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UBC Theses and Dissertations

Studies on the hormonal regulation of ion resorption in Schistocerca gregaria Spring, Jeffrey Herbert 1979

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TODIES ON THE HORMONAL REGULATION OF ION RESORPTION IN SCHXSTOCERCA GREGARIA by J e f f r e y Herbert Spring B- Sc., U n i v e r s i t y of Waterloo, 1973 Mo Sc., U n i v e r s i t y of Waterloo, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOB OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES {Department of Zoology) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1979 © J e f f r e y Herbert S p r i n g In present ing t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree tha t the 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 re fe rence and s tudy. I f u r t h e r agree tha t permiss ion f o r ex tens ive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s en t a t i v e s . I t i s understood tha t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n pe rm iss i on . Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P lace Vancouver, Canada V6T 1W5 Date DE-6 BP 75-51 1 E ABSTRACT When i s o l a t e d l o c u s t r e c t a are s h o r t - c i r c u i t e d i n *Ussing-type' chambers, they e x h i b i t an i n i t i a l d e c l i n e i n s h o r t - r c i r c u i t c u r r e n t (Isc) and t r a n s e p i t h e l i a l e l e c t r o p o t e n t i a l d i f f e r e n c e (PD) over the f i r s t two hours. F o l l o w i n g t h i s d e c l i n e , r e c t a remain i n a s t e a d y - s t a t e c o n d i t i o n f o r at l e a s t 4 h, d u r i n g which these parameters d e c l i n e only s l i g h t l y . There i s a f a c t o r present i n the corpora c a r d i a c a (CC) of s c h i s t o c e r c a g r e g a r i a which causes the I s c and PD of s h o r t -c i r c u i t e d r e c t a to i n c r e a s e . Maximum s t i m u l a t i o n r e s t o r e s r e c t a l I s c and PD to the l e v e l s observed immediately a f t e r removing t h i s organ from l o c u s t s . C y c l i c AMP causes a s i m i l a r maximum i n c r e a s e i n I s c and PD although the response e x h i b i t s a much s h o r t e r lag-time and a f a s t e r r a t e . o f i n c r e a s e than with CC. The r e l a t i o n s h i p between the l o g a r i t h m of CC dose or cAMP c o n c e n t r a t i o n and the maximum A I s c i s l i n e a r . Maximum s t i m u l a t i o n i s achieved with 0.05 pr CC or 0.3 mM cAMP, and the d e c l i n e i n A l s c i s dose-dependent and occurs over a matter of hours. I n h i b i t o r s of HCOJ/H+ and C l - t r a n s p o r t i n v e r t e b r a t e s do not i n h i b i t the s t i m u l a t i o n of r e c t a by CC or cAMP. P u t a t i v e n e u r o t r a n s m i t t e r substances and homogenates of f l i g h t muscle do not change r e c t a l I s c or PD.. Although homogenates of whole b r a i n s , v e n t r a l g a n g l i a and r e c t a l t i s s u e cause s m a l l i n c r e a s e s i n I s c , the c o n c e n t r a t i o n of a c t i v e f a c t o r i s n e a r l y three orders of magnitude g r e a t e r i n the CC. The u n i d i r e c t i o n a l and net f l u x e s of 2*Na+ ac r o s s s h o r t -c i r c u i t e d r e c t a remain constant with time and are un a f f e c t e d by the i n i t i a l d e c l i n e i n I s c or by s t i m u l a t i o n with CC or cAMP. 9 t II Net 3 6 C 1 - f l u x c l o s e l y matches the I s c over the e n t i r e experiment, f o l l o w i n g the i n i t i a l d e c l i n e i n Isc and r i s i n g when r e c t a are s t i m u l a t e d with CC or cAMP. The s t i m u l a t i o n of a c t i v e net CI— uptake i s s u f f i c i e n t t o account f o r the e n t i r e i n c r e a s e i n I s c . A d d i t i o n of CC to everted r e c t a l sacs causes the l e v e l s of cAMP i n t h i s t i s s u e to t r i p l e w i t h i n 15 minutes. Removal o f C l ~ from the bathing media i n h i b i t s ^ , any s t i m u l a t i o n by CC or cAMP, but the response r e t u r n s as soon as C I - i s r e s t o r e d . Experiments using complex C l - f r e e s a l i n e s suggest t h a t a l t e r n a t e e l e c t r o g e n i c t r a n s p o r t processes can be turned on, and supported, by complex media. When s t a r v e d l o c u s t s are f e d l e t t u c e , t h e i r haemolymph s t i m u l a t e s r e c t a l I s c and PD i n a s i m i l a r manner t o a submaximal dose of CC and the i n c r e a s e i n I s c can be completely accounted f o r by the i n c r e a s e . i n net 3 6 C 1 ~ f l u x , as i s the.case with CC and cAMP. . Removal of the. CC from l i v e l o c u s t s reduces or e l i m i n a t e s t h i s f e e d i n g response. F u r t h e r experiments suggest t h a t the CC f a c t o r , which I have c a l l e d C h l o r i d e - T r a n s p o r t S t i m u l a t i n g Hormone (CTSH), i s not one of the known CC hormones. Although CC and haemolymph samples can i n c r e a s e water r e s o r p t i o n i n r e c t a l s a c s , the a c t i o n of CTSH does not seem t o be d i r e c t l y l i n k e d t o a n t i d i u r e s i s . S t r u c t u r a l l y , i t i s a s m a l l p r o t e i n (M.W. 10,000), water-s o l u b l e and r e l a t i v e l y h e a t - s t a b l e . To date, I cannot dete c t any d i f f e r e n c e between the CC and haemolymph f a c t o r s , although f u r t h e r experiments to p u r i f y CTSH from both sources are i n progress. iii TABLE OF CONTENTS I. INTRODUCTION 1 I I . STIMULANTS OF ELECTROGENIC ION TRANSPORT 8 I n t r o d u c t i o n 8 M a t e r i a l s and methods 11 E l e c t r i c a l measurements i n y i t r o 11 S o l u t i o n s 13 Homogenates of corpora c a r d i a c a 13 Other homogenates 14 E l e c t r i c a l s t i m u l a t i o n 15 Assay procedure 15 Resul t s 16 C h a r a c t e r i s t i c s of unstimulated r e c t a 16 E f f e c t of CC homogenate on e l e c t r o g e n i c t r a n s p o r t 19 Dose-response e f f e c t of CC homogenate on I s c 21 S t a b i l i t y of CC homogenate 26 E f f e c t s of other t i s s u e homogenates on e l e c t r o g e n i c t r a n s p o r t 26 E f f e c t s o f n e u r o t r a n s m i t t e r substances on I s c 29 S t i m u l a t i o n of e l e c t r o g e n i c t r a n s p o r t by cAMP 29 E f f e c t s of s p e c i f i c t r a n s p o r t b l o c k e r s 32 D i s c u s s i o n 33 I I I . IDENTIFICATION OF SPECIFIC ION TRANSPOET PROCESSES REGULATED BY CORPORA CARDIAC A AND CYCLIC-AMP 33 I n t r o d u c t i o n 38 M a t e r i a l s and methods 39 Measurement of i o n f l u x e s 40 C l - F r e e s a l i n e s 42 T i s s u e cAMP assay 44 Re s u l t s 45 E f f e c t s of CC homogenate on Na+ and C l ~ F l u x e s 45 E f f e c t s of cAMP on Na + and C l ~ Fluxes 54 E f f e c t s o f anion s u b s t i t u t i o n s on r e c t a l response to CC or cAMP 55 E f f e c t of CC homogenate on t i s s u e cAMP 65 D i s c u s s i o n 67 IV-EVIDENCE FOR REGULATORY SUBSTANCES IN THE HAEMOLYMPH 71 I n t r o d u c t i o n 71 M a t e r i a l s and methods 72 P r e p a r a t i o n of CC homogenate 72 P r e p a r a t i o n of haemolymph f o r assay 72 Car d i a t e c t o m i e s 74 Measurement of e l e c t r i c a l parameters and u n i d i r e c t i o n a l C l - f l u x e s 76 P r e p a r a t i o n of r e c t a l sacs 76 Res u l t s 77 E f f e c t o f a c t i v e haemolymph on e l e c t r o g e n i c t r a n s p o r t 77 E f f e c t of a c t i v e haemolymph on 3 6 C l _ t r a n s p o r t 80 E f f e c t s of c a r d i a t e c t o m i e s on haemolymph a c t i v i t y 83 Haemolymph CTSH l e v e l s 84 Water uptake by r e c t a l sacs 90 D i s c u s s i o n 93 V.GENERAL DISCUSSION 97 iv LITERATURE CITED 107 V LIST OF FIGURES F i g u r e 1 The method used to voltage-clamp r e c t a 12 Fi g u r e 2 V i a b i l i t y o f unstimulated s h o r t - c i r c u i t e d r e c t a 18 Figu r e 3 E f f e c t s of CC and cAMP on the e l e c t r i c a l parameters of s h o r t - c i r c u i t e d r e c t a 21 F i g u r e 4 Peak response of r e c t a l I s c t o i n c r e a s i n g doses of CC 22 Figu r e 5 I n d i v i d u a l t r a c e s of I s c with time f o r r e c t a s t i m u l a t e d with CC 25 F i g u r e 6 E f f e c t s of t i s s u e homogenates, e l e c t r i c a l s t i m u l a t i o n and i n h i b i t o r s on I s c . 28 Fi g u r e 7 Peak response of I s c to i n c r e a s i n g doses of cAMP 31 Figu r e 8 U n i d i r e c t i o n a l 3 6 C 1 _ f l u x e s with time f o r s h o r t - c i r c u i t e d r e c t a s t i m u l a t e d with CC and cAMP 47 Figure.9 I s c and net 3 6 C 1 _ f l u x e s f o r s h o r t - c i r c u i t e d r e c t a s t i m u l a t e d with CC and cAMP 50 Fi g u r e 10 U n i d i r e c t i o n a l 2 2Na+ f l u x e s with time f o r s h o r t - c i r c u i t e d r e c t a s t i m u l a t e d with CC and cAMP 52 Figu r e 11 I s c and net 2 2 N a + f l u x e s f o r s h o r t - c i r c u i t e d r e c t a s t i m u l a t e d with CC and cAMP 54 Figu r e 12 I n d i v i d u a l t r a c e s of I s c with time f o r r e c t a bathed i n simple N 0 3 - s a l i n e , SO^-saline-2, or a c e t a t e s a l i n e 59 Fi g u r e 13 E l e c t r i c a l parameters with time f o r s h o r t -c i r c u i t e d r e c t a bathed i n simple C l - s a l i n e , and simple and complex SO ^ - s a l i n e s 63 Figure 14 E f f e c t s of haemolymph on the e l e c t r i c a l parameters of s h o r t - c i r c u i t e d r e c t a 79 F i g u r e 15 U n i d i r e c t i o n a l and net 3 6 C 1 - f l u x e s with time f o r s h o r t - c i r c u i t e d r e c t a s t i m u l a t e d with haemolymph 82 VI F i g u r e 16 S t i m u l a t i o n of I s c by haemolymph. and c a l c u l a t e d CTSH l e v e l s f o r c a r d i a t e c t o m i z e d , sham-operated and normal l o c u s t s 84 F i g u r e 17 Maximum A I s c and c a l c u l a t e d haemolymph CTSH l e v e l s f o r i n d i v i d u a l and pooled haemolymph samples 86 Figure 18 D i s t r i b u t i o n of haemolymph CTSH a c t i v i t y i n samples from i n d i v i d u a l l o c u s t s 89 F i g u r e 19 Hater a b s o r p t i o n with time by everted r e c t a l sacs s t i m u l a t e d with CC 91 Figu r e 20 Water a b s o r p t i o n with time by everted r e c t a l sacs s t i m u l a t e d with haemolymph 92 vii LIST OF TABLES Table 1 Composition of s a l i n e s used i n t h i s study 43 Table 2 Average values of I s c and PD f o r r e c t a bathed i n 4 d i f f e r e n t simple s a l i n e s 57 Table 3 E f f e c t of CC homogenate on t i s s u e l e v e l s of cAMP i n l o c u s t r e c t a 66 « *-• VIII ABBREVIATIONS AND DEFINITIONS ADH A n t i d i u r e t i c hormone cAMP Adenosine 3 , : 5 ' - c y c l i c monphosphoric a c i d Cardiatectomy Removal of the corpora c a r d i a c a from l i v e l o c u s t s CC Corpora c a r d i a c a CCEq CC-equivalents per 300 Jul of haemolymph CTSH C h l o r i d e - t r a n s p o r t s t i m u l a t i n g hormone DH D i u r e t i c hormone h Hour I s c S h o r t - c i r c u i t c u r r e n t L:D Light:Dark min Minute PD T r a n s e p i t h e l i a l e l e c t r o p o t e n t i a l d i f f e r e n c e pr P a i r R T r a n s e p i t h e l i a l d . . c . . r e s i s t a n c e RH R e l a t i v e humidity sec Second DEDICATION -This t h e s i s i s dedicated t o Jo Matthews, k i l l e d on May 29,1976. For the f r i e n d s h i p , f o r the help, f o r the c o n f i d e n c e t o go on. ACKNOWLEDGEMENTS I wish t o thank Dr. John P h i l l i p s f o r h i s encouragement, support, and e x t r a o r d i n a r y p a t i e n c e with me throughout t h i s study. I am g r a t e f u l t o my committee f o r t h e i r time and h e l p f u l comments. My s p e c i a l thanks t o Joan M a r t i n , both f o r our i n t e r e s t i n g d i s c u s s i o n s , and f o r keeping me i n l i n e . I a l s o wish t o thank a l l my f r i e n d s and c o l l e a g u e s who brightened my stay at UBC. . XI 1 I INTRODUCTION A major problem f a c e d by x e r i c i n s e c t s , such as i n t e r n a l medium i n a h o s t i l e environment;. T h i s problem, accentuated i n i n s e c t s because o f t h e i r high s u r f a c e area to volume r a t i o , e f f e c t i v e l y c o n s i s t s of two p a r t s ; the c o n s e r v a t i o n of water, and the i o n i c homeostasis of the body f l u i d s i n the f a c e o f widely f l u c t u a t i n g volumes. Mechanisms f o r water c o n s e r v a t i o n i n c l u d e b e h a v i o u r a l avoidance of extreme c o n d i t i o n s * an impermeable c u t i c l e and the p r o d u c t i o n of e x c r e t a which are hyperosmotic t o the haemolymph (reviewed by P h i l l i p s , 1970)- In t h i s t h e s i s , I w i l l examine some of the f u n c t i o n s o f the e x c r e t o r y system i n S c h i s t o c e r e a and, more.importantly, how they are c o n t r o l l e d . The i n s e c t e x c r e t o r y system c o n s i s t s of two major components, the Malpighian t u b u l e s and the rectum. The t u b u l e s , which are o f t e n compared to the v e r t e b r a t e glomerulus, remove an i s o s m o t i c f l u i d from the haemolymph.. Phosphate i o n s , some s p e c i f i c dyes and other l a r g e o r g a n i c molecules can be a c t i v e l y t r a n s p o r t e d , but the primary d r i v i n g f o r c e f o r f l u i d s e c r e t i o n by the t u b u l e s i s provided by an a c t i v e K+ pump (Maddrell et a l . , 1974; Maddrell* 1977). Although t h i s i s o s m o t i c s e c r e t i o n i s i d e a l f o r the n o n - s e l e c t i v e removal of s o l u t e s from the haemocoel, i t i s c l e a r that t h i s system alone would r a p i d l y a l t e r the composition of the haemolymph. T h i s i s prevented by the a c t i o n of the rectum, which s e l e c t i v e l y r e s o r b s a l l the e s s e n t i a l molecules and thereby r e g u l a t e s the composition and volume of the haemolymph. i s the maintainence. of a s u i t a b l e 2 Over the l a s t two decades, a number of important s t u d i e s have.helped t o e l u c i d a t e r e c t a l f u n c t i o n and i t s c o n t r o l . In h i s p i o n e e r i n g work with the desert l o c u s t , S e h i s t o c e r e a g r e q a r i a , P h i l l i p s (1964a) showed t h a t water c o u l d be a c t i v e l y resorbed a g a i n s t extremely high c o n c e n t r a t i o n g r a d i e n t s i n v i v o , even i n the absence of net i o n a b s o r p t i o n . Water uptake was g r e a t l y enhanced i n dehydrated l o c u s t s i n d i c a t i n g that the r e s o r p t i v e process i s r e g u l a t e d i n some manner. P h i l l i p s (1964c) a l s o showed t h a t Na+, K+ and C l _ i o n s c o u l d be resorbed much more r a p i d l y from the rectum i n v i v o and without a p r o p o r t i o n a l i n c r e a s e i n water uptake, when l o c u s t s were hydrated by s u p p l y i n g tap water ad l i b . The f i r s t comprehensive study of the hormonal c o n t r o l o f d i u r e s i s was by M a d d r e l l (1963; 1964a,b), working with i s o l a t e d H a l p i g h i a n t u b u l e s from the blood-sucking bug Bhodnius prglixus> Tubule s e c r e t i o n was i n i t i a l l y r a p i d but q u i c k l y f e l l to a low l e v e l . Eapid s e c r e t i o n could be r e s t o r e d with the a d d i t i o n of haemolymph from r e c e n t l y - f e d Bhodnius l a r v a e . The d i u r e t i c f a c t o r was found i n the p o s t e r i o r neurosecretory c e l l s of the metathoracic g a n g l i o n i c mass and i t s release:was i n i t i a t e d by s t r e t c h r e c e p t o r s i n the t e r g o s t e r n a l muscles of the a n t e r i o r abdomen^ T h i s work was important i n i t s demonstration of a n e u r a l pathway which t r i g g e r e d the r e l e a s e i n t o the haemocoel o f a hormone . which i n t u r n i n i t i a t e d d i u r e s i s . Wall (1967) demonstrated t h a t i n v i t r o • r e c t a from dehydrated cockroaches absorbed water more q u i c k l y than those from hydrated ones. E x t r a c t s of c o r p o r a a l l a t a * and metathoracic and t e r m i n a l abdominal g a n g l i a from hydrated i n s e c t s produced an 3 i n c r e a s e i n the r a t e of water a b s o r p t i o n whereas these t i s s u e s had no e f f e c t when taken from dehydrated i n s e c t s . B r a i n e x t r a c t s from dehydrated cockroaches a l s o had an a n t i d i u r e t i c e f f e e t . . T h e q u a n t i t a t i v e values presented i n her study are i n some doubt because with each volume de t e r m i n a t i o n , s a l i n e was added to the rectum so t h a t a f t e r the f i r s t d e t e r m i n a t i o n the r e c t a l c o n t e n t s were v a r i a b l e and of unknown composition. Q u a l i t a t i v e l y , however, the data do present evidence f o r hormonal c o n t r o l o f water balance. Mordue (1969) presented evidence f o r a d i u r e t i c f a c t o r s y n t h e s i z e d i n the medial n e u r o s e c r e t o r y c e l l s of the d e s e r t l o c u s t and r e l e a s e d from the corpora c a r d i a c a (CC) d u r i n g f e e d i n g . T h i s d i u r e t i c f a c t o r i n c r e a s e d the r a t e of Malpighian t u b u l e s e c r e t i o n , and decreased the i n i t i a l r a t e of water abs o r p t i o n by e v e r t e d r e c t a l sacs i n - v i t r o . On t h e : b a s i s of t h i s dual a c t i o n , Mordue suggested t h a t there was no requirement f o r an a n t i d i u r e t i c hormone i n the d e s e r t l o c u s t . However, t h i s and other s t u d i e s have been s u b j e c t t o reassessment. Maddrell et a l . (1974) have shown t h a t amaranth, which was used as an i n d i c a t o r of f l u i d t r a n s p o r t by s e v e r a l workers, i s i t s e l f a c t i v e l y t r a n s p o r t e d and i s thus not n e c e s s a r i l y an accurate measure of f l u i d movementi Goh and P h i l l i p s (1978) have shown t h a t water t r a n s p o r t i n r e c t a l sacs decreases over the. f i r s t two hours f o l l o w i n g e x t i r p a t i o n , during which time the t i s s u e i t s e l f s w e l l s ; ftll of the e a r l i e r s t u d i e s concerned with the c o n t r o l of r e c t a l a b s o r p t i o n of water ( V i e t i n g h o f f , 1965; Wall, 1967; Mordue, 1969) were c a r r i e d out immediately a f t e r d i s s e c t i o n , i . e ; d u r i n g t h i s t r a n s i e n t phase, so t h a t v a r i a t i o n s i n the time 4 delay between e x t i r p a t i o n and measurement are undoubtedly a source of c o n s i d e r a b l e v a r i a b i l i t y i n the r e s u l t s . Cazal and G i r a r d i e (1968) found t h a t whole CC of Locusta had only an a n t i d i u r e t i c e f f e c t on Malpighian t u b u l e s and r e c t a . T h i s was l a t e r confirmed by Mordue (1970), who a l s o showed t h a t a d i u r e t i c f a c t o r i s predominant i n e x t r a c t s of the storage l o b e s of the CC i n both Locusta and S c h i s t o c e r e a ; The a n t i d i u r e t i c f a c t o r i s present i n the g l a n d u l a r l o b e s and i s more potent i n Locusta than S c h i s t o c e r e a . With the e x c e p t i o n of P h i l l i p s ' o r i g i n a l work (1964a,b,c), a l l the s t u d i e s done t o date have used i n v i t r o - r e c t a l Recently, Herrera e t a l . (1976,1977) have examined t h e . i n f l u e n c e of i o n s u b s t i t u t i o n s on the e l e c t r i c a l parameters of s h o r t - c i r c u i t e d l o c u s t r e c t a ; T h e i r p r e p a r a t i o n s remained v i a b l e f o r only 45 min, and i n some experiments they v i o l a t e d one. of the b a s i c requirements f o r s h o r t - c i r c u i t i n g e p i t h e l i a by having d i f f e r e n t s o l u t i o n s on the lumen and haemocoel-sides of the p r e p a r a t i o n s . The major c o n c l u s i o n s of t h e i r s t u d i e s were t h a t a c t i v e C l -t r a n s p o r t from lumen t o haemocoel was p r i m a r i l y r e s p o n s i b l e f o r the s h o r t - c i r c u i t c u r r e n t ( I s c ; a measure of net a c t i v e i o n f l u x ) and t r a n s e p i t h e l i a l e l e c t r o p o t e n t i a l d i f f e r e n c e (PD), and that HCO3- played an important r o l e i n a c t i v e C l ~ t r a n s p o r t ; . W i l l i a m s (1976) and W i l l i a m s e t a l , (1978), using q u i t e a d i f f e r e n t voltage-clamped p r e p a r a t i o n , measured the f l u x e s of 2 2Na+, * 2 K + and 3 6 C 1 ~ across l o c u s t r e c t a ; They found t h a t , as with everted sacs, t h e r e was an i n i t i a l two hour p e r i o d d u r i n g which the I s c and PD d e c l i n e d r a p i d l y , f o l l o w e d by a p e r i o d o f at l e a s t 8 hours d u r i n g which r e c t a remained i n an approximately 5 s t e a d y - s t a t e c o n d i t i o n . They a l s o showed t h a t w h i l e t h e i n i t i a l d e c l i n e i n I s c was due t o a d e c r e a s e i n a c t i v e C l - t r a n s p o r t , a s H e r r e r a e t a l . (1976) r e p o r t e d , t h e a c t i v e C l - t r a n s p o r t d u r i n g t h e s t e a d y - s t a t e phase was m a t c h e d b y a c t i v e Na+ u p t a k e , l e a v i n g t h e e n t i r e I s c u n a c c o u n t e d f o r . . W i l l i a m s e t a l . . p o s t u l a t e d t h a t HCOj, H + , o r o r g a n i c a c i d t r a n s p o r t was t h e s o u r c e o f t h e m i s s i n g I s c . T h e r e a r e e r r o r s i n h e r e n t i n t h e use o f a t w o -e l e c t r o d e method o f v o l t a g e - c l a m p i n g e p i t h e l i a , as p o i n t e d o u t by Wood and M o r e t o n ( 1 9 7 8 ) , b u t t h e s e e r r o r s r e p r e s e n t l e s s t h a n 5% o f t h e I s c f o r t h e s y s t e m u s e d by W i l l i a m s e t a l . ( 1 9 7 8 ) , and a r e n o t s u f f i c i e n t t o a f f e c t t h e m a j o r c o n c l u s i o n s o f t h e i r s t u d y . The o r i g i n a l s t u d i e s by P h i l l i p s (1964a,c) s u g g e s t e d t h a t w a t e r a n d i o n t r a n s p o r t i n l o c u s t r e c t a were n o t d i r e c t l y l i n k e d , a s i n d i c a t e d by t h e i n i t i a l u p t a k e o f w a t e r f r o m s u g a r s o l u t i o n s o v e r t h e 20-80 min e x p e r i m e n t a l p e r i o d i n s i t u . T h e s e r e s u l t s were i n d e p e n d e n t l y c o n f i r m e d f o r l o c u s t s ( S t o b b a r t , 1968) and c o c k r o a c h e s ( W a l l , 1 9 6 7 ) . Goh and P h i l l i p s (1978) h a v e shown, h o w e v e r , t h a t w h i l e i n v i t r o r e c t a l s a c s w i l l a b s o r b w a t e r f r o m i s o s m o t i c s u c r o s e s o l u t i o n s f o r 1 h , p r o l o n g e d w a t e r t r a n s p o r t (2 t o 5 h) r e q u i r e s t h e p r e s e n c e o f m o n o v a l e n t , i n o r g a n i c i o n s on t h e l u m e n , b u t n o t t h e h a e m o c o e l - s i d e . . T h e s e d a t a s u g g e s t t h a t some mechanism o f s o l u t e c o n s e r v a t i o n i s o p e r a t i n g ; S o l u t e r e c y c l i n g w i t h i n t h e r e c t a l e p i t h e l i u m h a s been s u g g e s t e d on u l t r a s t r u c t u r a l g r o u n d s ( B e r r i d g e , 1 970; M a d d r e l l , 1970) and a s a t h e r m o d y n a m i c a l l y a c c e p t a b l e e x p l a n a t i o n o f a c t i v e w a t e r r e s o r p t i o n ( r e v i e w e d by P h i l l i p s , 1 9 7 0 , 1977a; W a l l e t a l . , 1970; M a d d r e l l , 1971; W a l l , 6 1971; Wall and Oschman, 1975). R e c y c l i n g has a l s o been supported e x p e r i m e n t a l l y by micropuncture s t u d i e s of the. i n t r a c e l l u l a r spaces of the cockroach r e c t a l pads (Wall e t - a l * , 1970) and more r e c e n t l y by d e t a i l e d e l e c t r o n probe X-ray a n a l y s i s of C a l l i p h o r a s a l i v a r y g l a n d s , N a l p i g h i a n t u b u l e s and r e c t a l p a p i l l a e (Gupta et a l . , 1977). However, the work of Goh and P h i l l i p s , (1978) was the f i r s t t o demonstrate a d i r e c t l i n k between s o l u t e t r a n s p o r t and a c t i v e water uptake; Although there has been much a t t e n t i o n given to f a c t o r s a f f e c t i n g d i u r e s i s i n i n s e c t s , there were: no p r e v i o u s i n v e s t i g a t i o n s of the p o s s i b l e e f f e c t s of these f a c t o r s , or o t h e r s , on the r e g u l a t i o n of i o n r e s o r p t i o n i n the i n s e c t rectum. The r e s e a r c h presented i n Chapter I I was i n i t i a t e d using the voltage-clamping method of W i l liams e t a l . . (1978) to f o l l o w the I s c ( i . e . net a c t i v e i o n f l u x ) across r e c t a exposed to v a r i o u s t i s s u e homogenates. T i s s u e s with known or suspected d i u r e t i c and a n t i d i u r e t i c a c t i v i t y were.assayed to determine whether they contained any f a c t o r s which a l s o i n f l u e n c e d the net e l e c t r o g e n i c i o n f l u x . When the a c t i o n of these.homogenates had been e s t a b l i s h e d , f u r t h e r assays were performed t o see i f t h i s a c t i o n c o u l d be mimicked by known n e u r o t r a n s m i t t e r s or i n t r a c e l l u l a r mediators of hormone a c t i o n (e.g. cAMP). When CC homogenate and cAMP had both been shown to i n c r e a s e r e c t a l I s c and PD, the f l u x e s of i n d i v i d u a l i o n s were s t u d i e d to determine which t r a n s p o r t pathway was a f f e c t e d (Chapter I I I ) . Experiments were a l s o done to show t h a t the . f a c t o r which s t i m u l a t e d a c t i v e C l ~ t r a n s p o r t was indeed a n a t u r a l hormone which i s r e l e a s e d i n t o the. haemolymph and not j u s t a 7 pharmacological a c t i o n of some substance i n CC homogenates (Chapter IV) i F i n a l l y , I d i s c u s s the p o s s i b l e f u n c t i o n of the C h l o r i d e - T r a n s p o r t S t i m u l a t i n g Hormone (CTSH) i n the whole l o c u s t (Chapter V) . The major s e c t i o n s of t h i s t h e s i s (Chapter II-IV) r e p r e s e n t t h r e e papers, which have been submitted i n s l i g h t l y m o d i f i e d form to the J o u r n a l of Experimental B i o l o g y , 8 I I STIMULANTS OF ELEGTHOGENIG ION TRANSPORT I n t r o d u c t i o n Haemolymph composition i n most t e r r e s t r i a l i n s e c t s i s u l t i m a t e l y r e g u l a t e d by s e l e c t i v e r e s o r p t i o n i n the rectum from a f l u i d s e c r e t e d by the Malpighian t u b u l e s (reviewed by Maddrell, 1971; P h i l l i p s , 1977a,b). P h i l l i p s ( 1 9 6 4 a , b ) showed that the: r a t e s of i o n a b s o r p t i o n from r e c t a i n s i t u are c o n s i d e r a b l y reduced and the r a t e of water a b s o r p t i o n i n c r e a s e s when hydrated l o c u s t s are f e d concentrated s a l i n e s o l u t i o n s ; c l e a r l y these t r a n s p o r t processes are r e g u l a t e d . Recent s t u d i e s have demonstrated t h a t the s t e a d y - s t a t e t r a n s p o r t of water across t h i s e p i t h e l i u m can be d r i v e n by any one of C l - , Na+ o r K+ (Goh, 1971; P h i l l i p s , 1977a,b; Goh and P h i l l i p s , 1978). Therefore, d i u r e t i c or a n t i d i u r e t i c f a c t o r s might act by r e g u l a t i n g the t r a n s p o r t of these i o n s ; W i l l i a m s e t a l . (1978) noted a r a p i d d e c l i n e i n a c t i v e i o n t r a n s p o r t a c r o s s v o l t a g e -clamped r e c t a i n v i t r o before a s t e a d y - s t a t e was reached. They sp e c u l a t e d t h a t t h i s was a consequence of removing the r e c t a from a n e u r a l or humoral s t i m u l a n t which i s present i n v i v o . I am not aware of any p r e v i o u s r e p o r t s of n e u r a l or endocrine f a c t o r s which d i r e c t l y i n f l u e n c e r e c t a l t r a n s p o r t o f i o n s . There i s some evidence, however* f o r the presence o f d i u r e t i c and a n t i d i u r e t i c f a c t o r s i n v a r i o u s n e u r a l and endocrine t i s s u e s of i n s e c t s (reviewed by Gee,1975). These f a c t o r s a l t e r r a t e s of water t r a n s p o r t by i n v i t r o r e c t a d u r i n g the i n i t i a l t r a n s i t o r y stage (1.e..the f i r s t two hours f o l l o w i n g 9 removal from the i n s e c t ) ; . In l o c u s t s , the storage lobes of the CC c o n t a i n a d i u r e t i c f a c t o r which i n h i b i t s r e c t a l a b s o r p t i o n of water (Mordue,1969,1970), while an a n t i d i u r e t i c f a c t o r i s present i n the g l a n d u l a r l o b e s (Mordue, 1970). There i s some disagreement as to whether homogenates of whole CC e x e r t a d i u r e t i c (Mordue, 1970) or an a n t i d i u r e t i c (Cazal and Girardie,1968) e f f e c t on S c h i s t o c e r c a - r e c t a i n v i t r o : Spring et a l . (1978) have suggested a p o s s i b l e e x p l a n a t i o n f o r these a p p a r e n t l y c o n t r a d i c t o r y r e s u l t s . A n t i d i u r e t i c a c t i v i t y has a l s o been a s c r i b e d to the CC of Ajgis (Altmann, 1956), G r y l l u s , P e r i p l a n e t a and Clitumnus (de Besse.and Cazal,1968). Although the corpora a l l a t a (CA) have been c i t e d as the source of a d i u r e t i c f a c t o r i n Ajais (Altmann, 1956) and P e r i p l a n e t a ( M i l l s , 1 9 6 7 ) , they c o n t a i n n e i t h e r d i u r e t i c nor a n t i d i u r e t i c f a c t o r s i n S c h i s t o c e r c a (Mordue and Goldsworthy,1969). Such f a c t o r s a r e . not r e s t r i c t e d t o the neurohaemal organs... In p a r t i c u l a r , there i s evidence f o r the presence of a n t i d i u r e t i c f a c t o r s i n the v e n t r a l g a n g l i a of S c h i s t o c e r c a (see Gee, 1975). U l t r a s t r u c t u r a l s t u d i e s have r e v e a l e d t h a t n e u r o s e c r e t o r y axons i n n e r v a t e the hindgut of the aphid (Johnson, 1963, 1966), the r e c t a l p a p i l l a e of the b l o w f l y (Gupta and B e r r i d g e , 1966) and the r e c t a l pads of the cockroach (Oschman and H a l l , 1969). I t i s p o s s i b l e t h e r e f o r e t h a t r e c t a l a b s o r p t i o n i s modified by l o c a l r e l e a s e of n e u r o s e c r e t o r y products or t y p i c a l n e u r o t r a n s m i t t e r substances from these.neurons. W i l l i a m s e t a l . . (1978) and Herrera e t al> (1976, 1977) have s t u d i e d the l o c u s t rectum under s h o r t - c i r c u i t e d conditions;. Those t r a n s p o r t processes which have been i d e n t i f i e d i n v i v o are 10 maintained remarkably w e l l i n the case of the p r e p a r a t i o n s used by W i l l i a m s e t . a l ; They observed a l a r g e net f l u x of both Na + and C I- to the haemocoel s i d e . These f l u x e s were o f approximately e q u a l magnitude, r e s u l t i n g i n no net t r a n s f e r o f charge and l e a v i n g Williams e t a l . unable t o account f o r the s t e a d y - s t a t e Isc,.They t e n t a t i v e l y assigned t h i s I s c to H+ZHCOj t r a n s p o r t because the r e c t a l c o n t e n t s are r a p i d l y a c i d i f i e d i n vivo ( P h i l l i p s , 1964b; Speight, 1968). I t i s a l s o p o s s i b l e t h a t the s t e a d y - s t a t e I s c could have been maintained by the absorption of org a n i c i o n s or P0^~ from the complex s a l i n e used by W i l l i a m s e t a l . The voltage-clamped p r e p a r a t i o n of l o c u s t rectum developed by W i l l i a m s et a l ; , o f f e r s a r a p i d method of as s a y i n g f a c t o r s which may a f f e c t e l e c t r o g e n i c t r a n s p o r t p r o c e s s e s . In t h i s chapter, I r e p o r t the e f f e c t of v a r i o u s t i s s u e homogenates, i n c l u d i n g those reputed t o c o n t a i n d i u r e t i c and a n t i d i u r e t i c a c t i v i t i e s , on t h e . s t e a d y - s t a t e s h o r t - c i r c u i t c u r r e n t (Isc) and o p e n - c i r c u i t e l e c t r o p o t e n t i a l d i f f e r e n c e (PD) a c r o s s i n v i t r o p r e p a r a t i o n s of l o c u s t r e c t a . I a l s o r e p o r t the e f f e c t s on I s c of homogenized r e c t a l t i s s u e , s e v e r a l p u t a t i v e n e u r o t r a n s m i t t e r substances, i n h i b i t o r s of C l - and HCOg t r a n s p o r t processes, and d i r e c t e l e c t r i c a l s t i m u l a t i o n on voltage-clamped r e c t a l p r e p a r a t i o n s ; Such s t u d i e s by themselves do not provide c o n c l u s i v e evidence t h a t these s t i m u l a t o r y or i n h i b i t o r y agents normally act to c o n t r o l r e c t a l a b s o r p t i o n i n v i v o . Evidence t h a t some o f the f a c t o r s which we have detected i n t h i s way do c o n t r o l r e c t a l a c t i v i t y i n v i v o i s presented i n l a t e r chapters. A p r e l i m i n a r y 11 note summarizing the major c o n c l u s i o n s i n t h i s and subsequent chapters has been publ i s h e d (Spring e t a l . , 1978). M a t e r i a l s and-Methqds The experimental animals were mature S c h i s t o c e r e a g r e g a r i a , one t o three months past t h e i r f i n a l moult. They were reared at 28°C and 50% H.H; under a photoperiod of L:D 16:8 and fed a d i e t of l e t t u c e and a mixture of d r i e d g r a s s , bran, yeast and powdered milk;. Females were used f o r i n v i t r o p r e p a r a t i o n s because of t h e i r l a r g e r s i z e . T i s s u e homogenates to be assayed f o r t h e i r a b i l i t y t o i n f l u e n c e r e c t a l t r a n s p o r t were prepared from a d u l t males to avo i d c y c l i c changes a s s o c i a t e d with female r e p r o d u c t i o n . E l e c t r i c a l Measurements In V i t r o To measure e l e c t r o g e n i c i o n t r a n s p o r t by the v o l t a g e -clamping method, r e c t a were mounted as f l a t sheets between two •Ussing-type' perspex chambers as d e s c r i b e d by W i l l i a m s e t a l . (1978)..Each chamber contained 7.0 ml..of s a l i n e which was v i g o r o u s l y s t i r r e d by bubbling with 95% 0^ - 5% CO,.. A l l experiments were conducted at room temperature (22°C). The s h o r t - c i r c u i t c u r r e n t (Isc) was recorded c o n t i n u o u s l y on a • F i s h e r E e c o r d a l l 5000* c h a r t r e c o r d e r ; The o p e n - c i r c u i t t r a n s e p i t h e l i a l p o t e n t i a l d i f f e r e n c e (PD) was monitored at i n t e r v a l s by s t o p p i n g the v o l t a g e clamp f o r 30 sec and using an a l t e r n a t e c i r c u i t to r e c o r d the v o l t a g e ( s e e . F i g . 1 ) . Membrane d. c. r e s i s t a n c e (R) was c a l c u l a t e d from PD and Isc using Ohm's law. . T i g . 1.. The method u s e d t o v o l t a g e - c l a m p l o c u s t r e c t a , (a) S i d e -view o f the ' U s s i n g - t y p e • chambers-. Arrow i n d i c a t e s d i r e c t i o n of s w i r l i n g p r o d u c e d by g a s - l i f t pumps, (b) E n l a r g e m e n t o f a r e a - e n c l o s e d by d o t t e d l i n e s i n F i g . 1a. S t i p p l e d a r e a i n d i c a t e s r e c t a l t i s s u e ; T, t u n g s t e n p i n s ; -0 r 0 - r i n g s . (c) B l o c k d i a g r a m o f t h e c i r c u i t r y used i n t h i s s t u d y a s i t i s c o n n e c t e d t o t h e c h a m b e r s ( F i g . 1 a ) . A, and Ax a r e FE1 i n s t r u m e n t a t i o n a m p l i f i e r s ( T e l e d y n e P h i l b r i c k : Dedham, Mass.) s e t a t 2000x and 1x g a i n r e s p e c t i v e l y . S h o r t - c i r c u i t c u r r e n t i s a p p l i e d a c r o s s t h e r e c t u m £y means of two s i l v e r p l a t e - e l e c t r o d e s (E) c o n n e c t e d a t 3 and 4. I s c i s r e a d as t h e v o l t a g e d r o p a c r o s s a 10 ksi, r e s i s t o r (8) w i t h the yang s w i t c h (S) i n t h e c u r r e n t (I) p o s i t i o n (as s h o w n ) T h e PD a c r o s s t h e r e c t u m i s r e a d by means o f two c a l o m e l h a l f - c e l l s (C) c o n n e c t e d a t 1 and 2 t o 3 M KC1 ag a r b r i d g e s (B) w i t h t h e gang s w i t c h i n t h e v o l t a g e (V) p o s i t i o n . I s c o r PD.are r e c o r d e d g r a p h i c a l l y on a ' F i s h e r L t e c o r d a l l ' s t r i p c h a r t r e c o r d e r a t t a c h e d a t 6. A • K e i t h l e y 602* e l e c t r o m e t e r can be a t t a c h e d a c r o s s 1 and 2 t o c h e c k a n d . c a l i b r a t e I s c and PD measurements. From W i l l i a m s e t a l . ( 1 9 7 8 ) . 13 t S o l u t i o n s A simple i n s e c t s a l i n e was used t o bathe r e c t a i n a l l experiments: 185 mM NaCl, 11 mM KC1, 10 mM g l u c o s e , 3 mM L-glutamate, 2.5 mM MgSOM, 1 mM KH^PO^, 24 mM NaHC0 3 and 3 mM CaCl^_. Phenol red was added as a pH i n d i c a t o r . I n i t i a l pH was 7.4. Bubbling with the gas mixture reduced t h i s t o 7.0 w i t h i n 5 minutes. There was l i t t l e change i n pH over the next 4 t o 18 h as determined by the i n d i c a t o r . A l l chemicals were obtained from Sigma Chemical Co. I n h i b i t o r s and n e u r o t r a n s m i t t e r substances were d i s s o l v e d i n 200 mM NaCl to form c o n c e n t r a t e d s o l u t i o n s such t h a t 50-500 fil were s u f f i c i e n t to produce the r e q u i r e d c o n c e n t r a t i o n when added t o the bathing s a l i n e . . An e q u i v a l e n t amount of bathing s a l i n e was removed from the chamber p r i o r to the a d d i t i o n of d r u g s . , A l l s o l u t i o n s were s t o r e d f r o z e n . Homogenates of Corpora-Cardiaca Corpora c a r d i a c a were.removed from the heads of mature male l o c u s t s as f o l l o w s : Severed heads were pinned facerdown i n a wax d i s s e c t i n g d i s h and an i n c i s i o n made from the foramen magnum along the c o r o n a l suture t o the f r o n t a l sutures. Transverse c u t s were made to each eye and continued along the p o s t e r i o r margin of the eye to mid-gena, then extended h o r i z o n t a l l y back to the foramen at the l e v e l of the labium. The two l a r g e p l a t e s so formed were removed along with the a s s o c i a t e d musculature, and adjacent a i r sacs and f a t body c a r e f u l l y removed. The severed end of the oesophagus was extended as f a r as p o s s i b l e and pinned i n p l a c e . The exposed CC, c o n s i s t i n g o f the blue storage l o b e s and white g l a n d u l a r l o b e s , were removed with watch-maker's 14 f o r c e p s and homogenized i n simple i n s e c t s a l i n e using a P o t t e r -Elvehjem homogenizer,. Homogenates were prepared at a c o n c e n t r a t i o n of 1 pr CC per ml, and s t o r e d f r o z e n u n t i l use. Other Homogenates To prepare homogenates of v e n t r a l g a n g l i a , the headless bodies of mature male l o c u s t s were pinned d o r s a l - s i d e up on p l a s t i c e n e b l o c k s and opened from anus through thorax with a median d o r s a l i n c i s i o n . F o l l o w i n g removal of the e n t i r e d i g e s t i v e t r a c t , v a r i o u s g a n g l i a were d i s s e c t e d out and homogenized i n s a l i n e i n a Potter-Elvehjem homogenizer. Homogenates were made up each day, s t o r e d on i c e and assayed w i t h i n 3-5 hours. B r a i n homogenates were prepared by opening the head ca p s u l e of a mature male l o c u s t i n the same manner as d e s c r i b e d f o r removal o f the CC. In some i n s t a n c e s , the e n t i r e supra-oesophageal gangli o n was homogenized; a l t e r n a t i v e l y , one o f the v e n t r a l quadrants was removed t o avoid the i n c l u s i o n of any of the medial n e u r o s e c r e t o r y c e l l s which i n n e r v a t e the CC. B r a i n fragments were homogenized i n s a l i n e i n the same manner as des c r i b e d f o r other g a n g l i a . As a c o n t r o l t i s s u e , p i e c e s of f l i g h t muscle approximately twice the s i z e of v e n t r a l g a n g l i a were t r e a t e d i n the same manner as the g a n g l i a . Whole r e c t a were removed from mature male l o c u s t s as d e s c r i b e d by W i l l i a m s e t a l . ; (1978) and were homogenized and st o r e d i n the same manner as g a n g l i a . 15 E l e c t r i c a l S t i m u l a t i o n The i n v i t r o rectum was s t i m u l a t e d e l e c t r i c a l l y by d i s c o n n e c t i n g the volt a g e clamp and a t t a c h i n g a 'Grass 1 s t i m u l a t o r d i r e c t l y to the s i l v e r c u r r e n t - e l e c t r o d e s (see F i g . 1 ) . E l e c t r i c a l pulses of v a r y i n g v o l t a g e , frequency and du r a t i o n were a p p l i e d across the pre p a r a t i o n . . T h e : v o l t a g e clamp was subsequently r e a p p l i e d and the I s c measured. Assay Procedure j Small volumes (20-200 ul) of drugs and homogenates were added to the haemocoel-side of r e c t a when they had reached the s t e a d y - s t a t e phase ( i . e . 90-150 min a f t e r d i s s e c t i o n ) . A f t e r t e s t i n g t h e i r e f f e c t , the chamber was r i n s e d with 4 changes of f r e s h s a l i n e and the I s c across the p r e p a r a t i o n was allowed t o r e t u r n to a s t e a d y - s t a t e before f u r t h e r t e s t i n g . P r e p a r a t i o n s were not used f o r more than 3 assays because the response to standard doses of s t i m u l a n t s subsequently d e c l i n e d . A f t e r the f i n a l t e s t on each p r e p a r a t i o n , 0.05 pr CC were added to the p r e p a r a t i o n t o c o n f i r m t h a t the t i s s u e was s t i l l r e s p o n s i v e t o s t i m u l a t i o n . R e s u l t s from the few p r e p a r a t i o n s which f a i l e d t o respond to CC were d i s c a r d e d . L o c u s t s used f o r any one experiment u s u a l l y came from the same, cage; i . e . the po p u l a t i o n had a l i m i t e d parentage. V a r i a t i o n among cages was much g r e a t e r than v a r i a t i o n w i t h i n each cage. I a l s o observed that the i n i t i a l PD and Isc v a r i e d somewhat with time a f t e r the l a s t meal, age and other f a c t o r s , and appeared to be a f u n c t i o n of the p h y s i o l o g i c a l s t a t e of the animal ( J . Hanrahan, J . S p r i n g , unpub. obs.). I a t t r i b u t e 16 d i f f e r e n c e s i n the a b s o l u t e values o f the e l e c t r i c a l parameters between experiments done at d i f f e r e n t times (e.g. .Fig...2 vs. F i g . 3 , f i r s t 90 min) t o some of these f a c t o r s . T h i s v a r i a b i l i t y was not considered s e r i o u s i n the present study because t e s t s were conducted only d u r i n g the s t e a d y - s t a t e phase and each r e c t a l p r e p a r a t i o n served, i n e f f e c t , as i t s own c o n t r o l . R e s u l t s C h a r a c t e r i s t i c s - of_gnstimulated_Rgcta The v i a b i l i t y of unstimulated r e c t a under s h o r t - c i r c u i t e d c o n d i t i o n s , as i n d i c a t e d by e l e c t r i c a l parameters, i s shown i n Fig.2. The PD d e c l i n e s slowly from an i n i t i a l value of 32 ± 2 mV to 12 ± 3 mV a f t e r 4 hours. These values are s i m i l a r to those r e p o r t e d by Wi l l i a m s et a l . , (1978) using s i m i l a r i n v i t r o p r e p a r a t i o n s . The t r a n s e p i t h e l i a l membrane r e s i s t a n c e (170-220 A-crn 2) d i d not change s i g n i f i c a n t l y over the course of the experiment. I s c f e l l from 6.7 ± 0.3 nEq/cm 2/h i n i t i a l l y to 2.9 ± 0.3 uEq/cm 2/h a f t e r 2 hours. Over the 2nd to 4th h the; I s c d e c l i n e d o n l y 0.8 uEq/cm 2/h (to 2.1 ± 0 . 2 uEq/cm 2/h) so t h a t a s t e a d y - s t a t e c o n d i t i o n was approximated. Although the i n i t i a l I s c was 15% lower than t h a t reported by Williams e t a l . , the observed values i n the two s t u d i e s were i d e n t i c a l between 0.5 and 4 hours. T h i s agreement between r e s u l t s i s of some i n t e r e s t because W i l l i a m s e t a l . bathed t h e i r p r e p a r a t i o n s i n the complex s a l i n e of Berridge (1966) which c o n t a i n s many o r g a n i c c o n s t i t u e n t s whereas the simple s a l i n e used i n the the present 17 Fig*2; V i a b i l i t y of unstimulated s h o r t - c i r c u i t e d r e c t a i n simple C l - s a l i n e as i n d i c a t e d by e l e c t r i c a l parameters, (a) t r a n s e p i t h e l i a l PD (lumen p o s i t i v e ; mean ± SEM; n = i 4-8) . (b) T r a n s e p i t h e l i a l d; . C i r e s i s t a n c e (mean ± SEM; n = 4-8). (c) S h o r t - c i r c u i t c u r r e n t (mean ± SEM; n = 22), Dashed l i n e s on a l l graphs i n d i c a t e mean va l u e s r e p o r t e d by W i l l iams e t a l ; (1978) f o r s h o r t - c i r c u i t e d r e c t a i n complex C l - s a l i n e , . TIME (h) 19 study contained only glucose and glutamate as energy sources. Moreover, NaCl c o n c e n t r a t i o n s were twice as hig h i n the simple s a l i n e t o maintain osmotic c o n c e n t r a t i o n . E f f e c t of -CC Homogenate on E l e c t r o g e n i c ^ T r a n s ^ o r t The e f f e c t s of homogenates of whole CC on r e c t a l t r a n s p o r t a c t i v i t y i s shown i n F i g . 3 . One t e n t h of one gland p a i r of CC added t o the haemocoel-side of r e c t a caused the PD t o i n c r e a s e from 15 + 2 D V t o 25 i 2 mV a f t e r 80 minutes. The PD then s l o w l y d e c l i n e d , dropping t o 23 ± 2 mV over the next 40 minutes. Membrane r e s i s t a n c e (157-126 Stem2) showed a s l i g h t (7 J^-cm2) but s i g n i f i c a n t (P<0„ 05, p a i r e d t - t e s t ) decrease upon the a d d i t i o n o f CC homogenate and continued t o d e c l i n e s t e a d i l y with time. When CC homogenate was added, the I s c began to i n c r e a s e only a f t e r a delay of 5 minutes.. F o l l o w i n g t h i s l a g , I s c i n c r e a s e d r a p i d l y at a r a t e of 4.8 uEq/cm 2/h 2 f o r the next 30 min and continued t o r i s e at a s t e a d i l y d e c r e a s i n g r a t e . f o r a f u r t h e r 40 minutes. O v e r a l l , the I s c rose from an unstimulated value of 3.6 to a maximum of 7.0 /iEq/cm 2/h at 75 ± 5 min (n = 22) a f t e r the a d d i t i o n of the homogenate^ T h i s peak c u r r e n t was maintained or d e c l i n e d very s l o w l y u n t i l t h e . end of the experiment at 4 hours. amounts of CC a good l i n e a r dosage added maximum i n c r e a s e Dose-response . E f f e c t - of CGi- Homogenate on_Ise -The response of r e c t a l I s c to i n c r e a s i n g homogenate i s shown i n F i g . 4 . There i s r e l a t i o n s h i p between the l o g a r i t h m of the. (expressed as f r a c t i o n s of a pr of CC) and the. 20 F i g . 3... E f f e c t s of CC and cAMP on the e l e c t r i c a l parameters o f voltage-clamped r e c t a . . * 0.1 pr CC added at arrow, (n = 12) ; ° 0.3 mM f i n a l c o n c e n t r a t i o n cAMP added at arrow, (n = 9 ) . (a) T r a n s e p i t h e l i a l e l e c t r o p o t e n t i a l d i f f e r e n c e (lumen p o s i t i v e ; mean ± SEM). (b) T r a n s e p i t h e l i a l d. c. r e s i s t a n c e (mean ± SEM). (c) S h o r t - c i r c u i t c u r r e n t (mean ± SEM). 21 22 O .002 .005 .010 .020 L o g a r i t h m of C C ' D o s e ( f ract ion of .050 .100 1 g l a n d p a i r ) F i g . 4 . Peak response of r e c t a l I s c {uEq/cm*/h; mean ± SEil) to i n c r e a s i n g doses of CC- The r e g r e s s i o n l i n e i s exprpssed a s : Maximum A I s c = 2 .62 l o g (CC dose) + 6 . 8 ; n = 126; r = 0 . 5 3 ; P < 0 . 0 5 . Numbers i n parentheses i n d i c a t e n f o r ' e a c h p c i r.t. ' 23 i n I s c over the range of 0,002 to 0.100 pr CC. As l i t t l e as 0.005 of a CC pr i s s u f f i c i e n t to cause a s i g n i f i c a n t i n c r e a s e i n I s c (0.6 ± 0.3 ;iEg/cm 2/h, P<0.05) . I n d i v i d u a l t r a c e s of I s c f o l l o w i n g exposure of r e c t a to 3 very high doses of CC homogenate are shown i n Fig,. 5, The maximum s t i m u l a t i o n caused by 0,05 pr CC (3,5 ± 0,3 /iEq/cm 2/h) d i d not d i f f e r s i g n i f i c a n t l y from t h a t o f a 1 0 - f o l d (4.0 ± 0.3 uEg/cm 2/h) or 2 0 - f o l d (3,9 ± 0 , 4 uEq/cm 2/h) higher dose (P<0.05, Student's t - t e s t ) . . . C l e a r l y the maximum response of I s c to CC s t i m u l a t i o n i s reached at 0.05-0.10 pr CC i n 7.0 ml b a t h i n g s a l i n e . A maximum dose of 0.05 pr CC u s u a l l y r e s t o r e s the I s c t o approximately the value observed immediately a f t e r removing r e c t a from l o c u s t s . A second maximum s t i m u l a t i o n with up to 2.0 pr CC before the r e c t a l I s c had d e c l i n e d to the unstimulated s t a t e simply caused the I s c to r e t u r n t o and remain at the o r i g i n a l maximum value,. As with most b i o l o g i c a l assays, c o n s i d e r a b l e v a r i a t i o n was,.observed among i n d i v i d u a l i n v i t r o p r e p a r a t i o n s , both i n terms of lower d e t e c t i o n l i m i t and maximum response to CC s t i m u l a t i o n ; e.g.maximum s t i m u l a t e d A l s c ranged between 1.5 and 5 times the unstimulated values. While doses i n excess of 0.05 pr CC d i d not cause a d d i t i o n a l i n c r e a s e s i n I s c they d i d s i g n i f i c a n t l y a l t e r the time taken f o r the I s c to f a l l t o one-half the maximum st i m u l a t e d value (t a. t f) . The t 6 , f of 52 ± 6 min observed f o r 0.05 pr CC i n c r e a s e d t o 88 ± 14 min with 1.0 p a i r . T h i s presumably r e f l e c t s the g r e a t e r time t h a t i s r e q u i r e d f o r the d e s t r u c t i o n of the excess amounts of a c t i v e CC f a c t o r present i n the b a t h i n q s a l i n e . The t o i - observed f o l l o w i n g s t i m u l a t i o n with 0,05 pr CC 24 F i g . 5 . T y p i c a l i n d i v i d u a l t r a c e s of I s c with time f o r r e c t a s t i m u l a t e d by CC. Increase values (mean ± SEM) are f o r pooled data; n i s number of t r i a l s , (a) 0.05 pr CC added at arrow. . (b) 0.10 pr CC added at arrow; (c) 1-00 pr CC added a t arrow. T h e r e . i s no s i g n i f i c a n t d i f f e r e n c e among the mean i n c r e a s e values f o r a,b or c.. M e a n I n c r ea se 3.5i 0.3 yEq-cm* h"' n=63 T I M E IV) cn 26 i s s i m i l a r to the i n i t i a l d e c l i n e i n I s c a c r o s s unstimulated r e c t a ( t 0 ^ of about 1 h, F i g , 2 ) , S t a b i l i t y of CC-Homogenate Some o b s e r v a t i o n s were made on the s t a b i l i t y of the a c t i v e f a c t o r i n CC homogenate. B o i l i n g the homogenate f o r 2-3 min had no measurable e f f e c t on i t s potency (8 t r i a l s ) , although prolonged b o i l i n g (>10 min) destroyed a l l a c t i v i t y . A l l these assays i n v o l v e d l a r g e doses (0.05 pr) of pooled homogenate so that a s l i g h t l o s s of potency may have gone undetected. Homogenates were r o u t i n e l y s t o r e d f r o z e n f o r 4 to 6 weeks without s u b s t a n t i a l l o s s of a c t i v i t y . At room temperature, CC i n s a l i n e (pH 7.4) s t i l l r e t a i n e d approximately 1/2 t h e i r o r i g i n a l a c t i v i t y a f t e r 24 hours. E f f e c t s of Other T i s s u e Homogenates on E l e c t r o g e n i c . T r a n s p o r t To determine whether the a c t i v e f a c t o r was present e x c l u s i v e l y i n the CC, the e f f e c t s of v a r i o u s other t i s s u e homogenates on I s c were t e s t e d ( F i g . 6 ) . Homogenates of f l i g h t muscle caused no s t i m u l a t i o n even when used i n l a r g e q u a n t i t i e s (5-10 times the mass of a v e n t r a l g a n g l i o n ) , i n d i c a t i n g t h a t the a c t i v e f a c t o r was not a g e n e r a l metabolite present i n most t i s s u e s . Whole b r a i n s , v e n t r a l quadrants of b r a i n s , v e n t r a l g a n g l i a and whole r e c t a a l l gave some s t i m u l a t i o n but t h e i r maximum e f f e c t was only e q u i v a l e n t to t h a t caused by 0.005 t o 0.010 pr of CC. Moreover, l a r g e q u a n t i t i e s of these homogenates were r e q u i r e d ; anything l e s s than 0.5 gland had l i t t l e or no s t i m u l a t o r y e f f e c t . These homogenates, u n l i k e CC, were 27 F i g . 6 . E f f e c t s of t i s s u e homogenates* e l e c t r i c a l s t i m u l a t i o n and i n h i b i t o r s on I s c . T y p i c a l i n d i v i d u a l t r a c e s are shown as w e l l as mean A I s c ± SEM (/iEq/cm2/h) . Number of t r i a l s are i n parentheses. A l l substances added a t arrows, (a) F l i g h t muscle. . (b) 0.5 whole b r a i n . , (c) 1.0 metathoracic g a n g l i o n . (d) 0.5 t e r m i n a l abdominal g a n g l i o n , (e). 0.5 rectum, (f) E l e c t r i c a l s t i m u l a t i o n (2.0 V* 20 Hz, 5 msec d u r a t i o n * 30 sec s t i m u l a t i o n s a p p l i e d every min f o r 5 min). (g) 5 x 10~* M acetazolamide. (h) 10~ 2 M t h i o c y a n a t e . 29 completely i n a c t i v a t e d by b o i l i n g f o r 2 minutes. When allowance i s made f o r the f a c t t h a t the mass of even the s m a l l e s t v e n t r a l g a n g l i o n t e s t e d i s more than 5 times gre a t e r than t h a t of a pr of CC (25 /ag) / i t i s c l e a r that the I s c s t i m u l a t i n g f a c t o r i s a t l e a s t 500 times more concentrated i n CC than i n the other t i s s u e s which were t e s t e d . The s m a l l s t i m u l a t o r y e f f e c t of r e c t a l homogenate on I s c might be due to the presence of neur o s e c r e t o r y m a t e r i a l i n the axon endings which have been observed i n t h i s organ. I f so, d i r e c t e l e c t r i c a l s t i m u l a t i o n of i n v i t r o r e c t a might s t i m u l a t e the r e l e a s e of t h i s n e u r o s e c r e t o r y m a t e r i a l . A v a r i e t y o f e l e c t r i c a l s t i m u l i ((1-5 V o l t s , 5 msec d u r a t i o n , 20-50 Hz; 30 sec s t i m u l a t i o n s a p p l i e d every 1 min f o r 4-8 min) a p p l i e d t o r e c t a during the s t e a d y - s t a t e phase had no subsequent e f f e c t on the I s c (Fig.6) . E f f e c t s of Neurotransmitter Substances on I s c None of the f o l l o w i n g p u t a t i v e t r a n s m i t t e r substances, a p p l i e d at or below the given c o n c e n t r a t i o n s , had any e f f e c t on I s c or PD: 5-hydroxytryptamine (10-* M, n = 8), epinephrine (10~ 3 M, n = 4), norepinephrine (10~ 3 M, n = 2), a c e t y l c h o l i n e (10- 3 M, n = 6) , dopamine (10-* W, n = 4) , octopamine (10~* M, n = 4 ) , glutamate ( 1 0 - 2 M, normally present i n s a l i n e ) , g l y c i n e (10-3 M , n = 4) and J T-aminobutyric a c i d (10~* M, n = 8) . S t i m u l a t i o n o f E l e c t r o g e n i c Transport by cAMP Since the a c t i o n of peptide hormones i s commonly mediated i n t r a c e l l u l a r l y by cAWP, I s t u d i e d the e f f e c t of t h i s second 30 messenger on s h o r t - c i r c u i t e d r e c t a . The r e s u l t s are compared with those f o r CC s t i m u l a t i o n i n F i g . 3 . A d d i t i o n of n e u t r a l i z e d cAMP ( f i n a l c o n c e n t r a t i o n 0.3 mM) to the haemocoel-side of i n v i t r o p r e p a r a t i o n s caused the PD to i n c r e a s e from 15 ± 1 mV to 22 + 2 mV a f t e r 120 minutes. There was no measurable l a g i n the response time and most of t h i s i n c r e a s e (to 20 mV) occur r e d w i t h i n the f i r s t 20 minutes.. T h i s i s i n c o n t r a s t to the CC-s t i m u l a t e d i n c r e a s e i n PD which, a f t e r an i n i t i a l l a g , rose c o n t i n u o u s l y f o r 80 min and then began t o d e c l i n e . C y c l i c AMP a l s o caused a s l i g h t but s i g n i f i c a n t drop i n membrane r e s i s t a n c e (25 i l c m 2 , P<0.05, p a i r e d t - t e s t ) . T h i s was about 4 times g r e a t e r than the r e s i s t a n c e change caused by CC homogenate. C y c l i c AMP caused the Isc t o i n c r e a s e w i t h i n seconds of i t s a p p l i c a t i o n whereas there was a 5 min lag-time i n response to CC homogenate. The I s c rose l i n e a r l y at a r a t e of 12.6 jaEq/cm 2/h 2 to 80% of i t s f i n a l value w i t h i n 10 min of a p p l y i n g cAMP. T h e r e a f t e r , the I s c continued to r i s e s lowly over the course of the experiment to a maximum value of 6.9 uEq/cm 2/h. In summary, cAMP caused a s i m i l a r maximum i n c r e a s e i n I s c as did CC, but these s t i m u l a n t s d i f f e r e d i n the lag-time of the response f o l l o w i n g s t i m u l a t i o n and the r a t e o f r i s e i n I s c . As f o r CC homogenate, the response of r e c t a l I s c to the log a r i t h m of cAMP c o n c e n t r a t i o n i s l i n e a r ( F i g . 7 ) . . A c o n c e n t r a t i o n of 0.005 mM cAMP i n the bathing s a l i n e on the haemocoel-side e l i c i t e d a measurable response (0.2 ± 0.04 uEg/cm 2/h, n = 6) and 0.3 mM cAMP caused the same maximal s t i m u l a t i o n as 0.1 pr CC ( A l s c = 4.1 uEg/cm 2/h) . I observed much l e s s v a r i a b i l i t y i n the response of i n v i t r o r e c t a to cAMP f i g . 7. Peak r e s p o n s e o f i s c O i E q / c m V h • mean + i P n i n c r e a s i n g d o s e s of cAMP Th* C l n r - ' t 0 l i n e a r p o r t i o n o f t h e c u r v e * f s o ^ d ? i ^ f S • ° n - l i n e f ° r - t h e 5;"L P < 0 * 0 5 - N U m D e r S i a P*"»theies i n d l c a ' t e n ' f o r each" 32 than to CC homogenates. E f f e c t s of S p e c i f i c Transport B l o c k e r s The work of Williams e t a l . (1978) suggested to me t h a t the i n c r e a s e i n I s c upon s t i m u l a t i o n might be due to i n c r e a s e d C l - or H+ZHCOj- t r a n s p o r t . I t h e r e f o r e s t u d i e d the e f f e c t s o f well-known i n h i b i t o r s of these t r a n s p o r t processes on r e c t a l I s c . Acetazolamide, a p p l i e d at a c o n c e n t r a t i o n of 5 x 10-* M c o n s i s t e n t l y caused the I s c of unstimulated r e c t a to decrease by 0.63 ± 0.06 uEq/cm 2/h ( F i g . 6 ) . The s i z e o f t h i s i n h i b i t i o n remained unchanged when r e c t a were s t i m u l a t e d with CC homogenate or cAMP; i . e . t h i s i n h i b i t o r d i d not appear t o a l t e r the i n v i t r o response t o s t i m u l a t i o n . Adding a second a l i q u o t t o b r i n g the f i n a l c o n c e n t r a t i o n t o 10~ 3 M had no f u r t h e r e f f e c t on I s c . Acetazolamide appeared to be e q u a l l y e f f e c t i v e when added t o the lumen or haemocoel s i d e of the p r e p a r a t i o n . Thiocyanate, an i n h i b i t o r of C l - t r a n s p o r t i n many ve r t e b r a t e systems, caused no chanqe i n the. I s c or PD of unstimulated r e c t a when added at a f i n a l c o n c e n t r a t i o n o f l O - 2 M. Thiocyanate i n no way a l t e r e d the normal response of s h o r t - c i r c u i t e d r e c t a t o CC homogenate or cAMP, although the e l e c t r i c a l ' n o i s e' present i n the t r a c i n g s i n c r e a s e d . When acetazolamide and th i o c y a n a t e were added s i m u l t a n e o u s l y , the i n h i b i t i o n was the same as t h a t observed with acetazolamide alone.. In summary, these experiments suggest t h a t about 25% of the net s t e a d y - s t a t e I s c i s due to c a r b o n i c anhydrase-mediated H + or HCOj t r a n s p o r t . There i s no i n d i c a t i o n t h a t t h i s t r a n s p o r t component i s s t i m u l a t e d by CC homogenate or cAMP. 33 D i s c u s s i o n The e l e c t r i c a l p r o p e r t i e s of the unstimulated, v o l t a g e -clamped r e c t a used i n the present study (Fig.2) are q u a n t i t a t i v e l y s i m i l a r t o those r e p o r t e d by Williams et a l . (1978),. The o n l y d i f f e r e n c e i n method between the two s t u d i e s i s the composition of the bathing s a l i n e . The s i m i l a r i t y of the r e s u l t s i n d i c a t e s t h a t a simple i n o r g a n i c s a l i n e c o n t a i n i n g o n l y glucose and glutamate as energy sources w i l l s u s t a i n t r a n s p o r t a c t i v i t i e s ( for up to 36 h) almost as w e l l as the complex t i s s u e c u l t u r e medium of B e r r i d g e (1966). The use of simple s a l i n e reduces the problems a s s o c i a t e d with m i c r o b i a l growth i n the complex s a l i n e over the. course of an experiment. The s u b s t i t u t i o n of simple f o r complex s a l i n e i s of some i n t e r e s t because W i l l i a m s e t a l . p o s t u l a t e d t h a t the t r a n s p o r t o f unknown o r g a n i c i o n s , PO 3 - or H+ZHCOj accounts f o r most of the Is c across unstimulated r e c t a d u r i n g the s t e a d y - s t a t e phase. They f u r t h e r suggested t h a t these unknown processes may be s u s t a i n e d by v a r i o u s o r g a n i c c o n s t i t u e n t s of the complex s a l i n e . However, our s a l i n e c o n t a i n s r e l a t i v e l y l i t t l e P O 3 - (1 mM) and no o r g a n i c c o n s t i t u e n t s b e s i d e s glucose and glutamate. Therefore, o t h e r components of B e r r i d g e ' s s a l i n e are not necessary t o maintain the s t e a d y - s t a t e I s c , a t l e a s t i f C l - i s present (see Chapter I I I ) . I n h i b i t i o n of I s c by acetazolamide supports the s u g g e s t i o n by Williams et a l . t h a t p a r t of the I s c i s due t o H+/HCO|- t r a n s p o r t . CC homogenates c o n t a i n a f a c t o r which s t i m u l a t e s e l e c t r o g e n i c t r a n s p o r t of anions to the haemocoel or c a t i o n s to the lumen and hence makes the haemocoel more negative r e l a t i v e 34 to the lumen. From the work of Wil l i a m s e t a l . (1978) and Herrera et a l , (1976, 1977), i t i s most l i k e l y t h a t C l ~ or H+/HCOJ t r a n s p o r t i s s t i m u l a t e d . However, the f a i l u r e o f acetazolamide t o i n h i b i t the i n c r e a s e i n I s c caused by CC and CAMP suggest t h a t H+/HCOJ t r a n s p o r t i s not s t i m u l a t e d . Another p o s s i b i l i t y i s that the f a c t o r might decrease t h e . a c t i v e uptake (L=>H) of Na + and K + which has been demonstrated i n v i t r o by Williams e t a l . . P r e l i m i n a r y o b s e r v a t i o n s suggest that the a c t i v e f a c t o r i n CC homogenate might be a pep t i d e . Most known o r p u t a t i v e n e u r o t r a n s m i t t e r s have been excluded. The: a c t i o n of CC homogenate i s mimicked by cAMP, the second messenger f o r peptide hormones. The CC f a c t o r i s wa t e r - s o l u b l e , suggesting t h a t i t i s not a s t e r o i d and i t i s somewhat h e a t - s t a b l e which i m p l i e s t h a t i t i s not a l a r g e p r o t e i n . . F l i g h t muscle homogenates, even i n l a r g e g u a n t i t i e s , have no e f f e c t on I s c which i n d i c a t e s t h a t the a c t i v e f a c t o r i s not a ge n e r a l m e t a b o l i t e . A l l the neural t i s s u e i n the l o c u s t appears to have some s t i m u l a t o r y e f f e c t on I s c . However, the maximum i n c r e a s e i n I s c caused by these homogenates i s a t most 25% of that observed upon the a d d i t i o n of CC homogenate and the amount of t i s s u e r e g u i r e d t o achieve t h i s l e v e l of s t i m u l a t i o n i s 200 times g r e a t e r than f o r the CC. The c o n c e n t r a t i o n of the a c t i v e f a c t o r , t h e r e f o r e , i s approximately t h r e e orders of magnitude g r e a t e r i n the CC than i n other t i s s u e s . The a c t i v e f a c t o r i n v e n t r a l g a n g l i a may be d i f f e r e n t from t h a t i n CC as i t i s g u i c k l y destroyed by b o i l i n g f o r 2 min, whereas the a c t i v i t y i n CC i s not. 35 The p a r t i a l s t i m u l a t i o n of I s c by homogenates of r e c t a l t i s s u e l e n d s credence t o the sugge s t i o n t h a t c o n t r o l might be mediated by ne u r o s e c r e t o r y axon endings wi t h i n the r e c t a l w a l l (Johnson, 1963, 1966; Gupta and B e r r i d g e , 1966; Oschman and Wall, 1969). The f a i l u r e of e l e c t r i c a l s t i m u l a t i o n t o i n c r e a s e I s c suggests t h a t t h i s i s not the case; however, the neuro s e c r e t o r y matter may have been l o s t through the severed axon endings d u r i n g the two-hour p e r i o d r e q u i r e d f o r the p r e p a r a t i o n to reach the s t e a d y - s t a t e phase. I t i s c o n c e i v a b l e that the i n i t i a l d e c l i n e i n I s c i s due to the g r a d u a l l o s s of neuro s e c r e t o r y m a t e r i a l from the severed nerves r a t h e r than the slow degradation of a bound hormone. CC homogenate does not cause s i g n i f i c a n t e l e v a t i o n of I s c a c r o s s r e c e n t l y d i s s e c t e d r e c t a which suggests t h a t the high i n i t i a l I s c i s due to s t i m u l a t i o n of the rectum i n the i n t a c t animal. The d e c l i n e i n Isc i s probably caused by the removal of t h i s s t i m u l u s r a t h e r than the l o s s of t i s s u e v i a b i l i t y . T h i s i s f u r t h e r supported by the f a c t t h a t the I s c across i s o l a t e d r e c t a can be. r e s t o r e d t o the i n i t i a l value many hours l a t e r by the a d d i t i o n o f CC homogenate or cAMP. Both cAMP and CC homogenates show l o g a r i t h m i c dose-response curves. However the l e v e l s of cAMP r e q u i r e d f o r even minimal s t i m u l a t i o n are more than 10 6 times q r e a t e r than the normal l e v e l s o f t h i s c y c l i c n u c l e o t i d e i n mammalian t i s s u e s ( S t e i n e r et a l . , 1972; P e r k i n s , 1973). I t i s commonly assumed t h a t the high l e v e l s of cAMP re q u i r e d to mimic v a r i o u s hormone a c t i o n s r e f l e c t the low p e r m e a b i l i t y of the plasma membrane to c y c l i c n u c l e o t i d e s . I t f o l l o w s that the cAMP content of t i s s u e 36 homogenates used i n the present study are more than s i x orders of magnitude too low to s t i m u l a t e r e c t a l I s c . There are some q u a n t i t a t i v e d i f f e r e n c e s i n the response o f r e c t a to cAMP and CC. The maximum r a t e of I s c i n c r e a s e caused by cAMP s t i m u l a t i o n (12.6 /iEq/cm 2/h 2) i s nea r l y three times t h a t f o r CC homogenate (4,-8 uEq/cm 2/h 2). . Moreover, cAMP a c t s i n s t a n t l y on r e c t a whereas there i s a l a g i n response time f o l l o w i n g the a d d i t i o n of CC homogenate. These d i f f e r e n c e s may r e f l e c t the time r e q u i r e d f o r the CC f a c t o r to bind t o r e c e p t o r s i t e s on the r e c t a l e p i t h e l i a l c e l l s and s t i m u l a t e the production of cAMP.. A s i m i l a r l a g i n hormonal s t i m u l a t i o n compared to t h a t by c y c l i c n u c l e o t i d e s i s f r e q u e n t l y observed i n other systems (Murad, 1973; P e r k i n s , 1973). Presumably, f l o o d i n g the t i s s u e with cAMP r a i s e s the i n t r a c e l l u l a r l e v e l s of t h i s n u c l e o t i d e f a s t e r than can be achieved by the adenyl c y c l a s e system alone. Also the constant i n f l u x o f f r e s h cAMP would s a t u r a t e c e l l u l a r phosphodiesterase, which would account f o r the p e r s i s t e n c e of cAMP-induced s t i m u l a t i o n compared t o t h a t induced by CC homogenate. The very s m a l l amount of CC homogenate used i n our s t u d i e s i n s u r e d t h a t the supply of a c t i v e f a c t o r was l i m i t e d . The f i n a l d i s t i n c t i o n i s t h a t there i s c o n s i d e r a b l y l e s s v a r i a t i o n i n the response of r e c t a t o cAMP than there i s to CC homogenate. T h i s may r e f l e c t the a v a i l a b i l i t y of b i n d i n g s i t e s f o r the CC f a c t o r . The number of r e c e p t o r s i t e s i s known to vary g r e a t l y i n response to v a r i o u s s t i m u l i f o r other hormones (L. E i d d i f o r d , pers. comm.). . The work d e s c r i b e d i n t h i s chapter r a i s e s a number o f important questions i n c l u d i n g which s p e c i f i c t r a n s p o r t process 37 i s c o n t r o l l e d by the CC, what i s the e f f e c t of CC on i n t r a c e l l u l a r cAMP l e v e l s and whether the CC f a c t o r i s b l o o d -borne. These q u e s t i o n s are c o n s i d e r e d i n the subsequent chap t e r s . 38 I I I IDENTIFICATION OF - SPECIFIC ION TRANSPORT PROCESSES REGULATED BY CORPORA CARDIAL A AND CYCLIC-AMP I n t r o d u c t i o n There, i s a f a c t o r present i n the corpora c a r d i a c a (CC) of S c h i s t o c e r c a g r e q a r i a which causes l a r q e changes i n a c t i v e , i o n t r a n s p o r t a c r o s s r e c t a l e p i t h e l i a as i n d i c a t e d by a 2-3 f o l d i n c r e a s e i n t h e s h o r t - c i r c u i t c u r r e n t (Isc) and t r a n s e p i t h e l i a l e l e c t r o p o t e n t i a l d i f f e r e n c e (PD). The a c t i o n o f t h i s heat-s t a b l e , w a t e r - s o l u b l e f a c t o r i s mimicked by the c y c l i c n u c l e o t i d e cAMP.. The question remains as to which s p e c i f i c i o n t r a n s p o r t process i s a f f e c t e d by the CC f a c t o r and cAMP. Wi l l i a m s e t a l . (1978) showed t h a t under s t e a d y - s t a t e c o n d i t i o n s there i s a r a p i d net f l u x of both Na+ and C l - from the lumen s i d e of voltage-rclamped r e c t a . These two f l u x e s are of ne a r l y equal magnitude but being of opp o s i t e e l e c t r i c a l charge, they do not make a s u b s t a n t i a l net c o n t r i b u t i o n t o the t o t a l I s c . . W i l l i a m s e t a l . suggest t h a t the balance of the I s c i s due to H + s e c r e t i o n to the lumen or anion a b s o r p t i o n (HCOj, o r g a n i c anions, PO 3 -) to the haemocoel. Some evidence f o r a H+/HCOJ t r a n s p o r t component has been provided i n the pre v i o u s chapter and by Herrera et a l . (1977) who demonstrated t h a t acetazolamide, an i n h i b i t o r of c a r b o n i c anhydrase, decreases the s t e a d y - s t a t e I s c by approximately 25%. Williams et- a l . used r e c t a bathed i n B e r r i d g e ' s complex s a l i n e whereas the presen t study was done using a simple i n o r g a n i c s a l i n e c o n t a i n i n g o n l y one o r g a n i c anion, glutamate, which i s not t r a n s p o r t e d by i n 39 v i t r o r e c t a l sacs ( B a l s h i n , 1973). Therefore the s t i m u l a t i o n of Isc by CC and cAMP observed in t h i s study i s probably not due t o the t r a n s p o r t of o r g a n i c i o n s , unless these are s y n t h e s i z e d i n t r a c e l l u l a r l y . There are more compelling reasons to b e l i e v e t h a t these two s t i m u l a n t s a c t s p e c i f i c a l l y by i n c r e a s i n g e l e c t r o g e n i c C l _ t r a n s p o r t . The i n i t i a l r a p i d f a l l i n I s c f o l l o w i n g removal o f r e c t a from l o c u s t s i s due to a d e c l i n e i n a c t i v e . C l ~ uptake while Na + i s not s i m i l a r l y a f f e c t e d (Williams e t a l . , 1978). The subseguent r e s t o r a t i o n of the I s c to the o r i g i n a l high l e v e l s by CC suggested t h a t the i n i t i a l d e c l i n e . i s a conseguence of removing r e c t a from hormonal s t i m u l a t i o n of C I - t r a n s p o r t and t h a t the CC f a c t o r r e s t o r e s t h i s s t i m u l a t i o n , although i n h i b i t i o n of Nat uptake would have the same e f f e c t on I s c . In t h i s chapter I have d i s t i n g u i s h e d among these v a r i o u s p o s s i b i l i t i e s by s t u d y i n g the e f f e c t of cAMP and CC on i s o t o p i c f l u x e s of s p e c i f i c i o n s and on the I s c of voltage-clamped r e c t a bathed i n C l - f r e e s a l i n e s . F i n a l l y cAMP l e v e l s i n r e c t a l t i s s u e were measured before and a f t e r s t i m u l a t i o n by CC homogenate to determine whether the CC f a c t o r a c t s by e l e v a t i n g i n t r a c e l l u l a r l e v e l s o f t h i s c y c l i c n u c l e o t i d e . M a t e r i a l s - and Methods Experimental animals were mature S c h i s t o c e r e a g r e q a r i a , one to three months past t h e i r f i n a l moult. They-were reared at 28° C and 50% B.H. under a photoperiod of L:D 16:8 on a d i e t of f r e s h l e t t u c e and a mixture of powdered milk, yeast, bran and d r i e d g r a s s . Females were used f o r a l l i n v i t r o - p r e p a r a t i o n s of 40 r e c t a because o f t h e i r l a r g e r s i z e . Males were used f o r a l l CC homogenates to avoi d any changes a s s o c i a t e d with the female r e p r o d u c t i v e c y c l e . Measurement of Ion F l u x e s Eecta were mounted as f l a t sheets between two •Ossing-type' chambers each of which was f i l l e d with 7.0 ml s a l i n e and mixed v i g o r o u s l y by a gas l i f t pump, using 95% 0^- 5% C0 t (Williams et a l . , 1978). A l l experiments were conducted at room temperature (22°C). r U n i d i r e c t i o n a l f l u x e s of . * 3 6 G 1 - and 2 2 N a + were measured under s h o r t - c i r c u i t c o n d i t i o n s . . I s o t o p e s were obtained from New England N u c l e a r , Inc. i n the f o l l o w i n g forms : a 0.41 M N a 3 6 C l s o l u t i o n a t pH 7.0 (5,8 mCi.gm-i) and 2.4 mCi.ml-i of 2 2 N a C l i n Hj^ O at p.H 4.5 ( c a r r i e r f r e e ) . I sotopes were added t o s i d e 1, and 1 u l a l i g u o t s were removed and d i l u t e d with 2.0 ml s a l i n e to determine i n i t i a l r a d i o a c t i v i t y . At 20 minute i n t e r v a l s 2.0 ml a l i g u o t s of bathing s o l u t i o n were removed from s i d e 2 t o determine the amount of i s o t o p e which had c r o s s e d the membrane. The s o l u t i o n removed during sampling was r e p l a c e d with an equal volume of u n l a b e l l e d s a l i n e . T h i s i n s u r e d that the l e v e l of r a d i o a c t i v i t y on s i d e 2 remained l e s s than 0.5% of t h a t on s i d e 1, so that b a c k - f l u x of i s o t o p e was n e g l i g i b l e . U n i d i r e c t i o n a l f l u x e s over each 20 minute p e r i o d were estimated using the f o l l o w i n g equation: where J,^^ i s the u n i d i r e c t i o n a l f l u x i n ;iEq/cm 2/h, a, r e p r e s e n t s 41 the c o n c e n t r a t i o n of r a d i o a c t i v i t y (cpm/ml) on s i d e 1, a t i s the i n c r e a s e i n the c o n c e n t r a t i o n of r a d i o a c t i v i t y (cpm/ml) on s i d e 2 over the 20 minute i n t e r v a l , V i s the volume of side 2 i n ml, c i s the c o n c e n t r a t i o n of u n l a b e l l e d i on i n the s a l i n e (jiEq/ral) , T i s the time i n t e r v a l over which the f l u x occurred ( i . e . 0.33h) and A i s the area of the membrane ( i . e . 0.196 cm 2). During sampling, voltage-clamping was i n t e r r u p t e d f o r 30 sec to measure PD. Sample r a d i o a c t i v i t y was determined by p l a c i n g 2.0 ml a l i q u o t s o f bathing s a l i n e on s t a i n l e s s s t e e l p l a n c h e t s , e v a p o r a t i n g t o dryness under a heat lamp and counting on a •Nuclear Chicago Model 470' gas-flow d e t e c t o r and automatic sample changer. No c o r r e c t i o n f o r s e l f - a b s o r p t i o n was r e g u i r e d because t h i s f a c t o r remained co n s t a n t . F l u x e s i n o p p o s i t e d i r e c t i o n s were measured on d i f f e r e n t p r e p a r a t i o n s c o n c u r r e n t l y with I s c and PD. Average I s c and PD values were very s i m i l a r f o r i n f l u x and e f f l u x s t u d i e s and were consequently pooled;. Net f l u x was c a l c u l a t e d as the d i f f e r e n c e between the two mean u n i d i r e c t i o n a l f l u x e s . V a r i a n c e s f o r the net f l u x e s were c a l c u l a t e d u s i n q the formula f o r common va r i a n c e o f unequal sample s i z e s ( L a r k i n , 1976)... • CC homoqenate and cAMP were prepared as d e s c r i b e d i n Chapter I I . Ninety min a f t e r the i n i t i a t i o n of voltage-clamping, an a p p r o p r i a t e dose (0.1 pr CC> 0.3 mM cAMP) of s t i m u l a n t i n s a l i n e was added to the haemocoel-side of t h e ; p r e p a r a t i o n . . I n experiments where samples were being removed from the haemocoel-s i d e , no attempt was made to r e p l a c e the stim u l a n t l o s t through sampling, so t h a t the c o n c e n t r a t i o n of s t i m u l a n t i n the b a t h i n g s a l i n e f e l l stepwise with time a f t e r the f i r s t 20 min (see 42 F i g . 9). C l - F r e e S a l i n e s A number of C l - f r e e s a l i n e s were prepared by r e p l a c i n g a l l of the C l - i n simple s a l i n e with equimolar amounts of SO 2-, NOj or a c e t a t e . These are subsequently r e f e r r e d to as SO^ ;-, N 0 3 - o r ac e t a t e s a l i n e s r e s p e c t i v e l y (Table 1) . A l l , s a l i n e s are ;pf s i m i l a r osmotic c o n c e n t r a t i o n except f o r the S 0 H - s a l i n e - 2 , which was d i s t i n c t l y hyperosmotic to the o t h e r s . i To study the e f f e c t of anion s u b s t i t u t i o n s on s t e a d y - s t a t e I s c and the response of r e c t a so t r e a t e d to s t i m u l a t i o n , p r e p a r a t i o n s were i n i t i a l l y bathed i n the s u b s t i t u t e s a l i n e . f o r 1 hour. The bathing s a l i n e , which now contained 1 any C l ~ l o s t from the t i s s u e , was completely r e p l a c e d with 4 changes of the same s a l i n e before experiments were begun.. Subsequent changes from one s a l i n e to another a l s o i n v o l v e d 4 complete changes o f the s a l i n e i n each chamber. W i l l i a m s e t a l . (1978) reported t h a t complex C l - f r e e s a l i n e s supported s t e a d y - s t a t e I s c e q u a l l y as w e l l as complex C l - s a l i n e . We observed, however, t h a t simple N0 3- or SO.,-salines 5 would support a s t e a d y - s t a t e -Isc of l e s s than h a l f t h a t of p r e p a r a t i o n s bathed i n simple C l - s a l i n e . To determine whether the d i s c r e p a n c i e s between our o b s e r v a t i o n s and those of W i l l i a m s e t - a l . were due t o the d i f f e r e n c e s between simple, and complex s a l i n e s o r some other f a c t o r , I exposed i n d i v i d u a l r e c t a s e q u e n t i a l l y to simple C l - s a l i n e and to simple and complex SO/j-salines i n d i f f e r e n t o r d e r s . Complex SO^-saline was prepared using the formula of Williams et a l . (1978), and because t h e i r 43 TABLE 1. Composition of s a l i n e s used i n t h i s study. 'Specific resistance of Type of simple s a l i n e Major s a l t s (mM) sal i n e (20"C) (JL .cm) C l - s a l i n e 185 NaCl, 11 KC1, 3 C a C l 2 48.8 (Normal s a l i n e ) N 0 3 - s a l i n e . 185 NaN03, 11KNC>3, 3 Ca(N0 3)' 2 51.4 Aceta t e - s a l i n e . ' 185 Na acetate, 11 K acetate, 71.5 3 Ca ( a c e t a t e ) 0 SO.-saline-1 93 Na„SO. , 5.5 K oS0., 3 CaSO. 59.5 4 2 4 2 4 4 S0 4-saline-2 185 Na2SO , 11 K 2S0 4, 3 CaS0 4 • 34.8 Complex SO.-saline* 12.3 Na.SO.,.4.3 K„S0., 2 CaSO. 174.0 4 2 4 2 4 , 4 A l l of the simple s a l i n e s contained 10 mM glucose, 3 mM L-glutamate, 2.5 mM MgS04, 1 mM KH 2P0 4, 24 mM NaHCO The major s a l t s i n each saline d i f f e r e d as indic a t e d above. * Complex'SO^-saline also contained (mM): 10.5 NaHCOj, 13 MgS04, 7.4 disodium succinate, 1.87 trisodium c i t r a t e , 12.8 malic acid, 16.6 glucose, 5.56 maltose, 128.3 sucrose, 2.67 glycine, 4.61 proline, 2.64 glutamine, 12.3 glutamic a c i d , 30 mg/1 p e n i c i l l i n and 100 mg/1 streptomycin sulphate. The pH was adjusted to 7.00 with NaOH, and t o t a l osmotic concentration was 317 mOsm. 44 C l - f r e e s a l i n e s were e q u i v a l e n t r a t h e r than equimolar, simple SO^-saline was prepared t h a t was e q u i v a l e n t to simple C l - s a l i n e ; i . e . with h a l f the Na xSO M and K xSO H c o n c e n t r a t i o n s of S0H-s a l i n e - 2 (Table 1). T h i s i s subsequently r e f e r r e d to as S 0 M -s a l i n e - 1 . R e c t a l p r e p a r a t i o n s were i n i t i a l l y bathed i n simple C l - s a l i n e , which was r e p l a c e d by simple or complex SO H--saline d u r i n g the s t e a d y - s t a t e phase. The remaining S 0 M - s a l i n e was then t e s t e d and the p r e p a r a t i o n s returned to simple C l - s a l i n e . The two s t i m u l a n t s , CC homogenate and cAMP were prepared as stock s o l u t i o n s i n S0«,-saline-2 s i n c e a l l C l - f r e e s a l i n e s c o n t a i n e d some S O 2 - and the sm a l l volume of s t i m u l a n t s o l u t i o n added d i d not s u b s t a n t i a l l y change the composition of the bathing media. Tis s u e cAMP Assay To assay t i s s u e l e v e l s of cAMP, everted r e c t a l sacs were prepared from a d u l t female l o c u s t s as d e s c r i b e d by Goh and P h i l l i p s (1978). They were weighed and incubated at 37° C f o r at l e a s t 2 h i n simple s a l i n e so t h a t r e c t a were, i n the steady-s t a t e phase of f l u i d t r a n s p o r t (Goh and P h i l l i p s , 1978). The sacs were then emptied, reweighed, and r e f i l l e d with .10 u l o f simple C l - s a l i n e ( c o n t r o l ) or 10 .ul of s a l i n e c o n t a i n i n g 0.25 pr CC and incubated f o r 15, 30 or 60 minutes.,Controls were incubated f o r the whole 60 minutes. The sacs were d r a i n e d , b l o t t e d dry, weighed and then g u i c k l y f r o z e n on p i e c e s o f aluminum f o i l on dry i c e . C y c l i c AMP l e v e l s were assayed using a com p e t i t i v e b i n d i n g assay k i t (TRK 432) from Amersham/Searle Lt d . . ( O a k v i l l e , Ont.). Frozen r e c t a were homogenized and 45 d e p r o t e i n i z e d using HClO^ and assayed as recommended i n the k i t i n s t r u c t i o n s . T r i t i u m a c t i v i t y was measured by p l a c i n g samples i n 10.0 ml of commercial s c i n t i l l a t i o n f l u i d and counting i n a •Nuclear Chicago Isocap 300* l i q u i d s c i n t i l l a t i o n counter using the c h a n n e l s - r a t i o method of quench c o r r e c t i o n . . R e s u l t s E f f e c t s of-CC Homogenate on_Na+ and C l ~ Fluxes The I s c and PD across unstimulated r e c t a bathed i n simple s a l i n e have already been d e s c r i b e d i n Chapter I I ( F i g . 2 ) . The mean va l u e s with time are shown by the dashed l i n e s i n F i g . ? and F i g - 1 1 W i l l i a m s e t a l . (1978) have f o l l o w e d the f l u x e s of Na + and C l _ a c r o s s voltage-clamped r e c t a bathed with a complex s a l i n e using the same method as i n the present study. They showed t h a t the i n i t i a l f a l l i n I s c was caused by a d e c l i n e i n net C l - uptake from the lumen and t h a t the s t e a d y - s t a t e phase (2 to 4 h) was c h a r a c t e r i z e d by c o n s t a n t u n i d i r e c t i o n a l and net f l u x e s of C l ~ , Na+ and K + with time; i . e . . l a r g e spontaneous i n c r e a s e s i n f l u x e s are never observed d u r i n g t h i s phase.. The normal t r e n d during s t e a d y - s t a t e i s towards a slow d e c l i n e i n the t r a n s p o r t of these i o n s . The e f f e c t of s t i m u l a t i o n by CC homogenate on u n i d i r e c t i o n a l 3 6 C 1 - f l u x e s a c r o s s voltage-clamped r e c t a i s shown i n F i g . 8 a . _As p r e v i o u s l y observed by W i l l i a m s e t a l . , , t h e r e was an i n i t i a l d e c l i n e i n net i n f l u x (L=>H) but no change i n b a c k f l u x (H=>L) over the f i r s t 90 minutes. When CC homogenate 4 6 F i g . 8 . U n i d i r e c t i o n a l c l - f l u x e s with time f o r s h o r t - c i r c u i t e d r e c t a bathed i n simple C l - s a l i n e (uEq/cm 2/h; mean ± SEM) . (a) 0.1 pr CC added at arrows. • i n d i c a t e s i n f l u x (L=>H; n = 12); ° i n d i c a t e s b a c k f l u x (H=>L; n = 10). (b) 0.3 mM f i n a l c o n c e n t r a t i o n cAMP added at arrows. • i n d i c a t e s i n f l u x (L=>H; n =11); ° i n d i c a t e s b a c k f l u x (H=>L; n = 11) -48 was added to the haemocoel-side, there was a r a p i d i n c r e a s e i n the i n f l u x of C l - (L=>H) . There was no corresponding change i n bac k f l u x (H=>L) f o l l o w i n g s t i m u l a t i o n . The magnitude of the response can be b e t t e r gauged from F i g . 9b, which compares net C l - f l u x and I s c . The net C l ~ f l u x equals or exceeds the I s c and c l o s e l y p a r a l l e l s the i n c r e a s e i n I s c f o l l o w i n g the a d d i t i o n o f CC homogenate. The i n i t i a l d e c l i n e i n I s c over the f i r s t 90 min i s a l s o c l o s e l y matched by the i n i t i a l decrease i n net C l -uptake. T h i s confirms the e a r l i e r work of Williams et a l . (1978), who used a complex s a l i n e , and f u r t h e r emphasizes t h a t l o c u s t r e c t a f u n c t i o n e g u a l l y w e l l i n simple or complex C l -s a l i n e . CC homogenate has no e f f e c t on e i t h e r the u n i d i r e c t i o n a l (Fig.10) or net f l u x e s of Na+ (Fig.11) under s h o r t - c i r c u i t e d c o n d i t i o n s . The net f l u x remains r e l a t i v e l y c onstant at 2.1 ± 0.2 uEg/cm 2/h (mean ± SEM; L=>H) over the e n t i r e : c o u r s e of the experiment. Williams e t a l . a l s o observed no change i n Na + f l u x over the course of t h e i r experiments, although t h e i r a b s o l u t e values f o r Na+ uptake (4.4 ± 0.5 uEg/cm 2/h) were somewhat hi g h e r . The f l u x r a t i o s , c a l c u l a t e d as mean i n f l u x over mean b a c k f l u x , are 3:1 f o r C l ^ and 2:1 f o r Na+..The C l ~ r a t i o i s i d e n t i c a l to t h a t obtained by Wi l l i a m s et a l . , while the Na + r a t i o i s o n l y h a l f t h e i r c a l c u l a t e d value. E f f e c t s of c AMP on_Na+ and C l - F l u x e s The e f f e c t of f l u x e s i s i l l u s t r a t e d F i g . 9 c . The r e s u l t s s t i m u l a t i o n by cAMP on u n i d i r e c t i o n a l C l -i n F i g . 8b, and net C l - f l u x and I s c i n are s i m i l a r to those d e s c r i b e d f o r CC 4 9 F i g . 9 . I s c and net C l ~ f l u x e s f o r s h o r t - c i r c u i t e d r e c t a bathed i n simple C l - s a l i n e (uEq/cm 2/h; mean ± SEM) ; Net f l u x data obtained from F i g ; 8 . • i n d i c a t e s net C l - i n f l u x (L=>H); ° i n d i c a t e s I s c ; dashed l i n e i n d i c a t e s mean I s c f o r unstimulated r e c t a (from Fig;2) (a) Histogram i n d i c a t e s decreasing c o n c e n t r a t i o n of s t i m u l a n t (expressed as % of the o r i g i n a l dose) with time due t o sampling procedure,. . (b) 0,. 1 pr CC added at arrow (n = 22). (c) 0.3 mM f i n a l c o n c e n t r a t i o n cAMP added at arrow (n = 22) . 51 Fig.10.. U n i d i r e c t i o n a l Na + f l u x e s with time f o r s h o r t - c i r c u i t e d r e c t a bathed i n simple C l - s a l i n e (uEg/cm 2/h; mean ± SEM). (a) 0.1 pr CC added at arrows. • i n d i c a t e s i n f l u x (L=>H; n = 9-11); ° i n d i c a t e s b a c k f l u x (H=>L; n = 11).. (b) 0.3 mM f i n a l c o n c e n t r a t i o n cAMP added at arrows. • i n d i c a t e s i n f l u x (L=>H; n = 11); ° i n d i c a t e s b a c k f l u x (H=>L; n = 11). Experiments (a) and (b) were conducted using l o c u s t s from d i f f e r e n t cages. UNIDIRECTIONAL 2W FLUXES CjuEq • crfi2 • H1) o o cn -—I m r u co O © 1 \ / KD KD H D ©H \ / O cn cr I \ - O § V / ©—i @—i t - O © H <H>/ i-O ©—l I—O ©—i i—O © - T KD ©H t - O 6M O —i cn ro 53 F i g . 11- I s c and net Na+ f l u x e s f o r s h o r t - c i r c u i t e d r e c t a bathed ' i n simple C l - s a l i n e (uEg/cm 2/h; mean ± SEM). Net f l u x data obtained from Fig;10. • i n d i c a t e s net Na+ i n f l u x (L=>H) ; ° i n d i c a t e s I s c ; s o l i d l i n e i n d i c a t e s O i l pr CC added at arrows (n = 20-22); dashed l i n e i n d i c a t e s 0-3 mM f i n a l c o n c e n t r a t i o n cAMP added at arrows (n = 22); dashed l i n e without p o i n t s i n d i c a t e s mean I s c f o r unstimulated r e c t a (from F i g . 2 ) . Experiments with CC and cAMP were conducted using l o c u s t s from d i f f e r e n t cages. 22. Isc AND NET Na FLUXES QuEq • cm 2-FT 1) O cn O I cn O i—© co \\<-MS ©H HO ©H I® ©H / <D '< KD O—l KD O—I 1J HD /OH <7 / HD CM / i KD t i / i KD I I I ; i i MD \ O—• O-f / O—I 7 O—I / O-i t-O o—< cn 55 homogenate but with the following differences..There i s a small but abrupt increase in the backflux (H=>L) of C l - when the recta are stimulated by cAMP. The increases i n both Isc and net C l ~ flux are much more rapid following the addition of cAMP compared to CC homogenate (Fig.9b,c). As with CC homogenate, the increase in net C l - flux i s more than s u f f i c i e n t to account for the entire increase i n Isc upon stimulation with cAMP._ Cyc l i c AMP has no e f f e c t on either u n i d i r e c t i o n a l (Fig.10) or net Na + fluxes (Fig.11). These remain r e l a t i v e l y constant (net f l u x 3.0 ± 0.2 uEg/cm2/h) over the course, of the experiment and the net value i s closer to that reported by Williams et a l . than observed with CC-stimulated recta (Fig,11). Effects of Anion Substitutions on Rectal Response to_CC or cAMP The response of recta to stimulants when bathed i n normal Cl - s a l i n e and various Cl-free s a l i n e s are compared i n Table 2 and Fig.12. Recta bathed in either hyperosmotic S0 M-saline-2 or isosmotic N0 3-saline respond i n s i m i l a r ways. The i n i t i a l Isc i s low, and the i n i t i a l decline i s greatly reduced compared to recta i n normal C l - s a l i n e (compare with F i g . 2 ) . A s l i g h t decrease i n Isc was observed when CC homogenate was added. Preparations i n N0 3-saline did not respond to cAMP stimulation while those i n S0 H-saline-2 showed only a s l i g h t increase i n Isc.. The . Isc across both preparations increased very r a p i d l y when the Cl-free salines were replaced with normal Cl - s a l i n e and subsequently decreased even more rapidly when recta were again exposed to either N0 3- or SO,,-salines. The steady-state Isc for preparations bathed f o r a second time with either S0 H-saline-2 56 TABLE 2. Average values (± SEM, n = 4-8) for a l l recta treated as shown i n . F i g . 12, i n d i c a t i n g changes i n Isc and PD due to various s t i m u l i for s h o r t - c i r c u i t e d recta bathed i n 4 d i f f e r e n t simple s a l i n e s , i d e n t i f i e d by major anion. Isc expressed as mean ± SEM. Only mean PD values are. shown ( i n brackets). In a l l .'cases Isc ind i c a t e s L=^H movement of anions, and H-side i s always negative to L-side under open-circuit conditions. Sequential Events Chloride a) I n i t i a l I (PD) at 10 min 6.7 ± 0 sc p o s t - d i s s e c t i o n (24) b) Steady-state I • (PD) 2.9 ± 0 t = 2 h '• (15) c) A l s c (APD) following +3.6 + 0 additi o n of 0.1 pr CC - (+10) d) A I (APD)- following +3.4 + 0 sc addition of 0.3 mM cAMP (+6) e) A I (APD) when placed sc i n simple C l - s a l i n e f) Time (min) for hal f of change -i n I to occur during (e) •. sc g) A Isc (APD) when returned from -C l - s a l i n e to C l - f r e e s a l i n e h) Time (min) f o r half of change -i n I to occur during (g) I) F i n a l (4.5 h) steady-state I g c 2.1 (PD) i n o r i g i n a l s a l i n e 2 I (pEq/cm /h) and PD (mV) I n i t i a l Bathing Saline Sulphate N i t r a t e Acetate 1.8 ± 0.3 • • 2.0 ± 0.6 4.3 ± 0.7 (17) 0 4 ) (30) 1.5 ± 0.2 0.8 ± 0.5 3.5 +0.7 (9) (5) (27) -0.7 ± 0.1 -0.1 ± 0.1 0 C-4) (0) (0) +0.7 ± 0.1 0 0 (+0.5) (0) (0) +2.2 ± 0.3 +3.6 ± 0.8 -2.7 ± 0.2 (+11) (+13) (-25) 8.5+2.3 8.5+2.6 1 7 + 4 -4.3 + 0.6 -2.5 + 0.4 +2.6 ± 0.2 (-12) (-10) (+13) 5.5 ± 0.5 3.8 ± 0.8 2 3 + 4 0.4 ± 0.2 . 0.8 ± 0 . 1 3.7 ± 0.6 58 Fig.12. I n d i v i d u a l t r a c e s of I s c with time f o r r e c t a bathed i n simple NO - s a l i n e , SO - s a l i n e - 2 , or a c e t a t e ; s a l i n e . . S o l i d bar i n d i c a t e s time p e r i o d during which r e c t a were bathed i n C l - f r e e s a l i n e ; open bar i n d i c a t e s simple C l - s a l i n e present. (a) i n d i c a t e s i n i t i a l change of C l - f r e e s a l i n e to f r e s h C l - f r e e s a l i n e , (b) 0.3 mM f i n a l c o n c e n t r a t i o n cAMP added at arrow, (c) 0 .1 pr CC added at arrow, (d) 0.3 mM f i n a l c o n c e n t r a t i o n cAMP added at arrow when normal C l - s a l i n e was present. Mean v a l u e s f o r s i m i l a r experiments are given i n Table 2. I — I C , y --z\xxo. • b g r f i ) 60 or N 0 3 - s a l i n e was very low. These o b s e r v a t i o n s d i f f e r from those of W i l l i a m s et a l . who found no change i n s t e a d y - s t a t e I s c when complex Cl--saline was r e p l a c e d by complex S0 H- or N 0 3 - s a l i n e s . In our s t u d i e s , when simple S 0 S - s a l i n e - 2 or N 0 3 - s a l i n e were re p l a c e d with C l - s a l i n e , the p r e p a r a t i o n s were once again responsive t o s t i m u l a t i o n by CC homogenate or cAMP, a l b e i t to a l e s s e r degree than u s u a l . T h i s responsiveness disappeared a g a i n as soon as the C l - was removed from the bathing s a l i n e (Fig.12, Table 2 ) . C l e a r l y , the I s c only i n c r e a s e s s u b s t a n t i a l l y f o l l o w i n g the a d d i t i o n of e i t h e r CC or cAMP i f C l - i s present i n the s a l i n e i The PD valu e s are . a l s o much lower f o r r e c t a i n SO*,-saline-2 or N 0 3 - s a l i n e compared to those i n simple C l - s a l i n e (Table 2 ) . Steady-state PD's f o r r e c t a bathed i n S0 H- and N 0 3 - s a l i n e s are 9 and 5 mV r e s p e c t i v e l y versus 15 mV i n C l - s a l i n e . One tenth pr CC caused a decrease of 4 mV a c r o s s r e c t a i n SO^-saline-2 but had no e f f e c t on p r e p a r a t i o n s bathed i n N0 3-saline,. . A d d i t i o n of 0.3 mM cAMP caused the PD t o i n c r e a s e very s l i g h t l y (0.5 mV) i n SO4 -s a l i n e - 2 and had no e f f e c t i n N0 3-saline;. . In c o n t r a s t * c o n t r o l p r e p a r a t i o n s i n C l - s a l i n e showed a PD i n c r e a s e of 10 mV when st i m u l a t e d with CC and 6 mV with cAMP. When C l - f r e e p r e p a r a t i o n s were, ret u r n e d to C l - s a l i n e , the PD q u i c k l y rose to 16 mV f o r p r e p a r a t i o n s p r e v i o u s l y bathed i n S O 2 - and to 18 mV i n i t i a l l y f o r p r e p a r a t i o n s p r e v i o u s l y bathed i n NO3, but the l a t t e r PD d e c l i n e d t o 9 mV a f t e r 1 hour.. When these p r e p a r a t i o n s were returned to t h e i r o r i g i n a l C l - f r e e bathing s a l i n e , PD f e l l to 2 mV f o r both S 0 K - s a l i n e - 2 and N 0 3 - s a l i n e . In summary, these changes i n PD p a r a l l e l the changes i n I s c and are a p p a r e n t l y 61 a s s o c i a t e d with C l - t r a n s p o r t . The s u b s t i t u t i o n of a c e t a t e f o r C l - causes a q u a n t i t a t i v e l y d i f f e r e n t response than do S O z , - s a l i n e - 2 or N 0 3 - s a l i n e (Fig.12, Table 2). The i n i t i a l I s c f o r r e c t a i n acetate s a l i n e decreases only very s l i g h t l y with time, with the r e s u l t t h a t the steady-s t a t e I s c i s very much higher than f o r any of the other s a l i n e s , i n c l u d i n g normal C l - s a l i n e . . The a d d i t i o n of CC homogenate or cAMP has no e f f e c t on the I s c . S u b s t i t u t i o n of C l - f o r a c e t a t e causes a r e l a t i v e l y slow decrease i n I s c of unstimulated r e c t a . R e p l a c i n g the C l - s a l i n e with a c e t a t e s a l i n e causes the I s c t o r a p i d l y i n c r e a s e t o the o r i g i n a l l e v e l f o r acetate..These o b s e r v a t i o n s confirm a p r e l i m i n a r y r e p o r t by W i l l i a m s et a l . (1978) using complex C l - and a c e t a t e s a l i n e s . The s t e a d y - s t a t e membrane r e s i s t a n c e (264 ± 7 Si-cm2, n=8) i s c o n s i d e r a b l y higher i n a c e t a t e s a l i n e compared to normal C l - s a l i n e (194 ± 6 Jl-cm2, n=15). As i s the case f o r I s c , PD i s g r e a t l y e l e v a t e d when a c e t a t e i s s u b s t i t u t e d f o r C l - (Table 2 ) . The s t e a d y - s t a t e PD was n e a r l y twice t h a t f o r C l - s a l i n e (27 vs 15 mV). Neither CC nor cAMP had any e f f e c t on PD when acetate s a l i n e was present. Returning the p r e p a r a t i o n s to C l - s a l i n e caused the PD to d e c l i n e d r a m a t i c a l l y to 2 mV. This value i n c r e a s e d to 15 mV when the p r e p a r a t i o n s were r e t u r n e d t o a c e t a t e s a l i n e a second time. In summary, ac e t a t e s a l i n e causes a much l a r g e r I s c and PD a c r o s s r e c t a than does normal C l - s a l i n e but th e r e i s no response t o CC or cAMP when t h i s o r g a n i c a c i d i s the major anion. The r e s u l t s o f s u b s t i t u t i n g simple S O * , - s a l i n e - 1 and complex SOu-saline f o r simple C l - s a l i n e are presented i n Fig.13. One 62 Fig.13. E l e c t r i c a l parameters (PD, fi, Isc) with time f o r ' s h o r t -c i r c u i t e d r e c t a bathed i n simple C l - s a l i n e and simple and complex S G \ s a l i n e s (mean ± SEM) . a • I n d i c a t e s p r e p a r a t i o n s bathed i n simple C l - s a l i n e . o o i n d i c a t e s p r e p a r a t i o n s bathed i n simple SO^-s a l i n e - 1 . o o i n d i c a t e s p r e p a r a t i o n s bathed i n complex SO^ - s a l i n e . Arrows i n d i c a t e time of s a l i n e change..n = 4-8 f o r each p o i n t . 63 64 u n e x p e c t e d o b s e r v a t i o n r e s u l t i n g f r o m t h e s e e x p e r i m e n t s i s t h a t t h e o r d e r o f s u b s t i t u t i o n i s i m p o r t a n t . P r e p a r a t i o n s t r a n s f e r r e d f r o m C l - s a l i n e i n t o s i m p l e S 0 H - s a l i n e - 1 show a l a r g e d e c r e a s e i n PD. T r a n s e p i t h e l i a l membrane d, . c. r e s i s t a n c e (R) i n c r e a s e s 7 0 % b u t d e c l i n e s w i t h t i m e a l t h o u g h i t c o n t i n u e s t o r e m a i n e l e v a t e d a b o v e t h e s t e a d y - s t a t e C l - v a l u e s (200 v s 155 &• cm 2) . I s c u n d e r g o e s an i n i t i a l t r a n s i e n t i n c r e a s e b u t t h e n s t e a d i l y d e c l i n e s t o o n e - h a l f t h e s t e a d y - s t a t e I s c i n C l - s a l i n e . . When s i m p l e S O t ^ - s a l i n e - 1 i s r e p l a c e d by c o m p l e x S O ^ - s a l i n e , PD i n c r e a s e s r a p i d l y t o 3 t i m e s t h e s t e a d y - s t a t e C l - v a l u e . R i n c r e a s e s 2-3 f o l d o v e r p r e p a r a t i o n s i n s i m p l e . S O ^ - s a l i n e - 1 , o r 3-4 f o l d o v e r t h e s t e a d y - s t a t e v a l u e f o r C l - s a l i n e . C o n c u r r e n t w i t h t h e s e e v e n t s , I s c shows an i n i t i a l t r a n s i e n t d e c l i n e t o n e a r z e r o , t h e n r i s e s s l o w l y o v e r t h e s u b s e g u e n t 90 m i n , s t a b i l i z i n g a t t h e same l e v e l (2.1 juEg/cm 2/h) a s f o r s t e a d y -s t a t e p r e p a r a t i o n s i n C l - s a l i n e , . W h e n t h e s e , p r e p a r a t i o n s were a g a i n p l a c e d i n C l - s a l i n e , a l l t h r e e e l e c t r i c a l p a r a m e t e r s r e t u r n e d t o t h e p r e - s u b s t i t u t i o n l e v e l s f o r C l - s a l i n e , a l t h o u g h I s c shows a s h a r p t r a n s i t o r y i n c r e a s e b e f o r e : r e t u r n i n g t o t h e s t e a d y - s t a t e . R e s p o n s e t o cAMP a t t h i s p o i n t i s n o r m a l . F o r p r e p a r a t i o n s t r e a t e d i n t h e r e v e r s e o r d e r ( i . e . C l - t o c o m p l e x SO^- t o s i m p l e S 0 4 - s a l i n e - 1 ) , t h e i n i t i a l t r a n s f e r f r o m C l - t o c o m p l e x SO*, - s a l i n e c a u s e s t h e PD t o i n c r e a s e 2-3 f o l d a s d o e s R. I s c shows a t r a n s i e n t d e c r e a s e , t h e n s t a b i l i z e s a t t h e v a l u e f o r s t e a d y - s t a t e p r e p a r a t i o n s i n s i m p l e Cl-saline.„ T h i s o b s e r v a t i o n i s i d e n t i c a l t o t h a t r e p o r t e d by W i l l i a m s e t a l . (1 9 7 8 ) . S u b s e g u e n t t r a n s f e r t o s i m p l e S 0 ^ - s a l i n e - 1 c a u s e s PD and R t o d e c l i n e w i t h t i m e t o l e v e l s s l i g h t l y a b o v e t h o s e f o r 65 s t e a d y - s t a t e p r e p a r a t i o n s i n C l - s a l i n e . I s c i n c r e a s e s to a l e v e l 35% higher than f o r p r e p a r a t i o n s i n e i t h e r C l - or complex S O 2 , -but t h i s d e c l i n e s t o a lower l e v e l with time. Returning these p r e p a r a t i o n s t o simple C l - s a l i n e causes PD and R to d e c l i n e t o very low l e v e l s . I s c a l s o d e c l i n e s to approximately h a l f the previous s t e a d y - s t a t e C l - value. Response t o cAMP at t h i s p o i n t i s g r e a t l y reduced. In summary, complex SC\| - s a l i n e s u s t a i n s the s t e a d y - s t a t e I s c a c r o s s l o c u s t r e c t a i n e x a c t l y the manner re p o r t e d by Williams e t a l . (1978). The present study shows t h a t simple S O t , - s a l i n e - 1 decreases PD and i n c r e a s e s R by 70% while complex S O t , - s a l i n e i n c r e a s e s both PD and R 2-4 f o l d . There i s a s y n e r g i s t i c e f f e c t such t h a t p r e p a r a t i o n s bathed f i r s t i n complex S O H - s a l i n e show enhanced values f o r I s c , PD and R when subsequently exposed to simple SO,,-saline-1. The d i f f e r e n t response t o anion s u b s t i t u t i o n s between that i n Fig.12 and experiments by Wi l l i a m s e t a l . . (1978) i s l i k e l y due to the use of simple i n s t e a d of complex s a l i n e s . E f f e c t of CC Homogenate pn T i s s u e c A MP When CC homogenate i s added to the haemocoel-side of everted r e c t a l s a c s , the t i s s u e cAMP l e v e l s i n c r e a s e 2-3 f o l d w i t h i n 15 min and remain e l e v a t e d f o r at l e a s t 1 h (Table 3 ) . This suggests t h a t the CC homogenate does i n f a c t a c t on I s c and PD by e l e v a t i n g i n t r a c e l l u l a r cAMP l e v e l s . 66 TABLE 3. Ef f e c t ' o f CC Homogenate Added to Haemoco'el-Side of Everted Rectal Sacs on Tissue Levels of cAMP i n Locust Recta Treatment Incubation Time (min) cAMP Content of n Rectal Tissue (pM/gm wet wt + SEM) Control (10 u l saline) 60 1.2 ± 0.4 (a) CC (10 u l sal i n e + 0.25 p r CC) 15 3.1 ± 1.0 (b) CC (10 u l sal i n e + 0.25 pr CC) 30 2.5.± 0.8 (c) CC (10 u l s a l i n e + 0.25 pr CC) 60 3.1 ± 1.1 (d) (b) and (d) d i f f e r s i g n i f i c a n t l y from the co n t r o l (a) at P<0.05. (c) i s not s i g n i f i c a n t l y d i f f e r e n t , from the c o n t r o l (a). 67 D i s c u s s i o n The data presented i n Fig.9 demonstrate t h a t the i n c r e a s e i n I s c caused by the CC f a c t o r and cAMP can be accounted f o r by the s t i m u l a t i o n o f a c t i v e C l - uptake from the r e c t a l lumen. The net C l - f l u x matches the I s c over the e n t i r e time course of the experiments, even though the response t o s t i m u l a t i o n i s much more r a p i d with cAMP than with CC, The c o n c l u s i o n t h a t only e l e c t r o g e n i c C l - t r a n s p o r t i s s t i m u l a t e d i s supported by the obs e r v a t i o n s t h a t n e i t h e r of the s t i m u l a n t s a f f e c t s Na + f l u x e s , nor do they i n c r e a s e the I s c or PD of pre p a r a t i o n s . b a t h e d i n N0 3- or ac e t a t e s a l i n e s , , CC homogenate app a r e n t l y a c t s by enhancing the i n f l u x of C l - from lumen to haemolymph. B a c k f l u x remains completely unperturbed and co n t i n u e s t o i n c r e a s e s l o w l y with time over the course of the experiment, .In c o n t r a s t , when cAMP i s added t o p r e p a r a t i o n s , not only does i t cause a l a r g e i n c r e a s e i n C l - i n f l u x but the r e i s a l s o a s m a l l but abrupt i n c r e a s e i n the C l - b a c k f l u x as we l l ( F i g . 9 ) * T h i s may be a t t r i b u t a b l e to the decrease i n membrane r e s i s t a n c e ( i . e . an i o n p e r m e a b i l i t y increase) which i s observed when cAMP i s added t o i n v i t r o p r e p a r a t i o n s (Chapter I I ) . . The cAMP l e v e l s i n whole, unstimulated r e c t a (Table 3) agree w e l l with published values of approximately 1 pM/mg wet wt f o r v a r i o u s t i s s u e s ( S t e i n e r e t a l , , 1972)..Stimulation with CC homogenate r a i s e s t h i s value to 3 pM/mg suggesting that the CC f a c t o r a c t s by s t i m u l a t i n g the adenyl c y c l a s e system or by i n h i b i t i n g phosphodiesterase, so t h a t the i n t r a c e l l u l a r cAMP l e v e l s are r a i s e d . Although the l e v e l s of cAMP r e q u i r e d i n the bathing medium to s t i m u l a t e r e c t a are more than one m i l l i o n 68 times g r e a t e r than normal t i s s u e l e v e l s , i t i s known that cAMP does not e a s i l y penetrate c e l l membranes and t h a t these l a r g e e x t e r n a l doses are r e q u i r e d to s t i m u l a t e most c y c l i c n u c l e o t i d e -mediated processes. The average net f l u x of Na* observed i n t h i s study (2.6 jiEq/cm 2/h) i s somewhat lower than the value (4.4 juEq/cra 2/h) re p o r t e d by Wi l l i a m s e t a l . (1978). The d i f f e r e n c e , i f r e a l , may be a consequence of using simple r a t h e r than complex s a l i n e . The use of simple s a l i n e , however, does not a f f e c t I s c or C l -f l u x e s : the values observed i n Fig.8 and 9 agree very c l o s e l y with those r e p o r t e d by Williams e t a l . Simple SO*, - and N Q 3 - s a l i n e s w i l l not support s t e a d y - s t a t e I s c as w e l l as simple C l - s a l i n e (Table 2, F i g , 1 2 ) ; however, the re p o r t by Wi l l i a m s et a l . that complex C l - f r e e s a l i n e s w i l l maintain the s t e a d y - s t a t e I s c has been comfirmed ( F i g . 13) . Apparently t h e r e i s some component of complex s a l i n e t h a t enhances i o n t r a n s p o r t by a c t i n g e i t h e r as an energy source, a st i m u l a n t , or t h a t i s i t s e l f t r a n s p o r t e d . The d i f f e r e n t p a t t e r n s observed when complex and simple S O ^ - s a l i n e s a r e . s u b s t i t u t e d f o r C l - s a l i n e (Fig.13) suggest t h a t there may be. an a l t e r n a t e t r a n s p o r t mechanism which can be s l o w l y turned on w i t h i n the t i s s u e over a p e r i o d of 60-90 min when C l - i s removed. T h i s would e x p l a i n the i n i t i a l drop and subsequent complete recovery of I s c when p r e p a r a t i o n s are t r a n s f e r r e d from C l - to complex SOti-saline. The time lag may r e f l e c t the slow r a t e of d i f f u s i o n of the r e s p o n s i b l e substance i n t o the r e c t a l t i s s u e , or the time r e q u i r e d f o r i n t r a c e l l u l a r r e g u l a t o r y events t o occur. Such a l a g would a l s o e x p l a i n why simple S O ^ - s a l i n e - l w i l l t e m p o r a r i l y 69 s u s t a i n the I s c b e t t e r i f r e c t a are p r e v i o u s l y exposed to complex S O i , - s a l i n e f o r some time. Williams e t a l . suggested t h a t HCOj might be t r a n s p o r t e d by the C l - pump when the l a t t e r i o n was present i n low c o n c e n t r a t i o n s , or t h a t there was a compensatory i n c r e a s e i n the t r a n s p o r t of o r g a n i c i o n s or PO 3 -. I used the same HCO^/CO-^buffer system as d i d Williams et a l . and at the same pH; t h e r e f o r e d i s c r e p a n c i e s between the two s t u d i e s would seem u n l i k e l y to be a s s o c i a t e d with HCOj t r a n s p o r t . Phosphate l e v e l s i n simple s a l i n e (1 mM) are probably too low to allow P0 3~ t r a n s p o r t t o account f o r the net I s c . The discr e p a n c y c o u l d be due to org a n i c i o n s present i n complex s a l i n e . T h i s i s supported by the r e s u l t s i n Table 2 which i n d i c a t e t h a t at l e a s t one organic anion, a c e t a t e , may be t r a n s p o r t e d : i . e . simple a c e t a t e s a l i n e s u s t a i n s a much high e r s t e a d y - s t a t e I s c than does simple C l - s a l i n e . The t r a n s p o r t o f ace t a t e by l o c u s t rectum has been c l e a r l y demonstrated by f l u x s t u d i e s a c r o s s s h o r t - c i r c u i t e d r e c t a (T.. Baumeister, unpub. obs.); however, t h i s t r a n s p o r t system may normally c a r r y some other o r g a n i c substance..The exact organic c o n s t i t u e n t s of complex s a l i n e which are r e s p o n s i b l e f o r enhanced I s c have yet to be i d e n t i f i e d , as do t h e i r mode of a c t i o n . I t i s a l s o p o s s i b l e t h a t HCOj- or H+ t r a n s p o r t might be st i m u l a t e d or f a c i l i t a t e d by some o r g a n i c a c i d present i n the complex s a l i n e . In Chapter I I , I showed t h a t there i s a component of the s t e a d y - s t a t e I s c which r e q u i r e s c a r b o n i c anhydrase (measured by acetazolamide i n h i b i t i o n ) and which might t h e r e f o r e be a s s o c i a t e d with H+/HCOJ t r a n s p o r t . I n t e r e s t i n g l y , the s t e a d y - s t a t e c u r r e n t which remains when r e c t a are bathed i n 70 simple N 0 3 - s a l i n e i s of a s i m i l a r magnitude t o the decrease i n I s c caused by the acetazolamide i n h i b i t i o n . T h i s i n d i c a t e s t h a t t h i s r e s i d u a l I s c a c r o s s r e c t a bathed i n simple C l - f r e e s a l i n e s may be completely due to H+/HCC-3 t r a n s p o r t . Although simple S0 H- and N 0 3 - s a l i n e s w i l l maintain a low s t e a d y - s t a t e I s c , they a b o l i s h the re s p o n s i v e n e s s of r e c t a to CC homogenate or cAMP which i s observed when C l - i s present. In f a c t , 0.1 pr CC added t o a p r e p a r a t i o n i n S 0 4 - s a l i n e - 2 a c t u a l l y caused a s l i g h t d e c l i n e i n the I s c . C y c l i c AMP however, d i d have a very s l i g h t s t i m u l a t o r y e f f e c t on p r e p a r a t i o n s maintained i n SO ^ - s a l i n e . I have observed a s m a l l i n c r e a s e i n the e p i t h e l i a l p e r m e a b i l i t y (as i n d i c a t e d by a decrease i n membrane r e s i s t a n c e ) when r e c t a are s t i m u l a t e d with high doses of cAMP (see Chapter I I ) . P o s s i b l y t h i s permits more r a p i d i n f l u x of SO2- to the C l -pump, which might t r a n s p o r t t h i s d i v a l e n t i o n a t very low r a t e s . In summary, I have demonstrated t h a t the f a c t o r i n CC homogenate which s t i m u l a t e s I s c and PD acr o s s the r e c t a l e p i t h e l i u m a c t s s p e c i f i c a l l y by i n c r e a s i n g the e l e c t r o g e n i c t r a n s p o r t of C l - . I t i s unnecessary at present t o p o s t u l a t e e f f e c t s on any other t r a n s p o r t process. Experimental evidence i s a l s o presented t o suggest t h a t CC homogenatelacts by r a i s i n g i n t r a c e l l u l a r l e v e l s of cAMP, which i n t u r n s t i m u l a t e s the C l -pump,. The. e f f e c t of cAMP on t r a n s p o r t may be d i r e c t , by i n c r e a s i n g the a c t i v i t y or number of C l - pump s i t e s , or i n d i r e c t by i n c r e a s i n g the pas s i v e entry of C l - i n t o the c e l l from the lumen and thus r a i s i n g the i n t r a c e l l u l a r l e v e l s of t h i s anion which are a v a i l a b l e t o the pump. 71 IV EVIDENCE - FOR REGULATORY SUBSTANCES IN - THE HAEMOLYMPH I n t r o d u c t i o n In Chapters I I and I I I , I have reported evidence f o r a f a c t o r present i n the corpora c a r d i a c a (CC) of S c h i s t o c e r c a g£§g§ g 4 a which i n c r e a s e s the s h o r t - c i r c u i t c u r r e n t (Isc) and t r a n s e p i t h e l i a l e l e c t r o p o t e n t i a l d i f f e r e n c e (PD) a c r o s s i n v i t r o p r e p a r a t i o n s of l o c u s t r e c t a . T h i s a c t i o n i s mimicked by cAMP. Both these agents a c t by s t i m u l a t i n g a c t i v e C l - uptake from the r e c t a l lumen; Since a b s o r p t i o n of water by t h i s organ a g a i n s t osmotic c o n c e n t r a t i o n d i f f e r e n c e s can be d r i v e n by C l - t r a n s p o r t (Goh and P h i l l i p s , 1978), the CC f a c t o r might a l s o i n c r e a s e r e c t a l water a b s o r p t i o n ; i . e . have an a n t i d i u r e t i c e f f e c t (Spring et a l . , 1978). The question remains as t o whether r e c t a l r e s o r p t i o n of C l - i s normally c o n t r o l l e d by the: r e l e a s e of a hormone from the CC i n t o the haemocoel or whether the a c t i o n o f homogenates of t h i s gland i s s t r i c t l y p harmacological. In t h i s chapter I present evidence t h a t the: haemolymph of r e c e n t l y - f e d l o c u s t s normally c o n t a i n s a f a c t o r , C h l o r i d e -Transport S t i m u l a t i n g Hormone (CTSH), which s t i m u l a t e s the same r e c t a l response as do CC homogenate and cAMP. . I removed the CC from l o c u s t s (cardiatectomy) to determine. whether t h i s e l i m i n a t e s the s t i m u l a t o r y f a c t o r from the haemolymph. F i n a l l y , I t e s t e d CC homogenate and haemolymph samples f o r t h e i r a b i l i t y to i n f l u e n c e water a b s o r p t i o n by everted r e c t a l sacs, to determine whether CTSH might be the d i u r e t i c or a n t i d i u r e t i c , f a c t o r p r e v i o u s l y detected i n l o c u s t CC (Cazal and G i r a r d i e , 72 1968; Mordue, 1969, 1970; reviewed by Gee, 1977). M a t e r i a l s - and_Methods The experimental animals were mature S c M s t o e e r c a g r e g a r i a , one to t h r e e months past t h e i r f i n a l moult. They were r e a r e d a t 28°C and 50% E.H.. under a photoperiod of L:D 16:8 and f e d a d i e t of f r e s h l e t t u c e and a mixture of d r i e d g r a s s , bran, yeast and powdered milk. Female l o c u s t s were used f o r voltage-clamped p r e p a r a t i o n s of r e c t a , haemolymph samples and c a r d i a t e c t o m i e s because of t h e i r l a r g e r s i z e and haemolymph volume. Ev e r t e d r e c t a l sacs were prepared from both sexes and water uptake was c a l c u l a t e d per u n i t wet weight t o c o r r e c t f o r v a r i a t i o n s i n s i z e . P r e p a r a t i o n of CC Homogenate Homogenates of whole CC were prepared from mature male l o c u s t s as d e s c r i b e d i n Chapter I I . , Dehydrated donors were maintained i n the l o c u s t colony with dry food a v a i l a b l e but without l e t t u c e f o r 5 to 7 days before use. Hydrated donors were a l s o maintained i n the colony with dry food but were fed f r e s h l e t t u c e i n excess twice d a i l y f o r 5 to 7 days b e f o r e use. P r e p a r a t i o n of Haemolymph for.-Assay Adult female l o c u s t s were dehydrated i n a d e s s i c a t o r over c o n c e n t r a t e d H^SCy f o r 24 h, f o l l o w i n g which they were moved to a l a r g e cage and allowed t o feed on f r e s h l e t t u c e . Haemolymph was c o l l e c t e d 90 t o 120 min a f t e r the s t a r t of f e e d i n g by one of two methods. 73 The f i r s t method of c o l l e c t i o n was t h a t of Wall (1970)..The l e g s were amputated and the i n s e c t placed i n a wax d i s h . The abdomen and thorax were opened with a median d o r s a l i n c i s i o n and the f l a p s of t i s s u e pinned open. Haemolymph was removed by a d s o r p t i o n onto t r i a n g l e s of Whatman #1 f i l t e r paper. Papers c o u l d be weighed before and a f t e r a d s o r p t i o n to determine the amount of haemolymph c o l l e c t e d . To assay the haemolymph, i n d i v i d u a l or pooled f i l t e r papers were placed i n f l a s k s with 5.0 ml of simple C l - s a l i n e per i n s e c t and shaken i n a cold-water bath (12°C) f o r 90-120 min using a B u r r e l l • W r i s t - A c t i o n ' shaker ( B u r r e l l Corp., P i t t s b u r g h , Pa.). Four ml ( i n the case o f i n d i v i d u a l samples) or 5.0 ml (from pooled samples) of s a l i n e were assayed f o r CTSH a c t i v i t y . . T h e s a l i n e was s t o r e d f r o z e n f o r up to 4 weeks with no d e t e c t a b l e l o s s of CTSH a c t i v i t y . T h i s method of c o l l e c t i o n was used to o b t a i n the l a r g e q u a n t i t y of pooled haemolymph f o r the f l u x experiments.. The second method of haemolymph c o l l e c t i o n (Mordue and Goldsworthy, 1969) avoided a problem a s s o c i a t e d with the f i r s t method, namely foaminq of the s a l i n e i n the *Ussing-type* chambers caused by haemolymph p r o t e i n s . The thoraxes o f i n d i v i d u a l l o c u s t s were squeezed q e n t l y and r e p e a t e d l y t o encourage crop emptying. When no f u r t h e r pigmented f l u i d c o u l d be e x p e l l e d , a small hole was s l i c e d i n the f r o n s . The l o c u s t was i n s e r t e d head down i n a c h i l l e d , c o n s t r i c t e d c e n t r i f u g e . t u b e and spun q e n t l y i n a c l i n i c a l c e n t r i f u g e f o r s e v e r a l minutes. The l o c u s t was d i s c a r d e d and the haemolymph t r a n s f e r r e d to a p o l y e t h y l e n e u l t r a c e n t r i f u g e tube. Any samples contaminated with the pigmented crop f l u i d were d i s c a r d e d . . The volume of 74 haemolymph was measured, and 4 volumes of methanol added. Each tube, c o n t a i n i n g the haemolymph-methanol s o l u t i o n from one l o c u s t , was homogenized u l a t r a s o n i c a l l y f o r 30, sec using a 'Sonic 300' s o n i c a t o r with a m i c r o - t i p (Artek Systems Corp., Farmingdale,N.Y.). The methanol e x t r a c t was then c e n t r i f u g e d at 12,800 x g i n a Brinkman ' C e n t r i f u g e 3200' f o r 2 minutes. The supernatant was saved and the methanol removed by e v a p o r a t i o n t o near dryness a t room temperature (22°C) with a stream o f f i l t e r e d N^. The r e s i d u e was resuspended i n 500 j i l of s a l i n e and c o u l d be used d i r e c t l y or r e f r o z e n . T h i s method was used to ob t a i n haemolymph samples from i n d i v i d u a l c a r d i a t e c t o m i z e d and sham-operated l o c u s t s . Samples were sometimes pooled and s t o r e d f r o z e n e i t h e r i n methanol or a f t e r r esuspension i n s a l i n e . To examine the e f f e c t s of haemolymph on water r e s o r p t i o n , i n s e c t s were dehydrated f o r 24 h, as d e s c r i b e d above, and d i v i d e d i n t o 2 groups. One group was then fed l e t t u c e f o r 90-120 minutes. Haemolymph samples (10 jul) were c o l l e c t e d from both the f e d and unfed groups by neck punctures and assayed on r e c t a l sacs as d e s c r i b e d below. Car d i a t e c t o m i e s Adult female l o c u s t s were dehydrated over H 3 wS0 H i n a d e s s i c a t o r f o r 24 h. They were then placed i n a j a r and c h i l l e d a t 4°C u n t i l immobile. A c h i l l e d l o c u s t , r e s t r a i n e d i n a s h o r t p i e c e . o f 1.5 cm diameter p o l y e t h y l e n e t u b i n g , was secured d o r s a l - s i d e up i n a t r a y f i l l e d with p l a s t i c e n e so t h a t the l o c u s t ' s head protruded from the t u b i n g . Two i n s e c t p i n s were placed j u s t a n t e r i o r to the pronotum to prevent the l o c u s t from 75 s l i d i n g out of the tube when i t s head was s t r e t c h e d as f a r forward as p o s s i b l e and secured with a s t r i p of p l a s t i c e n e . . A n L-shaped i n c i s i o n , with i t s base p a r a l l e l to the o c c i p u t , was cut i n the c e r v i c a l membrane and the t r i a n g l u l a r f l a p so formed was f o l d e d to one s i d e . A i r sacs and f a t body were:removed from the c e r v i c a l r e g i o n , and the a o r t a p u l l e d g e n t l y backward u n t i l the CC c o u l d be seen. These were then grasped with sharpened watch-maker's f o r c e p s and cut f r e e with iridectomy s c i s s o r s . . T h e normal p o s i t i o n of the head under the pronotum s e a l e d the wound with no v i s i b l e haemolymph l o s s . C o n t r o l l o c u s t s (sham-operated) were t r e a t e d e x a c t l y as the experimentals with the e x c e p t i o n t h a t once the a i r sacs and f a t body were removed and the a o r t a severed, the l o c u s t was r e l e a s e d . P o s t - o p e r a t i v e l y , l o c u s t s were t r a n s f e r r e d t o i n d i v i d u a l g l a s s j a r s and held without food or water at 28°C f o r 24 hours, . A f t e r 24 h, the l o c u s t s were:fed f r e s h l e t t u c e and 90 to 120 min t h e r e a f t e r the: haemolymph was e x t r a c t e d with methanol. Dehydrating l o c u s t s p r i o r to cardiatectomy ensured t h a t i n s e c t s weakened by the o p e r a t i o n d i d not have to be subjected subseguently to the r i g o u r s o f dehydration over a c i d . T h i s was e s s e n t i a l i n view of the p o t e n t i a l f o r water l o s s through the damaged c e r v i c a l membrane. The reduced haemolymph volume i n dehydrated l o c u s t s a l s o minimized haemolymph l o s s during o p e r a t i o n s and f a c i l i t a t e d the removal of the CC. A f t e r e x t e n s i v e experience with t h i s o p e r a t i o n , m o r t a l i t y among operated and sham-operated i n s e c t s during the 24 h post-o p e r a t i v e p e r i o d was reduced to l e s s than 10%. I f l o c u s t s were hel d f o r a f u r t h e r 24 h, m o r t a l i t y rose t o 30-50% i n both 76 groups. Measurement of E l e c t r i c a l Parameters a n d _ g n i d i r e c t i p n a l ^ G l 3 Fluxes I s c and PD a c r o s s i s o l a t e d r e c t a were c o n t i n u o u s l y monitored as d e s c r i b e d i n Chapter I I * U n i d i r e c t i o n a l and net C l ~ f l u x e s under s h o r t - c i r c u i t e d c o n d i t i o n s were determined as i n Chapter I I I with the f o l l o w i n g m o d i f i c a t i o n s : the:'Ussing-type' chambers were f i l l e d with only 5.0 ml of bathing s a l i n e and 1.0 ml samples of r a d i o a c t i v e s a l i n e were counted i n 10.0 ml of commercial s c i n t i l l a t i o n c o c k t a i l using a 'Nuclear Chicago Isocap 300' l i q u i d s c i n t l l a t i o n counter. Simple C l - s a l i n e (Chapter II) was used t o bathe r e c t a i n a l l experiments. P r e p a r a t i o n of R e c t a l . S a c s E v e r t e d , cannulated r e c t a l sacs were prepared as d e s c r i b e d by Goh and P h i l l i p s (1978). Sacs were weiqhed immediately upon removal from the l o c u s t s and then incubated i n oxyqenated simple s a l i n e at 30°C f o r a t l e a s t 2 h to allow the p r e p a r a t i o n s to reach the s t e a d y - s t a t e phase..To s t a r t the experiment, sacs were emptied, b l o t t e d dry and weighed usin q a 200 mg 'August Sauter' t o r s i o n balance-. C o n t r o l sacs were f i l l e d with 10 / i l of simple s a l i n e (480 mOsm) and run c o n c u r r e n t l y with experimental p r e p a r a t i o n s which were f i l l e d with 10 p.1 of s a l i n e c o n t a i n i n g 1 pr homogenized CC or 10 ;ul of freshly-drawn haemolymph (440 mOsm). Sacs were r e t u r n e d to the bath and weighed every 30 min f o r the f o l l o w i n g 2 hours. Water uptake was c a l c u l a t e d on the 77 b a s i s of /ul water uptake per g wet weight of t i s s u e per h f o r each 30 min i n t e r v a l . A l l s t a t i s t i c a l t e s t s and c a l c u l a t i o n s were performed as suggested by L a r k i n (1976). Supplementary t e s t s and t a b l e s were obtained from Freund (1967) and Johnson (1973). Resu l t s -Hf£ec_t of - A c t i v e Haemolymph on, E l e e t r o q e n i c T r a n s p o r t -The e f f e c t s of haemolymph from r e c e n t l y f e d l o c u s t s on the e l e c t r i c a l parameters of i n v i t r o r e c t a l p r e p a r a t i o n s are shown i n F ig.14. Haemolymph from 25 l o c u s t s was c o l l e c t e d on f i l t e r paper* The haemolymph, which averaged c l o s e , to 200 jul per i n s e c t , was e l u t e d i n t o 125 ml of saline,. The e g u i v a l e n t of the t o t a l haemolymph c o l l e c t e d from one l o c u s t (5 ml), when added to the haemocoel-side of s h o r t - c i r c u i t e d r e c t a , caused the PD t o i n c r e a s e from 8 ± 1 mV to 15 ± 2 mV a f t e r 60 minutes. A f t e r 80 min the PD began to d e c l i n e , dropping to 11 ± 2 mV over the succeeding 60 minutes. T r a n s e p i t h e l i a l membrane.d. c. r e s i s t a n c e (R; 118-<-138 &'cm2) d i d not change s i g n i f i c a n t l y f o l l o w i n g the a d d i t i o n o f a c t i v e haemolymph. When a c t i v e haemolymph was added, the. I s c began to i n c r e a s e w i t h i n a few seconds. Most p r e p a r a t i o n s (15 out of 19) e x h i b i t e d an i n i t i a l s m a l l b i p h a s i c response, c o n s i s t i n g of a r i s e (0.8 /iEq/cm2/h) over the f i r s t 9 min f o l l o w e d by a d e c l i n e (0.4 nEq/cm 2/h) over the next 6 min (Fig.14c). A l l p r e p a r a t i o n s then responded as thouqh they had been s t i m u l a t e d with CC homoqenate, r i s i n q from an averaqe 7 8 Fig.14. E f f e c t s of haemolymph on the e l e c t r i c a l parameters o f s h o r t - c i r c u i t e d r e c t a (mean ± SEM; n = 1 1 ) . . S a l i n e c o n t a i n i n g the haemolymph from one l o c u s t added at arrow, (a) T r a n s e p i t h e l i a l PD (lumen p o s i t i v e ) . (b) T r a n s e p i t h e l i a l d. c. r e s i s t a n c e . (c) S h o r t - c i r c u i t c u r r e n t , i n d i c a t i n g net t r a n s f e r o f anions L=>H.. Dashed l i n e i n d i c a t e s I s c f o r unstimulated r e c t a of l o c u s t s from a d i f f e r e n t cage (from F i g . 2 ) . Ol « 1 1 1 O I 2 3 4 TIME (h) 80 i n i t i a l value of 2.6 to a maximum I s c of 4.6 juEg/cm 2/h. A f t e r r e a c h i n g i t s maximum value a t 80 min the I s c d e c l i n e d g r a d u a l l y t o 3.1 juEg/cm 2/h over the succeeding 60 minutes. The i n i t i a l r a t e of i n c r e a s e i n I s c (0-9 min) was 5.2 uEg/cm 2/h 2. The second l i n e a r r a t e of i n c r e a s e (15-40 min) was 3.5 uEg/cm 2/h 2. These values should he compared to the maximum r a t e of i n c r e a s e i n I s c of 4.8 and 12.6 /iEg/cm 2/h 2 f o r r e c t a s t i m u l a t e d by 0.1 pr CC and 0.3 mM cAMP r e s p e c t i v e l y (see Chapter I I ) . In summary, except f o r the i n i t i a l s m a l l b i p h a s i c change i n I s c , the . g e n e r a l response to haemolymph was g u a n t i t a t i v e l y s i m i l a r to t h a t observed f o l l o w i n g the a d d i t i o n of a submaximal dose of CC homogenate. E f f e c t of A c t i v e Haemolymph on 3 * G l ~ T r a n s p o r t The e f f e c t of a c t i v e haemolymph on u n i d i r e c t i o n a l f l u x e s o f 3 6 c i — a c r o s s s h o r t - c i r c u i t e d r e c t a i s shown i n Fig,15a. There i s a t i m e - l a g of approximately 10 min f o l l o w i n g s t i m u l a t i o n d u r i n g which the C l - i n f l u x (L=>H) conti n u e s t o f a l l . The C l - i n f l u x then i n c r e a s e s r a p i d l y over the next 40 min to a peak value of 10 juEg/cm 2/h. From 70 min u n t i l the end of the experiment C l ~ i n f l u x decreases a t a r a t e s i m i l a r to the i n i t i a l d e c l i n e observed f o r unstimulated r e c t a . Backflux (H=>L) a l s o i n c r e a s e s f o l l o w i n g s t i m u l a t i o n from 3.0 t o 4,6 jaEg/cm 2/h, peaking 50 min a f t e r the a d d i t i o n of a c t i v e haemolymph then d e c l i n i n g s l o w l y t o 3.9 jiEq/cm 2/h over the course of the experiment. Net C l - f l u x , c a l c u l a t e d from u n i d i r e c t i o n a l f l u x e s , and the simultaneous I s c are compared i n Fig.15b. When r e c t a are s t i m u l a t e d by a c t i v e haemolymph, the e n t i r e i n c r e a s e i n I s c can 81 Fig.15. U n i d i r e c t i o n a l and net C l - f l u x e s with time f o r s h o r t -c i r c u i t e d r e c t a bathed i n simple C l - s a l i n e (uEq/cm 2/h; mean ± SEM). S a l i n e c o n t a i n i n g the haemolymph c o l l e c t e d from one l o c u s t added at arrow._ (a) u n i d i r e c t i o n a l 3 6 C 1 -f l u x e s ; • i n f l u x (L=>H; n = 10); ° b a c k f l u x (H=>L; n = 9). (b) Net C l - f l u x and I s c . ° i n d i c a t e s net 3 6 C l - f l u x (L=>H) c a l c u l a t e d from net f l u x e s ; • i n d i c a t e s I s c (mean ± SEM; n = 19). 82 83 be accounted f o r by the i n c r e a s e i n net C l - t r a n s p o r t and t h i s i s a l s o comparable with CC and cAMP s t i m u l a t i o n . E f f e c t s of Gardiatectomies on_Haemolymph- A c t i v i t y The e f f e c t of c a r d i a t e c t o m i e s and sham-operations on the s t i m u l a t o r y a c t i v i t y of haemolymph from a homogeneous group of r e c e n t l y - f e d l o c u s t s i s shown i n Fig.16a..Haemolymph from 4 out of 6 c a r d i a t e c t o m i z e d l o c u s t s had no s t i m u l a t o r y e f f e c t on r e c t a l I s c ; the remaining 2 caused a s l i g h t s t i m u l a t i o n . A l l of the sham-operated c o n t r o l s e l i c i t e d a p o s i t i v e s t i m u l a t i o n o f the I s c of t e s t r e c t a . While there was c o n s i d e r a b l e v a r i a b i l i t y i n response, the average i n c r e a s e i n A I s c was 7 - f o l d g r e a t e r ( s i g n i f i c a n t a t P<0.05; B a r t l e t t ' s t e s t ) than t h a t f o r the haemolymph of c a r d i a t e c t o m i z e d l o c u s t s . However, the A I s c o f 0.7 ± 0.1 uEq/cm2/h (mean ± SEM) f o r sham-operated l o c u s t s was 22% lower than f o r unoperated l o c u s t s p r e - t r e a t e d i n a s i m i l a r manner (0.9 ± 0 . 4 uEg/cm 2/h), although t h i s d i f f e r e n c e i s non-s i g n i f i c a n t (P>0.05; B a r t l e t t ' s t e s t ) . Haemolymph ;CTSH L e v e l s The average CTSH a c t i v i t y i n haemolymph c o l l e c t e d from i n d i v i d u a l normal ( i . e . unoperated) l o c u s t s using methanol e x t r a c t i o n (Fig.16a) appears to be c o n s i d e r a b l y lower than the average v a l u e p r e v i o u s l y obtained f o r pooled haemolymph samples using f i l t e r paper e x t r a c t i o n (Fig.14,15). This d i s c r e p a n c y i s caused by s e v e r a l f a c t o r s . Since the A I s c i s an e x p o n e n t i a l f u n c t i o n of the s t i m u l a n t dose, the comparison of mean i n c r e a s e i n A I s c caused by pooled SM I.O 'I <\' '2 o 6 UJ 3 2 0.5 2 < 2 (a) •—^ 10 ' o X —I 3. ELS OO > n UJ _ l Q: LU Q . 5 LO LO H t -U z UJ _J I < > 2 OLY a UJ 1 2 U UJ U < (b) O C A R D I A T E C T O M I Z E D S H A ' M - O P E R A T E D N O R M A L F i g . 16. S t i m u l a t i o n o f I s c by haemolymph and CTSH l e v e l s c a l c u l a t e d from t h i s d a t a t o r c a r d i a t e c t o m i z e d (n . =. 6) , sham-ope r a t e d (n = 6) , and n o r m a l (n - 8) l o c u s t s . Haemolymph c o l l e c t e d by m e t h a n o l e x t r a c t i o n . V e r t i c a l b a r s i n d i c a t e SEM - (a) A v e r a g e ' o f maximum A I s c o £ r e c t a t r e a t e d w i t h haenolymph from 1 i n s e c t . (b) haemolymph CTSH l e v e l s c a l c u l a t e d from ( a ) . F o r c a l c u l a t i o n s , s e e t e x t . 85 and i n d i v i d u a l haemolymph samples g i v e s misleading r e s u l t s . The c o r r e c t procedure i s to f i r s t convert the A I s c values f o r each i n d i v i d u a l sample t o e q u i v a l e n t amounts of CC be f o r e c a l c u l a t i n g mean CTSH content. From the s e m i - l o g a r i t h m i c dose-response curve f o r r e c t a l s t i m u l a t i o n by CC homogenate [ A Isc (uEg/cm 2/h) = 2,. 62 i l o g (CC dose) + 6.8; Chapter II{], the A I s c was f i r s t c onverted to e q u i v a l e n t u n i t s of CC and then c o r r e c t i o n was made f o r the volume of haemolymph i n each assay, so t h a t each sample c o u l d be expressed i n terms of CC-equivalents per 300 ;ul of haemolymph (CCEq). The values obtained using these c a l c u l a t i o n s are shown i n Fig;16b and 17b. The e r r o r caused by f a i l u r e to f o l l o w t h i s procedure i s shown i n Fig;17a which shows the mean A I s c f o r i n d i v i d u a l and pooled samples of haemolymph e x t r a c t e d using f i l t e r paper. Although the pooled samples give a A I s c which i s ne a r l y 3 times g r e a t e r than t h a t c a l c u l a t e d from the A I s c of the i n d i v i d u a l samples, c a l c u l a t i o n of the c i r c u l a t i n g CTSH l e v e l s shows on l y a s l i g h t d i f f e r e n c e (F i g ; 17b); C l e a r l y , comparing the mean A I s c of pooled and i n d i v i d u a l samples can lead t o unwarranted c o n c l u s i o n s about r e l a t i v e potency u n l e s s the c o r r e c t method of c a l c u l a t i o n i s f o l l o w e d . The methanol e x t r a c t i o n used i n the cardiatectomy s t u d i e s appears to be only 30% as e f f i c i e n t as the f i l t e r paper e x t r a c t i o n used to o b t a i n the pooled haemolymph samples used f o r the f l u x s t u d i e s ; T h i s was demonstrated by a s s a y i n g the t o t a l haemolymph c o l l e c t e d from i n d i v i d u a l s o f the same a n c e s t r y . . The c e n t r i f u g a t i o n step d u r i n g methanol e x t r a c t i o n removes 50% more haemolymph (300 ^ i l per i n s e c t ) than does f i l t e r paper a d s o r p t i o n (a) 2.0 < 2 x < 2 LO ^ . 0 4 0 O o .030 O u >• > ,020 f-t— o < I S) t -u I a 2 5. O 2 u < (b) .OIO I N D I V I D U A L S A M P L E S P O O L E D S A M P L E S 17. Maximum A I s c and c a l c u l a t e d haemolymph' CTSH l e v e l s f o r i n d i v i d u a l (n = 8) and p o o l e d (n = 9). haemolymph s a m p l e s c o l l e c t e d on f i l t e r p a p e r . B a r s i n d i c a t e SEM. (a) A v - r a a e o f maximum A I s c . (b) haemolymph CTSH l e v e l s c a l c u l a t e d f o r e a c h A I s c d e t e r m i n a t i o n i n (a) as d e s c r i b e d i n t e x t 87 (200 u l per i n s e c t ) ; n e v e r - t h e - l e s s the mean A I s c caused by samples o b t a i n e d by both e x t r a c t i o n methods was very low (0.9 ± 0.4 uEq/cmz/h; F i g 16a,17a) . The c a l c u l a t e d values f o r CTSH a c t i v i t y per 300 u l haemolymph (mean ± SEM) are 0.0296 ± 0.0166 CCEq using f i l t e r paper and 0.0092 ± 0.0033 CCEg using methanol. These v a l u e s c l e a r l y i n d i c a t e t h a t methanol e x t r a c t i o n i s only one t h i r d as e f f e c t i v e as f i l t e r paper a d s o r p t i o n i n c o l l e c t i n g haemolymph CTSH. Assay of the methanol p r e c i p i t a t e using voltage-clamped r e c t a i n d i c a t e d the presence of some CTSH a c t i v i t y . . Spontaneous d e s t r u c t i o n of CTSH might a l s o occur during the long p e r i o d r e g u i r e d f o r the ev a p o r a t i o n of methanol from the haemolymph samples. The l e v e l of CTSH a c t i v i t y i n d i f f e r e n t i n d i v i d u a l s v a r i e s c o n s i d e r a b l y . In one cage, f u l l y 25% of the samples t e s t e d had no d e t e c t a b l e CTSH (Fi g . 1 8 ) . T h i s does not n e c e s s a r i l y mean t h a t t h e r e was no CTSH i n the haemolymph; indeed CTSH c o u l d be detected i n these i n d i v i d u a l s by p o o l i n g t h e i r haemolymph.. The absolute minimum amount of CTSH which our assay can d e t e c t i s 0.0025 CC-equivalents per sample and i n p r a c t i c e n e a r l y 50% of assay p r e p a r a t i o n s exposed t o 0.005 pr CC showed no i n c r e a s e i n Is c (see Chapter II) . C l e a r l y , the amount of c i r c u l a t i n q CTSH present i n any one l o c u s t may r e f l e c t i t s own p h y s i o l o g i c a l s t a t e and i t s s e n s i t i v i t y to endogenous l e v e l s o f CTSH. The data i n Fig.18 i n d i c a t e t h a t CTSH a c t i v i t y i n pooled samples may be due. to very high l e v e l s i n a few l o c u s t s . I t i s my impression that there i s a l s o c o n s i d e r a b l e v a r i a b i l i t y among animals from d i f f e r e n t cages used at v a r i o u s times. Some of the v a r i a b i l i t y 88 Fig.18. D i s t r i b u t i o n of haemolymph CTSH a c t i v i t y i n samples from i n d i v i d u a l l o c u s t s . Open columns are f i l t e r paper e x t r a c t s of haemolymph; hatched columns are methanol e x t r a c t s of haemolymph. A l l i n d i v i d u a l s were from one cage. N U M B E R O F I N D I V I D U A L S 6 8 90 i s p o s s i b l y due to the l i m i t e d parentage of each cage and some to aging and the changes a s s o c i a t e d with r e p r o d u c t i v e c y c l e s over the 2 months duri n g which a cage i s used. These f a c t o r s a f f e c t not on l y the haemolymph donors but the assay r e c t a as w e l l ; Assays on d i f f e r e n t cages from time t o time support t h i s view but the data are i n s u f f i c i e n t t o r e p o r t q u a n t i t a t i v e . v a l u e s at present. In summary, f i l t e r paper i s 3 times more e f f e c t i v e than methanol i n e x t r a c t i n g haemolymph CTSH. Pooled samples of haemolymph can give higher mean i n c r e a s e s i n A I s c than do i n d i v i d u a l samples because the dose-response curve i s l o g a r i t h m i c ; Sham-operated l o c u s t s have CTSH l e v e l s only 25% lower than unoperated l o c u s t s when the same method of haemolymph e x t r a c t i o n i s used; Although many l o c u s t s appear t o have very low t o t a l haemolymph CTSH l e v e l s (50% had l e s s than 0.010 CCEg), the mean value f o r the i n s e c t s t e s t e d (0.03 CCEg) i s high c o n s i d e r i n g t h a t 0.05-0.10 pr CC w i l l produce, a maximum s t i m u l a t i o n of I s c ; Water Uptake by Rectal, Sacs Rates of f l u i d a b s o r p t i o n remained r e l a t i v e l y constant with time across everted r e c t a l sacs bathed e x t e r n a l l y i n simple C l -s a l i n e and i n i t i a l l y c o n t a i n i n g 10 u l d i s t i l l e d water ( c o n t r o l s ) or 10 u l d i s t i l l e d water p l u s homogenized CC (experimentals) as shown i n F i g ;1 9 * CC homogenate from hydrated l o c u s t s d i d not s i g n i f i c a n t l y i n c r e a s e f l u i d a b s o r p t i o n (Fig.19a)..CC homogenate from dehydrated donors, however, caused a s i g n i f i c a n t i n c r e a s e i n a b s o r p t i o n (P<0;05; B a r t l e t t ' s test) of 47% compared to CD tooo (a) UJ a 3 I 5 C O LLI I (b) l O O O 7 0 0 Y Y XL JL 0.5 . ; S_ U). 1.5 TIME C M —J 2.0 Fig. 1 9 . Hater a b s o r p t i o n with time by everted r e c t a l sacs bathed i n simple C l - s a l i n e (mean ± SEM). (a) ° i n d i c a t e s 10 u l d i s t i l l e d :Hr0 i n i t i a l l y added to haemocoel-side ( c o n t r o l s ; n = 7) ; « i n d i c a t e s 1.0 pr CC from .hydrated donor i n 10 jul d i s t i l l e d H x0 i n i t i a l l y added to haeniocoel-sid e (exper iaien ta I s ; n = 14). (a) ° i n d i c a t e s 10 u l d i s t i l l e d tixO i n i t i a l l y added to haemocoel-side ( c o n t r o l s ; n = d) ; • i n d i c a t e s 1.0 pr CC from dehydrated donor i n 10 n l d i s t i l l e d Hj.0 i n i t i a l l y added to haemocoel-side (experiraentals; n = 13). 92. »-< O * » . : 8 • : 8 : ' » 0 0.5 I.O 1.5 2.0 T I M E ( h ) Fig.20. Water a b s o r p t i o n with time by everted r e c t a l sacs cathed i n simple C l - s a l i n e (mean ± SEM). 0 i n d i c a t e s 10 u l s a l i n e i n t i a l l y added t o haemocoel-side ( c o n t r o l s ; n = 13) ; • i n d i c a t e s 10 n l haemolymph from recently-£eri donor i n i t i a l l y added to haemocoel-side (n = 25); A i n d i c a t e s 10 u l haemolymph from s t a r v e d donor i n i t i a l l y added to haemocoel-side (n - 19). 93 c o n t r o l values ( F i g , 19b),. When 10 u l samples of haemolymph from l o c u s t s were p l a c e d i n s i d e everted r e c t a l sacs, water uptake was i n c r e a s e d compared t o c o n t r o l s c o n t a i n i n g s a l i n e ( F i g . 2 0 ) . Haemolymph from dehydrated and r e c e n t l y - f e d donors i n c r e a s e d water uptake 45% and 52% r e s p e c t i v e l y . There was no s i g n i f i c a n t d i f f e r e n c e between these 2 valu e s although they were both s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l s (P<0.05; Student's t - ^ t e s t ) . D i s c u s s i o n The e f f e c t of haemolymph from r e c e n t l y f ed l o c u s t s on r e c t a l PD i s s i m i l a r to t h a t of CC homogenate although the absolute magnitude of the i n c r e a s e i s not as l a r g e (7 vs 10 mV) , nor does t h e i n c r e a s e p e r s i s t as long. The time course of the change i n I s c (e.g. maximum rate of r i s e ) when r e c t a are s t i m u l a t e d by a c t i v e haemolymph i s s i m i l a r t o t h a t f o l l o w i n g s t i m u l a t i o n of r e c t a by CC, although, as with PD, the a b s o l u t e i n c r e a s e i n I s c i s not as great (2.4 vs 3.4 nEg/cm 2/h) . Moreover, the e f f e c t s of a c t i v e haemolymph do not p e r s i s t as lon g ; haemolymph-stimulated Isc begins t o d e c l i n e a f t e r 80 min while r e c t a s t i m u l a t e d with 0.1 pr CC show no d e c l i n e i n I s c a f t e r 120 minutes. T h i s l e s s e r s t i m u l a t i o n of I s c and PD by a c t i v e haemolymph i s not unexpected..Most hormones have a very s h o r t h a l f - l i f e i n the haemolymph and c i r c u l a t i n g l e v e l s o f hormone are g e n e r a l l y f a r lower than those i n the s e c r e t i n g gland (Goldsworthy and Mordue, 1974),. a l l these d i f f e r e n c e s can be e x p l a i n e d by a lower dosage of a s i n g l e s t i m u l a n t i n the haemolymph. 94 A c t i v e haemolymph, l i k e CC homogenate and cAMP, a c t s p r i m a r i l y by i n c r e a s i n g the u n i d i r e c t i o n a l i n f l u x of C l - (L=>H) although t h e r e i s a s l i g h t i n c r e a s e i n ba c k f l u x (H=>L) as w e l l . For a l l 3 s t i m u l a n t s , the i n c r e a s e i n Isc can be completely accounted f o r by the r e s u l t i n g i n c r e a s e i n net C l - i n f l u x from the lumen. Since the s t i m u l a n t i s at times present i n the haemolymph i n c o n c e n t r a t i o n s h i g h enough to i n f l u e n c e the r a t e of r e c t a l t r a n s p o r t of C l - i n v i t r o , i t i s c l e a r l y a n a t u r a l hormone which I have c a l l e d C h l o r i d e - T r a n s p o r t S t i m u l a t i n g Hormone. Most samples of a c t i v e haemolymph produce a t r a n s i t o r y b i p h a s i c change i n I s c and continuous r e c o r d i n g s of o p e n - c i r c u i t PD show a s i m i l a r b i p h a s i c response (J. Hanrahan, pers._comm.). The net C l - uptake does not appear to i n c r e a s e u n t i l the second prolonged phase of s t i m u l a t i o n suggesting t h a t the b i p h a s i c change i n I s c may be due t o d i f f e r e n t haemolymph f a c t o r s . A l t e r n a t i v e l y the haemolymph CTSH may be r e l e a s e d i n a d i f f e r e n t form from that present i n th e . s t o r a g e organ or substances i n the haemolymph may modify the a c t i o n of CC f a c t o r . The l o s s of two t h i r d s of the haemolymph CTSH i n 80% methanol suggests t h a t CTSH may be present i n the . haemolymph l a r g e l y i n bound form, i . e . attached t o a c a r r i e r m o l e c u l e . , I t i s a l s o p o s s i b l e t h a t the CTSH may simply become p h y s i c a l l y trapped i n the p r o t e i n p r e c i p i t a t e which forms when the methanol i s added. To d i s t i n g u i s h between these p o s s i b i l i t i e s , p u r i f i c a t i o n of haemolymph and CC f a c t o r s are i n progress. Evidence t h a t CTSH i n the haemolymph i s d e r i v e d from the CC i s provided by the cardiatectomy experiment ( F i g . 16a,b). 95 Although l o c u s t s which have been sham-operated show a non-s i g n i f i c a n t 25% decrease i n c i r c u l a t i n g CTSH l e v e l s compared to normal l o c u s t s , 100% of the i n d i v i d u a l samples produced some s t i m u l a t i o n of the r e c t a l I s c whereas 67% of the c a r d i a t e c t o m i z e d l o c u s t s caused no s t i m u l a t i o n . The presence o f some CTSH a c t i v i t y i n the remaining l o c u s t s might be. e x p l a i n e d i n s e v e r a l ways. P o s s i b l y the CC i n these l o c u s t s were not t o t a l l y e x t i r p a t e d , or the f a c t o r might continue t o be r e l e a s e d from the. severed n e u r o s e c r e t o r y axons. Severed n e r v i c o r p o r i s c a r d i a c i w i l l h e a l and form de novo CC w i t h i n 72 h (Highnam and Goldsworthy, 1972; reviewed by Goldsworthy and Mordue, 1974). Such de novo glands w i l l f u n c t i o n e x a c t l y as the CC do, although with much lower i n t r i n s i c l e v e l s of n e u r o s e c r e t o r y products. Great care was taken t o completely remove the e n t i r e CC and the l o c u s t s were assayed 24 h post-cardiatectomy, t o minimize the p o s s i b i l i t y of de novo CC f o r m a t i o n . However t h i s process may occur more q u i c k l y i n some i n d i v i d u a l s . F i n a l l y , I have shown i n Chapter I I t h a t other n e u r a l t i s s u e s a l s o c o n t a i n , s m a l l q u a n t i t i e s of a f a c t o r which s t i m u l a t e s I s c , and the p o s s i b i l i t y of the a d d i t i o n a l r e l e a s e of an I s c - s t i m u l a t i n q f a c t o r from these s i t e s cannot be ignored. The mean c i r c u l a t i n g l e v e l s of CTSH i n the haemolymph of a l l r e c e n t l y - f e d l o c u s t s assayed were approximately 0.03 CCEg but t h e r e i s c o n s i d e r a b l e v a r i a t i o n among i n d i v i d u a l s . When compared to the f i n d i n g that 0.05-0.10 pr CC i s s u f f i c i e n t t o produce a maximal s t i m u l a t i o n of r e c t a l I s c (Chapter I I ) , i t i s c l e a r t h a t fed l o c u s t s r e l e a s e l a r g e g u a n t i t i e s of CTSH i n t o the haemocoel. Both the average content and the percentage of 96 i n d i v i d u a l s with d e t e c t a b l e l e v e l s of CTSH i n the haemolymph i s much lower i n dehydrated than i n r e c e n t l y - f e d l o c u s t s (Hanrahan, 1978). I have no i n d i c a t i o n of the r a t e at which CTSH i s i n a c t i v a t e d i n v i v o nor have I e x t e n s i v e l y examined the time course of CTSH i n l o c u s t haemolymph a f t e r f e e d i n g . I am t h e r e f o r e unable a t present to c a l c u l a t e the tr u e turnover r a t e of CTSH i n v i v o (see Mordue, 1969). The data presented i n Fig.19 i n d i c a t e a l a c k o f a n t i d i u r e t i c a c t i v i t y i n the CC of hydrated l o c u s t s although these same glands c o n t a i n l a r g e amounts of CTSH. S i m i l a r l y , Fig.20 shows a n t i d i u r e t i c a c t i v i t y i n the haemolymph of both dehydrated and r e c e n t l y - f e d l o c u s t s , although haemolymph from l o c u s t s c o n t a i n s r e l a t i v e l y l i t t l e CTSH a c t i v i t y . . Furthermore, P h i l l i p s (1964a,b) has shown t h a t i n v i v o l o c u s t s are capable o f r e s o r b i n g most of the io n s from very d i l u t e f a e c a l m a t e r i a l without a p r o p o r t i o n a l r e s o r p t i o n of water. ,1 t h e r e f o r e suggest that the a c t i o n of CTSH i s not p r i m a r i l y a n t i d i u r e t i c although any i n c r e a s e i n s a l t t r a n s p o r t might be expected t o enhance water a b s o r p t i o n t o some extent. CTSH may f u n c t i o n more as an • i n s e c t a l d o s t e r o n e ' , i n t h a t i t s primary f u n c t i o n may be to r e g u l a t e the r e s o r p t i o n and haemolymph c o n c e n t r a t i o n of Cl'- and i t s counter i o n s , Na + and K +. By analogy t o the r o l e of aldosterone i n the mammalian kidney (Hoar, 1966), CTSH might, however, enhance the a c t i o n of an a n t i d i u r e t i c f a c t o r when they are present i n the haemocoel t o g e t h e r . , C l e a r l y t h i s q u e s t i o n r e q u i r e s f u r t h e r study. 97 V GENERAL DISCUSSION In the p r e v i o u s c h a p t e r s , I have provided evidence t h a t CTSH i s a f a c t o r which occurs n a t u r a l l y i n l o c u s t haemolymph. I t i s apparently r e l e a s e d from the CC d u r i n g f e e d i n g (Hanrahan, 1978) and s t i m u l a t e s the a c t i v e r e s o r p t i o n of C l - from the r e c t a l lumen (Chapter IV). The a c t i o n of CTSH can be g u a n t i t a t i v e l y mimicked by the c y c l i c n u c l e o t i d e , cAMP. Although the broad range of chemical c o o r d i n a t o r s i n animals p r e s e n t s us with some d i f f i c u l t y i n s t r i c t l y d e f i n i n g the term "hormone", a p h y s i o l o g i c a l d e s c r i p t i o n can provide a u s e f u l b a s i s f o r d i s c u s s i o n . . Hormones s e c r e t e d by c e l l s o f n e u r a l o r i g i n are c l a s s i f i e d as neurohormones, and i n most i n s t a n c e s these are t r a n s p o r t e d to a neurohaemal organ where they are s t o r e d u n t i l r e l e a s e d i n t o the blood (Hoar, 1966). Although i t c o n t a i n s a number of i n t r i n s i c n eurosecretory c e l l s , the corpus cardiacum of i n s e c t s i s p r i m a r i l y c o n s i d e r e d to be a neurohaemal organ. A neurohormone (Hoar, 1966; Turner, 1968; Novales et a l . , 1973) i s considered to be that which: (1) i s a s p e c i f i c chemical substance, u s u a l l y a peptide (2) i s s e c r e t e d by s p e c i f i c c e l l s w i t h i n a s p e c i f i c , d u c t l e s s gland of n e u r a l o r i g i n (3) i s discharged d i r e c t l y i n t o the c i r c u l a t i n g f l u i d (4) i s e f f e c t i v e i n t r a c e g u a n t i t i e s (5) i s t r a n s p o r t e d to a d i s t a n t t i s s u e (6) e x e r t s a s p e c i f i c e f f e c t on a t a r g e t organ (7) i s normally destroyed i n the blood or at the 98 t a r g e t organ (8) a c t s i n the o v e r a l l c o o r d i n a t i o n of the animal T h i s d i s c u s s i o n w i l l examine CTSH f o l l o w i n g t h e s e . c r i t e r i a . . As neurohaemal organs, the CC c o n t a i n many d i f f e r e n t hormones, mostly s m a l l peptides (Mordue and Goldsworthy, 1972; Goldsworthy and Mordue, 1974)..This makes the i s o l a t i o n of any s i n g l e component very d i f f i c u l t , and presents the p o s s i b i l i t y t h a t the s t i m u l a t i o n of e l e c t r o g e n i c C l - t r a n s p o r t by CC i s not due to a new substance, but r a t h e r i s a s i d e - e f f e c t of some p r e v i o u s l y d i s c o v e r e d hormone. I compared the a c t i v i t y of CTSH with t h a t of the few p u r i f i e d or p a r t i a l l y - p u r i f i e d CC hormones a v a i l a b l e . The only hormone from l o c u s t CC which has been completely p u r i f i e d i s the a d i p o k i n e t i c hormone (AKH) which c o n t r o l s l i p i d m o b i l i z a t i o n i n these i n s e c t s . Samples of t h i s hormone, obtained from Dr. W. Mordue of I m p e r i a l C o l l e g e , had no e f f e c t on I s c (n=6) even at dosages of up t o 10 times those r e g u i r e d to provide maximum a d i p o k i n e t i c response ( i . e . w e l l above the normal c i r c u l a t i n g l e v e l s i n l o c u s t haemolymph)..Dr. Mordue a l s o k i n d l y provided me with p a r t i a l l y p u r i f i e d samples of the d i u r e t i c hormone (DH) e x t r a c t e d from the storage lobes of S c h i s t o c e r c a . DH had no e f f e c t on I s c (n=5) although the amount of d i u r e t i c a c t i v i t y l o s t i n t r a n s i t i s unknown. DH and CTSH do not co-chromatograph on c e l l u l o s e - a c e t a t e s t r i p s (DH value, W. Mordue, pers. comm.). At room temperature (21°C) DH has a h a l f -l i f e of 1.5 h (W. Mordue, pers. comm.) whereas CTSH has a h a l f -l i f e of approximately 24 h (Chapter I I ) . P r o c t o l i n , a smooth muscle s t i m u l a n t e x t r a c t e d from cockroach v e n t r a l g a n g l i a (Brown, 1967) and provided by Dr. B.E.Brown of the . U n i v e r s i t y of 99 Western O n t a r i o , a l s o had no e f f e c t on I s c (n=3). CTSH, t h e r e f o r e , appears t o be an e n t i r e l y new neurohumoural f a c t o r . . F u r t h e r work has been done towards the determination o f the chemical nature of CTSH. The i n i t i a l s t e p s i n the p u r i f i c a t i o n of CTSH were performed i n c o l l a b o r a t i o n with Dr. P h i l l i p s and Dr. Mordue i n 1978. Whole CC, separated storage and g l a n d u l a r l o b e s , and one pooled haemolymph sample were e x t r a c t e d with 80% methanol and chromatographed on a Sephadex G75 column (20 cm x 1.5 cm dia.) using e i t h e r d i s t i l l e d water or simple C l - s a l i n e as the e l u e n t . S a l i n e - e l u t e d samples (5.0 ml) were assayed d i r e c t l y on s h o r t - c i r c u i t e d r e c t a while, d i s t i l l e d water samples were f l a s h - e v a p o r a t e d to dryness and resuspended i n simple s a l i n e . In these few experiments, mostly d u p l i c a t e runs, the CTSH a c t i v i t y chromatographed i n the same e l u e n t volumes i n a l l cases and only one peak of CTSH a c t i v i t y was observed. T h i s substance appeared to be a molecule of approximately 10,000 M.W. by comparison with marker molecules chromatographed on the same columns..Using the same methods, Dr. Mordue (pers..comm.) d i s c o v e r e d t h a t both AKH and DH had molecular weights of approximately 2,000. A l l CTSH a c t i v i t y was destroyed by t r y p s i n d i g e s t i o n , suggesting t h a t the molecule i s a peptide or a s m a l l p r o t e i n . Although cockroach t e r m i n a l abdominal g a n g l i a c o n t a i n an a n t i d i u r e t i c peptide o f s i m i l a r s i z e (M.W. 8,000; Goldbard e t a l . , 1970), t h i s i s not present i n homogenates o f the CC ( M i l l s , 1967); y e t i n the 1978 study, P h i l l i p s demonstrated t h a t homogenates of whole cockroach CC have a s t i m u l a t o r y e f f e c t on s h o r t - c i r c u i t e d l o c u s t r e c t a (n=4). T h i s i n d i c a t e s t h a t the a n t i d i u r e t i c p r i n c i p l e o f the former authors i s not CTSH. 100 In Orthopterans, the CC are e a s i l y separated i n t o two d i s t i n c t r e g i o n s , the storage l o b e s , which serve as a neurohaemal organ f o r the medial n e u r o s e c r e t o r y c e l l s of the b r a i n , and the . g l a n d u l a r l o b e s which s y n t h e s i z e i n t r i n s i c hormones (Goldsworthy and Mordue, 1974). CTSH i s present i n both l o b e s although p r e l i m i n a r y experiments suggest t h a t n e a r l y 80% of the t o t a l a c t i v i t y i s present i n the storage l o b e s . By c o n t r a s t , i n both S c h i s t o c e r c a and Loousta, DH i s present o n l y i n the s t o r a g e l o b e s of the CC whereas the ADH i s present o n l y i n the g l a n d u l a r l o b e s (Mordue, 1970). The r e l e a s e of CTSH i n t o the haemolymph from the CC has been i n v e s t i g a t e d . Using the i n c r e a s e i n o p e n - c i r c u i t PD, r a t h e r than I s c , t o monitor CTSH a c t i v i t y , Hanrahan (1978)/ i n our l a b o r a t o r y , has shown t h a t the haemolymph CTSH l e v e l s i n c r e a s e by at l e a s t 40% a f t e r feeding and h i s p r e l i m i n a r y i n v e s t i g a t i o n suggests t h a t haemolymph CTSH a c t i v i t y peaks approximately 2 h a f t e r , f e e d i n g . In Chapter IV, I have shown that haemolymph from r e c e n t l y - f e d l o c u s t s c o n t a i n s a f a c t o r which, i n i t s e f f e c t s on i n v i t r o r e c t a , i s i n d i s t i n g u i s h a b l e from the f a c t o r present i n l o c u s t CC t h a t s t i m u l a t e s C l - r e s o r p t i o n . Levels of hormone i n these i n s e c t s are q u i t e high (0.03 pr CC per 300 ;al haemolymph). When p r e v i o u s l y starved l o c u s t s are f e d , s t a i n a b l e neurosecretory m a t e r i a l i s r e l e a s e d from the CC (Highnam, H i l l and Mordue, 1966; reviewed by Goldsworthy and Mordue, 1974) and t h i s may provide, h i s t o l o g i c a l evidence of CTSH r e l e a s e . F u r t h e r s t u d i e s on the c o n t r o l of r e l e a s e of CTSH are being conducted by J . Hanrahan. The dose-response curve f o r CC s t i m u l a t i o n (Fig.4) shows 101 t h a t the t h r e s h o l d response of the rectum t o CC homogenate occurs with approximately 0.0025 p a i r . Since CC weigh only about 25 ug the amount of t i s s u e r e g u i r e d f o r t h r e s h o l d s t i m u l a t i o n i s 0.0625 ;ug, p r o v i d i n g i n v i t r o c o n c e n t r a t i o n s of 12.5 ng/ml. The a c t u a l amount of hormone would of course be much l e s s , p o s s i b l y by s e v e r a l orders of magnitude, thus c l e a r l y s a t i s f y i n g c r i t e r i o n (4) above. Both CC and haemolymph i n c r e a s e a c t i v e C l - t r a n s p o r t i n v i t r o , showing t h a t although n e u r o s e c r e t o r y axons may i n n e r v a t e and act on the rectum, they are not r e q u i r e d f o r t h i s s t i m u l a t i o n to occur (see Chapter I I ) . The d e t e c t i o n of high l e v e l s of CTSH a c t i v i t y i n the haemolymph a l s o s t r o n g l y suggests that t h i s i s the primary pathway f o r CTSH d i s t r i b u t i o n . . CTSH e x e r t s a s p e c i f i c e f f e c t on i t s t a r g e t organ, the rectum, i n c r e a s i n g the a c t i v e uptake o f C l - from lumen to haemocoel..This a c t i o n i s i d e n t i c a l with CC homogenate (Fig,9) and haemolymph (Fig.15) and i s g u a n t i t a t i v e l y mimicked by the second messenger cAMP (Fig.9)..CC homogenate has been shown to t r i p l e , i n t r a c e l l u l a r cAMP l e v e l s i n the rectum (Table 3) which a l s o suggests t h a t CTSH a c t s v i a t h i s second messenger system.'. CTSH may a f f e c t other t r a n s p o r t or second messenger systems i n the rectum. Peacock (1976) showed t h a t homogenates of whole CC i n c r e a s e d Mg ATPase a c t i v i t y 5 - f o l d i n microsomal p r e p a r a t i o n s from l o c u s t rectum. In the same p r e p a r a t i o n s Na:K ATPase was s t i m u l a t e d 2 - f o l d at suboptimal (low Na +, K+) c o n d i t i o n s but l e s s than 15% a t optimal ( i . e . simple s a l i n e ) c o n d i t i o n s , which may e x p l a i n why no change i n a c t i v e Na + t r a n s p o r t was observed i n the presence of CC. Peacock (1976) s p e c u l a t e s that the Mg 102 ATPase may a l t e r the p e r m e a b i l i t y of the e p i t h e l i a l c e l l s t o C a 2 + . T h i s hypothesis would c o r r e l a t e w e l l with the observed s t i m u l a t i o n of C l - t r a n s p o r t by cAMP, si n c e Ca 2+ i s u s u a l l y i m p l i c a t e d i n second messenger systems u t i l i z i n g cAWP (Be r r i d g e , 1975). Furthermore, Hanrahan (1978) has shown t h a t both cGMP and the p r o s t a g l a n d i n p r e c u r s o r , arachadonic a c i d , s t i m u l a t e r e c t a l I s c and PD. Kachur et a l . (1979) have proposed t h a t i n the r a b b i t ileum, the s t i m u l a t o r y a c t i o n of a l l these compounds and some peptide gut hormones on C l - s e c r e t i o n i s i n f a c t mediated by high l e v e l s of i n t r a c e l l u l a r C a 2 + , each chemical a c t i n g t o r a i s e [ C a 2 + J i n a d i f f e r e n t way. T h i s may a l s o occur i n the l o c u s t rectum. From the present study, i t i s apparent that CTSH i s most l i k e l y i n a c t i v a t e d p r i m a r i l y by the rectum. Both haemolymph and CC homogenate samples are r e l a t i v e l y s t a b l e at 21°C, with a h a l f - l i f e of more than 24 h. .When haemolymph or submaximal doses of CC are added to voltage-clamped r e c t a , Isc and PD peak 60 to 80 min a f t e r s t i m u l a t i o n , but then begin to d e c l i n e w i t h i n 10 to 20 minutes. As noted i n Chapter I I , supramaximal doses of CC do not i n c r e a s e A I s c beyond the maximum value but do prolong the maximum s t i m u l a t i o n , suggesting t h a t CTSH i s i n a c t i v a t e d when i t binds to t h e membrane r e c e p t o r s . , CTSH may f u l f i l s e v e r a l r o l e s i n the i n t a c t animal. Hanrahan (pers. comm.) noted that when st a r v e d l o c u s t s are f e d l e t t u c e , which i s low i n C l - , haemolymph volume doubles and Malpighian tubule s e c r e t i o n t r i p l e s , but haemolymph C l - l e v e l s d e c l i n e o n l y 40%. T h i s i n d i c a t e s t h a t a l l of the.haemolymph C l -i s being conserved, presumably by the i n c r e a s e d r e s o r p t i o n o f 10 3 t h i s i o n from the r e c t a l lumen (Hanrahan, 1978).. Hanrahan (see S p r i n g et a l . , 1978) has a l s o d i s c o v e r e d t h a t the i n i t i a l PD of r e c t a immediately f o l l o w i n g d i s s e c t i o n i s much higher i n f e d than unfed l o c u s t s , which i m p l i e s t h a t CTSH i s a c t i n g i n v i v o . Wall (1970) showed t h a t when cockroaches were dehydrated and rehydrated, haemolymph o s m o l a r i t y and i o n c o n c e n t r a t i o n s f l u c t u a t e d only s l i g h t l y compared to haemolymph volume, i n d i c a t i n g t h a t i o n s were r a p i d l y being removed from and r e s t o r e d to the haemolymph. Hyatt and M a r s h a l l (1977) have shown t h a t Na+ and K+ are seguestered as u r a t e s i n the cockroach f a t body d u r i n g dehydration, but no such mechanism f o r C l _ c o n s e r v a t i o n has yet been d i s c o v e r e d . Wall (1970) has suggested t h a t i o n s , i n c l u d i n g C1-, are r e t a i n e d i n the rectum with the f a e c a l p e l l e t , and when the i n s e c t i s rehydrated, the C l - i n the rectum i s resorbed to r a i s e the haemolymph l e v e l s . The r e l a t i v e l y l a r g e amounts of C l - r e g u i r e d ( x 3 jiMoles) , p l u s the low l e v e l s of counter i o n s i n the f a e c e s (Na + + K + ^ 0 . 6 juMoles) , make t h i s p o s s i b i l i t y u n l i k e l y , but i t c o u l d e a s i l y be examined by measuring the C l - content of f a e c e s d u r i n g d e h y d r a t i o n . C e r t a i n l y Hanrahan's work (1978) suggests t h a t CTSH i s very important i n m a i n t a i n i n g the haemolymph C l - balance. The evidence accumulated to date suggests t h a t CTSH i s not i n f a c t an ADH but i s s p e c i f i c a l l y a C l - r e g u l a t i n g mechanism. Goh and P h i l l i p s (1978) have shown t h a t i n v i t r o water t r a n s p o r t can be maintained by any one.of Na +, K+ or C l - i o n s . C h l o r i d e alone supports water uptake from r e c t a l sacs l e s s than h a l f as w e l l as Na + or K+, but t h i s may be l a r g e l y a f u n c t i o n of the a b s o r p t i o n r a t e of i t s counter i o n ( c h o l i n e ) , s i n c e the 104 s u b s t i t u t i o n of S O 2 - f o r NOj a l s o decreases water uptake by sacs i n Na + and K + s a l i n e s . L i k e the mammalian hormone a l d o s t e r o n e , CTSH may serve . to enhance water r e s o r p t i o n when c o - t r a n s p o r t pathways.for water are opened by ADH. In v i v o , K + i s t r a n s p o r t e d 2-10 times more r a p i d l y than Na+ ( P h i l l i p s , 1964b), whereas i n v i t r o , K + t r a n s p o r t i s approximately equal to t h a t of Na+ (Goh, 1971; P h i l l i p s , 1977b). v i v o , the rectum sees, on the lumen-side, a f l u i d composed p r i m a r i l y of K + (140 mM) and C l - (93 mM) while Na + i s much lower (20 mM), and v i r t u a l l y a l l of the K + i s resorbed ( P h i l l i p s , 1970,1977a,b). I t i s evident t h a t much of the C l - i s absorbed as KCl and when CTSH i s r e l e a s e d by f e e d i n g , one of i t s a c t i o n s may be to i n c r e a s e the p e r m e a b i l i t y of the rectum to K+. T h i s would serve to i n c r e a s e K+ r e s o r p t i o n , at a time when more K + i s appearing i n the hindgut f l u i d as a r e s u l t of i n c r e a s e d Malpighian t u b u l e s e c r e t i o n . I t w i l l be necessary to f o l l o w the f l u x e s of Na + and K+ i n K-free and Na-free s a l i n e s r e s p e c t i v e l y , using GTSH-stimulated r e c t a , to determine whether CTSH a c t s i n t h i s manner. To o b t a i n a b e t t e r understanding of the a c t i o n of t h i s hormone i n i n t a c t l o c u s t s , f u r t h e r experiments w i l l be r e q u i r e d . I n i t i a l l y , l o c u s t s with c h r o n i c a l l y implanted agar bridges c o u l d be used to determine whether f e e d i n g i n c r e a s e s PD i n v i v o ; however, the gut l i g a t i o n may i n h i b i t f e e d i n g ( P h i l l i p s , 1964a). P h i l l i p s ' (1964a) p r e p a r a t i o n of l o c u s t r e c t a l i g a t e d i n s i t u c o u l d be used to determine whether r e c t a l C l - r e s o r p t i o n i n c r e a s e s a f t e r f e e d i n g , and a f t e r i n j e c t i o n of' CC homogenate.' C l - t r a n s p o r t has been shown to be hormonally c o n t r o l l e d , i n 105 v e r t e b r a t e s . There i s i n d i r e c t evidence f o r the r e g u l a t i o n of C l - t r a n s p o r t i n f i s h g i l l s (Maetz, 1971) , and cAMP s t i m u l a t e s a c t i v e C l - t r a n s p o r t i n shark r e c t a l glands ( S i l v a e t a l . , 1977) and k i l l i f i s h o p e r c u l a (Degnan et a l . . 1 977).. Cho l e r a e n t e r o t o x i n has a hormone-like a c t i o n on i n t e s t i n a l c e l l s and ac t s v i a cAMP t o s t i m u l a t e the a c t i v e s e c r e t i o n of C l ~ t o the mucosal-side and i n h i b i t Na + r e s o r p t i o n (Davis e t a l . , 1973). In the c u r r e n t model f o r e l e c t r o g e n i c C l ~ t r a n s p o r t i n most v e r t e b r a t e s ( F i e l d , 1974; Kachur et a l . , 1979) , j coupled NaCl uptake i n t o the c e l l i s d r i v e n by the Na+ g r a d i e n t provided by Na:K ATPase. C h l o r i d e then d i f f u s e s across the c o n t r a l a t e r a l membrane, while Na+ i s pumped back a c r o s s the s e r o s a l membrane. For both s e c r e t o r y and a b s o r p t i v e t i s s u e s , the c o n t r o l s i t e i s on the lumen-facing membrane and i s e i t h e r the coupled NaCl en t r y (absorption) or the. uncoupled C l - e x i t ( s e c r e t i o n ) . Although the C l - t r a n s p o r t i s e l e c t r o g e n i c , i t i s Na-dependent. I n s o f a r as we have shown t h a t the i n c r e a s e i n PD caused by cAMP occurs a c r o s s the lumen-facing membrane (i.e.. .the entry s i t e ) , the l o c u s t rectum f i t s t h i s c u r r e n t v e r t e b r a t e dogma (Spring e t a l . , 1978). However, p r e l i m i n a r y o b s e r v a t i o n s on s h o r t - c i r c u i t e d r e c t a i n d i c a t e t h a t C l - uptake i s not i n h i b i t e d by ouabain ( i . e . not Na-dependent). Goh (1971) found that water uptake by everted r e c t a l sacs was r e v e r s i b l y decreased by 50% with 1 0 - 3 M ouabain. A s i m i l a r decrease i s found when Na + and K + are removed from the bathing s a l i n e , s u g g e s t i n g t h a t C l ~ i s s t i l l b eing t r a n s p o r t e d to maintain water a b s o r p t i o n . In a d d i t i o n t o observing the a c t i o n of ouabain on more p r e p a r a t i o n s , determining whether CTSH can s t i m u l a t e C l - t r a n s p o r t i n Na-free 106 s a l i n e w i l l demonstrate u n e q u i v o c a l l y whether C l - t r a n s p o r t i n the l o c u s t rectum f i t s t h i s Na-dependent model. . Havinq examined the i n f o r m a t i o n concerning CTSH under the precepts of c l a s s i c a l endocrinology, the present study has provided the f i r s t demonstration of an i n s e c t hormone whose primary a c t i o n i s the c o n t r o l of a c t i v e C l ~ t r a n s p o r t . CTSH i s a neurohormone whose major f u n c t i o n appears to be the r e g u l a t i o n of haemolymph C l - l e v e l s , presumably by a l t e r i n g the r a t e s o f C l - a b s o r p t i o n from the rectum. The multitude of t r a n s p o r t s t u d i e s c u r r e n t l y i n progress r e v e a l t h a t a c t i v e C l - t r a n s p o r t i s more p r e v a l e n t i n the animal kingdom than p r e v i o u s l y b e l i e v e d * and i t s importance and r e g u l a t i o n w i l l provide many areas f o r f u r t h e r study. 107 LITERATURE CITED Altmann, G.. (19b6), Die r e g u l a t i o n des wasserhaushaltes der honigbiene. I n s e c t e s sociaux 3, 33-40. B a l s h i n , M. (1973). Absorption of amino a c i d s i n - v i t r o - by the rectum of the d e s e r t l o c u s t ( S c h i s t o c e r e a £regaria). Ph.D. T h e s i s , U n i v e r s i t y of B r i t i s h Columbia, i Vancouver, B.C. B e r r i d g e , M.J. (1966). 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