Science, Faculty of
Zoology, Department of
DSpace
UBCV
Spring, Jeffrey Herbert
Doctor of Philosophy - PhD
University of British Columbia
When isolated locust recta are short-circuited in 'Ussing-type' chambers, they exhibit an initial decline in short-circuit current (Isc) and transepithelial electropotential difference (PD) over the first two hours. Following this decline, recta remain in a steady-state condition for at least 4 h, during which these parameters decline only slightly. There is a factor present in the corpora cardiaca (CC) of schistocerca gregaria which causes the Isc and PD of short-circuited recta to increase. Maximum stimulation restores rectal Isc and PD to the levels observed immediately after removing this organ from locusts. Cyclic AMP causes a similar maximum increase in Isc and PD although the response exhibits a much shorter lag-time and a faster rate.of increase than with CC. The relationship between the logarithm of CC dose or cAMP concentration and the maximum ΔIsc is linear. Maximum stimulation is achieved with 0.05 pr CC or 0.3 mM cAMP, and the decline in Δlsc is dose-dependent and occurs over a matter of hours. Inhibitors of HCO₋₃/H+ and Cl- transport in vertebrates do not inhibit the stimulation of recta by CC or cAMP. Putative neurotransmitter substances and homogenates of flight muscle do not change rectal Isc or PD. Although homogenates of whole brains, ventral ganglia and rectal tissue cause small increases in Isc, the concentration of active factor is nearly three orders of magnitude greater in the CC. The unidirectional and net fluxes of ²²Na+ across short-circuited recta remain constant with time and are unaffected by the initial decline in Isc or by stimulation with CC or cAMP. Net ³⁶Cl- flux closely matches the Isc over the entire experiment, following the initial decline in Isc and rising when recta are stimulated with CC or cAMP. The stimulation of active net Cl- uptake is sufficient to account for the entire increase in Isc. Addition of CC to everted rectal sacs causes the levels of cAMP in this tissue to triple within 15 minutes. Removal of Cl- from the bathing media inhibits any stimulation by CC or cAMP, but the response returns as soon as Cl- is restored. Experiments using complex Cl-free salines suggest that alternate electrogenic transport processes can be turned on, and supported, by complex media. When starved locusts are fed lettuce, their haemolymph stimulates rectal Isc and PD in a similar manner to a submaximal dose of CC and the increase in Isc can be completely accounted for by the increase.in net ³⁶Cl- flux, as is the case with CC and cAMP. Removal of the CC from live locusts reduces or eliminates this feeding response. Further experiments suggest that the CC factor, which I have called Chloride-Transport Stimulating Hormone (CTSH), is not one of the known CC hormones. Although CC and haemolymph samples can increase water resorption in rectal sacs, the action of CTSH does not seem to be directly linked to antidiuresis. Structurally, it is a small protein (M.W. 10,000), water-soluble and relatively heat-stable. To date, I cannot detect any difference between the CC and haemolymph factors, although further experiments to purify CTSH from both sources are in progress.
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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Zoology
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Studies on the hormonal regulation of ion resorption in Schistocerca gregaria
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