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Ion transport and short circuit current in the rectum of the desert locust, Schistocerca gregaria Williams, Douglas Lloyd 1976

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ION TRANSPORT AND SHORT CIRCUIT CURRENT IN THE RECTUM OF THE DESERT LOCUST, SCHISTOCERCA GREGARIA by Douglas L l o y d W i l l i a m s B. Sc., Wayne St a t e U n i v e r s i t y , 1973 A T h e s i s Submitted i n P a r t i a l F u l f i l l m e n t The Requirements f o r the Degree o f Master o f Science i n The F a c u l t y o f Graduate S t u d i e s Department o f Zoology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard The U n i v e r s i t y o f B r i t i s h Columbia September, 1976 (g) Douglas L l o y d W i l l i a m s , 1976. In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h Co 1umb i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Z O O L O G Y The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D a t e 25 JUNE 1976 i ABSTRACT An i n v i t r o p r e p a r a t i o n of the rectum o f the d e s e r t l o c u s t S c h i s t o c e r c a g r e g a r i a i s d e s c r i b e d and c h a r a c t e r i z e d . The r e c t a l e p i t h e l i u m was mounted as a f l a t sheet s e p a r a t i n g two w e l l s t i r r e d chambers and the trans-membrane p o t e n t i a l d i f f e r e n c e (PD), membrane r e s i s t a n c e , and s h o r t c i r c u i t c u r r e n t (SCC) were monitored. The PD and membrane r e s i s t a n c e remained r e l a t i v e l y constant f o r a t l e a s t s i x hours a t 35 mV (lumen p o s i t i v e ) and 6000 Ohms . cm , r e s p e c t i v e l y . A f t e r an i n i t i a l two hour t r a n s i e n t p e r i o d d u r i n g which the SCC -2 -1 -2 -1 dropped from 8 uMoles of charge .cm .hr to 3.5 uMoles.cm .h an approximate s t e a d y - s t a t e c o n d i t i o n was reached and maintained f o r a t l e a s t another f o u r hours. The SCC was c o n s i s t a n t w i t h e i t h e r a net c a t i o n t r a n s p o r t from the hemolymph t o the lumen of the rectum or a net anion t r a n s p o r t i n the o p p o s i t e d i r e c t i o n . Since C l was the o n l y major anion i n the b a t h i n g media some experiments were c a r r i e d out to e v a l u a t e the con-t r i b u t i o n of a c t i v e C l t r a n s p o r t to the SCC. S u b s t i t u t i o n of SO~ o f NO~ d u r i n g the t h i r d and f o u r t h hours i n v i t r o had no e f f e c t on the SCC, although they a b o l i s h e d the i n i t i a l two hours t r a n s i e n t . S i m i l a r experiments i n which C l was s u b s t i t u t e d by a c e t a t e had a complex s t i m u l a t o r y e f f e c t on the SCC. Measure-36 — ments o f C l f l u x e s under SCC c o n d i t i o n s d u r i n g the same time i i p e r i o d demonstrated a mean net f l u x of 1.52 uMoles of C l .cm "".hr from the r e c t a l lumen to the hemolymph. T h i s value i s e q u i v a l e n t t o one h a l f the simultaneous SCC. These experiments c l e a r l y demonstrate t h a t the r e c t a l e p i t h e l i u m a c t i v e l y t r a n s p o r t s C l i n a d i r e c t i o n c o n s i s t e n t w i t h the observed SCC but t h a t t h i s a c t i v e t r a n s p o r t o f C l does not c o n t r i b u t e t o the SCC d u r i n g the t h i r d and f o u r t h hours i n v i t r o . S i nce r e c t a l Na + and K + t r a n s p o r t r e p o r t e d by o t h e r workers i s i n the wrong d i r e c t i o n t o account f o r the observed SCC, i t seems necessary t o propose (1) a C l exchange pump (probably w i t h HCO^) perhaps l o c a t e d on the l u m i n a l membrane to account f o r the f a c t t h a t a c t i v e C l t r a n s p o r t does not c o n t r i -bute t o the SCC and (2) a H + and/or HCO^ pump to account f o r the SCC. These p r o p o s a l s are i n c o r p o r a t e d i n a model f o r o r g a n i z a -t i o n o f i o n t r a n s p o r t processes i n the l o c u s t rectum. TABLE OF CONTENTS A b s t r a c t Table of Contents L i s t o f F i g u r e s and I l l u s t r a t i o n s Acknowledgement I n t r o d u c t i o n M a t e r i a l Methods R e s u l t s E l e c t r i c a l Parameters and V i a b i l i t y i n the In V i t r o P r e p a r a t i o n C h l o r i d e T r a n s p o r t and the Source o f the SCC C h l o r i d e F l u x S t u d i e s Under SCC C o n d i t i o n s D i s c u s s i o n B i b l i o g r a p h y i v LIST OF FIGURES AND ILLUSTRATIONS F i g u r e s Page 1 Schematized Drawing o f R e c t a l Morphology 3 2 Experimental Chambers 9 3 E l e c t r i c a l C i r c u i t r y 15 4 Time Course of. T r a n s - R e c t a l PD 24 5 Time Course o f Open C i r c u i t T r a n s - R e c t a l 26 R e s i s t a n c e 6 Time Course o f Short C i r c u i t T r a n s - R e c t a l 28 R e s i s t a n c e 7 Time Course of R e c t a l SCC 31 8 Time Course of R e c t a l SCC A f t e r an I n i t i a l Two 34 Hours under Open C i r c u i t C o n d i t i o n s 9 E f f e c t o f Temperature on R e c t a l SCC 36 10 E f f e c t o f 1 mM KCN on R e c t a l SCC 39 11 SO^ S u b s t i t u t i o n f o r C l " (T=2:15 to T=5:00): 42 E f f e c t on SCC 12 C l ~ E f f l u x from R e c t a l T i s s u e i n SO^ Ringer 45 13 Time Course o f R e c t a l SCC i n SO^ Ringer 48 14 Time Course o f R e c t a l SCC i n NO^ Ringer 50 15 Time Course of R e c t a l SCC i n Acetate Ringer 52 — 36 — 16 U n i - d i r e c t i o n a l C l Fluxes Measured w i t h C l 56 and Simultaneous Mean SCC 17 Net C l " F l u x Measured w i t h 3 6 C 1 ~ and 59 Simultaneous SCC 18 Model o f Ion T r a n s p o r t Processes i n Rectum 70 19 In V i t r o Apparatus o f H e r r e r a e t a l . (19 76) 74 V ACKNOWLEDGEMENT The author wishes to thank h i s s u p e r v i s o r , Dr. John E. P h i l l i p s , f o r much h e l p f u l a d v i c e and encouragement and Dr. John G o s l i n e f o r h i s kindness i n s e r v i n g as a c t i n g super-v i s o r d u r i n g the f i n a l p r e p a r a t i o n o f t h i s t h e s i s . He f u r t h e r wishes t o thank Joan M a r t i n , Nancy Scherer, and, e s p e c i a l l y , Tim Bradley f o r t h e i r f r i e n d s h i p and many hours o f f r u i t f u l d i s c u s s i o n . The author i s a l s o g r a t e f u l f o r the f i n a n c i a l support a f f o r d e d by a U n i v e r s i t y Graduate F e l l o w s h i p and a K i l l a m Memorial F e l l o w s h i p w h i l e working on t h i s t h e s i s . -1-INTRODUCTION Since i t s i n t r o d u c t i o n i n 1951 by Uss i n g and Zerahn, the technique o f s h o r t - c i r c u i t c u r r e n t (SCC) has been a power-f u l t o o l i n the e l u c i d a t i o n o f i o n t r a n s p o r t mechanisms i n e p i t h e l i a . F i r s t a p p l i e d t o the study o f a v a r i e t y o f v e r t e b r a t e e p i t h e l i a (Ussing & Zerahn, 1951; Uss i n g & Anderson, 1955; Hogben, 1955; Leaf & Renshaw, 1958; C o o p e r s t e i n & Hogben, 1959; Durbin & Heinz, 1959) i t s use has r e c e n t l y been extended to the study o f i n s e c t e p i t h e l i a (O'Riordan, 1969; Wood, 1972; Wood & Harvey, 1975). I wish t o r e p o r t here the a p p l i c a t i o n o f t h i s technique t o the study o f i o n t r a n s p o r t i n the rectum o f the d e s e r t S c h i s t o c e r c a g r e g a r i a F o r s k a l . The morphology and h i s t o l o g y o f the l o c u s t a l i m e n t a r y c a n a l were o u t l i n e d i n d e t a i l by P h i l l i p s (1964). B r i e f l y , the l o c u s t rectum i s an e x t e n s i b l e tube 5-7mm. long i n the c o n t r a c t e d s t a t e l o c a t e d a t the p o s t e r i o r end of the a l i m e n t a r y c a n a l (Figure 1). The lumen of the rectum i s l i n e d w i t h a c h i t i n o u s c u t i c l e o r i n t i m a which has been shown t o a c t as a molecular s i e v e w i t h a pore r a d i u s o f 6.5-8 A ( P h i l l i p s & D o c k r i l l , 1968). Beneath the i n t i m a the r e c t a l e p i t h e l i u m i s thrown up i n t o s i x l o n g i t u d i n a l f o l d s o r pads composed o f columnar e p i t h e l i a l c e l l s . The e p i t h e l i a l c e l l l a y e r i s surrounded by a continuous sheath o f c i r c u l a r muscle o u t s i d e -2-F i g u r e 1. Schematized drawing of r e c t a l morphology, (a) Cross s e c t i o n o f rectum. (b) T r a n s p o r t i n g e p i t h e l i a l c e l l l a y e r . P, r e c t a l pad. C, c u t i c u l a r i n t i m a . EC, t r a n s p o r t i n g e p i t h e l i a l c e l l l a y e r . MC, c i r c u l a r muscle l a y e r . ML, l o n g i t u d i n a l muscle bundles. T, tracheae and t r a c h e o l e s . N, c e l l n u c l e i . IC, i n t e r c e l l u l a r channels. 3-( a ) ( b ) I C -4-of which are s i x e q u i d i s t a n t l o n g i t u d i n a l muscle bands. The rectum i s the most h e a v i l y t r a c h e a t e d p a r t o f the a l i m e n t a r y c a n a l w i t h most of the tracheae e n t e r i n g the r e c t a l t i s s u e along the base of the r e c t a l pads. When the rectum i s d i s -tended w i t h f e c a l matter o r when i t i s s t r e t c h e d out as a f l a t sheet s e p a r a t i n g two chambers as i n most experiments i n t h i s study, the r e c t a l e p i t h e l i u m has a uniform t h i c k n e s s o f about 1 mm. and the r e c t a l pads appear simply as l o n g s i l v e r -white bands beneath the l u m i n a l c u t i c l e . C o n s i d e r a b l e i n f o r m a t i o n on r e c t a l a b s o r p t i o n o f i o n s was a v a i l a b l e f o r a t l e a s t one i n s e c t , the d e s e r t l o c u s t , b e f o r e commencing t h i s study. In the e a r l y 1960's, P h i l l i p s (1961, 1964) showed t h a t the i n v i v o l o c u s t rectum t r a n s p o r t s H or HCOj/ Na , K , C l , and B^O a g a i n s t e l e c t r o c h e m i c a l g r a d i e n t s . T r a n s p o r t o f the l a t t e r f o u r s p e c i e s was l a t e r shown t o occur i n an i n v i t r o p r e p a r a t i o n by Goh, (1971); P h i l l i p s and Goh, i n p r e p a r a t i o n ; and Meredith and P h i l l i p s , unpublished data. In 1968, Speight showed t h a t the i n v i v o -2 -1 + rectum t r a n s p o r t s a minimum of 2.2 uMoles cm hr of H and/or HCO^. Though the f o r e g o i n g s t u d i e s amply demonstrate t h a t i o n t r a n s p o r t processes occur i n the r e c t a l e p i t h e l i u m , f o r the most p a r t p r e v i o u s s t u d i e s have focused on water t r a n s p o r t . L i t t l e was known of the i n t e r r e l a t i o n s h i p s b e t --5-ween the d i f f e r e n t i o n pumps i n the rectum nor of t h e i r k i n e t i c and o t h e r p r o p e r t i e s . In a d d i t i o n t o c h a r a c t e r i z i n g a new i n v i t r o p r e p a r a t i o n , t h i s paper demonstrates t h a t the m a j o r i t y of the e l e c t r o g e n i c i o n t r a n s p o r t ( t o t a l anions minus t o t a l c a t i o n s ; i . e . , the SCC) i s c a r r i e d by n e i t h e r Na +, K +, or C l but i s probably supported by H + or HCO^ t r a n s p o r t . While the presen t paper was i n p r e p a r a t i o n the work o f H e r r e r a , Jordana, and Ponz (1976) came t o my a t t e n t i o n . T h i s paper, which w i l l be c o n s i d e r e d i n the D i s c u s s i o n , a l s o d e s c r i b e s the a p p l i c a t i o n o f the SCC technique t o the study of i o n t r a n s p o r t i n the l o c u s t rectum. The q u i t e d i f f e r e n t c o n c l u s i o n s which these authors r e p o r t can be a t t r i b u t e d t o the use of c o n d i t i o n s which f a i l e d t o s u s t a i n t r a n s p o r t ac-t i v i t y t o the same degree as t h a t r e p o r t e d i n the pr e s e n t study. -6-MATERIALS AND METHODS MATERIAL A d u l t female S c h i s t o c e r c a g r e g a r i a 1-3 months p a s t t h e i r f i n a l molt and f e d on l e t t u c e o r spinach and a mixture of d r i e d g r a s s , bran, y e a s t and powdered milk were used i n a l l experiments. The animals were maintained i n a cons t a n t temperature room a t 28°C and 50% R.H. i n cages each c o n t a i n -i n g a 60 watt incandescent bulb f o r a d d i t i o n a l heat and I l -l u m i n a t i o n . A photoperiod c y c l e o f 16 hours l i g h t and 8 hours dark was used. METHODS To study i o n t r a n s p o r t u s i n g the SCC method r e c t a were removed from the l o c u s t by the f o l l o w i n g procedure. The head and a l l the appendages were c u t o f f and the animal was pinned down on a P l a s t i c e n e b l o c k under a d i s s e c t i n g microscope w i t h the l e f t s i d e f a c i n g upward. A U-shaped c u t was made i n the l e f t s i d e o f the abdomen s t a r t i n g a t the l a s t segment and extending forward 3 segments. The r e s u l t i n g f l a p o f c u t i c l e was f o l d e d back d o r s a l l y and pinned down t o the P l a s t i c e n e b l o c k . T i s s u e s were kept moist d u r i n g subsequent d i s s e c t i o n w i t h the a p p r o p r i a t e Ringer's s o l u t i o n . The gut was grasped w i t h a p a i r o f f i n e watch-makers f o r c e p s j u s t -7-a n t e r i o r t o the rectum, and the f a t body, M a l p i g h i a n t u b u l e s and tracheae were a l l c u t and p u l l e d away from the rectum u s i n g another p a i r o f f o r c e p s . The rectum was then s l i t lengthwise u s i n g i r i d e c t o m y s c i s s o r s i n s e r t e d through the anus. A f t e r removing the r e c t a l contents the rectum was severed a t i t s a n t e r i o r and p o s t e r i o r b o r ders. The r e s u l t i n g square o f t i s -sue (about 0.8 cm. on a side) was t r a n s f e r r e d w i t h the f o r c e p s and mounted on an u p r i g h t h a l f chamber as d e s c r i b e d below. The chambers used i n these experiments (Figure 2) were m o d i f i e d from those d e s c r i b e d by Wood, (1972). The f l u i d r e s e r v o i r s were i n c l i n e d a t an angle o f 45° t o the h o r i z o n t a l t o permit mounting of the t i s s u e w h i l e the r i g h t chamber h a l f was f i l l e d w i t h Ringer's s o l u t i o n . The chamber halv e s were h e l d t o g e t h e r and supported by a v i s e - l i k e frame as shown i n F i g u r e 2. The diameter of the c i r c u l a r o r i f i c e over which the rectum was mounted was 0.5 cm. g i v i n g an exposed s u r f a c e area 2 of 0.196 cm . The r e s e r v o i r s had loo s e removeable caps t o prevent s o l u t i o n l o s s by s p a t t e r i n g and to a l l o w easy access to change s o l u t i o n s and conduct t r a c e r s t u d i e s . Each chamber h a l f and a s s o c i a t e d r e s e r v o i r was f i l l e d w i t h equal volumes of Ringer's s o l u t i o n : 20 ml. i n most experiments. F l u i d i n each chamber h a l f was c i r c u l a t e d by a gas-l i f t pump so as to d e l i v e r a flow o f s a l i n e from the l a r g e r e s e r v o i r a g a i n s t the s u r f a c e o f the r e c t a l w a l l . A gas -8-F i g u r e 2. Experimental chambers. R e s e r v o i r s H o l e Frame -10-mixture of 95% 0^ and 5% C 0 2 was used t o p r o v i d e adequate oxygenation i n the absence o f i n t a c t t r a c h e a l c o n n e c t i o n s . F l u i d c i r c u l a t i o n was both r a p i d and t u r b u l e n t i n s i d e the chambers e s p e c i a l l y near the membrane as determined by i n -j e c t i o n o f dye a t v a r i o u s p o i n t s i n the chamber and r e s e r -v o i r s . I n j e c t e d dye r e t u r n e d t o i t s s t a r t i n g p o i n t i n much l e s s than a second and f l u i d i n the r e s e r v o i r s and chambers became u n i f o r m l y c o l o r e d by the dye w i t h i n a few seconds. S t i r r i n g r a t e and oxygenation of the s o l u t i o n are i n t i m a t e l y r e l a t e d when u s i n g g a s - l i f t pumps. S t i r r i n g r a t e was kept c o n s t a n t i n these experiments by m a i n t a i n i n g the gas flow constant (using a s m a l l gas flow r e g u l a t o r ) a t about twice the r a t e a t which i t begins t o l i m i t r e c t a l SCC. The s t i r r i n g gas was r a i s e d t o 100% R.H. by b u b b l i n g through water b e f o r e being i n t r o d u c e d t o the chambers i n order t o minimize e v a p o r a t i o n of the Ringer's s o l u t i o n d u r i n g long experiments. Most experiments were performed a t room temperature. T h i s v a r i e d from day to day between 20° - 25°C but the change d u r i n g any one experiment was l e s s than 2°C. However, t o determine hhe e f f e c t o f temperature on SCC a c o i l e d g l a s s tube was i n s e r t e d i n t o each r e s e r v o i r and a h e a t i n g o r c o o l i n g f l u i d passed through i t from a "Haake" model FE con s t a n t temperature water c i r c u l a t i n g pump. Temperatures i n the - l i -re s e r v o i r were monitored by i n s e r t i n g a thermometer i n t o the top of each r e s e r v o i r . When i t was d e s i r e d t o change the Ringer's s o l u t i o n d u r i n g the course o f an experiment a l a r g e volume of new s o l u t i o n (about 250 ml.) was added through the top o f each r e s e r v o i r w h i l e the s o l u t i o n l e v e l was maintained by i n s e r t i n g a f i x e d - l e v e l vacuum l i n e i n t o the top o f each r e s e r v o i r . Changing both s o l u -t i o n s took about 90 seconds and was 99.9% e f f e c t i v e as measured by 3 6 C 1 ~ d i l u t i o n . A f t e r the r e c t a l e p i t h e l i u m was removed from the l o c u s t as d e s c r i b e d above i t was s t r e t c h e d over the mouth of the r i g h t chamber h a l f , lumen s i d e up, us i n g two p a i r s o f f o r c e p s . The t i s s u e was h e l d down by i m p a l i n g the p e r i p h e r a l areas o f t i s s u e on f i n e metal p i n s permanently mounted around the o r i f i c e o f the chamber h a l f . A rubber 0 - r i n g was then f i t t e d over the l i p o f the chamber above the rows of p i n s s e a l i n g o f f the o r i f i c e o f the r i g h t chamber. The r i g h t and l e f t chamber halves were then f i t t e d t o g ether, turned t o the h o r i z o n t a l p o s i t i o n , mounted i n the s u p p o r t i n g frame, and clamped f i r m l y t o g e t h e r . Then the l e f t chamber h a l f was f i l l e d w i t h Ringer's s o l u t i o n and the gas l i n e s f o r s t i r r i n g were at t a c h e d . The v o l t a g e and c u r r e n t e l e c t r o d e s were connected l a s t as d e s c r i b e d below. The i n i t i a l membrane p o t e n t i a l d i f f e r e n c e (PD) r e a d i n g was u s u a l l y taken w i t h i n 13-15 m. o f s t a r t i n g the d i s s e c t i o n o f the l o c u s t . -12-The b a s i c Ringer's s o l u t i o n used i n these experiments (Normal Ringer's) was adapted from B e r r i d g e (1966). I t co n t a i n e d 24.5 mM NaCl, 10.5 mM NaHC0 3, 8.5 mM KC1, 2 mM C a C l 2 , 13 mM MgCl 2, 7.4 mM disodium s u c c i n a t e , 1.87 mM T r i s o d i u m c i t r a t e , 12.8 mM ma l i c a c i d , 1.6. mM glucose, 5.56 mM maltose 79.8 mM sucrose, 2.67 mM g l y c i n e , 4.61 mM p r o l i n e , 2.64 mM glutaraine, 12.3 mM glutamic a c i d , 30 mg/1 p e n i c i l l i n , and 100 mg/1 streptomycin s u l -phate. The r e s u l t i n g s o l u t i o n was a d j u s t e d t o pH=7.00 wit h NaOH. The f r e e z i n g p o i n t o f the Ringer's s o l u t i o n was -0.587°C as mea-sured w i t h a " C l i f t o n " N a n o l i t e r Osmometer. In some e a r l y ex-periments Normal Ringer a l s o c o n t a i n e d 8.0 g/1 of l a c t a l b u m i n h y d r o l y s a t e and 4.0 g/1 of y e a s t e x t r a c t . The f r e e z i n g p o i n t i n t h i s case was maintained a t -0.587°C by r e d u c i n g the sucrose con-c e n t r a t i o n t o 11.7 mM. O m i t t i n g the l a c t a l b u m i n h y d r o l y s a t e and yeast e x t r a c t decreased the SCC by about 20% (see Re s u l t s ) but had no o t h e r observable e f f e c t s on the p r e p a r a t i o n . Normal Ringer w i t h l a c t a l b u m i n h y d r o l y s a t e and y e a s t e x t r a c t had a s p e c i f i c r e s i s t a n c e o f 77.5 Ohm. cm. S e v e r a l o t h e r Ringer's s o l u t i o n s were used i n t h i s study t o i n v e s t i g a t e the p r o p e r t i e s o f the C l t r a n s p o r t i n g system o f the rectum. In these s o l u t i o n s a l l the C l was r e p l a c e d w i t h e i t h e r SO^, NO^, or a c e t a t e . These Ringer's s o l u t i o n s , which w i l l be subsequently r e f e r r e d t o as "S0~-Ringer", "NO~-Ringer", and "Acetate Ringer" r e s p e c t i v e l y , -13-were otherwise i d e n t i c a l to Normal Ringer, except that SO^-Ringer contained 128.3 mM sucrose instead of the normal 79.8 mM i n order to keep the freezing point at -0.587°C. Another SO^-Ringer's solution contained lactalbumin hydro-lysate and yeast extract which introduced small amounts of C l ~ (3 mM) and i s referred to as "Low C l ~ SO~-Ringer". The C l ~ concentrations of a l l solutions were measured d i r e c t l y with a "Radiometer" CMT 10 Chloride T i t r a t o r . The C l content of the r e c t a l tissue was measured both immediately af t e r removal from the animal and af t e r varying lengths of time i n SO^-Ringer. The following pro-cedure was used: r e c t a l preparations were quickly dipped ten times i n sucrose solution having a freezing point of -0.587°C to wash of the majority of ions adhering to them, l i g h t l y blotted dry on tissue paper and transferred to i n -d i v i d u a l capped v i a l s containing 1 ml. of d i s t i l l e d water. The recta were then stored at room temperature for 24 hours by which time a l l the c e l l s had lysed releasing t h e i r con-tents into the solution. Then a f t e r vigorous shaking to ensure complete mixing, the C l concentration of the solution i n each v i a l was measured as previously described. The following c i r c u i t r y (Figure 3) which was modified from Wood (1972) was used to measure the PD and SCC of the i n v i t r o preparation. "M" represents the r e c t a l -14-F i g u r e 3. E l e c t r i c a l c i r c u i t r y . See t e x t f o r e x p l a n a t i o n s o f symbols. B 2 S l Chart Recorder -16-p r e p a r a t i o n . R 2, R^' a n c " ^4 a r e P r e c i s i o n 10° ohm +1% r e s i s t o r s . 3 R 5 i s a s e l e c t e d 10 ohm carbon r e s i s t o r . R^ i s a H e a t h k i t decade r e s i s t a n c e box, model 1N-17. and B 2 are 9 v o l t b a t -t e r i e s . The sw i t c h S 2 i s c l o s e d when a p p l y i n g a s h o r t c i r c u i t c u r r e n t a c r o s s the r e c t a l w a l l . The sw i t c h i s c l o s e d momen-t a r i l y t o pass a c u r r e n t p u l s e through the p r e p a r a t i o n t o measure t r a n s e p i t h e l i a l r e s i s t a n c e e i t h e r under s h o r t c i r c u i t o r open c i r c u i t c o n d i t i o n s . The v o l t m e t e r (a K i e t h l e y 602 ele c t r o m e t e r ) measures one t h i r d o f the t r u e membrane PD. The SCC i s read as a v o l t a g e a c r o s s R 5 u s i n g a K e i t h l e y 616 d i g i t a l e l e c t r o m e t e r . The membrane PD and SCC are recor d e d g r a p h i c a l l y on a 2 pen " F i s h e r R 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 . E 2 , E^, and are Calomel e l e c t r o d e s connected t o the two h a l f -chambers c o n t a i n i n g the r e c t a l e p i t h e l i u m v i a s a l t b r i d g e s con-s i s t i n g o f p o l y e t h l y e n e t u b i n g (PE-50) wi t h 3 cm. long g l a s s c a p i l l a r y t i p s and f i l l e d w i t h 3M KC1. i n 3% agar. The g l a s s c a p i l l a r y tubes, which are i n s e r t e d i n t o the chambers are shown i n F i g u r e s 2 and 3, were used because they are more r i g i d than p o l y e t h l y e n e t u b i n g . T h i s r i g i d i t y was e s s e n t i a l f o r accurate v o l t a g e readings because, when p a s s i n g c u r r e n t through the chambers, E 2 and E^ must be e q u i d i s t a n t from E^. Cu r r e n t i s a p p l i e d a c r o s s the chambers through E^ and E^ which are s i l v e r -s i l v e r c h l o r i d e e l e c t r o d e s cut from 0.004 i n c h s i l v e r f o i l . In a c t u a l p a a c t i c e t h r e e chambers w i t h i d e n t i c a l c i r c u i t s t o -17-that described above were used simultaneously. The two meters and the recorder were switched to each preparation when read-ings were required. The three electrode system of voltage measurement was used instead of the usual two electrode system because when E 2 and are equidistant from E 3 the voltage gradients set up through the Ringer's solution i n the two chamber halves by passing a SCC are automatically cancelled out across R^ and so the only voltage seen across R^ i s one t h i r d of the true membrane voltage plus the asymmetry po t e n t i a l . With a two electrode system, the voltage gradients are not cancelled out at the voltmeter. This complicates the measurement of voltage under s h o r t - c i r c u i t conditions considerably and t h i s , along with technical d i f f i c u l t i e s r elated to chamber construction, was considered s u f f i c i e n t reason to use the three electrode system. The error i n voltage measurement introduced by placing E 2 and E^ such a large distance ( i . e . , 0.5 cm.) from the membrane can be shown to be n e g l i g i b l e . Assuming a mem-brane PD of 100 mV (over three times the average i n i t i a l reading i n the present study) and considering that the r e s i s -tance of the voltage measuring c i r c u i t (R 2 +(R3 x R 4 ) / p (R^ + R^)) i s 1.5 x 10 ohms, the current through the c i r c u i t -9 would be 0.66 x 10 amperes. The resistance of the Ringer's -18-s o l u t i o n between E 2 and E^ i s about 60 Ohms. A c u r r e n t o f -9 0.66 x 10 amperes through a r e s i s t a n c e o f 60 Ohms would i n t r o d u c e a v o l t a g e drop o f o n l y 0.02 m i c r o v o l t s , which i s n e g l i g i b l e and a l s o w e l l below the i n t e r n a l n o i s e l e v e l o f the K i e t h l e y 602. For f u r t h e r d i s c u s s i o n o f e l e c t r i c a l measurements u s i n g t h i s experimental system see Wood (1972). P r i o r t o mounting the rectum i n the chambers the p o s i t i o n o f the v o l t a g e e l e c t r o d e s was ad j u s t e d so t h a t E 2 and E^ were e q u i d i s t a n t from E^. T h i s was accomplished by f i r s t f i l l i n g the chambers wit h Ringer's s o l u t i o n , i n s e r t i n g the v o l t a g e e l e c t r o d e s , and measuring the asymmetry p o t e n t i a l . Then a constant c u r r e n t o f 50 microamperes was passed through the s o l u t i o n v i a the c u r r e n t e l e c t r o d e s . I f E_ and E. were 2 4 e q u i d i s t a n t from E^ the v o l t a g e g r a d i e n t s i n t r o d u c e d i n t o the s o l u t i o n by t h i s c u r r e n t c a n c e l l e d a c r o s s R^ and o n l y the o r i g i n a l asymmetry PD was recorded. I f the observed v o l t a g e was o t h e r than the asymmetry p o t e n t i a l , E 2 , which i n t e r s e c t s the l o n g i t u d i n a l chamber a x i s a t about a 35° angle, was moved i n o r out o f i t s socket as shown i n F i g u r e 3 u n t i l o n l y the asymmetry p o t e n t i a l was observed on the vo l t m e t e r . At t h i s p o i n t the t i p o f E^ was the same d i s t a n c e from E 2 and E^. The c u r r e n t e l e c t r o d e s were then d i s c o n n e c t e d and the chambers d i s -assembled without d i s t u r b i n g the p o s i t i o n o f the v o l t a g e e l e c -t r o d e s . The r e c t a l e p i t h e l i u m was then mounted i n the chambers as p r e v i o u s l y d e s c r i b e d . 36 — U n i d i r e c t i o n a l f l u x s t u d i e s of C l a c r o s s the r e c t a l w a l l were measured to determine the net f l u x of c h l o r i d e under s h o r t c i r c u i t c o n d i t i o n s . C l was o b t a i n e d as a 2.41 M HCl s o l u t i o n w i t h a s p e c i f i c a c t i v i t y o f 4.4 mCi/gm. from New England Nuclear Inc., Boston, Mass., U.S.A. The s o l u t i o n was n e u t r a l i z e d t o pH=9 by adding the a p p r o p r i a t e amount of a NaOH s o l u t i o n o f equal m o l a r i t y and was f r o z e n u n t i l ready f o r use. Approximately 0.31 ml. o f the n e u t r a l i z e d s o l u t i o n was added t o e i t h e r the lumen o r the hemolymph r e s e r v o i r each of which c o n t a i n e d 20 ml. of Normal Ringer a t the b e g i n n i n g o f the t h i r d hour (T = 2:00) a f t e r mounting the rectum i n the chambers. An equal volume of " c o l d " NaCl s o l u t i o n of equal m o l a r i t y was added to the o p p o s i t e r e s e r v o i r a t the same time. The t o t a l amount o f c h l o r i d e added t o each s i d e d u r i n g t h i s procedure was never more than 1 mMole. T h i s i n c r e a s e d the C l c o n c e n t r a t i o n o f the b a t h i n g media by 17 mM t o g i v e a f i n a l C l ~ c o n c e n t r a t i o n of 84 mM. F i f t e e n minutes a f t e r adding the i s o t o p e f o u r 1 u l samples were withdrawn from the l a b e l l e d s i d e and each was added to 1 ml. o f d i s t i l l e d water i n a s t a i n l e s s 36 — s t e e l p l a n c h e t t e . S p e c i f i c a c t i v i t y of C l on the l a b e l l e d s i d e o f the membrane was c a l c u l a t e d from these r e a d i n g s . At the same time and every 15 m. t h e r e a f t e r u n t i l the end of the f o u r t h hour a 500 u l sample was removed from the i n i t i a l l y -20-u n l a b e l l e d s i d e and t r a n s f e r r e d to another p l a n c h e t t e . The p l a n c h e t t e s were then evaporated to dryness under an i n f r a r e d lamp and counted t o 10,000 counts w i t h a "Nuclear Chicago" Gas Flow De t e c t o r , Model 470 coupled to an Automatic Plan c h e t Sample Changer, Model 1042 and a S e r i e s 8703 Decade S c a l e r and L i s t e r . A t o t a l of 4 ml. (20%) o f the b a t h i n g s o l u t i o n was withdrawn d u r i n g each experiment. Since t h i s s o l u t i o n was not r e p l a c e d , a g r a d u a l l y i n c r e a s i n g h y d r o s t a t i c p r e s s u r e g r a d i e n t from the l a b e l l e d to the i n i t i a l l y u n l a b e l l e d s i d e developed. T h i s g r a d i e n t never amounted to more than 0.5 cm. of water and s i n c e i t was p r e s e n t f o r f l u x experiments i n both d i r e c t i o n s i t s i n f l u e n c e on net C l f l u x was assumed to be n e g l i g i b l e . I t probably accounts f o r the s m a l l p o s i t i v e slope o f the C l ~ i n f l u x time course i n F i g u r e 16. U n i d i r e c -t i o n a l C l f l u x e s d u r i n g each 15 minute i n t e r v a l between samples were computed from the f o l l o w i n g formula, a n . V . C adapted from Shaw (1955) where - -2 -1 J c ^ - = F l u x of C l i n uMoles cm hr 36 — a l f a 2 = R a d i o a c t i v i t y of C l i n counts/min. x ml. on s i d e 1 ( i n i t i a l l y l a b e l l e d ) and s i d e 2 ( i n i t i a l l y u n l a b e l l e d ) r e s p e c t i v e l y . -21-V = Volume of f l u i d on s i d e 2. C = C o n c e n t r a t i o n o f C l on s i d e 1. t = Time p e r i o d over which f l u x o c c u r r e d (6.25 h r ) . 2 A = Area of membrane (0.196 cm ). Due to t h e i r extreme v a r i a b i l i t y , f l u x v a l u e s f o r the f i r s t 15 minutes a f t e r adding the i s o t o p e (T = 2:15) were d i s c a r d e d . -22-RESULTS ELECTRICAL PARAMETERS AND VIABILITY OF THE IN VITRO PREPARATION Measurements of e l e c t r i c a l parameters i n d i c a t e t h a t the i n v i t r o p r e p a r a t i o n of the l o c u s t rectum used i n t h i s study s u r v i v e s f o r more than 6 hours and t h a t a s i t u a t i o n approximating a steady s t a t e e x i s t s d u r i n g the normal experimental p e r i o d ( t h i r d and f o u r t h hour) when f l u x s t u d i e s were conducted. The average PD a c r o s s the rectum (34 mV, lumen p o s i t i v e ) remains r e l a t i v e l y c onstant f o r a t l e a s t 6 hours (Figure 4) and i s i n good agreement w i t h the value ( 15 - 32 mV) observed i n v i t r o ( P h i l l i p s 1964). The i n i t i a l t r a n s - e p i t h e l i a l r e s i s t a n c e o f the open-2 c i r c u i t e d rectum was 5500 Ohms/cm. . A f t e r 2 hours the r e s i s -2 tance had i n c r e a s e d t o 6700 Ohms/cm where i t remained r e l a -t i v e l y c o nstant f o r the next three hours; t h e r e a f t e r a slow d e c l i n e was apparent (Figure 5 ) . The time course of the r e s i s t a n c e changes under SCC c o n d i t i o n s was q u i t e s i m i l a r (Figure 6). T h i s suggests t h a t the major r e s i s t i v e elements i n the r e c t a l e p i t h e l i u m are not s e n s i t i v e t o membrane PD as are axonal membranes. -2 -1 The SCC i s i n i t i a l l y h i g h (7.5 uMoles.cm .hr ), -23-F i g u r e 4. Time course of t r a n s - r e c t a l PD. In v i t r o p r e p a r a t i o n o f Herrera e t a l . (1976) . #,111 v i t r o p r e p a r a t i o n d e s c r i b e d i n t h i s study. Mean ± S.E.M. n=14-16 f o r the f i r s t 6 hours, and 2-9 t h e r e a f t e r . -25-F i g u r e 5. Time course o f t r a n s - r e c t a l r e s i s t a n c e under open c i r c u i t c o n d i t i o n s . Mean ± S.E.M. n=14-16 f o r a l l p o i n t s . I e o to o o e •P to •H to $ Pi 7000 6000 5000 4000 3000 2000 1000 I I L 3 a a Time i n hours -27-F i g u r e 6. Time course of t r a n s - r e c t a l r e s i s t a n c e under SCC c o n d i t i o n s . Mean ± S.E.M. n=3. CM I CO e <s c •H CD o c fC 4-) CO •W CO CD Pi 7000 6000 u 5000 4000 3000 2000 1000 0 I I . i_ 2 3 4 Time i n hours -29-drops r a p i d l y to a value o f 3 uMoles.cm .hr w i t h i n two hours, and t h e r e a f t e r f a l l s s l o w l y over s e v e r a l hours (Figure 7). The l a t t e r v alue f o r SCC i s w i t h i n the range expected from p r e v i o u s r e p o r t s of t o t a l t r a n s p o r t o f i o n s i n o t h e r i n v i t r o p r e p a r a t i o n s o f the l o c u s t rectum under v a r i o u s experimental c o n d i t i o n s (see D i s c u s s i o n ) . The Normal Ringer used i n some e a r l y experiments c o n t a i n e d l a c t a l b u m i n h y d r o l y s a t e and y e a s t e x t r a c t . These were omitted i n l a t e r experiments where a c h l o r i d e - f r e e Ringer's s o l u t i o n was d e s i r e d . S u r p r i s i n g l y , t h i s omission caused a s m a l l but s i g n i f i c a n t decrease i n the average SCC (Figure 7). However, these two c o n s t i t u e n t s probably con-t a i n s u b s t a n t i a l amounts o f o r g a n i c anions which i t w i l l be shown c o u l d have marked e f f e c t on SCC. I c o n s i d e r e d the p o s s i b i l i t y t h a t the sharp d e c l i n e i n SCC and the concomittant sharp r i s e i n r e s i s t a n c e d u r i n g the i n i t i a l two hour p e r i o d were a s s o c i a t e d w i t h rearrangements o f i o n i c g r a d i e n t s d u r i n g adjustments of the t i s s u e t o SCC c o n d i t i o n s , r a t h e r than a d e c l i n e i n m e t a b o l i c a c t i v i t y . A f u r t h e r p o s s i b i l i t y was t h a t the r a t e o f a v o l t a g e - s e n s i t i v e i o n t r a n s p o r t process decreased f o l l o w i n g removal o f the normal PD under SCC c o n d i t i o n s . However, the f a c t t h a t o p e n - c i r c u i t and s h o r t - c i r c u i t r e s i s t a n c e s were o f s i m i l a r magnitude and f o l l o w s i m i l a r time courses argues a g a i n s t the l a t t e r -30-F i g u r e 7. Time course of r e c t a l SCC. A , In. v i t r o p r e p a r a t i o n of H e r r e r a e t a l . (1976). In v i t r o p r e p a r a t i o n d e s c r i b e d i n t h i s study: rectum bathed i n Normal Ringer; rectum bathed i n Normal Ringer without l a c t a l b u m i n h y d r o l y s a t e and ye a s t e x t r a c t . Mean ± S.E.M.; n=8; •, n = l l . -31 -32-a l t e r n a t i v e . I f readjustments of i o n i c g r a d i e n t s i n the t i s s u e were the e x p l a n a t i o n , then the time course o f the SCC should remain the same even i f the i n v i t r o p r e p a r a t i o n i s l e f t f o r two hours i n the o p e n - c i r c u i t s t a t e b e f o r e a p p l y i n g the SCC c o n d i t i o n . F i g u r e 8 i n d i c a t e s t h a t t h i s i s not the case. The i n i t i a l SCC observed a f t e r two hours i n the o p e n - c i r c u i t s t a t e i s almost i d e n -t i c a l to the SCC a t t h a t time i n p r e p a r a t i o n s which were i n the SCC c o n d i t i o n from time zero. O b v i o u s l y the d e c l i n e i n SCC i n the i n i t i a l two hour p e r i o d i s due to a l o s s o f me t a b o l i c o r t r a n s p o r t a c t i v i t y . T h i s i s probably a consequence o f e l i m i n a t i n g the very heavy t r a c h e a l supply t o the rectum or p o s s i b l y i t r e -f l e c t s the inadequacy of Normal Ringer as a s u b s t i t u t e f o r hemo-lymph. I t i s a l s o p o s s i b l e t h a t removing the rectum from the animal e l i m i n a t e s some n a t u r a l n e u r a l o r hormonal s t i m u l a t i o n o f i o n t r a n s p o r t . To demonstrate the met a b o l i c dependence of the SCC generated by the i n v i t r o p r e p a r a t i o n , I i n v e s t i g a t e d the i n -f l u e n c e o f temperature and m e t a b o l i c i n h i b i t o r s on the SCC. The r e c t a l SCC i s markedly dependent on temperature (Figure 9 ) . Decreasing the temperature of the b a t h i n g s o l u t i o n ' from 25°C t o 10°C decreases the SCC by more than 66%. The Q 1 Q f o r the temperature range 10°-20°C was e s t i m a t e d t o be 2.5 and t h a t f o r the range 25°-35°C was 2.25. These v a l u e s o f are c o n s i s t a n t w i t h a metabolic dependence o f the SCC (Prosser, 1973). -33-F i g u r e 8 . Time course o f r e c t a l SCC a f t e r an i n i t i a l two hours under open c i r c u i t c o n d i t i o n s : Arrow i n d i c a t e s time when s h o r t c i r c u i t i n g was i n i t i a t e d . •, c o n t r o l SCC time course. Mean ± S.E.M. n= 2 . -34--35-F i g u r e 9. E f f e c t o f temperature on r e c t a l SCC i n a t y p i c a l p r e p a r a t i o n . Time i n hours -37-The rectvun was a l s o found to be very s e n s i t i v e to oxygen d e p r i v a t i o n . Because the oxygen supply t o the b a t h i n g s o l u t i o n a l s o f u n c t i o n e d as the s t i r r i n g gas, the e f f e c t s of oxygen d e p r i v a t i o n i n t h i s i n v i t r o p r e p a r a t i o n were more e a s i l y demonstrated by adding an i n h i b i t o r of o x i d a t i v e metabolism t o the b a t h i n g s o l u t i o n s . A d d i t i o n o f 1 mM KCN ( f i n a l c o n c e n t r a t i o n ) caused a r a p i d r e d u c t i o n of over 80% i n the SCC (Figure 10). C l e a r l y , most of the SCC i s f u e l e d by a e r o b i c metabolism though a sm a l l anaerobic component cannot be excluded. CHLORIDE TRANSPORT AND THE SOURCE OF THE SCC The d i r e c t i o n o f the observed SCC i n d i c a t e s e i t h e r a net t r a n s p o r t ( t o t a l c a t i o n s minus t o t a l anions) o f n e g a t i v e charges t o the hemolymph or p o s i t i v e charges to the lumen s i d e . I t i s known t h a t the l o c u s t rectum i n s i t u ( P h i l l i p s , 1964) and i n v i t r o (Goh, 1971; Goh and P h i l l i p s , i n p r e p a r a t i o n ) absorbs Na +, K +, and C l from the lumen a g a i n s t e l e c t r o c h e m i c a l g r a d i e n t s . Rapid a c i d i f i c a t i o n o f the lumen contents i n d i c a t e s e i t h e r t r a n s -p o r t o f H + i o n s to the lumen or HCO^ i o n s t o the hemolymph ( P h i l l i p s , 1961, 1965; Speight, 1968). Since C l ~ was the o n l y major i n o r g a n i c anion i n the Ringer's s o l u t i o n used i n the p r e s e n t study, t r a n s p o r t of t h i s i o n seemed a l i k e l y source of the observed SCC. I f so, the complete replacement o f C l ~ i o n s i n the b a t h i n g s o l u t i o n s by -38-F i g u r e 1 0 . E f f e c t o f 1 mM KCN on r e c t a l SCC. Arrow i n -d i c a t e s time a t which KCN was added t o the b a t h i n g media. Mean ± S.E.M. n = 4 . Dashed l i n e i n d i c a t e s c o n t r o l . 0 1 2 3 4 Time i n hours -40-other anions might be expected t o cause a r e d u c t i o n i n , or r e v e r s a l of the SCC or i t s complete disappearance i f o n l y C l i s a c t i v e l y t r a n s p o r t e d . To t h i s end, a Low c h l o r i d e SO^-Ringer s o l u t i o n was prepared i n which n e a r l y a l l the C l normally p r e s e n t was r e p l a c e d w i t h SO^ t o g i v e a f i n a l measured C l c o n c e n t r a t i o n of l e s s than 3 mM as compared to 67 mM C l i n Normal Ringer. R e c t a l p r e p a r a t i o n s were mounted i n the chambers i n Normal Ringer s o l u t i o n and a SCC was a p p l i e d i n the u s u a l manner f o r 2.25 hours. Then the b a t h i n g media were washed out and r e p l a c e d w i t h more than ten times the chamber volume o f Low c h l o r i d e SO^-Ringer's s o l u t i o n as d e s c r i b e d p r e v i o u s l y . At the s t a r t of the f i f t h hour the r e c t a were again exposed to Normal Ringer's s o l u t i o n . F i g u r e 11 i n d i c a t e s t h a t the SCC was u n a f f e c t e d by s u b s t i t u t i o n of SO^ f o r C l ~ i n the Ringer's s o l u t i o n . The r e s u l t s of t h i s experiment were unexpected s i n c e the l o c u s t rectum has been shown to t r a n s p o r t C l i n v i v o and i n v i t r o ( P h i l l i p s , 1961, 1964; Goh, 1971; P h i l l i p s and Goh, i n p r e p a r a t i o n ; Meredith and P h i l l i p s , unpublished data) and s i n c e the l a r g e SCC which I observed d u r i n g t h i s time p e r i o d i n d i c a t e d the teiability o f the p r e s e n t i n v i t r o p r e p a r a t i o n . S e v e r a l p o s s i b l e e x p l a n a t i o n s of these data occured to me: (1) Large s t o r e s of t i s s u e C l ~ might m a i n t a i n the SCC -41-F i g u r e 11. Time course of SCC d u r i n g a t y p i c a l experiment i n which r e c t a were exposed t o b a t h i n g media i n which a l l the C l ~ had been r e p l a c e d w i t h S0~. Arrows i n d i c a t e time o f exposure to SOT media and normal media r e s p e c t i v e l y . 8 r --43-f o r a c o n s i d e r a b l e time a f t e r p l a c i n g the rectum i n SO^-Ringer*s s o l u t i o n . (2) The C l " pump, i f p r e s e n t , might have a very low a f f i n i t y c onstant (K ); i . e . , w e l l below the l e v e l o f 3 mM C l " m pre s e n t i n Low-Cl SO^-Ringer's s o l u t i o n . (3) The C l pump might be a r a t h e r n o n - s p e c i f i c pump wMch can t r a n s f e r S0~ as w e l l as C l " . (4) P o s s i b l y t h i s i n v i t r o p r e p a r a t i o n does not a c t i v e l y t r a n s p o r t C l . (5) The C l pump might be t i g h t l y coupled t o N a + and K + t r a n s p o r t i n the same d i r e c t i o n such t h a t c a t i o n t r a n s p o r t stops when C l t r a n s p o r t ceases. To t e s t the f i r s t h y p o t h e s i s , t h a t t i s s u e C l " might s u s t a i n the SCC, the l e v e l o f t h i s anion i n r e c t a f r e s h l y d i s s e c t e d from the l o c u s t was measured and found t o be 1± 0.04 uMoles rectum. When r e c t a were mounted f o r v a r i o u s l e n g t h s o f time i n C l " f r e e SO^-Ringer's s o l u t i o n i t was found t h a t more than 90% of the t i s s u e C l was l o s t w i t h i n the f i r s t two hours (Figure 12). C l e a r l y , t i s s u e C l c o u l d not be m a i n t a i n i n g the SCC d u r i n g the 3rd t o 5th hour i n F i g u r e 11. Moreover the rectum pumps more C l acro s s i t s e l f i n one hour than i s presen t i n the whole o f the r e c t a l e p i t h e l i u m (see below). The second p o s s i b i l i t y , t h a t a C l pump i n the r e c t a l e p i t h e l i u m might have a K m value w e l l below 3 mM was t e s t e d by p r e p a r i n g a " C l - f r e e " SO^-Ringer 1s s o l u t i o n from which l a c t a l b u m i n h y d r o l y s a t e and ye a s t e x t r a c t were omitted -44-F i g u r e 12. C l e f f l u x from r e c t a incubated f o r v a r y i n g times i n SO^ -Ringer. Mean ± S.E.M. S.E.M. f o r 2nd and 4th hours was s m a l l e r than the p o i n t . n=10. -46-because they c o n t a i n e d some C l i o n s . Sucrose was added t o maintain the same osmotic p r e s s u r e . The c h l o r i d e c o n c e n t r a t i o n of t h i s SO~-Ringer's s o l u t i o n was l e s s than 0.1 mM ( i . e . , below d e t e c t a b l e l i m i t s ) . Recta were bathed i n t h i s C l ~ - f r e e Ringer's s o l u t i o n both d u r i n g d i s s e c t i o n and subsequently d u r i n g SCC experiments. The SCC d u r i n g the t h i r d and f o u r t h hours (Figure 13) was i n d i s t i n g u i s h a b l e from t h a t observed i n p r e v i o u s experiments (Figure 11). C l e a r l y , a low K m value f o r a c h l o r i d e pump does not e x p l a i n the r e s u l t s i n F i g u r e 11. However, one s u r p r i s i n g d i f f e r e n c e between the two experiments (Figures 11 and 13) was observed. The i n i t i a l l a r g e d e c l i n e i n SCC d u r i n g the f i r s t two hours i n Normal Ringer d i d not occur when p r e p a r a t i o n s were bathed i n SO~-Ringer. Rather a v a l u e -2 -1 of 2.6 uMoles.cm *hr was a t t a i n e d w i t h i n 0.5 hours and was maintained f o r a t l e a s t another 3.5 hours. The t h i r d p o s s i b i l i t y which might e x p l a i n the r e s u l t s i n F i g u r e 11 was t h a t a C l c a r r i e r a l s o t r a n s p o r t e d SO^. T h i s has been suggested f o r mammalian r e d b l o o d c e l l s (Gunn, 1972). The e f f e c t s of r e p l a c i n g the C l " i n Normal Ringer w i t h o t h e r anions on SCC was t h e r e f o r e t e s t e d . The magnitude and d i r e c t i o n of the SCC a c r o s s r e c t a exposed to NO-j-Ringer (Figure 14) were almost i d e n t i c a l t o those observed w i t h SO^-Ringer (Figure 13). S i m i l a r experiments w i t h Acetate Ringer's s o l u t i o n produced very i n t e r e s t i n g and unexpected r e s u l t s (Figure 15). The i n i t i a l SCC -47-F i g u r e 13. Time course of r e c t a l SCC i n S0 4 Ringer. Mean ± S.E.M. n=10. Dashed l i n e i n d i c a t e s c o n t r o l . - 4 8 --49-F i g u r e 14. Time course of r e c t a l SCC i n NO^ Ringer. Mean ± S.E.M., n=6. Dashed l i n e i n d i c a t e s c o n t r o l . -51-F i g u r e 15. Time course o f r e c t a l SCC i n Acet a t e Ringer. Mean ± S.E.M., n=6. Dashed l i n e i n d i c a t e s c o n t r o l . -53-i n A c e t a t e Ringer (6 uMoles .cm . hr ) was s i m i l a r t o t h a t i n i t i a l l y observed f o r r e c t a bathed i n Normal Ringer (Figure 7). However the subsequent r a p i d d e c l i n e which f o l l o w e d i n r e c t a bathed i n Normal Ringer was not observed. A f t e r a s m a l l drop w i t h i n the f i r s t hour, the SCC i n c r e a s e d g r a d u a l l y to the i n i t i a l v a lue by the t h i r d hour and d e c l i n e d s l o w l y t h e r e a f t e r . I n d i v i d u a l p r e p a r a t i o n s a l l showed t h i s p a t t e r n . On the average the SCC d u r i n g the t h i r d and f o u r t h hours was two t o t h r e e times h i g h e r f o r r e c t a bathed i n A c e t a t e Ringer than f o r those bathed i n Normal, NO^, o£ SO^-Ringer's s o l u t i o n s . P o s s i b l e e x p l a n a t i o n f o r t h i s s t i m u l a t i o n o f SCC by a c e t a t e are con-s i d e r e d i n the D i s c u s s i o n . C l e a r l y none of these experiments i n which C l ~ i s r e p l a c e d w i t h a c e t a t e , NO^, or SO^ f u l f i l l the p r e -d i c t i o n t h a t removal of C l from the b a t h i n g media should lower the SCC d u r i n g the t h i r d and f o u r t h hours. N e i t h e r do they ex-clude the p o s s i b i l i t y of a g e n e r a l anion pump which can t r a n s f e r - - = 35 = C l , NO^, SO^, or a c e t a t e . T h e r e f o r e the net f l u x o f SO^ was measured and found to be very low (Spring, unpublished r e s u l t s ) . C l e a r l y the SCC across r e c t a d u r i n g the t h i r d and f o u r t h hours i n SO~-Ringer's s o l u t i o n (Figure 11 and 13) was not maintained by a s u b s t i t u t i o n o f SO^ f o r C l t r a n s p o r t . These r e s u l t s l e d me t o q u e s t i o n whether s i g n i f i c a n t C l ~ t r a n s p o r t o c c u r r e d i n t h i s i n v i t r o p r e p a r a t i o n ( f o u r t h p o s s i b i l i t y ) , even though i t has been demonstrated both i n v i v d -54-( P h i l l i p s 1964) and i n another type o f i n v i t r o p r e p a r a t i o n (Goh, 1971; Meredith & P h i l l i p s , unpublished d a t a ) . Loss o f C l t r a n s p o r t i n i n v i t r o p r e p a r a t i o n s has been r e p o r t e d f o r f r o g s k i n ( H a r r i s , 1960) and i n another i n v i t r o p r e p a r a t i o n of the l o c u s t rectum ( I r v i n e & P h i l l i p s , 1971). Since f u r t h e r anion s u b s t i t u t i o n s t u d i e s seemed u n l i k e l y to answer t h i s 36 — q u e s t i o n I turned to f l u x s t u d i e s u s i n g C l under SCC con-d i t i o n s t o determine i f C l i s a c t u a l l y t r a n s p o r t e d i n t h i s i n v i t r o p r e p a r a t i o n . CHLORIDE FLUX STUDIES UNDER SCC CONDITIONS Recta were bathed i n Normal Ringer under SCC con-36 — d i t i o n s and u n i d i r e c t i o n a l f l u x e s o f C l were measured d u r i n g the t h i r d and f o u r t h hours i n v i t r o by adding the i s o t o p e t o one s i d e a t the end o f the second hour and o b s e r v i n g the subsequent r a t e a t which a c t i v i t y appeared on the o p p o s i t e s i d e . Mean u n i -d i r e c t i o n a l C l ~ f l u x e s and the mean simultaneous SCC are shown i n F i g u r e 16. Under SCC c o n d i t i o n s the mean hemocoel to lumen C l " f l u x ( i n f l u x ) remained r e l a t i v e l y c onstant throughout the course of the f l u x measurement experiments a t about 0.6 uMoles. - 2 - 1 -cm . hr The mean lumen to hemocoel C l f l u x ( e f f l u x ) d u r i n g the same time p e r i o d d e c l i n e d d u r i n g the course of the e x p e r i -- 2 ments from an i n i t i a l value (at T = 2:30) of 2.34 uMoles . cm -55-F i g u r e 16. U n i d i r e c t i o n a l C l f l u x e s measured wi t h J b C l and simultaneous mean SCC. Mean ± S.E.M., n=9 f o r both i n f l u x i n t o the r e c t a l lumen and e f f l u x from the lumen. SCC; •, e f f l u x ; •, i n f l u x . -57-hr 1 t o a f i n a l v alue o f 1.95 uMoles.cm '.hr ^ c l o s e l y p a r a l l e l -i n g the d e c l i n e i n SCC d u r i n g the same time p e r i o d . S u b t r a c t i n g these two u n i - d i r e c t i o n a l f l u x e s (Figure 17), an i n i t i a l (T = 2:30) net a c t i v e t r a n s p o r t of C l from the r e c t a l lumen to -2 -1 . the hemocoel of 1.9 uMoles.cm .hr xs i n d i c a t e d . By the end o f the f l u x experiments (T = 4:00) t h i s a c t i v e C l t r a n s p o r t has -2 -1 decreased to 1.25 uMoles.cm .hr , c l o s e l y p a r a l l e l t o the decay i n SCC d u r i n g t h i s time p e r i o d . The i n i t i a l v a lue (T = 2:30) f o r net C l t r a n s p o r t i s e q u i v a l e n t t o about 60% o f the simultaneous SCC and i s 110-220% h i g h e r than p r e v i o u s l y r e p o r t e d v a l u e s ( P h i l l i p s , 1964; Goh, 1971; Meredith and P h i l l i p s , u npublished r e s u l t s ) f o r C l t r a n s p o r t by the r e c t a l e p i t h e l i u m under open c i r c u i t c o n d i t i o n s where net a b s o r p t i o n i s l e s s than t r u e t r a n s p o r t due t o back d i f f u s i o n (see D i s c u s s i o n ) . The o n l y p o s s i b l e c o n c l u s i o n from these data seems to be t h a t C l f l u x i n v i t r o i s t i g h t l y coupled to the t r a n s p o r t o f some o t h e r i o n o r i o n s whose charge i s such as to e l e c t r i c a l l y c a n c e l the c h l o r i d e f l u x . T h i s might be achieved by anion exchange or c o - t r a n s p o r t o f c a t i o n s such as Na + o r K +. Since p r e v i o u s l y observed t r a n s p o r t of Na + or K + across the l o c u s t rectum i s i n the wrong d i r e c t i o n to e x p l a i n the observed SCC and s i n c e the SCC p e r s i s t s i n the absence o f C l , the whole o f the SCC remains unaccounted f o r by the t r a n s p o r t of these three i o n s . Amino a c i d s are known to be a c t i v e l y absorbed by the i n v i t r o l o c u s t -58-F i g u r e 17. Net C l f l u x measured w i t h C l c a l c u l a t e d from the date o f F i g u r e 16 and simultaneous SCC. Mean ± S.E.M., n=9 f o r C l " f l u x s t u d i e s and n=18 f o r SCC. SCC; • , net C l " f l u x . 8 . 0 6.0 -60-rectura ( B a l s h i n , 1973; B a l s h i n and P h i l l i p s , 1971) but t h e i r t o t a l c o n c e n t r a t i o n i n the b a t h i n g media i s too low to con-t r i b u t e s i g n i f i c a n t l y t o the SCC. The o n l y o t h e r ion- known to be t r a n s p o r t e d i n any s i g n i f i c a n t q u a n t i t y by the rectum i s hydrogen and/or b i c a r b o n a t e . T h e i r t r a n s p o r t i s i n the c o r r e c t d i r e c t i o n t o account f o r the observed SCC. -61-DISCUSSION To be u s e f u l , the a c t i v i t y o f an i n v i t r o p r e p a r a t i o n should approximate a s t e a d y - s t a t e o f s u f f i c i e n t d u r a t i o n t o p er-mit the r e q u i r e d experimentation and the p r o p e r t i e s of the p r e -p a r a t i o n should be r e l a t i v e l y c l o s e to those i n vivo,/ The r e c -t a l p r e p a r a t i o n used i n t h i s study meets these c r i t e r i a , as i n d i c a t e d by measurements f o r PD, SCC and membrane r e s i s t a n c e . PD and r e s i s t a n c e change very l i t t l e over a 6 h r . p e r i o d . The SCC d e c l i n e s o n l y s l o w l y between the 2nd and 6th h r . The s i z e o f the c u r r e n t over t h i s p e r i o d i s 4x g r e a t e r than p r e d i c t e d from + + — + e stimates of Na , K , C l and H t r a n s p o r t by the l o c u s t rectum i n v i v o ( P h i l l i p s , 1964; i . e . net charge t r a n s f e r c a l c u l a t e d as the d i f f e r e n c e between t o t a l anion and t o t a l c a t i o n t r a n s f e r ) . However, P h i l l i p s (1961) p o i n t e d out t h a t h i s value f o r H + t r a n s p o r t was an u n d e r - e s t i m a t i o n s i n c e i t was made from measure-ments o f l a r g e pH change. I f the more acc u r a t e measurement o f the i n i t i a l r a t e o f pH change by Speight (1968) i s used i n the c a l c u l a t i o n s , the SCC p r e d i c t e d from i n v i v o measurements -2 -1 (2.16 uMoles.cm .hr ) i s almost i d e n t i c a l t o the value observed i n v i t r o i n t h i s study d u r i n g the 2nd to 4th h r . I t f o l l o w s t h a t the i n i t i a l SCC i s 4x g r e a t e r i n v i t r o than p r e d i c t e d from p r e v i o u s i n v i v o s t u d i e s . However, the i n v i v o v a l u e s which are a v a i l a b l e are a l l f o r net a b s o r p t i o n under open c i r c u i t c o n d i t i o n s , i . e . -62-when both e l e c t r i c a l p o t e n t i a l and c o n c e n t r a t i o n d i f f e r e n c e e x i s t a c r o s s the r e c t a l w a l l . Under these c o n d i t i o n s net i o n a b s o r p t i o n i s d i f f e r e n t from t r u e a c t i v e t r a n s p o r t due to d i f f u s i o n o f i o n s down e l e c t r o c h e m i c a l p o t e n t i a l g r a d i e n t s . Since the r e c t a l lumen i s e l e c t r i c a l l y p o s i t i v e to the hemocoel i n v i v o , which would favour some a b s o r p t i o n o f Na + and K + by net d i f f u s i o n , the t r u e a c t i v e t r a n s p o r t o f these c a t i o n s would be somewhat l e s s than net a b s o r p t i o n v a l u e s . Conversely, the t r u e a c t i v e t r a n s p o r t o f c h l o r i d e , which i s normally opposed by an e l e c t r i c a l p o t e n t i a l d i f f e r e n c e , would be somewhat g r e a t e r than net a b s o r p t i o n of t h i s anion as measured i n v i v o . The PD under open c i r c u i t c o n d i t i o n s would a l s o tend t o decrease b i c a r b o n a t e f l u x from the lumen t o the hemocoel and Hydrogen f l u x from the hemocoel t o the lumen. In summary, i f allowance i s made f o r the PD observed i n v i v o , then the t r u e r a t e o f net charge t r a n s f e r due to t r a n s p o r t o f Na +, K +, C l ~ and H + o r HCO^ i n v i v o would be somewhat h i g h e r than i n d i c a t e d by the data on net a b s o r p t i o n by P h i l l i p s (1961, 1964) and Speight (1968). In s h o r t , the SCC produced by my in v i t r o p r e p a r a t i o n o f the l o c u s t rectum i s i n reasonable agree-ment w i t h t h a t p r e d i c t e d from s t u d i e s o f i o n a b s o r p t i o n i n v i v o , s u g g e s t i n g t h a t most of the i o n t r a n s p o r t a c t i v i t y i s r e t a i n e d i n v i t r o . Measurements of net f l u x i n v i t r o p r o v i d e d i r e c t evidence t h a t t h i s i s indeed the case f o r c h l o r i d e (see p. 57). -63 -The time course of the r e c t a l SCC i n v i t r o (Figure 8) i n d i c a t e s a t l e a s t two components; a r a p i d d e c l i n e d u r i n g the f i r s t two hours which i s followed-by the much slower d e c l i n e t h e r e a f t e r . The second phase might r e f l e c t a slow d e c l i n e i n a g e n e r a l v i a b i l i t y o f the t i s s u e . I t i s more i n t e r e s t i n g t o s p e c u l a t e on the nature o f the f i r s t phase. Some evidence e x i s t s t h a t t h i s i n i t i a l r a p i d d e c l i n e i n SCC i s a s s o c i a t e d w i t h a de-crease i n c h l o r i d e t r a n s p o r t . As demonstrated by F i g u r e s 14 and 15, the absence o f c h l o r i d e from the b a t h i n g media d u r i n g the i n i t i a l two hour p e r i o d a b o l i s h e s the i n i t i a l r a p i d d e c l i n e i n SCC. H e r r e r a e t a l . (19 76) a l s o observed t h a t replacement o f c h l o r i d e w i t h sulphate d u r i n g the f i r s t hour i n v i t r o causes a l a r g e d e c l i n e i n SCC i n d i c a t i n g t h a t most o f the SCC d u r i n g t h i s p e r i o d i s c a r r i e d by c h l o r i d e t r a n s p o r t . T h i s does not neces-s a r i l y c o n t r a d i c t my c o n c l u s i o n t h a t c h l o r i d e c a r r i e s l i t t l e o r 36 — none o f the SCC, s i n c e the s t u d i e s o f C l f l u x i n the p r e s e n t study were performed d u r i n g the t h i r d and f o u r t h hours, by which time the i n i t i a l r a p i d d e c l i n e i n SCC had ended. The q u e s t i o n o f what a c t i v e t r a n s p o r t a c t i v i t i e s a c t u a l l y d e c l i n e to produce t h i s r a p i d i n i t i a l decrease i n SCC remains unresolved. E x t e n s i o n of f l u x s t u d i e s to t h i s p e r i o d would be u s e f u l . I t i s c l e a r however from F i g u r e s 14 and 15 t h a t though a l a r g e p a r t o f the SCC d u r i n g the f i r s t two hours may be c a r r i e d by c h l o r i d e t r a n s p o r t , a l a r g e p a r t ( i n i t i a l l y 50%) d e f i n i t e l y i s not. -64-Th i s o b s e r v a t i o n i s c o n t r a r y t o the r e s u l t s o f H e r r e r a e t a l . (1976). The s t i m u l a t i o n o f r e c t a l SCC by a c e t a t e (Figure 16) i s o b v i o u s l y a complex phenomenon. The sharp d e c l i n e i n SCC d u r i n g the f i r s t 15 minutes i n v i t r o i s very r e m i n i s c e n t o f the s i m i l a r d e c l i n e o f the c u r r e n t f o r r e c t a bathed i n the SOT and NO, 4 3 Ringers (Figures 14 and 15). The obvious e x p l a n a t i o n i s t h a t some i o n t r a n s p o r t p r o c e s s o t h e r than t h a t o f c h l o r i d e decays f o l l o w -i n g removal o f r e c t a from the l o c u s t . T h i s i n i t i a l d e c l i n e appears t o be a r r e s t e d by some process t h a t tends to i n c r e a s e the SCC i n the presence o f a c e t a t e . T h i s may r e f l e c t the time r e q u i r e d f o r ac e t a t e t o en t e r the t i s s u e . Any one or more o f the f o l l o w i n g hypotheses c o u l d account f o r t h i s subsequent and gradu a l i n c r e a s e i n SCC: (1) Acetate i s the anion o f a weak a c i d and i t s presence i n h i g h c o n c e n t r a t i o n s i n the t i s s u e might pr o v i d e a source o f H +, r e s u l t i n g i n s t i m u l a t i o n o f H + t r a n s p o r t i n t o the lumen. (2) S i n c e a c e t a t e i s r e a d i l y m e t a b o l i z e d i n many animal t i s s u e s , i t might e n t e r the r e c t a l e p i t h e l i u m and pr o v i d e a favo u r a b l e energy source t o s t i m u l a t e a more vi g o r o u s a c t i v e t r a n s p o r t o f oth e r i o n s . (3) Acetate might i t s e l f be t r a n s -p o r t e d a c r o s s the r e c t a l e p i t h e l i u m i n t o the hemocoel. T h i s would i n c r e a s e SCC. The d e c l i n e i n SCC a f t e r the t h i r d hour (Figure 16) may r e f l e c t a d e c l i n e o f g e n e r a l t i s s u e a c t i v i t y w i t h time. Regardless -65-o f the e x p l a n a t i o n o f the time course, the experiments wi t h a c e t a t e Ringer again demonstrate t h a t much o r most o f the SCC i s not n e c e s s a r i l y due to t r a n s p o r t o f C l " . Moreover, a c e t a t e Ringer s u s t a i n s SCC c u r r e n t a t near the i n i t i a l v a l u e , about 4x h i g h e r than observed w i t h o t h e r Ringers d u r i n g the 2nd to the 4th hr. T h i s again r e f u t e s the c o n c l u s i o n o f H e r r e r a e t a l . t h a t C l t r a n s p o r t accounts f o r most o f the SCC i n l o c u s t rectum. These o b s e r v a t i o n s might l e a d one to q u e s t i o n the c a p a c i t y o f the p r e s e n t r e c t a l p r e p a r a t i o n to s u s t a i n C l " t r a n s -p o r t . However, net f l u x o f C l d u r i n g the 2nd to the 4th h r . -2 -1 (Figure 17) i n d i c a t e s a t r a n s p o r t r a t e o f 1.52 uMoles.cm .hr , a value g r e a t e r than was p r e v i o u s l y measured both i n v i v o and i n v i t r o ( P h i l l i p s , 1961, 1964; Goh, 1971; Meredith and P h i l l i p s , unpublished data; H e r r e r a e t a l . , 1976). T h i s c h l o r i d e t r a n s p o r t (at a r a t e e q u i v a l e n t to one t h i r d o f the SCC) i s i n the r i g h t d i r e c t i o n t o c o n t r i b u t e to the SCC, and y e t absence o f c h l o r i d e does not change the SCC over the 2nd to the 4th h r . A necessary c o n c l u s i o n from these f a c t s i s t h a t a t l e a s t two o t h e r i o n s are a c t i v e l y t r a n s p o r t e d by my i n v i t r o p r e p a r a t i o n ; one o r more ion s coupled to the c h l o r i d e f l u x t o c a n c e l i t s e l e c t r i c a l charge, and one or more io n s to account f o r the SCC. T i g h t coup-l i n g of c a t i o n and c h l o r i d e a b s o r p t i o n i s one e x p l a n a t i o n . How-ever, Goh (1971) and Meredith and P h i l l i p s (unpublished data) -66-concluded t h a t c h l o r i d e t r a n s p o r t was independent from sodium and potassium t r a n s p o r t under open c i r c u i t c o n d i t i o n s i s another i n v i t r o p r e p a r a t i o n o f the rectum, s i n c e c h l o r i d e was s t i l l t r a n s p o r t e d a g a i n s t an e l e c t r o c h e m i c a l p o t e n t i a l d i f f e r e n c e when sodium and potassium i n the b a t h i n g Ringer were r e p l a c e d w i t h c h o l i n e . Since there are no monovalent c a t i o n s i n the b a t h i n g media t h a t c o u l d be c o - t r a n s p o r t e d with c h l o r i d e t o n e u t r a l i z e i t s charge (except p o s s i b l y c h o l i n e ) , i t seems more reasonable to p o s t u l a t e an anion exchange pump, most l i k e l y i n v o l v i n g HCO^, t o account f o r the e x i s t i n g data. A second e x p l a n a t i o n i s t h a t C l ~ and HCO^ share a common t r a n s p o r t process and t h a t i n the absence of C l ~ , enhanced movement of HCO^ occurs from the lumen t o the hemocoel s i d e . Goh (19 71) a l s o concluded t h a t a c h l o r i d e pump i s l o c a t e d on e i t h e r o r both the a p i c a l membrane and the membrane f a c i n g the i n t e r c e l l u l a r channels o f the r e c t a l e p i t h e l i u m . The i n t e r i o r o f e p i t h e l i a l c e l l s i s neg a t i v e t o the lumen ( P h i l l i p s , 1961; V i e t i n g h o f f e t a l . , 1969) so t h a t a c t i v e t r a n s p o r t o f C l ~ may be r e q u i r e d a c r o s s the a p i c a l membrane f o r t h i s anion to en t e r the c e l l . I t i s c o n s i s t e n t w i t h the p r e v i o u s l y mentioned data t o propose t h a t a C l "HCO^ exchange pump i s l o c a t e d a t t h i s a p i c a l membrane i n s t e a d o f a simple C l pump. Goh (19 71) proposed t h a t a c h l o r i d e pump i s l o c a t e d on the membrane f a c i n g the i n t e r -c e l l u l a r channels as w e l l because when the rectum was inc u b a t e d -67-i n v i t r o i n c h o l i n e Ringer (Na + and K + f r e e ) , not o n l y d i d the t i s s u e not s w e l l as i t should i f a C l pump were l o c a t e d on the a p i c a l membrane o n l y , but the f i n a l t i s s u e C l c o n c e n t r a t i o n was lower than t h a t o f the b a t h i n g media. I t i s p o s s i b l e t h a t t h i s pump a l s o t r a n s p o r t s t i s s u e HCO3 i n t o the i n t e r c e l l u l a r channels o r e l s e t h a t a separate HC0 3 pump i s l o c a t e d on the same membrane. T h i s h y p o t h e s i s would e x p l a i n the anion d e f i c i t noted by Meredith and P h i l l i p s (unpublished data) i n absorbate c o l l e c t e d from t h e i r i n v i t r o p r e p a r a t i o n s o f the l o c u s t rectum. They found t h a t the t o t a l c a t i o n c o n c e n t r a t i o n o f the absorbate e n t e r i n g the hemocoel was 146 mM wh i l e the c h l o r i d e c o n c e n t r a t i o n was o n l y 109 mM. The d i f f e r e n c e o f 37 mM o f anion c o u l d be accounted f o r by HCO^ t r a n s p o r t i n t o the hemocoel. T h i s i s i n the proper d i r e c t i o n t o c o n t r i b u t e t o the SCC. F i n a l l y by analogy t o the v e r t e b r a t e kidney and g a s t r i c mucosa H + s e c r e t i o n i n t o the lumen should y i e l d c e l l u l a r HCO^ which i s a v a i l a b l e f o r t r a n s p o r t t o the blo o d s i d e . Though the hypothesis o f a C l -HCO-j exchange pump on the a p i c a l membrane accounts f o r the l a c k o f e f f e c t o f C l removal on the SCC d u r i n g the t h i r d and f o u r t h hours i n v i t r o , i t seems i n -c o n s i s t e n t w i t h the f a c t t h a t the rectum q u i c k l y a c i d i f i e s any s o l u t i o n i n j e c t e d i n t o i t s lumen ( P h i l l i p s , 1961) and a l s o w i t h my e a r l i e r c o n c l u s i o n t h a t the m a j o r i t y o f the SCC d u r i n g t h i s p e r i o d i n v i t r o i s c a r r i e d by H + o f HCO^ t r a n s p o r t . However. -68-s i n c e the r a t e o f c h l o r i d e t r a n s p o r t i s probably much lower than the H + t r a n s p o r t , the r e q u i r e d movement o f HCO^ i n t o the lumen by an exchange pump would on l y s l i g h t l y decrease net H + s e c r e t i o n , so t h a t the lumen content c o u l d s t i l l be a c i d i f i e d , as observed by P h i l l i p s (1961) and Speight (1968). These idea s on C l , H + and HCO^ t r a n s p o r t have been i n c o r p o r a t e d i n t o a model (Figure 18) f o r o r g a n i z a t i o n o f i o n t r a n s p o r t processes i n the l o c u s t rectum, which i s m o d i f i e d from t h a t o f Goh (1971). Goh (1971) proposes a c a t i o n pump w i t h a much hi g h e r a f f i n i t y f o r Na + than K + l o c a t e d on the membrane f a c i n g the i n t e r c e l l u l a r channels, t r a n s p o r t i n g c a t i o n s i n t o these channels. The a p i c a l membrane i s very permeable to K + and i t i s l i k e l y t h a t a t y p i c a l N a + - K + ATPase maintains the l a r g e c o n c e n t r a t i o n d i f f e r e n c e s which can e x i s t a c r o s s t h i s membrane. Furthermore, s i n c e the rectum produces hyposmotic absorbate, i o n r e a b s o r p t i o n i s p o s t u l a t e d i n the membrane f a c i n g the i n t e r -c e l l u l a r channel. While t h i s t h e s i s was i n p r e p a r a t i o n , H e r r e r a , Jordana and Ponz (1976) d e s c r i b e d the e f f e c t s o f v a r i o u s i o n s u b s t i t u t i o n s ©n the PD and SCC i n an i n v i t r o p r e p a r a t i o n o f the l o c u s t rectum. As r e f e r r e d t o above, t h e i r c o n c l u s i o n s do not agree w i t h those o f t h i s study. I b e l i e v e t h i s i s because these workers used an inadequate i n v i t r o p r e p a r a t i o n , imprecise e l e c t r i c a l methods, and observed o n l y t r a n s i e n t events over the f i r s t 40 minutes -69-F i g u r e 18. Model o f i o n t r a n s p o r t processes i n the l o c u s t rectum. See t e x t f o r e x p l a n a t i o n . -70-LUMEN C 0 2 + H 2 0 - ^ H + + H C O ; c r . K ^ N a HEMOCOEL -71-i n v i t r o . Over t h i s p e r i o d the PD and SCC o f t h e i r c o n t r o l p r e p a r a t i o n f e l l t o zero ( F i g u r e s 5 and 8). Toward the end o f t h i s p e r i o d , when t i s s u e a c t i v i t y was reduced t o a f r a c t i o n o f the i n i t i a l v a l u e , exposure o f r e c t a l p r e p a r a t i o n s t o c h l o r i d e -f r e e Ringer a b o l i s h e d the remaining SCC but oth e r i o n sub-s t i t u t i o n s d i d not. These workers concluded t h a t C l t r a n s p o r t accounted f o r a l l the SCC and t r a n s p o r t o f oth e r i o n was neg-l i g i b l e . Three major d i f f e r e n c e s i n the experimental method used by Her r e r a e t a l . (19 76), as compared t o the pr e s e n t study, c o u l d e x p l a i n the f a i l u r e o f t h e i r i n v i t r o p r e p a r a t i o n , as i n d i c a t e d by a r a p i d disappearance o f PD and SCC: (1) These workers bathed t h e i r p r e p a r a t i o n s i n simple Ringer, whereas I used the complex t i s s u e c u l t u r e media of B e r r i d g e , which may s u s t a i n r e c t a l metabolism much b e t t e r . (2) Good oxygenation i s c r i t i c a l t o s u r v i v a l o f i n -s e c t e p i t h e l i a i n v i t r o when t r a c h e a l connections are broken (Maddrell, 1971; Bradley, 1976). H e r r e r a e t a l . (1976) used cannulated sacs s t i r r e d by b u b b l i n g on the o u t s i d e o n l y . I used the r e c t a mounted as a f l a t sheet, w i t h vigorous s t i r r i n g and oxygenation on both s i d e s by a method t h a t minimized u n s t i r -r e d l a y e r s . (3) D i s s e c t i o n o r mounting o f the cannulated r e c t a by l i g a t i o n may have damaged the t i s s u e (eg. edge damage by l i g a t i o n ) . -72-The method used t o mount the rectum i n the pr e s e n t study avoided a p p l i c a t i o n o f pressure which i s known to cause edge damage i n othe r e p i t h e l i a . (Dobson and Kidder, 1968). I a l s o q u e s t i o n c o n c l u s i o n s based on t r a n s i e n t events immediately f o l l o w i n g d i s s e c t i o n o f r e c t a from l o c u s t s . The t i s s u e i s normally exposed t o a very hyperosmotic content w i t h i n the lumen and then a b r u p t l y exposed t o an i s o s m o t i c Ringer o f d i f f e r e n t com-p o s i t i o n i n v i t r o . Higher t i s s u e c o n c e n t r a t i o n s o f ions and oth e r s o l u t e s a s s o c i a t e d w i t h the p r e v i o u s hyperosmotic s t a t e may s u s t a i n p a r t i c u l a r t r a n s p o r t a c t i v i t i e s t e m p o r a r i l y a t unusual r a t e s . Moreover, the t i s s u e may a d j u s t c e r t a i n t r a n s p o r t processes t o achieve some volume r e g u l a t i o n f o l l o w i n g exposure t o the more d i l u t e Ringer. F i n a l l y , as F i g u r e 19 i n d i c a t e s , the g e o m e t r i c a l arrangement o f e l e c t r o d e s used by H e r r e r a e t a l . (1976) to measure v o l t a g e and c u r r e n t makes s u b s t a n t i a l e r r o r s i n mea-surement o f PD and SCC almost i n e v i t a b l e . In p a r t i c u l a r , c u r -r e n t d e n s i t y i s not uniform across the r e c t a l w a l l and c o r r e c -t i o n f o r v o l t a g e drop through the s o l u t i o n i s not i n d i c a t e d . Both f a c t o r s i n t r o d u c e an e r r o r o f unknown magnitude i n t o t h e i r estimate o f SCC. Furthermore, the l a s t h a l f o f t h e i r paper c i t e d ex-periments i n which r e c t a l SCC was measured wi t h s o l u t i o n s o f r a d i c a l l y d i f f e r e n t i o n i c composition on the two s i d e s o f the -73 F i g u r e 19. In v i t r o apparatus o f H e r r e r a e t a l . (1976). V, v o l t m e t e r . A, c u r r e n t meter. E v , v o l t a g e e l e c t r o d e s . E j , c u r r e n t e l e c t r o d e s . M, r e c t a l . p r e p a r a t i o n . G, s t i r r i n g gas e n t r y tube. -74--75-membrane. These c o n d i t i o n s do not s a t i s f y the p r e r e q u i s i t e s f o r measurement of SCC and consequently the data are i r r e l e v a n t . T h e i r c o n c l u s i o n t h a t C l c a r r i e s a l l o f the SCC then r e s t s en-t i r e l y on t h e i r o b s e r v a t i o n t h a t C l ~ removal reduces the SCC t o zero when p r e p a r a t i o n s are c l e a r l y near death. H e r r e r a e t a l . (1976) a l s o concluded on the b a s i s o f pH changes of unbuffered b a t h i n g media t h a t the amount o f SCC + —2 —1 c a r r i e d by H i s n e g l i g i b l e ( i . e . 0.025 uMoles.cm .hr ). T h e i r value f o r net H + f l u x i s 25 times s m a l l e r than the r a t e observed i n v i v o by P h i l l i p s (1961) and 90 times s m a l l e r than t h a t observed by Speight (1968), and i s probably not r e l i a b l e c o n s i d e r i n g t h a t any estimate o f net H + f l u x based on pH changes of a s o l u t i o n can o n l y be a minimum v a l u e . The o n l y r e l i a b l e method o f measuring net H + f l u x under these c o n d i t i o n s i s by continuous automatic t i t r a t i o n o f the l u m i n a l b a t h i n g medium with a standard base s o l u t i o n . F i n a l l y , f a i l u r e o f H e r r e r a e t a l . (1976) t o observe s u b s t a n t i a l a c i d i f i c a t i o n may simply r e -f l e c t the decreased v i a b i l i t y o f t h e i r p r e p a r a t i o n (evident from SCC v a l u e s ) . In c o n c l u s i o n then, the development o f the i n v i t r o p r e p a r a t i o n used i n the pr e s e n t study should g r e a t l y f a c i l i t a t e the study o f i o n t r a n s p o r t i n the l o c u s t rectum. The most im-p o r t a n t task i s to determine the r a t e o f Na +, K +, and H + and HCCU t r a n s p o r t under SCC c o n d i t i o n s . Only a f t e r these ques--76-t i o n s are answered can the i n t e r - r e l a t i o n s h i p s between the v a r i o u s i o n t r a n s p o r t processes be u n r a v e l l e d and the charac-t e r i s t i c s o f each mechanism s t u d i e d . -77-BIBLIOGRAPHY B a l s h i n , M. (1973). A b s o r p t i o n o f 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 c a g r e g a r i a } Ph.D. T h e s i s , U n i v e r s i t y o f B r i t i s h Columbia. B a l s h i n , M. and J.E. P h i l l i p s , (1971). A c t i v e a b s o r p t i o n o f amino a c i d s i n the rectum o f the d e s e r t l o c u s t ( S c h i s t o c e r c a g r e g a r i a ) . Nature New B i o l o g y 233: 53-55. B e r r i d g e , M.J. (1966). M e t a b o l i c pathways o f i s o l a t e d M a l p i g h i a n t u b u l e s o f the b l o w f l y f u n c t i o n i n g i n an a r t i f i c i a l medium. J . I n s e c t P h y s i o l . 12: 1523-1538. Bradley, T . J . 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A t i t r a t a b l e c a r r i e r model f o r both mono-and d i - v a l e n t anion transport-in.human r e d blo o d c e l l s . In Oxygen A f f i n i t y o f Hemoglobin and Red C e l l  A c i d Base S t a t u s , ed. Rorth, M. and P. Ast r u p pp. 823-827. Copenhagen: Munksgaard H a r r i s , E . J . (1960). T r a n s p o r t and accumulation i n b i o l o g i c a l systems. Butterworth's London. -78-Herrera, L., R. Jordana, and F. Ponz (1976). C h l o r i d e dependent transmural p o t e n t i a l i n the r e c t a l w a l l o f S c h i s t o c e r c a g r e g a r i a . J . I n s e c t P h y s i o l . 22: 291-297. Hogben, C.A.M. (1955). A c t i v e t r a n s p o r t o f c h l o r i d e by i s o l a t e d f r o g g a s t r i c e p i t h e l i u m . O r i g i n o f the g a s t r i c mucosal p o t e n t i a l . Amer. Jour . P h y s i o l . 180: 641-649. I r v i n e , H.B. and J.E. P h i l l i p s (1971). 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The r e l a t i o n between s o l v e n t drag and a c t i v e t r a n s p o r t o f i o n s . Abs. 3rd I n t e r n a t . Cong. Biochem., B r u s s e l s , p. 343. Ussing, H.H. and K. Zerahn (1951). A c t i v e t r a n s p o r t o f sodium as the source o f e l e c t r i c c u r r e n t i n the s h o r t - c i r c u i t e d i s o l a t e d f r o g s k i n . A c t a . Phys. Scandinav. 23: 110-127. V i e t i n g h o f f , U., E. Olszewska and L. Ja n i z e w s k i (1969). Measurements o f b i o e l e c t r i c p o t e n t i a l s i n the rectum o f L o c u s t a m i g r a t o r i a and Carausius  morosus i n i n v i t r o p r e p a r a t i o n s . J . I n s e c t  P h y s i o l . 15: 1273-1277. Wood, J.L. (19 72). Some aspects o f a c t i v e potassium t r a n s p o r t by the midgut o f the silkworm, Antheraea p e r n y i . Ph. D. T h e s i s , U n i v e r s i t y o f Cambridge. Wood, J.L. and W.R. Harvey (1975). A c t i v e t r a n s p o r t o f potassium by the C e c r o p i a midgut; t r a c e r k i n e t i c theory and t r a n s p o r t p o o l s i z e . J . exp. B i o l . 63: 301-311. 

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