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UBC Theses and Dissertations

Metabolic studies on the locust rectum Chamberlin, Mary Ella 1981

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J METABOLIC STUDIES ON THE LOCUST RECTUM by MARY ELLA CHAMBERLIN B.S., U n i v e r s i t y of C a l i f o r n i a , Davis, 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In THE FACULTY OF GRADUATE STUDIES (Zoology) We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA November 1981 Mary E l l a Chamberlin, 1981 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library 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 reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or pu b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Zoology  The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D a t e December 23, 1981 i i i ABSTRACT The i n v i t r o , s h o r t - c i r c u i t e d l o c u s t rectum a c t i v e l y t r a n s p o r t s c h l o r i d e . This e l e c t r o g e n i c t r a n s p o r t i s energized by aerobic metab-o l i s m , based p r i m a r i l y upon p r o l i n e o x i d a t i o n . I s o l a t e d r e c t a l mito-c h o n d r i a l o x i d i z e a v a r i e t y of s u b s t r a t e s , but the highest s t a t e 3 r a t e of 0^ consumption occurs when p r o l i n e i s the s t u b s t r a t e . An enzyme pro-f i l e of the r e c t a l t i s s u e i n d i c a t e s that the rectum can o x i d i z e amino acids and carbohydrates but has a l i m i t e d c a p a c i t y f o r l i p i d o x i d a -t i o n . A n a l y s i s of the two f l u i d s which bathe the rectum i n v i v o , the hemolymph and the M alpighian tubule f l u i d , r e v e a l s that p r o l i n e i s present i n bothethe hemolymph (12-15 mM) and tubule f l u i d (38-43 mMt). Although g l y c i n e i s a l s o found i n high concentration i n the hemolymph (13-17 mM), f a r lower concentrations of t h i s and other amino acid s are found i n the Malpighian tubule f l u i d . Glucose i s a l s o found i n the hemolymph (2 mM) and Malpighian tubule f l u i d (4 mM). The high concentraion of p r o l i n e i n the tubule f l u i d i n d i c a t e d that t h i s amino a c i d may be a c t i v e l y transported by tubules. This p r e d i c t i o n was confirmed by experiments w i t h i n v i t r o tubules. This i s the f i r s t evidence of a e t i v e s e c r e t i o n of a m e t a b o l i c a l l y u s e f u l compound by i n s e c t Malpighian tubules. Experiments i n v o l v i n g the measurement of s h o r t - c i r c u i t current (Isc) across the l o c u s t rectum were a l s o performed. The r e s u l t s of these experiments i n d i c a t e that high l e v e l s (50 mM') of p r o l i n e s t i m u l a t e the I s c of substrate-depleted r e c t a b e t t e r than high l e v e l s of any other s u b s t r a t e t e s t e d . P h y s i o l o g i c a l l e v e l s of p r o l i n e a l s o cause a l a r g e i n c r e a s e i n the I s c of substrate-depleted r e c t a , w h i l e p h y s i o l o g i c a l l e v e l s of glucose produce a much smaller s t i m u l a t i o n . Over 90% of the I s c s t i m u l a -t i o n can be produced by adding p r o l i n e (15 mM) s o l e l y to the lumen s i d e of the t i s s u e . Other s t u d i e s were performed to estimate the metabolic cost of a c t i v e l y t r a n s p o r t i n g c h l o r i d e . The oxygen consumption of ch<loride-depleted r e c t a were measured before and a f t e r r e i n t r o d u c t i o n of c h l o r i d e . From these data a CI /ATP r a t i o of 3-4 was obtained. Further c a l c u l a t i o n s i n d i c a t e d that t i s s u e p r o l i n e o x i d a t i o n i s s u f f i c i e n t to energize a c t i v e c h l o r i d e t r a n s p o r t . The r e s u l t s o u t l i n e d here suggest that i n v i v o , the: r e c t a l lumen i s bathed w i t h a high concentration of p r o l i n e which can be r e a d i l y o x i d i z e d by the r e c t a l mitochondria to support the work of a c t i v e l y t r a n s p o r t i n g c h l o r i d e . TABLE OF CONTENTS i v Chapter I Chapter I I Ab s t r a c t L i s t of Tables L i s t of F i g u r e s Acknowledgements Ab b r e v i a t i o n s General I n t r o d u c t i o n O x i d a t i v e Metabolism i n the Locust Rectum I n t r o d u c t i o n M a t e r i a l s and Methods Animals Chemicals R e c t a l Glycogen and Glucose Amino Acids i n R e c t a l Tissue I s o l a t i o n of Mitochondria Polarography M i t o c h o n d r i a l P r o t e i n Determination Tissue P r e p a r a t i o n f o r Enzyme Assays Enzyme Assays L o c a l i z a t i o n of Enzymes 14 C-pro l i n e to Conversion of W c o 2 End-products of P r o l i n e Metabolism R e s u l t s Page i i i v i i v i i i •x x i 1 7 7 8 8 8 8 9 9 9 10 10 11 12 13 16 16 Tissue Content of Amino Acids and Carbohydrates Substrate O xidation by I s o l a t e d R e c t a l Mitochondria I n h i b i t o r s Enzyme A c t i v i t i e s i n Locust Recta and F l i g h t Muscle 14 C0 2 Production from l ^ c - p r o l i n e End-products'of P r o l i n e Metabolism 16 20 24 24 28 28 V D i s c u s s i o n 28 Chapter I I I P o t e n t i a l Exogenous Substrates f o r the Locust Rectum 41 I n t r o d u c t i o n 4 i M a t e r i a l s and Methods 42 Animals 42 Pre-treatment of Dehydrated and Hydrated Animals 42 Determination of Hemolymph Volume 42 Hemolymph Amino Acids 43 C o l l e c t i o n of Malpighian Tubule F l u i d In Vivo 43 Amino A c i d S e c r e t i o n by In V i t r o Malpighian Tubules 44 Results 45 Hemolymph Volume and Free Amino Acids 45 Experiments w i t h Malpighian Tubules In Vivo 45 Experiments w i t h Malpighian Tubules In V i t r o 49 D i s c u s s i o n 57 Chapter IV Metabolic Support of the S h o r t - C i r c u i t Current Across the Locust Rectum 60 I n t r o d u c t i o n 60 M a t e r i a l s and Methods 61 Animals 61 S h o r t - C i r c u i t Current Measurements 61 S a l i n e s 61 Experimental P r o t o c o l 61 I n h i b i t o r s 64 Results 64 D i s c u s s i o n 91 Chapter V Oxygen Consumption of R e c t a l Tissue 95 I n t r o d u c t i o n 95 M a t e r i a l s and Methods 96 Animals 96 v i S a l i n e s 96 D i s s e c t i o n and Incubation of Recta 96 Oxygen Consumption Measurements 96 S t a t i s t i c s 98 Re s u l t s 98 D i s c u s s i o n 103 Chapter VI < -General. D i s c u s s i o n 107 L i t e r a t u r e C i t e d 111 Appendix A 119 Appendix B 120 - v i i Table I Table I I Table I I I Table IV Table V Table VI Table V I I Table V I I I Table IX Table X Table XI Table X I I Table X I I I Table XIV Table XV Table XVI Table XVII LIST OF TABLES R e s p i r a t o r y c h a r a c t e r i s t i c s of i s o l a t e d r e c t a l mitochondria Enzyme a c t i v i t i e s of l o c u s t f l i g h t muscle and rectum A c t i v i t i e s of enzymes i n mitochondria i s o l a t e d from l o c u s t rectum 14 14 Oxidation of C-p r o l i n e to CO^ by l o c u s t rectum and colon Page 19 23 25 Levels of ammonia and a l a n i n e i n the r e c t a l t i s s u e a f t e r p r o l i n e o x i d a t i o n Oxygen consumption of mitochondria i s o l a t e d from l o c u s t rectum and f l i g h t muscles of d i f f e r e n t i n s e c t s A c t i v i t i e s of m i t o c h o n d r i a l enzymes from l o c u s t rectum and f l i g h t muscle of the Japanese b e e t l e , P o p i l l i a j a p o n i c a Hemolymph volume of l o c u s t s i n d i f f e r e n t s t a t e s of h y d r a t i o n Concentrations of amino a c i d s i n l o c u s t hemolymph Concentrations of f r e e amino aci d s and glucose i n the hemolymph and Malpighian tubule f l u i d of l o c u s t s Free amino a c i d s i n the hemolymph of l o c u s t s before and a f t e r sham operations Free amino a c i d s i n midgut f l u i d A comparison of p r o l i n e concentrations i n Malpighian tubule f l u i d of l o c u s t s c o l l e c t e d by l i g a t i o n and c a n n u l a t i o n methods Composition of s a l i n e s used i n Chapter IV Composition of s a l i n e s used i n 0^ consumption s t u d i e s Oxygen consumption of r e c t a incubated i n d i f f e r e n t s a l i n e s Oxygen consumption before and a f t e r i o n a d d i t i o n to r e c t a pre-incubated i n d i f f e r e n t s a l i n e s 26 27 31 32 46 47 48 50 51 52 62 97 99 102 v i i i LIST OF FIGURES Page 56 Fi g u r e 1 Levels of amino a c i d s , glucose and glycogen i n the r e c t a l t i s s u e 15 Figure 2 Oxidation of 30 mM p r o l i n e by i s o l a t e d r e c t a l mitochondria 18 Fi g u r e 3 T y p i c a l , t r a c e s of m i t o c h o n d r i a l r e s p i r a t i o n i n the presence of i n h i b i t o r s 22 Fi g u r e 4 P r e v i o u s l y proposed pathways f o r p r o l i n e o x i d a t i o n i n i n s e c t f l i g h t muscle 35 Fi g u r e 5 Proposed model f o r p r o l i n e o x i d a t i o n by l o c u s t rectum 38 Fi g u r e 6 U/P r a t i o s generated by i n v i t r o Malpighian tubules exposed to 15 mM amino a c i d 54 Fi g u r e 7 U/P r a t i o s generated by i n v i t r o Malpighian tubules exposed to d i f f e r e n t p r o l i n e concentrations Figure 8 T y p i c a l t r a c e s of I s c w i t h time a f t e r d i s s e c t i o n f o r i n d i v i d u a l r e c t a 4 66 Figure 9 Mean.Isc w i t h time a f t e r d i s s e c t i o n f o r r e c t a bathed i n two d i f f e r e n t s a l i n e s 68 Fi g u r e 10 The e f f e c t s on I s c of adding i n d i v i d u a l amino a c i d s to both s i d e s of s u b s t r a t e -depleted r e c t a 71 Fi g u r e 11 The e f f e c t s on I s c of adding carbohydrates and organic a c i d s to both sides of s u b s t r a t e -depleted r e c t a 73 Figure 12 The e f f e c t s on I s c of adding d i o l e i n to both sides of substrate-depleted r e c t a 75 Figure 13 The e f f e c t s of adding p h y s i o l o g i c a l l e v e l s of p r o l i n e or glucose on the I s c of s u b s t r a t e -depleted r e c t a 77 Fi g u r e 14 T y p i c a l t r a c e s of I s c w i t h time f o r i n d i v i d u a l substrate-depleted r e c t a a f t e r a d d i t i o n of 15 mM p r o l i n e to one s i d e of the t i s s u e 81 Fi g u r e 15 The f r a c t i o n a l i n c r e a s e i n I s c of s u b s t r a t e -depleted r e c t a a f t e r a d d i t i o n of 15 mM p r o l i n e to e i t h e r the lumen or hemolymph s i d e of the t i s s u e 82 Fi g u r e 16 The e f f e c t of p r o l i n e on the I s c of s u b s t r a t e -depleted r e c t a bathed i n c h l o r i d e - f r e e . s a l i n e 84 i x Page Fi g u r e 17 The e f f e c t of p r o l i n e on the I s c of substrate-depleted r e c t a bathed i n sodium-f r e e s a l i n e 86 F i g u r e 18 The e f f e c t s of 10 mM 2-deoxy-d-glucose on the I s c of r e c t a exposed to p r o l i n e or no exogenous substrate 88 F i g u r e 19 The e f f e c t s of 10 mM aminooxyacetate on the I s c of r e c t a exposed to p r o l i n e , a l a n i n e or no exogenous substrates 90 F i g u r e 20 T y p i c a l t r a c e s of r e c t a l oxygen consump-t i o n of r e c t a bathed i n c h l o r i d e - f r e e s a l i n e 101 ACKNOWLEDGEMENTS I wish to thank John P h i l l i p s f o r p r o v i d i n g the opportunity and freedom to pursue t h i s study. I thank Peter Hochachka f o r generous use of h i s equipment and f a c i l i t i e s . I a l s o wish to thank the f o l l o w i n g people: John Hanrahan f o r h e l p f u l d i s c u s s i o n s , generous use of unpublished data and " t u r n i n g o f f the a i r " ; Kevin Strange f o r measuring t i s s u e i o n s , g i v i n g me p r i c e l e s s g i f t s and c r e a t i n g a s t i m u l a t i n g atmosphere i n the l a b ; Joan M a r t i n f o r being a source -of q u i e t support, a companion on comestible excursions and p r o v i d i n g me w i t h s h e l t e r i n the l a s t months of my stay here; Tom Mommsen f o r h i s advice during the course of the experimentation, reading p a r t s of the t h e s i s and use of unpublished data; Sam Gopaul f o r p r o v i d i n g me w i t h robust l o c u s t s ; Bev Hymes f o r proof-reading a d r a f t of the t h e s i s ; Jim B a l l a n t y n e f o r showing me how to i s o l a t e mitochondria and a l s o f o r h i s friendship-, support-and p r o v i d i n g me w i t h an e x c e l l e n t b o t t l i n c e n t i v e to f i n i s h the t h e s i s ! x i ABBREVIATIONS hr hour min minute ml m i l l i l i t e r ( s ) u l m i c r o l i t e r ( s ) mg m i l l i g r a m ( s ) mM m i l l i m o l a r nmol nanomole(s) nmeters nanometers ;uCi m i c r o c u r i e /ieq microequivalent I s c s h o r t - c i r c u i t current p c o n c e n t r a t i o n of a s o l u t e i n the hemolymph or bathing s o l u t i o n c o ncentration of a s o l u t e U i n the f l u i d secreted by the Malpighian tubules SE standard e r r o r of mean cAMP 3''-5' - c y c l i c monophosphoric a c i d ATP adenosine 5'-triphosphate ADP adenosine 5'-diphosphate AMP adenosine 5'-monophosphate EDTA ethylenediamine t e t r a a c e t i c a c i d NAD /^-nicotinamide adenine dinucedtide ( o x i d i z e d ) ? NADH /3-nicotinamide adenine d i n u c l e o t i d e (reduced) NADP @ -nicotinamide adenine d i n u c l e o t i d e d i n u c l e o t i d e phosphate (oxid i z e d ) acetyl-Co A S- a c e t y l Coenzyme A BSA bovine serum albumin DNP d i n i t r o p h e n o l FCCP t r i f l u o r o m e t h o x y c a r b o n y l -cyanide-phenylhydrazone x i i SSA s u l p h o s a l i c y l i c a c i d PCA p e r c h l o r i c a c i d 1 CHAPTER I GENERAL INTRODUCTION Two general s t r a t e g i e s have evolved f o r c e l l volume r e g u l a t i o n i n the animal kingdom. Isosmotic r e g u l a t i o n occurs i n animals such. as. molluscs:or crustaceans. These animals a l l o w the blood i o n conc e n t r a t i o n to f l u c t u a t e -during osmotic s t r e s s w h i l e the i n t r a c e l l u l a r s o l u t e s are regulated to prevent c e l l dehydration or l y s i s (reviewed by G i l l e s , 1975; 1979). A n i s -osmotic r e g u l a t i o n has been adopted by the ve r t e b r a t e s and i n s e c t s . These animals r e g u l a t e the content of the e x t r a c e l l u l a r f l u i d (plasma, hemolymph, etc.) so that the t i s s u e s do not experience wide f l u c t u a t i o n s i n t h e i r environment. Well developed excretory organs are re q u i r e d i n these groups so that t h i s r e g u l a t i o n of e x t r a c e l l u l a r f l u i d can be achieved. In v e r t -ebrates the kidney i s the major organ of osmoregulation, w h i l e i n i n s e c t s s i m i l a r r e g u l a t o r y f u n c t i o n s occur i n the Malpighian tubules and rectum. The two-part excretory system of i n s e c t s has been described i n s e v e r a l reviews ( P h i l l i p s , 1970; 1977a, 1981; M a d d r e l l , 1971). In a t y p i c a l system the Malpighian tubules produce a primary " u r i n e " which i s isosmotic w i t h the hemolymph. This f l u i d s e c r e t i o n i s normally d r i v e n by a c t i v e t r a n s p o r t of potassium and th e r e f o r e the secreted f l u i d i s r i c h i n t h i s i o n . Other s o l u t e s such as sodiumj c h l o r i d e , phosphate, magnesium, s u l f a t e , PAH, a l k a l o i d s , dyes and c a r d i a c " g l y c o s i d e s are a l s o a c t i v e l y transported i n some i n s e c t tubules (reviewed by P h i l l i p s , 19.81). Amino a c i d s , sugars and other organic s o l u t e s apparently enter the tubules p a s s i v e l y and are found i n low concentrations i n the tubule f l u i d of Rhodnius (Maddrell and Gardiner, 1974§ 1980), S c h i s t o c e r c a and other i n s e c t s (Maddrell and Gardiner, 1974). The Malpighian tubule f l u i d passes i n t o the gut and flows p o s t e r i o r l y -i n t o . the ..rectum where s o l u t e s and water are s e l e c t i v e l y reabsorbed. The i n s e c t rectum has been most e x t e n s i v e l y s t u d i e d p h y s i o l o g i c a l l y i n the desert 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 (reviewed. by- P h i l l i p s . , .1965, 1970, 1977a, 1977b, 1981). Using an i n s i t u technique, P h i l l i p s (1964a) showed that the l o c u s t rectum could produce concentrated excreta by reabsorbing + water without p r o p o r t i o n a l uptake of s o l u t e s . However, l u m i n a l ions (Na , K +, C l ) are necessary f o r prolonged water transport by i n v i t r o r e c t a l sacs (Goh and P h i l l i p s , 1978). Monovalent ions are a c t i v e l y reabsorbed from the r e c t a l lumen i n s i t u 2 ( P h i l l i p s , 1964b). P h i l l i p s (1964b) noted that c h l o r i d e was absorbed more r a p i d l y from KC1 than from NaCl s o l u t i o n s . This observation was sub s t a n t i a t e d by the work of Hanrahan and P h i l l i p s (1980a) i n which i t was shown that a c t i v e c h l o r i d e t r a n s p o r t i n v i t r o i s sti m u l a t e d by potas-r sium but not sodium. P h i l l i p s (1964c) a l s o observed that i o n rea b s o r p t i o n by the l o c u s t rectum was dependent upon the hy d r a t i o n s t a t e of the i n s e c t . Ions were more r e a d i l y absorbed from the r e c t a l lumen of ion-depleted l o c u s t s com-pared to rea b s o r p t i o n by s a l t - l o a d e d i n d i v i d u a l s . The nature of t h i s r e g u l a t i o n of i o n rea b s o r p t i o n was i n v e s t i g a t e d by Spring et al., f1978) and Spring and P h i l l i p s (1980 a,bbc) usi n g s h o r t - c i r c u i t e d r e c t a l prepara-t i o n s described by Williams. _et a l . (1978). S a l i n e homogenates of the corpus cardiacum s t i m u l a t e the s h o r t - c i r c u i t current of the l o c u s t rectum by s e v e r a l f o l d (Spring el; a l . , 1978; Spring and P h i l l i p s , 1980a) and t h i s increase i n current i s due to a s t i m u l a t i o n of t r a n s e p i t h e l i a l c h l o r i d e t r a n s p o r t from the lumen to the hemocoel s i d e of the t i s s u e (Spring et a l . , 1978; Spring and P h i l l i p s , 1980b). Homogenates of the corpus cardiacum caused i n t r a c e l l u l a r cyclic-AMP (cAMP) l e v e l s to r i s e (Spring and P h i l l i p s , 1980a) and SAMP i s thought to act as a "second messenger" f o r the f a c t o r found i n the corpus cardiacum. In support of t h i s view, e x t e r n a l l y a p p l i e d 6AMP a l s o s t i m u l a t e s the s h o r t - c i r c u i t current and c h l o r i d e t r a n s p o r t (Spring &t_ - a l . $ 1978; Spring and P h i l l i p s , 1980a,b). The f a c t o r found i n the corpus cardiacum i s apparently r e l e a s e d upon feeding s i n c e hemolymph from r e c e n t l y - f e d i n s e c t s s t i m u l a t e s the s h o r t - c i r c u i t current (Spring et a l . , 1978; Spring and P h i l l i p s , 1980c) and t h i s hemolymph f a c t o r disappears i f the corpus cardiacum i s c a u t e r i z e d before feeding (Spring and P h i l l i p s , 1980c). In. a d d i t i o n . to c h l o r i d e , the i n v i t r o l o c u s t rectum a c t i v e l y t r a n s p o r t s other, s o l u t e s i n c l u d i n g acetate (Raumeister et a l . , 1981), f i v e n e u t r a l amino .acids ( B a l s h i n a n d . P h i l l i p s , 1971; B a l s h i n , 1973), Phosphate Andrusiak et a l . , 1980) and sodium (Williams et a l . , 1978; Spring and P h i l l i p s , 1980b). Although the l o c u s t rectum can a c t i v e l y t r a n s p o r t s e v e r a l s o l u t e s , the r a t e of a c t i v e c h l o r i d e t r a n s p o r t a f t e r s t i m u l a t i o n irt v i t r o exceeds that of other transported species by s e v e r a l f o l d (reviewed by P h i l l i p s , 1981). C l e a r l y c h l o r i d e t r a n s p o r t must be an important consumer of metabolic energy i n the l o c u s t rectum. E l e c t r o n micrographs of S c h i s t o c e r c a ( I r v i n e , 3 1966) and Locusta (Jonas and V i e t i n g h o f f , 1975) r e c t a i n d i c a t e that the a p i c a l membrane i s l i n e d w i t h mitochondria. Hanrahan and P h i l l i p s (1980b) used i n t r a c e l l u l a r i o n - s e n s i t i v e e l e c t r o d e s to demonstrate that the e l e c t r o -genic " c h l o r i d e pump" i s loc a t e d i n the a p i c a l plasma membrane so i t i s l i k e l y that these mitochondria supply the energy for. c h l o r i d e t r a n s p o r t . I n h i b i t o r s t u d i e s by Herrera et a l , s ( 1 9 7 7 ) 'indicate that o x i d a t i v e metabo-l i s m provides the energy f o r t h i s a c t i v e t r ansport s i n c e DNP, KCN or anoxia a l l a b o l i s h the chloride-dependent t r a n s e p i t h e l i a l p o t e n t i a l . S i m i l a r l y , anoxia, KCN or azide i n h i b i t the s h o r t - c i r c u i t current across l o c u s t rectum ( W i l l i a m s , et a l . , 1978; Baumeister et a l . , 1981). Although these s t u d i e s i n d i c a t e that m i t o c h o n d r i a l o x i d a t i v e metabolism energizes a c t i v e c h l o r i d e t r a n s p o r t , the substrates u t i l i z e d by the i n s e c t rectum are v i r t u a l l y unknown. Some data i s a v a i l a b l e on the metabolism i n other i n s e c t t r a n s p o r t i n g e p i t h e l i a , although these have u s u a l l y been obtained i n c i d e n t a l to the study of organic i o n t r a n s p o r t . O x i d a t i o n of amino a c i d s , or the presence of enzymes i n v o l v e d i n t h e i r metabolism, has been demonstrated i n s e v e r a l s p e c i e s . Murdock and K o i d l (1972a) showed that glutamate was o x i d i z e d to CO2 or converted to glutamine or a l a n i n e by the gut (probably midgut caeca) of Locusta. These workers (1972b) a l s o found that the gut d i d not metabolize g l y c i n e . G l y c i n e i s a c t i v e l y transported by the l o c u s t rectum but i t i s n e i t h e r o x i d i z e d to CO^ nor converted to other compounds by t h i s e p i t h e l i u m ( B a l s h i n , 1973). The four other amino acid s which are transported by the l o c u s t rectum are apparently not converted to other compounds but B a l s h i n (1973) d i d not "determine If. =;a. f r a c t i o n of these transported amino acid s might.~be o x i d i z e d to CO2. Aminotransferases have been i d e n t i f i e d i n s e v e r a l i n s e c t t r a n s p o r t i n g e p i t h e l i a . Both aspartate and al a n i n e aminotransferases are found i n the gut and Malpighian .tubules, of S c h i s t o c e r c a ( K i l b y and N e v i l l e , 1957; Mane and Mehotra, 1977), the.caeca, v e n t r i c u l u s and, M alpighian tubules of Locusta (Murdock and K o i d l , 1972b) and the Malpighian tubules of P e r i p l a n e t a (McAllen and Chefurka, 1961). Deaminating enzymes have a l s o been reported i n t r a n s p o r t i n g e p i t h e l i a . Glutamate dehydrogenase i s found i n Locusta caeca and v e n t r i c u l u s and amino oxidases have been i d e n t i f i e d i n Malpighian tubules from a v a r i e t y of i n s e c t s (reviewed by Cochran, 1975). The hindgut of Sarcbphaga probably has some deaminating enzymes sin c e a c t i v e ammonium s e c r e t i o n i n v i t r o 4 i s g r e a t l y enhanced when amino acid s are present i n the bathing s o l u t i o n on the hemocoel s i d e (Prusch, 1972). There i s l i m i t e d i n f o r m a t i o n on carbohydrate metabolism i n i n s e c t t r a n s p o r t i n g e p i t h e l i a . Trehalose i s the major sugar of hemolymph and the enzyme which hydrolases .this, d i s a c c h a r i d e y . t r e h a l a s e , i s found i n i n s e c t gut ( G i l b y et a l . , 1967). Trehalase a l s o occurs i n t t h e Malpighian tubules of Locusta (Mordue, 1969) and Knowles (1975) r e p o r t s that t r e h a l o s e , but not glucose, i s hydrolyzed by the tubules of C a l l i p h o r a . Glycogen may a l s o be a normal metabolic s u b s t r a t e . Murdock and K o i d l (1972b) found low a c t i v i t i e s of glycogen phosphorylase i n the Malpighian t u b u l e s , caeca and gut of LOcusta and Mordue (1969) r e p o r t s the presence of t h i s enzyme i n the rectum of Locusta. Glycogen and glycogen phosphorylase are a l s o found i n cockroach rectum and colon (Tolman and Steele ,1980a)', Phosphor-y l a s e may be under hormonal c o n t r o l i n both Locusta (Mordue, 1969) and P e r i p l a r t e t a (Tolman and S t e e l e , l ' 9 8 0 a ) " b e c a u s e e x t r a c t s of the corpus cardiacum (and corpus a l l a t u m i n Periplarteta) cause an increase i n the a c t i v e form of t h i s enzyme. L i t t l e i s known about f a t t y a c i d o x i d a t i o n i n i n s e c t t r a n s p o r t i n g e p i t h e l i a . Murdock and K o i d l (1972b) found 3-hydroxy-acyl CoA dehydrogenase i n the Malpighian t u b u l e s , caeca and v e n t r i c u l u s of Locusta. Short chain f a t t y a c i d s l i k e acetate and propionate are not o x i d i z e d by the cockroach colon (Bracke and Markovetz, 1980) and absorbed acetate i s minimally o x i -d i z e d by the l o c u s t rectum (Baumeister et a l . , 1981). Although the above s t u d i e s show that i n s e c t t r a n s p o r t i n g e p i t h e l i a are capable of m e t a b o l i z i n g s e v e r a l s u b s t r a t e s , few s t u d i e s have examined what substrates a c t u a l l y support the work of t r a n s p o r t i n g water or i o n s . B erridge (1966) added s i n g l e m e tabolites to i i i v i t r o Malpighian tubules °^ C a l l i p h o r a i n order.to see which, ones would support f l u i d s e c r e t i o n . H i s r e s u l t s suggested that the tubules possess a normal c i t r i c a c i d c y c l e and g l y c o l y t i c .pathway.. Exogenously a p p l i e d a l a n i n e , glutamine, pyruvate, and s e v e r a l sugars a l l supported f l u i d s e c r e t i o n . Giordana and Sacchi (1978) reported that pyruvate or a l a n i n e support the development of a t r a n s e p i t h e l i a l p o t e n t i a l across the midgut of Bombyx. Although i o n t r a n s p o r t i n i n s e c t e p i t h e l i a has been shown to be dependent upon metabolic energy, the e f f e c t of fion t r a n s p o r t on metabolic r a t e has been s p a r i n g l y s t u d i e d . Mandel and Balaban ( 1 9 8 1 ) have reviewed the l i t e r a t u r e on the coupling between tr a n s p o r t and o x i d a t i v e metabolismj 5 i n e p i t h e l i a and have concluded that the model of Whittam (1963) probably holds f o r a l l e p i t h e l i a s t u d i e d to date. This model proposes that when an " i o n pump" i s activated^ATP i s hydrolyzed to ADP and t h i s ADP s t i m u l a t e s o x i d a t i v e phosphorylation. The ADP i s phosphorylated to form ATP which, when hydrolyzed, can energize the "pump". Accompanying the phosphorylation of ADP i s an increased r a t e of oxygen consumption, which i s observed i n most e p i t h e l i a when i o n t r a n s p o r t i s i n i t i a t e d or s t i m u l a t e d . Tolman and S t e e l e ^ 1980b) showed that oxygen consumption by i n v i t r o cockroach rectum i s i n h i b i t e d by sodium s u b s t i t u t i o n or ouabain, suggesting that i n h i b i t i o n of sodium t r a n s p o r t leads to a decrease i n metabolic r a t e . However, potassium s u b s t i t u t i o n leads to an increase i n oxygen consumption. Since l i t t l e i s known about i o n transport i n t h i s e p i t h e l i u n ^ i n t e r p r e t a t i o n of these r e s u l t s are somewhat s p e c u l a t i v e . Coupling between i o n t r a n s p o r t and metabolic r a t e has a l s o been s t u d i e d i n Hyalophora and Mahduca midguts. Potassium t r a n s p o r t i n these e p i t h e l i a i s dependent upon oxygen, but i n h i b i t i o n of potassium t r a n s p o r t by decreasing the e x t e r n a l potassium co n c e n t r a t i o n does not lead to a decrease mn oxygen consumption (Harvey et a l . , 1967; Mandel et a l . , 1980a). However, Mandel et a l . (1980b) demonstrated that ATP l i n k s i o n t r a n s p o r t and metabolism i n Martduca. The i n s e n s i t i v i t y of oxygen consumption to io n t r a n s p o r t was explained by the low ATP/ADP: r a t i o found i n t h i s t i s s u e which would cause the mitochondria to c o n s t a n t l y r e s p i r e at an u n c o n t r o l l e d or s t a t e 3 (Chance and W i l l i a m s , 1956) rate.. 1 In order to understand more f u l l y the i n t e r a c t i o n between i o n t r a n s -port and metabolism i n i n s e c t e p i t h e l i a , i t i s h e l p f u l to study a t i s s u e where one of these two - phenomena :1is\«r'easonably^ we£ivundera£op^ .•i;*^ '.jptt tra n s p o r t by the l o c u s t rectum has been e x t e n s i v e l y s t u d i e d (reviewed by P h i l l i p s 1970, 1977a, 1977b, 1981), hut l i t t l e i s known about the metabolism i n t h i s or any other i n s e c t t r a n s p o r t i n g e p i t h e l i a . In t h i s t h e s i s I stu d i e d four areas i n order to e l u c i d a t e the r e l a t i o n s h i p between io n t r a n s p o r t and metabolism i n the l o c u s t rectum. Each of these t o p i c s i s considered i n a separate chapter (II-V) and an i n t e g r a t i o n of these r e s u l t s i s presented i n the General D i s c u s s i o n (Chapter V I ) . The a c t i v i t i e s of 15 enzymes from l o c u s t rectum are compared to those from f l i g h t muscle i n Chapter I I . The metabolism of the f l i g h t muscle i s w e l l documented so t h i s comparative approach attempts to p r e d i c t which metabolic pathways are important i n the rectum. In Chapter I I I a l s o 6 c h a r a c t e r i z e the r e s p i r a t o r y p r o p e r t i e s of mitochondria i s o l a t e d from the l o c u s t rectum. From the f i r s t p art of t h i s chapter i t i s evident that p r o l i n e metabolism i s important i n the l o c u s t rectum. The l a s t p a r t of t h i s study .'proposes a pathway f o r p r o l i n e o x i d a t i o n . Chapter I I I deals w i t h the a v a i l a b i l i t y of substrates to the rectum i n v i v o . The rectum i s a tube which i s bathed by two f l u i d s , the hemo-lymph and Malpighian tubule f l u i d . The concentrations of amino acid s and glucose i n the hemolymph and Malpighian tubuiLe f l u i d were measured and reported i n t h i s chapter. In Chapter IV the r e l a t i v e a b i l i t y of exogenously a p p l i e d substrates to support s h o r t - c i r c u i t current across the l o c u s t rectum i s i n v e s t i g a t e d . High concentrations of substrates were added to substrate-depleted r e c t a to see which ones s t i m u l a t e s h o r t - c i r c u i t c u r r e n t . Substrates were a l s o added to one s i d e of the t i s s u e to see i f the rectum p r e f e r e n t i a l l y o x i -d i z e s substrates bathing the lumen or hemolymph s i d e of the t i s s u e . F i n a l l y , i n h i b i t o r s t u d i e s were performed to t e s t the pathway proposed i n Chapter I I . Chapter V examines the e f f e c t of i o n s u b s t i t u t i o n s on oxygen con-sumption of r e c t a l t i s s u e . This represents a p r e l i m i n a r y study of the coupling between i o n t r a n s p o r t and metabolic r a t e i n the l o c u s t rectum. 7 CHAPTER I I OXIDATIVE METABOLISM IN THE LOCUST RECTUM I n t r o d u c t i o n Studies of c e l l u l a r metabolism i n i n s e c t s have focused on the f l i g h t muscle and the f a t body w h i l e the metabolic p r o p e r t i e s of other t i s s u e s are v i r t u a l l y unknown (reviewed i n Chapter I ) . The f a t body i s g e n e r a l l y considered to'be a s y n t h e t i c organ w h i l e f l i g h t muscle i s a very o x i d a t i v e ~ t i s s u e . The l o c u s t f l i g h t museiLe o x i d i z e s both carbohydrates and l i p i d s (Weis-Fogh, 1952). Amino acids are a l s o o x i d i z e d i n the e a r l y stages of f l i g h t (Worm and Beenakkers, 1980; Rowan and Newsholme, 1979) but cannot support extended periods of f l i g h t (Brosemer and Veerabhdrappa, 1965). Oxygen i s e f f i c i e n t l y s u p p l i e d to the f l i g h t muscle mitochondria v i a a h i g h l y developed t r a c h e a l system. Another organ which i s h e a v i l y tracheated i s the rectum, i n d i c a t i n g that t h i s t i s s u e a l s o has h i g h meta-b o l i c demands. The l o c u s t rectum a c t i v e l y reabsorbs ions and water and these processes can be i n h i b i t e d by cyanide, a z i d e , anoxia or d i n i t r o -phenol (Baumeister et a l . , 1981; Herrera et a l . , 1977; W i l l i a m s et a l . , 1978). These observations i n d i c a t e that a c t i v e i o n t r a n s p o r t by the l o c u s t rectum r e l i e s on o x i d a t i v e metabolism, however l i t t l e i s known about the metabolic pathways which supply reducing e q u i v a l e n t s to the e l e c t r o n t r a n s p o r t chain. Baumeister et a l . (1981) showed that acetate i n minimally o x i d i z e d by the rectum. F i v e n e u t r a l amino acid s ( g l y c i n e , s e r i n e , a l a n i n e , t h r e -onine, p r o l i n e ) are a c t i v e l y absorbed against l a r g e e l e c t r o c h e m i c a l gradients across the l o c u s t rectum i n v i t r o but there was no evidence that these substances were converted to other compounds as they traversed the e p i t h e l i u m ( B a l s h i n , 1973; reviewed by P h i l l i p s , 1981). However, B a l s h i n QL973.).. did. not .measure ^^CO^ evolved from. " ^ C ^ l a b e l l e d amino acid s except g l y c i n e . G l y c i n e was not o x i d i z e d to CO^ but some of the other absorbed amino a c i d s might be o x i d i z e d by the r e c t a l t i s s u e . P r o l i n e i s found i n high c o n c e n t r a t i o n (66 mM) i n the r e c t a l t i s s u e ( B a l s h i n , 1973)and" might .therefore be an energy source as i t i s i n some i n s e c t f l i g h t muscles (reviewed by Hansford and Sacktor,. 1971; Sacktor, 1973). To b e t t e r understand the o x i d a t i v e metabolism of the l o c u s t rectum, mitochondria were i s o l a t e d from t h i s t i s s u e and t h e i r metabolism charact-e r i z e d . Enzyme a c t i v i t i e s were a l s o measured and compared to the enzyme 8 p r o f i l e of f l i g h t muscle to see whicih metabolic pathways may be important i n r e c t a l t i s s u e . M a t e r i a l s; a n d Methods Animals Adult S c h i s t o c e r c a g r e g a f i a of bothesexes, two to four weeks past t h e i r f i n a l molt, were used i n a l l experiments. Animals were r a i s e d at 28 CCand 60% r e l a t i v e humidity on a 12:12 l i g h t : d a r k eyele. They were fed a mixture of d r i e d g rass, d r i e d m i l k and bran ad l i b i t u m . Fresh l e t t u c e was sup p l i e d d a i l y except on weekends. Chemicals A l l biochemicals and i n h i b i t o r s , except d i n i t r o p h e n o l , were purchased from Sigma. Inorganic chemicals and d i n i t r o p h e n o l were purchased from l o c a l s u p p l i e r s ( F i s h e r S c i e n t i f i c Co., B r i t i s h Drug House, North American S c i e n t i f i c Chemical L t d . ) . R e c t a l Glycogen and Glucose Glycogen and glucose were measured according to a modified method of Keppler and Decker (1974). Recta were d i s s e c t e d from l o c u s t s and-cleaned, of trachea, Malpighian tubules and gut contents. The r e c t a were q u i c k l y weighed, t r a n s f e r e d to 1 ml co l d 0.6 N p e r c h l o r i c a c i d (PCA) and homogen-i z e d f o r about 10 seconds w i t h a P o l y t r o n homogenizer ( d i s t r i b u t e d by Brinkman Instruments, Rexdale, O n t a r i o ) . Glycogen was analyzed by f i r s t n e u t r a l i z i n g 0.2 ml of the homogenate w i t h 1 M KHCOg. The glycogen was then digested w i t h amyloglucosidase (130 u n i t s per ml of 200 mM acetate b u f f e r , pH 4.8) f o r 2 hours at 37°C. The r e a c t i o n was stopped with :0.05ml 70% PCA.The a c i d i f i e d s o l u t i o n was then c e n t r i f u g e d at 14Q00g f o r 5 minutes, 0'.6 -ml of the supernatant was n e u t r a l i z e d , .with s o l i d KHCO^ and 0.2 t o 0 . 4 ml of t h i s e x t r a c t was analyzed f o r glucose u s i n g the method described by Keppler and Decker (1974). Standards were run w i t h known amounts of oyster glycogen d i s s o l v e d i n 0.6 N PCA. R e c t a l glucose was measured by c e n t r i f u g i n g the o r i g i n a l PCA homog-enate at 14000g f o r 5 minutes, 0.1 ml of the supernatant was n e u t r a l i z e d w i t h 1 M KHCO^ and 0.1 ml analyzed f o r glucose as described above. Stan-dards were run w i t h glucose d i s s o l v e d i n 0.6 N PCA. 9 Amino Acids i i i R e c t a l Tissue E x t r a c t s f o r amino a c i d a n a l y s i s were prepared by homogenizing i n d i -v i d u a l , weighed r e c t a i n 3.75% s u l p h o s a l i c y l i c a c i d (SSA), pH 2.2. The homogenate was c e n t r i f u g e d at 14000g f o r 5 minutes. A l i q u o t s of the super-natant were analyzed f o r amino acid s on a Beckman 118C automatic amino a c i d ana-iyzer u s i n g l i t h i u m c i t r a t e b u f f e r s to e l u t e the amino a c i d from the column. Standards were purchased from Beckman or Sigma. I s o l a t i o n of Mitochondria Ten r e c t a were d i s s e c t e d , pooled and placed i n 15 mis of c o l d b u f f e r (pH 7.4) c o n s i s t i n g of 10 mM potassium phosphate, 1 mM ethylenediamine t e t r a a c e t i c a c i d (EDTA), 400 mM sucrose and 3 mg protease (Sigma, Type V I I from B a c i l l u s a m y l o l i q u e f a c i e n s ) . The r e c t a were s t i r r e d i n t M s medium f o r 10 minutes. The treatment w i t h protease r e s u l t e d i n b e t t e r r e s p i r a t o r y c o n t r o l i n i s o l a t e d mitochondria. The r e c t a were homogenized i n protease-f r e e b u f f e r using a Potter-Elvehjem t e f l o n homogenizer. A f t e r 6 to 8 passes of the p e s t l e , the homogenate was t r a n s f e r e d to t e s t tubes and c e n t r i f u g e d at 300g f o r 10 minutes. The p e l l e t was discarded and the supernatant c e n t r i f u g e d at 3000g f o r 10 minutes. The r e s u l t i n g mitochon-d r i a l p e l l e t was resuspended i n 1 ml b u f f e r and 0.2 ml was reserved f o r p r o t e i n determination. The homogenate was kept c o l d (below 4°C) through-out the e n t i r e i s o l a t i o n procedure. B u f f e r s c o n t a i n i n g l e s s sucrose (200 mM) r e s u l t e d i n uncoupled mitochondria. A d d i t i o n of 10 mM MgC^ s l i g h t l y increased the s t a t e 3 r a t e of p r o l i n e o x i d a t i o n but produced poor r e s p i r a t o r y c o n t r o l r a t i o s (RCR). Polarography Oxygen consumption by i s o l a t e d mitochondria was monitored by a C l a r k -type; e l e c t r o d e (Yellow: Springs instrument Co., Inc., Yellow Springs, Colorado) .inserted i n t o a 2 ml, temperature c o n t r o l l e d chamber maintained at 25°C. The e l e c t r o d e was attached to a G i l s o n . oxygraph. f o r a continuous reading of oxygen concentrations. The oxygraph was c a l i b r a t e d w i t h a i r -saturated d i s t i l l e d water (260 u m o l / O ^ / l i t e r ) and a saturated NaSS s o l u t i o n (0 umol 0 2 / l i t e r ) . The oxygen te n s i o n was not allowed to f a l l below 50% s a t u r a t i o n during the f o l l o w i n g experiments. B u f f e r (pH 7.4) c o n s i s t i n g of 10 mM potassium phosphate, 1 mM EDTA, 400 mM sucrose and 1% bovine serum albumin (BSA) Sigma f r a c t i o n V, essetir t i a l l y f a t t y a c i d f r e e ) was put i n the chamber, s t i r r e d u n t i l e q u i l i b r a t e d w i t h a i r and 0.1 to 0.2 ml of the m i t o c h o n d r i a l suspension ( f i n a l p r o t e i n 10 co n c e n t r a t i o n , 0.05 to 0.44 mg/ml) was then added to the b u f f e r . The chamber was sealed w i t h a g l a s s stopper and s u b s t r a t e s , adenosine-5'-diphosphate (ADP) and i n h i b i t o r s were added w i t h a 10 jal Hamilton syringe through a small hole i n the stopper. Most substrates and ADP were d i s s o l v e d i n BSA-free b u f f e r . P a l m i t o y l c a r n i t i n e was d i s s o l v e d i n 95% ethanol. P r e l i m i n a r y experiments were performed-,to*3insure that s u b s t r a t e and ADP concentrations were s a t u r a t i n g . I n h i b i t o r s such as aminooxyacetate and NH^Cl were d i s s o l v e d i n BSA-free b u f f e r w h i l e rotenone, oligomycin, triflourmethoxycarbonyl-cyanide-phenyl-hydrazone (FCCP), d i n i t r o p h e n o l (DNP) and antimycin were d i s s o l v e d i n 95% ethanol. A d d i t i o n of 95% ethanol alone d i d not a f f e c t the mitochon-d r i a l performance. Experiments were u s u a l l y conducted by f i r s t adding s u b s t r a t e (10yul) to the m i t o c h o n d r i a l suspension i n the chamber and adding 200 nmol of ADP to e l i c i t an increase i n oxygen consumption. Oxygen consumption, r e s p i r a t o r y c o n t r o l r a t i o s (RCR, s t a t e 3 r a t e / s t a t e 4 r a t e ) , and ADP;0 r a t i o s (amount of ADP phosphorylated to oxygen consumed) were c a l c u l a t e d according to Estabrook (1967). R e s p i r a t o r y s t a t e s are defined according to Chance and W i l l i a m s (1956). M i t o c h o n d r i a l P r o t e i n Determination P r o t e i n was determined by a b i u r e t method descrived by G o r n a l l et_ a l . (D949). The method was standardized w i t h BSA d i s s o l v e d i n 10 mM potassium phosphate, pH 7.4. Tissue P r e p a r a t i o n f o r Enzyme Assays Two d i s s e c t e d r e c t a were weighed and placed i n 1 ml c o l d 50 mM t r i s -HC1 b u f f e r , ph 7.4. F l i g h t muscles were cleaned of trachea and f a t body and d i s s e c t e d from the thorax. The f l i g h t muscles from i n d i v i d u a l animals were weighed and placed i n 1 ml c o l d 50 mM T r i s - H C l b u f f e r . These t i s s u e s were homogenized f o r approximately 10 seconds w i t h a P o l y t r o n homogenizer and then c e n t r i f u g e d at 20000g f o r t h i r t y minutes. The t i s s u e s and homo-genates were kept below 4°0 throughout t h i s procedure. The homogenization d i s r u p t e d the mitochondria, as observed by the r e l e a s e of c i t r a t e synthase i n t o the supernatent, but the p e l l e t was a l s o t e s t e d f o r r e s i d u a l a c t i v i t y a f t e r resuspending i t i n 2 to 3 ml of T r i s b u f f e r . Reported enzyme a c t i v i t i e s are then summations of the a c t i v i t i e s found i n the supernatent and p e l l e t . 11 Enzyme Assays A l l r e a c t i o n s were c a r r i e d out at 25°C i n 1 ml cuvettes. The reac-t i o n s were monitored on a Unicam SB8-200 recording spectrophotometer at 340 nmeters, unless otherwise noted. Reaction r a t e s were f i r s t monitored i n the absence of one of the reagents. The r e a c t i o n was then s t a r t e d w i t h the a d d i t i o n of the reagent which was omitted during the c o n t r o l p e r i o d . A l l assays contained 20 to 50 j u l of the enzyme e x t r a c t . The f o l l o w i n g assays are m o d i f i c a t i o n s of methods of Storey and B a i l e y (1978a,b). A l l concentrations given are f i n a l concentrations and p r e l i m i n a r y experiments were performed to insure that these concen-t r a t i o n s produced maximal enzyme a c t i v i t y . , Glycogen phosphorylase (E.C. 2.4.1.1): 50 mM potassium phosphate b u f f e r , pH 6. 8 ; 1 mM EDTA; 10 mM MgCl 2; 0.4 mM NADP; 2mg/ml glycogen (omitted i n c o n t r o l ) ; 4/iM glucose 1,6-diphosphate; excess phosphoglu-comutase and g l u c o s e - 6 - phosphate dehydrogenase. T o t a l phosphorylase a c t i v i t y was monitored by adding AMP to a f i n a l concentation of 1.6 mM. Phosphoglucomutase (E.C.2.7.5.1): 50 mM t r i s b u f f e r , pH 7.4; 4 mM glucose-l-phosphate (omitted i n c o n t r o l ) ; 0.02 mM glucose-1,6-diphosphate; 1 mM EDTA; 2 mM MgCl'2; 0.2 mM NADP; excess glucose-6-phosphate dehy-drogenase . Hexokinase (E.C.2.7.1.1): 50 mM T r i s b u f f e r , pH 7.4; 10 mM MgCl 2; 4 mM ATP (omitted i n c o n t r o l ) ; 1 mM glucose; 1 mM NADP; excess glucose-6-phosphate dehydrogenase. Phosphoglucoisomerase (E.C.5.3.1.9): 50 mM T r i s b u f f e r , pH 7.4; 0.5 mM NADP; 7 mM MgCl 2; 2 mM fructose-6-phosphate (omitted i n c o n t r o l ) ; excess glucose-r6-phosphate dehydrogenase. Glucose^ T v p h o s p h a t e d e h y d r o g e n a s e (E.G. 1:1.1.49) : 50 mM T r i s b u f f e r , pH 7.4; 7 mM MgCl 2; 0.4 mM NADP; 1 mM glucose-*-6^phosphate (omitted i n c o n t r o l ) . L a c t a t e .dehydrogenase (E.C.1.1.1.27): 50 mM T r i s b u f f e r , pH 7.4; 0.2 mM NADH; 1 mM pyruvate (omitted i n c o n t r o l ) . R r e l i m i n a r y experiments i n d i c a t e d that the l a c t a t e dehydrogenase was L - l a c t a t e dehydrogenase. NAD-dependent glycerol-3-phosphate dehydrogenase (E.C.1.1.1.8): 50 mM t r i s b u f f e r , pH 7.4; 0.2 mM NADH; 0.4 mM dihydroxyacetone phosphate (omitted i n c o n t r o l ) . 12 A r g i n l n e kinase (E.C.2.7.3.3): 50 mM T r i s b u f f e r , pH 7.4; 17.5 mM a r g i n i n e (omitted i n c o n t r o l ) ; 10 mM MgCl 2; 100 mM KC1; 10 mM ATP; 2 mM phosphoenolpyruvate; 0.2 mM NADH; excess pyruvate kinase and l a c t a t e dehydrogenase. C i t r a t e synthase (E.C.4.1.3.7): 50 mM T r i s b u f f e r , pH 8.1; 0.1 mM 5 , 5 ' - d i t h i o b i s - ( 2 - n i t r o b e n z o i c acid) (DTNB); 0.3 mM acetyl-CoA; 0.5 mM oxaloacetate. The r e a c t i o n was s t a r t e d w i t h DTNB and acetyl-CoA to measure acetyl-CoA deacylase and then the oxaloacetate was added to measure c i t r a t e synthase. The r e a c t i o n was followed at 412 nmeters and the e x t i n c t i o n 3 -1 -1 c o e f f i c i e n t used was 13.6x10 mole cm Malate dehydrogenase (E.C.1.1.1.37): 50 mM T r i s b u f f e r , pH 7.4; 0.2 mM NADH; 0.5 mM oxaloacetate (omitted i n c o n t r o l ) . Glutamate dehydrogenase (E.C.1.4.1.2); 50 mM T r i s b u f f e r , pH 7.4; 250 mM NH^CI; 0.1 mM EDTA; 0.1 mM NADH; 15mM C*~ketoglutarate (omitted i n c o n t r o l ) . ADP a c t i v a t i o n was teste d by adding ADP to a f i n a l concen-t r a t i o n of 1 mM. Glutamate-oxaloacetate transaminase (E.C.2.6.1.1): 50 mM T r i s b u f f e r , pH 7.4; 40 mM aspartate (omitted i n c o n t r o l ) ; 7 mMoC-ketoglutarate; 0.2 mM, NADH; 0.05. mM p y r i d o x a l phosphate; excess malate dehydrogenase. Glutamate-pyruvate dehydrogenase (E.C.2.6.1.2); 50 mM T r i s b u f f e r , pH 7.4; 200 mM a l a n i n e ; 10 mMc?C-ketoglutarate (omitted i n c o n t r o l ) ; 0.2 mM NADH; 0.05 mM pyridoxalphosphate and excess l a c t a t e dehydrogenase.. Trehalase (E.C.3.2.1.28) was measured by a modified method of Strang and Clement (1980) i n which the f i n a l concentrations are 50 mM T r i s b u f f e r , pH 7.4; 10 mM MgCl 2; 4 mM ATP; 1 mM NADP; 5 mM trehalose.; (omitted.-in con-trol).,;/excess'; g l u e o s ^ and hexokinase. 3-hydrpxy-acyl-CoA dehydrogenase (E.C.1.1.1.35) was assayed by a modified method of Beenakkers. (1969). F i n a l concentrations were 50 mM T r i s b u f f e r , pH 7.4; 0.2 mM acetoacetyl-CoA (omitted i n c o n t r o l ) ; 0.1 mM NADH. L o c a l i z a t i o n of Enzymes Since I found that i s o l a t e d mitochondria o x i d i z e p r o l i n e , some enzymes known to be in v o l v e d i n p r o l i n e o x i d a t i o n were assayed i n i s o l a t e d mito-chondria. Recta were d i s s e c t e d and homogenized as described i n the s e c t i o n on m i t o c h o n d r i a l p r e p a r a t i o n ; however, no protease was used. The mito-c h o n d r i a l p e l l e t was resuspended as described e a r l i e r and assayed f o r m a l i c 13 dehydrogenase, glutamate dehydrogenase, glutamate-pyruvate transaminase and glutamate-oxaloacetate transaminase as p r e v i o u s l y described. NAD-and NADP-dependent malate dehydrogenase ( d e c a r b o x y l a t i n g , "malic enzyme") were assayed as f o l l o w s : NAD-dependent malate dehydrogenase (d e c a r b o x y l a t i n g , E.C.1.1.1.39) was assayed by a modified method of Weeda et a l . (1980). F i n a l concen-t r a t i o n s were 50 mM T r i s b u f f e r , pH 7.4; 1 mM NAD; 20 mM malate; excess malate dehydrogenase and 10 mM MgCl 2 (omitted i n c o n t r o l ) . The r e a c t i o n • without MgCl 2 was allowed to reach e q u i l i b r i u m and then the m a l i c enzyme r e a c t i o n was s t a r t e d w i t h MgCl 2. NADP-dependent malate dehydrogenase (decarb o x y l a t i n g , E.C.1.1.1.40): 50 mM T r i s b u f f e r , pH 7.4; 5 mM malate (omitted i n c o n t r o l ) ; 5 mM MgCl 2; 1 mM NADP (Storey and B a i l e y , 1978a). 14 14 Conversion of C - p r o l i n e to C0 2 Since the r e c t a l mitochondria r e a d i l y o x i d i z e p r o l i n e (see r e s u l t s ) i t was of i n t e r e s t to see i f the i n t a c t rectum can o x i d i z e t h i s amino a c i d . Recta were d i s s e c t e d from l o c u s t s as p r e v i o u s l y described and placed i n about 5 ml of p h y s i o l o g i c a l s a l i n e which lacked m e t a b o l i t e s ( s e e Chapter IV, Table XIV, s a l i n e A). This s a l i n e was bubbled w i t h 95% 0 2 and 5% C0 2 f o r 2 hours. This 2 hour i n c u b a t i o n was performed to deplete the t i s s u e of endogenous hormone (Spring et a l . , 1978) and to lower the t i s s u e metab-o l i t e content. The r e c t a were then t r a n s f e r r e d to f r e s h s a l i n e w i t h one 14 of the f o l l o w i n g three compositions: (1) 25 mM p r o l i n e and C-proline to y i e l d a s p e c i f i c a c t i v i t y of 35 yuCi/mmol; (2) 25 mM p r o l i n e & ^ C - p r o l i n e ( s p e c i f i c a c t i v i t y , 36 yuCi/mmol) and 1 mM cAMP; (3) 24 mM p r o l i n e and 14 C-p r o l i n e ( s p e c i f i c a c t i v i t y , 35 /iCi/mmol), 3 mM glucose and 1 mM cAMP. The r e c t a were preincubated i n these s a l i n e s f o r 1 hour. B a l s h i n (1973) 14 showed that 1 hour was adequate f o r e q u i l i b r a t i o n of e x t e r n a l C-amino a c i d s w i t h the t i s s u e pool of amino a c i d s . The r e c t a were then removed from t h i s s a l i n e , b l o t t e d dry, weighed and placed i n a t e s t tube c o n t a i n -i n g 1 ml of s a l i n e i d e n t i c a l to the l a b e l l e d p r e i n c u b a t i o n s a l i n e . This s a l i n e was gassed w i t h 95% 0 2 and 5% C0 2 f o r about 10 seconds and the ' tube capped w i t h a rubber stopper. The underside of the stopper con-t a i n e d a w e l l i n which was placed f o l d e d g l a s s f i b e r paper. The .tube', was shaken ,for 20 minutes at 25°C i n a metabolic shaker (Magni W h i r l Constant Temperature Bath, Blue M. E l e c t r i c Co., Blue I s l a n d , I l l i n o i s ) . The r e a c t i o n s were stopped by i n j e c t i n g 200 fil of 3.75% SSA i n t o the tube. 14 Figure 1. Levels of amino a c i d s , glycogen and glucose i n the r e c t a l t i s s u e . (a) Amino a c i d content expressed as nmol/mg f r e s h weight (x + SE where l a r g e r than bar l i n e s , n=3). (b) Glucose (nmol/mg f r e s h weight, x + SE, n = l l ) and g l y c o -gen (nmol glucose/mg f r e s h weight, x + SE, n = l l ) . The r e c t a l t i s s u e i s approximately 85% water (vol./wt, c a l c u l a t e d from B a l s h i n , 1973). Ab b r e v i a t i o n s used i n t h i s f i g u r e are: asp, aspa r t a t e ; s e r , s e r i n e ; g i n , glutamine; g l y , g l y c i n e ; a l a , a l a n i n e ; h i s , h i s t i d i n e ; a r g , a r g i n i n e ; g l y c , glycogen;_gluc,-glucose. o 8 nmol/mg 8 S 8 3 CD 3 3 (Q (D i 1 1 h 8 ^ o 1 1 1 0) 0) 0) (3 i 1 1 1 I 1 1 h CO SI 16 A 100 /JLI a l i q u o t of 10% hyamine hydroxide ( B r i t i s h Drug House)was i n j e c t e d i n t o the w e l l at the same time. The tube was shaken f o r another 30 mins 14 u t e s . C o n t r o l s were run by c o l l e c t i n g C0_ from 1 ml of l a b e l l e d s a l i n e 14 without a rectum present. The glass f i b e r paper w i t h absorbed CT^ was put i n t o 10 ml of toluene-base s c i n t i l l a t i o n f l u i d (Shoubridge, 1981) 14 and the C counted on a Beckman LS 9000 l i q u i d s c i n t i l l a t i o n counter. Quench c o r r e c t i o n was by the Compton curve method as described i n the Beckman LS 9000 s e r i e s manual. 14 C- p r o l i n e o x i d a t i o n was a l s o measured i n .locust colon prepared by the method described f o r r e c t a and incubated i n 25 mM p r o l i n e (35 yiCi/mmol) pl u s 1 mM cAMP. End-Products of P r o l i n e Metabolism Alanine and ammonia production by r e c t a l t i s s u e were measured i n experiments s i m i l a r to those described i n the previous s e c t i o n ; however 14 C- p r o l i n e was not included i n the e x t e r n a l s a l i n e . Recta were i n c u -bated f o r two hours i n m e t a b o l i t e - f r e e s a l i n e then t r a n s f e r r e d to t h i s same s a l i n e c o n t a i n i n g 1 mM cAMP or to a s a l i n e c o n t a i n i n g 25 mM p r o l i n e and 1 mM cAMP and preincubated f o r one hour. TTte r e c t a were then removed from the p r e i n c u b a t i o n s a l i n e , weighed and put i n a t e s t tube c o n t a i n i n g 1 ml of s a l i n e i d e n t i c a l to the p r e i n c u b a t i o n s a l i n e . The s a l i n e was gassed w i t h 95% 02/5.%*'C02 and the t e s t tube capped and shaken f o r 20 minutes at 25°C The r e a c t i o n was stopped w i t h SSA as p r e v i o u s l y described. The r e c t a were homogenized i n the a c i d i f i e d e x t e r n a l s a l i n e w i t h a P o l y t r o n homogenizer and the homogenate was c e n t r i f u g e d at 14000g f o r 5 minutes. The supernatant was analyzed f o r a l a n i n e on an automatic analyzer as p r e v i o u s l y described. Ammonia was measured i n the supernatant as described by Chaney and Marbach Q962). R e s u l t s Tissue Content of Amino Acids and Carbohydrates Figure l a shows the l e v e l s of amino acid s i n the r e c t a l t i s s u e . Other amino a c i d s , /threonine,; methionine j^,i:s6leucirie, l e u c i n e , t y r o s i n e , phenylalanine, o r n i t h i n e and l y s i n e , were'found i n t r a c e amounts ( l e s s than 0.5 nmol/mg). Note that p r o l i n e (66.3 + 10.6 nmol/mg, x + SE), glutamine (44.5 + 17.0 nmol/mg) and g l y c i n e (21.0 + 4.2 nmol/mg) c o n s t i -t u t e about 80% of the t o t a l f r e e amino a c i d p o o l . 17 Figure 2. O x i d a t i o n of 30 mM p r o l i n e by i s o l a t e d r e c t a l mitochon-d r i a (0.11 mg p r o t e i n / m l ) . 200 nmols ADP added where i n d i c a t e d . 18 19 TABLE I Res p i r a t o r y c h a r a c t e r i s t i c s of i s o l a t e d r e c t a l mitochondria S State 3 Substrate 0^ Consumption RCR ADP:0 n (nmol/min-mg p r o t e i n ) Endogenous 23.1 + 1.8 n.m. n.m. 3 0.1 mM NADH 34.3 + 3.9 ;ri.m. n.m. 3 30 mM P r o l i n e 104.5 ± 11.2 4.5 + 0.8 1.7 +0.8 5 5 mM P r o l i n e 45.4 + 12.4 4.5 + 0.5 1.6 + 0.2 3 5 mM Glutamate 58.4 ± 5.0 5.6 +0.4 2.2 +0.1 4 5 mM .Alanine 25.4 + 6.1 n.m. n.m. 3 5 mM Pyruvate 37.1 + 8.5 4.0 +0.3 2.0+0.2 3 5 mM Pyruvate + 0.5 mM Malate 57.6 + 2.4 3.5 +0.2 2.2 +0.1 3 0.5 mM Malate 25.9 + 2.6 n.m. n.m. 3 5 mM Malate 46.1 + 7.4 4.8 +0.2 2.4 + 0.1 3 5 mM Succinate 27.1 + 7.8 1.9 +0.1 1.5 + 0.1 3 25 mM Succinate 72.9 + 2.9 2.6 + 0.1 1.5 + 0.1 3 5 mMoi-keto- . g l u t a r a t e 45.4 + 3.9 3.6 + 1.0 1.8 + 0.1 3 5 mM ©(.-glycerol phosphate 28.6 + 7.3 uncoupled uncoupled 3 7.5 J U M E a l m i t o y l c a r n i t i n e 31.4 + 6.9 5.2* 1.9** • - 3 7.5 J U M E a l m i t o y l c a r n i t i n e + 47.4 + 2.8 3.4 + 0.6 1.7* •. < 3 0.5 mM Malate A l l values are X + SE except where i n d i c a t e d w i t h -* (n=2) or ** (n=l). The RCR i s the r e s p i r a t o r y c o n t r o l r a t i o and the ADP:0 i s the r a t i o of the amount of ADP phosphorylated to oxygen consumed, n.m. = not measured 20 The c o n c e n t r a t i o n of glycogen (6.2 + 0.8 nmol/mg) and glucose (4.4 + 1.8 nmol/mg) are shown i n f i g . l b and are r e l a t i v e l y low compared to the amino a c i d l e v e l s . No c o r r e c t i o n was made i n these c a l c u l a t i o n s of substrate concent-r a t i o n s f o r e x t r a c e l l u l a r space i n the l o c u s t rectum, which c o n s t i t u t e s 25% of the t o t a l t i s s u e water ( J . Hanrahan, personal communication). The concentrations of amino acid s and glucose i n the hemolymph and Malpighian tubule f l u i d of the desert l o c u s t are much lower than values estimated f o r the whole r e c t a l t i s s u e (see Chapter I I I ) so the values i n f i g . 1 are probably underestimates of i n t r a c e l l u l a r c o n centrations. Another source of e r r o r i s that the f r e s h weight of the t i s s u e i n c l u d e s the weight of the i n e r t c u t i c u l a r i n t i m a . However, the i n t i m a c o n s t i -t u t e s only 5% of the f r e s h weight of the t i s s u e ( J . Hanrahan, personal communication) and was ignored i n t h i s and other c a l c u l a t i o n s i n v o l v i n g r e c t a l weight. Substrate Oxidation by I s o l a t e d R e c t a l Mitochondria F i g u r e 2 shows the e f f e c t of s u c c e s s i v e l y adding ADP to r e c t a l mito-chondria using p r o l i n e as a s u b s t r a t e . ADP i n i t i a t e s an u n c o n t r o l l e d or s t a t e 3 r a t e of oxygen consumption. The r a t e then slows to a c o n t r o l l e d or s t a t e 4 r a t e . Subsequent a d d i t i o n s of ADP r e s u l t e d i n lower s t a t e 3 r a t e s . When c a l c u l a t i n g the s t a t e 3 r a t e f o r Table I,=only the s t a t e 3 r a t e i n i t i a t e d by the f i r s t ADP a d d i t i o n was used. Table I shows the s t a t e 3 r a t e of oxygen consumption of r e c t a l mito-chondria o x i d i z i n g v a r i o u s s u b s t r a t e s . A high c o n c e n t r a t i o n , 30 mM, of p r o l i n e was r e q u i r e d to y i e l d a maximal r a t e of oxygen consumption. 30 mM p r o l i n e was o x i d i z e d more r a p i d l y than s a t u r a t i n g l e v e l s of other s u b s t r a t e s , i n c l u d i n g pyruvate plus malate and p a l m i t o y l c a r n i t i n e plus malate. A l a n i n e and o ( - g l y c e r o l phosphate d i d not support oxygen c o n s u m p T t i o n at a r a t e greater than that of mitochondria, o x i d i z i n g endogenous s u b s t r a t e . Most RCR's were between 3 and 5, i n d i c a t i n g i n t a c t mitochon-r' d r i a . The ADP/0 r a t i o s ranged f o r 2.4 to 1.6 f o r NAD-linked substrates e*id was 1.5 f o r the FAD-linked succinate dehydrogenase r e a c t i o n . 21 Figure 3. T y p i c a l t r a c e s of m i t o c h o n d r i a l r e s p i r a t i o n i n the presence of i n h i b i t o r s . I n h i b i t o r s were added to the r e c t a l m i t o c h o n d r i a l suspension at the times i n d i c a t e d and at the f o l l o w i n g concentrations: rotenone, 1 /iM; oligomycin 10 /ig/ml; FCCP. 0.1 mM; DNP, 0.1 mM; a n t i -mycin, 2 /iM. (a) Mitochondria (0.15 mg protein/ml) o x i d i z i n g 16 mM p r o l i n e . (b) Mitochondira (0.05 mg/ml) o x i d i z i n g 30 mM p r o l i n e . (c) Mitochondria (0.13 mg/ml) o x i d i z i n g 5 mM c< - k e t o g l y t a r a t e . (d) Mitochondria (0.13 mg/ml) o x i d i z i n g 30 mM p r o l i n e . (e) Mitochondria (0.11 mg/ml) o x i d i z i n g 25 mM su c c i n a t e . The apparent in c r e a s e i n oxygen con c e n t r a t i o n a f t e r i n h i b i t o r a d d i -t i o n i s simply an a r t i f a c t created by adding ethanol to the chamber. Rotenone 23 TABLE I I Enzyme a c t i v i t i e s of l o c u s t f l i g h t muscle and rectum Enzyme Rectum F l i g h t Muscle F l i g h t Muscle ( L i t e r a t u r e ) GP -AMP "'.:.0;.,7-•+: .0.-2 8.71+.0.8 1 +AMP -2.2 0..-2 9.0 -+0.6 -•- . 7.5 1 PGM 36.2 ± .3.2 5.4.6 + .3.4 THL 0.2 ' + 0.01 0.1 + 0.01 1.23 HK 6.6 ± 1.4 5.9 + 0.7 11.5 1 PGI 53.4 ± 1.3 186 084.5-187.6)* G6PDH 2.2 + 0.2 1.1 + 0.3 LDH 3.2 + 0.5 1.4 + 0.4 2.9 1 NAD-G3PDH 6.8 + 1.0 94.6 + 10.8 141 1 PAK 200.4 + 1.2 145.5 + 2.8 CS 19.7 (15.8-23.6)* 82.2 (77.1-87,2)* 242 1 MDH 398.5 + 23.4 129.4 + 6.4 GDH 29.1 + 3.0 8.-1 + 0.8 4 2 GPT 63.7 + 4.0 37.2 + 2.2 34 2 GOT 188.3 + 11.2 79.6 + 6.7 48 2 HOAD 0.7 + 0.1 6.3 + 1.5 13 1 Enzyme a c t i v i t i e s are expressed as ^ lmol/min.mg f r e s h weight (x + SE, n=3). I f n<. 3 (*) then a c t i v i t y i s expressed as x (range). The source of the l i t e r a t u r e values are i n d i c a t e d by the s u p e r s c r i p t : 1. Crabtree and Newsholme(1975) 2. Crabtree and Newsholme (1970, Locusta m i g r a t b r i a ) 3. Candy (1974) Ab b r e v i a t i o n s f o r enzyme names are as f o l l o w s : GP, glycogen phosphorylase; PGM, phosphoglucomutase; THL, trehalase;HK, hexokinase; PGI, phosphoglucoisomerase; G6PDH, glucose-6-phosphate dehydrogenase; LDH, l a c t a t e dehydrogenase; NAD-G3PDH, NAD-dependent glycerol-3-phosphate dehydrogenase; PAK phosphoarginine k i n a s e ; CS, c i t r a t e synthase; MDH, malate dehydrogenase; GDH, glutamate dehydrogenase; GPT, glutamate-pyruvate transaminase; GOT, glutamate-oxaloacetate transaminase; HOAD, 3-0H-acyl CoA dehydrogenase. 24 I n h i b i t o r s I s o l a t e d r e c t a l mitochondria respond to i n h i b i t o r s i n a manner s i m i l a r to mitochondria from other animal sources. Oligomycin completely i n h i b i t s ADP-stimulated r e s p i r a t i o n ( f i g . 3a). Uncouplers of o x i d a t i v e phosphor-y l a t i o n , DNP and e s p e c i a l l y FCCP, r e l i e v e t h i s i n h i b i t i o n ( f i g . 3b). Oxygen consumption i s i n h i b i t e d by rotenone when the mitochondria are o x i d i z i n g a NAD-linked s u b s t r a t e , e ^ - k e t o g l u t a r a t e ( f i g . 3 c). However, thetrotenorie i n h i b i t i o n i s not 100% when the mitochondria are o x i d i z i n g p r o l i n e ( f i g . 3d) si n c e p r o l i n e oxidase i s FAD-linked. S i m i l a r l y , r o t e -none i s not an e f f e c t i v e i n h i b i t o r of FAD-linked succinate o x i d a t i o n ( f i g . 3 e ) . ' Antimycin, which blocks o x i d a t i o n of NAD- and FAD- l i n k e d sub-s t r a t e s completely* i n h i b i t s oxygen consumption ( f i g . 3a). The o x i d a t i o n of 5 mM p r o l i n e (n =3) was unaffected by a d d i t i o n of 1 to 2 mM aminooxyacetate, an i n h i b i t o r of amino t r a n s f e r a s e s (Webb, 1966). 40 mM NH^Cl a l s o f a i l e d to i n h i b i t o x i d a t i o n of 5 mM p r o l i n e (n = 3). Enzyme A c t i v i t i e s i n Locust Recta and F l i g h t Muscle The r e s u l t s of the enzyme assays appear i n Table I I . Estimates of enzyme a c t i v i t i e s i n l o c u s t f l i g h t muscle appear to be i n reasonable agreement w i t h l i t e r a t u r e v a l u e s . Any d i f f e r e n c e s are probably due to d i f f e r e n c e s i n assay procedures. The most s t r i k i n g d i f f e r e n c e between the f l i g h t muscle and the rectum of l o c u s t i s the higher a c t i v i t i e s of glutamate dehydrogenase, glutamate-pyruvate transaminase and glutamate-oxaloacetate transaminase and the much lower a c t i v i t i e s of NAD-dependent OQ-glycerol phosphate dehydrogenase and 3-hydroxy-acyl-CoA dehydrogenase i n ,the rectum. Both l o c u s t t i s s u e s have low a c t i v i t i e s of l a c t a t e dehydrogenase and s i m i l a r l e v e l s of other enzymes i n v o l v e d i n carbohydrate metabolism. U n l i k e f l i g h t muscle glycogen phosphorylase, r e c t a l phosphorylase may be under modulation s i n c e i t i s a c t i v a t e d 3 - f o l d by AMP. Enzyme a c t i v i t i e s i n i s o l a t e d r e c t a l mitochondria are shown i n Table I I I . There was no c y t o s o l i c contamination of the m i t o c h o n d r i a l p e l l e t s i n c e a c t i v i t y of NAD-dependento(-glycerol phosphate dehydrogenase, a cytoplasmic enzyme, was n i l i n the p e l l e t . Malate-dehydrogenase had the highest a c t i v i t y of the enzymes t e s t e d . The two transaminases were found i n approximately the same a c t i v i t y and glutamate dehydrogenase a c t i v i t y was s l i g h t l y a c t i v a t e d by the a d d i t i o n of ADP. 25 TABLE I I I A c t i v i t i e s of enzymes i n mitochondria i s o l a t e d from l o c u s t rectum Enzyme Enzyme A c t i v i t y (nmol/min.mg p r o t e i n ) Glutamate dehydrogenase -ADP +ADP Glutamate—Pyruvate Transaminase Glutamate-Oxaloacetate Transaminase Malate Dehydrogenase NAD-Malic Enzyme NADP-Malic Enzyme P r o l i n e Oxidase 182.2 + 22.4 211.2 + 15.4 563.1 + 58.4 497.3 + 61.7 931.0 + 117.9 13.6 +3.0 44.9 + 5.2 13.6 + 0.8 A l l values are x + SE f o r 3 m i t o c h o n d r i a l p r e p a r a t i o n s . P r o l i n e oxidase a c t i v i t y was estimated from the oxygen consumption of mitochondria o x i d i z i n g 30 mM p r o l i n e i n the presence of 1 /uM rotenone. 26 TABLE IV 14 14 Oxidation of C-proline to CO2 by l o c u s t rectum and colon Substrates CO- Production (nmol C0 0/hr'mg wet weight) Rectum Colon 25 mM P r o l i n e 25 mM P r o l i n e .+ 1 mM cAMP 25 mM P r o l i n e + 3 mM glucose++ 1 mMcAMP. • -- ••: • • 16.83 + 2.25 18.69 +1.77 20.43 +1.47 ;n.m. 22.50 + 5.94 n,m. A l l values are x + SE, n=6. n.m. = not measured. TABLE V Levels of ammonia and al a n i n e i n the r e c t a l t i s s u e a f t e r p r o l i n e o x i d a t i o n Tissue Content* Substrates Ammonia n Alanine n Endogenous-CO mM e x t e r n a l P r o l i n e ) 25 mM e x t e r n a l P r o l i n e A l l values are x + SE. * Values f o r ammonia and a l a n i n e are a c t u a l l y those found i n the r e c t a l t i s s u e p l u s the 1 ml s a l i n e "bathing the rectum (see M a t e r i a l s and Methods). 3.47 + 0.53 4 2.31 + 0.22 3 7.14 +1.04 5 7.31 + 1.05 5 28 1_^_C0„ Production from 1 4 C - p r o l i n e 14 The r e s u l t s of C-pr o l i n e o x i d a t i o n experiments i n d i c a t e that GAMP o r g l u c o s e do not s i g n i f i c a n t l y a l t e r the o x i d a t i o n of p r o l i n e by the r e c t a l t i s s u e (Table I V ) . The colon a l s o o x i d i z e s p r o l i n e at a r a t e equivalent to that of the rectum. End-products of P r o l i n e Metabolism Table V shows the concentrations of ammonia and a l a n i n e i n the r e c t a l t i s s u e during o x i d a t i o n of endogenous substrates or 25 mM p r o l i n e . I f p r o l i n e i s completely-oxidized to C0„, water and ammonium according to the s t o i c h i o m e t r y of 1 p r o l i n e — ) 5 C0 o + 1 NH. , then r e c t a producing 14 ^ 4 p r o l i n e - d e r i v e d CO at a r a t e of 18.69 nmol/mg hr (Table IV) should produce 3.73 nmol NH^ /mg hr i n the steady s t a t e . I f a l a n i n e , water and CO^ a r e the end-products of p r o l i n e o x i d a t i o n i n l o c u s t r e c t a , as shown i n the equation, 1 p r o l i n e - ^ 2 CO. + 1 a l a n i n e , then production of 18.69 14 nmol CO^/mg hr (Table IV) should y i e l d 9.34 nmol alanine/mg hr. An estimate of the average r a t e of net ammonium and a l a n i n e production f o r the 1.33 hours the t i s s u e i s exposed to 25 mM p r o l i n e i s 2.76 nmol/mg hr and 3.76 nmol/mg h r , r e s p e c t i v e l y ( c a l c u l a t e d from Table V). However, these r a t e s may be underestimates of the steady s t a t e r a t e s of ammonium al a n i n e production because they are c a l c u l a t e d from the t o t a l accumulation of end-product over the pr e i n c u b a t i o n p e r i o d plus the period of steady s t a t e p r o l i n e o x i d a t i o n . However, i t appears that ammonium production by the rectum during p r o l i n e o x i d a t i o n i s c l o s e r to the p r e d i c t e d value than i s a l a n i n e production„under the same c o n d i t i o n s . D i s c u s s i o n The procedure f o r i s o l a t i n g r e c t a l mitochondria used i n t h i s study produced m i t o c h i n d r i a w i t h reasonable RCR's which are capable of o x i d i z i n g most of the t e s t s u b s t r a t e s . R e c t a l RCR values are s i m i l a r to those of, obtained by Mandel et_ a l . (1980a) f o r mitochondria i s o l a t e d from Manduca midgut when pyruvate or o{ - k e t o g l u t a r a t e are s u b s t r a t e s . The ADP:0 r a t i o s i n Table I are lower than the t h e o r e t i c a l values of 3 f o r NAD-linked sub^ s t r a t e s or 2 f o r FAD-linked s u b s t r a t e s . However, H i n k l e and Yu (1979) b e l i e v e that these t h e o r e t i c a l values are too high since the hydrogen gradient across the m i t o c h o n d r i a l membranes can energize other processes besides the phosphorylation of ADP. The ADP:0 r a t i o s and RCR's obtained from r e c t a l mitochondria o x i d i z i n g pyruvate plus malate are s u b s t a n t i a l l y lower thant the RCR (eO) and ADP:0 r a t i o (2.7) reported f o r Locusta 29 f l i g h t muscle mitochondria (Khan and de K o r t , 1978). This d i f f e r e n c e may be due to the r e l a t i v e f r a g i l i t y of the r e c t a l mitochondria so that i t i s not p o s s i b l e to i s o l a t e h i g h l y coupled mitochondria from the l o c u s t r e c t a R e c t a l mitochondria have a s u b s t a n t i a l r a t e of endogenous r e s p i r r ^ . t i o n i n the presence of ADP, i n d i c a t i n g that metabolites are trapped w i t h i n an i n t a c t inner m i t o c h o n d r i a l membrane. Exogenous pyruvate i s oxidized,, suggesting that there i s s u f f i c i e n t endogenous oxaloacetate to p a r t i c i p a t e i n the c i t r a t e synthase r e a c t i o n . However, a higher r a t e of pyruvate o x i d a t i o n i s achieved when the TCA c y c l e i s "primed" w i t h malate (Table I.) . Priming of the TCA c y c l e f o r maximal pyruvate o x i d a t i o n i s necessary i n i s o l a t e d mitochondria from s e v e r a l i n s e c t f l i g h t muscles (reviewed by Hansford and Sacktor, 1971). However, Slack and B u r s e l l (1976) b e l i e v e that priming i s not necessary i n v i v o and i s only necessary i n v i t r o when i s o l a t i o n procedures cause the mitochondria to lea k TCA intermediates. The i s o l a t e d mitochondria from l o c u s t r e c t a are probably somewhat "l e a k y " sinde NADH, which presumably cannot penetrate through the inner m i t o c h o n d r i a l membrane, i s o x i d i z e d (Table I ) . The r e s u l t s of the experiments presented i n t h i s chapter i n d i c a t e that the l o c u s t rectum i s capable of o x i d i z i n g amino aci d s and carbo-hydrates, but has a l i m i t e d c a p a c i t y f o r l i p i d o x i d a t i o n . The rectum has a very low a c t i v i t y of t h e / 3 - o x i d a t i o n enzyme, 3-hydroxy-acyl-CoA dehy-drogenase, and the i s o l a t e d mitochondria do not r e a d i l y o x i d i z e p a l m i t o y l c a r n i t i n e . In contrast-, the l o c u s t f l i g h t muscle o x i d i z e s l i p i d s to support extended periods of f l i g h t (Weis-Fogh, 1952) and so i t i s not s u r p r i s i n g that 3-hydroxy-acyl-CoA dehydrogenase a c t i v i t y i s 10 times higher i n the f l i g h t muscle than that i n the rectum. In additon, mito-chondria i s o l a t e d from l o c u s t f l i g h t muscle consume oxygen at a high s t a t e - 3 r a t e when o x i d i z i n g p a l m i t o y l c a r n i t i n e plus malate (191 nmol 0^/ min mg. protein;- Khan and deKort, • 1978) . Both f l i g h t muscle and rectum of l o c u s t s have very low a c t i v i t i e s of l a c t a t e dehydrogenase i n d i c a t i n g l i m i t e d c a p a c i t y f o r anaerobic metabolism. This supports the observations that the rectum i n v i t r o cannot a c t i v e l y t r a n s p o r t ions when o x i d a t i v e metabolism i s i n h i b i t e d (Baumeister et a l . , 1981; Herrera et a l . , 1977; W i l l i a m s et a l . , 1978). In the absence of l a c t a t e dehydrogenase, i n s e c t f l i g h t muscles have developed a d i f f e r e n t pathway to regenerate the NAD reduced i n g l y c o l y s i s . 30 Zebe et a l . (1959) demonstrated the importance of the ° C-glycerol phos-phate c y c l e f o r t r a n s f e r r i n g hydrogens across the m i t o c h o n d r i a l membranes of l o c u s t f l i g h t muscles. This c y c l e appears to be absent i n the l o c u s t rectum because there i s l i t t l e c y t o s o l i c NAD-1 inked ©^-glycerol phosphate dehydrogenase and: the mitochondria do not. o x i d i z e ^ - . g l y c e r o l phosphate. The rectum may .possess a-malate-aspar-tate s h u t t l e , (see .Lehninger., 1975) s i n c e the rectum contains maihte dehydrogenase and glutamte-oxaloacetate ' transaminase^,; two enzymes inv o l v e d in^ t h i s s h u t t l e ^ Weis-Fogh (1952) showed that l o c u s t f l i g h t muscle o x i d i z e s carbohy-drates during the i n i t i a l periods of f l i g h t . A c c o r d i n g l y , the f l i g h t muscle has the enzymes necessary f o r carbohydrate degradation. The rectum , has glycogen phosphorylase, t r e h a l a s e and hexokinase a c t i v i t i e s s i m i l a r to those found i n the f l i g h t muscle. However, only 34% of the r e c t a l phosphorylase i s e x t r a c t e d i n a c t i v e form. This suggests that t h i s enzyme i s under modulation, p o s s i b l y i n v o l v i n g a hormonal c o n t r o l mech-anism. Tolman and S t e e l e (1980a) found that the glycogen phosphorylase i n cockroach rectum and colon was a c t i v a t e d by e x t r a c t s of corpus c a r -dicum. They suggested that a p u t a t i v e a n t i d i u r e t i c hormone could s t i m -u l a t e glycogen breakdown to support the work of a c t i v e i o n t r a n s p o r t , and hence f l u i d r e a b s o r p t i o n . A s i m i l a r system may work i n the l o c u s t rectum s i n c e a c t i v e t r a n s p o r t of c h l o r i d e i s under hormonal c o n t r o l (Spring et a l . , 1978; Spring and P h i l l i p s , 1980a,b,c). Although carbohydrates can be u t i l i z e d by the rectum, the i n t r a c e l l - _ • u l a r s t o r e s of glycogen and glucose are s u b s t a n t i a l l y l e s s than those of p r o l i n e ( f i g . 1). B a l s h i n (1973) f i r s t measured the l e v e l s of p r o l i n e (60-70 mM) i n the r e c t a l t i s s u e and measurements done i n t h i s study con-f i r m h i s observations ( f i g . 1). The high c o n c e n t r a t i o n of p r o l i n e i n the r e c t a l t i s s u e suggested that t h i s amino a c i d i s normally o x i d i z e d by the rectum. The f l i g h t muscles of i n s e c t s which o x i d i z e p r o l i n e (e.g. L e p t i n o t a r s a decemilineata) a l s o have high l e v e l s of i n t r a c e l l u l a r p r o l i n e (35 nmol/mg; Weeda et a l . , 1979). In c o n t r a s t , the l o c u s t f l i g h t muscle contains only 8 nmol/mg (Rowan and Newsholme, 1979) and i t has been, shown that p r o l i n e o x i d a t i o n could provide only 13-26% of the energy r e q u i r e d f o r l o c u s t f l i g h t (Brosemer and Veerabhadrappa, 1965). The p r e d i c t i o n that p r o l i n e i s o x i d i z e d by the r e c t a l t i s s u e i s supported by the r e s u l t s of the enzyme assays and s u b s t r a t e o x i d a t i o n by i s o l a t e d mitochondria. The rectum contains c o n s i d e r a b l y more glutamte dehydrogenase, glutamate-TABLE VI Oxygen consumption of mitochondria i s o l a t e d from l o c u s t rectum and f l i g h t muscles of d i f f e r e n t i n s e c t s Insect P r o l i n e Oxygen Consumption (nmol/min-mg p r o t e i n ) Pyruvate P a l m i t o y l C a r n i t i n e S c h i s t o c e r c a * (rectum) Locusta Phormia G l o s s i n a L e p t i n o t a r s a P o p i l l i a 104 2 5 l 95 2 1445 4 594 5 569 6 58 259 1 490 2 9 4 172 5 465 6 47 191 2 n.r. 0 4 4 2 5 n.r. Pyruvate and p a l m i t o y l c a r n i t i n e o x i d a t i o n i s u s u a l l y adided by the ad-d i t i o n of TAC intermediates or K H C O 3 plus ATP. Sources f o r the l i t e r a t u r e values are i n d i c a t e d by the s u p e r s c r i p t : 1. de Kort et a l . , 1973 2. Khan and dekort, 1978 3. Sactor, 1976 4. B u r s e l l and Slack, 1976 5. Weeda et a l . , 1980 6. Hansford and Johnson, 1975 8 t h i s study 32 TABLE V I I A c t i v i t i e s of m i t o c h o n d r i a l enzymes from l o c u s t rectum and f l i g h t muscle of the Japanese b e e t l e , P o p i l l i a j a p b r i i c a Tissue Source Enzyme A c t i v i t y (nmol/min'mg p r o t e i n ) Pro Ox GDH GPT. MDH NAD^ME NADP-ME Locust Rectum 14 211 563 931 14 45 Beet l e F l i g h t Muscle 107 4355 132 -n.r. 395 168 Values f o r Popdtllia are from Hansford and Sacktor, 1975 Abbre v i a t i o n s are Pro Ox, p r o l i n e oxidase; GDH, glutamate dehydrogenase; MDH, malate dehydrogenase; NAD-^ ME, NAD-dependent malic enzyme; NADP-ME, NADP-dependent ma l i c enzyme. n.r. = not reported 33 pyruvate transaminase and glutamate-oxaloacetate transaminase than the f l i g h t muscle (Table I I ) , suggesting that amino a c i d metabolism may be important source-of energy f o r the rectum. The r e c t a l mitochondria r e a d i l y o x i d i z e p r o l i n e and p r e f e r i t over other s u b s t r a t e s t e s t e d . P r o l i n e o x i d a t i o n i s maximal when the p r o l i n e c o n c e n t r a t i o n i s a t l e a s t 30 mM (see r e s u l t s ) . This i n d i c a t e s that p r o l i n e oxidase i s saturated i n v i v o s i n c e i n t r a c e l l u l a r p r o l i n e c o n c e n t -r a t i o n arercver'60 nmol/mg. The absolute r a t e of p r o l i n e u t i l i z a t i o n by i s o l a t e d r e c t a l mitochondria i s l e s s than that i n f l i g h t muscle mito-chondria i s o l a t e d from i n s e c t s which o x i d i z e p r o l i n e (Table VI) and, a c c o r d i n g l y , the a c t i v i t i e s of enzymes i n v o l v e d i n p r o l i n e metabolism (Table V I I ) are lower i n r e c t a l mitochondria. The high r a t e of ox i d a -t i o n by f l i g h t muscle mitochondria r e f l e c t s the l a r g e metabolic f l u x r e q u i r e d to support f l i g h t . Although f r e s h l y d i s s e c t e d r e c t a (2.6 nmol O^/min'mg f r e s h weight.; see Chapter V) and l o c u s t f l i g h t muscle (3.7 nmol 0^/ mining; Robinson and Goldsworthy, 1974) have s i m i l a r r a t e s of oxygen consumption, f l i g h t r e q u i r e s up to a 100-fold increase i n the meta-b o l i c r a t e of the muscle (Sacktor,. 1976; Kammer and H e i n r i c h , 1978). On the other hand ? s t i m u l a t i o n of c h l o r i d e transport only causes only a 20% i n oxygen consumption by the r e c t a l t i s s u e (see Chapter V ) . Brosemer and Veerabhdrappa (1965) s t u d i e d p r o l i n e o x i d a t i o n i n the f l i g h t muscles of three species of i n s e c t s . They found that p r o l i n e i s converted to glutamate v i a two s e q u e n t i a l enzymes, f l a v i n - l i n k e d p r o l i n e dehydrogenase and NAD-linked - p y r r o l i n e - 5 - c a r b o x y l a t e dehydrog-enase. The p r o l i n e oxidase of l o c u s t rectum a l s o appears to be f l a v i n -l i n k e d because rotenone does not completely i n h i b i t oxygen consumption of mitochondria o x i d i z i n g p r o l i n e ( f i g . 3 ). The f a t e of p r o l i n e - d e r i v e d glutamate d i f f e r s i n d i f f e r e n t i n s e c t s . In the b l o w f l y , Phormia r e g i n a , p r o l i n e increases'the rate, of pyruvate o x i d a t i o n i n i s o l a t e d sarcosomes (Sacktor and C h i l d r e s s , 1967). I t i s thought that p r o l i n e crosses the inner m i t o c h o n d r i a l membrane, i s con-v e r t e d to glutamate and t h i s glutamate enters,the TCA c y c l e v i a glutamate-pyruvate transaminase (see f i g . 4a). The oxaloacetate produced from p r o l i n e condenses w i t h acet.yl-CoA derived from g l y c o l y s i s and insures a maximal r a t e of pyruvate o x i d a t i o n . Other i n s e c t s u s e p r o l i n e as a primary source of energy f o r f l i g h t . 34 Figure 4. P r e v i o u s l y proposed pathways f o r p r o l i n e o x i d a t i o n i n i n s e c t f l i g h t muscles. (a) B l o w f l y (Phormia r e g i n a ) , Sacktor and C h i l d r e s s (1967). (b) Tsetse - f l y (Glossina  m o r s i t a n s ) , B u r s e l l (1977). (c) Japanese b e e t l e ( P o p i l l i a j a p o n i c a ) , Hansford and Sacktor (1975). A b b r e v i a t i o n s used i n t h i s f i g u r e are: GDH, glutamate dehydrogenase; GPT, glutamate-pyruvate transaminase; ME, "malic enzyme"; MDH, malate dehydrogenase; oaa, oxaloacetate; 0< -kg, cA-ketoglutarate; a c e t y l CoA; S-acetyi-Coenzyme A. glycolysis-Hsyruvate-^acetyl CoA proline proline a b pyruvate — • acetyl CoA ME malate 36 Tsetse f l i e s have very l i t t l e muscle glycogen (Norden< and Paterson, 1969) and r e l y on p r o l i n e o x i d a t i o n to supply energy during prolonged f l i g h t . The sarcosomes of t s e t s e f l i e s o x i d i z e e x c l u s i v e l y p r o l i n e ( B u r s e l l and Slack, 1976) and y i e l d a l a n i n e and some ammonia as end-products. The proposed pathway of p r o l i n e o x i d a t i o n i n tsetse f l y i s shown i f f i g . 4b ( B u r s e l l , 1977). B u r s e l l and Slack (1976) reported that 20% of the p r o l i n e enters the TCA c y c l e v i a glutamate dehydrogenase and the r e s t enters v i a the transaminase. The pyruvate necessary f o r the transamination i s derived from malate v i a "malic enzyme" (Hoek et al» 1976). The p r o l i n e ^ w h i c h enters the TCA c y c l e v i a glutamate dehydroge-nase i s probably f u l l y o x i d i z e d v i a pyruvate dehydrogenase (17% of the t o t a l p r o l i n e consumed, B u r s e l l and Slack, 1976). Enzyme p r o f i l e s f o r t s e t s e , f l y sarcosomes support t h i s model because enzymes not i n v o l v e d i n t h i s pathway ( c i t r a t e synthase, a c o n i t a s e , i s o c i t r a t e dehydrogenase, malate dehydrogenase) are found i n very low a c t i v i t y (Norden and Matanganyidze, 1979). Mitochondria i s o l a t e d from the f l i g h t muscles of two b e e t l e s , P o p i l l i a j a p o n i c a (Hansford and Jphnspnj 1975) and L e p t i n o t a r s a  d ecemilineata (Khan and deKort, 1978; de Kort ejt a l . , 1973) a l s o v i g -our s l y o x i d i z e p r o l i n e , but can a l s o o x i d i z e pyruvate. However, the end-product of m i t o c h o n d r i a l p r o l i n e o x i d a t i o n i n P o p i l l i a i s ammonia and i n L e p t i n o t a r s a i t i s a l a n i n e (Hansford and Johnson, 1975; Weeda et a l . , 1980). Hansford and Johnson (1975) suggest that p r o l i n e i s f u l l y o x i d i z e d i n P o p i l l i a mitochondria (see f i g . 4c) w h i l e Khan and de Kort (1978) propose that p r o l i n e i s only p a r t i a l l y o x i d i z e d i n L e p t i n o t a r s a (see f i g . 4b). P r o l i n e carbons could enter the TCA c y c l e v i a glutamate dehydrogenase or glutamate-pyruvate transaminase i n the l o c u s t rectum s i n c e both enzymes are present i n the mitochondria (Table I I I ) . P r o l i n e o x i d a t i o n by i s o l a t e d r e c t a l mitochondria i s not i n h i b i t e d by aminooxyacetate. In a d d i t i o n , aminooxyacetate does not i n h i b i t the s h o r t - c i r c u i t current of the ±a v i t r o rectum when i t i s o x i d i z i n g predominately p r o l i n e (Chapter I V ) . These observations suggest that p r o l i n e carbons must enter the TCA c y c l e v i a glutamate dehydrogenase (see f i g . 5 ) . However, p r o l i n e o x i d a t i o n by r e c t a l mitochondria i s not i n h i b i t e d by ammonium ( r e s u l t s ) as i t i s i n the tsetse f l y ( B u r s e l l and Slack, 1976). This may i n d i -cate that the r e c t a l glutamate dehydrogenase i s l e s s s e n s i t i v e to product 37 Figure 5. Proposed model f o r p r o l i n e o x i d a t i o n by l o c u s t rectum. Abb r e v i a t i o n s used i n t h i s f i g u r e are: GDH, glutamate dehydrogenase; GPT, glutamate-pyruvate transaminase; ME, "malic enzyme"; MDH, malate dehydrogenase; oaa, oxaloacetate; o< -kg; c< - k e t o g l u t a r a t e ; a c e t y l CoA; S-acetyl-Coenzyme A. glycolysis » , pyruvate A acetyl CoA ME oaa malate /M rate <*-kg pyruvate Jff glutamate glycolysis j proline 39 i n h i b i t i o n than the t s e t s e f l y enzyme ( B u r s e l l , 1975). Tissue o x i d a t i o n of p r o l i n e leads to a production of both a l a n i n e and ammonia (Table V). The amino acceptor, pyruvate, necessary f o r a l a n i n e production could be produced from p r o l i n e as i n the t s e t s e f l y (see f i g . 4b). However, u n l i k e the t s e t s e f l y , the l o c u s t rectum can produce pyruvate from g l y c o l y s i s and t h i s pyruvate would be a v a i l a b l e f o r ; t r a n s a m i n a t i o n . Weeda 'etz'al.- (1980) have a l s o suggested that g l y -c o l y t i c a l l y - d e r i v e d pyruvate could be an amino acceptor f o r a l a n i n e pro-d u c t i o n i n L e p t i n o t a r s a f l i g h t muscle. Because ammonia i s produced by the r e c t a l t i s s u e during p r o l i n e o x i d a t i o n , at l e a s t some of the p r o l i n e must be f u l l y o x i d i z e d . In the absence of g l y c o l y t i c a l l y - d e r i v e d pyruvate (as i n i s o l a t e d mitochondria), p r o l i n e must provide the carbons f o r acetyl-CoA so that the TCA c y c l e can continue to operate. This i s accomplished by producing pyruvate v i a m a l i c enzyme ( f i g . 5) and then o x i d i z i n g t h i s pyruvate v i a pyruvate dehydrogenase and the TCA c y c l e . The r e c t a l mitochondria c o n t a i n both NAD- and NADP-dependent ma l i c enzyme, w i t h the a c t i v i t y being 3 times higher w i t h NADP as the coenzyme. This i s d i f f e r e n t from the enzyme p r o f i l e s of sarcosomes of the t s e t s e f l y and b e e t l e s i n which the NAD-dependent enzyme i s found i n higher a c t i v i t y (Hoek et a l . , 1976; Weeda et a l . , 1980; Hansford and Johnson, 1975). Because-there' are two p o s s i b l e sources of pyruvate, g l y c o l y s i s and p r o l i n e , i n the l o c u s t rectum, there may be some competition f o r pyruvate dehydrogenase. There may be some c o n t r o l at malic enzyme to i n s u r e that pyruvate i s only produced from malate when pyruvate supply from g l y c o l y -s i s i s low. Hansford and Johnson (1975) showed that b e e t l e m a l i c enzyme i s i n h i b i t e d by pyruvate and perhaps the same c o n t r o l occurs i n the rectum. However, i n t e r e s t i n g l y , a d d i t i o n of u n l a b e l l e d glucose to r e c t a 14 14 o x i d i z i n g C - p r o l i n e does not i n h i b i t C0 2 production (Table I V ) . There i s a l s o very high a c t i v i t y of malate dehydrogenase i n the r e c t a l mitochondria. This suggests that p r o l i n e could supply oxaloacetate to condense w i t h acetyl-CoA derived from e i t h e r p r o l i n e or g l y c o l y s i s . The c o n t r o l processes a c t i n g on the m a l i c enzyme/malate dehydrogenase branch p o i n t were not i n v e s t i g a t e d so i s not p o s s i b l e to d i s c u s s q u a n t i -t a t i v e p a r t i t i o n i n g of p r o l i n e carbon at t h i s p o i n t . Pr oline o x i d a t i o n by the r e c t a l t i s s u e i s not s t i m u l a t e d by 1 mM 1 40 cAMP. This c o n c e n t r a t i o n of cAMP f u l l y s t i m u l a t e s a c t i v e c h l o r i d e t r a n s -port by the rectum (Spring et a l . . , 1978; Spring and P h i l l i p s , 1980a,b,c); however, t h i s increase i n a c t i v e c h l o r i d e t r a n s p o r t c o n s t i t u t e s only 20% of the t o t a l work of the t i s s u e (see Chapter V) and th e r e f o r e d i f f e r e n c e s 14 i n CO- e v o l u t i o n may not be d e t e c t a b l e . 14 14 The colon a l s o o x i d i z e s C - p r o l i n e to CO2-(Table IV)" and Mommsen (personal communication) r e p o r t s that l o c u s t Malpighian tubules v i g o u r o u s l y o x i d i z e p r o l i n e . These observations suggest that p r o l i n e may be an important me.taboMc---substrate f o r other l o c u s t e p i t h e l i a and warrents f u r t h e r i n v e s t i g a t i o n . 41 CHAPTER I I I POTENTIAL • EXOGENOUS SUBSTRATES-FOR • - • THE LOCUST RECTUM. . ' I n t r o d u c t i o n In the previous chapter i t was shown that the l o c u s t rectum i s cap-able of o x i d i z i n g carbohydrates and amino a c i d s , e s p e c i a l l y p r o l i n e . The rectum must u l t i m a t e l y r e l y on a supply of exogenous substrates to support a c t i v e i o n and water t r a n s p o r t . The rectum i s bathed by two f l u i d s which could supply these s u b s t r a t e s : the hemolymph and the Malpighian tubule f l u i d . The remains of the digested food passing p o s t e r i o r l y from the midgut might a l s o be u t i l i z e d by the rectum. However, glucose and amino a c i d s are l a r g e l y absorbed by the l o c u s t midgut (Treherne, 1958; 1959) so the f l u i d passing out of the midgut i s probably low i n these s u b s t r a t e s . Locust hemolymph has high l e v e l s of f r e e amino a c i d s and t r e h a l o s e (Rutherford and Webster, 1978; Benassi et a l . , 1961; Treherne, 1959) and could supply the rectum w i t h these metabolic s u b s t r a t e s . However, l i t t l e i s known about the content of sugars and amino acid s i n l o c u s t Malpighian tubule f l u i d . The i n v i t r o .tubules of S c h i s t o c e r c a ^ passively transport-sugars w i t h a ."urine to plasma" (U/P) r a t i o never exceeding 1 (Maddrell and Gardiner, 1974). Locusta tubules a c t i v e l y reabsorb glucose ( R a f a e l i -B e r n s t e i n and Mordue, 1979; Mordue and R a f a e l i - B e r n s t e i n , 1978). I f S c h i s t o c e r c a tubules are s i m i l a r to those of Locusta then probably very l i t t l e glucose would reach the r e c t a l lumen v i a the Malpighian tubule f l u i d . Amino a c i d s have been detected i n the Malpighian tubule f l u i d s of a v a r i e t y of i n s e c t s , but no evidence of a c t i v e s e c r e t i o n ( i . e . U/PJ^ 1) of these compounds has been reported. Berridge (1965) detected amino n i t r o g e n i n Dysdercus u r i n e in v i v o , but the u r i n e amino n i t r o g e n con-c e n t r a t i o n never exceeded that of the hemolymph. In v i t r o tubules of Carausius (then D i x i p p u s , Ramsay, 1958) and Rhodnius (Maddrell and Gardiner, 1974) a l s o p a s s i v e l y t r a n s p o r t amino a c i d s , although Rhodnius tubule c e l l s can a c t i v e l y accumulate these compounds under some c o n d i t i o n s (Maddrell and Gardiner, 1980). To determine normal l e v e l s of substrates a v a i l a b l e to r e c t a I mea-sured amino a c i d s and glucose i n the hemolymph and Malpighian tubule f l u i d of l o c u s t s . Amino a c i d measurements were c a r r i e d out w i t h animals 42 i n d i f f e r e n t s t a t e s of h y d r a t i o n to see i f amino a c i d l e v e l s are a l t e r e d during s t r e s s e s that t h i s desert i n s e c t normally experiences. In t h i s study I a l s o used i s o l a t e d Malpighian tubules to i n v e s t i g a t e whether amino aci d s move p a s s i v e l y or a c t i v e l y across t h i s e p i t h e l i u m . M a t e r i a l s and Methods Animals Adult male S c h i s t o c e r c a g r e g a r i a 2 to 4 weeks past t h e i r f i n a l molt were used i n a l l experiments. The colony environment and feeding regime were as described i n Chapter I I . However, the animals used f o r measurements of hemolymph volume (Table V I I I ) and hemolymph f r e e amino aci d s (Table IX) were fed a d i e t c o n s i s t i n g of a l f a l f a r a t h e r than grass. Pre-treatment of Dehydrated and Hydrated Animals Locusts p r e v i o u s l y fed t h e i r d a i l y l e t t u c e meal were placed i n d e s i c c a t o r s at 28°C f o r 48 hours without food. One d e s i c c a t o r contained water ( i . e . 100% r e l a t i v e humidity) and the l o c u s t s exposed to t h i s humid atmosphere are termed hydrated. Another d e s i c c a t o r contained concentra-ted s u l f u r i c a c i d ( i . e . 0% r e l a t i v e humidity) and the animals from t h i s a r i d atmosphere are termed dehydrated. Other l o c u s t s were dehydrated and starved f o r 48 hours as j u s t described and then placed i n a d e s i c c a t o r w i t h a humid atmosphere and f e d : l e t t u c e ad l i b i t u m f o r 24 hours. These animals are r e f e r r e d to as rehydrated. Determination of Hemolymph Volume To check that the treatments described above cause l a r g e changes i n the water balance of the animal, the hemolymph volume was measured i n hydrated, dehydrated and rehydrated l o c u s t s . Hemolymph volume was 3 estimated from the d i l u t i o n of H-methoxy i n u l i n i n j e c t e d i n t o the hemo-3 lymph. 5 / i l of H-methoxy i n u l i n ( t o t a l 25 nCi) was i n j e c t e d through one of the intersegmental membranes of the abdomen w i t h a Hamilton s y r i n g e . The animals were then placed i n a p l a s t i c tub which was mounted on a s p i n d l e of a kymograph motor. The tub and motor were surrounded w i t h a s t a t i o n a r y cardboard " w a l l " on which were drawn v e r t i c a l s t r i p e s . When the motor was turned on, the tub r o t a t e d and the l o c u s t s responded to the apparent motion of the s t r i p e s by walking. The l o c u s t s were walked 43 f o r 20 minutes to in s u r e that the i n u l i n was mixed throughout the l o c u s t s ' hemocoel. The neck was then punctured and 2 to 5 / i l a l i q u o t s of hemo-lymph were c o l l e c t e d w i t h a m i c r o c a p i l l a r y tube (Drummond Microcap) and d i l u t e d i n 10 ml s c i n t i l l a t i o n c o c k t a i l ( S c i n t a v e r s e , F i s h e r S c i e n t i f i c ) , 3 The H a c t i v i t y was determined w i t h a Nuclear Chicago Isocap l i q u i d s c i n t i -l a t i o n " c o u n t e r and quenching correc t e d by the channels r a t i o method. Hemolymph Amino Acids To determine i f hemolymph f r e e amino aci d s are regulated during osmotic stress,hemolymph was c o l l e c t e d from dehydrated, hydrated and rehydrated l o c u s t s . The neck was punctured w i t h a needle and the hemo-lymph c o l l e c t e d w i t h a m i c r o c a p i l l a r y tube. The hemolymph was then d i l u t e d i n 0.5 ml of 3.75% s u l p h o s a l i c y l i c a c i d (SSA) i n 0.2 N L i " c i t r a t e and analyzed f o r content of i n d i v i d u a l amino aci d s as described i n Chapter I I . C o l l e c t i o n of Malpighian Tubule F l u i d In Vivo Two methods f o r obt a i n i n g : Malpighian tubule f l u i d from hydrated and dehydrated animals were used. A 5 / i l g l a s s c a p i l l a r y tube (Corning) was dipped'in p a r a f f i n o i l and pushed through the anus of a r e s t r a i n e d l o c u s t . The tube was ge n t l y pushed a n t e r i o r l y u n t i l i t met r e s i s t a n c e at the k i n k i n the colon (about 1.5 cm from the anus). The Malpighian tubule f l u i d which flows i n t o the gut was sucked i n t o the tube by c a p i l -l a r y a c t i o n . However, t h i s method o f t e n , proved u n s u c c e s s f u l because f e c a l m a t e r i a l r e t a i n e d i n the rectum clogged the tube. The other method of Malpighian tubule f l u i d c o l l e c t i o n i n v o l v e d l i g a t i n g the ileum a n t e r i o r and p o s t e r i o r to the j u n c t i o n of the tubules w i t h the gut. The l i g a t i o n was performed by making a small i n c i s i o n i n the t h i r d abdominal segment, l i f t i n g up the gut w i t h a gl a s s hook and t y i n g the gut w i t h s u r g i c a l thread. A s i m i l a r o p e r ation was done on the gut l i f t e d throu,ghtan i n c i s i o n made i n the f i f t h abdominal segment. Before s e a l i n g the i n c i s i o n s w i t h Tackiwax (Cenco, C e n t r a l S c i e n t i f i c Company), a few c r y s t a l s of amaranth were put i n the hemocoel. Since l o c u s t tubules t r a n s p o r t t h i s dye (Lee, 1961), the presence of Malpighian tubule f l u i d i n the l i g a t e d gut segment and leaks beyond the l i g a t u r e s could be e a s i l y detected. A f t e r the operation the animal was kept at room temperature f o r 12 to 16 hours. The locusts were then idecapitated and the abdomen opened. The gut was then removed, the swollen ileum punctured 4 4 and the red Malpighian tubule f l u i d c o l l e c t e d w i t h a m i c r o c a p i l l a r y tube. This method proved more r e l i a b l e than the one i n v o l v i n g c a nnulation w i t h a g l a s s tube. The brown f l u i d found i n the midgut ahead of the a n t e r i o r l i g a t u r e was a l s o sampled at t h i s time. The midgut f l u i d and Malpighian tubule f l u i d were d i s s o l v e d i n SSA f o r amino a c i d a n a l y s i s or i n 0.6 N p e r c h l o r i c a c i d f o r glucose a n a l y s i s . The methods f o r glucose and amino a c i d analyses are described i n Chapter I I . Hemolymph samples were taken before l i g a t i o n or cannulation and prepared f o r glucose or amino a c i d a n a l y s i s as described above. To check that the l i g a t i o n o p e r ation i t s e l f d i d not a l t e r the hemolymph amino a c i d composition, and u l t i m a t e l y tubule f l u i d composition, hemo-lymph was c o l l e c t e d from hydrated.animals before and a f t e r sham operations. The hemolymph samples were analyzed as p r e v i o u s l y described. Amino A c i d S e c r e t i o n by In V i t r o Malpighian Tubules I n i t i a l i n vdivo s t u d i e s suggested that p r o l i n e was a c t i v e l y secreted by the Malpighian tubules. To see i f t h i s s e c r e t i o n could be d u p l i c a t e d i n v i t r o , thereby p e r m i t t i n g experimental p e r t u r b a t i o n s which might confiffimcthisiobservation, a method was designed to c o l l e c t i n v i t r o Malpighian tubule f l u i d f o r amino a c i d a n a l y s i s . The abdomen of the l o c u s t was cut open and a cannula of polyethylene tubing (PE 60) was i n s e r t e d through the r e c t a l w a l l and pushed i n t o the colon. The cannula was t i e d at the j u n c t i o n of the colon and rectum, the rectum was cut away from the cannula and the midgut severed a n t e r i o r to the tubule j u n c t i o n w i t h the gut. This gut s e c t i o n w i t h attached tubules was r i n s e d e x t e r n a l l y w i t h s a l i n e (Table XIV, s a l i n e A) and the contents of the lumen f l u s h e d out w i t h t h i s same s a l i n e . Excess f l u i d was removed w i t h f i l t e r paper and a i r was gently blown through the cannula to c l e a r the lumen of f l u i d . The gut was then l i g a t e d j u s t a n t e r i o r and p o s t e r i o r to the tubules and the cannula cut away. This small "sac" c o n s i s t i n g of a small segment of the ileum and the attached Malpighian tubules was put i n 2 ml of s a l i n e .containing 100 mM NaCl, 5 mM.KgSO^, 3.1 mM Na2HP0^, 1.9 mM NaH-PO., 25 mM NaHC0 o, 1-mM CaCl„, 93.9 mM sucrose,^15SM of a . 2 4 3 2 s i n g l e test.amino a c i d and t r a c e q u a n t i t i e s of amaranth. The s a l i n e was bubbled w i t h a gas mixture c o n s i s t i n g of 95% 0 2and 5% C0 2 and main-ta i n e d at 25°C. A f t e r 2 hours the sacs were removed from the s a l i n e , b l o t t e d dry and the secreted tubule f l u i d c o l l e c t e d and analyzed 45 as described i n the previous methods s e c t i o n . R e s u l t s Hemolymph Volume and Free Amino Acids Table V I I I shows the hemolymph volumes of l o c u s t s subjected to v a r i o u s treatments. Exposure to low humidity f o r 2 days without food (dehyrated s t a t e ) r e s u l t s i n almost a 50% drop i n hemolymph volume com-pared to hydrated animals. Consumption of l e t t u c e over 24 hours r e s t o r e s t h i s l o s t f l u i d (rehydrated s t a t e ) . Despite the almost 2 - f o l d change i n hemolymph volume during dehydration and subsequent r e h y d r a t i o n , the concentratiomof" i n d i v i d u a l f r e e amino a c i d s i n the hemolymph remain r e l a t i v e l y constant (Table I X ) . However, the l e v e l s of p r o l i n e and g l u t -amine -do f l u c t u a t e during the r e h y d r a t i o n process (Appendix A). Experiments w i t h Malpighian Tubules In Vivo Table X shows the amino a c i d l e v e l s i n Malpighian tubule f l u i d and hemolymph of dehydrated and hydrated l o c u s t s in v i v o . Amino a c i d s other than the ones shown were absent or found i n t r a c e amounts i n the tubule f l u i d . With the exception of glutamate and p r o l i n e , t h e concentrations of amino a c i d s i n the tubule f l u i d are g e n e r a l l y equal to or l e s s than those- found i n the hemolymph ( i . e . U/P 6 1 ) . P r o l i n e and glutamate have U/P r a t i o s much grea t e r than 1 suggesting that these amino a c i d s may be a c t i v e l y seer eted by l o c u s t Malpighian tubules (see D i s c u s s i o n ) . P r o l i n e , by f a r , i s the most abundant amino a c i d i n the tubule f l u i d , comprising 80% of the t o t a l amino a c i d c o n c e n t r a t i o n . The concentrations of amino a c i d s i n tubule f l u i d are s i m i l a r i n dehydrated and hydrated animals. However, the U/P r a t i o f o r p r o l i n e i s higher i n dehydrated animals probably because the hemolymph con c e n t r a t i o n of p r o l i n e i s s i g -n i f i c a n t l y lower (pfLo.05) than that i n hydrated animals. This i s q u i t e d i f f e r e n t from what i s shown i n Table IX where there i s no d i f f e r e n c e i n hemolymph p r o l i n e c o n c e n t r a t i o n between the two treatments. The reason f o r t h i s discrepancy i s unknown. Glucose i s a l s o found i n the Malpighian tubule f l u i d at a con c e n t r a t i o n higher than that i n the hemo-lymph (Table X). In c a l c u l a t i n g the U/P r a t i o f o r amino aci d s i n Table X, hemolymph concentrations were c a l c u l a t e d from samples taken before the l i g a t i o n 46 TABLE V I I I Hemolymph volume of l o c u s t s i n d i f f e r e n t s t a t e s of h y d r a t i o n Hydration State Hemolymph Volume (jul) Hydrated 134.2 + 11.3 (12) Dehydrated 70.7 + 8.0 (13) Rehydrated 122.4 + 8.7 '(5) Values are x + SE. Number i n parentheses i s the number of animals. For terminology regarding h y d r a t i o n s t a t e see t e x t . 47 TABLE IX Concentrations of amino a c i d s i n l o c u s t hemolymph Concentration (mM) Amino A c i d Hydrated Dehydrated Rehydrated Aspartate 0.13 + 0. 01 (14) 0. 37 + 0.11 (8) 0. 12 + 0. 03 (5) Threonine 0.36 + 0. 03 (14) 0. 46 + 0.11 (8) 0. 62 + 0. 15 (5) Serine 1.74 + 0. 22 (14) 3. 24 + 0.82 (8) 1. 65 + 0. 28 (5) Asparagine 0.79 + .0. 17 (13) 1. 39 + 0.38 (4) 1. 37 + 0. 48 (5) Glutamine 5.02 + 0. 45 (14) 7. 06 + 1.00 (8) 2. 77 + 0. 60 (4) Glutamate 0.09 + 0. 01 (11) 0. 11 + 0.04 (4) 0. 10 + 0. 02 (4) P r o l i n e 12.51 + 0. 77 * (14) 15. 17 + 3.30 (8) 12. 10 + 1. 37 (5) G l y c i n e 16.70 + 1. 61 (9) 13. 34 + 0.84 (7) 12. 65 + 1. 74 (4) A l a n i n e 0.69 + 0. 07 (14) 1. 20 + 0.18 (6) 0. 94 + 0. 26 (5) Valine. 0.46 + 0. 05 (13) 0. 58 + 0.15 (6) 0. 73 + 0. 15 (5) Methionine 0.32 + 0. 03 (14) 0. 58 + 0.11 (8) 0. 34 + 0. 06 (5) I s o l e u c i n e 0.27 + .0. 02 (13) 0. 41 + 0.06 (8) 0. 54 + 0. 16 (5) Leucine 0.27 + 0. 10 (11) 0. 46 + 0.09 (7) 0. 54 + 0. 15 (5) Tyrosine 0.74 + 0. 05 (14) 0. 88 + 0.12 (8) 1. 26 + 0. 21 (5) Phenylalanine 0.51 + 0. 06 (13) 0. 74 + 0.15 (8) 1. 00 + 0. 21 (5) L y s i n e 0.96 + 0. 12 (14) 0. 90 + 0.09 (8) 0. 97 + 0. 12 (5) H i s t i d i n e 1.14 + 0. 08 (14) 2. 12 + 0.64 (5) 1. 13 + 0. 20 (5) A r g i n i n e 1.33 + 0. 14 (14) 1. 69 + 0.21 (3) 1. 52 + 0. 28 (5) T o t a l 44.03 50.70 40.35 Values are x + SE. Number i n parentheses i s the number of anima l s . Values f o r the t o t a l amino a c i d content i s the sum of the mean values f o r the. indiv,idual:':amino" a c i d s . For terminology regarding the s t a t e of hy d r a t i o n see t e x t . 48 TABLE X Concentrations of f r e e amino a c i d s and glucose i n the hemolymph and Malpighian tubule f l u i d of l o c u s t s Concentration (mM) Aminos Acid'•or- n Malpighian Tubule Hemolymph (P) /U/P Sugar .r --, ' F l u i d (U) (a) Hydrated Locusts Aspartate 8 0. 54 + 0.84 0. 43 + 0.11 1. 85 + .0.52 Serine 4 0. 71 + 0.16 2. 29 + 0.45 0. 23 + 0.05 Glutamate 3 1. 13 + 0.23 0. 20 + 0.01 5. 79 + 1.25* Glutamine 6 0. 54 + 0.1>5 3. 41 + 0.60 0. 15 + 0.20 P r o l i n e 8 38. 02 + 3.53 12. 01 + 1.10 3. 21 + 0.21* G l y c i n e 8 3. 58 + 0.63 14. 27 + 1.28 0. 25 + 0.04 Alanine 7 0. 96 + 0.28 1. 25 + 0.35 1. 38 + 0.69 Glucose 6 4. 57 + 1.03 2. 44 + 0.30 1. 94 + 0.43* (b) Dehydrated Locus t s Aspartate 3 0. 53 + .0.27 0. 88 + 0.70 1. 13 + 0.45 Serine 3 1. 00 + 0.68 4. 50 + 3.90 0. 35 + 0.13 Glutamate 2 IJ). 58 (0, .43-0.74) 0. 99 (0. .78-1.19) . 0. 66 (0. .36-0.95) Glutamine 3 1. 11 + 0.74 2. 91 + 0.84 0. 32 + 0.14 P r o l i n e 10 43. 85 + 4.07 8. ,49 + 1.13 6. 20 + 1.15* G l y c i n e 9 3. 93 + 1.01 14. ,26 + 1.82 0. 29 + 0.08 A l a n i n e 2 0. ,74 (0 .72-0.76) 0. ,94 (0, .49-1.40) 1. ,02 (0, • 5 1f.S4) Values are x + SE unless,;.n<3 then values are x (range) . A l l values f o r Malpighian f l u i d content were obtained by gut l i g a t i o n except glucose which was obtained by ca n n u l a t i o n (see t e x t f o r methods). An a s t e r i s k denoted a U/P r a t i o which i s s i g n i f i c a n t l y greater than 1 (p£0.05). 49 operation. I f the operation i t s e l f elevated hemolymph amino a c i d l e v e l s , then the r e l a t i v e l y high concentrations of p r o l i n e and glutamate i n the tubule f l u i d might r e f l e c t p a s s i v e - d i f f u s i o n across the tubule w a l l from a r t i f i c i a l l y high l e v e l s of amino a c i d s i n the hemolymph. Such an a r t -i f a c t i s u n l i k e l y because sham operated l o c u s t s d i d not e x h i b i t , s i g n i f -i c a n t changes (except g l y c i n e ) i n the hemolymph amino a c i d c o n c e n t r a t i o n Table X I ) . Remnants of midgut f l u i d might contaminate the Malpighian f l u i d which accumulates i n the l i g a t e d gut segment and thereby c o n t r i b u t e to an e r r o r i n e s t i m a t i n g tubule f l u i d amino a c i d composition. Table X I I shows the c o n c e n t r a t i o n of amino acid s i n midgut f l u i d . I f some of t h i s f l u i d was i n i t i a l l y trapped between the l i g a t u r e s i t would be d i l u t e d by the Malpighian tubule s e c r e t i o n s . Since p r o l i n e i s r e l a t i v e l y low i n the midgut f l u i d (4.7 mM) the high concentration of p r o l i n e i n the tubule f l u i d (38-44 mM, Table X) could not be due to contamination from the midgut f l u i d . However, i t i s .possible that amino acid s found i n very low c o n c e n t r a t i o n i n the c o l l e c t e d Malpighian tubule f l u i d (evgy. s e r i n e and glutamine) might be p a r t i a l l y derived from the midgut f l u i d . I t i s p o s s i b l e that the ileum might reabsorb some water from the tubule f l u i d trapped between the l i g a t u r e s . I f p r o l i n e was a c t u a l l y found i n low c o n c e n t r a t i o n i n the tubule f l u i d and water was more r a p i d l y reabsorbed than p r o l i n e by the ileum, then a high c o n c e n t r a t i o n of p r o l i n e might be observed i n the remaining trapped f l u i d . However, t h i s e r r o r seems u n l i k e l y because the tubule f l u i d p r o l i n e concentrations are the same whether c o l l e c t e d by l i g a t i o n or c a n n u l a t i o n , which does not a l l o w pose s i b l e water r e a b s o r p t i o n by the ileum (Table X I I I ) . Hanrahan (personal communication) a l s o found that K +, N a + and' C l i n tubule f l u i d , , c o l l e c t e d by a l i g a t i o n method very s i m i l a r to the one described here, were detected i n c oncentrations very s i m i l a r to those found by P h i l l i p s (1964c) us i n g a c a n n u l a t i o n technique. F i n a l l y , Dow (1981) showed that the l o c u s t ileum _in v i t r o reabsorbs water to a very small extent. Experiments w i t h Malpighian Tubules In V i t r o F i g ure 6 shows the U/P r a t i o s obtained f o r _in v i t r o l o c u s t tubules exposed to 15 mM p r o l i n e , g l y c i n e or a l a n i n e . P r o l i n e i s the only amino a c i d which has a U/P r a t i o ( c o n c e n t r a t i o n i n secreted f l u i d / c o n c e n t r a t i o n i n bathing s a l i n e ) greater than 1. P r o l i n e trapped i n the tubules TABLE XI Free amino a c i d s i n the hemolymph of l o c u s t s before and a f t e r sham operations Concentration (mM) Amino A c i d Before A f t e r Aspartate 0.78 + .0.13 0.55 + 0.17 Threonine 1.11 + 0.10 0.69 + .0.09 Serine 4.19 + 0.81 2.85 + 0.47 Asparagine 1.52 + 0.54 0.83 ± 0.22 Glutamine 8.62 + 1.16 7.82 + 1.28 Glutamate 0.24 + 0.08 0.14 + 0.04 P r o l i n e 11.80 + 1.14 13.10 + .0.77 Gl y c i n e 13.06 + 0.78 15.74 + .1.06* Al a n i n e 4.15 + 0.69 2.70 + 0.44 Values are x + SE of s i x determinations. The a s t e r i s k denotes a s i g n i f i c a n t d i f f e r e n c e (p^O.05) between amino a c i d l e v e l s before and a f t e r the o p e r a t i o n . TABLE X I I Free amino acid s i n midgut f l u i d Amino A c i d Concentration (mM) Aspartate 0.51 + 0. 18 Threonine 0.20 Serine 1.37 + 0. 38 Asparagine 0.44 + 0. 07 Glutamine 3.44 + 1. 55 Glutamate absent P r o l i n e 4.67 + 2. 82 G l y c i n e 3.57 + 1. 83 Ala n i n e 0.43 + 0. 14 V a l i n e absent Cy s t i n e 1.42 + 0. 10 Methionine 1.89 + 0. 81 I s o l e u c i n e 0.20 + 0. 08 Leucine 0.43 + 0. 29 Tyrosine 0.45 + 0. 16 Phenylalanine 0.50 + 0. 12 Lysi n e 0.34 + 0. 11 H i s t i d i n e 0.51 + 0. 09 A r g i n i n e 0.62 + 0. 09 T o t a l 20.99 Values are x + SE of 3 determinations, except threonine where only one value was obtained. TABLE X I I I A comparison of p r o l i n e concentrations i n Malpighian tubule f l u i d of hydrated l o c u s t s c o l l e c t e d by two methods Concentration (mM) Malpighian t u b u l e s F l u i d Hemolymph U/P (U) (P) Cannulation (5) 35.76 + 7.61 14.02 + 1.82 2.50 + 0.35 L i g a t i o n (6) 38.02 + 3.53 12.01 + 1.10 3.21 + 0.21 S i g n i f i c a n t l y d i f f e r e n t at p 0.05? no no no Values are x+ SE. Numbers i n parenthese are the number of determinations. Values f o r l i g a t e d animals are from Table X. 53 Figure 6. U/P r a t i o s generated by i n v i t r o Malpighian tubules exposed to 15 mM amino a c i d (x + SE, n=6). 2.01 1.5+ 1.0f 0.5+ £ _ L pro g'y ala 55 Figure 7 . U /P r a t i o s generated by i n v i t r o Malpighian tubules exposed to d i f f e r e n t p r o l i n e concentrations (x + SE, n=6). 5 15 25 Concentration (mM) 57 at the time of d i s s e c t i o n could not produce such a high U/P ratio 1?. Tubules exposed to 15 mM glucose i n the absence of e x t e r n a l amino a c i d s produced a f l u i d which contained only 2 mM p r o l i n e . I f t h i s same amount of contamination was present i n the tubules exposed to 15 mM p r o l i n e , i t would c o n s t i t u t e only 8% of the p r o l i n e c o n c e n t r a t i o n . F i g u r e 7 shows the U/P r a t i o s obtained when the tubules were exposed to d i f f e r e n t concentrations of p r o l i n e . The t r a n s p o r t of p r o l i n e appears s a t u r a b l e s i n c e exposure of the tubules to 25 mM p r o l i n e y i e l d s a U/P r a t i o s i g n i f i c a n t l y smaller than those achieved w i t h 5 or 15 mM p r o l i n e . The U/P r a t i o s obtained f o r d i f f e r e n t concentrations of p r o l i n e could be due to l a r g e d i f f e r e n c e s i n f l u i d s e c r e t i o n r a t e (see D i s c u s s i o n ) . Although f l u i d s e c r e t i o n r a t e was not measured i n t h i s i n v i t r o prepara-t i o n , there d i d not appear to be a d i f f e r e n c e i n the f l u i d s e c r e t i o n r a t e when the tubules were exposed to d i f f e r e n t concentrations of p r o l i n e ( f i g ; ; 7) of-= when:exposed to a l a n i n e or g l y c i n e . D i s c u s s i o n The concentrations of hemolymph f r e e amino acid s i n S c h i s t o c e r c a hemolymph measured i n t h i s study are s i m i l a r to those reported by other workers (Rutherford and Webster, 1978; Benassi et a l . , 1961; Treherne, 1959), however, my values f o r p r o l i n e are higher (Table I X ) . During periods of dehydration and subsequent r e h y d r a t i o n , there i s a l a r g e change i n hemolymph volume,yet there i s l i t t l e change i n the amino a c i d con-c e n t r a t i o n i n t h i s body f l u i d . This i n d i c a t e s that amino a c i d s are ' removed from the hemolymph during dehydration and added back to the hemo-lymph upon r e h y d r a t i o n . Regulation of amino acid s i n t h i s manner would serve to r e g u l a t e hemolymph osmotic pressure during osmotic s t r e s s and has been observed i n other Qrthopterans (Woodring and Blakeney, 1980; Djajakusumah and M i l e s , 1966). The Malpighian tubule f l u i d a l s o contained amino a c i d s , although the p r o f i l e was q u i t e d i f f e r e n t from that of the hemolymph (Table X). Most amino a c i d s found i n the hemolymph were e i t h e r absent or found i n very low c o n c e n t r a t i o n i n the tubule f l u i d . However, p r o l i n e and g l u t -amate had a U/P r a t i o greater than 1 , i n d i c a t i n g that these amino a c i d s may be a c t i v e l y t ransported. Glutamate i s n e g a t i v e l y charged at physio-l o g i c a l pH and could be p a s s i v e l y transported down the e l e c t r i c a l gradient created by the t r a n s t u b u l a r p o t e n t i a l (16 mV, lumen p o s i t i v e , Maddrell-and .58 Klunsuwan, 1973). The Nernst equation p r e d i c t s that t h i s p o t e n t i a l could create a U/P r a t i o of 1.9 f o r glutamate, however, the U/P r a t i o i n v i v o i s 5.8 (Table X). Therefore, some of the glutamate t r a n s p o r t i s probably a c t i v e . P r o l i n e , on the other hand, has no net charge at p h y s i o l o g i c a l pH and t h e r e f o r e appears to be a c t i v e l y s e creted; by the l o c u s t tubules. I n t e r e s t i n g l y , glucose i s a l s o found i n higher concen-t r a t i o n i n the tubule f l u i d than i n the hemolymph i n v i v o (Table X). I t may be p o s s i b l e that glucose i s a c t i v e l y secreted, but t h i s aspect was not i n v e s t i g a t e d . The t r a n s p o r t of p r o l i n e by l o c u s t tubules was i n v e s t i g a t e d f u r -t h e r by using an i n v i t r o technique. Ramsay (1958) e s t a b l i s h e d four c r i = t e r i a which must be f u l f i l l e d i f a n e u t r a l s o l u t e i s p a s s i v e l y transported by Malpighian tubules: 1. The U/P r a t i o of the s o l u t e concentrations must never exceed 1. 2. The U/P r a t i o i s independent of P. 3. U/P = b/a+b,,where a = f l u i d s e c r e t i o n r a t e and b = the per-m e a b i l i t y of the tubule w a l l to the s o l u t e . 4. There i s no competition f o r t r a n s p o r t between s o l u t e s . The f i r s t two c r i t e r i a were used to i n v e s t i g a t e p r o l i n e t r a n s p o r t b,y. l o c u s t tubules. Both i n v-ivo (Table ~X) - and i n v i t r o ( f i g s . 6 and 7) } the U/P r a t i o i s greater than 1. However, the U/P r a t i o obtained i n the i n v i t r o experiments was co n s i d e r a b l y lower than that obtained i n the i n v i v o experiments. This i n d i c a t e s that the t r a n s p o r t processes may not be preserved i n the i n v i t r o p r e p a r a t i o n , perhaps due to l o s s of hemolymph-borne hormone or damage i n c u r r e d during sac p r e p a r a t i o n . Figure 7 i n d i c a t e s that c r i t e r i o n #2 i s a l s o v i o l a t e d s i n c e the U/P r a t i o , i s s i g n i f i c a n t l y " l o w e r at higher "P" concentrations of p r o l i n e . These r e s u l t s i n d i c a t e that p r o l i n e i s a c t i v e l y transported by l o c u s t tubules. This i s the f i r s t evidence of s e c r e t i o n of a m e t a b o l i c a l l y u s e f u l com-pound by i n s e c t Malpighian tubules . Locust Malpighian tubules are estimated to secrete i n v i v o at a minimum r a t e of 8 / i l / h o u r ( P h i l l i p s , 1964c). I f the secreted f l u i d c o ntains 38 mM p r o l i n e (Table X ) , then 304 nanomols enter the rectum every hour. P r o l i n e i s a c t i v e l y accumulated by the rectaib t i s s u e from the lumen at a r a t e of 356 nmol/hour*rectum i n v i t r o ( B a l s h i n , 1973) so a l l the secreted p r o l i n e can probably be recovered. In f a c t , l i t t l e 59 p r o l i n e i s l o s t i n the feces (5 nmol/hour'locust; c a l c u l a t e d ' .from data i n Appendix B ) • I t i s i n t e r e s t i n g to note that the co n c e n t r a t i o n of p r o l i n e i n the tubule f l u i d i n v i v o i s the same whether the animal i s hydrated or dehydrated. Locusts ,under hypertonic s t r e s s secrete tubule f l u i d at a minimum r a t e of 7 / i l / h o u r ( P h i l l i p s , 1964c). Assuming that the same s e c r e t i o n r a t e occurs i n dehydrated animals, the amount of p r o l i n e e n t e r i n g the rectum would be 308 nanomols/hour (see s Table X) , very c l o s e to that c a l c u l a t e d f o r dehydrated animals. However, dehy-d r a t i n g animals excrete more p r o l i n e (17 nmols/hour-locust, c a l c u l a t e d from Appendix B) than hydrated animals, suggesting that the rectum reab-sorbs l e s s p r o l i n e when the animal needs to lower i t s hemolymph s o l u t e content. 60 CHAPTER IV METABOLIC SUPPORT OF THE SHORT-CIRCUIT CURRENT ACROSS THE. LOCUST.RECTUM I n t r o d u c t i o n In Chapter I I i t was shown that the l o c u s t rectum i s capable 6f> o x i d i z i n g amino acid s and carbohydrates, but has a l i m i t e d c a p a c i t y f o r f a t t y a c i d o x i d a t i o n . Although enzyme p r o f i l e s i n d i c a t e which metabolic pathways are present i n the rectum, they do not t r u l y i n d i c a t e what path-waysis important i n supplying energy f o r a c t i v e t r a n s p o r t . I t i s p o s s i b l e that there i s p a r t i t i o n i n g of energy (presumably ATP) i n the c e l l so that a s p e c i f i c metabolic pathway provides the energy f o r a c t i v e i o n t r a n s p o r t . This phenomenon : i s .-observed i n v a s c u l a r smooth muscle where sodium t r a n s -port i s energized by g l y c o l y s i s but not o x i d a t i v e metabolism (Paul et a l . , 1980). The energyf f o r c h l o r i d e transport i n the rectum u l t i m a t e l y r e l i e s on the metabolism of exogenous s u b s t r a t e s . In Chapter I I I I showed that amino acids and glucose bathe both sides of the rectum irt v i v o . L i p i d s , t r e h a l o s e and organic a c i d s are present i n i n s e c t hemolymph (Altman, 1961). Any of these substrates could p r o v i d e energy f o r c h l o r i d e t r a n s p o r t pro-v i d i n g they can 1) cross the r e c t a l c e l l membrane(s) and 2) be metabo-l i z e d by the t i s s u e . A c t i v e i o n tr a n s p o r t i n the l o c u s t can be e a s i l y monitored by using a s h o r t - c i r c u i t technique developed by Wil l i a m s et a l . (1978). The s h o r t -c i r c u i t current (Isc) i s stimulated by cAMP and t h i s increase i n I s c i s completely due to i n c r e a s e d - c h l o r i d e transport from the lumen to hemo-lymph (reviewed i n Chapter I ) . In t h i s study- p o t e n t i a l metabolites were added to cAMP^stimulated r e c t a , previously- depleted of endogenous substrate to determine what s u b s t r a t e s support a c t i v e c h l o r i d e t r a n s p o r t . Experiments were a l s o undertaken to determine i f one m e t a b o l i t e , p r o l i n e , supports the I s c e q u a l l y w e l l from the hemolymph or lumen-side of the t i s s u e . 61. M a t e r i a l s and Methods Animals Adult female l o c u s t s two to four weeks past t h e i r f i n a l molt were used i n a l l experiments. Their colony environment and feeding regime were as described, i n Chapter I I . Sho r t — G I f cuir-'Gu ft en t Mea sufemeri t s Locust r e c t a were mounted as f l a t sheets between two modified Ussing chambers (Williams et a l . • , 1978) and bathed on both sides i n s a l i n e s which are described below. S a l i n e s were bubbled w i t h a. gas mixture'of r95% O^and 5% CC^. T r a n s e p i t h e l i a l p o t e n t i a l was clamped at 0 mV ( s h o r t -c i r c u i t e d c o n d i t i o n ) w i t h compensation f o r s e r i e s r e s i s t a n c e of the e x t e r n a l s a l i n e . Rothe _et a l . (1969) provides a d e t a i l e d d e s c r i p t i o n of the c i r -c u i t r y and Hanrahan (1981) discusses the importance of compensation f ° r s a l i n e r e s i s t a n c e i n t h i s p r e p a r a t i o n . The I s c was monitored w i t h a S o l t e c 220 recorder. S a l i n e s The composition of the s a l i n e s used i n t h i s chapter are given i n Table XIV. Sodium, potassium, c h l o r i d e and magnesium are based on measure-ments of hemolymph concentrations ( J . Hanrahan, personal communication). The amino a c i d concentrations i n s a l i n e C (Table XIV) are based upon measurements of female l o c u s t hemolymph ( f o r methods see Chapter I I I ) . P u blished values f o r glucose (Mayer and Candy, 1969) and tr e h a l o s e (van dercHorst et a l . , 1978).concentrations were used i n s a l i n e C. The f i n a l osmotic pressure of' 420 mOsm was achieved by adding sucrose to the s a l i n e s . P r e l i m i n a r y experiments demonstrated that sucrose was 14 not o x i d i z e d by the l o c u s t rectum s i n c e C-sucrose was not metabolized 14 to C0 2. Experimental P r o t o c o l The rectum, was mounted on the Ussing chamber and bathed b i l a t e r a l l y w i t h s a l i n e A (Table XIV) which lacked any metabolic s u b s t r a t e s . The rectum was r i n s e d twice w i t h t h i s s a l i n e f o r one to three hours. cAMP was then added to the hemolymph s i d e of the t i s s u e ( f i n a l c o ncentration 1 mM) which caused an increase i n I s c . The I s c d e c l i n e d over s e v e r a l 2 hours and,iwhen the I s c dropped to 0.95 to 1.90 jueq/cm "hr the rectum was considered "s u b s t r a t e - d e p l e t e d " (see f i g . 8). P r e l i m i n a r y experiments i n d i c a t e d that i f the I s c f e l l below t h i s l e v e l before metabolite a d d i t i o n TABLE XIV Composition of s a l i n e s used i n t h i s study. Compound A • • B ' G S a l i n e •"' D~ E ' ' ' F ' ' G H NaCl 100.05. 100.0. 100. 0 75.0 - - - -K 2 S 0 4 5.0 5.0 5. 0 - - - - -MgS0 4 10.0 10.0 10. 0 - - - - -NaH oP0. 2 4 1.9 1.9 1. 9 - 1.9 1.9 - -Na„HPO. 2 4 3.1 3.1 3. 1 - 3.1 3.1 - -NaHC0 3 25.0 25.0 25. 0 25.0 25.0 25.0 - -C a C l 2 1.0 1.0 1. 0 1.0 - - 1.0 1.0 NaCH_SO. 3 4 - - - 100.0 100.0 - -KCH oS0, J 4 - - - 5.0 5.0 - -Mg(N0 3) 2 - - - 10.0 10.0 - -CaN0 3 - - - 1.0 1.0 - -Choline C l - - - - - 80.0 80.0 MgCl 2 - - 10.0 - - 10.0 10.0 KH 2P0 4 - - 1.9 - - 1.9 1.9 K 2HP0 4 - - 3.1 - - 3.1 3.1 Choline HC0 3 - - - - - 25.0 25.0 KC1 - - 5.0 - - - '-Glutamine - - 5 T •0 - - - - -P r o l i n e - - 18. 0 - - - -G l y c i n e - -• 15. 0 - - -• -Serine -• 2. 0 -A l a n i n e ^ - • • ^ - 2. .0 H i s t i d i n e 2. 0 ' Glucose - 1. 0 • - • Trehalose - - ' 20. 0 ' • -Na Succinate - - 50.0 - -Sucrose 119.0 69.0 49. 0 14.0 118.0 68.0 164.0 114.0 A l l values are i n mM. Sucrose was added to a l l s a l i n e s to b r i n g the f i n a l o s m o l a r i t y to 420 mOsm. S a l i n e s were bubbled w i t h 95% 0 2and 5% CO and the pH adjusted to 7.4. -63 the p r e p a r a t i o n o f t e n d i e d . I f metabolites were added when the I s c was above t h i s l e v e l they o f t e n f a i l e d to a f f e c t the I s c presumably because of high l e v e l s of endogenous s u b s t r a t e s . A f t e r the rectum was substrate depleted^ the s a l i n e was changed bilatT,. e r a l l y to s a l i n e A . t o which 50 mM of a s i n g l e m etabolite was added. The s a l i n e c o n t a i n i n g 50 mM succinate had a d i f f e r e n t composition which i s given i n Table XIV ( s a l i n e D). These s a l i n e s a l s o contained 1 mM cAMP on the hemolymph si d e of the rectum. The high metabolite con-c e n t r a t i o n of 50 mM was chosen i n an attempt to overcome any b a r r i e r to the metabolite's entry i n t o the t i s s u e such as low p e r m e a b i l i t y or low c a r r i e r a f f i n i t y . However, d i o l e i n was added at the p h y s i o l o g i c a l con-c e n t r a t i o n of 10 mg/ml (Jutsum and Goldsworthy, 1976). Bovine serum albumin (1% weight/volume, Sigma f r a c t i o n V, e s s e n t i a l l y f a t t y a c i d free) was added along w i t h the d i o l e i n . For comparative purposes^, the I s c was measured i n a c o n t r o l s a l i n e which contained the major amino acids and sugars found i n the hemolymph ( s a l i n e C, Table XIV). The rectum was exposed to t h i s s a l i n e from d i s -s e c t i o n to the end of the experiment. The r e s u l t s of the experiments o u t l i n e d above ind i c a t e d , that pro-l i n e caused the l a r g e s t increase i n I s c of a l l s ubstrates t e s t e d . I then i n v e s t i g a t e d whether t h i s s ubstrate s t i m u l a t e d the I s c e q u a l l y w e l l when added to e i t h e r the hemolymph or lumen s i d e of the t i s s u e . For these experiments^concentrations of p r o l i n e (15 mM).similar to those found i n hemolymph (see Chapter I I I ) were used. Experiments were a l s o conducted i n which p h y s i o l o g i c a l concentrations of glucose or p r o l i n e (see Chapter III)we:c\eT added to the substrate-depleted t i s s u e . To confirm that the p r o l i n e s t i m u l a t i o n of I s c was due to an increase i n c h l o r i d e t r a n s p o r t and not that of other ions^experiments i n v o l v i n g chlorides-free s a l i n e s were performed. The r e c t a were depleted of endoge^-nous su b s t r a t e s as described above and then the bathing s a l i n e was changed b i l a t e r a l l y to a c h l o r i d e ^ f r e e s a l i n e ( s a l i n e E, Table XIV) which had 1 mM cAMP but no m e t a b o l i t e s . The t i s s u e was exposed to s e v e r a l changes of t h i s s a l i n e f o r one to two hours. W i l l i a m s et a l , (1978) showed that t h i s treatment v i r t u a l l y depletes the r e c t a l t i s s u e of c h l o r i d e . The bathing s a l i n e was then changed to s a l i n e F (Table XIV) which contained no c h l o r i d e , but contained 50 mM p r o l i n e and 1 mM cAMP. C h l o r i d e was added at the end of the experiment i n the form of NaCl so that the f i n a l 6 4 c h l o r i d e c o n c e n t r a t i o n was 100 mM. G l y c i n e transport i n the l o c u s t rectum i s p a r t i a l l y coupled to sodium tra n s p o r t ( B a l s h i n and P h i l l i p s , 1971; B a l s h i n , 1973). To see i f sodium was required f o r the p r o l i n e - s t i m u l a t e d I s c , experiments using sodium-f r e e s a l i n e s were conducted. The p r o t o c o l f o r these experiments was the same as that i n the c h l o r i d e - f r e e experiments, except that s a l i n e s G and H replaced E and F r e s p e c t i v e l y (see Table XIV). NaCl was added at the end of the experiment so that the f i n a l sodium concentration was 100 mM. A l l experiments were ^conducted at room/ temperature (22-25°C). I n h i b i t o r s Metabolic i n h i b i t o r s were added to both sides of the s h o r t - c i r c u i t e d preparations to d i s c e r n the nature of the endogenous substrates and to i n v e s t i g a t e the pathway of p r o l i n e o x i d a t i o n i n the rectum. 2-deoxy-d-glucose, an i n h i b i t o r of g l y c o l y s i s , and aminooxyacetate, an i n h i b i t o r of transaminases, were d i s s o l v e d i n s a l i n e A and added to the s a l i n e bathing s h o r t - c i r c u i t e d preparations to a f i n a l c o n c e n t r a t i o n of 10 mM. R e s u l t s Figure 8 shows the t y p i c a l behavior of the I s c across r e c t a when no exogenous su b s t r a t e s are present. There i s s u b s t a n t i a l v a r i a b i l i t y i n the magnitude;,'; of the I s c response to cAMP s t i m u l a t i o n and i n the d u r a t i o n of the increased I s c . In most preparations the I s c d e c l i n e d to low l e v e l s 2 (0.95 - 1.90/ieq/ cm *hr) w i t h i n a few hours of s t i m u l a t i o n w i t h cAMP. However, some preparations t e n a c i o u s l y maintained a h i g h I s c i o r many hours, suggesting a greater s t o r e of endogenous s u b s t r a t e s . ( f i g . 8c). Preparations such as F i g . 8c were used i n i n h i b i t o r s t u d i e s . Figure 9 shows the mean time course of the I s c s t i m u l a t i o n by cAMP. Note the gradual d e c l i n e i n I s c f o r preparations w i t h no exogenous substrates comp-pared to the sustained h i g h I s c when a p h y s i o l o g i c a l complement of amino a c i d s and sugars; are provided. Again i t can be seen that i n the absence of e x t e r n a l s u b s t r a t e s , the •^icM^V''jT;^^€i^Ta.%X-o^:i's-iitess• than when the rectum i s exposed to amino acid s and sugars.. Figures 10 through 12 show the e f f e c t s of adding d i f f e r e n t substrates to both s i d e s of substrate-depleted r e c t a . A d d i t i o n of p r o l i n e to the preparations caused a 5 - f o l d increase i n I s c which was 2.5-to 5 - f o l d greater than I s c s t i m u l a t i o n produced by,."other amino acid s ( f i g . 10). G l y c i n e , the other major hemolymph amino a c i d besides p r o l i n e .(see Chapter 65 F i g u r e 8. T y p i c a l traces of Isc w i t h time a f t e r d i s s e c t i o n f o r i n d i v i d u a l r e c t a . Recta were bathed w i t h s a l i n e A (Table XIV), c o n t a i n i n g no m e t a b o l i t e s , and s t i m u l a t e d w i t h 1 mM cAMP. (jJMgUiobart) 67 Figure 9. Mean Isc w i t h time a f t e r d i s s e c t i o n for, r e c t a bathed i n two d i f f e r e n t s a l i n e s (x + SE, where l a r g e r than s y m b o l ) . 9 , s a l i n e A (Table XIV), c o n t a i n i n g no me t a b o l i t e s , n=7. O , s a l i n e C (Table XIV), con-t a i n i n g p h y s i o l o g i c a l l e v e l s of the main hemolymph amino a c i d s and sugars, n=6. 69 I I I ) , f a i l e d to s t i m u l a t e the I s c . In f a c t , w i t h g l y c i n e present, the I s c f e l l below c o n t r o l l e v e l s Cfig. 10). Glucose st i m u l a t e d the I s c 4 times b e t t e r than t r e h a l o s e , succinate or pyruvate ( f i g . 11), but glucose ( & I s c , 1 hour a f t e r glucose a d d i t i o n = 5.54 + 0.36) d i d not s t i m u l a t e the I s c as w e l l as 50 mM p r o l i n e ( f i g . 10;&Isc 1 hour a f t e r p r o l i n e a d d i t i o n ' = ;7.58 #. 0.81). A d d i t i o n of d i o l e i n to s u b s t r a t e -depleted r e c t a d i d not change the I s c ( f i g . 12). A l t h o u g h a d d i t i o n of 50 mM glucose to substrate^depleted r e c t a caused, a 4 - f o l d increase .in I s c Cfig. 11), a d d i t i o n s of p h y s i o l o g i c a l l e v e l s of .glucose produced a much smaller s t i m u l a t i o n Cfig. 13). A d d i -t i o n s of p h y s i o l o g i c a l l e v e l s of p r o l i n e to substrate-depleted r e c t a produced a more than 4 - f o l d increase i n I s c . The I s c one hour a f t e r 2 p r o l i n e a d d i t i o n was smaller (7.67 + 0.41/ieq/cm *hr) i n f i g . 13 than 2 i n f i g . 10 (9.36 + 0.85/ueq/cm " h r ) , but these two values are not s i g n i f -i c a n t l y d i f f e r e n t C p ^ 0 . 0 5 ; -tr--=lv7-8 ) • F i g ure 14a shows the e f f e c t of adding 15 mM p r o l i n e f i r s t to the hemolymph and then to the lumen si d e of the t i s s u e . A d d i t i o n of p r o l i n e to the hemolymph s i d e caused only a small increase i n I s c of 2.00 + 0.41 2 jueq/cm 'hr; subsequent a d d i t i o n or p r o l i n e to the lumen caused a f u r t h e r 2 increase i n I s c of 4.81 + 1.6;ueq/cm .hr. I f 15 mM p r o l i n e i s added to the lumen s i d e of substrate-depleted r e c t a f i r s t , there i s a l a r g e increase i n I s c C4.83 + 0.77 zieq/cm 'hr) w h i l e subsequent a d d i t i o n s of p r o l i n e to the hemolymph s i d e of the same preparations caused the Is c to increase 2 by only 0.34 + 0.13>ueq/cm 'hr (see f i g . 14). The f r a c t i o n a l increase of the t o t a l I s c s t i m u l a t i o n due to hemo-lymph or l u m i n a l a d d i t i o n of 15 mM p r o l i n e i s shown i f f i g . 15. P r o l i n e added f i r s t to the hemolymph s i d e of the rectum produces only 35% of the t o t a l i n c r e a s e i n I s c ( f i g . 15a). However, p r o l i n e added to the lumen si d e f i r s t produces 93% of the t o t a l I s c s t i m u l a t i o n ( f i g . 15b). C l e a r l y p r o l i n e s u p p l i e d on the lumen s i d e i s much more e f f e c t i v e i n s t i m u l a t i n g the I s c than p r o l i n e on the hemocoel s i d e . Spring et a l . (1978) and Spring and P h i l l i p s (1980b) showed that the cAMP- sti m u l a t e d increase i n I s c was completely due to an increase i n c h l o r i d e t r a n s p o r t . This observation i s supported by the r e s u l t s i n f i g . 16. P r o l i n e a d d i t i o n does not s t i m u l a t e the I s c i f c h l o r i d e i s not present i n the s a l i n e . This i n d i c a t e s that the p r o l i n e - s t i m u l a t e d I s c i s due to an i n c r e a s e i n c h l o r i d e absorption and.not s e c r e t i o n - o f c a t i o n s 70 Figure 10. The e f f e c t s on I s c of adding i n d i v i d u a l amino a c i d s to both s i d e s of substrate-depleted r e c t a (x + SE, where l a r g e r than symbol, n=6). V e r t i c a l arrow i n d i c a t e s a d d i t i o n of 50 mM amino a c i d ; A , p r o l i n e ; # , a l a n i n e ; Jk , g l y c i n e ; O, glutamate; • , glutamine. Amino acid s were added 5.3 + 1.0 to 6.2 + 0.2 hours (x + SE) a f t e r d i s s e c t i o n . The dashed l i n e represents the time course of the I s c f o r substrate-depleted r e c t a when no exogenous substrates were added (taken from f i g . 9). H o r i z o n t a l arrow i n d i c a t e s the maximal s t i m u l a t i o n of Isc by cAMP when r e c t a are exposed to s a l ine C (Table XIV) which contains amino acid s and sugars (taken from f i g . 9 ) j 71 72 Figure 11. The e f f e c t s on Isc of adding carbohydrates and organic a c i d s to both sides of substrate-depleted r e c t a (X + SE, where l a r g e r than symbol; n=6 e x c e p tO n=5). V e r t i c a l arrow i n d i c a t e s a d d i t i o n of 50 mM carbohy-drate or a c i d : 6 , glucose; O , su c c i n a t e ; Jk , t r e -halose; A , pyruvate. M e t a b o l i t e s were added 4.6 + 0.6 to 6.1 + 1.0 hours (x + SE) a f t e r d i s s e c t i o n . The dashed l i n e represents the time course of the I s c f o r substrate-depleted r e c t a when no exogenous sub-s t r a t e s are added (taken from f i g . 9 ) . H o r i z o n t a l arrow i n d i c a t e s the maximal s t i m u l a t i o n of I s c by cAMP when r e c t a are exposed to s a l i n e C (Table XIV) •' which contains amino a c i d s and sugars (taken from f i g . 9 ) . O O" a> a5 f 30 45 60 75~ Time (minj sfo 105 1&) 74 Figure 12. The e f f e c t s on I s c of adding d i o l e i n to both sides of, substrata-depleted recta..(x .+ SE; n=3). V e r t i c a l arrow i n d i c a t e s addi'tiori of 10 mg/ml d i o l e i n .plus 1% BSA. D i o l e i n was added 10.3 +0.2 hours (x + SE) a f t e r d i s s e c t i o n . The dashed l i n e represents the time course of the I s c f o r substrate-depleted r e c t a when no exogenous substrates are added (taken from f i g . 9 ) . H o r i z o n t a l arrow i n d i c a t e s the maximal s t i m u l a t i o n of I s c by cAMP when r e c t a are exposed to s a l i n e C (Table XIV) which contains amino a c i d s and sugars (taken from f i g . 9 ) . 75 76 Figure 13. The e f f e c t s of adding p h y s i o l o g i c a l , l e v e l s (see Chapter I I I ) of p r o l i n e _ o r glucose on the Isc of sub-s t r a t e - d e p l e t e d r e c t a (x + SE, where l a r g e r than symbol). 38 mM p r o l i n e was added to the lumen s i d e and 12mM p r o l i n e was added to the hemolymph si d e of the t i s s u e ( & , n=4). In a second experiment 4 mM glucose was added to the lumen si d e and 2 mM glucose was added to the hemolymph si d e of the rectum ( # , n=4). P r o l i n e and_glucose were added 3.9 + 0.6 and 3.2 + 0.3 hours (x + SE) a f t e r d i s s e c t i o n r e s p e c t i v e l y . The dashed l i n e represents the time course of the I s c f o r substrate-depleted r e c t a when no exogenous sub-s t r a t e s are added (taken from f i g . 9). H o r i z o n t a l arrow i n d i c a t e s the maximal s t i m u l a t i o n of I s c by cAMP when r e c t a are exposed to s a l i n e C (Table XIVX which contains amino a c i d s and sugars (taken from f i g . 9 ). 78 i n t o the lumen ( i . e . sodium or hydrogen) or absorption of other anions ( i . e . b i c a r b o n a t e ) . The a d d i t i o n of NaCl at the end of the experiment produced an increase i n the I s c ( f i g . 16), but t h i s s t i m u l a t i o n was smal-l e r than that seen i n f i g . 10. The r e c t a used i n the experiment shown 2 i n f-ig.16 were kept at a substrate-depleted l e v e l (0.95 - 1.90 ueq/cm .hr) longer than those shown i n f i g . 1 0 . This may have damaged the t i s s u e so that i t became l e s s responsive to s t i m u l a t i o n . Transport of g l y c i n e , and probably other amino acid s by the l o c u s t rectum i n v i t c p i . i s p a r t i a l l y - dependent upon lu m i n a l sodium ( B a l s h i n , 1973). F i g u r e 17 i n d i c a t e s that. 50 mM. p r o l i n e can s t i m u l a t e the I s c of s u b s t r a t e -depleted r e c t a i n the absence of e x t e r n a l sodium; moreover a d d i t i o n of e x t e r n a l sodium does not p o t e n t i a t e the s t i m u l a t i o n by p r o l i n e . However, the s t i m u l a t i o n produced by the a d d i t i o n of p r o l i n e i n f i g . 17 i s not as great as i n f i g . 10. As mentioned above, t h i s may be due to t i s s u e • damage incurred, by •tj.athiftgj.v.the-, reeta? - i n s u b s t r a t e - f r e e s a l i n e longer than those shown i n f i g . 10. F i g u r e 18 shows that the a d d i t i o n of 10 mM 2-deoxy-d-glucose, an i n h i b i t o r of g l y c o l y s i s , i n h i b i t s the I s c of cAMP-stimulated r e c t a o x i d i z -i n g endogenous s u b s t r a t e s . This i n d i c a t e s that the endogenous substrate i s carbohydrate, probably n o n - d i f f u s i b l e glycogen, which has been seen i n e l e c t r o n micrographs of l o c u s t r e c t a l t i s s u e ( J . P h i l l i p s , personal communication). The a d d i t i o n of t h i s i n h i b i t o r to cAHEastimulated r e c t a exposed b i l a t e r a l l y to 50 mM p r o l i n e causes a s l i g h t i n i t i a l decrease i n I s c , but subsequently the I s c r i s e s toward the p r e - i n h i b i t o r l e v e l . This r e s u l t suggests that p r o l i n e does not simply augment o x i d a t i o n of endogenous carbohydrate, but o x i d a t i o n of p r o l i n e , alone, can support M g h l e v e l s of I s c . Aminooxyacetate i n h i b i t s amino t r a n s f e r a s e s and the b i l a t e r a l a d d i -t i o n of 10 mM aminooxyacetate g r e a t l y i n h i b i t s the I s c of cAMP-stimulated r e c t a exposed to 50 mM a l a n i n e ( f i g . 19). This i n d i c a t e s that a l a n i n e metabolism occurs v i a an amino t r a n s f e r a s e , probably glutamate-pyruvate transaminase... Recta o x i d i z i n g endogenous substrates or s t i m u l a t e d by 50 mM p r o l i n e are not i n h i b i t e d by aminooxyacetate,indicating that amino t r a n s f e r a s e s may not be important i n p r o l i n e o x i d a t i o n or o x i d a t i o n of endogenous s u b s t r a t e s . 79 Figure 14. T y p i c a l t r a c e s of I s c w i t h time f o r i n d i v i d u a l sub-s t r a t e - d e p l e t e d .recta a f t e r a d d i t i o n of 15 mM p r o l i n e to one s i d e of the t i s s u e . (a) P r o l i n e added f i r s t to the hemolymph s i d e then to lumen s i d e of the t i s s u e , (b) P r o l i n e added f i r s t to the lumen s i d e then hemo^ lymph s i d e of the t i s s u e . P r o l i n e was added to (a) 4.2 hours and to (b) 8.2 hours a f t e r d i s s e c t i o n . 81 Figure 15. The f r a c t i o n a l increase i n I s c of substrate-depleted r e c t a a f t e r . a d d i t i o n of 15 mM p r o l i n e to e i t h e r the lumen or hemolymph si d e of the t i s s u e . 1.0 i n d i c a t e s the maximal steady s t a t e I s c a f t e r 15 mM p r o l i n e has been added to both sides of the rectum. (a) P r o l i n e was f i r s t added to the hemolymph side (H) of the t i s s u e and then added to the lumen side (L) (n=6). P r o l i n e was added 5.3 +0.9 hours (x + SE) a f t e r d i s s e c t i o n , (b) P r o l i n e was f i r s t added to the lumen si d e (L) of the t i s s u e and then added to the hemo-lymph si d e (H) (n=6). P r o l i n e was added 6.8 +0.7 hours (x + SE) a f t e r d i s s e c t i o n . Fractional Increase in l s c P — JjL 0) 83 F i g u r e 16. The e f f e c t of p r o l i n e on the I s c of substrate-depleted r e c t a bathed i n c h l o r i d e - f r e e s a l i n e (x + SE where l a r g e r than symbol). 50 mM p r o l i n e was added b i l a t -e r a l l y at the.time i n d i c a t e d by the f i r s t arrow. C h l o r i d e was l a t e r added at the. second arrow to a f i n a l concentration*of 100 mM. C h l o r i d e was added 5.5 +0.5 hours (x-J. SE) a f t e r d i s s e c t i o n . 85 17. The e f f e c t of p r o l i n e on the I s c of su b s t r a t e - d e p l e t i r e c t a bathed:;in sodium-free s a l i n e (x + SE). 50 mM p r o l i n e was added b i l a t e r a l l y at the time i n d i c a t e d by the f i r s t arrow. Sodium c h l o r i d e was l a t e r added at the.second arrow to a f i n a l c o n c e n t r a t i o n of 100 mM. Sodium was added 8.2 +0.6 hours (x + SE) a f t e r d i s s e c t i o n . 86 87 Figure 18. The e f f e c t s of 10 mM 2-deoxy-d-glucose on the I s c of r e c t a exposed to p r o l i n e or no exogenous s u b s t r a t e s . 2-deoxy-d-glucose was added to r e c t a stimulated by 50 mM p r o l i n e ( A , n=4; see f i g . 10) or to r e c t a w i t h no exogenous substrates ( Wk, n=6; see f i g . 8c). . I n h i b i t o r , was added b i l a t e r a l l y 10.4 + 0.7 ( A ) or 6.2 + 0.9 (•) hours (x + SE) a f t e r d i s s e c t i o n . 89 Figure 19. The e f f e c t s of 10 mM aminooxyacetate on the I s c of r e c t a exposed to p r o l i n e , a l a n i n e or no exogenous su b s t r a t e s . Aminooxyacetate was added to r e c t a w i t h no exogenous sub s t r a t e ( • , n=4; see f i g . 8c) or to recta, s t i m u l a t e d by 50 mM p r o l i n e ( A ,n=7; see f i g . 10) or 50 mM a l a n i n e ( • , n=6; see f i g . 10). Aminooxyacetate' was added b i l a t e r a l l y 4.6 + 1.1 ( • ) , 8.2 + 0,3 ( A ) or 6.4 + 0.5 (•) hours (x + SE) a f t e r d i s s e c t i o n . 91 D i s c u s s i o n The r e s u l t s of t h i s study i n d i c a t e that exogenously a p p l i e d amino acids and carbohydrates support the I s c of the l o c u s t rectum, w h i l e d i o l e i n does not. The endogenous sub s t r a t e which i s present a f t e r many hours i n m e t a b o l i t e - f r e e s a l i n e i s probably carbohydrate s i n c e the I s c of r e c t a without supply of exogenous substrates i s i n h i b i t e d by the ..Inhibitor of g l y c o l y s i s , 2-deoxy-d-glucose C f i g . 18). I t - i s unlike'ly^.th^-t.lihe.-.end^gr enous sub s t r a t e .is .amino a c i d because an i n h i b i t o r o£ aminotransferases, aminooxyacetate, had no e f f e c t on c o n t r o l I s c C f i g . 19). However, oxi^-d a t i o n by amino oxidases or deaminases cannot r u l e d out. F i n a l l y , the endogenous sub s t r a t e could be l i p i d . However, t h i s i s u n l i k e l y because the t i s s u e has l i t t l e c a p a c i t y f o r l i p i d o x i d a t i o n (.see Chapter I I ) and l i p i d deposits are not apparent i n e l e c t r o n micrographs of the r e c t a l t i s s u e ( I r v i n e , 1966). In order f o r a metabolite to s t i m u l a t e the I s c of substrate-depleted r e c t a , i t must be able to f i r s t reach the c e l l membranes. A r e s t r i c t i v e b a r r i e r e x i s t s on the a p i c a l s i d e of the t i s s u e i n the form of the c u t i c u -l a r i n t i m a . The i n t i m a prevents compounds the s i z e of d i s a c c h a r i d e s from reaching the r e c t a l t i s s u e from the lumen at s i g n i f i c a n t r a t e s ( P h i l l i p s and D o c k r i l l , 1968). Access to the b a s a l membranes i s r e s t r i c t e d by a muscle l a y e r and secondary c e l l s ( - P h i l l i p s , 1964a), as w e l l as a flow of water from the lumen to the hemolymph (reviewed by P h i l l i p s , 1970). Once a coumpound has reached a membrane then i t could d i f f u s e i n t o the c e l l or enter the r e c t a l c e l l v i a a c a r r i e r mediated process. High con-c e n t r a t i o n s of glucose produce a 4 - f o l d s t i m u l a t i o n of I s c i n s u b s t r a t e -depleted r e c t a and could enter the c e l l by d i f f u s i o n or a c t i v e t r a n s p o r t . Although B a l s h i n (1973) found no t r a n s e p i t h e l i a l t r a n s p o r t of the glucose analogue, 3-0-methylglucose, he s t a t e d that he had p r e l i m i n a r y evidence 14 f o r a c t i v e C-glucose absorption by the rectum.. Glucose i s found i n low concentrations i n l o c u s t hemolymph,aMalpighian tubule f l u i d (2 and 4 mMm r e s p e c t i v e l y ; Chapter I I I ) and midgut f l u i d (2 mM; Dow, 1981). When glucose i s added to substrate-depleted r e c t a at these n a t u r a l low concentrations, i t s t i l l s t i m u l a t e s the I s c but the s t i m u l a t i o n i s smaller than w i t h 50 mM glucose and the s t i m u l a t i o n soon- decays. Hanrahan (per-s o n a l communication) r e p o r t s that i n v i t r o r e c t a exposed to a s a l i n e con-t a i n i n g only 10 mM glucose as an energy source f a i l to give l a r g e , 92 sustained I s c s t i m u l a t i o n s w i t h cAMP. These r e s u l t s i n d i c a t e that exoge-nous glucose i n v i v o probably s u p p l i e s some energy f o r the rectum, but other compounds are probably more important i n f u e l i n g the rectum. • •-  Trehalose -'slightly s t imulated the I s c of substrate-depleted r e c t a when added at a c o n c e n t r a t i o n of 50 mM. 50 mM i s not a pharmaco-l o g i c a l c o n c e n t r a t i o n s i n c e concentrations t h i s h i g h have been reported i n l o c u s t hemolymph (Strang and Clement, 1980). However, tre h a l o s e must enter the r e c t a l c e l l from the b a s a l s i d e s i n c e t h i s sugar cannot pene-t r a t e the Intima ( P h i l l i p s and D o c k r i l l , 1968). Pyruvate and succinate f a i l e d to s t i m u l a t e the I s c of the l o c u s t rectum, although the r e c t a l mitochondria o x i d i z e these organic a c i d s (see Chapter I I ) . Roth of these compounds are n e g a t i v e l y charged at p h y s i o l o g i c a l pH and may not be able to d i f f u s e i n t o the r e c t a l c e l l s . Raumeister et a l . (1981) showed that other organic a c i d s (oxaloacetafce, fumarate, malate, c i t r a t e , propionate) 'are••net'..transported;.by. the.-locust rectum:-ih v i t ro and" this'may a l s o be the cassei; f o r ; pyruvate and s u c c i n a t e . D i o l e i n f a i l e d to s t i m u l a t e the I s c . This was not s u r p r i s i n g s i n c e the rectum appears to have l i m i t e d l i p i d - o x i d i z i n g c a p a b i l i t i e s . Ketone bodies were not t e s t e d i n t h i s study, but i t i s p o s s i b l e that they may be reabsorbed and o x i d i z e d by the r e c t a l t i s s u e as suggested by Baumeister et a l . ( 1 9 8 1 ) . However, l e v e l s of ketone bodies i n l o c u s t hemolymph are q u i t e low (Baumeister et a l . , 1981). A l l amino acid s t e s t e d except g l y c i n e stimulated the I s c of s u b s t r a t e -depleted r e c t a to some extent. Balshin, (1973) and B a l s h i n and P h i l l i p s (1971) showed that g l y c i n e i s accumulated i n r e c t a l c e l l s of l o c u s t s but not metabolized by the r e c t a l t i s s u e . Therefore i t i s not unexpected that g l y c i n e d i d not s t i m u l a t e the I s c . In f a c t , the a d d i t i o n of g l y c i n e to substrate-depleted r e c t a caused a decrease i n the I s c . A c t i v e t r a n s -port of g l y c i n e across the l o c u s t r e c t a l w a l l i s at l e a s t p a r t i a l l y coupled to sodium t r a n s p o r t . (Bal'shin, 1973; B a l s h i n and P h i l l i p s , 1971) so the t r a n s p o r t of n e u t r a l l y - c h a r g e d g l y c i n e from the lumen to the hemolymph s i d e of the t i s s u e would be accompanied by a movement of sodium i n the same d i r e c t i o n . In the presence of cAMP} the I s c i s due to a c t i v e c h l o r i d e absorption (Spring et a l . , 1978; Spring and P h i l l i p s , 1980);% t h e r e f o r e t h i s g l y c i n e - s t i m u l a t e d movement of p o s i t i v e charge (sodium) would r e s u l t i n a decrease i n Is c which was observed i n f i g . 10. B a l s h i n (1973) reported that 10 mM glutamate was not transported 93 by the rectum. However, 50 mM glutamate s t i m u l a t e s the Iscxof s u b s t r a t e -depleted recta, suggesting at "this c o n c e n t r a t i o n i t can enter the c e l l and be metabolized.nyGlutamate, however, occurs at trac e l e v e l s i n the hemolymph and i n v e r y low- concentrations i n the Malpighian tubule f l u i d (Chapter I I I ) so the c o n t r i b u t i o n of exogenous glutamate to r e c t a l metab-olism i s probably n e g l i g i b l e , Glutamine.,is found i n the r e c t a t i s s u e at h i g h c o n c e n t r a t i o n (Chapter II;"amides",. B a l s h i n , 1973) and may be metabolized by the rectum. Although glutaminase was not measured i n . t h e l o c u s t rectum, i t i s l i k e l y that t h i s enzyme i s present, s i n c e glutamine stimulated the I s c . Alanine caused a two^fold increase i n the I s c of substrate-depleted r e c t a . This .amino a c i d i s found in.hemolymph and Malpighian tubule f l u i d (Capter I I I ) and i s a c t i v e l y transported by the rectum ( B a l s h i n , 1973). The alanine-dependent s t i m u l a t i o n i s blocked by aminooxyacetate ( f i g . 19) suggesting that an amino t r a n s f e r a s e , l i k e l y glutamate-pyruvate t r a n s -aminase, i s inv o l v e d i n a l a n i n e metabolism.. A l a n i n e , i t s e l f , i s not o x i d i z e d by the r e c t a l mitochondria (Chapter I I ) , but i f ammonia from a l a n i n e could be transferred* 'to endogenous .©(-ketoglutarate v i a glutamate-pyruvate transaminase then the product, pyruvate, could be o x i d i z e d by the mitochondria. Alanine i s a l s o thought to s t i m u l a t e the transepthe-l i a l p o t e n t i a l of Bombyx mori midgut v i a a metabolic e f f e c t (Giordana and S a c c h i , 1978, 1980; Sacchi and Giordana, 1980). .The. g r e a t e s t . s t i m u l a t i o n of. the Jlsc'.-was- observed with:the - a d d i t i o n of 5.0. mM p r o l i n e to the s a l i n e s bathing the rectum.. Even w i t h physio-l o g i c a l concentrations of pr o l i n e , there' i s a 4-f old/increase., -in I s c .which was much l a r g e r than the change i n I s c w i t h p h y s i o l o g i c a l l e v e l s of glucose ( f i g . 13). P r o l i n e i s a c t i v e l y accumulated from the lumen by the r e c t a l t i s s u e i n v i t r o ( B a l s h i n , 1973) and i s found i n high concentrations i n t r a -c e l l u l a r ^ (over 60 mM; Chapter I I ; , B a l s h i n , 1973). Although the p r o l i n e -dependent I s c s t i m u l a t i o n i s not dependent upon the presence of e x t e r n a l sodium ( f i g . 17), a d d i t i o n of p r o l i n e to the lumen s i d e of the t i s s u e caused a t r a n s i e n t drop i n the I s c ( f i g . 14) c o n s i s t e n t w i t h c a t i o n coupled entry. I f p r o l i n e transport i s coupled to sodium t r a n s p o r t , as is: g lycine-t r a n s p o r t ( 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), the drop i n I s c may be due to an inward f l o w of sodium ions accompanying the entry of p r o l i n e . Over 90% of t o t a l -•- proline-dependent I s c s t i m u l a t i o n can be achieved by adding p r o l i n e s o l e l y to the lumen s i d e of the r e c t u m . ( f i g 15). In v i v o , the Malpighian tubules a c t i v e l y t r a n s p o r t p r o l i n e so the lumen s i d e of the rectum i s normally- bathed i n a f l u i d h i g h i n p r o l i n e (Chapter I I I ) . This scheme has the advantage of presenting the a p i c a l l y l o c a t e d mitochondria .with a substrate which they r e a d i l y - o x i d i z e . P r o l i n e added to the hemolymph si d e of t h i s " t i s s u e can s t i m u l a t e the tissue,, but i t s e n t r y i n t o the t i s s u e may be r e s t r i c t e d by the b a s a l p e r m e a b i l i t y b a r r i e r s mentioned e a r l i e r . : The proline-dependent s t i m u l a t i o n of I s c i s s l i g h t l y i n h i b i t e d by 2-deoxy-d-glucose ( f i g . 18). This suggests that some of the p r o l i n e may augment endogenous carbohydrate o x i d a t i o n , and when t h i s carbohydrate metabolism i s i n h i b i t e d theirel'i's va-"tJ:ans-lent'X4r6p»v-in;:the~»Ise.., However, the I s c begins to recover suggesting that p r o l i n e metabolism alone can support the I s c . U n l i k e a l a n i n e - dependent Isc s t i m u l a t i o n , the p r o l i n e - s t i m u l a t e d I s c i s unaffected by aminooxyacetate ( f i g . 19). This i n d i c a t e s that the aminotransferase, glutamate-pyruvate transaminase, may. not be important i n p r o l i n e o x i d a t i o n . In Chapter I I i t was suggested that another.enzyme, glutamate dehydrogenase, may be important i n d e l i v e r i n g p r o l i n e carbons to the c i t r i c a c i d c y c l e s The r e s u l t s of t h i s i n h i b i t o r experiment support t h i s p r e d i c t i o n . Although l u m i n a l p r o l i n e s t i m u l a t e s the I s c of substrate-depleted r e c t a much b e t t e r than other s u b s t r a t e s , the rectum i s capable of o x i d i z -i n g a wide v a r i e t y of carbohydrate and amino a c i d s . In v e r t e b r a t e s t r a n s -p o r t i n g e p i t h e l i a , carbohydrates and l i p i d s are important sources of energy w h i l e amino a c i d s seem to be l e s s important" ( K e l l y et a l . , 1980; Hersey, 1974; Cohen and Kamm,1976). Berridge (1966); reported that another i n s e c t t r a n s p o r t i n g e p i t h e l i a , the Malpighian tubules of C a l l i p h o r a , r e l i e s on carbohydrate and amino a c i d metabolism to support f l u i d s e c r e t i o n . Amino ac i d s are found i n much higher c o n c e n t r a t i o n i n i n s e c t hemolymph than i n e x t r a c e l l u l a r , f l u i d s of v e r t e b r a t e s (Altman, T96i'), t h e r e f o r e they may be> a r e a d i l y a v a i l a b l e source of energy i n i n s e c t s . A l s o by o x i d i z i n g p r i m a r i l y amino aci d s and carbohydrates, the l o c u s t rectum does not have to compete w i t h the more m e t a b o l i c a l l y demanding, l i p i d -burning f l i g h t muscle f o r the same s u b s t r a t e . 95 CHAPTER V OXYGEN CONSUMPTION OF RECTAL TISSUE I n t r o d u c t i o n The l o c u s t rectum a c t i v e l y t r a n s p o r t s c h l o r i d e and t h i s t r a n s p o r t can be s t i m u l a t e d by cAMP i n v i t r o (Spring and P h i l l i p s , 1980a,b; Spring et a l 1978). The mechanism of t h i s c h l o r i d e t ransport i s q u i t e d i f f e r e n t from those described f o r v e r t e b r a t e t i s s u e s (reviewed by F r i z z e l et a l . , 1979) sinc e i t i s not sti m u l a t e d by sodium or bicarbonate, but i s sti m u l a t e d by potassium (Hanrahan and P h i l l i p s , 1980a). Because i t i s a c t i v e , c h l o r i d e t r a n s p o r t i s dependent upon metabolic energy. This energy must come from o x i d a t i v e phosphorylation s i n c e i n h i b i t i o n of t h i s process completely stops c h l o r i d e t r a n s p o r t by the rectum (Baumeister e t a l . , 1981, Herrera et a l . , 1977; Wi l l i a m s et a l . , 1978). These e a r l i e r s t u d i e s i n d i c a t e d that p e r t u r b a t i o n s of c e l l u l a r metabolism a f f e c t c h l o r i d e t r a n s p o r t ! However, i t i s not known whether changes i n c h l o r i d e t r a n s p o r t a f f e c t the metabolic r a t e of the l o c u s t rectum. In most e p i t h e l i a a change i n the r a t e of i o n tra n s p o r t i s accompanied by a change i n oxygen consumption (reviewed by Mandel and Balaban, 1981). One i n s e c t e p i t h e l i u m , the cockroach rectum, appears to folloxv t h i s p a t t e r n . Sodium removal or ouabain a d d i t i o n i n h i b i t s oxygen consumption by the cockroach rectum, presumably by i n h i b i t i n g sodium tr a n s p o r t (Tolman and S t e e l , 1980b). However, i n the midgut of Manduca, i n h i b i t i o n of potassium t r a n s p o r t does not a f f e c t r e s p i r a t i o n rate(Mandel et a l . , 1980a). Because i o n tra n s p o r t by the l o c u s t rectum has been w e l l s t u d i e d (see review Chapter I ) , t h i s t i s s u e provides a good system to study the i n t e r a c t i o n between the r a t e of i o n tr a n s p o r t and metabolic r a t e i n an i n s e c t e p i t h e l i u m . I f the l o c u s t rectum i s s i m i l a r to most other e p i t h e l i a , then changes i n the r a t e of a c t i v e c h l o r i d e t r a n s p o r t should a f f e c t the metabolic r a t e . The work presented i n t h i s chapter represents a p r e l i m i n a r y study of the coupling between a c t i v e i o n tr a n s p o r t and metabolism i n the l o c u s t rectum. M a t e r i a l s and Methods Animals Adult male S c h i s t o c e r c a g r e g a r i a 2 to 4 weeks past t h e i r f i n a l molt were used i n a l l experiments. The feeding regime and r e a r i n g c o n d i t i o n s were the same as those described i n Chapter I I . S a l i n e s The compositions of the s a l i n e s used i n t h i s study are shown i n Table XV. The c o n t r o l and c h l o r i d e - f r e e s a l i n e s are the same as those i n Table.-'XIV ( s a l i n e s B arid E, r e s p e c t i v e l y ) . A l l s a l i n e s were bubbled w i t h a gas mixture c o n s i s t i n g of 95% G^and 5% CC^ and the pH adjusted to 7.4 w i t h the appropriate base or a c i d . D i s s e c t i o n and Incubation of Recta Recta were ..cut. open, removed '=f rom'"loeusts- and ^placed i n 5 ml of one of the s a l i n e s shown i n Table XV ( w i t h or without cAMP). This p r e i n c u b a t i o n s a l i n e was bubbled w i t h 95%002/5% CO^ and the r e c t a incubated f o r 3 hours at room temperature (22-25°C). This period of i n c u b a t i o n i s s u f -f i c i e n t to remove endogenous hormones that a f f e c t r e c t a l i o n t r a n s p o r t (Spring et^ a l . , 1978). P r e l i m i n a r y experiments i n d i c a t e d that a 3 hour i n c u b a t i o n i n s p e c i f i c i o n - f r e e s a l i n e s r e s u l t e d i n a greater than 10-f o l d decrease i n t i s s u e i o n content (ueq/mg f r e s h weight) of the deleted i o n . Oxygen Consumption Measurements The apparatus used to monitor r e c t a l t i s s u e oxygen consumption was the same as the one used to measure m i t o c h o n d r i a l r e s p i r a t i o n i n Chapter I I . The chamber contained 2 ml of sa3iine i d e n t i c a l to that i n which the r e c t a were pre-incubated and t h i s was e q u i l i b r a t e d w i t h 95% 0^/5% CO2 and maintained at 25°C. A rectum was then hooked on an i n s e c t p i n and the upper p o r t i o n of the p i n sealed i n the hole of the g l a s s stopper. The g l a s s stopper was then put i n the chamber so that the rectum was suspended i n the s a l i n e which was c o n s t a n t l y s t i r r e d by a magnetic s t i r r e r . C o n t r o l experiments w i t h no rectum hooked on the p i n showed no l o s s of oxygen from the chamber. .The oxy gen-: consumption was monitored f o r at l e a s t 5 minutes.*. A"' s m a l l amount' (50-100 /ul) of NaCl, KC1, KCH^SO^ or c h o l i n e CI, d i s s o l v e d i n water, was then - inje c t e d - -into the; chamber-•iwi:t-h^.a:AHami€'t0n"syringe and 97> TABLE XV Composition of s a l i n e s used i n 0 2 consumption s t u d i e s Compound Co n t r o l Concentration S a l i n e s C l .-free : ... (mM) K C l - f r e e Na/K-fr NaCl 100.0 - - -K 2 S 0 4 5.0 - - -MgS0 4 10.0 - - 15.0 NaH oP0. 2 4 1.9 1.9 1.9 -Na„HP0. 2 4 3.1 3.1 3.1 -sucrose 69.0 68.0 65.0 87.0 NaHC0 3 25.0 25.0 25.0 -C a C l 2 1.0 - - 1.0 NaCH oS0. 3 4 - 100.0 105.0 -KCH-SO. 3 4 - 5.0 - -Mg(N0 3) 2 - 10.0 10.0 -CaN0 3 -= 1.0 1.0 -c h o l i n e C l - - - 100.0 c h o l i n e HC0 3 - - - 25.0 p r o l i n e 50.0 50.0 50.0 50.0 Sucrose was added to b r i n g the f i n a l o s m o l a r i t y to 420 mOsm. pH was adjusted to 7.4 w i t h the appropiate a c i d or base. .98 the oxygen consumption monitored. A f t e r the experiments the r e c t a were d r i e d i n an oven at 60°C overnight and weighed to o b t a i n the dry weight of the t i s s u e . S t a t i s t i c s The r a t e s of oxygen consumption of r e c t a incubated i n d i f f e r e n t s a l i n e s were compared by Student's t - t e s t . Rates of oxygen consumption before and a f t e r i o n i n j e c t i o n s i n t o the chamber were compared usi n g a p a i r e d t - t e s t . R e s u l t s Table XVI shows the mean r a t e s of oxygen consumption f o r r e c t a incubated i n d i f f e r e n t s a l i n e s . Recta incubated i n v a r i o u s i o n - f r e e s a l i n e s a l l consumed oxygen at r a t e s s i g n i f i c a n t l y l e s s than those i n c o n t r o l s a l i n e . R e c t a l 0„ consumption was more d r a s t i c a l l y reduced by + + removal of the main c a t i o n s , Na and K , than the main anion, C l . T y p i c a l t r a c e s of oxygen consumption of r e c t a incubated i n c h l o r i d e -f r e e s a l i n e i n the presence ( f i g . 20b)or absence ( f i g . 20a) of cAMP are shown i n f i g . 20. A d d i t i o n of NaCl to r e c t a i n c h l o r i d e - f r e e s a l i n e without cAMP produces no change i n oxygen consumption ( f i g . 20a). However, when cAMP i s present, oxygen consumption i s stimulated by NaCl ( f i g . 20b). The oxygen consumption a f t e r r e i n t r o d u c t i o n of c h l o r i d e i s l i n e a r over the e n t i r e measurement pe r i o d (10 to 15 minutes). Table XVII shows the e f f e c t s on 0^ consumption of adding ions to ion-depleted r e c t a . A d d i t i o n of NaCl or KC1 to unstimulated r e c t a bathed i n c o n t r o l s a l i n e does not s i g n i f i c a n t l y a f f e c t the oxygen.consumption. This i n d i c a t e s that these i o n a d d i t i o n s do not o s m o t i c a l l y d i s r u p t the t i s s u e . A d d i t i o n of NaCl or KC1 to chJoride-depleted r e c t a causes a 20% increase i n oxygen consumption, but only when 1 mM cAMP i s present. The a d d i t i o n of c h l o r i d e ( c h o l i n e s a l t ) to KCl-depleted t i s s u e exposed to cAMP produces a 15% increase i n oxygen consumption. However, a 50% incr e a s e i s observed i f KC1 i s added to r e c t a under i d e n t i c a l c o n d i t i o n s . These r e s u l t s i n d i c a t e that the chloride-dependent s t i m u l a t i o n of oxygen consumption i s manifested p r i m a r i l y when cAMP i s present and i s augmented by the presence of potassium. TABLE XVI Oxygen consumption of r e c t a incubated i n d i f f e r e n t s a l i n e s Treatment Oxygen Consumption (nmol 0^' min '''•mg '''dry weight) C o n t r o l S a l i n e C o n t r o l S a l i n e + 1 mM cAMP CI - f r e e S a l i n e CI - f r e e S a l i n e + 1 mM cAMP K C l - f r e e S a l i n e K C l - f r e e S a l i n e + 1 mM cAMP Na + / K + - f r e e S a l i n e 16 16 16 15 24 24 7 16.07 +0.87 14.58 + 0.51 12.82 + 0.68* 10.60 + 0.44* 9.29 + 0.34* 9.60 + 0.40* 6.26 + 0.46* Values are x + SE. A s t e r i s k denotes a s i g n i f i c a n t d i f f e r e n c e between the 0^ consumption of r e c t a incubated i n c o n t r o l s a l i n e and r e c t a incubated i n the i n d i c a t e d s a l i n e ( p ^ 0 . 0 5 ) . 100 Fi g u r e 20. T y p i c a l t r a c e s of r e c t a l oxygen consumption of r e c t a incubated i n c h l o r i d e - f r e e s a l i n e . (a) a d d i t i o n of NaCl. to c h l o r i d e - d e p l e t e d r e c t a i n the absence of cAMP. (b) a d d i t i o n of NaCl to c h l o r i d e - d e p l e t e d r e c t a i n the presence of cAMP. The s l i g h t upward jump i n the tr a c e at the p o i n t of NaCl a d d i t i o n i s simply mechanical disturbance of the oxygen e l e c t r o d e . The dotted l i n e represents the course of oxygen con-aumption without NaCl a d d i t i o n . Time (min) 102 TABLE XVII Oxygen consumption before and a f t e r i o n a d d i t i o n to r e c t a pre-incubated i n d i f f e r e n t s a l i n e s 0„ Consumption S a l i n e Added Ions* (nmol 02«min -mg d r y w e i g h t ) Before A f t e r C o n t r o l NaCl 18.74 + 0.92 19.03 + 1.14 Co n t r o l + cAMP NaCl 15.25 + 0.87 15.70 + 0.92 Co n t r o l KCl 13.39 + 0.60 13.87 + 0.67 Co n t r o l + cAMP KC1 13.92 + 0.49 14.07 + 0.55 C l ~ - f r e e NaCl 10.60 + 0.79 10.97 + 0.66 C l ~ - f r e e + cAMP NaCl 11.15 + 0.71 13.55 + 0.92** C l ~ - f r e e KCl 14.87 + 0.51 15.62 + 0.64 C l ~ - f r e e + cAMP KCl 10.23 + 0.41 12.20 + 0.37** K C l - f r e e c h o l i n e C I — 8.41 + 0.59 8.00 + 0.61 K C l - f r e e KCl 10.16 + 0.42 12.42 + 0.37** K C l - f r e e KCH^SO, 9.20 + 0.61 14.12 + 0.83** 3 4 K C l - f r e e + cAMP ch o l i n e CI 9.77 + 0.34 11.24 + 0.48** K C l - f r e e + cAMP KCl 9.93 + 0.85 15.28 + 0.97** K C l - f r e e + cAMP KCH-SO. 9.12 + 0.84 12.37 + 0.90** 3 4 Na + / K + - f r e e KCH„S0. 3 4 6.26 + 0.46 7.45 + 0.29** Values are x + SE, n = 8. * , f i n a l c o n c e n t r a t i o n of added ions was 100 mM. * * , s i g n i f i c a n t d i f f e r e n c e (p£ 0.05) between 0^ consumption before and a f t e r i o n a d d i t i o n . 103 D i s c u s s i o n Omission of the main monovalent ions from the s a l i n e bathing l o c u s t r e c t a r e s u l t s i n lower r a t e s of oxygen consumption compared to those i n c o n t r o l s a l i n e (Table XVI). These low r e s p i r a t i o n r a t e s could be i n d i c a t i v e of t i s s u e damage due to problems i n r e g u l a t i n g c e l l volume i n i o n d e f i c i e n t s a l i n e s . A l t e r n a t i v e l y , the low r a t e of oxygen con-sumption could r e f l e c t a decreased work load due to d e l e t i o n of ions which are normally a c t i v e l y transported. This second .explanation seems l i k e l y s i n c e oxygen consumption of c h l o r i d e - d e p l e t e d r e c t a i s stimulated by c h l o r i d e , but only when cAMP i s present (Table X V I I ) . This r e s u l t suggests t h a t the i n c r e a s e i n oxygen consumption i s r e l a t e d to i o n t r a n s -port since- '; ^ '.active c h l o r i d e t r a n s p o r t i s a l s o s t i m u l a t e d s e v e r a l f o l d by cAMP (Spring and P h i l l i p s , 1980a,b; Spring et a l . , 1978). The a d d i t i o n of potassium to KCl-depleted r e c t a a&so s t i m u l a t e d oxygen consumption by 35-53%. This may i n d i c a t e that the Na-K-ATPase i d e n t i f i e d i n t h i s t i s s u e (Peacock, 1977) i s a c t i v a t e d under these con-d i t i o n s . C h l o r i d e t r a n s p o r t i n the l o c u s t rectum i s sti m u l a t e d 4=-to 6-fold by potassium (Hanrahan and P h i l l i p s , 1980a) and the r e s u l t s of t h i s study support t h i s o b s e r v a t i o n . When KCl i s added to KCl-depleted r e c t a i n the presence of cAMP, there i s a 54% increase i n oxygen consumption. However, i f c h o l i n e c h l o r i d e i s added to r e c t a p r e - t r e a t e d i n the same way, the oxygen consumption increases only 15%. I t i s of i n t e r e s t to c a l c u l a t e whether the observed changes i n oxygen, consumption due to r e i n t r o d u c t i o n of ions to ion-depleted r e c t a t r u l y r e f l e c t s the work necessary to a c t i v e l y t r a n s p o r t c h l o r i d e . The work (0^ consumption) needed to a c t i v e l y t r a n s p o r t c h l o r i d e can be e s t i -mated from the f l u x r a t i o of c h l o r i d e across s h o r t - c i r c u i t e d r e c t a l p r e p a r a t i o n s . This e s t i m a t i o n can then be compared to the oxygen con-sumption of r e c t a suspended i n s a l i n e i f the f o l l o w i n g assumptions are accepted: a) The rectum suspended i n s a l i n e i s v i r t u a l l y s h o r t - c i r c u i t e d and t h e r e f o r e the t r a n s e p i t h e l i a l p o t e n t i a l i s zero. b) The oxygen consumption of c h l o r i d e - d e p l e t e d r e c t a a f t e r NaCl -addition i s r e p r e s e n t a t i v e of a new steady s t a t e of oxygen i:Q4 consumption and c h l o r i d e t r a n s p o r t . The f i r s t assumption i s j u s t i f i e d because the area of the r e c t a l t i s s u e i s s m a ll r e l a t i v e to the l a r g e volume of e l e c t r i c a l l y conductive s a l i n e surrounding i t . Therefore an e l e c t r o c h e m i c a l gradient could not be e s t a b l i s h e d when the rectum i s exposed to t h i s l a r g e volume of s t i r r e d s a l i n e . There i s an e r r o r i n the second assumption. Oxygen consumption i s measured f o r 5 to 10 minutes a f t e r NaCl r e i n t r o d u c t i o n . However, when NaCl i s added to c h l o r i d e - d e p l e t e d , s h o r t - c i r c u i t e d r e c t a , steady s t a t e c h l o r i d e t r a n s p o r t i s not achieved u n t i l 15 minutes a f t e r NaCl a d d i t i o n ( J . Hanrahan, personal communication). Therefore, i n the f o l -lowing c a l c u l a t i o n s } , the steady s t a t e v a l u e used f o r the r a t e of c h l o r i d e t r a n s p o r t i s an overestimate of the c h l o r i d e t r a n s p o r t o c c u r r i n g i n the f i r s t 5 to 10 minutes f o l l o w i n g NaCl r e i n t r o d u c t i o n . According to Zerahn (1956) the work required to a c t i v e l y t r a n s p o r t an i o n can be reduced to three components: a) work r e q u i r e d to overcome a c o n c e n t r a t i o n gradient b) work re q u i r e d to overcome an e l e c t r i c a l gradient c) work re q u i r e d to overcome the i n t e r n a l r e s i s t a n c e of the i o n pump. Both the s h o r t - c i r c u i t e d and suspended r e c t a experience no c o n c e n t r a t i o n or e l e c t r i c a l g radient so the f i r s t two work components can be ignored. The t h i r d component can be c a l c u l a t e d from the f o l l o w i n g equation: W = RT ln(Mp/Mg) where W i s work, R i s the gas constant, T i s temper-ature (°K) and M^.-arid are the forward and back f l u x e s , r e s p e c t i v e l y . Mp/Mg i s 6.47 f o r s h o r t - c i r c u i t e d r e c t a i n c o n t r o l s a l i n e i n the presence ofcAMP ( J . Hanrahan, personal communication) so / W= 1.987(298) i n 6.47 = 1,105.6 c a l o r i e s / C l ~ e q u i v a l e n t (1). When sti m u l a t e d w i t h cAMP, a s h o r t - c i r c u i t e d rectum t r a n s p o r t s c h l o r i d e 2 at a r a t e of about 9 /ieq/cm hr (see Chapter I V ) . M u l t i p l y i n g value (1) by t h i s number we o b t a i n a value of ,' 9950.4 x l O - 6 -calories/cm 2hr (2). This number can be expressed per dry weight of r e c t a l t i s s u e by using 2 a conversion f a c t o r of 0.32 cm /mg dry weight ( J . Hanrahan, personal communication) to o b t a i n —6 3184 x 10 calories/mg hr ( 3 ) . The AG° of ATP h y d r o l y s i s i s -7.7 Kcal/mol (Mahler and Cordes, 1971). 105 Therefore i f consumption of 1 0„ y i e l d s 6 ATP, then each 0 - i s ^ e q u l v a l e n t -2 -to 46,200 c a l o r i e s (or 1 jumol 0„ = 4.6 x 10 c a l o r i e s ) . D i v i d i n g value -2 (3) by 4.6 x 10 c a l o r i e s and converting from hours to minutes we a r r i v e at the p r e d i c t e d v a l u e f o r work r e q u i r e d to a c t i v e l y t r a n s p o r t c h l o r i d e across s t i m u l a t e d l o c u s t rectum i n the. s h o r t - c i r c u i t e d s t a t e > ; 1.2 nmol 0 2/min ' iug dry weight. Note that the A G of ATP h y d r o l y s i s i n the c e l l i s probably greater (up to -10 Kcal/mol, Mahler and Corder, 1971) than t h e A G ° used here, so t h i s estimate of oxygen consumption may be h i g h . The actual, change i n oxygen, consumption due to adding NaCl to c h l o r i d e - d e p l e t e d r e c t a i n the presence of cAMP i s 2.4 nmol 02/min-mg dry weight (Table X V I I ) . This i s i n reasonable agreement w i t h the p r e d i c t e d v a l u e , although s l i g h t l y h i gher. *' One- can a l s o p r e d i c t the s t o i c h i o m e t r y between numbers of c h l o r i d e ions transported to oxygen consumed i f assumptions "a" and "b" on page 103 are accepted. A stimulated s h o r t - c i r c u i t e d rectum t r a n s p o r t s c h l o r i d e 2 at 9peq/cm hr (Chapter 9) and t M s r a t e can be converted to /ieq/min mg dry weight as, described e a r l i e r , - t o y i e l d 48 nanoeq/min-mg. I f c h l o r i d e t r a n s p o r t r e q u i r e s 2.4 nanomols 02/min.mg (Table XVII) then 20 c h l o r i d e ions are transported f o r every O2 consumed. This value can a l s o be expressed as 3-4 C1~/ATP. I t should be emphasized that t h i s i s - o n l y a p r e d i c t i o n of the s t o i c h i o m e t r y . Rigorous experiments using i d e n t i c a l c o n d i t i o n s f o r oxygen consumption and i o n t r a n s p o r t measurements must be performed i n the f u t u r e to confirm t h i s p r e l i m i n a r y estimate of the s t o i c h i o m e t r y . A C l r a t i o has only been measured i n one other e p i -thelium, d o g f i s h r e c t a l gland ( S i l v a e t . al.,1980). The measured r a t i o i n the l a t t e r t i s s u e i s 30 C l /O2. Apparently c h l o r i d e t r a n s p o r t c o n s t i t u t e s only 20% of the t o t a l work of the t i s s u e , based upon the percent increase i n 0^ consumption of c h l o r i d e - d e p l e t e d t i s s u e when NaCl i s added (Table X V I I ) . Other processes such as a c t i v e t r a n s p o r t of other ions or s y n t h e t i c a c t i v i t i e s must use the ATP produced by the mitochondria. A Na*-K +-ATPase has been i d e n t i f i e d i n the rectum of S c h i s t o c e r c a (Peacock, 1977), but i t s l o c a -t i o n i n e p i t h e l i a l r a t h e r than muscle c e l l s i s u n c e r t a i n . However, P h y s i o l o g i c a l - evidence i n d i c a t e s a c t i v e Na + t r a n s p o r t at the b a s o l a t -e r a l border. In f a c t , most of the r e c t a l mitochondria are s i t u a t e d 106 along t h i s border i n S c h i s t o c e r c a ( I r v i n e , 1966). The Na —K -ATPase may perform, much., of the work of the t i s s u e s i n c e i n h i b i t i o n of t h i s enzyme by sodium or potassium d e l e t i o n leads.to the l a r g e s t drop i n t i s s u e oxygen consumption (Table X V I ) . The f u n c t i o n of the Na +K +-ATPase may be to produce the sodium gradients which energize secondary a c t i v e t r a n s p o r t processes such as water and amino a c i d t r a n s p o r t (reviewed by P h i l l i p s , 1980). However, l a r g e t r a n s e p i t h e l i a l f l u x e s of sodium across the rectum are not observed i n , v i t r o (Williams e t ^ a l . , 1978; Spring and P h i l l i p s , 1980b). This may be due to r e c y c l i n g of "this i on w i t h i n the r e c t a l t i s s u e (reviewed by P h i l l i p s , 1980). With the exception of Manduca and Hyalophora midguts (Mandel j|t a l . , 1980a; Harvey et a l . , 1967), a l l e p i t h e l i a s t u d i e d d i s p l a y changes i n oxygen consumption when a c t i v e i o n t r a n s p o r t i s a l t e r e d (reviewed by Mandel and Balaban, .1981). The l o c u s t rectum a l s o appears to f o l l o w t h i s general p a t t e r n because the oxygen consumption of ion-depleted r e c t a i s s e n s i t i v e to the r e i n t r o d u c t i o n of i o n s . ATP i s the l i n k between a c t i v e i o n t r a n s p o r t and metabolism i n a l l e p i t h e l i a s t u d i e d to date (reviewed by Mandel and Balaban, 1981). Further experimentation w i l l i n d i c a t e whether the lopust rectum resembles, other e p i t h e l i a i n t h i s respect. 107 CHAPTER VI GENERAL DISCUSSION The results presented in the previous chapters indicate that proline metabolism is an important source of energy for chloride transport in the locust rectum. This conclusion is supported by the following obser-vations: ' 1) The rectal tissue contains proline at a concentration much higher than that for other amino' acids or carbohydrates (Chapter II). 2) The enzyme profile of the locust rectum indicates that this tissue can oxidize carbohydrates and amino acids, but has limited capacity for l i p i d oxidation (Chapter II). 3) Mitochondria isolated from the locust rectum oxidize proline more rapidly than any other substrate tested (Chapter II). 4) The locust Malpighian tubules actively transport proline so that the lumen of the rectum is bathed with a high concentra-tion of proline.(Chapter^III), an amino acid the rectum actively reabsorbs (Balshin, 1973). 5) The Isc of substrate-depleted recta is stimulated by physio-logical concentrations of proline and this stimulation can be achieved by adding proline to only the luminal side of the rectum (Chapter IV). Although these observation indicate, qualitatively, that proline oxidation can support chloride transport, one can question whether the measured rate of proline oxidation i s sufficient to energize active chloride transport. In Chapter V i t was estimated that 4 /ueq of chloride were transported for every Aimol of ATP hydrolyzed. If proline is partially oxidized to alanine then 14 ATP are formed (for pathway see f i g . 4b). If proline i s completely oxidized to CO^ and NH^+, then 32 ATP are formed (for pathway see f i g . 4c). Therefore the theoretical value for the amount of proline oxidized per chloride transported By these two pathways (4b or 4c) i s : 1 jumol proline 1 Aimol ATP 17.9 nmol proline' (la) 14 jumol ATP X 4 ;ueq CI" ~ ;ueq C l -or 1 Aimol proline x 1 Aimol ATP = 7.8 nmol proline (lb). 32 ATP 4 Aieq CI" Aieq C l ~ 108 A st i m u l a t e d rectum t r a n s p o r t s c h l o r i d e at about 9 Aieq/cm "hr (Chapter IV).which i s equivalent to 2.88 Aieq/hr-mg dry weight (conversion f a c t o r of 0.32, J . Hanarahan, personal communication). Assuming 85% t i s s u e water ( B a l s h i n , 1973) t h i s value can be converted to ' 0.43/ueq-/-hr'mg wet weight (-2) By m u l t i p l y i n g value (2) by ( l a ) or (l b ) f S L t h e o r e t i c a l r a t e of p r o l i n e o x i d a t i o n necessary to support t h i s r a t e of c h l o r i d e t r a n s p o r t i s calculated: 7.70 nmol p r o l i n e / hr-mg wet weight ( p a r t i a l o x i d a t i o n ) (3a) 0r 3.35 nmol p r o l i n e / hr^mg wet weight (complete o x i d a t i o n ) . (3b) The a c t u a l r a t e of p r o l i n e o x i d a t i o n by r e c t a i n s a l i n e exposed to 25 mM p r o l i n e and 1 mM cAMP i s 3.73 nmol p r o l i n e / hr-mg wet weight ( c a l c u l a t e d from " ^ C - p r o l i n e - d e r i v e d "^C02» Table 4 ) . This value c l o s e l y agrees w i t h the t h e o r e t i c a l r a t e c a l c u l a t e d f o r r e c t a completely o x i d i z i n g p r o l i n e . Results i n Chapter I I i n d i c a t e that some, i f not a l l , the p r o l i n e i s completely o x i d i z e d by the r e c t a l t i s s u e s i n c e NH^ + i s produced during p r o l i n e o x i d a t i o n . I f p r o l i n e i s completely o x i d i z e d at a r a t e of 3.73 nmol p r o l i n e / hr* mg wet weight, then ammonium i s a l s o produced at t h i s r a t e . The f r e s h weight of a rectum i s 10.3 + 0.29 mg (x + SE, n = 36, unpublished data) so the p r e d i c t e d r a t e of ammonium production i s 38.4 nmol ammonium/hr-rectum. I n - v i v o , the measured r a t e of ammon-ium e x c r e t i o n by whole l o c u s t s i s 39.6 nmol/hr ( c a l c u l a t e d from Appendix B), However, i t i s u n l i k e l y that a l l the ammonium excreted by the •locust i s der i v e d from p r o l i n e o x i d i z e d by the rectum because probably not a l l the p r o l i n e i s completely o x i d i z e d (see f i g . 5) and some of the p r o l i n e - d e r i v e d ammonium could be l o s t to the hemolymph. In a d d i -t i o n , o x i d a t i o n of other amino acid s by the gut or m i c r o b i a l fermen-t a t i o n .could produce ammonium. Ammonium has been detected i n the feces of other i n s e c t s (reviewed by B u r s e l l , 1967), but u r i c a c i d i s considered to be the primary n i t r o -gen e x c r e t o r y product of i n s e c t s . Brown (1937) showed that there was about 57 times more u r i c a c i d than ammonium i n the feces of the grass-hopper, Melanoplus b i v i t t a t u s . However, i t i s i n t e r e s t i n g to note that ammonium and not u r i c a c i d i s the major n i t r o g e n waste product i n cockroaches ( M u l l i n s and Cochran, 1972). P e r i p l a n e t a excretes about 433 nmols ammonium/hr-animal .(calculated from M u l l i n s and Cochran, 109 1972) which i s more than 10 times the r a t e of e x c r e t i o n observed i n S c h i s t o c e r c a (see preceeding page). This suggests that ammonium excre-t i o n i n the l a r g e r l o c u s t may not be the primary n i t r o g e n waste pro-duct i n l o c u s t s . However, measurements of u r i c a c i d and other n i t r o -genous compounds i n l o c u s t feces are necessary before the r e l a t i v e c o n t r i b u t i o n of ammonium to t o t a l n i t r o g e n e x c r e t i o n i n the l o c u s t can be assessed. Another question which should be answered i s why p r o l i n e r a t h e r than any other s u b s t r a t e i s u t i l i z e d by the rectum. The answer may l i e i n the metabolic and p h y s i c a l p r o p e r t i e s of p r o l i n e . P r o l i n e i s a n o n - e s s e n t i a l amino a c i d f o r l o c u s t s ( W i l l i a m s , 1980) and may be synthesized by the f a t body which synthesizes the precursor of p r o l i n e , glutamate ( K i l b y and N e v i l l e , 1957). Orthopterans are a l s o a t t r a c t e d to d r o u g h t - r e s i s t a n t p l a n t s which contain p r o l i n e so the desert l o c u s t may consume l a r g e amounts of t h i s amino a c i d (Haglund, 1980). There-f o r e o x i d a t i o n of p r o l i n e by l o c u s t t i s s u e s would not l i k e l y deplete the animal of t h i s amino a c i d and i n t e r f e r e w i t h the s y n t h e s i s of pro-t e i n s or peptides which r e q u i r e p r o l i n e . P r o l i n e i s extremely s o l u b l e i n water '(«162g/100 ml, Stecher et a l . , 1968) so high concentrations of t h i s amino a c i d can be stored i n body f l u i d s or i n t r a c e l l u l a r ! y . Once i n the c e l l p r o l i n e i s u n l i k e l y to d i f f u s e out r a p i d l y because p r o l i n e i s not s o l u b l e i n non-polar s o l -vents (Stecher et a l . , 1968) and-thus could not pass e a s i l y through the • l i p i d tb.ilayer of the plasma membranes. Transport of p r o l i n e across membranes would be unaffected by e l e c t r i c a l g r a dients (unless coupled to a charged sp e c i e s , i . e . Na +) because p r o l i n e 13 ^neut-ral at p h y s i o l o g i c a l pH. In a d d i t i o n , accumu-l a t i o n of t h i s n e u t r a l amino a c i d w i t h i n a t i s s u e would not a f f e c t i n t r a c e l l u l a r pH. Complete o x i d a t i o n of p r o l i n e leads to a production of ammonium ra t h e r than ammonia sin c e ammonium i s one.of the products of the g l u t -amate dehydrogenase r e a c t i o n (Lehninger, 1975; see f i g . 5). The ammon-ium produced does not d i s s o c i a t e s i g n i f i c a n t l y at p h y s i o l o g i c a l pH. Complete o x i d a t i o n of p r o l i n e y i e l d s 32 ATP and i s comparable to the 36 ATP produced during glucose o x i d a t i o n . Glucose, however, i s probably not u t i l i z e d to a great extent by the rectum because t h i s sugar i s found• in.low c o n c e n t r a t i o n i n l o c u s t hemolymph and r e c t a l t i s s u e (Chapter TL'Cand I I I ) . Trehalose i s the main sugar found i n locust' hemolymph and i s Sound i n high concentration (over 50 mM; Rutherford and Webster, 1978), but does not appear to be an important source of energy f o r a c t i v e c h l o r i d e t r a n s p o r t by the rectum (Chapter I V ) . The f l i g h t muscles r e a d i l y o x i d i z e glucose and t r e h a l o s e and may out-compete the rectum f o r these carbohydrates. I t may be advantageous f o r the rectum to o x i d i z e p r o l i n e which the f l i g h t muscles only moderately o x i d i z e (Brosemer and Veerabhdrappa, 1965). The ammonium produced by r e c t a l p r o l i n e o x i d a t i o n could then be e a s i l y excreted w i t h the fec e s . I l l LITERATURE CITED Altman, P.L. (1961). Blood and Other Body F l u i d s , (ed. D.S. D i t t m e r ) , Federation of the American Society f o r Experimental B i o l o g y , Washington, D.C., 540 pp. Andrusiak, E.W., J.E. P h i l l i p s and J . Speight (1980). Phosphate t r a n s -port by the l o c u s t rectum i n v i t r o . Can. J . Zool. 58:1518-1523. B a l s h i n , M. (1973). 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APPENDIX A Hemolymph volume and hemolymph co n c e n t r a t i o n of Amino acid s of r e c e n t l y rehydrated l o c u s t s Time a f t e r Hemolymph Concentration (mM) Feeding (hr) Volume ( u l ) P r o l i n e G l y c i n e Glutamine 0* 70.7 + 8.0 (13) 15.17 + 3.30 (8) 13.34 + 0.84 (7) 7.06 + : 1.00 (8) 1 . 89.2 + 5.7 (5) 6.76 + 0.65 10.74 + 0.85 (5) 6.94 + 0.52 (5) 2 85.1 + 7.5 (5) 12.58 + 1.58 (9) 10.53 + 0.70 (9) 10:20 Or74 (9) 3 90.4 + 12.5 (4) 15.09 + 1.53 (8) 12.60 + 0.95 (8) 13.14 + 0.86 (7) 8 112.5 + 9.6 (4) 18.15 + 3.61 (8) 12.69 + 1.48 (8) 11.18 + 3.05 (8) 24* .122.4 + 8.7 (5) 12.10 + 1.37 (5) 12.65 + 1.74 (4) 2.77 + 0.60 (4) Values are x + SE. The numbers i n parentheses are the number of determinations. * denotes data taken from Tables V I I I and XI i n Chapter I I I . Animals were dehydrated as described i n Chapter I I I and then fed f r e s h l e t t u c e ad l i b i t u m f o r the time i n d i c a t e d . APPENDIX B Ex c r e t i o n of amino acid s and ammonium i n the feces of l o c u s t s Amount Excreted (umol/48 hr l o c u s t ) Hydration State P r o l i n e G l y c i n e Ammonium Dehydrating .0.84 + 0.20 (13) 0.20+0.04 (13) 1.87+0.18 (6) Hydrating 0.27 + 0.07 (8) 0.12 + 0.02 (8) 1.31 + 1.60 (6) Values are x + SE. The number i n parentheses- i n the number of determinations. Animals were placed i n syringes and s t o o d - v e r t i c a l l y so the anus was s i t u a t e d over a s a m l l container of 3.75% s u l p h o s a l i -c y l i c a c i d . The e x p e l l e d feces were c o l l e c t e d i n t h i s a c i d . Dehydrating animals were placed i n a d e s i c c a t o r over c o n c e n t r a t e d ' s u l f u r i c a c i d , while hydrating animals were placed i n a d e s i c c a t o r over water. 

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