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Mechanism of water and salt absorption in the in vitro locust rectum Goh , Soon Leong 1971

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MECHANISM OF WATER AND SALT ABSORPTION IN THE IN VITRO LOCUST RECTUM by SOON LEONG GOH B.Sc., Uni v e r s i t y of B r i t i s h Columbia, I969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER-OF SCIENCE i n the Department of Zoology V/e accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1971 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I 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. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Zoology The University of Brit ish Columbia Vancouver 8, Canada Date July 28th 1971 i ABSTRACT A method i s described f o r the preparation of an everted r e c t a l sac of the desert l o c u s t . Water and solute absorption by the rectum was determined by measuring changes i n hemocoel f l u i d and r e c t a l t i s s u e . I n i t i a l absorption rates of Na, K, CT, water and t r a n s - r e c t a l p o t e n t i a l are comparable to those i n vivo under s i m i l a r conditions. A f t e r an i n i t i a l t r a n s i e n t period (1 hour), transport a c t i v i t y of the i n v i t r o rectum remained i n a steady state f o r at l e a s t 4- hours. The r e l a t i o n s h i p between osmotic gradient and steady state rate of net water movement across the r e c t a l wall was determined. Absorption of water i s p a r t i a l l y -2 -3 i n h i b i t e d by anoxia, malonate (10 M), dinitrophenol (10~ J M), potassium -3 3 —3 cyanide (10 M) plus iodoacetate (10 M) and ouabain (10 M). Tissue ions and water are secreted i n t o the hemocoel compartment when the r e c t a l sac i s incubated i n isosmotic pure sucrose s o l u t i o n . Dependence of water movement on solute transport i s i n d i c a t e d by the requirement of lumen ions f o r prolonged maintenance of water absorption. E f f e c t s of d i f f e r e n t ions (Na, K and Cl) i n bathing media on absorption rate of water and ions, absorbate concentrations, t r a n s - e p i t h e l i a l e l e c t r o - p o t e n t i a l d i f f e r e n c e s , and t i s s u e compositions were determined. Observed properties of water and solute movement i n v i t r o are discussed and evaluated i n r e l a t i o n to possible mechanisms f o r a c t i v e absorption of water. Possible l o c a t i o n s of transport s i t e s are suggested i n a h y p o t h e t i c a l scheme based on the u l t r a s t r u c t u r e of r e c t a l epithelium. i i TABLE OF CONTENTS TITLE PAGE INTRODUCTION 1 MATERIALS AND METHODS Material 6 Methods (a) Preparation of Everted Rectal Sac 6 (b) Weighing Procedure 8 (c) Detection of Leakage 9 (d) Temperature Control . 10 (e) Composition of Bathing Media • 10 (f) Determination of Ion Concentrations and Freezing-Point Depressions 12 (g) Determinations of Ion Concentration i n Rectal Tissue 12 (h) pH of Solutions 14 ( i ) Measurement of Electro-Potential Differences 14 ( j ) Glucose Concentration Determination 15 (k) Treatment of Results 15 RESULTS CHAPTER I - CHARACTERISTICS OF RECTAL ACTIVITY (a) Introduction 17 (b) A b i l i t y of Media to Support I n i t i a l Water uptake 17 (c) Conditions f o r Optimum A c t i v i t y 19 (d) Importance of Hydrostatic Pressure 20 (e) Steady State Condition 22 (f) Effect of Osmotic Gradient on Net Water Movement 25 i i i TITLE (g (h ( i (j (k (1 CHAPTER (a (b (c (d (e CHAPTER (a (b (c (d (e CHAPTER (a (b PAGE Absorption from I n i t i a l l y Pure Sucrose Solution 28 Absorption with no Hemocoel F l u i d I n i t i a l l y 31 Glucose Concentration i n Absorbate 32 E f f e c t of pH 32 E f f e c t of I n h i b i t o r s 32 Summary 35 I I - EFFECT OF IONS ON WATER ABSORPTION Introduction 37 E f f e c t of Cations - Na, K, L i , Choline 37 E f f e c t of Anions - CI, NO^, SO^ 39 Absorption i n Absence of Na, K and CI 40 Summary 41 I I I - CHANGES IN ION CONCENTRATION AND VOLUME OF RECTAL TISSUE DURING ABSORPTION Introduction Ionic Composition of Rectal Tissue Incubated i n D i f f e r e n t Media Rectal Composition A f t e r 3 Hours Incubation i n Ringer With Modified Ion Concentration Tissue Volume Changes Summary IV - ABSORBATE CONCENTRATIONS, RATE OF ION ABSORPTION, ELECTRO-POTENTIAL DIFFERENCES Introduction 5^ Absorption i n Complex Medium and Simple Ringer .54 42 43 45 48 52 i v TITLE PAGE (c) Subsequent Effect of 1. Hour Pre-Incubation i n Sucrose Solution 57 (d) Absorption from Modified Ringer Solution 60 (e) Absorption Against an Osmotic Gradient 67 (f) Loss of Ions to Lumen Solution 68 (g) Summary 69 GENERAL DISCUSSION 71 SUMMARY 86 LITERATURE CITED 88 ACKNOWLEDGEMENTS I am most grateful to Dr. J.E. P h i l l i p s f o r guidance and helpful discussion during the preparation of t h i s thesis. I wish to thank Dr. D. Jones and Dr. R. Keeler who ki n d l y read the manuscript and, especially, Dr. D. Jones for being my acting supervisor i n the absence of Dr. J.E. Ph i l l i p s . . I am grateful to Miss P. Collen f o r determining the freezing-point depressions i n t h i s study. I am also deeply indebted to my wife, Christine, f o r her encouragement and the typing of the thesis . This work was supported by grants from the National Research Council of Canada to Dr. J.E. P h i l l i p s and the McLean Fraser Memorial Fellowship. INTRODUCTION The evolutionary success of i n s e c t s i s p a r t l y a t t r i b u t a b l e to a h i g h l y e f f i c i e n t excretory system which can maintain blood concentrations w i t h i n narrow l i m i t s i n the face of extreme f l u c t u a t i o n s i n environ-mental conditions associated with the t e r r e s t r i a l h a b i t a t . In p a r t i c u l a r water conservation i s of paramount importance because of small body s i z e of in s e c t s and hence r e l a t i v e l y large surface area f o r water l o s s (Wigglesworth, I 9 6 6 ) . This has re s u l t e d i n the evolution of e f f i c i e n t mechanisms f o r the conservation of water i n t h i s group. In most i n s e c t s , excretion i s accomplished by the combined actions of malpighian tubules and rectum. Recent studies have demonstrated that the main function of the rectum i s the s e l e c t i v e reabsorption of water and solutes secreted by the malpighian tubules (reviewed by Craig, I 96O; Shaw and Stobbart, 1965 ; Wigglesworth, I 9 6 5 ) . In t h i s sense the rectum i s u l t i m a t e l y responsible f o r reg u l a t i o n of blood concentrations and, e s p e c i a l l y , the r e t e n t i o n of water. The a b i l i t y of the rec t a of many i n s e c t s to absorb water from the feces was f i r s t observed by Wigglesworth ( 1932)„ Recent studies on in s e c t s deprived of water f o r extended periods have shown that the i n s e c t rectum can produce st r o n g l y hyperosmotic excreta (Ramsay, 1955? P h i l l i p s , 1 9 6 4 a ; Wall and Oschman, 1 9 7 0 ) . The desert l o c u s t , (Schistocerca  gregaria ForskSl) with more st r i n g e n t requirements f o r continuous water conservation, has developed a very e f f i c i e n t r e c t a l reabsorptive system. I n t h i s species, P h i l l i p s (1964a,b,c) has shown that Na, K and Cl are reabsorbed from the r e c t a l lumen against electro-chemical gradients and 2 independent of solvent drag. Thus by d e f i n i t i o n (Anderson and Ussing, i960) these ions are a c t i v e l y transported. Hyperosmotic excreta i s formed by active absorption of water from the rectum rather than by solute secretion into the lumen. ( I t should be noted that no d i s t i n c t i o n between primary and secondary transport i s implied by the term 'active transport of water'.) P h i l l i p s (1964a; also Wall, 196?; Stobbart, I968) has found that net water uptake can continue against an increasing osmotic gradient without concomitant ion uptake and i s not abolished by reversing the t r a n s - e p i t h e l i a l p o tential difference ( i . e . electro-osmosis i s u n l i k e l y ) . Besides mammals and some bird s , only insects and other t e r r e s t r i a l arthropods are known to have the a b i l i t y to conserve water by the produc-t i o n of hyperosmotic urine. The insects do not possess the anatomical equivalent of the counter-current system found i n the vertebrate kidney and yet they can accomplish the same l e v e l of urine osmolalities. Furthermore, r e c t a l water absorption i s unlike that i n the vertebrate ileum (Curran, i960; Fordtran and Dietschy, I966) and g a l l bladder (Diamond, 1962c; Dietschy, 1964; Kaye et a l , I966). In these systems, water movement i s c l o s e l y correlated with net solute transport and does not lead to increasing osmotic gradients. Analysis of absorbate indicates that the transported f l u i d i s hyperosmotic or at most isosmotic to the f l u i d from which absorption occurs. Consequently, water movement i s interpreted as a secondary process as proposed i n the double-membrane hypothesis (Curran and Mcintosh, I962) and the standing-gradient osmotic flow model (Diamond and Bossert, I967). In the locust rectum, water absorption can occur from an i n i t i a l l y 3 pure sucrose s o l u t i o n and the absorbate i s hyposmotic. The observed transport rates of ions are too low to account f o r the net f l u x of water, although b a c k - d i f f u s i o n of ions i s continuously occurring. P h i l l i p s (1965, 1969, 1970), Oschman and Wall (I969) and Wall and Oschman (1970) therefore suggested that r e c t a l water movement could be driven by a c t i v e r e - c i r c u l a t i o n of ions, thus not i n v o l v i n g net solute movement across the r e c t a l w a l l as a whole. U l t r a s t r u c t u r a l observations of the r e c t a l epithelium i n the cockroach (Oschman and Wall, I 9 6 9 ) and the l o c u s t ( I r v i n e , I 9 6 6 ) revealed that the r e c t a l p a p i l l a e contain an extensive system of i n t e r c e l l u l a r spaces ( 2 0 0 8. wide) formed by i n t e r d i g i t a t i n g membranes at the l a t e r a l boundaries of the c e l l s . On u l t r a s t r u c t u r a l grounds, Berridge and Gupta ( I 9 6 7 ) suggested that i o n transport i n t o the l a t e r a l i n t e r c e l l u l a r spaces causes f l u i d absorption from the lumen by l o c a l osmosis. P h i l l i p s (1969» 1 9 7 0 ) and Oschman and Wall ( I 9 6 9 ) have pointed out that acceptance of the former hypothesis a l s o requires that ions must be absorbed from the l a t e r a l spaces to account f o r the hyposmosity of absorbate (measured d i r e c t l y by Wall and Oschman, 1 9 7 0 ) l e a v i n g the r e c t a l pads. In support of t h i s hypothesis of l o c a l osmosis followed by solute reabsorption, Wall et a l ( 1 9 7 0 ) found f l u i d ( c o l l e c t e d by micropuncture) from the a p i c a l sinuses to be hyperosmotic to the lumen content. However, c l e a r demonstration that water transport i n the i n s e c t rectum i s coupled to and dependent upon solute transport ( i . e . r e - c y c l i n g ) has not yet been achieved. Since t h i s type of thermodynamic evidence i s the p r i n c i p a l b a s i s f o r assuming that water transport i s secondary to i o n transport across vertebrate e p i t h e l i a (reviewed by Schultz and Curran, I 9 6 8 ) , 4 a s i m i l a r demonstration of water-ion coupling i s required to t e s t the v a l i d i t y of t h i s hypothesis i n the case of the i n s e c t rectum ( P h i l l i p s , 1969). The present study was undertaken to i n v e s t i g a t e some of the pr o p e r t i e s of the r e c t a l reabsorption process using an i n v i t r o preparation of the l o c u s t rectum. In v i t r o preparations have played a major r o l e i n e l u c i d i n g mechanisms of solute and water transport across other e p i t h e l i a such as vertebrate i n t e s t i n e (reviewed by Schultz and Curran, I968), g a l l bladder (reviewed by Diamond, I968) and amphibian membranes (Ussing, 195^)• The advantages of i n v i t r o preparations are that i t permits the c o n t r o l and manipulation of conditions (thus enhancing experimental design) and allows study of the d i r e c t e f f e c t s (as opposed to i n d i r e c t v i a endocrine system, etc.) of metabolic i n h i b i t o r s and various media on absorption rate, absorbate concentration and t i s s u e composition. Recently, several workers have t r i e d to develop i n v i t r o preparations of the i n s e c t rectum; e.g. l o c u s t ( I r v i n e , I966; I r v i n e and P h i l l i p s , 1971; Mordue, 1969)1 cockroach (Wall, I967), and s t i c k i n s e c t ( Vietinghoff, I966). However, unambiguous conclusions concerning mechanisms of r e c t a l reabsorption have been v i r t u a l l y impossible since none of these workers considered how t h e i r preparations changed with time. An exact d e s c r i p t i o n of changes i n a c t i v i t y of i n v i t r o preparations with regard to water and i o n transport and maintenance of p o t e n t i a l d i f f e r e n c e s with time i s necessary. In the present study, evaluation of the performance of the i n v i t r o preparation was p o s s i b l e by comparison with i n vivo studies of 5 P h i l l i p s (I964a,b,c) and Stobbart ( I 9 6 8 ) . Unlike most previous prepa-rations, an everted rectal sac was employed. The f i r s t part of this thesis i s concerned with development and evaluation of the i n vitro preparation. Some characteristics of the i n vitro preparation and the change i n water transport activity with time i n different media are described. Some factors that could cause or influence movement (such as hydrostatic pressure, osmotic gradients, volume of external f l u i d , aeration of bathing media, presence of ions on lumen or hemocoel sides and effects of inhibitors) are examined. The main objective of the present study was to investigate the possible dependence of water transport on solute transport. The lat t e r part of the thesis, therefore, deals with the direct effects of various media containing different concentrations of ions (such as Na, K, choline, L i , CI, NO^ , SO^) on such parameters as water transport, tissue volume and composition, absorbate concentrations, solute absorption rates and trans-rectal potentials. These experiments were designed to evaluate the relationship between ion transport and net water movement. The importance of tissue ions and circulation of ions via the lumen or within the rectal pad were also examined. 6 MATERIALS AND METHODS Material Adult male Schistocerca gregaria which had past t h e i r f i n a l moult were used i n a l l the experiments. The locusts were reared i n cages at 28°C and 50$ r e l a t i v e humidity and fed on bran, lettuce and spinach. Before the s t a r t of experiments, locusts were i s o l a t e d and supplied with only hyperosmotic saline (300 mM/l NaCl; 150 mM/l KCI; 30 mM/l MgCl 2.6H 20; 30 mM/l CaCl2.2R"20) for 2 to 4 days, since saline-fed locust reabsorb water i n the rectum against greater osmotic gradients than those fed on tap water ( P h i l l i p s , 1964a). Methods (a) Preparation of Everted Rectal Sac. The locust was anaesthetized with a mixture of C0 2 and ether vapour and then secured dorsal side up on a p l a s t i c i n e block. (The following operation was carried out under a dissecting microscope.) A U-shaped i n c i s i o n (8 mm2) was made do r s o - l a t e r a l l y on the fourth to s i x t h abdominal segments and the r e s u l t i n g f l a p of c u t i c l e was pinned back. The hind-gut was raised s l i g h t l y and a clean human h a i r was passed under i t to provide a l i g a t u r e . Care was taken not to touch or damage the rectum. A short piece (3 cm length) of P.E. 160 polyethylene tubing with a s l i g h t l y f l a r e d end was inserted through the anus into the rectum u n t i l the f l a r e d end of the tubing had just passed the anterior end of the r e c t a l pads. (Some of the early experiments were performed using P.E. 90 polyethylene tubing.) A l i g a t u r e was then t i e d between the anterior end of the r e c t a l pads 7 and the end of the tubing. The hind-gut and the connecting trachea were cut away from the rectum and the cannulated rectum was then everted by slowly withdrawing the tubing p o s t e r i o r l y away from the anus. Extreme care was taken to prevent extensive s t r e t c h i n g as t h i s was found to damage the transport function of the rectum. The everted rectum was then withdrawn u n t i l the p o s t e r i o r ends of the r e c t a l pads had emerged from the anus. The sac was then cut away from the animal between the anus and the p o s t e r i o r ends of the r e c t a l pads. The cannulated r e c t a l sac was placed i n a small polyethylene v i a l containing 8 ml of Ringer with the lumen ( i . e . c u t i c l e side) of the rectum now fa c i n g outward. The hemocoel side (or i n s i d e ) of the everted r e c t a l sac was then r i n s e d by i n j e c t i n g about 1 ml of the Ringer using a syringe attached to P.E. 10 polyethylene tubing, which could be i n s e r t e d i n t o the sac through the cannula. This r i n s e f lushed out any hemolymph and f e c a l m a t e r i a l trapped during the cannulating and everting procedure. Using another piece of human h a i r , a l i g a t u r e was t i e d a t the p o s t e r i o r end of the r e c t a l pads to close the r e c t a l sac. The hemocoel side of the r e c t a l sac was again r i n s e d with about 0.5 nil of the Ringer (or other modified experimental s o l u t i o n s ) and any remaining f l u i d was withdrawn completely (+ 0.25 u l i by weighing) with the syringe. The Ringer or experimental s o l u t i o n (10 u l a l i q u o t ) could then be introduced on the hemocoel side of the rectum by i n j e c t i o n through the cannula with a 'Hamilton 1 syringe. The s o l u t i o n i n the sac generally rose about one-third the way up the cannula so that a s l i g h t h y d r o s t a t i c pressure was set up from' the hemocoel to lumen s i d e . A hook was made from a d i s s e c t i n g p i n and Fig. 1. In vitro rectal sac with lumen side facing the external incubating solution (25 ml) and the hemocoel side containing 10 p i of experimental solution. A hook i s attached to the cannula and i s used for suspending the rectal sac during incubation and when weighed on " a torsion balance. weighing hook cap cannula (P.E. 160) hemocoel fluid everted rectal sac ligature lumen (external) solution 8 the head inserted into the top of the cannula to block i t . The cannula was then punctured to create several tiny holes made just under the head of the hook. The hook was used to support the rectal sac during weighing and incubation (Fig. 1). The glass t i p of the hook f i t t e d t i g h t l y to the cannula and this served to prevent excessive evaporation of the hemocoel solution while the small perforations permitted equali-zation of a i r pressure as a i r was displaced by f l u i d absorbed during the experiment. The entire procedure described above was usually completed i n less than 5 minutes. During experiments, a l l hemocoel f l u i d i n the rectal sac was removed at the end of every hour and replaced, after rinsing out the sac, with another 10 ul aliquot of the experimental solution. Removal of f l u i d was accomplished by use of P.E. 10 tubing attached to a disposable syringe or a tapered 30 u l micropipette when the f l u i d was kept for chemical analysis. A total of 0,5 ml of fresh experimental solution was used i n the rinse procedure. The replacement of the hemocoel f l u i d was completed within 30 seconds. (b) Weighing Procedure. Net transfer of f l u i d across the rectal wall was determined from changes i n weight of the cannulated rectal sac. An increase i n weight indicated absorption by the rectum; whereas, a decrease indicated net loss of f l u i d to the lumen side. I n i t i a l experiments demonstrated that rectal sacs could be repeatedly re-weighed to within + 0.25 mg using a 200 mg 'August Sauter' torsion balance. Before each reading was taken, the cannulated rectal sac was l i g h t l y wiped with absorbent tissue. Since the surface of rectal cuticle i s hydrophobic, 9 removal of external f l u i d i s f a c i l i t a t e d . The entire weighing procedure took less than 30 seconds. Any changes i n weight of the cannulated rectal sacs normally reflected changes i n the hemocoel volume. However, the p o s s i b i l i t y that hydration or dehydration of the rectal tissue, occurred during incubation (especially following transfer to different experimental solutions) was considered. Changes i n tissue volume were estimated by comparing the i n i t i a l weight of a given rectal sac at the start of an hour with the i n i t i a l weight of the same rectal sac at the start of the next hour. A.t both times the internal volume was constant (+ 0,25 ul) at 10 u l . Therefore, differences between the i n i t i a l , weights indicated changes of the rectal tissue volume which exceeded 0.5 u l . In cases where significant hydration or dehydration did occur, the net changes i n hemocoel volume were determined by subtracting or adding the tissue volume changes to or from the observed changes i n weight of the cannulated rectal sacs. (c) Detection of Leakage. On termination of the experiment, the cannulated rectal sac was tested for possible leakage by placing i t i n a concentrated solution of amaranth for more than 12 hours. Any appearance of this dye on the hemocoel side indicated leakage since the rectal wall i s impermeable to amaranth due to the cuticular intiraa (Phillips and Dockrill, 1968), In most of the l a t e r experiments where the rectal sacs were removed and used for determinations of tissue ions, the test for leakage was not performed. However, control preparations were tested for leakage occasionally and i n a l l these cases no leakage was observed. TABLE 1. Composition of Complex Medium. Compound mg/lOO ml NaCl 143.0 NaHCOo 88.6 KCI 63.5 NaOH 112.5 CaCl2.2H20 29.0 MgCl2.6H20 264.0 Na succinate 200.0 Na citrate 55.0 Na malate 200.0 Glucose 200.0 Trehalose 200.0 Maltose 200.0 Sucrose 400.0 Glycine 20.0 Proline 53.0 Glutamine 40.0 Na glutamate 235.0 Lactalbumin hydrolysate 800.0 T.C. Yeastolate 400.0 P e n i c i l l i n 3.0 Streptomycin 10.0 10 (d) Temperature Control. The experimental solutions and rectal preparations contained i n 25 ml Erlenmeyer flasks were a l l kept at 30 + 0.5°C i n a small constant temperature bath. When solutions were oxygenated or aerated, the gas temperature was allowed to equilibrate by passing 1 foot of the gas lead (P.E. 10 polyethylene tubing) through the water bath. External solutions (facing the cuticular side of rectal sacs) were continuously mixed by the bubbling gas i n a l l experiments. (e) Composition of Bathing Media 0 In the preliminary experiments a number of media were tested for their a b i l i t y to sustain transport ac t i v i t y of cannulated rectal sacs. Hemolymph obtained from the neck of locusts was tested by placing i t on hemocoel side and a Ringer (NaCl, 650 mg/lOOmlj KCI, 14.0 mg/lOOml; CaCl2.2R"20, 15.9 mg/lOOml; NaHgPO^, 1.0 mg/lOOml; and NaHCO-j, 20.0 mg/lOOml) with added sucrose on the lumen side, A complex incubation medium described by Berridge (1966) was next tested. The composition of this medium i s shown i n Table 1. The absorption rate of f l u i d at the end of the f i r s t hour was found to be comparable to i n vivo rates. However, using this medium, i t was relatively d i f f i c u l t to alter the ionic composition as required by the experimental design and consideration would have to be given to possible influence on rectal a c t i v i t y of various organic compounds such as amino acids and albumin, A simple Ringer used by Mordue (1969) was reported to support (at least for short periods) a comparable rate of f l u i d absorption to those observed i n vivo or i n vitro using the complex incubation medium, • TABLE 2. Composition of Simple Ringer and Various Modified Ringers (mM/l). Compound Simple K-free Na-free L i Choline NOV SO^ Sucrose NaCl KC1 MgCl 2 C a C l 2 NaH2P04, NaHC03 Glucose KH2PO^. KHCO3 L i C l Choline CI H3PO^ TEA. OH NaN03 KNO3 M g ( & 3 ) 2 Ca(N03) 2 Na2S0i). K 2S0^ - - - 3.2 MgSOzj. - - - 3.6 CaSOzj. - - - 2.2 Sucrose - - - - - - 293.0 Choline HCOo - - - - 2.1 168.0 174.4 - - - - - -6 .4 - 174.4 - - - - -3.6 3.6 3.6 3.6 3.6 - - -2.2 2.2 2.2 2.2 2.2 • - - -6.1 6.1 - - - 6.1 6.1 -2.1 2.1 — - - 2.1 2.1 -16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 — — 6.1 6.1 - - - -— — 2.1 2.1 - - - -— - - 174.4 - - - -— - - - 174.4 - - -— _ — - 6.1 - - 6.1 _ — — — 6.1 - - 6.1 — _ — — - 168.0 - -— — - - — 6.4 - -- - - - - 3.6 - 3.6 — _ — — - 2.2 - 2.2 — - - - - - 84.0 -Na:K Ringer was a 1:1 mixture of K-free and Na-free Ringers. pH of a l l Ringers (except sucrose Ringer) was adjusted to 7.0. The unad-justed pH of simple Ringer was 5.9. The freezing-point depression of a l l media above (except SO^ Ringer) was -0.7l3°C. S0^ Ringer has freezing-point depression of -0,49l°C. 11 The composition of this medium i s shown i n Table 2. The ionic requirements for water transport were studied by replacing Na, K or Cl i n the simple Ringer with various other inorganic or organic cations and anions depending on the objective of the experiment. The compositions of these media are shown i n Table 2, The osmotic pressure of the various modified media was kept constant (with 1 exception noted i n Table 2) by replacing any ion with an osmotically equivalent quantity of the substituted ion or sucrose. For example, i n the sucrose Ringer, Na, K and Cl were replaced with an amount of sucrose calculated to give the same osmolarity as the substituted ions. The only exception was SO^ Ringer where the cations of the SO^ salts were kept at the same molarity whereas the SO^ replaced half the molarity of the substituted Cl. Sucrose was added to the simple Ringer to make up solutions with higher osmolarities thereby permitting osmotic gradients to be established between the hemocoel and the lumen sides. The osmotic gradients across the rectal wall were calculated from the freezing-point determinations made on the various solutions. These solutions were prepared by diluting a stock solution (Ringer plus 0.7 M/l sucrose) with normal simple Ringer. The various inhibitors of transport and respiration tested were dinitrophenol (DNP), ouabain, KCN and iodoacetate (IAA). They -3 were added to the experimental solutions at a concentration of 10 M. Solution with inhibitor was applied to either side or both sides of the rectal sac after i n i t i a l one hour incubation i n the same solution without inhibitor to test the i n i t i a l a c t i v i t y of each 12 rectal sac. Control sacs were incubated in the test solution without inhibitor over the whole experimental period. (f) Determination of Ion Concentrations and Freezing-Point Depressions. The Na and K concentrations in the lumen and hemocoel solutions were determined by flame emission spectrophotometry using a 'Techtron A A 120' flame spectrophotometer. Samples (1 ul) were collected using disposable 'Drumrnond' micropipettes and diluted in either 6 ml of distilled water or 3 ml of a 500 ppm of Na solution (swamp) for Na or K determinations respectively. The solutions (contained in polyethylene vials) were then frozen and stored for determinations as convenient. Chloride concentrations were measured by electrometric titration of 1 ul samples with 0,2$ AgNO^  (Ramsay, Brown and Croghan, 1955)* Samples for Cl determinations (0,5 to 10 ul) were stored frozen under liquid paraffin (Phillips, 1964b). Osmotic pressure determinations were made by the cryoscopic method of Ramsay and Brown (1955) with the following modification. Disposable 'Drummond' micropipettes (1 ul) were used to contain the aqueous samples between liquid paraffin bubbles. If determinations could not be carried out immediately, fluid samples were kept frozen in the micropipettes until use. Osmotic pressures are expressed as freezing-point depressions (A C) or in osmoles/l (AC/l.86). (g) Determinations of Ion Concentration in Rectal Tissue, Concentrations of Na, K and Cl in rectal tissue were measured using the methods mentioned above. To prepare the tissues 13 for these determinations, recta obtained after various incubation conditions were rinsed b r i e f l y (5 seconds) i n 0,335 M/l sucrose solution to remove most of the ions i n the adhering superficial layer of external solution. Recta were then blotted l i g h t l y with clean absorbent tissue and weighed. Some of the recta were analysed immediately and the rest were stored frozen i n clean polyethylene v i a l s . For Na and K determinations, individual recta were placed i n weighed platinum boats and dried i n an oven at 90°C for 24 hours. The boats with the dried tissues were re-weighed to obtain the tissue dry weight and then placed i n the oven at 460°C for 12 hours (Phillips, 1964b), The ashed tissues were then dissolved i n 6 or 8 ml of d i s t i l l e d water, Aliquots (3 ml) of the l a t t e r solution were pipetted into 3 ml of a 1,000 ppm Na solution (swamp) for K measurement. The remainder of the solution of ashed tissue i n d i s t i l l e d water was analysed directly for Na, In some of the l a t t e r experiments, 5 u l aliquots of 20$ HC1 solution were added to the dried recta tissue before ashing. Control experiments for possible contamination were performed by treating platinum boats without recta i n similar manner. Ion losses during drying and ashing procedures were tested by pipetting 4 u l samples of Ringer to platinum boats and then treating them i n the same way as rectal tissues. For determinations of tissue CI, the recta were homogenized i n a test tube (with a teflon pestle) and then 0,5 ml of 50$ glac i a l acid was added to precipitate the protein (Phillips, 1964b). Test tube and contents were centrifuged and 0.05 or 0,1 ml of supernatant was titra t e d for CI as previously described. Controls for contamination were obtained by repeating above procedure without recta or with Fig. 2. Arrangement of apparatus used for measuring i n vitro trans-rectal potentials. Asymmetry p.d. i s measured by removing the hemocoel end of 1.5 M KC1 agar bridge and immersing i t i n the external incubating solution. 14 known aliquots of Ringer. (h) pH of Solutions. The pH of a l l the media (except complex media and unadjusted simple Ringer) was adjusted to 7 .0 with various acids or bases as shown i n Table 2 , I n i t i a l experiments were carried out using the complex media (pH not measured) and i n some cases unadjusted simple Ringer (pH 5 . 9 1 ) . The transport of water by r e c t a l sacs incubated i n Ringer at either pH 7.0 or pH 5 .91 was not s i g n i f i c a n t l y d i f f e r e n t . However, pH of the experimental solutions were adjusted to 7 .0 (except sucrose Ringer) since t h i s value i s closer to physiological pH of locust hemo-lymph (7 .1) as reported by P h i l l i p s ( 1 9 6 l ) . ( i ) Measurement of ELectro-potential Differences. Preliminary determinations of the potential difference (p.d.) across the r e c t a l w a l l of preparations bathed i n complex medium were performed using Ag-AgCl electrodes embedded i n 3$ agar bridges made up with complex medium. In l a t e r studies which involved incubation of r e c t a l sacs i n d i f f e r e n t media, calomel electrodes (Radiometer) i n series with 1 .5 M/l KCI-agar bridges made up i n P.E. 10 tubing were used. The apparatus arrangement i s shown i n F i g . 2 , P.d.'s were measured using a 'Keithley model 6 0 2 ' electrometer. The asymmetry p.d. of the c i r c u i t recording system was measured with the t i p of the agar bridge (normally inserted into hemocoel compartment for p.d. measure-ment) immersed i n the external incubation medium containing the calomel reference electrode. The asymmetry p.d. was generally 0 to 3 mV, except when pure sucrose solution was used as external incubation 15 media (10 to 20 mV)„ This asymmetry p.d. was either backed off or subtracted from trans-epithelial p.d. determinations. (j) Glucose Concentration Determination. In 2 experiments glucose movement into the hemocoel compartment during f l u i d absorption was studied. Aliquots (5 ul) of absorbate (i.e. f l u i d entering hemocoel) were tested for glucose concentration using glucose-sensitive strips ('Tes-tape'). Glucose concentrations were measured to the nearest 0.25 g/l by observing colour changes i n the strips. (k) Treatment of Results. The absorption rate (X) i n uEq/hr/rectum (R) for any given ion i s given by the following equation: (10+C)B - 10.A X = 1000 where C i s the absorption rate of water (in ul/hr/R), and A and B are the i n i t i a l and f i n a l concentrations (in mEq/l) of the ion i n the hemocoel compartment respectively over the same 1 hour interval. A positive value indicates a net movement of ions into the hemocoel compartment. Negative values would indicate a net loss of ions from the hemocoel. Absorbate concentration (Y) i n mEq/l i s derived from the rate of absorption. The equation used i s given below: Y = 1000.X/C where X i s the rate of ion absorption i n uEq/hr/R and C i s the rate of water absorption i n ul/hr/R, 16 In certain instances, net loss of ions from the hemocoel solution occurred even after incubation. Using the above equation, net loss leads to calculation of a negative absorbate concentration. While the la t t e r terminology would seem contradictory, these negative values are l i s t e d to indicate that net loss of ions had occurred. Freezing-point depressions of absorbate were calculated using the same equation used i n determining absorbate concentration of ions. In some experiments, when no f l u i d was i n i t i a l l y placed i n the hemocoel compartment, absorbate which accumulated i n the l a t t e r space was collected at hourly intervals and the absorbate composition directly determined. Differences i n results were analysed s t a t i s t i c a l l y for significance by using the Student's t-tests. Unless stated otherwise, the term 'significantly different' has a probability level less than or equal to 0.05. 17 RESULTS CHAPTER I CHARACTERISTICS OF RECTAL ACTIVITY (a) Introduction. Preliminary experiments were designed to find a medium and establish conditions (e.g. oxygenation and volume of lumen solution) required to support active transport of water across the rectal wall. Criteria of success were the i n i t i a l rate of fluid absorption and the rate of decline of activity with time. The ultimate objective was to achieve, in vitro, transport rates which were relatively constant for several hours and which were comparable to those measured in the intact animal, (b) Ability of Media to Support Initial Water Uptake, In some preliminary experiments, hemolymph (10 ul) obtained from the neck of locusts was placed on the hemocoel side (on the assumption that normal blood should give the best results). Either Ringer with 150 mM/l of sucrose added or Ringer with 350 mM/l of sucrose added was placed on the lumen side. The net absorption of water with time is shown in Fig, 3, Net absorption rates in both experi-ments declined rapidly with time. Rectal activity during the second half hour declined to about 15$ of activity during the f i r s t half hour. Mean absorption rates over the f i r s t hour for Ringer with 150 raM/l sucrose and for Ringer with 350 mM/l sucrose on the lumen side w e r e 9.2 ul/hr/R and 7.1 ul/hr/R respectively. Fig. 3. Net absorption of water from lumen to hemocoel side of rectal sacs with time. Hemolymph was placed on hemocoel side and external solution consisted of Ringer + 150 mM/l sucrose (triangles) or Ringer + 350 mM/l sucrose ( c i r c l e s ) . Bars indicate ± S.D. and subscript figures show number of preparations. 12 T i m e ( m i n ) Fig. 4. Net absorption of water with Ringer + 150 mM/l sucrose on both sides. Bars indicate ± S.D. (4 preparations). 1 2 r T i m e ( m i n ) Fig. 5» Net absorption of water with complex medium on both sides of rectum. Bars indicate ± S.D. (12 preparations). T i m e ( m i n ) 18 The calculated freezing-point depression of Ringer with 150 mM/l sucrose i s -0.68°C and Ringer with 350 mM/l sucrose i s -0.97°C. Since hemolymph from saline-fed locust has an average freezing-point depression of -0.92°C ( P h i l l i p s , 1964a) , the hemocoel side of the r e c t a l sac was hyperosmotic to the lumen side fo r experiments using Ringer with 150 mM/l sucrose and hyposmotic for Ringer with 350 mM/l sucrose. The net absorption of water observed under the l a t t e r condition could not be due to simple d i f f u s i o n but must involve an active mechanism which can transport water against an osmotic gradient. Since i t i s d i f f i c u l t to obtain locust blood and manipulate i t s concentration, a r t i f i c i a l media were tested f o r t h e i r a b i l i t y to maintain transport. Ringer (with 150 mM/l sucrose added) was placed on both sides of the r e c t a l sac and the absorption rate of water i s shown i n F i g . 4 . In t h i s isosmotic condition, the absorption rate during the second h a l f hour declined to 30% of that f o r the f i r s t h a l f hour (rate of 8 . 9 ul/hr/R). In v i t r o performance was apparently lower than f o r the i n vivo rectum ( P h i l l i p s , 1 9 6 4 a ) . However, t h i s experiment suggested that a r t i f i c i a l media could be substituted f o r hemolymph although r e c t a l a c t i v i t y was not adequately maintained. Berridge (1966) reported excellent maintenance of secretion by blowfly malpighian tubules i n v i t r o using a complex tissue culture medium. This medium was placed on both sides of r e c t a l preparations. Net absorption rates under isosmotic conditions are shown i n Fig. 5« Rectal a c t i v i t y declined much less (40$) during the second h a l f hour. The mean absorption rate with complex medium was s i g n i f i c a n t l y higher (P<0.01) at the end of the hour than the absorption rates i n a l l Fig. 6. Initial absorption rates of water by rectal sacs incubated in simple Ringer (solid circles) as compared to rates in complex medium (open circles). Bars indicate + S.D. and subscript figures show number of preparations. T i m e ( m i n ) F i g . 7. Comparison of mean absorption rates i n simple Ringer with recta from insects of d i f f e r e n t ages a f t e r f i n a l moult ( s o l i d c i r c l e s - 5 days; triangles - 14 to 21 days; open c i r c l e s - 41 to 49 days). Subscript figures indicate number of preparations. 1 6 T i m e ( m i n ) 19 the previous experiments ( F i g , 3 and 4) and was equal to the absorption rate (17,ul/hr/R) i n vivo ( P h i l l i p s , 1964a) following i n j e c t i o n of a pure isosmotic sucrose s o l u t i o n i n t o the l o c u s t rectum, A simple Ringer was used by Mordue (1969) i n studies of the i n v i t r o rectum and the rates of water absorption f o r the i n i t i a l hour (the t o t a l p e r i o d studied) were comparable to rates i n vivo or using complex medium. I t would be preferable to study i o n i c e f f e c t s on water absorption using the simple Ringer as i o n i c concentrations are e a s i l y a l t e r e d compared to complex medium which contains various organic compounds. Preliminary experiments were performed p l a c i n g the simple Ringer of Mordue on both sides of r e c t a l sacs. The uptake rates are. shown i n F i g . 6 i n comparison with uptake i n complex medium. The r e s u l t s suggested only a 20$ d i f f e r e n c e between the a c t i v i t y of r e c t a i n the two media. Moreover, maintenance of a c t i v i t y , as i n d i c a t e d by rate of de c l i n e , was adequate and comparable i n both media, (c) Conditions f o r Optimum A c t i v i t y , The r e l a t i o n s h i p between r e c t a l a c t i v i t y and age of locusts has seldom been considered i n the study of the i n s e c t rectum. I t would be reassuring i n i n t e r p r e t a t i o n of r e s u l t s to determine i f any d i f f e r e n c e s i n a c t i v i t y occur using r e c t a from adult l o c u s t s of d i f f e r e n t age. F i g , 7 shows the water absorption with time f o r 3 groups of re c t a (incubated i n simple Ringer) from adult l o c u s t s which were 5 to 49 days past the f i n a l moult. There was no apparent d i f f e r e n c e i n uptake with age. To reduce p o s s i b l e v a r i a t i o n , a l l l a t e r experiments were performed with recta from l o c u s t s 5 to 21 days past the f i n a l moult. F i g . 8. E f f e c t of external (lumen) volume and oxygenation on rate of water absorption. Large external volume (25 ml) i s i n d i c a t e d by open c i r c l e s (with oxygenation) and s o l i d c i r c l e s (without oxygenation). Triangles i n d i c a t e small external volume (50 u l ) with or without oxygenation (pooled). Arrow i n d i c a t e s time of t r a n s f e r of r e c t a l sac to a l a r g e oxygenated external volume (25 ml). Bars i n d i c a t e ± S.D. and subscript f i g u r e s show number o f preparations. T i m e ( m i n ) 20 In insects, the tissues are supplied with oxygen by the tracheal system. Tracheation of the rectum, especially along the region of the rectal pads, i s probably the heaviest of any tissue i n the locust. In a l l the previous experiments, only the solution bathing the lumen was oxygenated. Experiments were undertaken to establish whether oxygenation enhanced or inhibited uptake, since poisoning due to oxygen has been reported for many tissues. Fig. 8 shows effect of oxygenation on the absorption with normal complex medium placed on both sides of the rectal sac. Uptake i n unoxygenated solution over the f i r s t hour was 11.2 ul/hr/R. This value i s significantly lower (P<0.01) than uptake i n oxygenated medium (17 ul/hr/R). Essential components (e.g. ions or glucose) of the media may be consumed during transport or inhibitory end-products might possibly accumulate. The effect of external volume on rectal a c t i v i t y was therefore studied (Fig. 8). With a small external volume (0.05 ml) of complex medium, uptake rate decreased to zero after the f i r s t half hour. Observations indicated that the low uptake was due to the small external volume rather than deterioration of tissue since transfer to a larger volume (25 ml) of complex medium after 40 minutes led to resumption of uptake. Presence or lack of oxygenation has no apparent effect on uptake from small volumes. (d) Importance of Hydrostatic Pressure. For many natural epithelia and a r t i f i c i a l membranes, (with reflection coefficient close to unity) equal rates of water movement are produced by osmotic and hydrostatic pressure gradients of equal magnitude (Pappenheimer, 1953). This can also be reasonably applied 21 to the rectal wall (Phillips, 1964a). In the intact animal, the po s s i b i l i t y of net absorption from the rectum by u l t r a - f i l t r a t i o n under a hydrostatic pressure gradient (produced i n vivo by muscular contractions of the rectal wall) must be considered. The advantage of an everted rectal sac preparation i s that any absorption which occurs from the lumen to the hemocoel side could not be accounted for by hydrostatic pressure produced by contractions. In a l l the experiments performed so far (except with small external volumes), there was no i n i t i a l hydrostatic pressure since the level of the external solution was adjusted to the same lev e l as the hemocoel f l u i d i n the cannula of the preparations. Subsequently, absorption that occurred was against an increasing hydrostatic pressure gradient as the absorbed f l u i d extended up the cannula. The maximum hydrostatic pressure gradient was approximately 2 cm IL^ O or 0,003 atmospheres after absorption for an hour when complex medium was on both sides of rectal wall. The maximum hydrostatic pressure gradient measured i n the intact rectum of the desert locust was less than 12 cm 1^0 or 0,02 atmospheres (Phillips, 1964a), This pressure was not considered important since rectal absorption can s t i l l occur against an osmotic pressure gradient of approximately 11.2 atmospheres. In vitro studies with everted recta demonstrated conclusively that absorption (in the absence of an osmotic gradient) can occur against a slight hydrostatic pressure gradient. This factor i s therefore not responsible for producing the net water movement observed across the rectal wall. F i g . 9. Net absorption of water f o r more than 1 hour during incubation i n complex medium (open c i r c l e s ) and simple Ringer ( s o l i d c i r c l e s ) . Bars indicate + S.D. and subscript figures show number of preparations. N e t A b s o r p t i o n o f H 2 0 ( u l / R ) 22 (e) Steady State Condition. In a l l previous experiments, rectal absorption was studied during the f i r s t hour. The results indicated that the rectal a c t i v i t y declined s l i g h t l y with time over this period. Some of the earlie r experiments were repeated to obtain measurements beyond the f i r s t hour period using both complex and simple Ringer as incubation media (Fig, 9)• At the end of every hour, the hemocoel f l u i d was replaced with fresh solution. With both types of media, rectal absorption remained relatively constant (at 50$ of the rate for the f i r s t hour) for at least 4 hours after the i n i t i a l high transient rate. Steady state a c t i v i t y of rectal sacs remained s l i g h t l y less i n simple Ringer than i n complex medium. Rectal sacs incubated Overnight (over 20 hours) i n oxygenated simple Ringer showed only a slight decline (50$) i n a c t i v i t y . However, a l l l a t e r experiments involving rates of ions and water movement at the steady state were normally completed within 4 hours. In vivo trans-rectal p.d.'s i n the locust have been reported by Phi l l i p s (1964c) to range from 15 to 32 mV (lumen positive with respect to the hemocoel side). These observations have been confirmed by Vietinghoff et a l (1969) on the i n vitro rectum of the locust. Trans-epithelial p.d.'s were observed to f a l l with time and approached zero after 2 hours with the preparation used by the l a t t e r author. Since maintenance of a p.d. i s one indication of the steady state a c t i v i t y for any solute transporting epithelia, the above situation was re-examined with the present i n vitro preparation. Trans-rectal p.d.'s were measured during incubation i n complex Fig. 10. P.d. across rectal wall with time when complex medium was placed on both sides of rectal sac. (Open squares - with oxygenation; solid squares - no oxygenation.) Arrow indicates time of transfer to a non-aerated lumen solution. • • • D -m o H 43 • m • • • • • • • o oo • D D • • • • • D D • • • a • - \ CM 0 a • m i-n-a. o C O o " 3 -o CM (A « i ) - Q ' d Fig. 11. Trans-rectal p.d. with time during incubation i n simple Ringer. (Solid circles - oxygenated; open triangles - aerated; so l i d triangles - no aeration.) © CM © © 0 © © © <30 • @ ® <3jg-J M 9 9 4* O o 00 -e-© © i — o co © 9 o © © ® © <30 -«*d 0 G© <J » « o J3 CU CM CM 23 medium or simple Ringer (isosmotic conditions) for up to 6 hours. Fig, 10 shows the p.d. with time during incubation i n complex medium with or without oxygenation. In the oxygenated medium, the i n i t i a l trans-rectal p.d.'s ranged from 55 to 75 fflV (with lumen side positive). P.d.'s remained within this range for up to 6 hours except for a few readings of up to 86 mV. In one such experiment, a rectal sac was transferred after 4 hours to a fresh incubating medium which had not been oxygenated or aerated. In this case, p.d, f e l l abruptly from 70 mV down to 23 mV at the rate of approximately 2.3 mV/min, Rectal sacs that were not oxygenated i n i t i a l l y had high i n i t i a l p.d.'s which decreased rapidly with time and approached zero after 3 .5 hours. Apparently, oxygenation i s required for maintenance of trans-rectal p.d.'s. These experiments show that the i n v i t r o sacs i n oxygenated com-plex medium can maintain trans-epithelial p.d.'s at a relatively steady l e v e l . The p.d.'s were much higher than i n the intact animal (Phillips, 1964c) but the differences are possible due to the different experimental conditions, In the simple Ringer with oxygenation (Fig, l l ) , the trans-rectal p.d.'s were also high i n i t i a l l y (60 to 90 mV, lumen positive to hemocoel). The p.d.'s f e l l rapidly for 1 ,5 hours and then remained relatively constant at 14 to 28 mV for 5.5 hours with a few values as low as 8 mV. Four rectal sacs were l e f t overnight incubated i n simple Ringer with oxygenation and the p.d.'s measured (after 20 hours from start of experiment) ranged between 7 and 20 mV (lumen positive). This confirms earlier observations on water absorption rates that the i n i t i a l high rates are transient and that a c t i v i t y remains reasonably steady Figo 12. Effect of hemolymph, complex medium, simple Ringer and d i s t i l l e d water on the trans-rectal p.d. measured i n vivo. Prior to injection of above solutions, the normal i n vivo rectal p.d.'s are shown (solid c i r c l e s ) . P.d. with time was measured after injection (10 ul aliquot) of hemolymph (open c i r c l e s ) , complex medium (open squares), simple Ringer (solid squares) or d i s t i l l e d water (triangles) into the ligated rectal lumen. Arrows indicate time of injection. 120 T i m e ( m i n ) 24 a f t e r the t r a n s i e n t stage. The steady p.d.'s measured i n v i t r o are comparable to p.d.'s i n the i n t a c t animal. Under d i f f e r e n t conditions (aeration or no a e r a t i o n ) , p.d.'s dropped r a p i d l y to l e s s than 10 mV i n 1 .5 hours and were near zero a f t e r 2 . 5 hours. Obviously, t i s s u e requires oxygenation to maintain i t s a c t i v i t y . Results with aerated medium suggest tha t the low p 0d.'s reported f o r l o c u s t recta by V i e t i n g h o f f et a l (1969) were p o s s i b l y due to i n s u f f i c i e n t oxygenation. The high oxygen requirement of r e c t a l t i s s u e was suggested by the heavy trac h e a t i o n of r e c t a l pads observed during d i s s e c t i o n . The t r a n s - r e c t a l p.d. observed with incubation i n complex medium i s almost 3 - f o l d higher than that observed i n the i n t a c t animal. Since i t i s p o s s i b l e that the high p.d. i s a consequence of d e t e r i o r a t i o n of t i s s u e or i n h i b i t i o n of some a c t i v e process, some preliminary experiments were performed i n vivo to observe the e f f e c t s of d i f f e r e n t experimental solutions ( i n j e c t e d i n l i g a t e d rectum) on t r a n s - r e c t a l p.d.'s. The technique used to measure i n vivo p.d. was s i m i l a r to that described by P h i l l i p s ( 1964c) . The solutions i n j e c t e d (via anus) were d i s t i l l e d water, simple Ringer, complex medium, and hemolymph. The r e s u l t s are shown i n F i g . 12 . The normal i n v i v o p.d.'s determined p r i o r to i n j e c t i o n s of experimental solutions (10 u l a l i q u o t ) were 21 , 25, and 2 7 . 5 mV (lumen p o s i t i v e with respect to hemocoel). These p.d.'s are comparable to i n  vivo p.d.'s reported by P h i l l i p s (1964c) and represent the s i t u a t i o n when a l a r g e osmotic gradient has developed across the r e c t a l w a l l . With i n j e c t i o n of 10 u l of hemolymph in t o the lumen, t r a n s - r e c t a l p.d, increased r a p i d l y to more than 50 mV a f t e r 10 min. Using complex Fig. 13. Net transfer of water with time (during the f i r s t hour) under various osmotic gradients; 0o226 (solid c i r c l e s ) , 0,457 (open triangles), 0.683 (solid triangles), 1,145 (open squares), and 1,757 (solid squares) osmolar gradients. Zero gradient (open circles) were taken from Fig. 5. I n i t i a l net water movements were measured for the f i r s t 10 minutes (dotted l i n e s ) . Bars indicate ± S.D. and subscript figures indicate number of preparations. 25 medium, the p.d, reached 63 mV at 6 min and then decreased sl i g h t l y to 55 mV. When the rectal f l u i d was removed and re-injected with simple Ringer, the p.d, increased to 88 mV within 4 min. Using another animal, the rectum was injected with simple Ringer and then d i s t i l l e d water. With simple Ringer, the p.d. rose sharply and when replaced with d i s t i l l e d water, p.d. increased to 115 mV. Obviously, i n vivo trans-rectal p.d.'s can be greatly increased by placing iosomotic or hyposmotic solutions i n the lumen. This suggests high p.d.'s are due to rapid f l u i d and ions moving into the rectum from dilute media during the build up of the steady state osmotic gradient. These observations also suggest that the high p.d.'s observed in vitro with complex medium are not unnatural to the rectum i n vivo. (f) Effect of Osmotic Gradient on Net Water Movement. Net absorption (under isosmotic conditions) indicated that an active process was involved i n transport of water since net movement by simple osmosis was eliminated. In vivo studies (Phillips, 1964a) indicated that the rate of net water movement across the recta i s dependent on the osmotic gradient and that this epithelium might exhibit r e c t i f i c a t i o n of osmotic flow. Exact i n i t i a l rates of water movement were d i f f i c u l t to measure accurately i n the intact animal. In vitro experiments were therefore undertaken to re-examine this relationship and to test the r e a l i t y of the reported re c t i f i c a t i o n . Preliminary studies were performed i n which the time course of net absorption was followed over the f i r s t hour for various trans-rectal osmotic pressure gradients (Fig, 13), In these experiments ionic concentration of the complex medium (on both sides) Fig. 14. The relationship between rate of water movement over the f i r s t hour (solid circles) and the i n i t i a l osmotic pressure gradient (osmolar) across the rectal wall. Positive values indicate net absorption and negative values indicate net loss from the hemocoel side. Open circles show the relationship between i n i t i a l rate (taken for first 10 minutes) and the osmotic pressure gradient. Broken line shows the relationship in vivo (taken from Phillips, 1964a) where xylose solutions of different molality were injected into the ligated rectum. Bars indicate ± S.D. and subscript figures show number of preparations. C o n c n G r a d i e n t ( o s m o l a r ) 26 was constant and only sucrose was added to increase the osmolarity of the external s o l u t i o n . The relationship between mean absorption rates (over f i r s t hour and also i n i t i a l l i n e a r rates) and the i n i t i a l trans-r e c t a l osmotic pressure gradients i s shown i n F i g . 14, "When there was no osmotic gradient, the absorption rate was 16.7 ul/hr/R and the rate declined s t e a d i l y as the osmotic pressure gradient was increased. When the osmotic pressure of the external solution exceeded that of the hemocoel solution by 0.76 osmolar, there was no net movement of water, and beyond t h i s value, the movement of water was reversed leading to a net water loss from the hemocoel s i d e . The relationship between rate of net water movement and osmotic gradient i s approximately l i n e a r from zero to 0.683 osmolar gradients. At higher osmotic gradients, there i s a change i n relationship such that the slope decreases. This suggests that at high osmotic gradients (0.76 osmolar) the minimum osmotic permeability of the r e c t a l wall to water (or the passive net f l u x of water) i s 5.55 ul/hr/A osmolar and increases to a maximum of 23.7 ul/hr/A osmolar f o r smaller osmotic gradients. The above results are very s i m i l a r to those observed f o r the absorption within the f i r s t hour a f t e r i n j e c t i o n i n t o the rectum i n vivo ( P h i l l i p s , 1964a). In the absence of an osmotic gradient ( F i g . 14), there i s an active absorption i n vivo of 17 ul/hr/R. In the presence of an osmotic gradient i n v i v o , the active uptake component i s superimposed on a passive water movement which depends on the osmotic gradient. There i s an apparent difference i n permeability of the r e c t a l wall depending on the d i r e c t i o n of net water movement. This comparison indicates that over comparable time periods water absorption F i g . 1 5 . Net absorption of water with time during incubation i n simple Ringer under various osmotic pressure gradients. Osmotic pressure gradients ( i n osmolar) are shown by s u b s c r i p t f i g u r e s (lumen with respect to hemocoel). Bars i n d i c a t e ± S.D. f o r 5 preparations, 60 J 1 ; I L 1 2 3 4 T i m e (hr) F i g . 16. Relationship between rate of water movement and osmotic pressure gradient (osmolar) i n the steady state during incubation i n simple Ringer. Bars indicate ± S.D. f o r 5 preparations. C o n c n G r a d i e n t ( o s m o l a r ) 27 by the i n vitro preparation i s similar to that i n vivo. It should be stressed that the absorption rates measured previously i n vivo (Phillips, 1964a) were i n i t i a l rates immediately following injections of solutions of different osmolarities into the lumen and, possibly, did not represent the steady state situation. Steady state a c t i v i t y i s a prime requisite for the application of irreversible thermodynamics to the study of water and ion transport (e.g. Diamond, 1962a,b,c). One of the required parameters for application of irreversible thermodynamic to a study of water and solute coupling i s the osmotic permeability of the rectal wall to water i n the steady state. Therefore, the preliminary study mentioned above was repeated using simple Ringer (as used i n a l l l a t e r studies) with observation extended to 4 hours. After the f i r s t hour, constant rates of water transport were observed for different osmotic gradients across the rectal wall (Fig. 15). Additional experiments using different gradients were also conducted to establish more clearly the situation when net water movement was from hemocoel to lumen side. In one such experiment, the hemocoel compartment was made hyperosmotic to lumen by addition of sucrose to simple Ringer, In another, the external Ringer was diluted 50$ with d i s t i l l e d water. The stea.dy state rate of water absorption (between 1 and 4 hours) for each osmotic gradient i s shown i n F i g 0 16. Basically, the relationship between steady state rate of water movement and osmotic gradient i s similar to that observed i n earlier studies (of rates for the f i r s t hour with complex medium and the i n vivo studies). Fig. 16 shows that the rate of net water absorption 28 was 6 ul/hr/R i n the absence of an osmotic gradient and the rate declined-linearly as the osmotic gradient was increased to 0,33 osmolar. No net absorption of water occurred when the lumen solutions was hyperosmotic to the hemocoel solution by more than 0.33 osmolar. Beyond this value, the net movement of water was reversed ( i . e . net loss of water from the hemocoel side). At higher gradients when the water movement was reversed, the slope of the relationship between osmotic gradient and rate decreased. The calculated osmotic permeability of rectal wall (from the slope of the graph i n Fig. 16) i s 21,3 ul/hr/R/A osmolar for absorption and 7.5^ ul/hr/R/A osmolar for flow i n the opposite direction (hemocoel to lumen). These values are very close to values obtained from earlier studies using f i r s t hour rates with rectal sacs incubated i n complex medium (Fig, 14). The maximum and minimum osmotic permeability i n the previous study were 23.7 and 5.55 ul/hr/R/A osmolar. This suggests that the osmotic permeability of the rectal wall remains constant with time and i s independent of the type of media used. The only difference seems to be a f a l l i n the active component (from 16,7 to 6 ul/hr/R) of water movement during the f i r s t hour. (g) Absorption from I n i t i a l l y Pure Sucrose Solution. Net water movement under isosmotic conditions across a l l vertebrate epithelia studied to date i s dependent on simultaneous net active transport of solutes (e.g. intestine, reviewed by Curran, 19655 Fordtran and Dietschy, 1966) which i s considered the primary transport. This i s not the case for the locust and cockroach i n vivo (Phillips, 1964a,b; Wall, 1967: Stobbard, 1968) i n which absorption F i g . 1 7 . Net absorption of water during incubation with i n i t i a l l y pure sucrose s o l u t i o n ( c a l c u l a t e d f.p. - 0 , 76 ° C ) placed on the external side (with complex medium on hemocoel side) of the r e c t a l sac ( t r i a n g l e s ) compared with the absorption rate with complex medium on both sides ( c i r c l e s ) . Bars i n d i c a t e dt S . D . and subsc r i p t f i g u r e s show number of preparations. 18 16 14 v^ i—i A '—' o 12 CM a O on 10 •r-l +-> CU u o CC 8 <J -4-> o> 55 .612 4 6 10 2 0 30 40 T i m e ( m i n ) 50 60 29 can occur from an i n i t i a l l y pure sucrose solution. This water absorption from an i n i t a l l y pure sucrose solution was not accompanied by net solute absorption since the rectal cuticle i s impermeable to sucrose (Phil l i p s , 1964a), A c r i t i c a l test of the v i a b i l i t y of any i n vitro preparation of the locust rectum i s to determine whether the capacity to absorb from isosmotic sucrose i s preserved. To test for this capacity, a sli g h t l y hyperosmotic sucrose solution (calculated freezing-point depression of -0.76°C) was placed on the external side and complex medium on the lumen side. Any absorp-tion of water would then be against a small osmotic gradient (0.069 osmolar) across the rectal wall. Fig. 17 shows that absorption of 7.9 u l of water occurred from the sucrose solution during f i r s t hour. This uptake was one half of the uptake rate with Ringer on both sides. The mean uptake from sucrose solution was 13.9 ul/hr/R during the f i r s t half hour but the rate declined rapidly to 1,9 ul/hr/R i n the second half hour. This uptake from sucrose was of the magnitude observed i n vivo (P h i l l i p s , 1964a). Obviously neither simultaneous net solute absorption nor the presence of ions on the lumen side are absolutely required for water transport against an osmotic gradient although ion absorption enhances water transfer. The results with an in vitro preparation confirmed the i n vivo observations but are more convincing since the external volume (25 ml) i s very large, excluding slight accumulations of ions on the lumen side as a result of back-diffusion across the rectal wall. The previous experiment was repeated using simple Ringer solution on the hemocoel side and pure sucrose solution (measured F i g . 18. Net absorption of water during incubation with isosmotic sucrose solution on lumen and simple Ringer on hemocoel side ( s o l i d c i r c l e s ) . Replacement of external sucrose with simple Ringer a f t e r 1, 2 and 4 hours i s indicated by arrows. Open c i r c l e s show rate of water absorption with simple Ringer oh both sides. Dotted l i n e shows absorption rate i n simple Ringer without pre-treatment i n sucrose. Bars indicate + S.D. and subscript figures show number of preparations, N e t A b s o r p t i o n o f H 2 O ( u l / R ) 30 freezing-point depression -0.723 C) on the lumen. A slight osmotic gradient (0.004 osmolar) was thereby set up-with the lumen hyperosmotic to the hemocoel. The long-term effect of incubation i n pure sucrose solution on the a c t i v i t y of rectal sacs was tested by replacing the external sucrose solution with simple Ringer after 1, 2 or 4 hours. The results are shown i n Fig, 18, Water absorption with sucrose on the lumen side and simple Ringer on the hemocoel side was 6,38 ul/hr/R during the f i r s t hour with the rate declining from 9,26 ul/hr/R i n the f i r s t half hour to 3.5 ul/hr/R i n the second half hour. When incubation i n sucrose was continued for the second hour, the uptake rate decreased to almost zero after 1.5 hours. However, replacement of the sucrose solution with Ringer immediately restored absorption. When replacement of sucrose solution with Ringer was carried out after 1 hour incubation i n sucrose, the uptake rate showed a high transient rate which declined to a steady rate similar to that recorded (Fig, 9) when preparations were not pre-incubated i n sucrose. But replacement with Ringer after 2 hours i n sucrose resulted i n a less dramatic rise, with the rate remaining steady at 4,4 ul/hr/R, The uptake by rectal sacs that were incubated i n sucrose continuously was almost abolished after 1,5 hours. After 4 hours i n sucrose solution, uptake rate was also p a r t i a l l y restored when the sucrose was replaced with Ringer, These results suggest that decline of rectal a c t i v i t y i n sucrose i s probably due to absence of ions on the lumen side. Since uptake rates could be restored to normal by replacing external sucrose solution with simple Ringer, the rectal tissue was not permanently impaired by absence of ions. However, after prolonged absence of ions (2 hours F i g . 19. Net absorption of water during incubation i n simple Ringer with no hemocoel f l u i d i n i t i a l l y -present (open c i r c l e s ) . S o l i d c i r c l e s show e f f e c t of simple Ringer at pH 5.9 placed on both sides of r e c t a l sac. Absorption rate i n normal simple Ringer (pH 7.0) on both sides of r e c t a l sac i s i n d i c a t e d by dotted l i n e . Bars i n d i c a t e ± S.D. and subscript f i g u r e s show-number of preparations. 31 sucrose), the rectal absorption could only be restored to 70$ of the normal rate. .These experiments indicated that while rectal sacs can absorb from pure sucrose solutions, the presence of ions on the lumen side i s required for prolonged maintenance of rectal absorption of water. (h) Absorption with no Hemocoel Fluid I n i t i a l l y , In a l l previous experiments, 10 u l of experimental medium was always placed on the hemocoel side of the rectal sac before incubation. Using this method to study the mechanism of water transport, the absor-bate concentration of ions must be calculated indirectly from the i n i t i a l and f i n a l hemocoel f l u i d concentrations and the rate of water absorption. Experimental errors are thereby magnified. Collection of absorbate, direc t l y by placing no f l u i d on the hemocoel side would provide a useful check of the former method. An experiment was run to test the effect of an i n i t i a l absence of hemocoel f l u i d on water absorption rates. Direct absorbate concentrations could then be compared with calculated values. Fig. 19 shows absorption with time using simple Ringer on the lumen side only. The hemocoel side was rinsed with pure sucrose solution (0.335 M/l) and remaining f l u i d withdrawn before incubation. This procedure ensured that ions and f l u i d were not present on the hemocoel surface of the rectal sac i n significant quantities i n i t i a l l y . Uptake rates were not significantly different from rates with simple Ringer on the hemocoel side. This suggests that the i n i t i a l presence of ions and f l u i d on the hemocoel side i s not required for the absorp-tion of water. Fig. 20. The absorbate concentration of glucose with time when incubated i n simple Ringer with no i n i t i a l hemocoel f l u i d , (2 preparations.) Arrow indicates media concentration of glucose. A b s o r b a t e G l u c o s e C o n c n ( g / l ) 8- O 9 32 ( i ) Glucose Concentration i n Absorbateo Absorbate obtained i n previous experiment w i t h no i n i t i a l hemocoel f l u i d was t e s t e d f o r glucose c o n c e n t r a t i o n . The r e s u l t ( F i g . 20) shows t h a t glucose c o n c e n t r a t i o n i n absorbate was present but i n much lower c o n c e n t r a t i o n than i n the lumen s o l u t i o n . This suggests t h a t glucose a b s o r p t i o n could be due t o p a s s i v e movement. I t does not r u l e d out the p o s s i b i l i t y t h a t glucose i s consumed by r e c t a l t i s s u e or c h e m i c a l l y converted t o another compound (e.g. t r e h a l o s e - Treherne, 1957)» nor does t h i s o b s e r v a t i o n excluded a c t i v e t r a n s p o r t but a t a slower r a t e than f o r water. ( j ) E f f e c t o f pH. A c t i v e s e c r e t i o n of hydrogen ions i n exchange f o r K a b s o r p t i o n (at the lumen-facing membrane) has been p o s t u l a t e d by P h i l l i p s (1965) s i n c e i t was found t h a t the i n v i v o r e c t a l lumen i s maintained a c i d i c (pH 4,7) t o hemolymph (pH 7,1). I n the i n v i t r o experiments, the pH of simple Ringer and i t s m o d i f i e d forms ( i . e . K-free, Na-free, L i , c h o l i n e , NO-j and S0^ Ringers) was maintained a t 7.0. The p o s s i b i l i t y t h a t h i g h pH i n the lumen (compared t o t h a t i n v i v o ) of experimental s o l u t i o n has an e f f e c t on the i n v i t r o r e c t a l a b s o r p t i o n was t e s t e d u s i n g simple Ringer a t pH 5.9. F i g . 19 shows t h a t net water a b s o r p t i o n w i t h b a t h i n g medium a t pH 5.9 was comparable t o uptake i n medium a t pH 7.0, I t appears t h a t lumen pH (over t h i s range) has no e f f e c t on the r e c t a l a b s o r p t i o n of water, (k) E f f e c t of I n h i b i t o r s , One c r i t e r i o n o f a c t i v e t r a n s p o r t processes i s t h a t they are Fig. 21, Net absorption of water i n complex medium with 10"^ M malonate (added to both sides of rectal sac); with oxygenation (triangles) and without oxy-genation (dotted l i n e ) . Oxygenated controls without malonate are shown (c i r c l e s ) . Bars indicate ± S.D. and subscript figures show number of preparations. 18 10 20 30 40 50 60 T i m e ( m i n ) Fig. 22. Effect of 10~3 M DNP (solid circles) and 10~3 M KCN + IC-"3 M IAA ( open circles) added to simple Ringer on both sides of rectal sac after pre-treatment in external isosmotic sucrose solution for an hour without inhibitor. Arrow shows the time of removal of KCN + IAA, Dotted lines indicate control without inhibitor. Bars indicate ± S.D. and subscript figures show number of preparations. T i m e ( h r ) 33 reduced or abolished by respiratory inhibitors. The effect of presence or absence of oxygen on uptake rates i n complex medium was discussed earl i e r (Fig. 8). Fig. 21 shows the effect of malonate (an inhibitor of the c i t r i c acid cycle), i n the presence cn. absence of oxygen. Isos-motic conditions were maintained by placing complex medium with sodium malonate (2 g/l) on both sides of the rectum. Mean uptake rate i n oxygenated solution was 14.5 ul/hr/R i n the f i r s t half hour dropping to 2.2 ul/hr/R i n the second half hour. With the inhibitor present, mean uptake rate was not significantly different with or without oxygenation. In both cases, absorption rates declined very rapidly toward zero i n the second half hour. In the presence of 10"^ M malonate, the mean uptake rate over the f i r s t hour i s only 50$ of that i n complex medium without malonate. This indicates that absorption i s at least p a r t i a l l y dependent on aerobic respiration. The delay i n inhibition probably represents the time required for diffusion of malonate into the c e l l s . Recently, inhibition of water absorption i n the cockroach rectum by DNP (Wall, 1967) and i n the stick insect by cyanide (Vietinghoff, I965) have been reported. Preliminary studies have indicated that water absorption i n the locust could be p a r t i a l l y inhibited by malonate or lack of oxygen. Other respiratory inhibitors such as KCN with IAA and DNP were tested using simple Ringer as the incubation medium. The inhibitors (at 10"3 M) were added after a 1 hour pre-incubation with sucrose solution on the lumen side. Conditions were kept isosmotic by adding the inhibitors to both sides. Water absorption with time i s shown i n Fig. 22. In the isosmotic condition, a mixture of 10"-^  M KCN F i g . 23. Effect of 10~J> M KCN + 10"^  M IAA added to either hemocoel (open tri a n g l e s ) or lumen ( s o l i d t r i a n g l e s ) side of r e c t a l sac during absorption against a s l i g h t osmotic gradient (0.116 osmolar). -3 10 M ouabain (squares) was added to lumen side only. In a l l cases, i n h i b i t o r s were added from hours 1 to 3. Control without any i n h i b i t o r i s shown by dotted l i n e . Bars indicate ± S.D. and subscript figures show number of preparations. -L 2 3 T i m e ( h r ) 4 34 and 10*"3 M IAA inhibited 40$ of uptake rate as compared to rate im simple Ringer. On removal of inhibitors, uptake of water was restored to normal. Whereas, under similar conditions, 10"3 M DNP decreased the uptake rate to 4.5 ul/hr/R. The calculated inhibition with the. DNP is 24$ of normal. Partial inhibition (40$) with both KCN and IAA added together suggests that water uptake is in part dependent on aerobic respiration and glycolysis. However, i t is more likely that the inhibitors could not diffuse completely into the rectal tissue under isosmotic condition and act on a l l the active transport sites. Effect of inhibitors (KCN and IAA) was further re-examined during absorption against a slight osmotic gradient (0,116 osmolar) with the lumen side made hyperosmotic by addition of sucrose to the simple Ringer. Fig. 23 shows the effect of KCN and IAA (both at 10"3 M) added to either the lumen or hemocoel side after the f i r s t hour incubation without inhibitor. Addition of inhibitors on the hemocoel side has no apparent effect on the uptake rate as compared to the i n i t i a l control rate (without any inhibitor). On the other case, with KCN and IAA added on the lumen side, about 90$ inhibition was achieved after half an hour and this inhibition was apparently irreversible since removal of inhi-bitors after 2 hours (i.e. at the third hour) did not restore the uptake rate. The delay in inhibition (30 minutes) probably represents the time for the inhibitors to reach the active site. When a slight osmotic gradient is applied, the extra load imposed on the uptake mechanism may somehow facilitated the movement of inhibitors into the tissue. The absence of inhibition with the poisons placed on the hemocoel side appeared contradictory, but i t is possible that the continuous secretion 35 of water from the lumen to the hemocoel side could hinder the diffusion of the inhibitors into the rectal tissue. This suggestion is substan-tiated by the observation (Balshin, personal communication) that the concentration of KCN and IAA required to inhibit amino acid transport by the same rectal preparation decreases as the osmotic gradient increa-ses, thus reducing water movement. The effect of ouabain (10~-^  M) added to the lumen side only was tested in a similar fashion. Ouabain is a specific inhibitor acting on linked Na and K transport (Glynn, 1964). Inhibition of water uptake rate by the latter would give some indication of the dependence of the water movement on Na and K transport. Fig. 23 shows that addition of ouabain decreased uptake rate of water by 50$ a"d removal of poison restored the rate to almost 91$ of normal rate. This suggests that the water, uptake is partially dependent on a linked Na/K transport system. While the concentration of ouabain used is several orders of magnitude greater than that required to inhibit Na transport in vertebrates systems, this level may be required to overcome the permeability barrier to diffusion of large molecules into the rectal tissue via the cuticular intima (Phillips and Dockrill, 1968), Neil and Scudder (personal commu-nication) have found that 24 hours incubation in 10"^ M/l of cardiac glycosides is required to inhibit contraction of insect muscle, whereas the same inhibitor at 10~3 M is effective within 1 hour. (l) Summary, 1, Quantitative similarities between the in vitro and in vivo recta are indicated by the rates of water absorption under isosmotic conditions, i n i t i a l absorption rates in response to various osmotic 36 gradients, absorption from pure sucrose s o l u t i o n , and, the t r a n s - r e c t a l p.d.'s. 2. The constant absorption rate of water and t r a n s - r e c t a l p.d. (between 1 and 5 hours) i n d i c a t e that the a c t i v i t y of the i n v i t r o r e c t a l preparation approaches a steady state adequate f o r studies of i o n and water absorption. 3. Water absorption from isosmotic sucrose s o l u t i o n stops a f t e r 1,5 hours. Apparently, a supply of ions on the lumen (but not the hemocoel) side i s required f o r prolonged maintenance of water transport. This suggests that the water transport i s secondary to and dependent on the absorption of io n s . 4. The r e l a t i o n s h i p between osmotic gradient across the r e c t a l w a l l and the steady state rate of water movement was determined i n v i t r o . At high osmotic gradients, the osmotic permeability of r e c t a l wall decreased f o r water movement i n the opposite d i r e c t i o n (hemocoel to lumen). 5. The a c t i v e nature of r e c t a l absorption process i s i n d i c a t e d by i n h i b i t i o n of i o n and water transport by anoxia, r e s p i r a t o r y i n h i b i t o r s (malonate, KCN + IAA) and a s p e c i f i c i n h i b i t o r of Na-K transport (ouabain). 37 CHAPTER II EFFECT OF IONS ON WATER ABSORPTION (a) Introduction. Preliminary studies indicated that absorption of water by the rectum with isosmotic sucrose solution on the lumen side stops after 1.5 hours but i s restored after replacing the external sucrose solution with simple Ringer. Analyses of the hemocoel f l u i d after incubation for 1 hour i n sucrose (Chapter IV) indicated that i n the absence of ions on the lumen side (replaced by sucrose), tissue ions were transported to the hemocoel side with water. The dependence of water movement on ion transport could therefore be studied by depleting rectal tissue of ions (by incubation i n isosmotic sucrose solution for an hour) and then studying the a b i l i t y of media with different ionic compositions (but constant osmolarity) to restore water absorption. (b) Effect of Cations - Na, K, L i , Choline. This technique was used to study the effect of completely replacing the cations Na and K i n the simple Ringer on the water absorption rates (after pre-incubated i n isosmotic sucrose solution for an hour). In the Na-free Ringer, a l l the Na i n the various salts was replaced with an equivalent amount of K. The K i n the K-free Ringer, was similarly replaced with Na, Na:K Ringer was a 1:1 mixture of Na-free and K-free Ringers, A l l the Na, but not the K, was replaced by L i i n the L i Ringer, (The composition of different media i s l i s t e d i n Table 2.) The effects of replacement of cations (Na and K) using 5 different isosmotic media (Na-free, K-free, Na:K, L i , and choline Ringers) on the F i g . 24. Net absorption of water during incubation i n modified media (on both sides of r e c t a l sac) of d i f f e r i n g cation concentrations (afte r i n i t i a l pre-treatment f o r f i r s t hour i n external isosmotic sucrose solution with simple Ringer on hemocoel side ) . The media were K-free (open c i r c l e s ) , Na-free ( s o l i d c i r c l e s ) , L i ( s o l i d t r i a n g l e s ) , Na:K (inverted s o l i d t r i a n g l e s ) , and choline (open tr i a n g l e s ) Ringers. Dotted l i n e shows the net absorption rate i n control (simple Ringer). Bars indicate ± S.D. and subscript figures show number of preparations. T i m e ( h r ) 38 rectal absorption of water with time are shown i n Fig. 24. In a l l the experiments the same modified media was placed on both sides of the rectal preparation. In the absence of Na (replaced with K), the uptake remained linear up to the end of experiment (3 hours). The mean rate (5.5 ul/hr/R) differed by less than 7$ from that for normal simple Ringer, Surprisingly, the absorption rate i n K-free Ringer (Fig, 24) also was constant and showed no significant difference from the normal rate. In this case, the mean absorption rate was 5«53 ul/hr/R (i.e. about 94$ of normal rate). It appears that water movement can be equally well driven i n the t o t a l absence of either Na or K. Alternatively, the transport of CT (the other major component of Ringer) or other ions (such as Mg, Ca, phosphate, bicarbonate and glucose which were a l l present at the same low concentrations as i n simple Ringer), restored absorption of water. The absence of both Na and K was therefore tested using choline Ringer. The effect on water absorption with time i s shown i n Fig. 24. Water absorption i n the absence of Na and K decreased to 2.73 ul/hr/R, which i s only 46$ of normal rate but remains relatively constant after the f i r s t hour. That i s , the uptake rate was only p a r t i a l l y inhibited i n the absence of Na and K. This suggests that Na and K are required for maintenance of maximum water uptake. Other ions such as CI could account for the parti a l uptake from choline Ringer. The relative importance of Na and K concentration i n the incubation media on water uptake i s indicated (Fig. 24) using Na:K Ringer. In this case, the rate increased to 7.43 ul/hr/R which i s 126$ of normal state. This indicates that water absorption i s sl i g h t l y enhanced by NaiK Fig. 25. Effect of replacing Cl with either NO^  (solid circles) or SO^ (open circles) on net absorption of water after i n i t i a l pre-treatment (over f i r s t hour) i n external sucrose. Dotted lin e shows absorption i n control (simple Ringer). Bars indicate + S.D. and subscript figures show number of preparations. 39 concentration ratios close to unity but overall water transport i s remarkably independent of Na:K ratios. The specific requirement of the water transport system for Ma was tested using the L i Ringer (with K concentration kept near the normal l e v e l ) . As shown i n Fig. 24, mean uptake after the f i r s t hour was constant (up to 6 hours) at 3.5 ul/hr/R (less than 60$ of normal rate). The results may indicate that L i cannot substitute completely for Na, as i n other Na transport systems. This would suggests that the Na transport mechanism i s reasonably specific such that Na cannot be replaced by similar or closely related cations. It i s also possible that (in the absence of Na) low levels of K i n L i Ringer could account for the observed uptake. (c) Effect of Anions - CT, NCy SO^, • The effect of completely replacing CI was tested using 2 types of media (NO^ and SO^ Ringers). In these media, the cation concentrations were the same as i n simple Ringer. NO3 was selected because i t has almost the same hydrated size and the same valency as CI. The absorption of water from NO^  Ringer was constant after the f i r s t hour (Fig. 25). Mean uptake rate was 5.67 ul/hr/R, which i s not significantly different from normal (5,9 ul/hr/R). Replacement of CI with SO^ (Fig. 25) decreased the mean uptake rate significantly to 2.96 ul/hr/R, i . e . 50$ of normal rate. Thus S0^ i s unable to replace CI i n maintaining normal water uptake. This could be related to the large hydrated size of SO^ which i s nearly twice that of CI and NO-jj that i s , transport of cations may be retarded by the slow passive movement of a counter-ion (S0^), Many membranes have been Fig. 26. Net absorption of water i n sucrose Ringer (open circles) after pre-treatraent i n external isosmotic sucrose solution (over f i r s t hour). Prolonged incubation i n external sucrose solution (solid triangles) was taken from Fig, 18. Arrows indicate replacement of experimental solutions (on lumen and hemocoel sides) with simple Ringer, Dotted lin e shows control (simple Ringer), Bars indicate + S.D. and subscript figures show number of preparations. ko reported to be impermeable to SO^ ions. Lewis (personal communication) found that the rectal intima of the desert locust was relatively imper-meable to SO^ compared to Cl. These results suggest that Cl i s not s p e c i f i c a l l y required and that maximum uptake of water i s primarily dependent on the presence of permeant anion (Cl or NO^). However,, experiments with choline Ringer indicate Cl transport can p a r t i a l l y restore water transport. (d) Absorption i n Absence of Na, K and Cl. In previous experiments, a l l the media (except isosmotic sucrose solution) contained at least one of the 3 ions - Na, K or Cl. An experiment was performed i n which a l l 3 of these ions were absent ( i . e . with sucrose Ringer) to exclude the p o s s i b i l i t y that transport of Mg, Ca, phosphate, bicarbonate or glucose can support water absorption by the rectal sac. The absorption (after one hour pre-incubation i n isosmotic sucrose solution) with time i s shown i n Fig. 26. The mean uptake rate of water from sucrose Ringer decreased to 0.72 ul/hr/R after one hour. This rate i s not significantly different from the mean steady state uptake rate with pure sucrose solution on the lumen side (0.75 ul/hr/R; Fig, 18), It should be noted however, that i n the experiments using pure sucrose solution, simple Ringer was always placed on the hemocoel side. This suggests that water absorption i s only supported by the presence of Na, K or Cl on the lumen side. Despite the. prolonged incubation i n medium devoid of Na, K and Cl, the uptake rate from sucrose Ringer remained low (0.72 ul/hr/R) and never dropped to zero. This experiment also indicates that p a r t i a l restoration of water uptake i n choline Ringer (Fig. 2k) i s due to presence of Cl 41 rather than other components of the Ringer, (e) Summary. Clearly, the only components of simple Ringer which can restore a rapid rate of water absorption a f t e r pre-incubation i n pure sucrose s o l u t i o n are Na, K and CT. Of the l a t t e r , f u l l a c t i v i t y can be restored by either Na or K alone, but CI alone can only maintain water absorption at h a l f the normal rate. The rate of s a l t and water transport may be reduced i n media containing a single counter-ion (e.g. SO^ and choline) to which the r e c t a l w a l l i s r e l a t i v e l y impermeable. 42 CHAPTER III CHANGES IN ION CONCENTRATION AND VOLUME OF RECTAL TISSUE DURING ABSORPTION (a) Introduction. Tissue ion concentrations were of considerable interest, since they permit the individual concentration gradients (approximate only) which existed across the apical and basal plasma membranes of the rectal epithelium i n vitro to be established. The l a t t e r information together with measurements of electro-potential differences (Chapter IV) and profiles (Phillips, 1964b; Vietinghoff et a l , I969) might permit tentative localization of active transport sites on specific c e l l membranes and hence lead to c e l l models for trans-epithelial transport. Observing the changes i n tissue volume and ion content when rectal sac are incubated i n various media of widely different composition could provide a variety of experimental perturbations to test such c e l l models. Secondly, i t i s important to distinguish between transport across the whole rectal wall (i . e . from external to hemocoel compartments) as opposed to transient movements of intracellular constituents from the tissue to either side. Calculations of absorption rates (Chapter IV) from changes i n the concentrations of hemocoel f l u i d alone do not permit such a distinction to be made. Determination of changes i n tissue com-position and volume during absorption, however, do allow such a separation of ion and water movements. With these thoughts i n mind, ion and water contents of rectal sacs were determined (1) immediately after preparation of the l a t t e r ( i . e . prior to use of rectal sacs i n any experiment), (2) after 1 hour TABLE 3 . Ionic composition of rectal tissue (before and after incubation) in complex medium, simple Ringer, external isosmotic sucrose solution and hyperosmotic lumen solution. (Mean ± S.D., number of observations are indicated in brackets.) Dry Wt (mg) Water Content Total Ionic Content (uEq/R) Ionic Concn (mEq/kg tissue ^ 0 ) ($ wet wt) Na K ' Cl Na K Cl Initial content (rinsed) 2.2+0.4 (10) 78.0±3.6 d o ) 0.78+0.11 (6) 0.65+0.07 (6) 0.6910.07 (5) 9 9 . U 1 2 . 3 (6) 8 5 . 4 H 1 . 7 (6) 85.7± 5.9 (5) Initial content (not rinsed) 1.710.1 ( 6 ) 82.5±1.3 (6) 1 .18+0.12 (6) 0.55+0.09 (6) 1 5 1 . 6H6.1 (6) 69.7+ 5.0 (6) Complex medium (1 hr) 2.3+0.2 (6) 80.4±1.4 (6) 0.59±0.15 (4) 0.73+0.13 (4) 60.8±10.7 (4) 81.0+11.9 (4) Simple Ringer 1.8+0.4 (9 ) 80.2+2.8 (9) 0.85+0.15 (7) 0.53+0.12 (7) 0 .64±0.09 (3) U 9 . H 2 6 . 7 (7) 76.3+14.5 (7) 100.4+ 8.2 (3) External sucrose solution (1 hr) 1.9+0.4 (5) 7 8 . 3±L 6 (5) 0.54+0.11 (5) 0.52+0.09 (5) 78.0+12.6 (5) 75.0+ 8.0 (5) External sucrose solution (2 hr) 2.0 (2) 73.2±0.9 (2) 0.33 (1) 0.25 (1) • 69.6 (1) 42.2 (1) Hyperosmotic lumen solution 2.0+0.4 (3) 80.4±1.6 (3) 1.23+0.34 (3) 0.83+0.19 (3) 0.89+0.02 ( 2 ) 153.2118.0 (3) 103.1±10.8 (3) 116.4±11.5 (2) 43 incubation with isosmotic sucrose solution on the external side and simple Ringer on the hemocoel side (i.e. prior to transfer to modified Ringers), (3) after 1 hour incubation with complex medium on both sides and (4) at the end (i.e. 3 hours) of incubation i n modified Ringer solutions. Results are shown i n Tables 3 and 4 . (b) Ionic Composition of Rectal Tissue Incubated i n Different Media 0 Since a l l recta were rinsed b r i e f l y i n sucrose solution (0 .335 M/l) before analysing for tissue ions, there was a p o s s i b i l i t y that this procedure removed significant quantities of tissue ions from the intracellular compartment, (The object of the rinsing was to remove ions i n extracellular f l u i d adhering to the tissue surfaces.) This p o s s i b i l i t y was examined by comparing the results with those for recta not rinsed with sucrose (Table 3)» Tissue Na content (per rectum) i n rinsed recta was 66$ of that i n unrinsed recta. Since the K concen-tration was i n fact s l i g h t l y higher (15$) i n rinsed than i n unrinsed recta, this suggests that tissue ions were not l i k e l y washed out by the rinsing. The decrease of Na (and increase i n K) i n the rinsed tissue i s probably due to removal of extracellular Na present on hemo-coel and lumen surfaces since the extracellular (i.e. dissecting) f l u i d had a very high Na concentration but low K concentration. The volumes and ion contents of rectal tissue after incubation i n complex medium (both sides) for 1 hour, isosmotic sucrose solution (external) for either 1 or 2 hours, and simple Ringer (both sides) for 3 to 5 hours (pooled) are shown i n Table 3 . (Some of the l a t t e r had been pre-incubated i n sucrose, on lumen side only, before transfer to simple Ringer but the tissue ions remained at same level irrespective 44 of i n i t i a l pre-incubation.) Water content of recta incubated under isosmotic conditions -with Ringer on both sides (80.4$ and 80.0$ of wet weight for complex medium and simple Ringer respectively) showed l i t t l e difference from recta after 1 hour i n sucrose (78,3$). However, water content of recta after 2 hours i n sucrose decreased significantly (P<0,005) to 73.2$, During prolonged absence of lumen ions (2 hours sucrose), tissue became dehydrated even under isosmotic conditions. The tissue ion concentrations show wide individual variations so that few significant differences were observed. Total rectal K content was relatively unchanged after incubation i n complex medium, simple Ringer and after 1 hour i n external sucrose solution. This i s clearly i l l u s t r a t e d by comparing the K concentrations i n tissue water (mean values of 75.0 to 81.0 mEq/kg tissue HgO). Total tissue Na content (mean values 0.52 to 0.73 uEq/R) under the above conditions appeared to be lowest after absorption from isosmotic sucrose solution for 1 hour (31$ below i n i t i a l ) and highest after 3 to 5 hours absorption from simple Ringer (no significant change). Incubation i n complex medium, however, led to a decrease i n tissue Na. Since the Na concen-tration of complex medium i s only 65$ of that i n simple Ringer, the difference i n f i n a l rectal Na content i s probably related to differences i n Na levels of the 2 incubation media. Concentration gradients for K appear similar to those for most animal c e l l s . More specifically, the K concentrations of simple Ringer and complex medium were 6.7 and 9.2 mEq/l respectively while the K lev e l i n tissue water (76.3 to 81.0 mEq/l) i s about 10 times higher. Measurements after 3 to 5 hours incubation i n simple Ringer suggests 45 that rectal tissue i s able to retain this K level i n spite of high concentration gradients at both apical and basal surfaces. It i s possible that tissue K level i s maintained by an active uptake mechanism on one or both sides of the rectal wall. Changes i n rectal ion concentration during absorption against a slight osmotic gradient (0.116 osmolar) were observed i n experiments i n which simple Ringer made hyperosmotic with sucrose (70 mM/l) was placed on the external side. The results are also shown i n Table 3o Compared to the values following incubation i n simple Ringer (under isosmotic conditions), the t o t a l tissue content of Na, K and Cl increased 45, 56 and 40$ respectively. Na, K and Cl concentrations i n tissue water also showed a similar trend but the increases were smaller. The t o t a l tissue content of a l l 3 ions increased from 2.02 uEq/R (isosmotic condition) to 2.95 uEq/R (with hyperosmotic lumen solution). Since media concentrations of ions were the same under these 2 conditions, tissue ion changes may reflect the requirement for increased cellular osmotic pressure during water uptake from hyperosmotic solutions. (c) Rectal Composition After 3 Hours Incubation i n Ringer with Modified Ion Concentrations. In the absence of any one of the 3 ions (Na, K or Cl), rectal absorption of water s t i l l occurred at almost the same rate as i n simple Ringer (Chapter I I ) . Rectal ionic concentrations after 3 hours incubation i n such media were examined to determine the f i n a l level of the omitted ion i n the tissue and observe compensatory adjustments i n the levels of other ions. The effect of replacing Na, K or Cl i n the incubation media on TABLE 4. Ionic composition of rectal tissue after incubation i n various modified Ringers. (Mean ± S.D., number of experiments are indicated i n brackets.) Dry weight and Cl concentration of recta incubated i n Na:K Ringer were calculated with assumed water content of 80$ (mean normal wet wt). Dry Wt (mg) Water Content ($ wet wt) Total Ionic Content (uEq/R) Ionic Concn (mEq/kg tissue HgO) Na K ' Cl Na K Cl K-free Ringer 1.810.3 (5) 78.812.5 (5) 1.36+0.30 (5) 0.06+0.04 (5) 0.81+0.30 (3) 205.1H7.9 (5) 7.915.3 (5) 112.1+12.1 (3) Na-free Ringer 1.510.1 (5) 84.7*0.4 (5) o,03±o.ol (5) 1.78+0.19 (5) 0.98+0.09 (3) 3.31 0.7 (5) 213.0+6.7 (5) 128.01 1,1 (3) L i Ringer 1.6±0.2 (5) 80.812.3 (5) 0.03+0.02 (5) 0.17+0.06 (5) 0.7710.11 (3) 4.81 3.7 (5) 24,5+5.9 (5) 110.31 9.4 (3) Choline Ringer 2.0+0.4 (5) 78.4+2.7 (5) 0.01+0.01 (5) 0.0210.02 (5) 0.75±0.09 (3) I . 6 1 0.7 (5) 3.313.1 (5) 115.21 2,3 (3) Wj Ringer 1.7+0.3 (5) 79.2+0.5 (5) 0.95±0.20 (5) 0.50+0.09 (5) 0.02+0.02 (3) 145.7120.2 (5) 75.5+5.3 (5) 2.81 2.1 (3) SO4. Ringer 1.910.2 (5) 78.5±L0 (5) 0.90+0.12 (5) 0.4510.08 (5) 132.9H9.1 (5) 66.119.0 (5) Sucrose Ringer 2.0+0.1 (5) 75.4±1.8 (5) 0.01+0.00 (5) 0.02+0.01 (5) 1.7± 0.5 (5) 2.9+0.9 (5) Na:K Ringer 1.8+0.2 (3) 80.0 0.76+0.12 (3) 108.6± 5.7 (3) 4 6 tissue composition are summarized i n Table 4 , In a l l these cases, the f i n a l r e c t a l compositions should be compared with that of recta following 1 hour pre-incubation i n isosmotic sucrose solution ( i . e . i n i t i a l recta l e v e l s ) since a l l these experiments involved t h i s pre-treatment. Tissue water contents i n a l l cases (except i n Na-free and sucrose Ringers) remained unchanged. Tissue hydration i n the Na-free ( i . e . high K) Ringer was s i g n i f i c a n t l y higher ( 6 $ above normal) than for a l l other treatments, whereas tissues i n sucrose Ringer were s i g n i f i c a n t l y (P<0.005) dehydrated ( 5 . 7 $ below normal). In the l a t t e r case, dehydration probably resulted from the dr a s t i c depletion of tissue ions (Na, K). This was accompanied by loss of a b i l i t y to transport water. Conversely, the greatly increased hydration i n Na-free Ringer was accompanied by a very large increase i n tissue K ( t o t a l cation content 38$ greater than normal). Such an increase might indicate that either K was being absorbed by tissue at a greater rate or more K was retained ( i . e . l e s s secreted to hemocoel compartment or more re-cycling involved). A. d r a s t i c a l l y increased rate of secretion of K into the hemocoel compartment (Chapter IV) indicated that the former, at l e a s t , does occur, Na concentrations i n tissue incubated i n K-free, SO^ and NO-j Ringers appeared to be the same as i n simple Ringer (Table 3)« In medium with no Na (Na-free, choline, sucrose and L i Ringers), the tissues were almost depleted of Na ( i . e . l o s s of 90$ or more of i n i t i a l content). However, with normal Na and K concentrations (but no Cl) i n the media ( S 0 ^ and NO-j Ringers), tissue Na concentrations were not much d i f f e r e n t from recta placed i n simple Ringer (Table 3 ) . In the absence of K (K-free Ringer), tissue content of Na increased 47 significantly (43$) above that i n tissue incubated i n a l l other media under isosmotic conditions. This suggests that Na i n the tissue increased to compensate for the drastic loss of K. Concentrations of K i n tissues incubated i n SO^ and NO^  Ringers were the same as i n i t i a l values (i.e. after 1 hour i n external sucrose) and those after 5 hours i n simple Ringer. In media free of K (K-free, choline and sucrose Ringers), the tissues were almost depleted of K (9$ or less of i n i t i a l content remaining). In high K medium (Na-free Ringer), K concentration i n the tissue increased to more than 300$ of normal, which more than compensates (osmotically) for loss of tissue Na, In L i Ringer, tissue Na was presumably replaced by L i , Surpri-singly, tissue content of K i n the L i Ringer was also significantly lowered to approximately 30$ of the level i n a l l other media where K was present. Previous observations (Chapter II) had already indicated that L i could not completely replaced Na i n maintenance of water absorption rate. However, the low tissue content of K suggests that at normal concentrations of K i n media, the presence of Na i s required for maintenance of normal tissue K and water uptake. Alternatively, L i may inhibit K transport (Chapter IV). Cl concentrations of tissues incubated i n media with normal (i . e . simple Ringer) concentrations of Cl (K-free, choline, L i and Na:K Ringers) remained the same (after 3 hours). In Na-free (high K) Ringer, tissue Cl was higher than i n a l l other media. This serves to balance e l e c t r i c a l l y the greatly increased K (and t o t a l cation) concentrations of tissues under this treatment. Tissue i n NO^  Ringer l o s t most of their Cl (3.2$ of i n i t i a l remaining) after 3 hours. Fig. 27. Tissue volume changes with time during incubation i n simple Ringer under different osmotic gradients across rectal wall, (a) Isosmotic condition with Ringer on both sides, (b) Hemocoel hyperosmotic to lumen by 0,178 osmolar. (c) Lumen hyperosmotic by 0.116 osmolar. (d) Lumen hyperosmotic by O.I78 osmolar. (e) Lumen hyperosmotic by 0,256 osmolar. (f) Lumen hyperosmotic by 0.3^ 3 osmolar. (g) Lumen hyperosmotic by 0.5^ 2 osmolar. (h) Lumen hyperosmotic by 0.772 osmolar. (i) Lumen hyperosmotic by 1.005 osmolar. Sucrose i s added to increase osmolarity of the simple Ringer. (Circles represent individual readings.) C h a n g e i n T i s s u e V o l u m e ( u l / h r / R ) 48 Since the water absorption rate was normal, t h i s indicates that NO-^  had probably replaced Cl i n the tissues and that high tissue KO^ i s not detrimental to water transport. I t i s also possible that the Cl transport system does not discriminate between anions with s i m i l a r hydrated size and charge. (d) Tissue Volume Changes, Tissue volume changes were checked i n a l l the experiments to ensure that the change i n weight of the r e c t a l sac preparations was a true i n d i c a t i o n of f l u i d entering the hemocoel compartment and not tissue swelling or dehydration. The method used for determining changes i n tissue volume was outlined e a r l i e r . In cases where consistent and s i g n i f i c a n t hydration or dehydration did occurred, ( i . e . exceeding 0 .5 u l ) the net changes i n tissue volume of i n d i v i d u a l preparation were subtracted or added to the observed changes i n weight. Fig . 27a-i show the ef f e c t of d i f f e r e n t osmotic gradients (by addition of sucrose to simple Ringer placed on the hemocoel side) on the tissue volume changes. During incubation under isosmotic conditions (Fig. 2?a), r e c t a l tissues generally swelled (mean 2.5 u l or about 40$ of i n i t i a l tissue R^O) during the f i r s t hour of incubation and then remained r e l a t i v e l y constant with further incubation (for 3 hours). I n i t i a l swelling of tissue i n simple Ringer i s not surprising since i n the normal i n t a c t animal (fed on hypertonic s a l i n e ) , the rectum i s subjected to osmotic gradients as high as 1.350 osmolal (lumen f l u i d hyperosmotic to hemolymph; P h i l l i p s , 1964c). Probably the freshly dissected r e c t a l tissue i s hyperosmotic to simple Ringer leading to rapid osmotic flow into the t i s s u e s . However, the i n i t i a l swelling i s Fig, 2 8 . (a) Tissue volume changes with time i n simple Ringer (on both sides) after pre-incubation i n 0.335 M sucrose (on lumen) for an hour, (b) Effect of 10 M _3 KCN + 10 M I M added to both sides during incubation i n simple Ringer (after i n i t i a l treatment i n sucrose), (c) Effect of 10~3 M DNP added to both sides during incubation i n simple Ringer (after i n i t i a l treatment i n sucrose), (Circles represent individual readings.) sucrose 4 2 -2 ST -4 u •simple Ringer © 1 © e _©_ 0) a o > CQ .2 -2 H fl -4 CD bD fl .fl U 10"3 M KCN & 10"3 M IAA-simple Ringer b 4 2 -0 -2 -simple Ringer + 10"3 M DNP o © 0 O o 0-1 1-2 2-3 T i m e ( h r ) e o o 3-4 49 a transient feature r e s t r i c t e d to the f i r s t hour. In Fig. 27b, with the osmotic gradient reversed ( i . e . hemocoel made hyperosmotic to lumen by d i l u t i o n of external solution), the tissue volume changes followed the same trend as under isosmotic conditions. When the lumen was hyperosmotic to the hemocoel side (Fig. 2 7 c - i ) , there was always an i n i t i a l loss of water during the f i r s t hour and then tissue volume remained stable on continued incubation. As the osmotic gradient was increased (from 0.116 to 1 .005 osmolar), the i n i t i a l losses of tissue water also increased (from a mean of 1 .35 to 4 .13 ul/hr/R respectively). During dissection from the locust and p r i o r to incuba-t i o n , a l l r e c t a l sacs were exposed to simple Ringer f o r a b r i e f period. This probably caused a s l i g h t hydration of ti s s u e . Subsequently, dehydration would occur when the tissue was subjected to an osmotic gradient (with lumen hyperosmotic). In a l l cases, no s i g n i f i c a n t tissue volume changes occurred a f t e r the i n i t i a l hour. E s s e n t i a l l y , r e c t a l tissue equilibrated under the di f f e r e n t conditions a f t e r t h i s transient stage. In the absence of ions on the lumen side (with isosmotic sucrose solution outside; F i g . 28a), r e c t a l tissue was consistently dehydrated (ranging from -2 .0 to - 4 . 7 5 ul/hr/R during the f i r s t hour). On trans-f e r r i n g the sacs to simple Ringer (on both sides) hydration of tissue occurred. The amount of swelling during the second hour almost compensated for the i n i t i a l water loss i n sucrose during the f i r s t hour. Thereafter, tissue volume changes were i n s i g n i f i c a n t . Apparently, i n the absence of ions on the lumen side, a large proportion (approximately 30 to 60$) of the t o t a l water transfer i s due to secretion of tissue F i g . 29. Tissue volume changes with time during absorption against a s l i g h t osmotic gradient (0.116 osmolar) with the lumen made hyperosmotic by a d d i t i o n of sucrose to simple Ringer, (a) Tissue volume changes -3 with no i n h i b i t o r s (as c o n t r o l ) , (b) 10 M KCN + -3 10 M IAA added to lumen side from the f i r s t to t h i r d -3 hour, (c) 10 M ouabain added to lumen side from the f i r s t to t h i r d hour, ( C i r c l e s represent i n d i v i d u a l readings.) 0 - 2 - 4 -tt \ 2 u xi \ 0 "—' - 2 s - 4 r—1 O > o 2 CQ CQ • i H 0 H fl • i - i - 2 CO ng - 4 a Xi O © -inhibitor pre sent-•inhibitor present-© 4 2 0 6 o o © - 2 © - 4 - f -inhibitor present-.±. e 0 - 1 1 - 2 2 - 3 T i m e ( h r ) o e 3 - < 50 water along with ions into the hemocoel. Fig, 28b,c show the tissue volume changes i n the presence of inhibitors (10~ 3 M KCN + 10~ 3 M IAA and 10" 3 M DNP) added to the simple Ringer on both sides of the rectum after pre-incubation i n sucrose. With a mixture of 10~3 M KCN and l O - 3 M IAA (Fig. 28b), kO% inhibition of water uptake was shown earlier (Chapter I ) . I t was observed that hydration of the tissue continued (at rate of 2.25 ul/hr/R) i n the presence of the inhibitors over the whole 3 hours incubation period. On removal of inhibitors (at the fourth hour), the tissue volume decreased to sl i g h t l y below normal. This suggests that an active process which removes ions from the tissue and maintains both water transport and tissue volume was p a r t i a l l y inhibited. The result with 10 M DNP i s shown i n Fig. 28c. There was no apparent difference i n tissue volume changes with or without DNP (Fig, 28c and 28a respectively) during the f i r s t 2 hours. However, from the second to third hour, consistent hydration of tissue occurred. This could indicate p a r t i a l inhibition as postulated for KCN + IAA. The delay i n hydration may represent the time required for DNP to reach the active sites. However, -3 water transport rate was not significantly inhibited i n 10 M DNP, Further studies on the effect of DNP should be done before any con-clusion can be made on i t s action i n rectal reabsorption. Fig. 29b,c,d show that inhibitors added during incubation i n the presence of an osmotic gradient (0.116 osmolar) have no apparent effect on the normal course of tissue volume changes observed when no inhibitor (Fig, 29a) was added. Water uptake rates (Chapter I) -3 have indicated that inhibition occurred with 10 M KCN + IAA and Fig. 30. Tissue volume changes during absorption i n various modified Ringers (on both sides) after pre-treatment i n isosmotic sucrose solution (on lumen) for an hour. Tissue volume changes i n normal simple Ringer (from Fig. 28a) were used as control, (a) K-free Ringer (K replaced by Na). (b) Na-free Ringer (Na replaced by K), (c) Na:K Ringer (1:1 mixture of K-free and Na-free Ringers), (d) Choline Ringer (Na and K replaced by choline), (e) L i Ringer (Na replaced by L i ) , (f) NO3 Ringer (Cl replaced by NO^). (g) SCTJ. Ringer (Cl replaced by SO^). (h) Sucrose Ringer (Na, K and Cl replaced by sucrose). (Circles represent individual readings.) sucrose-4 2 0 - 2 -4 u a r—< O > o CQ co •r-l H • r H CD fafi a U 4 2 0 - 2 -4 2 0 - 2 -^—simple Ringer © © -K-free Ringer 0 o 3 -«-Na-free Ringer o o © 0-1 -©-o 1-2 2 - 3 T i m e (hr) control © 3 - 4 4 - 5 sucrose- NOg Ringer -4 u Xi \ 1—1 s— a 4 t—^ o 2 > CP 0 CQ CQ -2 H -4 : SO4 Ringer g © G9 • © 1 O © Q® € S3 • ..,.,,,-.,1,, CU « rt 43 U 10 8 6 4 2 0 -2 sucrose Ringer-0-1 1-2 2-3 T i m e ( h r ) simple Ringer e 3-4 4-5 51 10 M ouabain (added to the lumen solution). It i s possible that the hyperosmotic medium of the lumen side prevented hydration of tissue that would normally have occurred under isosmotic conditions. Tissue volume changes during incubation i n modified media (after pre-incubation i n isosmotic sucrose for the i n i t i a l hour) are shown i n Fig. 30a-g. The control used for comparison.was the volume change during incubation i n normal simple Ringer (Fig. 28a), Tissue volume changes were similar to those of the control i n K-free (Fig. 3 0 a ) , NajK (Fig. 3 0 c ) , choline (Fig. 3 0 d ) , L i (Fig. 30e) and NO^  (Fig. 3 0 f ) Ringers. With recta incubated i n Na-free (high K) Ringer (Fig. 3 0 b ) , hydration of tissue continued (mean value of 4 ul/hr/R) for 2 hours after sucrose pre-incubation (compared to 1 hour only for the control). Extreme hydration was confirmed by the direct analysis of water content of recta (Table 4). In the Na-free Ringer, water content of recta (as $ of tissue wet weight) was significantly higher than i n a l l other tissue analysed. Recta placed i n SO^ Ringer (Fig. 30g) after sucrose pre-incubation showed less hydration (15$ of control; Fig. 28a) during the f i r s t hour i n SO^  Ringer. During the second hour, recta were consistently dehydrated ( -1 ,0 ul/hr/R). It appears that the recta were unable to regain tissue water which was l o s t during pre-incubation i n sucrose. SO^  i s known to penetrate membranes at a slow rate (relative to Cl), so, i t i s possible that i n the absence of a accompanying anion (e.g. Cl), the osmolarity of the tissue was decreased by one third since 33$ of total ionic (Na, K and Cl; Table 3) content i n control tissue i s Cl. Unlike the above treatments, dehydration of tissue i n sucrose Ringer (Fig. 30h) continued after the i n i t i a l sucrose pre-treatment. 52 When such recta were t r a n s f e r r e d a f t e r 3 hours to normal simple Ringer, the t i s s u e gained almost 90$ of t h e i r wet weight within an hour at a rate of 8 ul/hr/R, As i n pure isosmotic sucrose s o l u t i o n , t i s s u e ions and water were also secreted i n t o the hemocoel during incubation i n sucrose Ringer so that f i n a l t i s s u e i o n content was very low (Table 4), When placed i n simple Ringer, these t i s s u e s presumably swelled as ions were absorbed from the lumen side i n t o the t i s s u e . (e) Summary. 1. Drastic hydration of t i s s u e (under isosmotic condition) occurred -1 -3 during i n h i b i t i o n with 10 J M KCN + 10 M IAA on both sides and during incubation i n Na-free (high K) Ringer. In Na-free treated recta, an increase (38$ of normal) i n t o t a l i o n i c content i n d i c a t e d that K was absorbed at an increased rate or more K was retained ( i . e . re-cycled) by the t i s s u e , 2. Almost complete depletion of t i s s u e Na, K or C l content was observed a f t e r 3 hours incubation i n the various Ringers that were free of a p a r t i c u l a r i o n . In the absence of K, t i s s u e Na concentration increased to maintain t i s s u e osmolarity. S i m i l a r l y , t i s s u e K increased tremendously (over-compensated) i n the absence of Na (Na-free Ringer). In the absence of C l , NO^ presumably replaced C l i n maintaining t i s s u e osmolarity or e l e c t r i c a l balance, whereas, SO^ was a poor substitute f o r C l . 3. In the completed absence of I fe , K and Cl ( i . e . i n pure sucrose s o l u t i o n and sucrose Ringer) on lumen side, t i s s u e water and ions were presumably secreted i n t o the hemocoel or lumen compartments. The deple t i o n of t i s s u e ions was accompanied by severe l o s s of t i s s u e 53 water. The depleted t i s s u e , when t r a n s f e r r e d to simple Ringer, regained 90$ of the o r i g i n a l wet weight wi t h i n an hour. 54 CHAPTER IV ABSORBATE CONCENTRATIONS, RATE OF ION ABSORPTION, ELECTRO-POTENTIAL DIFFERENCES (a) Introduction, In studying the mechanism of water reabsorption, i t i s of prime importance to know the absorbate concentration and rate of ion transport into the hemocoel compartment. With these observations, i t i s then possible to correlate trans-epithelial solute transport and tissue ion levels (Chapter III) with water uptake under a variety of conditions. This might permit a hypothesis for f l u i d and ion transport to be developed (see General Discussion), (b) Absorption i n Complex Medium and Simple Ringer. The absorption rate and absorbate concentrations of ions entering the hemocoel at hourly intervals during incubation of rectal sacs with either isosmotic sucrose solution on the lumen side or with normal Ringer (both complex and simple) on both sides (i.e. isosmotic and isotonic) are shown i n Fig. 31 and 32. In the absence of ions on the lumen side (i . e . with pure sucrose solution as external bathing media), the absor-bate entering the hemocoel compartment s t i l l contain ions. In fact, with complex medium inside (Fig. 31), the absorbate concentrations of Na and Cl remained unchanged regardless of whether sucrose solution or Ringer was on the outside, whereas K concentrations significantly (P<0.005) decreased 73$ i n the absence of an external source of ions. The absorbates, under both conditions, were hyposmotic to the bathing media; however, recta incubated i n external sucrose solution produced F i g . 31» Absorbate concentrations and absorption rate during incubation (over the f i r s t hour) i n complex medium; with 0.335 M sucrose s o l u t i o n on lumen side and with complex medium on both s i d e s . Osmotic pressures of external bathing solutions are i n d i c a t e d by f i n e dotted l i n e s . Bars i n d i c a t e ± S.D. f o r 6 preparations. A b s o r p t i o n R a t e of I o n ( u E q / h r / R ) © p o © ro a> oo "1 I i r a to O ca -* —*• ro cj) A b s o r p t i o n R a t e of H 2 0 ( u l / h r / R ) A b s o r b a t e C o n c n ( m E q / l ) ro cyj co o o o o T i—<$>—i o o o p [\J C75 0 3 A b s o r b a t e F . P . (-°C) Fig. 32. Absorbate concentrations and absorption rate during incubation (over f i r s t hour) i n simple Ringer; with isosmotic sucrose solution on lumen side (mean ± S.D., 8 preparations) and with Ringer on both sides (mean + S.D., 4 preparations). Osmotic pressures of external bathing solutions are indicated by fine dotted l i n e s . A b s o r p t i o n R a t e of I o n ( u E q / h r / R ) o o CO p o 3* a; p o I to o CO a to o ro P 2 p A b s o r p t i o n R a t e of H 2 O ( u l / h r / R ) A b s o r b a t e C o n c n ( m E q / l ) o 00 o r o o O 13a| I <§> 1 —1 l-h •a 4—• P p p p r o i» o> bo A b s o r b a t e F . P . (-°C) 55 a more hyposmotic absorbate (36$ below that of lumen solution) than with Ringer on both sides (17$ below). Since mean absorption of water was 50$ higher with complex medium on both sides, rates of Na and C l s e c r e t i o n i n t o the hemocoel compartment were proportionately increased as compared to those during incubation i n pure sucrose s o l u t i o n . From these r e s u l t s , i t appears that Na and C l absorption and water movement are coupled. Reduced K concentration i n the absorbate during experiments with external sucrose s o l u t i o n c o r r e l a t e d with the greater hyposmosity of the absorbate r e l a t i v e to the bathing media. When the experiment was repeated using simple Ringer ( F i g . 32), the nature of the external s o l u t i o n (isosmotic sucrose s o l u t i o n or Ringer) had l i t t l e e f f e c t on Na and K concentrations i n the absorbate but the absorbate Cl concentration was s l i g h t l y lower (25$ below that with Ringer) i n the presence of external sucrose s o l u t i o n . Again more ions were absorbed during incubation i n simple Ringer than i n pure sucrose s o l u t i o n where water uptake rate had decreased by 40$ (compared to rate with Ringer on both s i d e s ) . In t h i s experiment, no d i f f e r e n c e i n apparent reabsorption of i o n ( i . e . r e - c y c l i n g ) from primary absorbate was i n d i c a t e d . This could be r e l a t e d to the d i f f e r e n c e i n K to C l r a t i o s of the 2 Ringers, In complex medium (see Tables 1 and 2 f o r compositions of media) the K:C1 r a t i o i s 0.6:1 whereas i n simple Ringer t h i s r a t i o i s 0.035*1. Therefore i n high concentrations of C l , the postulated r e - c y c l i n g mechanism may be switched over to reabsorption of C l . These 2 experiments demonstrated c o n c l u s i v e l y that i n the absence of ions on the lumen side, t i s s u e ions were secreted i n t o the hemocoel compartment during the uptake of water. This i s an important Fig. 33« (a) Absorbate concentrations with time during incubation i n simple Ringer, (b) Absorption rate and trans-rectal p.d. with time during incubation i n simple Ringer. Solid lines indicate isosmotic condition with Ringer on both sides (mean ± S.E., 4 preparations) and broken lines indicate no i n i t i a l hemocoel solution (mean + S.E., 5 preparations). Circles represent average values for p.d, (lumen with respect to hemocoel). Fine dotted line indicates freezing-point depression (f.p.) of external bathing medium. J ' O ' ! L ro •£*> o) oo -* o A b s o r p t i o n R a t e of H £ 0 ( u l / h r / R ) A b s o r p t i o n R a t e o f H ? Q ( j i l / h r / R ) 56 observation since previous reports ( P h i l l i p s , 1964a; Wall, 1967; Stobbart, 1968) that water absorption from the i n s e c t rectum can occur from hyperosmotic pure sucrose solutions i n the absence of net i o n transport across the r e c t a l w a l l were based s o l e l y on changes i n the composition of f l u i d i n the lumen. The l a t t e r experiments would not have detected i o n movement from the t i s s u e . Absorbate concentrations and absorption rates were a l l c a l c u l a t e d i n d i r e c t l y from the i n i t i a l and f i n a l concentrations of hemocoel f l u i d and mean water uptake r a t e s . The r e l i a b i l i t y of r e s u l t s obtained by such c a l c u l a t i o n s was tested by comparison with those obtained by d i r e c t analyses of absorbate ( c o l l e c t e d f o l lowing incubation without any f l u i d i n s i d e r e c t a l sacs i n i t i a l l y ) . D i r e c t and c a l c u l a t e d estimates of Na, K, C l and osmotic pressure are compared i n F i g . 3 3 aib. No large or s i g n i f i c a n t d i f f e r e n c e s are evident. However, the apparent Na concen-t r a t i o n i n the 'calculated' absorbate appeared to be s l i g h t l y higher than that obtained by d i r e c t a n a l y s i s . This decrease i n Na concentration could be due to p a r t i a l d e p l e t i o n of t i s s u e Na since the i n t e r i o r s of r e c t a l sac were r i n s e d i n i t i a l l y with pure sucrose s o l u t i o n i n the ' d i r e c t a n a l y s i s ' experiments. Steady state a c t i v i t y of the i n v i t r o r e c t a l sac preparation was i n d i c a t e d by water absorption rates and the t r a n s - r e c t a l p.d.'s (Chapter I ) . Other c r i t e r i a that should be considered (before steady state conditions are assumed) are the i o n absorption rates and absorbate concentrations. From F i g . 3 3 a i b again, i t i s evident that the absorbate concentrations of Ife and C l increased s l i g h t l y a f t e r the f i r s t hour of incubation whereas that of K decreased. The combined absorption rates Fig. ( a) Absorbate concentrations with time during incubation i n simple Ringer (on both sides) after treatment i n external 0.335 M sucrose solution for an hour, (b) Absorption rate and trans-rectal p.d. with time under the above conditions. Circles represent average values for p.d. (lumen with respect to hemocoel). Fine dotted line indicates f.p. of external bathing media, (Mean ± S.E., 4 preparations.) u o ^0.8 co 0.4 rt x> u o CO JL 2 T i m e ( h r ) 57 of a l l 3 ions d i d not change s i g n i f i c a n t l y (P>0„10) a f t e r the f i r s t hour. E s s e n t i a l l y , i o n i c movements approximated a steady state c o n d i t i o n a f t e r the f i r s t hour. The osmotic pressure o f the absorbate throughout the experiment (up to 5 hours) remained s l i g h t l y hyposmotic to the incuba-t i n g media i n d i c a t i n g that the i n v i t r o preparation was capable of producing hyposmotic absorbate as the rectum does i n v i v o . These obser-vations again suggest that the i n v i t r o r e c t a l preparations were v i a b l e and that observation made with the l a t t e r r e f l e c t normal processes i n the i n t a c t rectum. (c) Subsequent E f f e c t s of 1 Hour Pre-incubation i n Sucrose S o l u t i o n . In the absence of ions on lumen side, t i s s u e i o c s were secreted Into the hemocoel during the f i r s t hour. Analyses a f t e r 1 hour incuba-t i o n i n isosmotic sucrose s o l u t i o n (Chapter I I I ) i n d i c a t e d that quite a l a r g e proportion of the i n i t i a l Na, K and C l s t i l l remained i n r e c t a l t i s s u e , even though net water movement was approaching zero. Therefore, i t was of great i n t e r e s t to continue sucrose incubation f o r an a d d i t i o n a l hour and observe the movement of ions between the t i s s u e and the hemocoel. E f f e c t of pre-incubation i n sucrose s o l u t i o n on subsequent i o n movements and e l e c t r i c a l a c t i v i t y with simple Ringer on both sides were also of i n t e r e s t as a c o n t r o l f o r l a t e r s t u d ies. The r e s u l t s are summarized i n F i g . 3 4 a , b and F i g , 35 a,b f o r experiments i n v o l v i n g 1 and 2 hours pre-incubation with sucrose s o l u t i o n r e s p e c t i v e l y . F i g , 3 4 a ,b show that, a f t e r the second hour, Na absorbate concen-t r a t i o n s and transport rates d i d not change s i g n i f i c a n t l y with time (2 to 5 hours) and were remarkably s i m i l a r to values ( F i g , 33) f o r recta which were not pre-incubated i n sucrose. There was a s l i g h t l y higher 58 transient rate of Na absorption i n the second hour. Absorbate i n the pre-incubated recta was hyposmotic i n i t i a l l y but tends to approach isosmosity with the bathing media by the f i f t h hour. This suggests that the tissue gradually l o s t i t s capacity to form hyposmotic absorbate following pre-treatraent with sucrose; however, the s i t u a t i o n should be studied i n more d e t a i l on absorbate collected d i r e c t l y before any firm conclusions regarding t h i s trend are made. Absorbate concentrations and absorption rates f o r K were r e l a t i v e l y constant with time and did not show high i n i t i a l transient values. High i n i t i a l absorbate concentra-tions of K were expected since, i n vivo, the normal r e c t a l f l u i d contains 241 mEq/l of K ( P h i l l i p s , 1964c). The t r a n s - r e c t a l p.d. (Fig. 34b) i n sucrose solution was i n i t i a l l y very high (l5lmV, lumen positive) but declined to 116 mV at the end of f i r s t hour. This high p.d. could not be explained by Cl secreted into the hemocoel since comparison of t o t a l ions inward movement shows a higher t o t a l f o r the cations (Na and K) than C l . However, i t could be accounted by passive movement of the cations from tissue to lumen especially i f the membrane were highly permeable to Na or K r e l a t i v e to tissue anion. Such a d i f f u s i o n p.d. would decay with time as the cations were continually l o s t from the tissue to lumen side, thereby reducing the concentration gradients f o r Na and K across the a p i c a l plasma membrane. This i s i n agreement with the conclusion by P h i l l i p s (1964b) that the high p.d. across the a p i c a l border of r e c t a l epithelium i s l a r g e l y a K d i f f u s i o n p.d. since the size of the measured p.d. depends d i r e c t l y on the concentration of K (but not Na or Cl) on the lumen side i n vivo. The large concentration gradient (10-fold) for K which can be maintained F i g . 35» (a) Absorbate concentrations with time during incubation i n simple Ringer (on both sides) a f t e r treatment i n external 0.335 M sucrose s o l u t i o n f o r 2 hours, (b) Absorption rate and t r a n s - r e c t a l p.d, with time under the above conditions. C i r c l e s represent average values f o r p.d, (lumen with respect to hemocoel). Fine dotted l i n e i n d i c a t e s f.p. o f external bathing medium. (Mean + S.E., 3 preparations.) T i m e ( h r ) A b s o r p t i o n Rate of Ion (uEq/hr/R) o o p p * K J o> co o I 8 ; I ! =J ro a> oo -* o A b s o r p t i o n Rate of (u l / h r / R ) 59 between r e c t a l t i s s u e and the lumen ( P h i l l i p s , 1964b; and Table 3) i n the presence of continued K leak to the lumen (see Table 5)» suggests the l o c a t i o n of an inward K pump on the a p i c a l membrane ( P h i l l i p s , I965). A f t e r the f i r s t hour i n sucrose, r e c t a t r a n s f e r r e d to the simple Ringer underwent a t r a n s i e n t r e v e r s a l of p.d, to -7.5 mV and then regained the normal p o s i t i v e p.d. (with respect to lumen side) w i t h i n 1 minute ( F i g . 34b), Since the main ca t i o n i n the Ringer was Na, apparently t h i s r e v e r s a l of p.d. was due to i n i t i a l r a p i d Na movement (note high Na absorption rate, second hour; F i g . 34b) i n t o the ion-depleted t i s s u e down a g r e a t l y increased electro-chemical gradient. A f t e r t h i s t r a n s i e n t stage and with renewed a c t i v i t y of the postulated K pump, the t i s s u e probably regained i t s previous complement of ions and hence the normal p.d, was restored. F i g . 35 aib show absorbate concentrations and absorption rates during pre-incubation i n sucrose s o l u t i o n f o r 2 hours followed by t r a n s f e r to the normal simple Ringer. During the second hour i n sucrose, the average absorbate concentrations of Na and K were a c t u a l l y s l i g h t l y negative values. This suggests that Na and K were p o s s i b l y removed from the f r e s h hemocoel f l u i d during the second hour incubation i n sucrose, or at the very l e a s t , that there was no net s e c r e t i o n of t i s s u e ions. Mean absorbate concentrations of C l decreased s l i g h t l y but wide v a r i a t i o n s were observed f o r t h i s i o n and Na, The wide d i f f e r e n c e s between i n d i v i -dual values were undoubtedly the consequence of applying the i n d i r e c t method f o r obtaining absorbate concentrations and rates when very small net movement of ions and water were involved. The absorbate during the second hour i n sucrose s o l u t i o n was s l i g h t l y more hyposmotic than that 60 during the f i r s t hour. On t r a n s f e r r i n g to the Ringer solution,' ( a f t e r 2 hour pre-incubation), Na absorption was immediately restored to normal ( i . e . c o n t r o l with no pre-incubation i n sucrose, F i g . 33) . Absorbate con-centrations and absorption rates of K and C l took more than an hour to regain t h e i r normal l e v e l s . These r e s u l t s i n d i c a t e that r e c t a l t i s s u e s l o s t more K and C l ( e s p e c i a l l y K; but not to the hemocoel side; Table 5) during the second hour i n sucrose s o l u t i o n so that 1 to 2 hours of incubation i n simple Ringer was required before ions from the lumen side could r e - e s t a b l i s h the normal rate of s e c r e t i o n i n t o the hemocoel compartment. The t r a n s - r e c t a l p.d. ( F i g . 35b) during the second hour of pre-incubation was s t i l l high and decayed s t e a d i l y . On t r a n s f e r r i n g to Ringer, the p.d. reversed as p r e v i o u s l y observed ( F i g . 3^b) and then slowly regained p o s i t i v e p.d. a f t e r more than 20 minutes. The nature of these changes i s probably s i m i l a r to those suggested above ( i . e . f o r 1 hour pre-incubation i n sucrose s o l u t i o n ) . (d) Absorption from Modified Ringer S o l u t i o n . In an attempt to deplete r e c t a l t i s s u e of ions that might be e s s e n t i a l f o r a c t i v e absorption of water, a l l r e c t a l preparations d i s -cussed i n t h i s s e c t i o n were pre-incubated i n pure sucrose s o l u t i o n (vrith normal simple Ringer on hemocoel side) f o r an hour, since e a r l y experiments i n d i c a t e d that water uptake could subsequently be restored to normal (Chapter I I ) . The absorbate concentrations, absorption rates and t r a n s - r e c t a l p.d.'s were followed with time f o r 3 a d d i t i o n a l hours i n order to observe changes when Na, K or C l were replaced i n the bathing Fig. 36. (a) Absorbate concentrations with time during incubation i n K-free Ringer (on both sides) after treatment i n external 0.335 M sucrose solution for an hour, (b) Absorption rate and trans-rectal p.d, with time under the above conditions. Simple Ringer i s placed on hemocoel side during pre-incubation. Circles represent average values for p.d, (lumen with respect to hemocoel). Fine dotted lin e indicates f.p. of external bathing medium, (Mean ± S.E., 4 preparations.) & I l I ! l ^ 1 2 3 4 T i m e (hr) 61 media. In a l l cases the same media was placed on both sides a f t e r pre-incubation. The results obtained from recta incubated i n K-free Ringer (6.7 mM/l K replaced by Na) are shown i n Fig. 36a,b. For comparison of Na, K, Cl and osmotic pressure values, recta incubated i n simple Ringer with or without pre-incubation i n external sucrose solution (Fig. 33a,b and Fig . 34a,b) were used. In the absence of K, the absorbate concentration of Na increased s i g n i f i c a n t l y (P<0.01) to a higher steady l e v e l (120$ of Na transfer i n normal simple Ringer, Fig. 34a). S i m i l a r l y , rate of Na secretion ( t h i r d and fourth hour) into hemocoel compartment increased s l i g h t l y (115$ of Na secretion rate i n simple Ringer F i g . 33b and 3^b. The absorbate concentration of K decreased to 0,83 mEq/l and rate of absorption dropped to 0.004 uEq K/hr/R (5$ of control) a f t e r 3 hours. Absorbate concentration and absorption rate of Cl remained approximately the same. Since a normal rate of water absorption was maintained over the whole period, apparently, K absorption was not essential f o r water uptake and could be compensated for by increased Na transport. However, there was an important difference i n the osmotic pressure of the absor-bate, which was now hyperosmotic to the lumen solution. This indicates that without K i n the medium, absorption of Na and Cl could not support formation of normally hyposmotic absorbate. In F i g . 36b, the tr a n s - r e c t a l p.d. i n K-free Ringer was i n i t i a l l y at 14 mV (lumen p o s i t i v e ) and i t decreased slowly to steady p.d. of 5 mV within 2 hours. This could probably be accounted f o r by the reduced passive movement of K from tissue to lumen solution and hence reduced K d i f f u s i o n p.d, across the a p i c a l membrane as the tissue became depleted Fig. 37. (a) Absorbate concentrations with time during incubation i n Na-free (high K) Ringer (on both sides) after treatment i n external 0.335 M sucrose solution for an hour, (b) Absorption rate and trans-rectal p.d. with time under the above conditions. Simple Ringer i s placed on hemocoel side during pre-incubation. Circles represent average values for p.d, (lumen with respect to hemocoel). Fine dotted l i n e indicates f.p. of external bathing medium, (Mean ± S.E., 4 preparations.) sucrose—>\< Na-free Ringer a T i m e (hr) A b s o r p t i o n R a t e of H 2 O ( u l / h r / R ) 62 of K i n the K-free Ringer (see Table 4). Another possible reason was the increased Na transport to the hemocoel side which would make the l a t t e r l e s s negative ( r e l a t i v e to lumen s i d e ) . Absorbate concentrations and absorption rates i n Na-free Ringer (Na replaced by 180 mM/l K) are shown i n F i g . 37aib. In the absence of Na, the absorbate concentrations and absorption rates of Na and Cl showed s i m i l a r trend to K and Cl movements i n the K-free Ringer. That i s , Na concentration and absorption rate decreased d r a s t i c a l l y to 9«46 mEq/l and to 0,057 uEq/hr/R respectively over the t h i r d and fourth hours whereas Cl remained at normal l e v e l s . With the greatly increased K concentration (27X normal) of the medium, the absorbate concentration and absorption rate of t h i s ion increased (14X normal) to value compa-rable to those found for Na i n the normal simple Ringer. However, i n Na-free (high K) Ringer, the osmotic pressure of the absorbate was c l e a r l y hyposmotic to lumen side and highly comparable to the absorbate i n simple Ringer. This suggests that water transport can be driven equally w e l l by either Na or K but that the formation of hyposmotic absorbate ( i . e . re-cycling) i s dependent on presence of K. The t o t a l secretion of cations (Na + K) remains r e l a t i v e l y constant with time i n both Na-free and K-free Ringers and only the r a t i o of the 2 cations changes, Fig. 37b shows the t r a n s - r e c t a l p.d. i n Na-free (high K) Ringer I n i t i a l l y , the p.d. increased sharply from 17 nV (lumen p o s i t i v e ) to 40 mV i n 6 minutes. After that, the p.d, dropped exponentially to 3 mV a f t e r 3 hours. The high i n i t i a l transient appears to be correlated with the rapid changes i n the r e l a t i v e absorption rates of Na and K, The Fig. 38. (a) Absorbate concentrations with time during incubation i n Na:K Ringer (on both sides) after treatment i n 0.335 M sucrose solution for an hour, (b) Absorption rate with time under the above conditions. Simple Ringer i s placed on hemocoel side during pre-incubation. (Mean ± S.E., 4 preparations.) T i m e (hr) Fig. 39» Relationship of absorbate concentrations and absorption rate as a function of Na and K concentrations i n the medium. Values represent mean of concentrations and rates during the f i n a l 2 hours incubation i n the Ringers. M e d i a C o n c n ( m M / l ) 63 sharp r i s e i n the peak could be explained by the d i f f u s i o n of Na from t i s s u e to lumen ( i . e . Na d i f f u s i o n p.d.) while the K gradient (and hence K d i f f u s i o n p.d,, which i s normally dominant) was i n i t i a l l y reduced close to zero by the high external K concentration. As Na i s secreted to the hemocoel, t h i s Na concentration gradient and hence d i f f u s i o n (see Table 4) p.d, should decrease while the a p i c a l K pump bu i l d s up a smaller gradient across the a p i c a l border again (Table 4). The smaller f i n a l K d i f f u s i o n p.d. during the l a t t e r stages of t h i s experiment could then be responsible f o r the lower (than normal) p.d. ( F i g . 34b). Absorbate concentrations and absorption rates of ions with recta incubated i n Na:K Ringer are shown i n F i g . 38a,b. This shows that absor-bate concentration and absorption rate ( t h i r d and fourth hour) of Na decreased 45$ and 20$ (re s p e c t i v e l y ) with a decrease of 44$ i n medium concentration while K values increased about 400$ as medium concentration o f K was increased to 13X normal l e v e l s . The absorbate concentration of Na showed a s l i g h t decrease with time whereas that of K increased. The C l concentration of absorbate was s i m i l a r to that of controls but there was an increase (40$) i n t o t a l absorption rate. This increase c o r r e l a t e d with the increase (26$) i n the water uptake rates of recta incubated i n the Na:K Ringer. Absorbate concentrations and rates of i o n absorption f o r media having d i f f e r e n t r a t i o s of Na to K concentrations (keeping C l and osmotic pressure constant) are depicted i n F i g , 39. The values i n the l a t t e r f i g u r e are the means of observed values during the l a s t 2 hours since these best approximate steady state a c t i v i t y . An increase Fig. 40. (a) Absorbate concentrations with time during incubation i n choline Ringer (on both sides) after treatment i n external 0.335 M sucrose solution for an hour, (b) Absorption rate and trans-rectal p.d. with time under the above conditions. Simple Ringer i s placed on hemocoel side during pre-incubation. Circles represent average values for p.d. (lumen with respect to hemocoel). Fine dotted line indicates f.p. of external bathing medium, (Mean ± S.E., 4 preparations.) sucrose K ^0.8 W fl 0.6 o <4-l O ® 0 4 -M • K fl ° 0.2 a. bsor pi « -fl O CM a O CU tf C O •rH -*-> o CQ rQ < 64 i n either Na or K In the incubating media i s associated with a r i s e i n the absorbate concentration and absorption rate of the respective i o n . Within experimental errors, t h i s relationship i s approximately-l i n e a r for both ions. Absorbate concentrations and absorption rates of Cl appeared to be r e l a t i v e l y constant and independent of medium K:Na concentration r a t i o , possible being more d i r e c t l y related to t o t a l cation movement ( i . e . Na + K). These observations suggest that 2 separate transport systems are involved for Na and K absorption so that when both ions are present i n high concentrations (e.g. Na:K Ringer), the 2 transport systems can somehow drive net water movement at higher rate than i n simple Ringer. The effects of replacing both Na and K i n the incubating medium simultaneously (substituting with choline) are shown i n Fig. 40a,b. Absorbate concentration and absorption rate of Na decreased d r a s t i c a l l y with time and during the fourth hour, there was no net transfer of Na, Potassium i n the absorbate decreased s l i g h t l y during the i n i t i a l hour and then appeared to maintain t h i s lower l e v e l (at 50$ of values i n normal simple Ringer). The absorbate concentration and absorption rate of C l increased s i g n i f i c a n t l y (PO.Ol) with each additional hour of incubation i n the choline Ringer. In the f i n a l hour, absorbate concen-t r a t i o n of Cl was almost i s o t o n i c with the incubating medium. The absorption rate of Cl had decreased to 85$ of normal rates and the absorbate remained hyperosmotic to the bathing medium. The results indicate that 50$ of normal water uptake can be maintained by Cl absorption but the absorbate i s not hyposmotic (Fig. 40a) as i n the control. The absorption of Cl i n the absence of s i g n i f i c a n t net Na Fig. 41. (a) Absorbate concentrations with time during incubation in NO-j Ringer (on both sides) after treatment in external 0,335 M sucrose solution for an hour, (b) Absorption rate and trans-rectal p.d, under the above conditions. Simple Ringer is placed on hemocoel side during pre-incubation. Circles represent average values for p.d. (lumen with respect to hemocoel). Fine dotted line indicates f.p. of external bathing medium. (Mean ± S.E., 4 preparations.) A b s o r p t i o n R a t e of H 2 O ( u l / h r / R ) 65 and K transfer suggests that there i s an independent Cl pump which a c t i v e l y transports Cl to the hemocoel since the l a t t e r movement i s against a large electro-potential gradient. (This conclusion assumes that choline transport i s u n l i k e l y . ) Fig. 40b shows that the t r a n s - r e c t a l p.d, was i n i t a l l y higher (44 mV, lumen positive) than normal and decayed slowly to 20 mV at the end of the experiment. In the absence of Na and K, the active transport of Cl without a r a p i d l y permeating cation would explain the i n i t i a l high p.d, across the r e c t a l w a l l , (Also removal of external K would increase the a p i c a l K d i f f u s i o n p.d, i n i t i a l l y u n t i l t i s s u e becomes depleted of t h i s ion, Table 4). This high p.d. favours the passive movement of choline from lumen to tissue and hemocoel. Increased movement of choline a f t e r t h i s i o n accumulated within the tissue could explain the subsequent reduction i n the p.d, across the r e c t a l w a l l . Comparing the results using NO^  Ringer (Fig , 41a,b) with the controls ( F i g . 3 4 a ,b), the absorbate concentrations of Na, K and osmotic pressure show no large (or s i g n i f i c a n t ) differences. However, the mean values suggest i d e n t i c a l absorption rate f o r Na, whereas the K rate decreased to about 60$ of the control rate. Absorption rate of Cl decreased progressively from an i n i t i a l rate of 0.2 to 0.01 uEq/hr/R during the fourth hour. The osmotic pressure of absorbate was hyposmotic (consistently) to the bathing medium but on average higher than values from simple Ringer, The t r a n s - r e c t a l p.d, remained unchanged at a very low value (4 mV) and the i n i t i a l reversal of p.d, was not observed. Since the replacement of Cl with N0^ had no apparent ef f e c t on water absorption, i t i s obvious that the Cl i s not s p e c i f i c a l l y required f o r Fig, 42. (a) Absorbate concentrations with time during incubation i n SO^ Ringer (on both sides) after treatment i n external 0.335 M sucrose solution for an hour, (b) Absorption rate and trans-rectal p.d. under the above conditions. Simple Ringer i s placed on hemocoel side during pre-incubation. Circles represent average values for p.d, (lumen with respect to hemocoel). Fine dotted lines indicate f.p.'s of external bathing medium. (Mean ± S.E., 4 preparations.) D. ( m V ) A b s o r p t i o n R a t e of I o n ( u E q / h r / R ) A b s o r p t i o n R a t e of H 2 O ( u l / h r / R ) F i g . 4 3 . (a) Trans-rectal p.d. with time during incubation i n L i Ringer a f t e r i n i t i a l treatment i n external 0.335 M sucrose s o l u t i o n over the f i r s t hour, (b) T r a n s - r e c t a l p.d. with r e c t a l sacs incubated i n sucrose Ringer a f t e r the above i n i t i a l treatment. C i r c l e s represent average values f o r p.d. (lumen with respect to hemocoel). Mean + S.E. f o r 4 prepa-r a t i o n s . 1 2 3 T i m e ( h r ) T i m e ( h r ) 66 the l a t t e r and can be replaced by an anion (NO3) of s i m i l a r hydrated s i z e . During incubation i n the SO^ Ringer (Fig. 42a,b), the Ife and Cl concentrations i n the absorbate were s i m i l a r to those f o r experiments with NO^  Ringer, However, with the reduction i n water uptake (50$ of normal), the absorption rates of the ions were s i m i l a r i l y reduced. The absorbate obtained with SO^ Ringer was hyperosmotic (17 to 35$) to the bathing medium. This suggests the i n a b i l i t y of recta to produce hypos-motic absorbate i n the absence of a r a p i d l y penetrating anion such as C l . The t r a n s - r e c t a l p.d, was 13 mV (lumen pos i t i v e ) i n i t i a l l y , then reversed i n about 15 minutes and remained at -8 to -5 mV (lumen with respect to hemocoel) throughout the experiment. The i n a b i l i t y of SO^ (replacing Cl) to support normal uptake of water i s not surprising since many b i o l o g i c a l membranes are known to be r e l a t i v e l y impermeable to the SOij. due to i t s large hydrated s i z e . This has been d i r e c t l y demonstrated for the r e c t a l c u t i c l e of the locust (Lewis, personal communication). The reversal of the p.d. i s due presumably to the active transport of Na into the hemocoel compartment i n the absence of a rap i d l y permeating anion. The absorbates during incubation i n L i and sucrose Ringers were not analysed. The t r a n s - r e c t a l p.d.'s i n these media are shown i n Fig. 43. On transf e r r i n g to L i Ringer from isosmotic sucrose solution, the p.d. reversed immediately (-4 mV) and then quickly changed back to a p o s i t i v e value. I t reached i t s peak p.d. 24 mV (lumen positive) i n 40 minutes and then decayed slowly. I n i t i a l reversal i s probably caused by i n f l u x of L i ions from the lumen into the ti s s u e . Decreased secretion Fig. 44. Absorbate concentrations and absorption rate during absorption against an osmotic gradient (0.116 osmolar) with simple Ringer on both sides (lumen made hyperosmotic with sucrose), (a) Absorbate concentrations, (b) Absorption rate. Fine dotted line indicates f.p. of external bathing medium. (Mean ± S.E., 5 preparations.) A b s o r b a t e F . P . (- C) o T 00 T 3 CD tr v-A-t ro t-^-i: ro w U CO A b s o r b a t e C o n c n ( m E q / l ) ro ro as co ~* o A b s o r p t i o n R a t e of H £ 0 ( u l / h r / R ) A b s o r p t i o n R a t e o f I o n ( ; i E q / h r / R ) r o cn co o A b s o r p t i o n R a t e of H 2 0 ( ; i l / h r / R ) 67 of Na from t i s s u e to hemocoel r e l a t i v e to C l , and i n i t i a l enhancement of the K d i f f u s i o n p.d, across the a p i c a l border could both cause the trans-r e c t a l p.d. to r i s e w e ll above normal. The higher e l e c t r o - p o t e n t i a l gradient favours movement of L i i n t o hemocoel and t i s s u e and t h i s could account f o r reduction of the p.d. with time. In the absence of Na, K and C l , the p.d. i n sucrose Ringer ( F i g , 43) remained high i n i t i a l l y (63 mV) and slowly decreased to 26 mV at the end of experiment. The small decrease at the beginning might be caused by i n f l u x i n t o the c e l l of cations (e.g. Mg and Ca) present i n the medium. Otherwise, the s i t u a t i o n i s p o s s i b l y s i m i l a r to that during pre-incubation i s isosmotic sucrose s o l u t i o n . (e) Absorption Against an Osmotic Gradient. A l l previous experiments were undertaken with the lumen i n i t i a l l y isosmotic to the hemocoel s i d e . This s e c t i o n deals with experiments using simple Ringer (with no pre-incubation i n sucrose solution) i n which (70 mM/l) sucrose was added to the lumen s o l u t i o n to create an osmotic gradient (0.116 osmolar) across r e c t a l w a l l . The absorbate concentrations and absorption rates are shown i n F i g . 44a,b. With the lumen hyperosmotic to the hemocoel, Na and K s e c r e t i o n i n t o the hemocoel remained r e l a t i v e l y steady with time at rates not s i g n i f i -c a n t l y (PX).lO) d i f f e r e n t - f r o m those observed i n the absence of an osmotic gradient ( F i g . 33 a n d 34). However, the absorbate concentra-t i o n s of Na and C l appeared to increase s l i g h t l y but s t e a d i l y with time. The osmotic pressure of the absorbate was also hyposmotic to the lumen s o l u t i o n by 14$ over the whole experimental period, compared to 12$ under isosmotic conditions ( F i g , 33a). TABLE 5. Calculations f o r determining movement of Na and K from t i s s u e to lumen s o l u t i o n during incubation i n Na-free Ringer (A), K-free (C) and choline Ringer (B and D). Tissue content was obtained from Tables 3 and 4; t o t a l i o n secreted i n t o hemocoel was obtained from F i g , 37b (Na-free), F i g , 36b (K-free) and F i g . 40b ( c h o l i n e ) . Figures i n brackets i n d i c a t e ± S.D. (4 observations each). Tissue Ion Cone (uEq/R) — — — — — — — — — — , Na K A. B C D 1. I n i t i a l tissue content 0.536 0.536 0.517 0.517 2. F i n a l tissue content 0.028 0.011 0.058 0.021 3. Loss (1-2) from tissue 0.508 0.525 0.459 0.496 4, Amount secreted into hemocoel 0.398 (±0.057) 0.287 (±0.013) 0.040 (±0.006) 0.073 (+0.014) Estimated loss (3-4) from tissue to lumen solution 0.110 (+0.057) 0.236 (±0.012) 0.419 (+0.006) 0.424 (±0.014) $ of t o t a l loss from tissue to lumen side 21.7 (+11.2) 44.9 (±2.3) 91.4 (±1.3) 85.4 Mean loss as $ 33.3 (±14.5) 88.4 (±3.8) 68 (f) Loss of Ions to Lumen Solution. During some experimental conditions (e.g. incubation i n K-free Ringer), large concentration gradients (for K i n th i s case) between the r e c t a l tissues and both the hemocoel and the lumen sides were created. Net loss of ions from the tissues to external solution ultimately occurred (Table 4), Repeated references have also been made to large K d i f f u s i o n p.d.'s across the a p i c a l border of r e c t a l epithelium ( P h i l l i p s , 1964b). In t h i s section, an attempt w i l l be made to examine t h i s question and i n p a r t i c u l a r , to determine the r e l a t i v e rates of Na and K loss across the ap i c a l and basal borders of r e c t a l tissue. Loss of K from tissue to the lumen side i n preparations incubated i n K-free and choline Ringers was calculated by subtracting the t o t a l K secreted to the hemocoel side (from F i g , 36b and 40b) from the actual loss of tissue K over the same 4 hour period (Table 4). I n i t i a l tissue K was that of tissue which had been pre-incubated f o r 1 hour i n sucrose before t r a n s f e r r i n g to the K-free or choline Ringer, Na loss was s i m i l a r l y calculated using recta incubated i n Na-free and choline Ringers. The results are shown i n Table 5« Over the 4 hour period i n Na-free or choline Ringer, 8,1 to 47.6$ of the i n i t i a l tissue Na ions were l o s t to the lumen side and the remaining Na was secreted into the hemocoel compartment; that i s , the dominant movement was toward the hemocoel side. Over the same period i n K-free and choline Ringers, 85 to 91$ of the i n i t i a l tissue K ions were l o s t to the lumen side ( i . e . the dominant loss of K was to the lumen side ) . The mean r a t i o of K to Na loss from tissue to lumen side was 2,65 to 1. This suggests that the permeability of the a p i c a l border 69 (i . e . lumen border) to K i s probably at least 2 times that for Na since the i n i t i a l tissue content of both these cations was about the same. Since this loss of K does not occur when 6.7 mM/l K i s i n the bathing media, an apical located K pump which compensates for this loss i s postulated. Moreover, Na loss to the lumen side from recta i n Na-free Ringer (22$) i s significantly lower (P<0.01) than compared with that i n choline Ringer. Conversely, K loss to the lumen side i n K-free Ringer i s higher than i n choline Ringer, Apparently, i n the presence of external K, less Na i s l o s t from tissue to lumen solution whereas i n presence of external Na, more tissue K i s lost to lumen. This observation suggests a possible Na-K exchange system at the lumen (apical) surface of rectal wall which moves Na into tissue and K outward to lumen passively. Since exact electro-potential differences across the apical border under a l l these conditions were not measured, i t i s d i f f i c u l t to suggest a definite mechanism for such an exchange. (g) Summary. 1. In the absence of an external source of ions (i.e. sucrose bathing solution), tissue ions are secreted into the hemocoel compartment during the absorption of water. Ion transport (especially Na) and water movement appear to be coupled since absorbate concentration of Na remained at the same level with or without lumen ions. The p o s s i b i l i t y of reab-sorption (i.e. re-cycling) of K and Cl from the primary absorbate (before entering hemocoel) has been suggested. 2. In the absence of K and a permeating anion (e.g Cl or NO^), the rectum i s incapable of secreting a hyposmotic (to lumen bathing solution) 70 absorbate into the hemocoel compartment. However, the transport of any one of Na, K or Cl can drive net water movement across the rectal epithelium at normal rates when transport of the other two monovalent ions i s reduced to negligible levels. 3. There appears to be separate transport systems for Na and K absorption and a separate pump for Cl movement. The locations of the K pump at the apical and the Na pump at the basal borders have been suggested, 4. High permeability of the apical membrane to K (relative to Na) i s indicated by K and Na movements from tissue to lumen. The normal trans-rectal p.d, could be accounted for i n part by a K diffusion potential. 71 GENERAL DISCUSSION The transport a c t i v i t y of the i n v i t r o r e c t a l preparation used i n t h i s study i s compared with that i n vivo ( P h i l l i p s , 1964a,b,c) i n Table 6. Absorption rates were determined i n vivo by measuring changes i n volume and composition of f l u i d i n the r e c t a l lumen. However, the rates i n the i n v i t r o study were c a l c u l a t e d from changes i n concentra-t i o n or volume of hemocoel f l u i d . Thus differences i n rates between the two preparations may p a r t i a l l y r e f l e c t changes i n t i s s u e volume and i o n content during absorption. Moreover, experiments i n vivo were c a r r i e d out at 2°C below those i n v i t r o . Considering these d i f f e r e n c e s , there i s reasonably close agreement between i n v i t r o and i n vivo rates. The apparently higher rate of NaCl transport r e l a t i v e to water uptake i n v i t r o c o r r e l a t e s with the higher absorbate osmotic pressure i n v i t r o compared to that i n v i v o . This may be due to exposure of i n v i t r o recta to d i l u t e s o l u t i o n s during preparation ( i . e . s e l f - r e g u l a t i o n ) or to l a c k of hormonal i n f l u e n c e . Caution should be exercised i n any comparison of i n vivo and i n  v i t r o studies since hormones have been implicated i n the r e g u l a t i o n of excretory system i n i n s e c t s . In the l o c u s t , a d i u r e t i c hormone has been shown by Highnam, H i l l and G i n g e l l (I965). More recently, Mordue (I969) reported that f a c t o r s from the corpora cardiaca reduce water absorption i n i s o l a t e d r e c t a l sacs. ( I t i s known that the osmotic pressure of excreta of the desert l o c u s t can vary from very s l i g h t l y hyperosmotic to hemolymph to 2 , 5 - f o l d depending on water requirements of the animal; P h i l l i p s , 1964a.) In the s t i c k i n s e c t , V i e t i n g h o f f (I966 and I967) has claimed that neurohormone C reduces and neuro-TABLE 6 . Net absorption rates (mean ± S.D.) i n v i t r o over the f i r s t hour during incubation i n complex medium (6 preparations) or simple Ringer (5 preparations) compared to i n vivo rates f o r hydrated and dehydrated animals (taken from P h i l l i p s , l964a,b). Mean Absorption Rate ( u l H 2 0/hr/R and uEq Ion/hr/R) In vivo In v i t r o Hydrated Dehydrated Complex Medium H 2 0 17.0 17.0 1 7 . 2 5 ± 1 . 5 Na 0.22 0.36 0.75±0.15 K 0.83 0.56 0.61+0.20 ca 0.16 0.16 0.20+0.04 Simple Ringer H 2 0 17.0 17.0 11.5 ±2.2 Na 0.85 0.38 0.98±0.16 K 0.45 0.45 0.25+0.06 Cl 0.72 0.38 0.99±0.17 72 hormone D increases r e c t a l absorption. While these, f a c t o r s are ignored i n the present study, t h i s does not detract from the usefulness of i n v i t r o studies f o r determining c e l l u l a r mechanisms and properties of absorption processes (e.g. Diamond, I965» Smyth, 1965. Schultz and Curran, 1968) . For several e p i t h e l i a , reduction or absence of a c t i v e processes observed i n vivo has been reported f o r some i n v i t r o preparations. For example, water and Na transport i s reduced and C l transport i s absent i n the i n v i t r o f r o g s k i n (reviewed by H a r r i s , i 9 6 0 ) . Likewise, I r v i n e and P h i l l i p s (1971) using an i n v i t r o (not everted) preparation of the l o c u s t rectum (oxygenated on hemocoel side only) found that Na and water transport a c t i v i t y was retained but that of K and C l l o s t . The q u a l i t a t i v e d i f f e r e n c e i n a c t i v i t y between the l a t t e r and the present i n v i t r o preparation might be vigorous oxygenation of the lumen side of the everted r e c t a l sac. This might sustain the K and C l transport mechanisms pr e v i o u s l y postulated ( P h i l l i p s , I965) to be located at the a p i c a l side of the r e c t a l epithelium. Moreover, i n present i n v i t r o preparation of the l o c u s t rectum, the solute transport rates a c t u a l l y increased s l i g h t l y i n some cases. I f the 'ion r e - c y c l i n g ' hypothesis o f water transport (discussed l a t e r ) i s cor r e c t , increased i o n movement i n v i t r o could be due to reduced capacity to reabsorb ( i . e . re-cycle) ions ( e s p e c i a l l y Na) from the primary absorbate f l u i d before the l a t t e r emerges on hemocoel s i d e . The above a c t i o n could also explain the lower osmotic gradients against which water i s absorbed i n v i t r o . P a r t i a l dependence of water uptake on aerobic r e s p i r a t i o n was i n d i c a t e d i n preliminary studies during incubation i n complex medium 73 -2 ? containing 10 M malonate. S i m i l a r l y , lO"- 5 M DNP (an aerobic r e s p i r a -t i o n i n h i b i t o r ) and a mixture of 10~^ M KCN + 10~3 M I M both p a r t i a l l y i n h i b i t water absorption with the l a t t e r i n h i b i t o r s showing greater e f f e c t . Greater i n h i b i t i o n obtained with.KCN + I M mixture might i n d i c a t e the r e l a t i v e importance of g l y c o l y s i s i n v i t r o since I M i s an i n h i b i t o r of anaerobic r e s p i r a t i o n . However, the i n h i b i t o r s appeared to be e f f e c t i v e only when added on the lumen side . This i s not s u r p r i s i n g since i n h i b i t o r placed on the hemocoel side would have to penetrate a l a y e r of muscle and a l a y e r of secondary c e l l s to reach the r e c t a l epithelium proper (Irvine, 1966). D i f f u s i o n i n t o tissues from hemocoel side may be f u r t h e r hindered by water s e c r e t i o n to the blood s i d e . With the i n h i b i t o r placed on the lumen, permeation i n t o the t i s s u e may be f a c i l i t a t e d by water movement. The f a c t that ouabain (10 M) on the lumen side i n h i b i t e d water uptake suggests the l a t t e r movement i s dependent on solute absorption since t h i s cardiac glycoside i n h i b i t s Na-K membrane transport (Glynn, 1 9 6 4 ) . This confirms the observation of I r v i n e and P h i l l i p s (I97I). Several workers have reported that K transport by various i n s e c t epithe-l i a (e.g. midgut, Haskell et a l , I 9 6 5 ; malpighian tubule, Berridge, I 9 6 6 ) i s i n s e n s i t i v e to ouabain. This could presumably be true f o r the r e c t a l epithelium since very high concentrations of ouabain were required f o r i n h i b i t i o n . However, i t i s a l s o possible that slow penetration of ouabain could reduce i t s e f f e c t i v e concentration at the transport s i t e s , thus explaining the requirement f o r high external concentrations. Further evidence (besides i n h i b i t i o n by ouabain) indicates" the dependence of water transport on i o n transport. Previous workers 74 ( P h i l l i p s , 1964a, 1969; Wall, 1967; Stobbart, I968) have reported that considerable amounts of water can be absorbed from the i n s e c t rectum i n vivo without net t r a n s f e r of solute across the r e c t a l w a l l as a. whole. Thus d i r e c t evidence f o r coupling of water t r a n s f e r to i o n transport ( i . e . secondary transport of water) was l a c k i n g . The current study confirmed the c a p a b i l i t y f o r absorption from isosmotic sucrose s o l u t i o n s but demonstrated that such uptake was accompanied by a c t i v e i o n s e c r e t i o n from the t i s s u e s to hemocoel side (but not across the whole r e c t a l w a l l ) ; i . e . water movement does not occur without i o n transport. Furthermore, t h i s study showed that prolonged maintenance of water transport required uptake of ions from the lumen side. The i n v i t r o l o c u s t rectum appears to absorb water at a normal rate i n the absence of e i t h e r Na or K i o n s . With equal concentration of Na and K i n the incubation medium, water uptake can be stimulated to 126$ of the normal rate. S i m i l a r l y , C l ions can be substituted by NO^ but the rate i s reduced when SO^ i s used. The hydrated s i z e of NO^ i s rather s i m i l a r to C l whereas membranes are r e l a t i v e l y impermeable to SO^ due to i t s l a r g e hydrated s i z e . Water uptake can also be main-tained, but at a lower rate, when Na i s replaced by L i or when both Na and K are replaced with choline. However, i n the complete absence of Na, K and C l (when replaced by sucrose) or with pure sucrose s o l u t i o n on lumen side, the absorption of water can be almost abolished. Obvi-ously, water absorption cannot be supported by Mg, Ca, phosphate, bicarbonate and glucose transport, at l e a s t at the concentration l e v e l s present i n simple Ringer. Active transport of both K and Na were i n d i c a t e d by net movement 75 against electro-chemical gradients i n experiments with SO^ Ringer i n which the normal p.d, was reversed. Apparently, there are separate pumps f o r Na and K since t h e i r absorption rates are l i n e a r l y r e l a t e d to media concentration of Na and K ( F i g , 39). The pump i s rather s p e c i f i c f o r Na since the rectum cannot use L i as e f f e c t i v e l y . The existence of a separate pump f o r C l (not associated with the monovalent cations) i s demonstrated with the replacement of both Na and K with c h o l i n e . In t h i s case, movement of C l i s against an electro-chemical gradient without s u b s t a n t i a l accompanying moveBient of Na and K cations (during the t h i r d and fourth hours). This suggests an electrogenic pump f o r C l since i n the choline Ringer, t r a n s - r e c t a l p.d. i s a c t u a l l y increased (with hemocoel side more negative to lumen). The a b i l i t y of NO^ to completely replace C l i n maintaining r e c t a l a c t i v i t y (e.g. water uptake and p.d.) suggests that t h i s C l pump may be u n s p e c i f i c and can t r a n s f e r other monovalent anions s i m i l a r i n s i z e to C l . On the other hand, NO^ may simply p a s s i v e l y follow Na transport due to a favourable e l e c t r o - p o t e n t i a l gradient, Requirements f o r formation of a hyposmotic absorbate appear d i f f e r e n t f o r those necessary to, maintain the absorption of water. Hyposmotic absorbate can be produced i n the absence o f Na (replaced with K) and p o s s i b l y C l (replaced with NO^). When the small amount of K i n normal Ringer i s replaced by Na, the rectum can absorb water at a normal rate but ana l y s i s of transported f l u i d shows that i t i s now hyperosmotic to lumen f l u i d . S i m i l a r l y , hyperosmotic absorbate i s formed i n the absence of both Na and K (when replaced with choline) and when Cl i s replaced with SO^. Apparently, the presence of K and 76 a r e a d i l y permeating counter-ion ( C l or NO^) are e s s e n t i a l f o r the formation of a hyposmotic absorbate. This i s ' n o t s u r p r i s i n g since high K (139 mEq/l) i s normally secreted from the malpighian tubules i n vivo ( P h i l l i p s , 1964c) , These observations suggest that water movement can be driven by Na, K and C l pumps. The a b i l i t y of a l l the 3 ions to move• water makes sense since the animal has to regulate r e l a t i v e uptake of i o n ( i . e . hemolymph i o n r a t i o s ) simultaneously with adjustment of o v e r a l l osmotic pressure. While there i s a requirement f o r K i n the formation of a hyposmotic absorbate, t h i s i s r e l a t i v e l y concentration independent. I t i s noteworthy that the absorbate concentration of Na (under most experimental conditions) was higher than the t i s s u e concentration whereas that of K was c o n s i s t e n t l y lower (10 to 50$ of K concentration i n t i s s u e ) . Since, i n high K (Na-free) Ringer, the rate of K se c r e t i o n does become very large, t h i s suggests that the low K concentration under other experimental conditions i s due to a c t i v e K retention rather than low permeability w i t h i n the r e c t a l epithelium. Likewise, since the hemocoel has been reported to be e l e c t r i c a l l y p o s i t i v e to the c e l l i n t e r i o r ( P h i l l i p s , 1964b; V i e t i n g h o f f et a l , 1969), the observed s e c r e t i o n of Na from t i s s u e to hemocoel against a concentration gradient must be an a c t i v e process. Net water movement against appreciable osmotic gradients have been shown i n vertebrate ileum (Fordtran and Dietschy, I966), f i s h g a l l bladder (Diamond, 1962a,b,c) and frog s k i n (Ussing, 195^)• In these systems, water movement i s c l o s e l y r e l a t e d to net solute transport and does not develop i n c r e a s i n g osmotic gradients. Analysis of absorbate F i g . 45. Three hypothetical schemes which might give r i s e to the a c t i v e transport of water i n the absence of net solute transport (taken from P h i l l i p s , 1970). For a f u l l explanation, see t e x t . LUMEN (1000 mOsm) diffusion H 2 0 solute transport \ r \ > 1000 mOsm ^ high hydrostatic pressure t I i back diffusion or solute transport I I laminar flow o f " solute and H 2 0 diffusion H 2 0 I I i > 1000 mOsm high hydrostatic pressure laminar flow H 2 0 I —I — V solute transport and back diffusion I Barrier I 6 = | back diffusion of cation Barrier II a & 0 H 2 0 by electroosmosis ^ transport of cation +• Intracellular ( 4 0 0 mOsm) ( 4 0 0 mOsm) HEMOLYMPH LOCAL OSMOSIS DOUBLE MEMBRANE ELECTRO "OSMOSIS and SOLUTE RETURN EFFECT 77 i n these systems has in d i c a t e d that the transported f l u i d i s hyperosmotic or at most isosmotic to the f l u i d from which absorption occurs„ Consequently, water movement i s i n t e r p r e t e d as a secondary process as proposed i n the double-membrane hypothesis (Curran, I 9 6 0 ; Curran and Mcintosh, 1962; Patlak et a l , I963) and the standing-gradient osmotic flow model (Diamond, I965» Diamond and Bossert, 1967. Diamond 1971). In i n s e c t s , the process involved i n net water absorption appears to be b a s i c a l l y d i f f e r e n t . In the l o c u s t rectum, net water absorption can occur from an i n i t i a l l y pure sucrose s o l u t i o n and the absorbate i s hyposmotic to the f l u i d which absorption occurs. Though transport of ions (Na, K and Cl) i s continuously occurring, the rates observed are too low to account f o r the net f l u x of water across the r e c t a l w a l l . S i m i l a r l y , P h i l l i p s (1964a,b) has shown that net water movement i s independent of net solute transport and leads to development of inc r e a s i n g osmotic gradients. Since transport and bac k - d i f f u s i o n of ions are continuously occurring, P h i l l i p s (1965» 1970) suggested that water movement could be driven by a c t i v e r e - c i r c u l a t i o n of ions, thus not i n v o l v i n g net solute movement across the r e c t a l w a l l . The 3 hypotheses to explain independent transport of water driven by a l o c a l s olute pump are shown i n F i g . 45 ( P h i l l i p s , 1970) . Each o f these 3 hypotheses requires an a c t i v e solute pump to dr i v e water movement. In the f i r s t hypothesis, l o c a l osmosis of water i s postulated by a c t i v e solute transport i n t o the i n t e r c e l l u l a r spaces. Net solute movement across the whole r e c t a l w a l l i s not involved since b a c k - d i f f u s i o n ( i . e . r e - c y c l i n g ) of solute occurs across the r e c t a l w a l l . This model requires 2 membranes with d i f f e r e n t permea-78 b i l i t y and transport p r o p e r t i e s . One of the membranes i s responsible f o r s e c r e t i n g solutes i n t o the l a t e r a l channel (high permeability to HgO) and the other f o r reabsorption (ion transport i n the reverse d i r e c t i o n and low permeability to 1^0). The l a t e r a l channels have been i d e n t i f i e d i n several vertebrate e p i t h e l i a as the s i t e of l o c a l osmosis by c o r r e l a t i n g f l u i d transport rate with the d i s t e n t i o n of the l a t e r a l spaces (Kaye et a l , I 9 6 6 ; Diamond and Bossert, 1967; Tormey and Diamond, 19&7). The double-membrane hypothesis ( F i g . 45) requires 2 membranes ( i n s e r i e s ) with d i f f e r e n t r e f l e c t i o n c o e f f i c i e n t s . One must have a high r e f l e c t i o n c o e f f i c i e n t ( i . e . h i g h l y permeable to water r e l a t i v e to solutes) while the other must have low r e f l e c t i o n c o e f f i c i e n t ( i . e . l e s s s e l e c t i v e to water vs. solute movement). I f the intermediate compartment (between the 2 membranes) were maintained hyperosmotic to the lumen side by a solute pump, then water d i f f u s i o n i n t o the hyperosmotic t i s s u e compartment could l e a d to b u i l d up of a high h y d r o s t a t i c pressure which i n turn might cause f i l t r a t i o n of water and solute through the l e s s s e l e c t i v e membrane. Local solute r e - c y c l i n g by transport of solute across the non-selective membrane back i n t o the c e l l could then maintain hyposmotic water movement against an osmotic gradient. These 2 hypotheses require that the concentration of solutes i n the c e l l s be hyperosmotic to the lumen f l u i d . However, P h i l l i p s ( l964a,b) found that the t o t a l Na, K and C l concentration i n the r e c t a l epithelium i s lower than that i n hemolymph, thus these hypo-theses require that a high concentration of other solutes or some F i g . 46. P o s s i b l e l o c a t i o n s of l o c a l osmosis hypothesis (cycles 1, 3» 5) and double-membrane model (cycles 2, 4, 6) as suggested by the u l t r a s t r u c t u r e of the l o c u s t rectum (from P h i l l i p s , 1970). Large arrows i n d i c a t e s i t e and d i r e c t i o n of solute transport and broken l i n e s with small arrows i n d i c a t e p o s s i b l e d i r e c t i o n and routes of solute return (by a c t i v e or passive mechanisms). Cycle 4 represents a continuous process which recurs throughout the e n t i r e length of the l a t e r a l spaces. Rectal Pad solute transport — > solute diffusion Lumen Cuticle Subintimal space Primary cells of pad Intercellular spaces Secondary cells of pad Hemocoel 79 other means of lowering water a c t i v i t y i n the t i s s u e . Stobbart (I968) a l s o suggested that the lower i o n i c concentration i n the r e c t a l epithe-lium need not c o n f l i c t with these theories since the regions of high i o n i c concentration i n the t i s s u e may be small and since some of the r e - c y c l i n g solutes may be organic i n nature. The present study, also i n d i c a t e s that the t o t a l concentration of fla, K and C l i n the t i s s u e water was lower than i n the bathing media. Recent micropuncture studies on cockroach rectum (Wall and Oschman, 1970; Wall et a l , 1970; Wall, 1971) have suggested that about 50$ of the r e c t a l pad t i s s u e and i n t e r -c e l l u l a r f l u i d osmolality i s made up of non-electrolytes. Analysis of f r e e amino acids content i n l o c u s t rectum (Balshin, personal communication) has shown that p r o l i n e e x i s t s a t high concentrations i n the t i s s u e . P o s s i b l y , the r e - c y c l i n g organic solute postulated i s an amino a c i d such as p r o l i n e . The t h i r d model of water transport by electro-osmosis (shown i n F i g . 4-5) seems u n l i k e l y since reversing the e l e c t r o - p o t e n t i a l gradient or varying the s h o r t - c i r c u i t i n g current across the r e c t a l w a l l has no marked e f f e c t on net water movement ( P h i l l i p s , 1964a). This i s again confirmed i n the present study where water absorption was reduced only 50$ while the t r a n s - r e c t a l p.d. was reversed during incubation i n SO^ Ringer. The p o s s i b l e l o c a t i o n s of l o c a l osmosis and double-membrane e f f e c t with solute r e - c y c l i n g , as suggested by u l t r a s t r u c t u r a l studies, are shown i n F i g . 46 (reviewed by P h i l l i p s , 1970) . Cycles 1, 3 and 5 are examples of l o c a l osmosis with solute return and cycles 2 , 4 and 6 show the double-membrane e f f e c t . Any one or more of these cycle might F i g . 47. A hypothetical scheme showing the l o c a t i o n of transport mechanism and p.d, within the l o c u s t r e c t a l epithelium. Concentrations of lumen bathing medium, t i s s u e water and absorbate (mEq Io n / l and £±C) represent observed values i n simple Ringer. For a f u l l d e s c r i p t i o n , see t e x t . 80 cause net water movement i n the lumen to hemocoel d i r e c t i o n . In t h i s model, P h i l l i p s (1970) predicted that cycles 2, 3 and 4 require a hyperosmotic i n t r a c e l l u l a r compartment (or at l e a s t equal to lumen content) to permit net d i f f u s i o n of water across the a p i c a l membrane. Cycles 3» 4 and 5 and a primary water pump should produce a strongly hyposmotic absorbate. In cycle 1, the absorbate would be hyperosmotic or isosmotic since l o c a l r e - c y c l i n g i s not involved. Cycle 6 represents a supplementary system f o r enhanced water absorption as i n l o c u s t rectum but i s not e s s e n t i a l f o r water transport (since the secondary c e l l s are absent i n the cockroach). The experimental observations are summarized i n an hypothetical model f o r the l o c a t i o n of transport mechanisms involved i n the absorp-t i o n of ions and water i n the l o c u s t rectum ( F i g . 4?). The s i t e and d i r e c t i o n of transport mechanisms are i n d i c a t e d by s o l i d arrows. Broken arrows i n d i c a t e the d i r e c t i o n of net d i f f u s i o n whereas open (white) arroxirs i n d i c a t e processes that are s t i l l questionable concerning the l o c a t i o n and nature (passive or a c t i v e ) . The d i r e c t i o n of osmotic flow of water i s i n d i c a t e d by dotted arrows. The i o n i c concentrations of lumen, t i s s u e water and absorbate were those observed during i n c u -b a t i o n i n simple Ringer. The mean absorbate concentrations are from the second to f i f t h hours of incubation. Trans-membrane p.d.'s are obtained from i n vivo ( P h i l l i p s , 1964b) and i n v i t r o ( V i e tinghoff et a l , I969) measurements of l o c u s t rectum. P h i l l i p s (1964b, 1965) suggested that r e c t a l absorption of C l i s an a c t i v e process since net C l movement occurred against an e l e c t r o -chemical gradient and i n the absence of net water movement ( i . e . solvent 81 drag i s unlikely). Present studies confirmed the above observations and demonstrated that net movement of Cl occurs without significant transport of cations (Na and K) and can lead to an increased electro-potential gradient. Thus, the po s s i b i l i t y of solute-solute drag for net Cl transfer i s unlikely and therefore, an electrogenic pump for Cl i s proposed. Active transport of Cl has been reported i n the vertebrates (reviewed by Ussing, 195^) a n < i i n some insects (e.g. i n malpighian tubules of Rhodnius; Maddrell, I969). Two possible locations of Cl transport are shown i n the hypothe-t i c a l model (Fig. 4 7 ) . Since the trans-membrane p.d, at the apical border i s negative (tissue with respect to lumen), Phillips (1964b, I965) suggested that the Cl pump i s located on the apical membrane. Present studies indicate the Cl pump could also be located within the la t e r a l plasma membrane and would secrete Cl into the intercellular channels to account for hyperosmotic secretion i n K and Na free Ringer (i . e . choline Ringer). This p o s s i b i l i t y i s further substantiated since, during incubation i n the choline Ringer, no hydration of tissue was observed and f i n a l tissue concentration of Cl was much lower than i n the bathing media, (The converse of the la t t e r situations would have indicated an apical ion pump.) However, present evidences cannot ruled out the p o s s i b i l i t y that there could be 2 active locations for transport of Cl, Active transport of Na i n the locust rectum has been suggested by Phillips (1964b, I965) and Irvine and Phillips (1971) since net transfer of Na i s not accounted for by the electro-chemical gradient. Present i n vitro experiments also indicated that an active process 82 i s involved i n the transport of Na. The Na transport system appears to be a separate and s p e c i f i c pump since net absorption of Na i s r e l a t i v e l y independent on K and C l movement (e.g. K-free, Na:K and SO^ Ringers) does not seem to be much a f f e c t e d by g r e a t l y increased K concentrations. P h i l l i p s (1965) postulates that the Na pump i s located on the hemocoel-facing membrane (or l a t e r a l membrane) since the electro-chemical gradient favours passive movement of the i o n across the lumen membrane. In the present studies, comparison between media and t i s s u e Na concen-t r a t i o n has shown ( F i g . 47) that the Na concentration gradient favours t h i s passive movement (from lumen to t i s s u e ) . The l o c a t i o n of Na pump ( i . e . s e c r e t i n g i n t o the i n t e r c e l l u l a r channel) i s supported by several observations. Water and NaCl s e c r e t i o n from t i s s u e to hemocoel side i s maintained when no ions are present e x t e r n a l l y ; the l a t t e r movement i s against a concentration gradient and, probably, an e l e c t r i c a l gradient and leads to t i s s u e dehydration. Moreover, Na i s p r e f e r e n t i a l l y l o s t to the hemocoel side i n the Na-free and choline Ringers. Production of a hyperosmotic absorbate i n the K-free Ringer excludes the p o s s i b i -l i t y of r e - c y c l i n g of Na and, thus, reabsorption o f Na from the i n t e r -c e l l u l a r channel i s not postulated. The a c t i v e nature of K movement across the r e c t a l wall was demonstrated i n vivo ( P h i l l i p s , 1964b) using the s i m i l a r c r i t e r i a as f o r Na transport. This was again confirmed i n the i n v i t r o studies where a separate transport system f o r K was i n d i c a t e d since net K t r a n s f e r can occur i n the absence of Na ( i n Na-free and L i Ringers), and absence of C l ( i n NO-j and SO^ Ringers). I t might be argued (from the present 83 experiments alone) that, i n the Na-free Ringer, K movement i s secondary transport of C l , The transport of K i n t o the t i s s u e a t the a p i c a l membrane with an opposing movement on the hemocoel side was suggested by P h i l l i p s (1965). In t h i s case, net movement of K to the blood side occurred since the rate of lumen to t i s s u e K transport exceeds the opposing K transport on the hemocoel s i d e . The a c t i v e K transport s i t e s a t the lumen and hemocoel-facing membranes were also i n d i c a t e d , i n the present studies, by the maintenance of normal rate of water uptake, formation of hypos-motic absorbate and the considerable t i s s u e hydration i n the absence o f Na (high K), Location of an a p i c a l K pump would account f o r the d r a s t i c t i s s u e hydration and g r e a t l y increased t i s s u e K content observed i n the absence of Na and, po s s i b l y , the net t r a n s f e r of water (by i n t e r n a l h y d r o s t a t i c pressure as postulated f o r the double-membrane model). The opposing transport of K from hemocoel to t i s s u e , that i s , reabsorp-t i o n or r e - c y c l i n g , would not only enhanced the above features ( f o r an a p i c a l K pump), but also r e s u l t e d i n the formation of a hyposmotic absorbate. This K pump appears to r e a ^ i r e the presence o f a accompanying permeating counter-ion ( C l or NO-j) since, i n i t s absence, recta l o s t t h e i r a b i l i t y to form a hyposmotic absorbate ( i n SO^ Ringer). Active reabsorption ( r e - c y c l i n g ) of K i s fu r t h e r substantiated by the a b i l i t y o f r e c t a to r e t a i n K ( r e l a t i v e to Na) ions despite prolonged incubation i n media free of K (e.g. isosmotic sucrose s o l u t i o n ) . I t w i l l be r e c a l l e d that i n K-free media, t i s s u e K i s p r e f e r e n t i a l l y l o s t to the lumen s i d e . In the i n v i t r o preparation, s e c r e t i o n of K i n t o the i n t e r c e l l u l a r channel from t i s s u e i s also postulated since a normal rate 84 of -water absorption was observed i n the absence of Na, Possibly, the K pump here i s not e s s e n t i a l since C l transport or the high i n t e r n a l hydr o s t a t ic pressure ( b u i l t up during t i s s u e hydration) could account f o r f l u i d movement i n t o the l a t e r a l spaces. The p.d. across the a p i c a l membrane (lumen p o s i t i v e to t i s s u e ) i s a t t r i b u t e d to b a c k - d i f f u s i o n of K i n t o the lumen since P h i l l i p s (1964b) found that t h i s p o t e n t i a l i s dependent on lumen concentration of K, Present studies confirmed the ra p i d d i f f u s i o n of K from t i s s u e to lumen bathing s o l u t i o n . The l o c a t i o n o f transport mechanisms can be co r r e l a t e d with the various i o n cycles ( e i t h e r l o c a l osmosis or double-membrane model with sol u t e return; F i g . 46) postulated by P h i l l i p s (1970). Cycle 5 could represent the K pump at the a p i c a l membrane with r a p i d b a c k - d i f f u s i o n . Cycles 3 and 4 are consistent with the Na (outward) and K (inward) transport l o c a t e d a t the l a t e r a l i n t e r c e l l u l a r channel. Cycle 1 could be eliminated i n the i n v i t r o rectum since water absorption d i d not occur from isosmotic sucrose s o l u t i o n ( i . e . a f t e r prolonged incubation) when ions could leak back from the hemocoel side. S i m i l a r l y , cycle 2 i s improbable since the rectum cannot u t i l i z e hemocoel ions f o r the mainte-nance of water absorption. In summary, present observations have demonstrated a dependence of net water uptake (under isosmotic conditions) on transport of solutes and have i n d i c a t e d the po s s i b l e ions involved i n solute r e - c y c l i n g . P o s s i b l e l o c a t i o n s of i o n transport mechanisms are also suggested. I t should be noted that the present study has not excluded the p o s s i b i -l i t y of a primary water pump (which has yet to be found i n any 85 transporting e p i t h e l i a ) . However, t h i s i n v i t r o preparation has shown i t s usefulness i n the studies of transport mechanism across the l o c u s t rectum. 86 SUMMARY 1. A method i s described for the preparation of an everted rectal sac of the desert locust„ Water and solute absorption by the rectum was determined by measuring changes i n hemocoel f l u i d and rectal tissue. 2. I n i t i a l absorption rates of Na, K, Cl and water and trans-rectal potential are comparable to those i n vivo under similar conditions. 3. After an i n i t i a l transient period (over 1 hour), transport a c t i v i t y of the i n vitro preparation remained i n a steady state condition for at least 4 hours. 4. The relationship between osmotic gradient and steady state rate of net water movement across the rectal wall was determined. 5. Net absorption of water i s p a r t i a l l y inhibited by anoxia, -2 —3 -3 malonate (10 M), dinitrophenol (10 M), potassium cyanide (10 J M) plus iodoacetate (10~3 M) and ouabain (10~3 M). 6. Tissue ions and water are secreted into the hemocoel compartment when rectal sac i s incubated i n isosmotic sucrose solution. 7. Dependence of water movement on solute transport i s indicated by the requirement of lumen ions for prolonged maintenance of water absorption. 8. Effects of different ions (Na, K and Cl) i n bathing media on absorption rate of water and ions, absorbate concentrations, trans-ep i t h e l i a l electro-potential differences and tissue composition were determined. 9. Observed properties of water and solute movement i n vitro 87 are discussed and evaluated i n r e l a t i o n to possible mechanisms f o r a c t i v e absorption of water. Possible l o c a t i o n s of transport s i t e s are suggested i n a hypothetical scheme based on the u l t r a s t r u c t u r e of r e c t a l epithelium. 88 LITERATURE CITED Anderson, B. and H.H. Ussing, i960. Active transport. In "Comparative  Biochemistry". Eds. M. Florkin and H.S. Mason. Academic Press: London. Berridge, M.J. I966. Metabolic pathways of isolated malpighian tubules of the blowfly functioning i n an a r t i f i c i a l medium, i i . Insect Physiol. 12: 1523-1538. Berridge, M.J. and B.L. Gupta. I967. 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