c neurons. The p r e s e n c e o f p r o c t o l i n i n t h e r e p r o d u c t i v e t r a c t o f a v a r i e t y o f i n s e c t s and t h e a c t i o n o f t h i s p e p t i d e on s u c h t i s s u e s has been a c u r r e n t f o c u s f o r many s t u d i e s . The v i s c e r a l m u s c l e s o f the o v i d u c t s o f L o c u s t a m i g r a t o r i a . f o r example, a r e r e m a r k a b l y s e n s i t i v e to p r o c t o l i n (30). Amounts o f t h e p e p t i d e as low as 2 f m o l induce a t o n i c c o n t r a c t i o n t h a t i s dose-dependent up t o 200 f m o l . A p r o c t o l i n - l i k e immunoreactive s u b s t a n c e was f o u n d i n the axons o f n e r v e s l e a d i n g t o the o v i d u c t as w e l l as i n a number o f c e l l b o d i e s i n t h e V I I t h abdominal g a n g l i o n . Moreover, r e v e r s e - p h a s e HPLC o f t i s s u e e x t r a c t s o f o v i d u c t s , o v i d u c a l n e r v e s , and t h e V l l t h abdominal g a n g l i a i n d i c a t e t h a t t h e p r o c t o l i n - l i k e b i o a c t i v e substance c o - e l u t e s with a u t h e n t i c p r o c t o l i n . The p e p t i d e was p r e s e n t i n a r e a s o f t h e o v i d u c t t h a t r e c e i v e e x t e n s i v e i n n e r v a t i o n w h i l e i t was t e n - f o l d l e s s i n a r e a s t h a t r e c e i v e l i t t l e o r no i n n e r v a t i o n . The o v i d u c t s o f t h e c o c k r o a c h e s L. maderae (31) and P. a m e r i c a n a (32) a l s o c o n t a i n p r o c t o l i n . In both instances, q u a n t i t a t i v e e s t i m a t e s o f p r o c t o l i n - l i k e b i o a c t i v i t y were made f o l l o w i n g t h e s e p a r a t i o n o f e x t r a c t s on r e v e r s e - p h a s e HPLC. A f t e r d e p o l a r i z a t i o n o f the t i s s u e i n h i g h potassium s a l i n e , the p r o c t o l i n - l i k e s u b s t a n c e i n P. americana was r e l e a s e d from o v i d u c t s i n a c a l c i u m dependent f a s h i o n . O v i d u c t s i n L. maderae (31) showed some r e s p o n s i v e n e s s t o p r o c t o l i n i n a c a l c i u m - f r e e medium and t h e p e p t i d e a l s o appeared t o f a c i l i t a t e t h e r e - e n t r y o f c a l c i u m i n t o muscle a f t e r d e p l e t e d p r e p a r a t i o n s were r e t u r n e d t o normal l e v e l s o f external calcium. Two r e c e n t s t u d i e s have shown t h a t p r o c t o l i n has a p o t e n t s t i m u l a t i n g e f f e c t on t h e v i s c e r a l muscles o f t h e o v i d u c t i n Rhodnius p r o l i x u s (33,34) and i n the s t a b l e f l y Stomoxys c a l c i t r a n s (35). I n b o t h o f these i n s e c t s , r e v e r s e - p h a s e HPLC and q u a n t i t a t i v e b i o a s s a y were u s e d t o demonstrate the p r e s e n c e o f a p r o c t o l i n - l i k e substance i n e x t r a c t s o f the r e p r o d u c t i v e t i s s u e s . Immunohistochemical p r e p a r a t i o n s r e v e a l e d i n t e n s e p r o c t o l i n - l i k e i m m u n o r e a c t i v i t y i n t e r m i n a l a r b o r i s a t i o n s o f n e r v e s on t h e spermathecae, common o v i d u c t and b u r s a o f Rhodnius. The muscles o f the o v a r i a n s h e a t h i n s t a b l y f l y r e s p o n d e d t o p r o c t o l i n , and 0.5 t o 0.7 p i c o m o l e s o f p r o c t o l i n - l i k e b i o a c t i v i t y p e r o v a r y were d e t e c t e d i n e x t r a c t s . O v i d u c t e x t r a c t s o f t h e s t a b l e f l y by c o m p a r i s o n c o n t a i n e d 1.2 t o 1.7 p i c o m o l e s o f p r o c t o l i n p e r o r g a n . The a c c e s s o r y g l a n d t u b u l e s o f t h e male c r i c k e t G r y l l u s b i m a c u l a t u s (36) c o n t r a c t s p o n t a n e o u s l y , b u t they a l s o r e c e i v e


56

INSECT NEUROPEPTIDES: CHEMISTRY, BIOLOGY, AND

i n n e r v a t i o n from d o r s a l u n p a i r e d median neurons (DUMR7) t h a t a r i s e from the t e r m i n a l g a n g l i o n . When these DUMR7 neurons were e l e c t r i c a l l y s t i m u l a t e d , c o n t r a c t i o n s were evoked i n muscles o f the gland tubules. Glutamate a t 10~ M, and p r o c t o l i n , a t low c o n c e n t r a t i o n s o f 10~ M p r o d u c e d s u s t a i n e d c o n t r a c t i o n s o f the accessory gland. T h i s i n c r e a s e i n b a s a l tonus w i t h each s u b s t a n c e was a l s o dose-dependent. By use o f r e v e r s e - p h a s e HPLC and b i o a s s a y , p r o c t o l i n was d e t e c t e d i n a c c e s s o r y g l a n d e x t r a c t s and i n the s a l i n e medium s u r r o u n d i n g g l a n d s s u b j e c t e d t o h i g h p o t a s s i u m s a l i n e . P r o c t o l i n appears t o p o t e n t i a t e s y n a p t i c t r a n s m i s s i o n i n the c e n t r a l n e r v o u s system o f the c o c k r o a c h P. americana ( 3 7 ) . Bursts o f s p i k e a c t i v i t y i n the v e n t r a l n e r v e c o r d o f t h i s i n s e c t were e l i c i t e d by m e c h a n i c a l s t i m u l a t i o n o f the c e r e a l o r g a n s . I n the p r e s e n c e o f m i c r o m o l a r p r o c t o l i n , the peak f r e q u e n c y and the d u r a t i o n o f b u r s t s were s l o w l y , b u t s i g n i f i c a n t l y , i n c r e a s e d . C a r b a c o l , by comparison, c a u s e d an immediate enhancement o f spontaneous a c t i v i t y , b u t p o t e n t i a t i o n o f b u r s t s was n o t o b s e r v e d . Thus, i t was c o n c l u d e d t h a t p r o c t o l i n might f u n c t i o n as a n e u r o m o d u l a t o r i n the t e r m i n a l g a n g l i o n . A l t h o u g h c o n v i n c i n g e v i d e n c e has been p r e s e n t e d f o r a s p i k e - m e d i a t e d mechanism o f e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g i n i n s e c t v i s c e r a l muscle (38-41) , nanomolar amounts o f the n e u r o p e p t i d e p r o c t o l i n can cause s i z e a b l e c o n t r a c t i o n s i n p o t a s s i u m d e p o l a r i z e d hindgut p r e p a r a t i o n s (42). This observation suggested t h a t p e p t i d e s may a f f e c t the c o n t r a c t i l e elements o f muscle d i r e c t l y w i t h o u t the i n t e r v e n t i o n o f membrane p o l a r i z a t i o n . Indeed, the f i r s t s e r i o u s c h a l l e n g e t o the e x c l u s i v e n e s s o f the e l e c t r o m e c h a n i c a l t h e o r y o f c o u p l i n g i n v e r t e b r a t e i n t e s t i n a l smooth muscle o c c u r r e d when i t was d i s c o v e r e d t h a t o x y t o c i n , as w e l l as a c e t y l c h o l i n e and h i s t a m i n e , c o u l d produce c o n t r a c t i o n s i n p o t a s s i u m - d e p o l a r i z e d smooth muscle ( 4 3 ) . More r e c e n t r e s e a r c h has shown t h a t a t l e a s t two d i s t i n c t c a l c i u m transmembrane c h a n n e l s e x i s t i n the v i s c e r a l muscles o f the h i n d g u t o f the c o c k r o a c h L. maderae (44) and the o v i d u c t o f the l o c u s t L. m i g r a t o r i a ( 4 5 ) : 1) a v o l t a g e dependent c h a n n e l which i n i t i a t e s p h a s i c c o n t r a c t i o n s i n t h e s e muscles t h r o u g h the g e n e r a t i o n o f a c t i o n p o t e n t i a l s ; and 2) an a g o n i s t - o p e r a t e d c h a n n e l which may be a s s o c i a t e d w i t h l i t t l e o r no change i n membrane p o t e n t i a l . A l t h o u g h c a l c i u m appears t o e n t e r the muscle c e l l s by two c h a n n e l s i n b o t h o f t h e s e p r e p a r a t i o n s , d i f f e r e n t i n t r a c e l l u l a r messenger systems a r e l i n k e d t o the membrane receptors for proctolin. I n the h i n d g u t o f L. maderae (46.47) c y c l i c n u c l e o t i d e s a r e i n v o l v e d , w h i l e i n the l o c u s t o v i d u c t (48) i n o s i t o l p h o s p h o l i p i d h y d r o l y s i s seems t o be the m e d i a t o r o f the physiological action of proctolin. An i n t e r e s t i n g d i s c o v e r y about the a d e n y l a t e c y c l a s e enzyme l o c a t e d i n the h i n d g u t o f L. maderae (49) was t h a t i t r e s p o n d e d t o i n c r e a s i n g amounts o f p r o c t o l i n i n a b i p h a s i c manner. Initially p r o c t o l i n (10 pM) c a u s e d a 50% s t i m u l a t i o n o f a d e n y l a t e c y c l a s e a c t i v i t y and as the p e p t i d e c o n c e n t r a t i o n r e a c h e d 50 pM, the enzyme a c t i v i t y r e a c h e d a v a l u e 700% above b a s a l a c t i v i t y . When a p e p t i d e c o n c e n t r a t i o n o f 100 pM was a t t a i n e d the enzyme a c t i v i t y dropped b a c k t o 50% above b a s a l a c t i v i t y b u t as the c o n c e n t r a t i o n o f p r o c t o l i n r e a c h e d 1 nM the enzyme a c t i v i t y a g a i n peaked a t a v a l u e o f 700% above the b a s a l l e v e l . These two peaks o f a d e n y l a t e c y c l a s e 4

9


ACTION



6.


COOK AND WAGNER

57

a c t i v i t y p a r a l l e l c l o s e l y the c o n c e n t r a t i o n o f p r o c t o l i n r e q u i r e d t o evoke two t y p e s o f m u s c u l a r - c o n t r a c t i o n i n the h i n d g u t : 1) an i n c r e a s e i n the f r e q u e n c y and a m p l i t u d e o f p h a s i c c o n t r a c t i o n s (50 pM); and 2) l a r g e i n c r e a s e s i n t o n i c c o n t r a c t i o n s (1 nM). The 14 y e a r s t h a t have e l a p s e d s i n c e the d i s c o v e r y o f p r o c t o l i n have p r o v i d e d many i n v e r t e b r a t e n e u r o b i o l o g i s t s the o p p o r t u n i t y t o a s s e s s the p h y s i o l o g i c a l a c t i o n s o f t h i s p e p t i d e ; i n many r e s p e c t s , t h e s e s t u d i e s have l e d the way i n i n c r e a s i n g our u n d e r s t a n d i n g o f how p e p t i d e s f u n c t i o n i n a l i v i n g system. The accomplishments o f t h i s p e r i o d c a n b e s t be summarized by the f o l l o w i n g b r i e f s t a t e m e n t s : 1) p r o c t o l i n i n subnanomolar amounts c a n a c t i v a t e b o t h v i s c e r a l and s k e l e t a l muscles f o r extended p e r i o d s o f time; 2) p r o c t o l i n can o f t e n c o e x i s t w i t h other chemical e f f e c t o r s i n a s i n g l e neuron, and as a c o t r a n s m i t t e r may impart i t s own unique c h a r a c t e r t o muscle a c t i o n ; 3) many o f the r e p o r t e d p h y s i o l o g i c a l a c t i o n s o f p r o c t o l i n v i o l a t e the t r a d i t i o n a l d e f i n i t i o n o f a t r a n s m i t t e r s u b s t a n c e ; 4) p r o c t o l i n c a n a c t i v a t e muscle c e l l s t h r o u g h v o l t a g e dependent as w e l l as r e c e p t o r o p e r a t e d c h a n n e l s ; and 5) the mode o f a c t i o n o f p r o c t o l i n i s n o t u n i v e r s a l b u t d i v e r s e and m u l t i p h a s i c w i t h each i n s e c t nerve-muscle r e s p o n d i n g i n i t s own u n i q u e way. Leucokinins The l e u c o k i n i n s (LK's) a r e a new c l a s s o f i n s e c t m y o t r o p i c n e u r o p e p t i d e s i s o l a t e d from head e x t r a c t s o f the c o c k r o a c h L. maderae. These o c t a p e p t i d e s a l l c o n t a i n a s i m i l a r c o r e sequence o f 5 amino a c i d s t h a t e x t e n d from p o s i t i o n 4 t h r o u g h 8. T h i s sequence Phe-X-Ser-Trp-Gly-NH seems t o be r e q u i r e d f o r h i n d g u t stimulation. The i n i t i a l r e s p o n s e o f the h i n d g u t t o the LK's was c h a r a c t e r i z e d by an i n c r e a s e i n the f r e q u e n c y and/or a m p l i t u d e o f p h a s i c c o n t r a c t i o n s (50,51). At higher peptide concentrations, a t o n i c component was g e n e r a l l y p r e s e n t . A l l o f the LK's showed a r e s p o n s e a t 3 x 10" M t h a t was 5-10% above the mean l e v e l o f spontaneous a c t i v i t y . The maximum r e s p o n s e f o r each o f the p e p t i d e s was r e c o r d e d a t a c o n c e n t r a t i o n 2.1 X 10" M. Thus, t h e i n t r i n s i c a c t i v i t i e s f o r the LK's a r e n e a r l y e q u a l because the d o s e - r e s p o n s e c u r v e s have about the same a s y m p t o t i c l i m i t s . A comparison o f the dose c o n c e n t r a t i o n s t h a t gave a h a l f maximal r e s p o n s e ( E D ) f o r the 8 p e p t i d e s i s shown i n T a b l e I . 2

19

7

50

In a d d i t i o n t o t h e s e r e s p o n s e s , each o f the LK's c a u s e d a p r o t r a c t e d e x c i t a t i o n o f c o n t r a c t i l e events i f hindgut p r e p a r a t i o n s were a l l o w e d t o remain i n s a l i n e s o l u t i o n s c o n t a i n i n g the p e p t i d e ( F i g u r e 1 ) . A l l e i g h t o f the LK's evoked c o n t r a c t i o n s from h i n d g u t s a f t e r membrane d e p o l a r i z a t i o n s w i t h 158 mM p o t a s s i u m . This offers c l e a r e v i d e n c e f o r the p r e s e n c e o f r e c e p t o r - o p e r a t e d c h a n n e l s f o r the LK's i n the h i n d g u t . Moreover, the f a c t t h a t h i n d g u t s exposed to a c a l c i u m f r e e medium f a i l e d t o r e s p o n d t o the p e p t i d e s s u g g e s t s t h a t t h e s e r e c e p t o r - o p e r a t e d c h a n n e l s a r e c a l c i u m dependent. A l t h o u g h the h i n d g u t showed h i g h s e n s i t i v i t y t o the LK's, o t h e r t y p e s o f v i s c e r a l muscle were l e s s r e s p o n s i v e . The f o r e g u t and o v i d u c t , f o r example, were 100-1000 f o l d l e s s s e n s i t i v e t h a n the hindgut. A c o n c e n t r a t i o n o f more t h a n 10" M o f each o f the p e p t i d e s was r e q u i r e d t o e l i c i t a d e t e c t a b l e r e s p o n s e from t h e s e 8


58 Table

INSECT NEUROPEPTIDES: CHEMISTRY, BIOLOGY, AND ACTION I.

C h e m i c a l p r o p e r t i e s o f t h e l e u c o k i n i n s from L. maderae and t h e i r p h a r m a c o l o g i c a l a c t i o n on t h e h i n d g u t

Threshold o f synthetic Leucokinins pmol/head* Structure* product* LK I 0.48 DPAFNSWG-NH 2.0 x 10" *! LK I I 0.35 DPGFSSWG-NH 1.6 x 10" M LK I I I 0.22 DQGFNSWG-NH 7.2 x 10- M LK IV 0.23 DASFHSWG-NH 1.4 x 10" *! LK V 0.17 GSGFSSWG-NH 5.2 x 10" *! LK V I 0.16 pQSSFHSWG-NH 5.6 x 10- M LK V I I 0.09 DPAFSSWG-NH 1.2 x 10" M LK V I I I 0.06 GADFYSWG-NH 2.9 x 10~ M * R e f e r e n c e s 19, 20, 22, and 23. f R e f e r e n c e s 50 and 51. 10

2

10

2

n

2

10

2

11


2

n

2

10

2

n

2

3.6 1.5 4.3 2.9 8.6 3.3 6.2 7.7

ED, x 10" Mt x lO^Mf x lO^Mf x 10" Mt x 10- Mt x 10" Mt x 10" Mt x 10- Mt 9

9

10

9

9

10

organs. The h e a r t b y comparison e i t h e r gave i n c o n s i s t e n t r e s p o n s e s or f a i l e d t o respond t o the peptides. An u n e x p e c t e d b i o l o g i c a l a c t i v i t y f o r t h e LK's was r e c e n t l y discovered i n studies o f i s o l a t e d malpighian tubules o f the yellow f e v e r mosquito Aedes a e g y p t i ( 5 2 ) . Each o f t h e LK's a t 100 pmol caused a d e p o l a r i z a t i o n o f the t r a n s e p i t h e l i a l v o l t a g e o f these tubules. L e u c o k i n i n V I I I , f o r example, i n h i b i t e d t r a n s e p i t h e l i a l f l u i d s e c r e t i o n a t low c o n c e n t r a t i o n s (10" M) and s t i m u l a t e d f l u i d s e c r e t i o n a t h i g h c o n c e n t r a t i o n s (3.5 X 10" M). Together, the d e p o l a r i z i n g e f f e c t s on v o l t a g e and t h e s t i m u l a t i o n o f f l u i d s e c r e t i o n s u g g e s t t h a t LK's i n c r e a s e t h e c h l o r i d e p e r m e a b i l i t y o f the t u b u l e w a l l . S t r u c t u r e - f u n c t i o n s t u d i e s suggest t h a t the a c t i v e r e g i o n o f the octapeptide i s l a r g e l y r e s t r i c t e d t o the C-terminal pentapeptide. n

9

Leucopyrokinin L e u c o p y r o k i n i n (pQTSFTPRL-NH ) was t h e most abundant o f t h e m y o t r o p i c n e u r o p e p t i d e s found i n head e x t r a c t s o f L. maderae. Each head e q u i v a l e n t c o n t a i n e d 1.36 pmol o f t h e p e p t i d e , a v a l u e much l e s s than that obtained f o r e i t h e r M o r M . When t h e h i n d g u t was exposed t o l e u c o p y r o k i n i n (LPK), a pronounced e l e v a t i o n i n t h e a m p l i t u d e and f r e q u e n c y o f p h a s i c c o n t r a c t i o n s was o f t e n e v i d e n t , and o n l y a s m a l l change i n tonus was d e t e c t e d even a t h i g h e r concentrations o f the peptide. T h i s was i n marked c o n t r a s t t o t h e more e v i d e n t changes i n tonus evoked b y p r o c t o l i n and t h e l e u c o k i n i n s i n a comparable c o n c e n t r a t i o n range. LPK c a u s e d an i n i t i a l b u r s t o f p o t e n t i a t i o n i n n e u r a l l y evoked c o n t r a c t i o n s o f t h e h i n d g u t b u t t h i s was f o l l o w e d b y an i n t e r m i t t e n t p a t t e r n o f p o t e n t i a t i o n s t h a t was q u i t e u n l i k e t h e u n i f o r m type o b s e r v e d w i t h p r o c t o l i n (Wagner and Cook, u n p u b l i s h e d information). A l t h o u g h t h e f o r e g u t and o v i d u c t o f L. maderae were about 100 times l e s s s e n s i t i v e t o LPK t h a n t h e h i n d g u t , t h e c h a r a c t e r o f t h e r e s p o n s e was s i m i l a r . When t h e s e t i s s u e s were exposed t o t h e p e p t i d e , t h e most e v i d e n t r e s p o n s e was an i n c r e a s e i n t h e a m p l i t u d e and f r e q u e n c y o f c o n t r a c t i o n s , w i t h l i t t l e i n d i c a t i o n o f any change 2

x

2


6.

COOK AND WAGNER


59

i n tonus. The r e s p o n s e o f t h e h e a r t t o LPK was f a i r l y c o n s i s t e n t . A p p l i c a t i o n o f t h e p e p t i d e i n c o n c e n t r a t i o n s between 10" M and 5 x 10" M c a u s e d an i n c r e a s e i n t h e a m p l i t u d e and f r e q u e n c y o f myocardial contractions. However, when t h e h e a r t was exposed t o 10" M LPK a marked s u p p r e s s i o n i n a m p l i t u d e was o f t e n o b s e r v e d . 9

8

7

Leucomyosuppressin L e u c o m y o s u p p r e s s i n (pQDVDHVFLRF-NH ) was t h e f i r s t s t r u c t u r a l l y i d e n t i f i e d n e u r o p e p t i d e t h a t i n h i b i t s muscular c o n t r a c t i o n s i n i n s e c t s ( 2 6 ) . The r e s p o n s e o f t h e i s o l a t e d h i n d g u t o f L. maderae t o l e u c o m y o s u p p r e s s i n (LMS) was dose dependent and r e v e r s i b l e . Both the f r e q u e n c y and a m p l i t u d e o f spontaneous c o n t r a c t i o n s were r e d u c e d n e a r t h e t h r e s h o l d c o n c e n t r a t i o n f o r t h e p e p t i d e (9 x 1 0 " M ) . N e u r a l l y evoked c o n t r a c t i o n s o f t h e h i n d g u t were a l s o s u p p r e s s e d by LMS i n a dose dependent manner (Cook and Wagner, u n p u b l i s h e d information). However, r e p e a t e d exposures o f t h e h i n d g u t t o t h e p e p t i d e o f t e n c a u s e d a d e s e n s i t i z a t i o n o f t h e p r e p a r a t i o n . As i s the c a s e w i t h t y r a m i n e , LMS p r o d u c e d an i n h i b i t i o n o f t h e h i n d g u t t o g l u t a m a t e , p r o c t o l i n , and LPK. Thus, IKS i s n o t a s p e c i f i c antagonist f o r peptides. The f o r e g u t o f L. maderae was as s e n s i t i v e as t h e h i n d g u t t o LMS, w i t h a t h r e s h o l d n e a r 5 x 10' M. The o v i d u c t and h e a r t by comparison were much l e s s s e n s i t i v e and c o n s i s t e n t i n t h e i r r e s p o n s e s t o t h e p e p t i d e . Most o v i d u c t p r e p a r a t i o n s showed a d e t e c t a b l e i n h i b i t i o n t o LMS a t 2.4 x 10' M, b u t i n some i n s t a n c e s a c o n c e n t r a t i o n o f 10" M p r o d u c e d a s t i m u l a t i o n . A l t h o u g h LMS on o c c a s i o n c a u s e d an i n h i b i t i o n o f spontaneous c o n t r a c t i o n s o f t h e h e a r t a t 10~ M, most p r e p a r a t i o n s t h a t d i d r e s p o n d r e q u i r e d a c o n c e n t r a t i o n i n e x c e s s o f 10~ M. The r e s p o n s e o f t h e h e a r t t o LMS was by no means c o n s i s t e n t ; many p r e p a r a t i o n s f a i l e d t o r e s p o n d t o t h e p e p t i d e a t a l l , w h i l e o t h e r s responded i n i t i a l l y b u t f a i l e d a f t e r a second exposure. Recent e x p e r i m e n t s have shown t h a t LMS a t c o n c e n t r a t i o n s h i g h e r t h a n 100 nM c a n reduce t h e a m p l i t u d e o f n e u r a l l y evoked EPSP i n t h e s k e l e t a l muscles o f t h e mealworm T e n e b r i o m o l i t e r ( 5 3 ) . However, the p e p t i d e has no e f f e c t on t h e g l u t a m a t e - i n d u c e d d e p o l a r i z a t i o n o f these muscles. M i c r o m o l a r amounts o f LMS have a l s o been shown t o i n h i b i t b o t h t h e f r e q u e n c y and a m p l i t u d e o f spontaneous h e a r t c o n t r a c t i o n s i n t h e l o c u s t S c h i s t o c e r c a g r e g a r i a i n a dose dependent (54) manner. Immunocytochemical s t u d i e s (55) have d e m o n s t r a t e d t h a t the major s o u r c e o f LMS i n L. maderae appears t o be l o c a l i z e d i n t h e p a r s i n t e r c e r e b r a l i s and i n t h e r e g i o n between t h e p r o t o c e r e b r a l and d e u t o c e r e b r a l lobes o f the b r a i n . A pair of intensely reacting c e l l s were f o u n d i n each o f t h e t h o r a c i c g a n g l i a . The abdominal g a n g l i a , b y comparison, c o n t a i n e d s e v e r a l c e l l s t h a t were o n l y moderately a c t i v e .


2

n

n

8

7

9

8

C o n c l u d i n g Remarks The f a c t t h a t t h e s t r u c t u r a l d e t e r m i n a t i o n o f t h e m a j o r i t y o f i n s e c t n e u r o p e p t i d e s , thus f a r d e s c r i b e d , has r e l i e d on some k i n d o f muscle a s s a y has r a i s e d s e v e r a l c o n c e r n s . F i r s t , i t h a s been p o i n t e d o u t t h a t muscle a c t i v a t i o n may n o t r e p r e s e n t t h e p r i m a r y f u n c t i o n a l r o l e




Ο

i

5

Ο

1

I

I

Menn et al.; Insect Neuropeptides ACS Symposium Series; American Chemical Society: Washington, DC, 1991. 7

7

7

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F i g u r e 1. P r o t r a c t e d r e s p o n s e o f t h e h i n d g u t t o l e u c o k i n i n I I (A) and t h e e f f e c t s o f p o t a s s i u m d e p o l a r i z a t i o n (B) and a c a l c i u m f r e e medium (C) on t h e a c t i o n o f l e u c o k i n i n I and p r o c t o l i n . (A) Response o f t h e h i n d g u t t o 2.7 x 10" M LK I I ( f i r s t a r r o w ) . (B) Response o f t h e h i n d g u t t o 2 x 10" M p r o c t o l i n ( f i r s t arrow) and 2 x 10" M LK I ( s e c o n d a r r o w ) . Downward arrows indicate a rinse i n fresh saline solution. A d d i t i o n o f 158 mM p o t a s s i u m w i t h 2 mM c a l c i u m a t f i r s t and s e c o n d b a r s . Response o f t h e h i n d g u t t o 2 x 10" M LK I ( t h i r d arrow) and 2 x 10" M p r o c t o l i n ( f o u r t h arrow) a f t e r p o t a s s i u m d e p o l a r i z a t i o n . (C) Response o f t h e h i n d g u t t o 2 x 10" M LK I ( f i r s t arrow) f o l l o w e d by t h e a d d i t i o n o f a c a l c i u m f r e e medium ( b a r ) . Failure o f t h e h i n d g u t t o r e s p o n d t o 2 x 10" M LK I ( s e c o n d arrow) . C a l c i u m c o n t r a c t u r e a f t e r t h e a d d i t i o n o f 2 mM c a l c i u m t o t h e p r e p a r a t i o n ( t h i r d arrow). D i s p l a c e m e n t c a l i b r a t i o n 1 mm. Time mark 1 min. (Reproduced from reference 50.)



62


f o r t h e s e p e p t i d e s . T h i s may w e l l prove t o be t r u e . However, t h e e x p r e s s e d c o n c e r n seems t o be r o o t e d i n attempts t o e s t a b l i s h i n s e c t p e p t i d e n o m e n c l a t u r e on t h e b a s i s o f p r i m a r y b i o l o g i c a l f u n c t i o n (56) b u t t h i s c o n c e p t appears i n a d e q u a t e f o r t h r e e r e a s o n s : 1) no unambiguous g u i d e l i n e s have been s e t t o determine t h e p r i m a r y b i o l o g i c a l f u n c t i o n f o r any p e p t i d e ; 2) i t has a l r e a d y been e s t a b l i s h e d t h a t most p e p t i d e s a r e m u l t i - f u n c t i o n a l e n t i t i e s ; and 3) when t h e s t r u c t u r e o f p e p t i d e s a r e f i r s t announced knowledge on t h e range o f t h e i r b i o l o g i c a l a c t i o n i s g e n e r a l l y r a t h e r l i m i t e d . A more e x p e d i e n t format f o r i n s e c t p e p t i d e nomenclature has r e c e n t l y been s u g g e s t e d by Robb e t a l . ( 5 2 ) . I n t h i s i n s t a n c e , t h e p e p t i d e was named on t h e b a s i s o f t h e i n s e c t s o u r c e and a few key n o t e s o f i t s chemistry. The q u e s t i o n "why a r e t h e r e so many m y o t r o p i n s ? " has echoed another concern. I f we a c c e p t t h e f a c t t h a t l i t e r a l l y hundreds o f muscles c o n s t i t u t e t h e b a s i s o f movement i n i n s e c t s and t h e premise t h a t t h i s movement i s t h e p r i n c i p l e avenue f o r e x p r e s s e d b e h a v i o r , c a n we t h e n be s u r p r i s e d t h a t many d i f f e r e n t c h e m i c a l messengers ( i . e . , neuropeptides) are i n v o l v e d i n r e g u l a t i n g the process? Moreover, c a n we r i g h t l y assume t h a t t h i s a c t i o n has no p h y s i o l o i g c a l s i g n i f i c a n c e s i m p l y because many d i f f e r e n t agents seem t o i n d u c e an e x c i t i t o r y o r i n h i b i t o r y response? C a r e f u l s t u d y has a l r e a d y shown t h a t s u b t l e d i f f e r e n c e s do e x i s t between t h e s e s u b s t a n c e s and t h e i r r e s p e c t i v e r e s p o n s e s even w i t h i n a s i n g l e muscle ( 5 8 ) . What we a r e l o o k i n g a t i s a continuum o f i n t e r c e l l u l a r communication and o n l y p a t i e n t and m e t i c u l o u s r e s e a r c h w i l l u n r a v e l i t s complexity. C e r t a i n l y the m u l t i f u n c t i o n a l nature o f neuropeptides p r o v i d e s a f a s c i n a t i n g l e a d i n our e f f o r t s t o u n d e r s t a n d t h e c h e m i c a l mechanisms t h a t r e g u l a t e i n s e c t motor b e h a v i o r sequences. However, t o a c c o m p l i s h t h i s end, we must have a b e t t e r comprehension o f t h e range o f messages t h a t t h e s e agents may w e l l i m p a r t w i t h i n l i v i n g systems. T h i s w i t h o u t doubt w i l l r e q u i r e much more r e s e a r c h on t h e c o m p a r a t i v e p h y s i o l o g i c a l and pharmacological a c t i o n o f the p e p t i d e s .

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Florey, E. Symp. Biol. Hung. 1988, 36, 743-750. Baker, J. L.; Smith, T. G. In Society for Neuroscience Symposium, Vol. II; Cowan, W. M.; Ferrendelli, J. A., Eds.; Society of Neuroscience: Bethesda, MD, 1977; pp 340-370. Mayeri, E.; Rothman, B. S. In Neurosecretion: Molecules. Cells. Systems; Farner, D. S.; Lederis, K., Eds.; Plenum Press: New York, 1981; pp 305-316. Hoyle, G. In Comprehensive Insect Physiology Biochemistry and Pharmacology. Vol. 5; Kerkut, G. A.; Gilbert, L. I., Eds.; Pergamon Press: New York, 1985; pp ix-xii. Evans, P. D.; Myers, C. M. J. Exp. Biol. 1986, 124, 143-176. Brown, B. E. Science 1967, 155, 595-597. Holman, G. M.; Cook, B. J. J. Insect Physiol. 1970, 16, 1891-1907. Davey, K. G. J. Exp. Biol. 1962, 39, 319-324. Brown, B. E. Gen. Comp. Endocrinol. 1965, 5, 387-401.



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