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In vitro rectal transport and rectal ultrastructure in the desert locust (Schistocerca gregaria) Irvine, H. Barry 1966

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IN VITRO RECTAL TRANSPORT AND RECTAL ULTRASTRUCTURE IN THE DESERT LOCUST (SCHISTOCERCA GREGARIA.) By H. Barry I r v i n e A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s t h e s i s as conforming to the requ i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1966. In presenting this thesis in part i a l fulfilment of the requirements for an advanced degree at the University of Br i t i s h Columbia,, I agree that the Library shall, make i t freely available for reference and study. 1 further agree that permission- for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT . /.: .' The r e c t a l pad of S c h l s t o c e r c a g r e g a r i a c o n s i s t s of a l a y e r of l a r g e columnar e p i t h e l i a l c e l l s and a l a y e r of smaller oval-shaped c e l l s . Both l a y e r s appear s p e c i a l i z e d f o r t r a n s p o r t , as judged by the l a r g e number of mitochondria and membrane i n f o l d i n g s w i t h i n the two c e l l types. The u l t r a s t r u c t u r e of the columnar e p i t h e l i u m and of the secondary c e l l s i s described as i t appears under the e l e c t r o n microscope. The a b i l i t y of the rectum to t r a n s p o r t water and s a l t s was t e s t e d _in v i t r o . U n l i k e the i n v i v o p r e p a r a t i o n , the rectum i n v i t r o does not t r a n s p o r t potassium and c h l o r i d e and has only a l i m i t e d a b i l i t y to t r a n s p o r t sodium and water against a gradient. D i n i t r o p h e n o l (lCT^M.), iodoacetate (10" 2M.) and ouabain ( 10 _ 2M.) a b o l i s h water and sodium t r a n s p o r t . Potassium cyanide (10~ 2M.) and ouabain (10~3M.) do not appear to i n h i b i t water or sodium t r a n s p o r t . Iodoacetate (lO'^M.) i n h i b i t s sodium transport but does not a f f e c t water t r a n s p o r t . The i n v i t r o rectum i s dependent upon anaerobic r e s p i r a t i o n . The r e s u l t s are discussed i n terms of a scheme presented f o r i n v i v o c e l l u l a r f u n c t i o n . ( P h i l l i p s , 1 9 6 5 ) . The studies of u l t r a s t r u c t u r e and t r a n s p o r t physiology of the l o c u s t rectum do not r e f u t e the h y p o t h e t i c a l schemes presented i n t h i s t h e s i s . i i TABLE OF CONTENTS Page GENERAL INTRODUCTION 1 I . ULTRASTRUCTURE OP THE LOCUST RECTUM I n t r o d u c t i o n 6 M a t e r i a l s and Methods 7 Observations fa) General Organization 7 (b) The Columnar Epi t h e l i u m 8 (c) Basement Membrane 11 Id) Secondary C e l l s 11 ( f ) The Sheath 11 D i s c u s s i o n 12 Key to Abbreviations i n Micrographs 17 I I . STUDIES ON IN VITRO RECTUM I n t r o d u c t i o n l 8 M a t e r i a l s and Methods (a) M a t e r i a l s 19 (b) E x t e r n a l and I n t e r n a l S o l u t i o n s , P r e p a r a t i o n 19 (c) P r e p a r a t i o n of In V i t r o Rectum 21 Id) Storage of Recta l P l u i d Sample 22 le) Analyses of Rectal P l u i d Sample 23 If) Treatment of Results 24 (g) Measurement of T r a n s r e c t a l P o t e n t i a l . . 25 Results (a) P r e l i m i n a r y Experiments 27 (b) E f f e c t of I n h i b i t o r s at 10"3M 29 f c j E f f e c t of I n h i b i t o r s at 10" 2M 32 Id) S t a t i s t i c a l Analyses of Results 3^ le) T r a n s r e c t a l P o t e n t i a l s 35 ( f ) Measurement of Recta l S w e l l i n g 36 D i s c u s s i o n 37 SUMMARY AND CONCLUSIONS '. 51 REFERENCES 53 ACKNOWLEDGEMENTS. I wish to thank Dr. J . E. P h i l l i p s f o r the valuable advice and p a t i e n t d i s c u s s i o n which c o n t r i b u t e d much, to the pr e p a r a t i o n of t h i s t h e s i s . I wish to thank Dr. C. V. Finnfcgan., Dr. P. 'A. Dehnel, Dr. G. G. Scudder, and Dr. D-. J. Randall f o r t h e i r c r i t i c i s m of the manuscript. I g r a t e f u l l y acknowledge the ass i s t a n c e of Dr. A. A. Acton and Mr. L e s l i e Veto i n the prepara-t i o n of the e l e c t r o n micrographs., and Miss A. Ansen and Mrs. C. Beaumont f o r a i d i n analyses of some of the r e s u l t s . GENERAL INTRODUCTION. Osmotic and i o n i c r e g u l a t i o n and the physiology of e x c r e t i o n i n i n s e c t s have been reviewed by Shaw and Stobbart ( 1 9 6 5 ). B r i e f l y , the excretory system c o n s i s t s of a number of blind-ended Malpighian t u b u l e s , with w a l l s one c e l l t h i c k , adjoined to the gut at the j u n c t i o n of the midgut and hindgut. These tubules pass a f i l t r a t e of the major blood components i n t o the gut and thence to the rectum. E a r l y i n v e s t i g a t o r s (e.g. Wigglesworth, 1932) suggested that the rectum reabsorbed water and s a l t s . Prom measurements of i o n concentrations i n various parts of the gut of seve r a l i n s e c t s , i t was suggested that sodium, potassium, c h l o r i d e , ammonium, and carbonate ions were a c t i v e l y absorbed i n the rectum, although conclusive evidence was l a c k i n g (reviewed by Shaw and Stobbart, 1965). Rectal i o n absorption was su b s t a n t i a t e d by Ramsay ( 1 9 5 0 , 1953)> working on mosquito l a r v a e , and by P h i l l i p s (1961) on 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 F o r s k a l . The l a t t e r author showed that the l o c u s t rectum a c t i v e l y transported sodium, potassium, and c h l o r i d e from the lumen against e l e c t r o c h e m i c a l gra-d i e n t s , independent of solvent flow. A p r i n c i p a l f u n c t i o n of the rectum i n most i n s e c t s t h e r e f o r e i s s e l e c t i v e r e a b s o r p t i o n of ions secreted by the Malpighian tubules, and the organ i s thus of c e n t r a l importance i n maintaining osmotic and i o n i c balance. L i k e many t e r r e s t r i a l i n s e c t s , the desert l o c u s t 2 produces . s t r o n g l y hypertonic excreta " ( P h i l l i p s , 1 9 6 4 ) . I n studying water absorption from the rectum of the l o c u s t , P h i l l i p s discovered that t h i s h y p e r t o n i c i t y was accomplished by water re a b s o r p t i o n from the r e c t a l l.umen against an i n c r e a s i n g osmotic gradient which could not be accounted f o r by a h y d r o s t a t i c pressure g r a d i e n t , electro-osmosis, or a net simultaneous uptake of s o l u t e . The conclusion was . that water was being a c t i v e l y moved across the r e c t a l e p i t h e l i u m . * In many in s t a n c e s , (eg. Curran, I960; Diamond, 1962) water movement against a gradient has been shown to accompany s a l t t r a n s p o r t by c o - d i f f u s i o n . In S c h i s t o c e r c a , a net sol u t e movement i s not apparently necessary f o r water movement in. v i v o , although t r a n s p o r t and back d i f f u s i o n of ions are continuously o c c u r r i n g ( P h i l l i p s , 1 9 6 4 ). I t i s p o s s i b l e that water movement, (17 u l . / h r . i n the absence of an osmotic gradient) i s being d r i v e n by t h i s a c t i v e c i r c u -l a t i o n of i o n s , thus not i n v o l v i n g net s a l t movement across the r e c t a l w a l l . Three hypotheses as to how an independent tr a n s p o r t of water might be d r i v e n by a l o c a l s a l t pump are shown i n P i g . 1 (courtesy J . E. P h i l l i p s ) . The f i r s t hypothesis i n v o l v e s reverse p i n o c y t o s i s . I f the c e l l i n t e r i o r were made very hypertonic by an i o n • ^ P h i l l i p s defines a c t i v e t r a n s p o r t of water as a net movement of water against an osmotic and h y d r o s t a t i c pressure g r a d i e n t , which r e s u l t s i n an increase i n the osmotic pressure c9? the s o l u t i o n from which the absorption takes p l a c e , and r e s u l t i n g i n an increase i n the osmotic pressure gradient across the membrane separating the two s o l u t i o n s . F i g . I . Three schemes which might give r i s e to a c t i v e t r a n s p o r t of water, i n absence of net s a l t t r a n s p o r t . (Courtesy J . E. P h i l l i p s ) . cr = r e f l e c t i o n c o e f f i c i e n t LUMEN (1000 mOsro) I diffusion | H20 N4/ >1000 mOsm [ <400 I diffusion|H 20 High Hydrostatic Pressure _ Solute Transport ~ laminar flow H2o Barrier I back diffusion of cation Barrier II transport ^ of cation Intracellular (400 raOsm) (400 mOsm) HAEMOLYMPH REVERSE PINOCYTOSIS CO-DIFFUSION ELECTRO-OSMOSIS pump l o c a t e d on the blood side of the c e l l , an Inward d l f f u -s i o n of water from the lumen could occur. I f hypertonic cytoplasmic f l u i d became enclosed by a membrane and only the s a l t s were pumped out of the v e s i c l e , then i f the v e s i c l e coalesced with the s e r o s a l or hemocoel border, water could be moved with no net movement of s a l t . This hypothesis i s s i m i l a r to the one proposed by Pappas and Brandt (1958) f o r the production of hypotonic f l u i d by c o n t r a c t i l e vacuoles of protozoa. The second hypothesis proposed i n P i g . 1 a l s o r e q u i r e s a hypertonic compartment maintained by a l o c a l i o n pump. I t i s s i m i l a r to a h y p o t h e t i c a l s i t u a t i o n discussed by P a t l a k ( 1 9 6 3 )• Under t h i s hypothesis of c o - d i f f u s i o n , water d i f f u s i n g i n t o the hypertonic compartment from the lumen would create a p o s i t i v e h y d r o s t a t i c pressure w i t h i n the compartment. I f the blood side of t h i s compartment had larg e pores with a low r e f l e c t i o n c o e f f i c i e n t (Staverman, 1 9 4 8 ) , while the lumen side had small pores, a s i t u a t i o n would a r i s e whereby the h y d r o s t a t i c pressure gradient would push the s o l u t i o n through the l a r g e pores i n t o the blood space. The ions would then be returned to the compartment by the l o c a l s a l t pump. An o b j e c t i o n to these f i r s t two t h e o r i e s i s t h e i r requirement f o r a hypertonic c e l l . P h i l l i p s (1961) found the r e c t a l t i s s u e to contain low i o n i c concentrations of sodium, potassium, and c h l o r i d e , t & t a l l i n g only 400 m i l l i -osmoles/l. P o s s i b l y h y p e r t o n i c i t y i s achieved by high 4 concentrations of ions or organic solutes other than those measured or the hypertonic compartment might not represent the whole of the r e c t a l e p i t h e l i u m . This l a t t e r p o s s i b i l i t y i s considered i n the s e c t i o n d e a l i n g with e l e c t r o n microscopy. The t h i r d theory does not invoke a hypertonic compartment. Since P h i l l i p s (1961) measured a net trans-r e c t a l p o t e n t i a l of 20 m i l l i v o l t s (lumen p o s i t i v e ) , c a t ions being pumped i n t o the lumen by the h y p o t h e t i c a l l o c a l i o n pump., would tend to d i f f u s e back down the p o t e n t i a l gradient to the blood s i d e , moving water by electro-osmosis. The ions could be r e c y c l e d by the i o n pump lo c a t e d at the luminal border. Each of these three hypotheses r e q u i r e s an a c t i v e s a l t pump to d r i v e the water movement. A t e s t of these suggestions might be achieved by determining the degree of dependence of water movement upon s a l t t r a n s p o r t , p o s s i b l y by i n h i b i t i n g s a l t t r a n s p o r t with a s p e c i f i c i n h i b i t o r of the sodium-potassium pump, such as ouabain, or by the d i f f e r e n -t i a l e f f e c t s of other i n h i b i t o r s (potassium cyanide, d i n i t r o p h e n o l , iodoacetate) upon the two processes of water and s a l t movement. I f water tr a n s p o r t occurred while i o n tr a n s p o r t was i n h i b i t e d by ouabain, then i t s dependence upon sodium-potassium t r a n s p o r t would be questioned. An object of t h i s t h e s i s was to study p o s s i b l e r e l a t i o n s h i p s between i o n and water absorption. There i s a general l a c k of in f o r m a t i o n on the u l t r a -s t r u c t u r e of the i n s e c t rectum. Before any d e t a i l e d d i s c u s s i o n 5 of the water tra n s p o r t mechanism can be documented, i t Is necessary to examine the u l t r a s t r u c t u r e of the rectum to discover what systems of membrane b a r r i e r s and compartments e x i s t between the r e c t a l lumen and the hemocoel across which l a r g e osmotic gradients may be maintained. A study of the general o r g a n i z a t i o n of the epithelium^ by e l e c t r o n microscopy was therefore undertaken. 6 SECTION I . ULTRASTRUCTURE OF THE LOCUST RECTUM. I n t r o d u c t i o n . The importance of the i n s e c t rectum i n maintaining water and s a l t balance has been known f o r some time (see I n t r o d u c t i o n ) . Only a l i m i t e d amount of work, however, has been done to r e l a t e the u l t r a s t r u c t u r e of the rectum to i t s physiology. C l e a r l y any r e a l i s t i c hypothesis concerning c e l l u l a r mechanisms of r e c t a l r e a b s o r p t i o n must consider the system of membranes and compartments making up t h i s organ. The s t u d i e s of N o i r o t and Noirot-Timoth.ee ( i 9 6 0 ) on Anoplotermes  sanctus S i l v . , Smith and L i t t a u ( i 9 6 0 ) on Macrosteles  f a s c i f r o n s S t a l , and B a c e t t i (1962) on Aiolopus strepens L a t r . , i n d i c a t e s that r e c t a l t i s s u e s possess many features common to other t i s s u e s known to be engaged i n t r a n s p o r t . Such t i s s u e s as the Malpighian tubules of i n s e c t s , mouse kidney t u b u l e , and c r a y f i s h nephridium, c o n s i s t of columnar c e l l s w i t h i n f o l d e d plasma membranes c l o s e l y a s s o c i a t e d with numerous mitochondria (reviewed by Schmidt-Nielsen, 1 9 6 3 ) . However, the photomicrographs of the above workers who have st u d i e d other i n s e c t r e c t a do not provide d e t a i l s u f f i c i e n t to support or r e f u t e any hypothesis regarding the mechanism of water t r a n s p o r t i n the l o c u s t , p a r t i c u l a r l y since i t Is s t i l l not known how c o n s i s t e n t r e c t a l u l t r a s t r u c t u r e i s throughout the i n s e c t a . The l o c u s t rectum produces a very hypertonic excreta. Any hypothesis must consider where the osmotic b a r r i e r i s l o c a t e d i n the t i s s u e . From l i g h t micro-7 scope s t u d i e s , a secondary l a y e r of c e l l s was noted at the base of the columnar c e l l s , but i t was not known whether these c e l l s formed a complete l a y e r ( P h i l l i p s , 1 9 6 4 ) , r e p r e s e n t i n g a second p e r m e a b i l i t y b a r r i e r . Nothing-was known of these c e l l s ' f u n c t i o n or u l t r a s t r u c t u r e . I t was hoped that i n f o r m a t i o n regarding c o n t i n u i t y and f u n c t i o n of these c e l l s might be obtained. A t h i r d object of the e l e c t r o n microscopic i n v e s t i -g a t i o n was to t e s t one of the hypotheses forwarded i n the i n t r o d u c t i o n . I f reverse p i n o c y t o s i s were i n v o l v e d i n water t r a n s p o r t , v e s i c l e s might be expected to occur at the s e r o s a l border of the columnar c e l l s , and should be apparent under the e l e c t r o n microscope. M a t e r i a l s and Methods. Recta l t i s s u e from f i f t h i n s t a r and a d u l t S c h i s -t o c e r c a was f i x e d f o r e l e c t r o n microscopy with C a u l f i e l d s 1 b u f f e r e d osmium (Kay, 1 9 6 5 ) , or with gluteraldehyde and post f i x i n g i n osmium ( S a b a t i n i , 1963). Dehydrated t i s s u e was embedded i n Maraglass and sectioned on a Porter-Blum u l t r a microtome. Thin s e c t i o n s were placed on acetone-cleaned, uncoated g r i d s , and s t a i n e d i n ur anyl acetate, (5 min.) and l e a d c i t r a t e (5 min.). The m a t e r i a l was observed under an H i t a c h i HU-ll-A e l e c t r o n microscope. Observations. (a) General o r g a n i z a t i o n . The rectum c o n s i s t s of s i x l o n g i t u d i n a l pads of 8 columnar t i s s u e , as seen i n the cutaway diagram i n F i g . I I and P l a t e 1. The pads are separated by low cuboidal e p i -thelium ( P h i l l i p s , 1 9 6 l ) . The organ i s s u p p l i e d with numerous la r g e tracheae which branch p r o f u s e l y w i t h i n the pads. The l i g h t micrograph, i n P l a t e 1 shows a cross s e c t i o n of the rectum. The columnar c e l l s and secondary c e l l s making up the r e c t a l pad are i n d i c a t e d . (b) The columnar e p i t h e l i u m . E l e c t r o n micrographs i n d i c a t e that the columnar t i s s u e c o n s i s t s of long (100 u) narrow ( 5 - 1 0 u) c e l l s , as seen, i n P l a t e 2 . The plasma-membrane forming the luminal border i s e x t e n s i v e l y folded., ( P l a t e 3 and 4 ) , g r e a t l y i n c r e a s i n g the surface area. The f o l d s , or cytoplasmic l a m e l l i , are 800 to 1200 A across and extend 3 or 4 u i n t o the c e l l . These measurements agree with those of Smith and L i t t a u ( i 9 6 0 ) on another i n s e c t , the leafhopper Macrosteles  f a s c i f r o n s . The cytoplasm In the l a m e l l i contains many mitochondria,, ( P l a t e 5 and 6 ) , some of which extend almost to the luminal surface. The mitochondria appear i n various c o n d i t i o n s , some having c r i s t a e others c o n t a i n i n g a s l i g h t l y granular matrix. The l a c k of c r i s t a e may be due to poor f i x a t i o n , but with e i t h e r gluteraldehyde or osmium, the appearance was the same. Mitochondria In the photographs of B a c e t t i (1962) had s i m i l a r c h a r a c t e r i s t i c s . Microtubules are a l s o seen w i t h i n the cytoplasm F i g . I I . Cut away diagram showing lumen of l o c u s t rectum. F i g . I I I . Diagram showing septate desmosome (taken from P l a t e 8) PAD OF COLUMNAR CELLS 9 of the l a m e l l i , extending d i s t a l l y f o r an undetermined d i s -tance i n t o the cytoplasm. P l a t e 3 shows the lar g e number of tubules present, and t h e i r extension i n t o the cytoplasm. o The e x t e r n a l diameter of 2 7 5 - 3 0 0 A agrees with measurements described by Pawcett ( 1 9 6 6 ). Although the true length was not determined, the tubules appeared at l e a s t 6-8 u long. The i n t e r c e l l u l a r border near the luminal border of the rectum shows c e l l contact phenomena s i m i l a r to that which Pawcett (1966) terms a j u n c t i o n a l complex. This con-s i s t s of a zonula •occludens and a macula adherens, f o l l o w e d by a region of desmosome. The desmosome i n t h i s case i s a septate desmosome which i s often found i n i n v e r t e b r a t e s (Locke, 1 9 6 5 ) . As shown i n P l a t e 3* 8 , and 9, the region of the septate desmosome takes a tortuous path, so that the borders of the two c e l l s i n t e r d i g i t a t e l i k e a d o v e t a i l e d j o i n t , l o c k -in g the c e l l s together. Mitochondria are often contained w i t h i n the i n t e r d i g i t a t i n g extensions of cytoplasm, and are r e g u l a r l y seen c l u s t e r e d along the i n t e r c e l l u l a r border. The septate desmosome, upon higher m a g n i f i c a t i o n ( P l a t e 8 . ) , shows an e l e c t r o n dense matrix between the two c e l l membranes. The c h a r a c t e r i s t i c s of the septate desmosome taken from Plate- 8 are shown i n P i g . I I I . p e r i o d i c a l l y the septa are absent f o r a few hundred Angstroms, whereupon the two u n i t membranes fuse i n what Pawcett (1966) c a l l s a maculae occludents. ( P l a t e 8 . ) . Beyond the reg i o n of the desmosome, towards the 10 hemocoel, the I n t e r c e l l u l a r border widens to 400 A and of t e n separates completely l e a v i n g ^ i n t e r c e l l u l a r gaps. ( P l a t e 3 and 1 0 ). These gaps are very prominent towards the haemocoel side of the c e l l . The border from the desmosome to the blood side i s very tortuous and has l a p g e numbers of mitochondria c l o s e l y a p p l i e d to i t . Although more evidence i s needed, i t i s strange that i n the twelve micrographs examined the i n t e r c e l l u l a r border has not been observed to j o i n the s e r o s a l or hemocoel border of the c e l l . I t often stops j u s t short of the border, and may even terminate i n a v e s i c l e or i n t e r c e l l u l a r gap. ( P l a t e 1 1 ) . At the s e r o s a l border, numerous oval v e s i c l e s are noted, u s u a l l y .5 to 1 .0 u long. These have not been observed to rupture, and always appear with a f r i n g e of cytoplasm between the v e s i c l e membrane and the plasma membrane. ( P l a t e l l ) . Other c e l l u l a r i n c l u s i o n s are lysosomes, s i m i l a r to those described by De Duve (1966) and Fawcett ( 1 9 6 6 ) , and t r a c h e o l e s . Lysosomes are present i n n e a r l y every plate., ( l a b e l l e d i n P l a t e 10) . The t r a c h e o l e s are seen i n P l a t e 3> but are found i n many regions of the c e l l , accompanied by t h e i r surrounding c e l l , c o n t a i n i n g mito-chondria and o c c a s i o n a l l y a nucleus. I t may be "to these small n u c l e i that P h i l l i p s (1961) and others r e f e r to as appearing i n the proximal cytoplasm of the columnar c e l l , near the lumen. 11 (c) Basement Membrane. Between the columnar e p i t h e l i u m and the secondary c e l l s i s a 1.6 u t h i c k basement membrane c o n s i s t i n g of 0 many 200 A f i b e r s i n a complex array. The length of the f i b e r s , though undetermined, i s greater than bO u. P e r i o d i c s t r i a t i o n s were not d i s c e r n a b l e . (d) Secondary C e l l s . Between columnar c e l l s and the muscle are the secondary c e l l s . These ovoid c e l l s (20 u x 10 u) have a p e c u l i a r border of spongy appearance, containing.many c a v i t i e s from which 300 A channels lead i n t o the c e l l . ( P l a t e 1 2 , 1 3 , and 14). The channels t w i s t back and f o r t h around many mitochondria, and extend i n t o the c e l l to the l e v e l of the nucleus. These channels and mitochondria completely f i l l the cytoplasm of the secondary c e l l on the side proximal toothe columnar c e l l s . D i s t a l l y the cytoplasm of the secondary c e l l contains some endoplasmic r e t i c u l u m and a few s c a t t e r e d mitochondria, but no e l a -b o r a t i o n s of the plasma membrane. (e) The Sheath. Between the secondary c e l l s and the hemocoel i s a basement membrane, the t r a c h e a l trunks, and a t h i n l a y e r of muscle. ( P l a t e 15-) 12 D i s c u s s i o n The g r e a t l y increased surface area of the cyto-plasmic l a m e l l i , i n conjunction with the lar g e number of mitochondria l o c a t e d at the luminal border, i n d i c a t e s that t h i s surface i s re s p o n s i b l e f o r some f u n c t i o n r e q u i r i n g energy and a lar g e working surface, a f u n c t i o n such as t r a n s p o r t . The observation that the luminal border has tigh't i n t e r c e l l u l a r j u n c t i o n s i s a f u r t h e r i n d i c a t i o n that t h i s surface i s a p e r m e a b i l i t y b a r r i e r . C l e a r l y , i f a membrane were c r e a t i n g and maintaining an e l e c t r o c h e m i c a l g r a d i e n t , i t would be advantageous to reduce the passive p e r m e a b i l i t y (back d i f f u s i o n ) v i a i n t e r c e l l u l a r channels. I t has been suggested that these j u n c t i o n a l complexes (Parquhar and Palade, 1965) bind a s e r i e s of i n d i v i d u a l c e l l s i n t o a s t r u c t u r a l l y continuous b a r r i e r , across which an e l e c t r o c h e m i c a l gradient can more e a s i l y be maintained. The zonula occludents, f o r example, could r e s t r i c t water movement along the i n t e r c e l l u l a r spaces. Parquhar and Palade a l s o suggest that the maculae occludents may rep-resent a region of low r e s i s t a n c e , a l l o w i n g r a p i d e q u i l i b -r a t i o n of sodium and potassium ions between the c e l l s , making the c e l l mass a f u n c t i o n a l l y continuous compartment. Lowenstein et a l . (1964) suggest that the septate des-mosome may al s o be a low r e s i s t a n c e pathway between c e l l s from observations of e l e c t r i c a l r e s i s t a n c e and passage of f l u o r e s c e i n dye between adjacent c e l l s . Of p o s s i b l e i n t e r e s t concerning the septate desiruosome i s the observation that the 13 septa appear to be separate i n c l u s i o n s between the u n i t c e l l membranes, i n agreement with the Coggeshall (1966) f i n d i n g s i n the earthworm epidermis, r a t h e r than continua-t i o n s of the plasma membrane as suggested by Locke ( 1 9 6 5 ) . Whatever other f u n c t i o n s the desmosome has, i t probably binds the c e l l s i n t o a t i g h t sheet, which i s of primary importance i f the c e l l s are r e s p o n s i b l e f o r osmotic work. The occurrence of mitochondria i n such c l o s e a s s o c i a t i o n with the wanderings of the i n t e r c e l l u l a r border i s of unknown s i g n i f i c a n c e . (At low m a g n i f i c a t i o n , one sees that almost a l l the mitochondria i n the c e l l are close to e i t h e r the i n f o l d e d luminal border or to the two i n t e r -c e l l u l a r borders of the c e l l . See P l a t e 2.) The f u n c t i o n of the microtubules i n t h i s t i s s u e i s unknown. Pawcett (1966) suggests that microtubules have a r o l e i n maintaining c e l l shape as a " c y t o s k e l e t a l e l e -ment". I f t r u e , t h i s would be of advantage i n shaping and maintaining the cytoplasmic l a m e l l i , thus maintaining the increased surface area f o r t r a n s p o r t . An a l t e r n a t i v e , though not mutually e x c l u s i v e suggestion i s that the tubules .may be important i n protoplasmic movements, as i m p l i e d by Porter ( 1 9 6 6 ). His observations suggest that t r a n s p o r t does not take place w i t h i n the tubules but he d i d observe, by cinephotography, cytoplasm and mitochondria streaming along the surface of the tubules. In a study of sp i n d l e f i b e r s and microtubules, Mazia (1966) analyzed the tubules 14 b i o c h e m i c a l l y and suggested that they might be composed of a c t i n . He a l s o s t a t e s that microtubules could be found nearly everywhere that movement occurs w i t h i n the c e l l . I f the f u n c t i o n of the tubules i s one of cytoplasmic movement1, w i t h i n the cytoplasmic l a m e l l i of these r e c t a l col-umnar c e l l s , then i t i s conceivable that the d i f f u s i o n r a t e of r e c e n t l y transported ions and water away from the membrane and deeper i n t o the cytoplasm, may be augmented by the presence of microtubules. This would reduce the p o s s i b i l i t y of back d i f f u s i o n of ions and would r e s u l t i n an increased e f f i c i e n c y of the t r a n s p o r t i n g system. The lysosomes, by t h e i r suggested f u n c t i o n as autophagic v e s i c l e s (De Duve, 1 9 6 6 ), could be s i g n i f i c a n t to these c e l l s which have a high r a t e of metabolism^- and make heavy demands on the mitochondria. Presumably the autophagic v e s i c l e s remove and d i g e s t o l d mitochondria, making t h e i r components a v a i l a b l e f o r r e i n c o r p o r a t i o n i n t o new mitochondria. Mito'chondrial fragments are seen i n lysosomes i n some of the micrographs. Several general s i m i l a r i t i e s e x i s t between r e c t a l t i s s u e of the l o c u s t and other t i s s u e s known to be engaged i m t r a n s p o r t . Osvaldo and Harrison (1966) have shown microtubules between the i n f o l d i n g s i n c e l l s of the loop of Henle i n mamalian kidney. Fawcett (1966) shows lysosomes i n kidney tubule c e l l s . A t i g h t j u n c t i o n a l complex i s seen i n Malpighian tubules (Tsubo and Brandt, 1 9 6 3 ) , amphibian s k i n (Farquhar and Palade, 1 9 6 5 ) , and hamster 15 i n t e s t i n a l e p i t h e l i u m (Fawcett, 1 9 6 6 ). A l l are t i s s u e s across which t r a n s p o r t takes place and which r e q u i r e a good t i g h t b a r r i e r to reduce p e r m e a b i l i t y . F i n a l l y , the i n f o l d e d border a s s o c i a t e d with numerous mitochondria i s , of course, common to t r a n s p o r t i n g surfaces. One apparent d i f f e r e n c e between r e c t a l columnar e p i t h e l i a l c e l l s and those of the kidney tubulej Malpighian tubule of i n s e c t s , c r a y f i s h n e p h r i d l a , and other excretory tubules, Is the l a c k of the i n f o l d i n g of the basal membrane. In many cases, t h i s Is the area of heaviest i n f o l d i n g of the plasma membrane, j u s t the opposite of what has been found In the l o c u s t . Of h i s t o r i c a l note, Pease (1956) observed that t i s s u e s engaged i n water tra n s p o r t had the d i s t i n c t i v e c h a r a c t e r i s t i c of the i n f o l d e d s e r o s a l border. Locust rectum, unusual i n i t s a b i l i t y to transport water, does not have these i n f o l d i n g s . I t i s now known that the t i s s u e s Pease chose f o r h i s comparison were t i s s u e s where water movement accompanies ion movements p a s s i v e l y and does not i n v o l v e water transport against an i n c r e a s i n g osmotic gradient. The i n t e r e s t i n g observation that the secondary c e l l s have a complex u l t r a s t r u c t u r e and e x h i b i t a p o l a r i t y of i n f o l d i n g s and mitochondria may be s i g n i f i c a n t i n the a b i l i t y of the rectum to t r a n s p o r t water. That the secondary c e l l s may form a p e r m e a b i l i t y b a r r i e r i s i n d i c a t e d but not proven by the micrographs. I f j u n c t i o n a l complexes such as the desmosome had been observed, one might have been able to suggest an o s m o t i c a l l y t i g h t b a r r i e r across these 16 secondary c e l l s . However, t h i s was not observed. The h i g h l y i n f o l d e d surface and the high concentration of mitochondria i n d i c a t e a tran s p o r t f u n c t i o n f o r these c e l l s . R e f e r r i n g to the i n t r o d u c t o r y model l a b e l l e d c o - d i f f u s i o n , i t i s p o s s i b l e that the hypertonic compartment could be the space between the columnar c e l l s and the secondary c e l l s . The secondary c e l l s could be the surface s e c r e t i n g s a l t i n t o the compartment to maintain the hyper-t o n i c i t y , while the t h i n region between adjacent secondary c e l l s might represent the area of large pores with a low r e f l e x i o n c o e f f i c i e n t . H y p o t h e t i c a l l y , t h i s could pump water as proposed i n the i n t r o d u c t i o n . An a l t e r n a t i v e h y p o t h e t i c a l f u n c t i o n of the secondary c e l l s might be as" a second stage i o n pump i n series' 7with the larg e columnar c e l l s . Of p o s s i b l e i n t e r e s t i s the f i n d i n g of J a r i a l (personal communication) that the c o r i x i d , Cenocorixa b i f i d a (Hung.), which excretes hypotonic urine, only,has no secondary c e l l s . In summary, t h i s study of the u l t r a s t r u c t u r e of the l o c u s t rectum does not r u l e out the hypothesis of water t r a n s p o r t by reverse p i n o c y t o s i s , given i n the i n t r o d u c t i o n , since v e s i c l e s are observed on the haemocoel side of the columnar c e l l s . The discovery of a complex secondary c e l l l a y e r with i t s p o l a r i t y of i n f o l d i n g s and d i s t r i b u t i o n of mitochondria, i n d i c a t e s that two c e l l l a y e r s i n s e r i e s are in v o l v e d i n t r a n s p o r t , but whether one or both are in v o l v e d i n t r a n s p o r t i n g water remains to be discovered. 17 KEY TO ABBREVIATIONS USED IN PLATES 1 - 1 5 . In order of appearance: L cytoplasmic lamellae D desmosome T microtubule IG i n t e r c e l l u l a r gap Tr tracheole M mitochondrion Ly lysosome V v e s i c l e B basement membrane C&M channels and mitochondria N nucleus Ep columnar e p i t h e l i u m Sec secondary c e l l Mus muscle PLATE 1. L i g h t micrograph, showing a cross s e c t i o n of the rectum of the desert l o c u s t . Fixed i n Bouin's, and embedded i n wax. ( x 8 0 ) PLATE 2. Diagram of r e c t a l e p i t h e l i a l and secondary c e l l s i n r egion of r e c t a l pads, composed from e l e c t r o n micrographs. ( x i 5 0 ) LUMEN INFOLDINGS OF PLASMA MEMBRANE SEPTATE DESMOSOME TRACHEOLE MITOCHONDRION WITH TWO PLASMA § MEMBRANES NUCLEUS LYSOSOME VESICLES BASEMENT MEMBRANE CELLS PLATE 3 . Micrograph showing the luminal border of the r e c t a l e p i t h e l i u m . L cytoplasmic l a m e l l i ; D desmosome; i T microtubules; IG i n t e r c e l l u l a r gap; Tr t r a c h e o l e . "(X 7,200) PLATE 4. Micrograph of cytoplasmic l a m e l l i . Note d e n s i t y of these i n f o l d i n g s . L cytoplasmic l a m e l l i . (X 75,000) PLATE 5. Micrograph showing the occurrence of mitochondria w i t h i n the cytoplasmic l a m e l l i ' . (X 22,000) • PLATE 6. Micrograph showing how some mitochondria appear have normal c r i s t a e . The d i f f e r e n c e between microtubules and the cytoplasmic i n f o l d i n g s can c l e a r l y be seen. (X 60,000) PLATE 7. Micrograph showing number of microtubules w i t h i n the cytoplasmic l a m e l l i . Note the s t r i a t e d appearance at l e f t . (X 4 0 , 0 0 0 ) PLATE 8. Micrograph showing septate desmosome. Septa may be seen at D and at arrow. A macula occludens may be seen below and to the r i g h t of D, where the two u n i t membranes fuse i n t o a 5 layered system. (X 5 0 , 0 0 0 ) PLATE 9. Micrograph showing how many of the mitochondria appear w i t h i n the i n t e r d i g i t a t i o n s of the i n t e r c e l l u l a r border. (X 7 5 , 0 0 0 ) PLATE 10. Micrograph showing the s e r o s a l or hemocoel border of the columnar c e l l s . Ly lysosome; IG i n t e r c e l l u l a r gap; V v e s i c l e ; M mitochondrion; B basement membrane; Tr t r a c h e o l e . (X 10,000) PLATE 1 1 . Micrograph showing the i n t e r c e l l u l a r border approaching the d i s t a l border of the c e l l . The v e s i c l e i s at the c e l l border but i t has not been e s t a b l i s h e d that these are secreted. (X 7 5 , 0 0 0 ) PLATE 12. Low power micrograph, of a secondary c e l l showing the channels and mitochondria (C & M) i n the spongy l i k e r e gion (top) of the c e l l , on the lumen si d e . The nucleus (N) and a tracheole are a l s o seen. (Tr.) (X 4 , 5 0 0 ) PLATE 1 3 . Micrograph showing the sponge-like appearance of the border of the secondary c e l l s . Ep columnar epithelium; B basement membrane Sec secondary c e l l . (X 18,000-) PLATE 14 . Micrograph showing some d e t a i l of the channels and mitochondria (M) i n the spongy border of th secondary c e l l . (X 76.,000) PLATE 15 . Micrograph g i v i n g the r e l a t i o n s h i p between the ep i t h e l i u m (Ep), the secondary c e l l (Sec), and the trachea (Tr) and muscle (Mus). Note the narrow j u n c t i o n of the two adjacent secondary c e l l s . This was from a s e c t i o n of the f i f t h i n s t a r rectum, so that the i n c l u s i o n s are some-what smaller than they appear i n the a d u l t . (X 4,000) 18 SECTION I I . STUDIES ON IN VITRO PREPARATION OP RECTUM OF LOCUST I n t r o d u c t i o n The object of the present study was to develop an in. v i t r o p r e p a r a t i o n of S c h i s t o c e r c a rectum, and to f o l l o w the movements of s o l u t e and water across the r e c t a l e p i t h e l i u m . The dependence of these movements upon c e l l u l a r metabolism, not p r e v i o u s l y shown, might then be assessed by use of i n h i b i t o r s such as potassium cyanide, iodoacetate, and d i n i t r o - p h e n o l . The advantage of an i n v i t r o prepara-t i o n i s that i t permits a study of the d i r e c t e f f e c t s of the i n h i b i t o r s on metabolism, and on i o n and water move-ments across the r e c t a l e p i t h e l i u m , independent of secondary e f f e c t s due to i n h i b i t i o n of r e s p i r a t o r y v e n t i -l a t i o n and c i r c u l a t i o n present i n the in. v i v o p r e p a r a t i o n . The present i n v e s t i g a t i o n presented an oppor-t u n i t y f o r comparing the _in v i t r o p r e p a r a t i o n with the i n v i v o studies of P h i l l i p s ( 1 9 6 4 ). The comparison of i n v i t r o with _in v i v o s t u d i e s , not often made i n the l i t e r a t u r e , allowed an e v a l u a t i o n of the performance of the _in v i t r o p r e p a r a t i o n of the rectum. These experiments were a l s o designed to confirm water t r a n s p o r t using a d i f f e r e n t volume i n d i c a t o r ( i n u l i n - C 1 ^ ) from that used by P h i l l i p s ( a l b u m i n - 1 1 ^ 1 ) . Development of a f u n c t i o n a l In v i t r o prepara-t i o n makes p o s s i b l e experiments such as those of Ussing 19 (1954) on s h o r t - c i r c u i t e d f r o g s k i n , and s i m p l i f i e s the study of i o n i c f l u x e s . Such studies would g r e a t l y f a c i l i t a t e the e l u c i d a t i o n of the c e l l u l a r mechanisms i n v o l v e d i n the water t r a n s p o r t by l o c u s t rectum. M a t e r i a l s and Methods (a) M a t e r i a l s . Mature, male S c h i s t o c e r c a g r e g a r i a were chosen from the l a b o r a t o r y colony, reared on a d i e t of bran, f r e s h l e t t u c e , grass, and water i n a c o n t r o l l e d environment of 83°P. and 5 0 $ r e l a t i v e humidity under continuous l i g h t . To reduce the v a r i a t i o n due to age, temperature, environmental h i s t o r y , and other v a r i a b l e s , i t was decided to run four t e s t s (no i n h i b i t o r plus three experimentals with i n h i b i t o r ) simultaneously on i n d i v i d u a l s from the same egg batch reared i n a; s i n g l e cage. The c o n t r o l and t e s t animals were roughly chosen f o r uniform s i z e and weight. (b) E x t e r n a l and i n t e r n a l s o l u t i o n s . For these i n v i t r o experiments, i t was d e s i r a b l e that the e x t e r n a l medium (the s o l u t i o n bathing the outside of the r e c t a l sac) be as s i m i l a r to l o c u s t blood as p o s s i b l e . S c h i s t o c e r c a hemolymph contains some eleven amino a c i d s , t r e h a l o s e , dextrose, and the main blood ions (Treherne, 1 9 5 8 ) . A medium s i m i l a r to that described by Treherne was prepared. This had an osmotic pressure comparable to that of the blood (380 m i l l i o s m o l a r ) . The pH of 5 . 4 was balanced to 7 . 0 (the pH of the hemolyraph). On balancing the pH, a p r e c i p i t a t e formed l e a v i n g the medium a m i l k y colour. This was not f i l t e r e d o f f . The i n h i b i t o r s were added to t h i s e x t e r n a l s o l u t i o n i n concentrations of 1 0 " 2 , or 10"3 Molar. I n h i b i t o r s were not added to the i n t e r n a l s o l u t i o n (the s o l u t i o n i n j e c t e d i n t o the lumen of the rectum) since the lumen of the rectum i s l i n e d with a c u t i c l e permeable only to small molecules ( P h i l l i p s , 1 9 6 5 ) . E a r l y s o l u t i o n s contained p e n i c i l l i n and streptomycin., but t h i s was l a t e r discarded with the f i n d -i n g that f r e e z i n g the stock s o l u t i o n adequately i n h i b i t e d b a c t e r i a l growth. Subsequently, a l l s o l u t i o n s were stored at - 1 0°C Since the experiments were designed to t e s t a c t i v e t r a n s p o r t out of the rectum, the composition of the i n t e r n a l s o l u t i o n was adjusted to set up e l e c t r o c h e m i c a l gradients favouring passive net f l u x of ions and water i n t o the rectum; hence any absorption out of the rectum would be a c t i v e . The i n t e r n a l s o l u t i o n c o n s i s t e d of 70 mM/l. sodium c h l o r i d e , made hypertonic to the e x t e r n a l s o l u t i o n by a d d i t i o n of 260 mM/l. sucrose. The f i n a l s o l u t i o n had a f r e e z i n g p o i n t of - ,0.75°C. and a pH of 6 . 5 . / 14 The volume i n d i c a t o r ( i n u l i n - C , New England Nuclear Corp.) was added to give the s o l u t i o n an a c t i v i t y of about 1000 cpm/ul. As a v i s u a l a i d i n d e t e c t i n g leakage of the 1 -4 \ rectum, the dye amaranth was added (3 x 10 M.). In the few cases of r e c t a l puncture, t h i s dye was immediately obvious i n the e x t e r n a l media. (c) I n v i t r o p r e p a r a t i o n of the rectum. S c h l s t o c e r c a were s a c r i f i c e d by removing the head. The abdomen was cut o f f near I t s base and placed i n a simple h o l d i n g device ( P i g . I V ) , c o n s i s t i n g of a short length, of 1/8 inch, d i a . s u r g i c a l tubing and p i n s , which spreads the abdomen, thus holding i t i n place on [the wax operating t a b l e . Cannulation of the rectum through the anus was accomplished by using a 20 mm length of a 30 u l p i p e t t e (Drummond Microcap) with the end annealed and l u b r i c a t e d with d i s t i l l e d water. This cannula was very e a s i l y I n s e r t e d 3-4 mm i n t o the anus, while holding the ep i p r o c t with f o r -ceps, and q u i c k l y f i x e d i n place w i t h a beeswax and r e s i n mixture ( F i g . I V ) . An oxygen supply was f i x e d i n t o the hemocoel by i n s e r t i n g a f i n e bore (#10) polyethylene tube through a small cut i n the c u t i c l e of the eighth segment. This was f i x e d In place with beeswax and r e s i n ( F i g . I V ) . The rectum was then q u i c k l y d i s s e c t e d from the abdomen by making a l a t e r a l cut from the eighth segment, a n t e r i o r through two segments ( P i g . I V ) . A f l a p of c u t i c l e was removed from the abdomen and the gut grasped with forceps and p u l l e d out through the opening. The rec-tum was then cut j u s t a n t e r i o r to the r e c t a l pads and the hindgut discarded. The r e c t a l contents were thoroughly 22 washed out by coupling an eye-dropper to the cannula and f l u s h i n g s a l i n e through the organ, followed by s e v e r a l a l i -quots of a i r . The cut end of the rectum was then l i g a t e d with cotton thread and the organ d i s s e c t e d out as c l e a n l y as p o s s i b l e . The sac was incubated i n the e x t e r n a l s a l i n e , with or without i n h i b i t o r , f o r one hour a f t e r which time the cannula was connected to the i n j e c t i o n apparatus ( P i g . V) and 30 or 50 u l of f l u i d i n j e c t e d , i n t o the l i g a t e d rectum. A 10 u l i n i t i a l sample was taken f o r a n a l y s i s a f t e r complete mixing of the i n j e c t i o n s o l u t i o n with any r e s i d u a l r e c t a l f l u i d . The sample could be e a s i l y removed by withdrawing the rod." from the syringe ( F i g . V) and i n s e r t i n g a f i n e p i p e t t e y down Into the f l u i d . The i n j e c t e d f l u i d was l e f t i n the rectum f o r 5 hours f o l l o w i n g i n c u b a t i o n , except where noted. The f i n a l sample was taken by disconnecting the coupling and cannula, and gently squeezing the remaining r e c t a l f l u i d from the rectum onto a p a r a f f i n surface. The squeezing was necessary i n the cases where volume r e d u c t i o n took p l a c e , since only about 2/3 ( l 4 u l ) of the i n i t i a l content remained. (d) Storage of r e c t a l f l u i d sample. The I n i t i a l and f i n a l samples of r e c t a l f l u i d were stored under l i q u i d p a r a f f i n , on wax-coated p o r c e l a i n spot p l a t e s . The spot p l a t e s were e a s i l y prepared by pouring molten wax i n t o the depressions, then q u i c k l y pouring the F i g . IV. Diagram showing cannula i n abdomen, p r i o r to removal rectum. F i g . V. Diagram showing i n j e c t i o n apparatus w i t h cannulated rectum i n v i t r o . ROD RUBBER "*0* RING SYRINGE RE J. COUPLING CANNULA SALINE RECTUM 23 excess out. The spot p l a t e s with the samples were kept at _10°C i f not analyzed immediately. (e) Analyses of r e c t a l f l u i d . The osmotic pressure was determined by the cryoscopic method of Ramsay and Brown ( 1955)* whereby the f r e e z i n g p o i n t on a l i q u o t s of l e s s than 1 u l were measured to w i t h i n - 0 . 0 1°C. S i n g l e determinations were made. Chloride i o n was measured by po t e n t i o m e t r i c t i t r a -t i o n of 1 u l a l i q u o t s of the sample with s i l v e r n i t r a t e , using a Radiometer (model 25 ES) pH meter, a f t e r the method of Ramsay et a l . ( 1 9 5 5 ) . S i n g l e determinations were made. The accuracy of t h i s method i s - 1 $ . Sodium was estimated on an E.E.L. or Unlearn SP 900 flame photometer with an,-,accuracy of - 1 - 2 $ . The sample was prepared by d i l u t i n g 1 u l a l i q u o t s ; i n 4 ml of d i s t i l l e d water so that the d e f l e c t i o n f e l l w i t h i n the most s e n s i t i v e range of the machine, 5 ppm f o r EEL, at which range there are no apparent i n t e r f e r e n c e e f f e c t s from other i o n s , ( C o l l i n s and Polkinhorne, 1952) . Several readings were made on each a l i q u o t . .Potassium was estimated on the Unlearn SP 9 0 0 , by d i l u t i n g 3 u l of sample with 6 ml of 500mM sodium c h l o r i d e swamping s o l u t i o n . Duplicate samples were prepared and analyzed. To measure pH, Radiometer microelectrodes with a sample working volume of 2 u l . were employed. Readings were 24 accurate to - 0 . 0 5 pH u n i t s on a l i q u o t s of 5 u l . of sample. The electrodes were coupled to a Radiometer /(25 ES) pH meter. E a r l y attempts at measuring volume of r e c t a l con-tents were made with P 3 2 l a b e l l e d radio-phosphate. P r e l i m i -nary experiments i n d i c a t e d that t h i s method was u n s a t i s f a c t o r y due to lo s s o o f P^ 2 a c t i v i t y . Subsequent experiments were c a r r i e d out with i n u l i n - C 1 ^ . I n u l i n does not penetrate the c u t i c u l a r i n t i m a of the l o c u s t ( P h i l l i p s , 1965). The i n j e c -t i o n f l u i d was prepared to contain roughly 1000 counts per minute per u l . A l i q u o t s (3 u l . ) were taken from the sample and added to 10 ml of s c i n t i l l a t i o n f l u i d (Bray, i 9 6 0 ) and counted on a Nuclear Chicago l i q u i d s c i n t i l l a t i o n counter (Model 720), quench^corrected by the channels r a t i o method. The extreme range of d u p l i c a t e determinations on a r e c t a l sample, i n c l u d i n g p i p e t t i n g e r r o r and counting e r r o r , amounted to * 3 $ . ( f ) Treatment of r e s u l t s To compute the net volume change of r e c t a l contents from the changes i n the inulin-C"'"'^ a c t i v i t y , the f o l l o w i n g equation was used: % water absorbed = 100 ( 1 - ^ i ) -Gf where i s the i n i t i a l c oncentration of the i n u l i n as measured i n counts per minute, and i s the f i n a l c oncentration of i n u l i n . The concurrent movement of water must be considered when determining the net absorption of i o n s . This was 25 c a l c u l a t e d as f o l l o w s : $ i o n absorbed - 100( 1- c i I f ) Cf I i where 1^ i s the f i n a l c o ncentration of i o n , and Ij_ i s the i n i t i a l c oncentration of i o n . The d i f f e r e n c e s i n absorption between c o n t r o l and i n h i b i t o r - t r e a t e d preparations were s t a t i s t i c a l l y analyzed f o r s i g n i f i c a n c e with a For t r a n computor program, using a random-i z e d block design f o r a n a l y s i s of varia n c e , and t e s t i n g with Dunett's Test as given by Steele and T o r r i e ( 1 9 6 5 ). C o r r e l a -t i o n s and regressions were t e s t e d using the T r i a n g u l a r Regres-s i o n package a v a i l a b l e at the computing center. (g) Measurement of t r a n s r e c t a l p o t e n t i a l . The average net p o t e n t i a l of 20 m i l l i v o l t s e x i s t i n g across the rectum, lumen p o s i t i v e ( P h i l l i p s , 1 9 6 4 b ) , was considered to be a p o s s i b l e i n d i c a t o r of the performance of the i n v i t r o p r e p a r a t i o n . This parameter was a l s o r e q u i r e d to determine which ions were a c t i v e l y transported. S i l v e r - s i l v e r c h l o r i d e e l e c t r o d e s were fashioned as described by P h i l l i p s ( 1 9 6 4 b ). These were i n s e r t e d i n t o bridges c o n s i s t i n g of 3$ agar s o l u t i o n s of e i t h e r the i n j e c t i o n f l u i d or the e x t e r n a l s a l i n e . The electrodes were used to record from the i n t e r n a l and e x t e r n a l f l u i d s r e s p e c t i v e l y . The p r e p a r a t i o n of the agar gels i n t h i s way avoids j u n c t i o n p o t e n t i a l s between d i f f e r e n t s o l u t i o n s . A l l measurements of p o t e n t i a l were made with a Radiometer (model 25 ES) pH meter. 4 26 The asymmetry p o t e n t i a l was measured before and a f t e r each r e c t a l p o t e n t i a l by p l a c i n g the t i p s of both elec t r o d e s i n 3 Molar potassiumechloride. The t r a n s r e c t a l p o t e n t i a l was co r r e c t e d f o r t h i s asymmetry p o t e n t i a l . T r a n s r e c t a l p o t e n t i a l s were measured by withdrawing the rod from the i n j e c t i o n apparatus ( F i g . V) and i n s e r t i n g the drawn out i n t e r n a l electrode:!into the r e c t a l f l u i d . The outer e l e c t r o d e was simply immersed i n the e x t e r n a l s a l i n e . 27 Results (a) P r e l i m i n a r y experiments. While developing the i n v i t r o p r e p a r a t i o n , s e v e r a l v a r i a t i o n s of method were t e s t e d . .Performance under various c o n d i t i o n s was judged from a b i l i t y of the p r e p a r a t i o n to concentrate the r e c t a l content against a gradient. T h i r t y u l . of a sodium c h l o r i d e s o l u t i o n (made hypertonic with sucrose, see methods) was i n j e c t e d i n t o the rectum and l e f t f o r 4 hours. The f l u i d remaining at the end of the experiment was analyzed f o r osmotic pressure and c h l o r i d e . The r e s u l t s appear i n Table 1. Comparison of the f r e e z i n g p o i n t s of the i n j e c t e d s o l u t i o n and the f i n a l samples of r e c t a l f l u i d c l e a r l y i n d i c a t e s that the i n v i t r o p r e p a r a t i o n as described i n the methods, and c o n d i t i o n s 1 and 2 of Table 1, was concentrating the i n j e c t e d f l u i d against an osmotic gradient. Unlike the i n v i v o s i t u a t i o n where c h l o r i d e i s a c t i v e l y absorbed." from the lumen against a concentration gradient ( P h i l l i p s , 1 9 6 4 ) , there i s a lar g e passive i n f l u x of c h l o r i d e down a concen-t r a t i o n gradient i n t o the lumen of the in. v i t r o rectum. Removing the abdominal integument from around the rectum might damage the t i s s u e by t e a r i n g some of the tracheo l e s out of the c e l l s . Several experiments were done wit h the c u t i c l e l e f t i n place (with the O2 l i n e i n t o the hemocoel to ensure c i r c u l a t i o n ) , but t h i s d i d not appear to change the r e s u l t s i n terms of the concentrating a b i l i t y of the organ. Since l e a v i n g the abdominal integument i n 28 place made d i s s e c t i o n e a s i e r and f a s t e r , and o f f e r e d l e s s chance of damage to the r e c t a l sac, t h i s procedure was adopted. To determine whether the a r t i f i c i a l s a l i n e was an adequate s u b s t i t u t e f o r hemolymph, the performance of the pre p a r a t i o n bathed i n S c h i s t o c e r c a hemolymph was test e d f o r comparison. Hemolymph was obtained from .water-fed l o c u s t s by removal of a hind l e g . The f l u i d could be c o l l e c t e d i n a p i p e t t e ( • 0 . 4 - 0 . 5 ml per animal) and was used as a bathing media f o r the i n v i t r o p r e p a r a t i o n . Since the r e s u l t s obtained d i d not i n d i c a t e a s u b s t a n t i a l l y improved concen-t r a t i n g a b i l i t y by the rectum and since f r o t h i n g during oxygenation was a problem, the a r t i f i c i a l s a l i n e ( e x t e r n a l s o l u t i o n ) was used as the standard bathing s o l u t i o n i n the remainder of the experiments. Insects have a t r a c h e a l system f o r supplying oxygen d i r e c t l y to the t i s s u e s . I t was considered that the bubbling of 0^ through the immersion media might not be adequate to supply oxygen to the luminal side of the rectum, since tracheoles penetrate deeply i n t o the t i s s u e . A crude " r e s p i r a t o r " was attached to the cut end of the abdomen to apply a small p o s i t i v e and negative blood pressure cy c l e to the hemocoel. Hopefully t h i s might simulate normal v e n t i -l a t i o n and assure proper oxygenation of the t i s s u e v i a the normal route. The r e s u l t s d i d not i n d i c a t e an improvement. In one experiment, the e x t e r n a l s a l i n e was not adjusted to pH 7 . 0 . Complete i n h i b i t i o n of the concentrating TABLE 1. SUMMARY OF PRELIMINARY EXPERIMENTS ON THE IN VITRO RECTUM Condition Freezing Point Depression (- ^ °C.) Chloride Concentration (m. e q u i v . / l . ) I . Rectal F l u i d at End of Experiment 1. Abdominal c u t i c l e completely removed l e a v i n g only r e c t a l sac 1 . 2 9 , 1 . 4 4 , 1 . 2 3 , 1 .33 1 4 4 , 1 3 7 , 119 2 . Abdominal c u t i c l e l e f t i n place, 0^ bubbled i n t o hemocoel 1 . 2 9 , 1 . 2 6 , 1 . 6 8 , 1 . 4 0 1 7 2 , 111 , 1 3 3 , 139 3 . Abdominal c u t i c l e l e f t i n place, connected to " r e s p i r a t o r " 1 . 1 8 , I . 7 6 , 1 . 0 6 , 1 .91 146 4 . Abdominal c u t i c l e l e f t i n place, immersed i n l o c u s t hemolymph ( £± °C .83) 1 . 2 8 , 1.11 1 0 0 , 125 5 . Abdominal c u t i c l e l e f t i n place, pH of e x t e r n a l s a l i n e 5 . 5 . 7 0 , . 7 5 , . 7 5 , .82 6 1 . 5 , 5 0 . 0 I I . I n i t i a l Values E x t e r n a l s a l i n e - 0 . 7 5 125 I n j e c t e d ( i n t e r n a l ) s a l i n e - 0 . 9 2 75 29 mechanism occurred? i n d i c a t i n g a s e n s i t i v i t y to a c i d pH of the outer bathing s o l u t i o n . To t e s t that the observed concentration was due to r e c t a l a c t i v i t y and not to evaporation w i t h i n the small space of the i n j e c t i o n apparatus, s e v e r a l preparations were made s u b s t i t u t i n g polyethylene tubing i n placec of the r e c t a l sac. The same procedures of i n j e c t i n g and sampling f l u i d were followed (see methods). The volume i n d i c a t o r showed a concentration change w i t h i n the 3$ counting e r r o r , while the osmotic pressure changes were l e s s than 5 m i l l i o s -m o l e s / l . The experiment with e x t e r n a l s a l i n e at pH 5«5 a l s o confirms that the concentrating e f f e c t was due to r e c t a l a c t i v i t y . Thus the _in v i t r o p r e p a r a t i o n i s capable of measureable metabolic a c t i v i t y over a 5 hour p e r i o d . (b) The e f f e c t of i n h i b i t o r s at 1 0 " 3 Molar. I n h i b i t o r s were used to t e s t the r e l a t i o n s h i p between i o n movements, water movements, and metabolism. Potassium cyanide (KCN), an i n h i b i t o r of e l e c t r o n t r a n s p o r t , was used to i n h i b i t aerobic r e s p i r a t i o n . Ouabain i s a s p e c i f i c i n h i b i t o r of membrane ATP'ase and the sodium-potassium pump, and was used to i n h i b i t t r a n s p o r t of these cat i o n s by the in. v i t r o r e c t a l sac. Recta were d i s s e c t e d out i n t o s a l i n e c o n t a i n i n g one of the i n h i b i t o r s , or i n t o the u n i n h i b i t e d c o n t r o l s a l i n e . A 30 u l . a l i q u o t of the p r e v i o u s l y described hypertonic s a l i n e s o l u t i o n was i n j e c t e d w i t h i n 5 minutes 30 of the operation and a 10 u l . sample withdrawn w i t h i n 1 minute. The f i n a l sample (10 - 15 u l . ) was removed after-f i v e hours. The i n i t i a l and f i n a l samples were then analyzed f o r volume, osmotic pressure, sodium and c h l o r i d e i o n s , and the net changes i n r e c t a l content of these para-meters computed. In a l l , 12 experiments, each i n v o l v i n g 3 preparations ( l c o n t r o l and 2 i n h i b i t e d ^ ) , were run. The mean net changes i n r e c t a l content appear In Table 2 . The c o n t r o l preparations show water absorption against an i n c r e a s i n g osmotic gr a d i e n t , since the osmotic pressure increases from 490 m i l l i o s m o l a r to 640 m i l l l o s -molar. The sodium i s a c t i v e l y absorbed by the rectum against a concentration gradient. This uptake represents a decrease In i n i t i a l c o ncentration of about 9 $ . Chloride i o n shows a passive f l u x i n t o the lumen down an e l e c t r i c a l and a concentration g r a d i e n t , i n c r e a s i n g the concentration by 3?o. The e f f e c t s of the 10"3M. ouabain and cyanide are not s i g n i f i c a n t when compared to the c o n t r o l . Since these preparations had not been soaked i n the i n h i b i t o r p r i o r to i n j e c t i o n of r e c t a l f l u i d , the experiment was repeated i n an i d e n t i c a l manner except that the preparations were pre-t r e a t e d with i n h i b i t o r f o r one hour to allow a d d i t i o n a l time f o r d i f f u s i o n to the a c t i v e s i t e s . In a d d i t i o n the e f f e c t of 10"3 Molar d i - n i t r o - p h e n o l (DNP), an uncoupler of o x i d a t i v e phosphorylation from e l e c t r o n t r a n s p o r t , was t e s t e d . A l s o , i o d o a c e t i c a c i d was added to the KCN s o l u -TABLE 2 THE NET CHANGE IN RECTAL CONTENTS OVER A 4 HOUR PERIOD. A negative sign i n d i c a t e s net absorption or a decrease i n r e c t a l concentration and a p o s i t i v e s i g n an increase i n r e c t a l concentration. Net Change i n Re c t a l Contents Control KCN 10 3M Ouabain 10 3M 3 61 Volume of i n i t i a l ) - 1 8 . 5 Osmotic Pressure (m.osmole/l.) +151 Na (u. equiv.) - 0 . 3 5 CI (u, equiv.) + 0 . 0 6 - 1 0 . 9 +91 - 0 . 0 7 + 0 . 3 5 -24.8 +209 + 0 . 2 5 + 0 . 3 0 - 3 . 4 ±56 + + .18 .15 31 t i o n since i t i s known to i n h i b i t anaerobic g l y c o l y s i s , and should t h e r e f o r e show whether the p r e p a r a t i o n was dependent on-anaerobic metabolism. F i f t y u l of i n j e c t i o n f l u i d were i n j e c t e d r a t h e r than 30 u l , a l l o w i n g a l a r g e r volume of f i n a l sample and hence d u p l i c a t e measurements of volume and sodium. In a d d i t i o n , potassium concentration was measured i n d u p l i c a t e . The composition of the f l u i d p r i o r to i n j e c t i o n i s given i n Table 3 , and immediately a f t e r i n j e c t i o n and at the end of the f i v e hour experiment i n Table 4 . The net changes i n the r e c t a l volume, osmotic pressure, and sodium, potassium and c h l o r i d e contents are presented i n Table 5 . These data are the means computed from 12 experiments, each i n v o l v i n g four preparations ( l c o n t r o l and 3 i n h i b i t e d ) . The c o n t r o l p r e p a r a t i o n performed as. described p r e v i o u s l y , pumping water and sodium out of the rectum against a concentration g r a d i e n t , while the osmotic pres-sure gradient increased. Chloride and potassium entered the rectum p a s s i v e l y , down a concentration gradient. Iodoacetate i n h i b i t s sodium t r a n s p o r t and leads to a small passive i n f l u x of t h i s i o n i n t o the lumen, yet water continues to be pumped out against a gradient. Ouabain has no s i g n i f i c a n t e f f e c t on the a c t i v i t y of the p r e p a r a t i o n , as shown p r e v i o u s l y , although s l i g h t i n h i b i t i o n of the sodium absorption might have occurred since many of the measurements i n d i c a t e d a sodium f l u x i n t o the rectum. There was much v a r i a t i o n and t h i s d i f f e r e n c e i s not s i g n i -TABLE 3 INITIAL COMPOSITION OP EXTERNAL SALINE, AND INJECTION FLUID (MADE HYPERTONIC WITH SUCROSE), PRIOR TO INJECTION. INHIBITORS (10 _3M) ARE ADDED TO EXTERNAL SALINE. O.P. Na CI K S o l u t i o n m.osmoles/l. m.equiv./l. m.equiv./l. m.equiv./l. E x t e r n a l S a l i n e 376 105 125 4.8 I n t e r n a l S a l i n e 420 65 45 0 TABLE 4 RECTAL PLUID CONCENTRATIONS OP INITIAL ( l MINUTE) AND FINAL (AFTER 5 HOURS) SAMPLES, IN THE IN VITRO RECTUM DURING EXPERIMENT WITH 10"3M INHIBITOR O.P. V o l . Na K CI m.osmole/l. C.P.M. m.equiv./l. m.equiv./l. m.equiv./l. Control .366 1 5 2 . 7 6 3 . 6 0 . 7 4 7 . 4 DNP .375 1 5 2 . 6 6 2 . 9 1 . 6 5 0 . 1 I n i t i a l Ouab. .377 1 5 6 . 2 6 3 . 3 1 .8 5 0 . 8 KCN+IAA . 3 8 0 1 5 7 . 6 6 6 . 2 J- - 5 51 . 3 S d ±.01 ± 4 . 0 ± 2 . 0 ± 0 . 6 ± 1 .9 Control .508 2 0 8 . 0 7 9 . 1 6 . 9 7 8 . 2 F i n a l DNP .462 1 3 7 . 0 7 5 - 6 6 . 5 7 0 . 5 Ouab. .501 1 9 0 . 6 7 9 . 7 4 . 7 7 2 . 9 KCN+IAA .573 2 0 5 . 2 112 .4 7 . 1 8 6 . 7 S-. ±.028 ± 1 0 . 1 ± 1 3 . 0 ± 1 . 6 9 - ± 5 . 2 Analyses of variance shows the v a r i a t i o n i n i n i t i a l O.P., V o l . , and Na to be due to r e p l i c a t i o n and not treatment. TABLE 5 . THE EFFECTS OF INHIBITORS AT 1 0 " 3 MOLAR CONCENTRATION ON IN VITRO RECTAL ABSORPTION OVER A PERIOD OF 5 HOURS, AFTER A PRE-INCUBATION OF - ONE HOUR IN THE INHIBITOR. A p o s i t i v e sign i n d i c a t e s . a net increase i n r e c t a l content, a negative sign i n d i c a t e s a net decrease. S i g n i f i c a n t d i f f e r e n c e s between c o n t r o l and i n h i b i t e d preparations are .indicated by -(**) f o r P=0.01 and (*) f o r P=.05 Net change i n r e c t a l contentoover 5 hours Control DNP KCN+IAA Ouabain S d Volume (% i n i t i a l ) -24 .4 +18.5** - 2 0 . 4 . - 1 4 . 6 Osmotic Pressure -(m.osmole/1.) + l 4 l +87 +193 ' +124 Sodium (u.equiv.) - 0 . 1 5 5 +0.108* + 0 . 0 8 8 * +0.012-Chloride. (u.equiv.) +0.047 +0.11* + 0 . 0 6 +.031 Potassium (u.equiv.) +0.175 +0.25 + 0 . 1 8 6 + 0 . 1 0 6 + Uo -0.005 ±0.02 ±0.03 32 f i c a n t . DNP completely i n h i b i t e d net water and sodium uptake, so that the d i r e c t i o n of movement was i n t o the rectum, the reverse d i r e c t i o n from that i n the c o n t r o l . C a l c u l a t i o n of the sodium concentration of the transported f l u i d i n the c o n t r o l p r e p a r a t i o n shows the concentration of t h i s f l u i d to be very low compared to the r e c t a l f l u i d : i n j e c t e d volume = 40 u l . 24.4$ of 40 u l . = 10 u l . of water absorbed. Since . 16 u, equiv. sodium absorbed, sodium concentration of transported s o l u t i o n = . 16 x 1 0 " = 0 . 0 1 6 M 10 x IO-61 or 16 m i l l i m o l a r . The i n i t i a l t o n i c i t y of the r e c t a l content was 420 m i l l i o s m o l a r or about 420 m i l l i m o l a r ( s l i g h t l y more due to a c t i v i t y c o e f . ) , while the apparent t o n i c i t y of the t r a n s r e c t a l f l u i d was 16 m i l l i m o l a r . C l e a r l y , the water movement i s not dependent on the c r e a t i o n of an osmotic gradient by sodium t r a n s p o r t . (c) E f f e c t of i n h i b i t o r s at 10" 2 Molar. The i n e f f e c t i v e n e s s of ouabain, KCN, and KCN plus iodoacetate^ at concentrations of 10~3 Molar, i n i n h i b i t i n g r e c t a l a c t i v i t y could be due to l a c k of pene-t r a t i o n from the s a l i n e to the a c t i v e s i t e s , or the r e c t a l t i s s u e i s i n s e n s i t i v e to these i n h i b i t o r s . In e i t h e r case i t was f e l t that higher concentrations of TABLE 6 . INITIAL COMPOSITION OP OUTER SALINE AND INJECTION PLUID (MADE HYPERTONIC WITH SUCROSE), PRIOR TO INJECTION. INHIBITORS (10-2M) WERE ADDED TO THE EXTERNAL SALINE. O.P. Na CI S o l u t i o n m.osmole/l. m.equiv. / l . m.equiv./l. pH Outer s a l i n e 398 I n t e r n a l s a l i n e 452 130 87 125 7 . 0 83 6 . 5 TABLE 7 . MEAN RECTAL FLUID CONCENTRATIONS OF INITIAL ( l MINUTE) AND FINAL SAMPLES (AFTER 5 HOURS) IN THE IN VITRO RECTUM DURING EXPERIMENT WITH 10" 2M INHIBITORS O.P. - A°C. V o l . C.P.M. Na m.equiv./l. CI m.equiv./l pH S a - . m 4 S a = 4 . 4 5 s a = 2 . 3 3 s r 3 . 3 S a = 0 . 0 6 7 I n i t i a l Control .838 2 3 7 . 5 8 6 . 5 8 2 . 7 4.142 KCN .831 2 3 0 . 2 84 . 8 8 5 . 1 4 . 1 5 6 KCN+IAA .816 2 3 2 . 8 8 2 . 8 84 . 8 4 . 2 7 1 Ouabain .844 245 .6 8 3 . 3 8 2 . 3 4 . 0 9 3 F i n a l Control 1.113 2 8 9 . 2 9 2 . 7 1 1 4 . 9 4 . 0 4 8 KCN 1.044 2 6 3 . 9 9 6 . 5 1 2 2 . 9 3 . 9 9 7 KCN+IAA .798 1 7 8 . 0 7 3 . 0 9 6 . 8 4 . 5 2 7 Ouabain .901 2 3 8 . 1 8 6 . 4 100-.7 4 . 1 8 6 S-;=.07l d S g = 1 9 . 7 S a = 7 . 9 s r 9 . 3 . S a = 0 . 5 3 6 TABLE 8. THE EFFECTS OF INHIBITORS AT CONCENTRATION OF 10" 2 MOLAR ON IN VITRO RECTAL ABSORPTION OVER A PERIOD OF 5 HOURS. THE PREPARATIONS WERE PRE-INCUBATED FOR ONE HOUR IN THE INHIBITOR, PRIOR TO BEGINNING THE EXPERIMENT. A p o s i t i v e sign i n d i c a t e s a net increase i n r e c t a l contents, a negative sign i n d i c a t e s a net decrease. S i g n i f i c a n t d i f f e r e n c e s between c o n t r o l and I n h i b i t o r t r e a t e d preparations are i n d i c a t e d by (**) f o r p=0.01 and by (*) f o r p=.05. Net change i n r e c t a l contents. Control KCN KCN+IAA Ouabain S^ Volume ($) -16 -9-7 +33** +6.6* -0 . 2 Osmotic Pressure (m.osmole/1.) +151 +114 -9.7** +31** -67 Sodium , (u. equiv.) -0 .25 +0.03 +0,37* +0 .31* -0 .25 Chloride , (u, equiv.) +0.40 +0.68 +1 .22* +O.78 - 0 . 3 0 33 the I n h i b i t o r might c.a.u,se i n h i b i t i o n of the _in v i t r o rectum. The experiments were c a r r i e d out. i n the same manner as p r e v i o u s l y • d e s c r i b e d . T h i r t y u l . of a hyper-t o n i c i n j e c t i o n f l u i d having the composition shown i n Table 6 and described i n the methods, was i n j e c t e d i n t o the rectum a f t e r an i n i t i a l one hour in c u b a t i o n i n the s a l i n e ( c o n t r o l ) or i n the s a l i n e plus i n h i b i t o r . An i n i t i a l 10 u l . sample was taken Immediately and a f i n a l sample a f t e r 5 hours. The mean concentrations of volume, i n d i c a t o r , sodium, c h l o r i d e , t o t a l s o l u t e , and pH immediately a f t e r i n j e c t i o n and a f t e r 5 hours, are given i n Table 7. The i n i t i a l v a r i a t i o n i s probably due to s a l t s and water present i n the rectum before i n j e c t i o n which then becomes mixed with the i n j e c t i o n f l u i d . None of t h i s small v a r i a t i o n i s s i g n i f i c a n t . C l e a r l y , the c o n t r o l and i n h i b i t e d preparations are s t a r t i n g at the same base l e v e l of i o n and volume i n d i c a t o r , . a n d the s i g n i f i c a n t changes are due to a c t i o n of the e p i t h e l i u m In t h i s i n v i t r o p r e p a r a t i o n . The net changes i n r e c t a l contents are given In Table 8. The c o n t r o l in. v i t r o rectum behaved as i n the previous two experiments. KCN was again found to be i n e f f e c t i v e i n i n h i b i t i n g r e c t a l a c t i v i t y , r e l a t i v e to the c o n t r o l . The a d d i t i o n of iodoacetate to the KCN completely i n h i b i t s both the a c t i v e sodium tran s p o r t against a concentration gradient, and the a c t i v e water 34 t r a n s p o r t against an i n c r e a s i n g osmotic gradient seen i n the c o n t r o l . Water a c t u a l l y moves down an a c t i v i t y gradient i n t o the rectum i n l a r g e q u a n t i t y , and the passive i n f l u x of c h l o r i d e i s s i g n i f i c a n t l y higher than i n the c o n t r o l , when the rectum i s i n h i b i t e d with iodoacetate. Ouabain appears to i n h i b i t the p r e p a r a t i o n to approxi-mately the same degree as iodoacetate,. • I f the sodium concen t r a t i o n of the s o l u t i o n transported- out of the c o n t r o l r e c t a l sac Is c a l c u l a t e d as In the previous s e c t i o n , the f l u i d Is again seen to be very hypotonic to the s o l u t i o n remaining i n the r e c -tum, confirming the e a r l i e r observation that sodium uptake by the rectum does not account f o r the water uptake. (d) S t a t i s t i c a l analyses of data; c o r r e l a t i o n and r e g r e s s i o n . I n d i v i d u a l preparations show considerable v a r i a -t i o n i n the previous experiments. I t was hoped that by t a k i n g advantage of t h i s v a r i a b i l i t y and r e g r e s s i n g one parameter with another i n the same p r e p a r a t i o n , some a d d i t i o n a l understanding of the i n t e r r e l a t i o n s h i p s between water movements, i o n f l u x e s and metabolism might be acquired. The data f o r the 10~3 Molar, and 1 0 " 2 Molar experiments were t e s t e d f o r a l l p o s s i b l e regressions between the measured parameters. The s i g n i f i c a n t regres-TABLE 9 . SIGNIFICANT CORRELATIONS DRAWN FROM THE EXPERIMENTS WITH IN VITRO RECTUM UNDER CONTROL AND INHIBITED CONDITIONS. .The c o r r e l a t i o n c o e f f i c i e n t " r " . i n d i c a t e s th.e goodness of f i t of the r e g r e s s i o n l i n e ( i f a l l p o i n t s f e l l on l i n e r= 1 . 0 ) The "F" Is the symbol f o r the F t e s t which assesses how much of the v a r i a t i o n i n Y can be accounted f o r by v a r i a t i o n i n X. A lar g e F i n d i c a t e s good c o r r e l a t i o n . Th.e s i g n i f i c a n c e i s expressed as p r o b a b i l i t y values and i n d i c a t e d by (**) f o r p=0.01 and by (*) f o r p= 0 . 0 5 . TABLE OF SIGNIFICANT CORRELATIONS AT 1 0 " 2 MOLAR CONCENTRATIONS OF INHIBITOR. Osmotic Pressure on Volume Equation f o r r e g r e s s i o n r P F P Control Y=.31 + . 0 0 2 3 x . 196 .35 KCN Y= . 1 2 5 + ( - . 0 0 9 x } - . 6 7 5 ' 7 . 5 3 ## Ouabain Y= . 0 7 1 + { - . 0 0 2 x ) - . 6 6 3 7 . 0 5 if KCN+IAA Y= . o 6 9 + ( - . 0 0 3 x ) - . 7 5 1 ** 1 1 . 6 6 ** Volume on Chloride Control Y=-23.13 + -42x .747 1 1 . 3 7 KCN Y= - l 6 . 9 + -27x • 393 - - 1 . 6 5 Ouabain Y=-13.0 + . 63x . 9 0 8 4 2 . 4 6 KCN+IAA Y=-25 .4 + l . l 9 x . 7 0 5 * 8 . 8 8 *+ Chloride on Sodium ' Control Y=26.53 + 1 . 0 4 x .795 15 -43 KCN Y=26.1 + . 7 9 * .831 2 0 . 0 1 Ouabain Y=17.7 + 1 . 1 2 x .799 1 5 . 9 2 KCN+IAA Y=40 .35 + 0 . 6 3 x .649 6 . 5 8 TABLE OF SIGNIFICANT CORRELATIONS AT 1 0 - 3 MOLAR CONCENTRATIONS OF INHIBITOR. Osmotic Pressure on Volume r P F P Control Y= - . 3 3 + f - . 0 0 7 x ) - . 6 7 * 8 . 0 9 24D Y= . 0 3 + . 0 0 3 * . 2 9 - .95 -Ouabain Y= . 0 5 + • ( - . 0 0 5 x ) - . 7 9 4 1 7 . 1 KCN+IAA Y= .14 + C - . 0 0 3 x ) - . 6 3 * _ 6 . 4 3 * _ Volume on Chloride -Control Y=-21:?l + (-.I3x) - . 2 5 . 7 2 24D Y= 1 2 . 8 + . 0 9 x . 2 6 .75 Ouabain Y=-21.1 + • 37x • 54 4 . 0 4 KCN+IAA Y=-36.9 + . 4 9 x . 9 3 6 1 . 3 ** Chloride on Sodium Cont r o l Y= 31-3 + . 8 6 x .757 13-41 24D Y= 45 .2. +. . 7 6 x • 30 - - .74 - -Ouabain Y= 1 5 . 0 + . 5 x . 3 9 2 .81 KCN+IAA Y= 3 3 . 2 + . 0 0 2 x .004 . 0 0 0 2 sions appear i n Table 9. The volume change i s n e g a t i v e l y c o r r e l a t e d with osmotic pressure; that i s , the b e t t e r the water pump p e r f o r -mance, the greater the increase i n osmotic pressure. Volume change i s p o s i t i v e l y c o r r e l a t e d with c h l o r i d e , i n d i c a t i n g that reduced water pump performance accompanies lar g e pas-s i v e c h l o r i d e i o n i n f l u x i n t o the rectum. Sodium absorption i s a l s o p o s i t i v e l y c o r r e l a t e d with c h l o r i d e i o n i n f l u x , i . e . l a r g e c h l o r i d e i n f l u x accompanies low sodium transport or high sodium i n f l u x . Sodium does not appear to be c o r r e l a t e d s i g n i f i c a n t l y with volume change. C o r r e l a t i o n s were not found i n every case, as seen i n Table 9. However, that they occurred at a 95$ l e v e l of s i g n i f i c a n c e s e v e r a l times and that n e a r l y a l l the c o r r e l a t i o n s showed s i m i l a r equa-t i o n s whether s i g n i f i c a n t or not, appears to i n d i c a t e that the i n t e r r e l a t i o n s h i p s discussed above e x i s t . (e) T r a n s r e c t a l p o t e n t i a l . To evaluate which i o n movements were a c t i v e and which were passive i n the previous experiments, the tran s -r e c t a l p o t e n t i a l was followed over the same p e r i o d of time (15 hours) and under the same co n d i t i o n s of c o n t r o l and i n h i b i t o r as i n the experiments with 1 0 - 2 Molar i n h i b i t o r . The experimental procedure was the same as i n previous experiments, the p o t e n t i a l d i f f e r e n c e being measured when the i n i t i a l and f i n a l samples would normally be taken. Some preparations were measured immediately upon d i s s e c t i o n , but most were measured a f t e r one hour i n c u b a t i o n . The TABLE 1 0 . THE MEAN TRANSRECTAL POTENTIAL AT THE BEGINNING AND END OP A DUPLICATE EXPERIMENT USING INHIBITORS AT 10" 2M. N.B. SIGN REFERS TO LUMEN RELATIVE TO HEMOCOEL. MEAN ± S.D. (NO. OF OBSERVATION) P o t e n t i a l D i f f e r e n c e ( M i l l i v o l t s ) P r e p a r a t i o n At I n j e c t i o n Time A f t e r 5 Hours KCN 10" ^ M KCN+IAA 10" 2M Ouabain 10" 2M Control 17 ± 16 (12) 12 ± 8 (12) 2 ± 5 (9) 3 ± 6 (9) 15 t 9 (12) 25 ± 17 (12) l ± 3 (9) 2 * 8 ( 9 ) 36 r e s u l t s are shown i n P i g . VI and P i g . V I I . Table 10 gives the means and standard d e v i a t i o n s at i n j e c t i o n time and 5 hours l a t e r . The curves i n the graphs are not s t a t i s t i c a l l y d i f f e r e n t so the i n h i b i t o r s do not seem to be e f f e c t i n g the t r a n s r e c t a l p o t e n t i a l r e l a t i v e to the c o n t r o l . ( f ) Measurements of r e c t a l s w e l l i n g . To check that the volume change was t r u l y i n d i c a -t i v e of t r a n s r e c t a l t r a n s p o r t of water, and not a t i s s u e s w e l l i n g , r e c t a were weighed on a 50 mgm t o r s i o n balance before and a f t e r the i n v i t r o experiments. The r e c t a l sac was q u i c k l y d i s s e c t e d out i n s a l i n e , b l o t t e d on f i l t e r paper and weighed. The very slow r a t e of weight change of the t i s s u e while on the balance suggest, by e x t r a p o l a t i o n , that weight change was n e g l i g i b l e during the i n t e r v a l between d i s s e c t i o n and weighing. The i n i t i a l average weight was 1 3 . 9 mgm (range 1 3 . 4 - 1 4 . 4 mgm). The f i n a l average weight was 1 2 . 1 mgm (range 1 0 .b - 1 4 . 0 mgm). None of the f i n a l weights exceeded the range of i n i t i a l weights, so s u b s t a n t i a l s w e l l i n g d i d not take p l a c e , and could not account f o r volume l o s s by the r e c t a l contents, which averaged 10 mgm i n the c o n t r o l p r e p a r a t i o n s . F i g . V I . Graph showing the change In t r a n s r e c t a l p o t e n t i a l with time, as measured under co n d i t i o n s of experiments at i n h i b i t o r _p concentrations of 10 Molar. P i g . V I I . Graph showing the change i n t r a n s r e c t a l p o t e n t i a l with the log-^Q time, as measured under c o n d i t i o n s of experiments at i n h i b i t o r concentrations of 10~ 2 Molar. 37 D i s c u s s i o n Unlike the i n v i v o rectum of S c h i s t o c e r c a , the c o n t r o l in. v i t r o p r e p a r a t i o n d i d not show any c a p a b i l i t y f o r c h l o r i d e or potassium t r a n s p o r t and only minimal sodium t r a n s p o r t , out of the lumen. For example, i n v i t r o , the f i n a l lumen concentrations of c h l o r i d e and potassium are very close to the concentration of these ions i n the bathing s o l u t i o n , while the i_n v i v o p r e p a r a t i o n reduces the c h l o r i d e i n the rectum to 5 $ and the potassium to l e s s than 2$ of the hemolymph, concentration. The i n v i v o rectum supports l a r g e Ion concentration g r a d i e n t s , the i n v i t r o rectum does not. The r a t e s of i o n movement i n the c o n t r o l _ln v i t r o rectum are compared with the ra t e s i r i v i v o under s i m i l a r c o n d i t i o n s i n Table 1 1 . The i n vivo r a t e s are taken from P h i l l i p s ( '1961). '£ABLE i i . Ion Mean net absorption rate,, (u equiv./hr./rectum) In v i v o In v i t r o Na . 16 ± . 0 6 .05 * . 0 2 CI . 2 0 ± . 0 8 - . 0 8 ± .04 K . 0 6 ± . o i - . 0 3 5 - . 0 0 7 H 2 0 6 u l ,/hr , / r e c t . 2 u l . / h r . / r e c t . Although these experiments were not done under JO e x a c t l y s i m i l a r c o n d i t i o n s (gradients) comparisons do i n d i c a t e the r a d i c a l d i f f e r e n c e s between the two prep a r a t i o n s . The 38 In v i t r o rectum p r e p a r a t i o n shows only about 30$ of the a c t i v i t y of the in. v i v o s i t u a t i o n , w i t h respect to sodium and water. C l e a r l y , c a u t i o n should be used when extending i n v i t r o r e s u l t s to the l i v i n g animal; however, the prepara-t i o n i s u s e f u l i n o b t a i n i n g a d d i t i o n a l i n f o r m a t i o n about the mechanism of the observed water t r a n s p o r t . I n v i t r o and i n v i v o preparations of other animal t i s s u e a l s o have been shown to d i f f e r q u a l i t a t i v e l y . Frog s k i n (reviewed by H a r r i s , i 9 6 0 ) , f o r example, shows reduced r a t e s of sodium and water tra n s p o r t when these are followed i n v i t r o . Chloride t r a n s p o r t has been demonstrated i_n v i v o but not i n the i n v i t r o f r o g s k i n . When comparison i s made between the i n v i t r o and i n v i v o p r e p a r a t i o n s , c o n s i d e r a t i o n of the i n s e c t hormonal system should be made. A d i u r e t i c hormone secreted by ga n g l i a i n the head of S c h i s t o c e r c a has been shown by HigViam, H i l l and G i n g e l l (1965) . Maddrell "(1962) discusses a d i u r e t i c hormone secreted by the abdominal ganglion of Rhodnius. What e f f e c t they might have on the rectum of S c h i s t o c e r c a i n v i v o , and the e f f e c t of t h e i r absence on the i n v i t r o p r e p a r a t i o n , i s unknown. The hormones could be r e g u l a t i n g the r e c t a l water and i o n re a b s o r p t i o n . The increase i n osmotic pressure of the r e c t a l f l u i d i n v i t r o i s due p r i m a r i l y to the absorption of water. This can be shown by the c a l c u l a t i o n that a 24$ water l o s s can account f o r ( . 2 4 x 420 m i l l i o s ) about 100 of the 140 m i l l i o s m o l a r increase i n osmotic pressure ( c o n t r o l data 39 from Table 5 )• The remaining 40 m i l l i o s m o l a r increase i s due to the i n f l u x of potassium, c h l o r i d e , and probably some amino a c i d s , Into the rectum. The i r i v i t r o prepara-t i o n c l e a r l y shows the a b i l i t y to concentrate .the s o l u t i o n i t c o n t a i n s , by pumping water against an i n c r e a s i n g osmotic gradient. Water t r a n s p o r t can occur when potassium and c h l o r i d e are moving i n t o the rectum, and i n the absence of an accompanying net e f f l u x of sodium i o n as seen i n the experiment using iodoacetate and KCN at 10 Molar. In the l a t t e r experiment sodium shows a net movement i n t o the rectum, while water i s pumped out against the Increasing osmotic gradient. P h i l l i p s ( l 9 6 l ) demonstrated that no net t r a n s p o r t of s a l t s need accompany water t r a n s p o r t . The present i n v i t r o study has i n d i c a t e d that water move-ment against a gradient can take place even when the major ions are moving i n the opposite d i r e c t i o n . The water t r a n s p o r t in. v i t r o i s i n agreement with the observations of P h i l l i p s , and supports h i s f i n d i n g s by a d i f f e r e n t technique of volume I n d i c a t i o n , i . e . by use of I n u l i n - C ^ ^ . In judging the. a c t i v i t y of the _in v i t r o rectum, c o n s i d e r a t i o n must be given to the net i o n f l u x e s i n terms of the e l e c t r o p o t e n t i a l gradient across the t i s s u e . While the t r a n s r e c t a l p o t e n t i a l measured i n the i n t a c t animal appears undiminished over 3 hours ( P h i l l i p s , 1 9 6 4 ) , the p o t e n t i a l shows a steady d e c l i n e toward zero i n the i n v i t r o experiment. I f the concentration gradient and the t r a n s r e c t a l p o t e n t i a l are considered together f o r each of sodium, potassium, and c h l o r i d e i o n , the e l e c t r o -40 chemical gradient i s seen to favour c h l o r i d e and potassium movement i n t o the rectum throughout the experiment. This gradient i s i n i t i a l l y zero f o r sodium, but as the p o t e n t i a l d e c l i n e s the concentration gradient favours movement of sodium i n t o the rectum. Sodium absorption i s th e r e f o r e probably a c t i v e , while the movements of potassium and c h l o r i d e i n t o the lumen are passi v e . The independence of water movement from accom-panying s a l t movement i s s u b s t a n t i a t e d by the c a l c u l a t i o n of the osmotic pressure of the transported f l u i d , which" has been shown to be very hypotonic to the r e c t a l f l u i d . .In a l l v e r tebrate systems which have been s t u d i e d , such as the f r o g s k i n (.Us s i n g , i 9 6 0 ) and f i s h g a l l - b l a d d e r (Diamond, 1 9 6 3 ) , water i s moved as an isosmotic or hyper-osmotic s o l u t i o n , i . e . s a l t t r a n s p o r t sets up a l o c a l osmotic pressure gradient. The independence of water tra n s p o r t from net s a l t t r a n s p o r t appears to be d i f f e r e n t from any vertebrate system so f a r i n v e s t i g a t e d . The p o s s i b i l i t y that the volume decrease was not due to a c t i v e r e c t a l absorption of water, but r a t h e r to s w e l l i n g of the r e c t a l t i s s u e was considered. The observed increase i n r e c t a l f l u i d potassium and decrease i n sodium i o n i s the type of exchange one might expect i f a c e l l u l a r sodium—potassium pump at the luminal border (extruding sodium, accumulating potassium), were i n h i b i t e d . Such an exchange would lead to 'some c e l l u l a r s w e l l i n g (Tosteson and Hoffman, i 9 6 0 ) . I f the i n v i t r o p r e p a r a t i o n 41 were not being p r o p e r l y oxygenated, the reduced a c t i v i t y of such a Na-K pump would be expected to r e s u l t from reduced energy supply. The amount of s w e l l i n g necessar.y to account f o r the 24$ volume red u c t i o n ( c o n t r o l i n 10~ 3M experiments) or 10 u l , would amount to an increase of 10 mgm., or-a doubling of r e c t a l weight. This was not found. The volume l o s s i s due to t r a n s r e c t a l t r a n s -p o r t . The performance of the In. v i t r o rectum can be i n t e r p r e t e d i n terms of the h y p o t h e t i c a l scheme ( F i g . V I I I ) f o r c e l l u l a r o r g a n i z a t i o n of r e c t a l t r a n s p o r t as suggested by P h i l l i p s ( 1 9 6 5 ) , i f two assumptions are made. 'One assumption i s that the r e c t a l t i s s u e was not being adequately oxygenated by d i f f u s i o n from the e x t e r n a l s a l i n e , an assumption supported by the observation that KCN 1 0 - 2M had no measurable e f f e c t on the i r i v i t r o r e c t a l a c t i v i t y . The second assumption i s that the l a c k of oxygen i s most acute at the luminal border, having the dense p o p u l a t i o n of mitochondria and being f a r t h e s t from the oxygen source. I f the metabolic t r a n s p o r t a c t i v i t y at t h i s membrane were g e n e r a l l y i n h i b i t e d , , i t can be seen that the passive movements would predominate and c h l o r i d e and potassium would d i f f u s e i n t o the r e c t a l lumen from the c e l l s and hemocoel. Since there i s evidence ( P h i l l i p s , 1 9 6 l ) that the t r a n s r e c t a l p o t e n t i a l i s a potassium d i f f u s i o n p o t e n t i a l , the d i f f u s i o n of the potassium i o n out of the r e c t a l c e l l s on the luminal F i g . V I I I . Scheme of P h i l l i p s (1965) f o r c e l l u l a r arrange- ' " ment of mechanism^for r e c t a l t r a n s p o r t of i o n s , as i n d i c a t e d by in. v i v o observations. The scheme i n d i c a t e s that a c t i v e net i o n movement i s the r e s u l t not only of an a c t i v e t r a n s p o r t component, ( s o l i d arrows), but a l s o a passive or back d i f f u s i o n component (broken arrows). An increase i n p e r m e a b i l i t y , i n c r e a s i n g the passive component, would have the same net r e s u l t as a decrease i n the a c t i v e pumping mechanism. Figures i n brackets i n d i c a t e i o n i c concentrations i n m.equiv./l., pH u n i t s , or f r e e z i n g - p o i n t depression (-F.P.) i n °C. Unbracketed values i n d i c a t e e l e c t r o -p o t e n t i a l d i f f e r e n c e s i n m i l l i v o l t s . 42 side would r e s u l t i n a l o g a r i t h m i c d e c l i n e of the p o t e n t i a l across t h i s membrane as the potassium concentration gra-dient decreases, so that the t r a n s r e c t a l p o t e n t i a l would approach, zero m i l l i v o l t s . Provided the hemocoel membrane fu n c t i o n s as proposed i n the scheme, while the lumen membrane i s d i s -rupted as j u s t described, sodium i o n could be expected to move p a s s i v e l y from the lumen i n t o th.e c e l l s and probably be pumped i n t o the hemocoel, e x p l a i n i n g the s l i g h t uptake of sodium by the c o n t r o l preparations; how-ever, sodium uptake might not be t r a n s r e c t a l . On the other hand when i n h i b i t o r s are a p p l i e d to the hemocoel s i d e , the sodium pump would be expected to be i n h i b i t e d (metabolical-ly or d i r e c t l y ) and sodium would then move from the hemocoel to the lumen of the rectum, as was observed. The c o r r e l a t i o n s presented i n Table 9 are con-s i s t e n t with the hypothesis presented thus f a r , i n r e l a t i o n to the scheme i n P i g . V I I I . The p o s i t i v e corre-l a t i o n between volume change and c h l o r i d e i o n i m p l i e s that when the membrane i s working w e l l , ( i . e . . when there i s an adequate energy supply f o r t r a n s p o r t mechanisms In ge n e r a l ) , the volume of r e c t a l f l u i d at the end of the experiment Is low, i n d i c a t i n g good water t r a n s p o r t , and • the i n f l u x of c h l o r i d e i s low since r e s i d u a l c h l o r i d e pump a c t i v i t y reduces net i n f l u x of c h l o r i d e . A general v a r i a t i o n i n membrane transport a c t i v i t y could a l s o ex-p l a i n the c o r r e l a t i o n of sodium and c h l o r i d e . By reducing 43 the a c t i v e component of i o n movement f o r both'ions, a c o r r e l a t i o n of low sodium tra n s p o r t and high c h l o r i d e i n f l u x would r e s u l t . The volume would be expected to c o r r e l a t e n e g a t i v e l y w i t h osmotic pressure -since the osmotic pressure increases as the water i s pumped out and the s a l t s " l e f t i n the lumen of the rectum, although the c o r r e l a t i o n i s not n e c e s s a r i l y exact because of the net i n f l u x of s o l u t e . Whether v a r i a t i o n i n net i o n i n f l u x i s due to i n h i b i t e d pump a c t i v i t y as j u s t described or to general changes i n the passive p e r m e a b i l i t y of the membranes i s not known. Both suggestions f i t a l l data e q u a l l y w e l l . An obvious t e s t f o r a passive p e r m e a b i l i t y increase be-tween c o n t r o l and i n h i b i t e d rectum i n v i t r o could be to study i o n i c f l u x e s by p l a c i n g l a b e l l e d sodium and c h l o r i d e on the s e r o s a l side of the rectum and measuring the r a t e of appearance of a c t i v i t y i n the lumen. .Cyanide i n h i b i t s o x i d a t i v e phosphorylation by binding i r r e v e r s i b l y w i t h cytochrome oxidase (reviewed by Harper, 1965)• As a r e s u l t , energy cannot be trapped i n the form of high energy phosphate bonds, since the aerobic r e s p i r a t o r y pathways are blocked. Vertebrate preparations such as f i s h g a l l - b l a d d e r are i n h i b i t e d by concentrations of 10"^ Molar cyanide (Diamond, 1 9 6 2 ) . E x t r u s i o n of sodium i o n by squid nerve i s i n h i b i t e d by 10"3 M. cyanide (Ca l d w e l l et a l . , i 9 6 0 ) . The i n v i t r o i n s e c t t i s s u e used i n the present experiments was 44 i n s e n s i t i v e to cyanide at l O - ^ Molar, r e l a t i v e to a c o n t r o l p r e p a r a t i o n . This i n s e n s i t i v i t y to th.e i n h i b i -t o r at such high concentrations could be due to e i t h e r of the f o l l o w i n g two suggestions. Th.e cyanide i s not g e t t i n g to the s i t e of i n h i b i t i o n or the c e l l s i n v i t r o are not u t i l i z i n g - p y a n i d e - s e n s i t i v e aerobic r e s p i r a t i o n . Since cyanide i s a small i o n which penetrates membranes r e a d i l y , i t Is d i f f i c u l t to accept the f i r s t suggestion, e s p e c i a l l y since iodoacetate appears to be g e t t i n g i n t o the c e l l s . The conclusion i s that the i n v i t r o prepara-t i o n i s not using aerobic r e s p i r a t i o n , p o s s i b l y due to inadequate t i s s u e oxygenation as p r e v i o u s l y discussed. I t i s seen from the e l e c t r o n micrographs i n the s e c t i o n on s t r u c t u r e that tracheoles penetrate the r e c t a l c e l l s almost to the lumen. Bubbling 0^ i n the e x t e r n a l s a l i n e does not appear to d u p l i c a t e t h i s system of d e l i v e r i n g C>2 to the t i s s u e . The i r i v i t r o rectum appears to be depending on anaerobic g l y c o l y s i s as a supply of energy. The a d d i t i o n of i o d o a c e t i c a c i d to the i n v i t r o p r e p a r a t i o n completely i n h i b i t s water and sodium t r a n s -port out of the rectum, and leads to passive i n f l u x . Iodoacetic a c i d i s known to i n h i b i t the enzyme glyceraldehyde-3-ph.osphate dehydrogenase, thus i n h i b i t -i n g g l y c o l y t i c formation of ATP (Harper, 1965). The I n h i b i t i o n of the a c t i v e mechanisms by iodoacetate therefore supports the assumption that the _in v i t r o rectum preparation i s depending upon anaerobic metabolism. 45 The previous d i s c u s s i o n has i n d i c a t e d that the i n v i t r o rectum i s f u n c t i o n i n g a n a e r o b i c a l l y . I t i s therefore s u r p r i s i n g that the i n h i b i t o r DNP(lO~3 Molar) should have such, a marked i n h i b i t o r y e f f e c t upon water and sodium t r a n s p o r t . DNP t y p i c a l l y acts to uncouple o x i d a t i v e phosphorylation from e l e c t r o n t r a n s p o r t system but i s i n e f f e c t i v e i n preventing g l y c o l y t i c production of ATP, (reviewed by Harper, 1 9 6 5 ) . A s i m i l a r anomaly has been observed f o r the t u r t l e bladder. B r i c k e r and Klahr (1966) showed that DNP (10~5M) i n h i b i t e d anaerobic sodium transport i n t h i s t i s s u e . These workers demonstrated b i o c h e m i c a l l y that the g l y c o l y t i c pathway was being completed. I n h i b i t i o n of ATP synthesis was not the cause, of the i n h i b i t e d . sodium t r a n s p o r t . A l s o , ATP stores w i t h i n the t i s s u e were only reduced s l i g h t l y . (DNP stimulates ATP'ase a c t i v i t y ; Q u a s t e l l , 1 9 6 4 ) . One of t h e i r hypotheses was that a high energy intermediate was i n v o l v e d between ATP and the sodium pump, and that t h i s was being uncoupled from the pump by DNP. Whether a s i m i l a r hypothesis may apply f o r DNP a c t i o n on the i n v i t r o rectum of S c h i s t o c e r c a i s unknown. One should f i r s t demonstrate that g l y c o l y s i s i s not i n h i b i t e d . Other p o s s i b i l i t i e s are that DNP i s i n h i b i t i n g the sodium and water pumps by d i r e c t l y a f f e c t i n g the transport mechanisms, or by changing the membrane pe r m e a b i l i t y , rather than by i n h i b i t i n g the energy source. 46 Ouabain i s a cardiac g l y c o s i d e and i s a s p e c i f i c i n h i b i t o r .of membrane ATP'ase. The compound prevents accumulation of potassium and e x t r u s i o n of sodium by red blood c e l l s , while not e f f e c t i n g g l y c o l y s i s (Kahn, 1 9 6 l ) . Since the i n h i b i t o r y a c t i o n i s antagonized by high con-ce n t r a t i o n s of e x t e r n a l potassium, i t i s b e l i e v e d that ouabain i s competing f o r the same membrane s i t e as potassium. That t h i s s i t e i s membrane ATP'ase i s suggested from the comparison of the concentration of ouabain required to i n h i b i t ATP'ase with, the concentra-t i o n r e q uired to i n h i b i t the sodium-potassium pump. A s t e r o i d molecule, ouabain i s b e l i e v e d to a t t a c h to the ATP'ase by the 2 3 r d carbon and may i n a c t i v a t e the s i t e simply by i t s large s i z e (Repke, 1965). . Ouabain blocks u p h i l l t ransport of sodium-potassium i n many t i s s u e s such as vertebrate kidney, s k i n , l e n s , red blood c e l l , t h y r o i d , nerve, muscle, and s a l i v a r y gland (Kahn, 1 9 6 1 ) , and such, i n v e r t e b r a t e t i s s u e as crab nerve (Skou, 1 9 5 7 ) . I t a l so changes the p e r m e a b i l i t y of f r o g s k i n to water and c h l o r i d e (MacRobbie and Ussing, 1 9 6 1 ) , blocks c h l o r i d e transport i n i n t e s t i n e (:Qooperstain, 1 9 5 9 ) , and blocks sodium-dependent t r a n s p o r t of n o n - e l e c t r o l y t e s such as sugars and amino acids i n i n t e s t i n e (Csaky, 1 9 6 3 ) . According to Csaky, ouabain normally i n h i b i t s at concentrations of lCT? - 10"5 Molar. Insect prepara-t i o n s have been found quite i n s e n s i t i v e to ouabain. H a s k e l l , Clemons and Harvey ( 1 9 6 5 ) , and Maddrell (1966) found sodium transport i n midgut and malpighian tubules to be unaffected by ouabain at concentrations of 10~3 Molar. Treherne • (1966) however, has demonstrated that ouabain at 10"^ M o l a r . i n h i b i t s sodium e x t r u s i o n from i n s e c t nerve. In th.e present experiments w i t h S c h i s t o c e r c a rectum, 10~3 Molar ouabain d i d not s i g n i f i c a n t l y i n h i b i t sodium t r a n s p o r t , and had no apparent e f f e c t _2 upon water transport out of the rectum. At 10 Molar both t r a n s p o r t s were I n h i b i t e d . The high, ouabain concentrations may have been necessary f o r several reasons. Repke (1965) has shown that ouabain i s only 1/5 as e f f e c t i v e an i n h i b i t o r of ATP'ase at pH 5 , when compared to pH 7 . I t i s known that r e c t a l c e l l s of S c h i s t o c e r c a secrete hydrogen i o n i n t o the lumen ( P h i l l i p s , 1 9 6 4 ) . The i n t r a c e l l u l a r pH i s unknown. I f th.e i n t r a c e l l u l a r pH were a c i d , and i f the ouabain were a c t i n g at th.e luminal membrane ( i t was a p p l i e d to the s e r o s a l s i d e ) , then a high concentration of the i n h i b i t o r would be required to counteract the e f f e c t of th.e a c i d pH. The la r g e molecular s i z e (M.W.=729) and th.e f a c t that i t has never been shown that ouabain enters c e l l s (Csaky, 1963) makes t h i s explanation u n l i k e l y . A second explanation f o r the high, concentration 48 r e q u i r e d , i n v o l v e s the p e r m e a b i l i t y of the r e c t a l t i s s u e to ouabain. Prom the e l e c t r o n micrographs, one sees that the large molecule would have to d i f f u s e through a l a y e r of muscle and probably a l a y e r of secondary c e l l s , as w e l l as two t h i c k basement membranes, to get to i t s s i t e of i n h i b i t o r y a c t i o n , assuming i n h i b i t i o n of the columnar c e l l s . C l e a r l y , a high s a l i n e concent-r a t i o n of ouabain increases the l i k e l i h o o d of the molecule a r r i v i n g at i t s tar g e t by d i f f u s i o n . I f a d i f f u s i o n b a r r i e r to ouabain i s present, then the high concentration of 1 0 ~ 2 Molar may be r e q u i r e d to reach an i n h i b i t i n g concentration of 10~7 - 10~5 Molar at the a c t i v e s i t e s . Supporting the low p e r m e a b i l i t y of i n s e c t t i s s u e , Larsen et a l . (1966) found c u r a r e - l i k e e f f e c t s ^ s i m i l a r to those f o r vertebrate neuro-muscular j u n c t i o n s , could be e l i c i t e d only at high concentration of the i n h i b i t o r (lO~ 2M.). The e f f e c t s were found r e v e r s i b l e . A t h i r d p o s s i b i l i t y i s that i n s e c t t i s s u e i s g e n e r a l l y i n s e n s i t i v e to ouabain, though Treherne-'s work puts t h i s i n doubt. Progesterone and testosterone antagonize the a c t i o n of ouabain (Csaky, 1963). Per-haps the i n s e c t t i s s u e i s protected from ouabain by i n s e c t s t e r o i d s i n the t i s s u e and -hemolymph. Whether ouabain at 1 0 " 2 Molar i n h i b i t s r e c t a l water transport by i n h i b i t i n g a sodium-potassium pump 49 ( l i n k i n g the water transport to i o n transport) or by causing general metabolic a r r e s t or p e r m e a b i l i t y change has not been determined. The separation of these a l t e r -n a tives would r e q u i r e considerably more experimentation. At present no known adverse metabolic e f f e c t s occur at high, ouabain concentrations, but from the work of Tosteson (1964) on volume r e g u l a t i o n and the maintenance of c e l l m i l i e u , i f the sodium-potassium pump were i n h i b i t e d at 1 0 ~ 2 Molar ouabain concentrations, one would expect the l o s s of potassium and increase i n sodium to lead to an increase i n osmotic pressure. This would r e s u l t i n increased water i n f l u x , i n c r e a s i n g c e l l volume, and causing a general p e r m e a b i l i t y i n c r e a s e . Conceivably, t h i s could r e s u l t i n an i n t e r r u p t i o n of c e l l metabolism. Th.e i n t e r p r e t a t i o n of the r e s u l t s with, ouabain must be approached with, caution since the e f f e c t s of the i n h i b i t o r at t h i s high concentration are unknown. The f a c t that i t i s a s t e r o i d and that s t e r o i d s are l i p i d s o l u b l e , suggests that ouabain could cause a change i n membrane p e r m e a b i l i t y . Whether the i n h i b i t i o n o f j t h e water pump i s due to i n h i b i t e d i o n t r a n s p o r t or increased p e r m e a b i l i t y to water and back d i f f u s i o n of i o n s , can not be st a t e d . The observation that u p h i l l water tra n s p o r t can occur out of the rectum when the s a l t movements are a l l i n t o the rectum, makes a very strong case f o r a c t i v e transport of water i n the absence of net accompanying 50 movement of s a l t s . The three schemes presented i n the i n t r o d u c t i o n suggested how a r e c y c l i n g l o c a l i o n pump might e x p l a i n the water tra n s p o r t i n the absence of s a l t t r a n s p o r t . I f the assumption i s made that t h i s pump i s the sodium-exchange pump, then the observation that ouabain, 1 0 ~ 2 Molar, i n h i b i t s water tr a n s p o r t sup-ports the three schemes' dependence on a l o c a l i o n tr a n s p o r t . However, w i t h 10~3\ Molar potassium cyanide and iodoacetate, sodium t r a n s p o r t appears to be e l i m i n a t e d , yet water movement against an i n c r e a s i n g gradient i s s t i l l observed. This evidence tends to separate the water pump from a dependence upon i o n t r a n s p o r t . A study of i o n i c f l u x e s and changes i n t i s s u e i o n i c concentrations during absorption in. v i t r o might provide more i n s i g h t i n t o the water t r a n s p o r t . The r e s u l t s , however, show the water movement to be dependent upon c e l l metabolism, a f a c t h i t h e r t o not c l e a r l y demonstrated. 51 SUMMARY, 1. The general u l t r a s t r u c t u r e of the columnar e p i t h e l i u m and secondary c e l l s of the r e c t a l pad i s described. 2. This t i s s u e appears to c o n s i s t of two c e l l l a y e r s . ( s p e c i a l i z e d f o r a c t i v e t r a n s c e l l u l a r t ransport) i n s e r i e s , r a t h e r than a s i n g l e l a y e r as p r e v i o u s l y supposed. 3 . U l t r a s t r u c t u r e ! . observations to date are c o n s i s t e n t w i t h a l l three hypotheses of water tra n s p o r t suggested i n t h i s t h e s i s . 4." An i n v i t r o p r e p a r a t i o n of the l o c u s t rectum was deve-loped and the e f f e c t s of various metabolic i n h i b i t o r s on water and i o n absorption from the pre p a r a t i o n were st u d i e d , using i n u l i n - C " ^ as a volume i n d i c a t o r . 5 . The _in v i t r o rectum r e t a i n s the ca p a c i t y to absorb sodium and water against an i n c r e a s i n g osmotic pressure gradient but, u n l i k e the in. v i v o rectum, does not maintain the normal t r a n s r e c t a l p o t e n t i a l or the a c t i v e absorption of c h l o r i d e and potassium i o n s . 6. Water absorption was observed when net movement of a l l monovalent ions was i n t o the lumen, as i n the presence of potassium cyanide '(.10~3M,) plus iodoacetate (10~3M.). 7 . Water and sodium transport i n v i t r o are probably dependent on anaerobic r e s p i r a t i o n and are completely i n h i b i t e d by iodoacetate (lO~ 2M.), ouabain ^(lO" 2M.) and d i n i t r o p h e n o l (10~3M.) but unaffected by potassium 52 cyanide r ( l 0 ~ 2 or 10~3M.) or by lower concentrations of ouabain (lO'^M.). 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