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Absorption of amino acids In vitro by the rectum of the desert locust (Schistocerca gregaria) Balshin, Michael 1973

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ABSORPTION OF AMINO ACIDS IN VITRO BY THE RECTUM OF THE DESERT LOCUST (SCHISTOCERCA GREGARIA) by MICHAEL BALSHIN M.Sc, The Hebrew University of Jerusalem, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t 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 "2 O O L~ O (Y The University of British Columbia Vancouver 8 , Canada ABSTRACT Rectal sacs of Schistocerca gregaria Forskal adults are capable of active absorption of those neutral amino acids naturally found in locust hemolymph. This absorption occurs against large electrochemical gradients, is inhibited by KCN, and is enhanced only slightly by solvent flow. Metabolic conversion of the amino acids during the translocation across the epithelium is not significant. The site of active absorption appears to be located on the apical border of the epithelium. Exit of the amino acids from the epithelium to the hemolymph may occur by passive diffusion. The absorptive process exhibits stereo-specificity, saturation kinetics, and competitive inhibition. Relative rates of absorption of different amino acids correlate approximately with their relative abundance in the hemolymph. A reciprocal correlation is found between the absorp-tion rate and the length of the hydrocarbon side chain of the transported amino acid. The glycine transport system across the rectal wall involves a large Na dependent and a small Na independent component. Partial inhibition of glycine uptake is observed when CI or are omitted from the incubation medium. The Na+ dependence of net x^C-glycine uptake into the rectal epithelium from the lumen is a consequence of both an increase in influx and decrease in efflux. The tissue concentration of Na+ is not affected by the presence or absence of external glycine. Net transfer of glycine across KCN poisoned rectal sacs is not reversed upon reversal of the Na+ concentration gradient. The present work suggests the involvement of a specific carrier in the mechanism of active transport of amino acids by the locust's rectum. It would be premature to assess, however, whether the trans-location of amino acids is a primary transport process, or a secondary one coupled to net Na+ fluxes across the rectal wall. Nonetheless, the presence of such a mechanism would suggest that the rectum plays an important role in the regulation of amino acid levels in the hemolymph of insects. iv TABLE OF CONTENTS Page ACKNOWLEDGMENTS XiV ABSTRACT i i i TABLE OF CONTENTS v LIST OF TABLES v i i i LIST OF FIGURES x LIST OF ABBREVIATIONS x i i i CHAPTER I General Introduction 1 CHAPTER II Materials and Methods 10 A. Experimental animals 10 B. Preparation of everted rectal sacs and 10 incubation procedures C. Experiments with radioactive tracers 14 D. Measurements of net water movement 15 and ion concentrations E. Measurements of electro-potential 15 difference across the rectal wall F. Amino acid analysis 16 G. Metabolism of amino acids by rectal tissue 16 1. Thin layer chromatography 17 2. High voltage paper electrophoresis 17 3. Incorporation of ^ C into C02 18 H. The accumulation of x^C-labelled amino 19 acids in the rectal tissue I. The effect of respiratory inhibitors on 20 amino acid transport v Page J. Efflux of 14C-labelled amino acids 2 0 Chapter III Transport of Amino Acids Against Concentration Gradients A. Introduction 22 B. Viability of jLn vitro preparations 22 C. The accumulation of l^rj-giycine against 2 3 an electrochemical gradient D. Tissue content and metabolism of glycine 29 i . Thin layer chromatography 29 i i . High voltage paper electrophoresis 32 i i i . Incorporation of l^c-activity into CO2 43 E. Transport of other amino acids 44 F. Summary 49 G. Discussion 50 CHAPTER IV Location and Characteristics of the Transport System A. Introduction 55 B. Tissue accumulation of ^4C-labelled amino acids 56 1. Glycine 56 2. Serine 56 3. Comparative survey 59 C. The amino acid composition of the rectal tissue and the bathing media during incubation 62 D. Specificity of absorption 69 E. Kinetics of -^C-glycine influx 73 F. Competition 79 G. Summary 83 H. Discussion 83 vi Page CHAPTER V Sodium Dependence of Glycine Transport A. Introduction B. The influence of monovalent ions on the net transport of glycine across the rectal wall C. The effect of Na+ on the tissue accumulation of glycine 1. Replacement with K+ 2. Replacement with choline D. The effect of external Na+ concentration on glycine influx E. Efflux of glycine F. The effect of external glycine on the accumulation of 22jja+ by tji e r e ct a l tissue G. The net transfer of 14c -glycine upon the reversal of the Na+ gradient H. Summary I. Discussion CHAPTER VI APPENDIX BIBLIOGRAPHY General Discussion 94 96 100 100 103 105 107 111 113 115 117 125 133 140 v n LIST OF TABLES Table Pa8fe Chemical composition of modified Ringers used in various experiments x x II Net rates of glycine and water absorption by in vitro recta in media of various composition III Concentration of free amino acids and urea in the rectal tissue using an amino acid analyser 28 30 IV Rf values for unlabelled amino acid standards, ninhydrin positive spots in external (E.M.) and internal (I.M.) media after 6 hr. incubation, and radioactive spots identified by autoradiography 38 V Estimated net rates of amino acid transfer and water absorption across the wall of in vitro recta 46 VI x^C-glycine ratios (H/L and T/L), net rates of glycine and water absorption, and % dry weight of rectal tissue after a two hour incubation in the presence or absence of 10~3 M KCN 58 VII Uptake of different L-amino acids by rectal tissue 61 VIII The T/L ratios of l^C-activity and the % dry weight of rectal sacs exposed to various 14C L-amino acids 63 IX The composition of free amino acids in the rectal tissue and in the incubation media when rectal sacs are exposed to different amino acids 66 X Net rates of organic solutes and water absorption by in vitro recta 70 XI The uptake of x^C-serine stereo-isomers by the rectal tissue - 72 XII The external inulin space of the rectal sacs 74 XIII Net rates of glycine and water absorption by jLn vitro recta in media of various compositions 98 v i i i Table Page XIV The effect of external glycine on 2% a+ levels in rectal tissue, 112 XV The H/L ratio of ^(J-glycine upon reversal of Na+ gradient 116 XVI Calculated secretion rates (R) of glycine by Malpighian tubules of the desert locust in vivo for various U/P ratios 127 XVII The relative average absorption rates of amino acids by in vitro recta, compared to relative concentrations of amino acids in the hemolymph in vivo 130 A Effect of Na+ on glycine influx 135 B Rates of l^C-glycine uptake by rectal tissue when different concentrations of serine or proline are present in the test solution 136 C Effect of different concentrations of Na+ on the influx of glycine 137 D Cell water and •'-'^ C-glycine content and cell glycine concentration after different periods of time in the presence or absence of external sodium in the incubation media 138 E Na+ content of rectal tissue after different periods of incubation in various media 139 xx LIST OF FIGURES Figure Page 1. The experimental set-up for measuring absorption by in vitro rectal sacs (Schematic) 13 2. The ^ C-glycine ratio (H/L) and the net movement of water across rectal sacs with time 24 3. The potential difference (H side negative) across everted rectal sacs with time 25 4. The 14C-glycine ratio (H/L) and the net rate of water movement across the rectal wall in the absence of an osmotic gradient . 27 5-9 Two-dimensional thin layer chromatography of samples withdrawn from the inside medium of the rectal sac, incubated with l^C-glycine 33 5i~9x Autoradiography of the above thin layer chromatograms 33 I O T . High voltage electrophoresis of samples from the internal medium of sacs exposed externally to 1 4c-glycine 42 10xi- Autoradiograph of the above electrophoretogram 42 11. The l^C-amino acid ratios (H/L) and the net movement of water across the rectal sacs with time of incubation 45 12j. High voltage paper electrophoresis separation of internal media bathing rectal sacs exposed to different l^C-araino acids 48 1 2 n . Autoradiograph of the above electrophoretograms 48 13. The ^ C-glycine ratios (H/L and T/L) developed across rectal sacs at two hours of incubation 57 14. The l^C L-serine content in rectal tissue and the flux rate of this amino acid into the internal bathing media during incubation 60 15. Comparison of accumulation ratios for eight •^ C-amino acids in tissue (T/L) and in internal bathing media (H/L) relative to the external media 64 x Page Figure 16. The entry of x^C-glycine into the rectal epithelium 76 17. The entry of x^C-glycine into the rectal epithelium at different concentrations of glycine 77 18. Double reciprocal plots of i n i t i a l rates of glycine influx into the rectal tissue as a function of external glycine concentration 78 19. The rate of x^C-glycine uptake by rectal tissue when different concentrations of L-serine or proline are present in the test solution 80 20. Double reciprocal plot of glycine influx into rectal tissue in the presence or absence of L-serine 82 21. A 'key' (binary classification) to the principal types of mechanisms which have been proposed to account for net transfer of amino acids across epithelial membranes against electrochemical gradients 85 22. A model illustrating the possible role of an extracellular compartment in the absorption of amino acid across insect rectal wall 87 23. The 14-C-glycine ratio (H/L) and the net movement of water across the rectal wall in media of various compositions 97 24. The effect of Na+ on the x^C-glycine accumulation by rectal tissue 102 25. The effect of Na+ on the 14c-glycine influx into the rectal tissue 104 26. The effect of different concentrations of Na+ on the glycine influx 106 27. The influence of the external Na+ concentration on the i n i t i a l rate of x^C-glycine influx into the rectal tissue 108 28. The efflux of x^C-glycine from rectal sacs 110 29. Schematic diagram of the co-transport model (Na+ gradient hypothesis) 119 xi Figure page A. The movement of glycine across rectal sacs upon reversal of the Na+ gradient 134 x i i LIST OF ABBREVIATIONS H Hemocoel hr. Hour L Lumen min. Minute H/L Hemocoel/Lumen Side sec. Second T/L Tissue/Lumen Side Kg. Kilogram yl Microliter mg. Milligram ml M i l l i l i t e r W/V Weight/Volume nmoles Nanomoles W.W Wet Weight mmoles Millimoles D.W. Dry Weight M Molar Concentration A.A. Amino Acid mM Millimolar Concentration M.W. Molecular Weight P.E. Polyethylene Tubing cm. Centimeter mequiv. Milliequivalents yC Microcurie yequiv. Microequivalents S.E. Standard Error mosm Milliosmoles E.M. External Medium mV Millivolts I.M. Internal Medium mA Milliampere P.D. Potential Difference U/P Concentration ratio of solute in the Malpighian tubule secretion over that in the hemolymph Krj, Substrate concentration for half-maximal unidirectional flux Vmax Maximal rate of unidirectional flux x i i i ACKNOWLEDGMENTS The author wishes to express his sincere gratitude to the following: Dr. J. E. Phillips, my advisor, for his helpful suggestions and insight, for his encouragement in times of difficulty, and for providing educational opportunities during the course of this work. Dr. A. Perks and Dr. E. Taylor for their helpful suggestions, as well as for the amino acid analysis. Miss J. Meredith and Mr. E. Andruziak for their help and friendship. Drs. I. Cabantchik and P. A. Knauf of the Research Institute of The Hospital for Sick Children, Toronto, for their advice and friendship. Mrs. Doris Wills for her skilful typing. My wife for her patience and understanding, and to whom i t is an honor to dedicate this work. This thesis is based on work performed under an operating grant from the National Research Council. Xiv CHAPTER I GENERAL INTRODUCTION The total concentration of amino acids in the hemolymph of insects is unusually high compared to blood levels in other groups of animals. Values for insects range from 10 to 38% of the total blood solutes, being in general higher amongst the endopterygotes. The level in the hemolymph of the desert locust (105 mM) is comparable to the NaCl content. Obviously in such an insect, amino acids must play a major role in osmotic regulation or variation ( reviewed by Florkin & Jeuniaux, 1964; Schoffeniels & Gilles, 1970a). There have been numerous studies of ionic and osmotic regulation in insects, but these have rarely been extended to consider amino acids (reviewed by Stobbart and Shaw, 1964). In some instances at least, the adjustment of the osmotic pressure in the hemolymph is achieved by altering amino acid rather than ion (e.g. Cl~)levels (reviewed by Florkin & Jeuniaux, 1964; Leader and Bedford, 1972). That both inorganic ion and amino acid levels are controlled when the insect is subjected to osmotic stress is well established in the case of the dragon fly larvae and for Dytiscus  marginalis adults ( Schoffeniels, 1950, 1960). However, the mechanisms by which the amino acid concentration in the hemolymph is controlled are not known ( Schoffeniels & Gilles, 1970b). Most measurements have been made under experimental conditions which are often not completely defined. The most complete study of this type is concerned with changes in blood amino acid composition during secretion of silk by silkworms (reviewed by Chen, 1966). Modification of amino acid patterns in hemolymph at different developmental stages have been extensively studies (reviewed by Chen, 1966 and Jeuniaux, 1971), but these throw l i t t l e light on the -1--2-regulation in response to various environmental parameters. It is well established that the nature of the diet largely contributes to the comp-osition of the free amino acid in the hemolymph, both quantitatively and qualitatively (reviewed by Jeuniaux, 1971). There are at least four major processes which should influence hemolymph levels of amino acids and might be sites of regulation; 1) Absorption in the midgut. 2) Net exchange with intracellular pools (including synthesis and degradation of amino acids). • 3) Hydrolysis of hemolymph proteins. 4) Removal or retention by the excretory system. Treherne (1959) concluded that ingested amino acids are absorbed mainly in the midgut of the desert locust by simple diffusion down a concentration gradient created by absorption of water and ions. This is supported by the observation (Shyamala and Bhat, 1966) that amino acids uptake in the midgut of Bombyx Mori is not reduced by metabolic inhibit-ors. There is relatively l i t t l e evidence to suggest that regulation of hemolymph amino acid levels might be achieved by controlling the absorp-tion in the midgut. However, differential absorption of amino acids has been noted in some insects (Bhatnagar, 1962; Bragdon and Mittler, 1963). In contrast, there is a considerable amount of literature indicat-ing elaborate control of ingestion and delivery of fluid from the crop to the midgut (reviewed by Dadd,1970) in relation to the physiological state of the insect. -3-Tissue content and metabolism of amino acids in insects and other arthropods have been reviewed recently (Schoffeniel and Gilles, 1970a). In contrast with the voluminous literature on the free amino acid content of crustacean tissues, there are only a few preliminary reports on this subject concerning insects and these are restricted to nervous tissue or changes in egg and larval contents during development. While metabolism of amino acids in insects has been extensively studied, there is l i t t l e information on regulation of their synthesis and de-gradation in relation to osmotic pressure, ion concentrations or amino acid levels of the hemolymph. Ravi^Rao and Bhat (1971) suggested that glycine is actively accum-ulated by the integument of silkworm Bombyx Mori by a saturable mechanism which is Na+ dependent. Price and Bosman (1966) and Stevenson and Wyatt (1962) have reported that l^C-leucine is incorporated into the fat body of Hyalophora cecropia larvae. This incorporation is enhanced by added magnesium chloride, but diminished by increasing levels of sodium and potassium. There are more compelling reasons for implicating the excretory system in amino acid regulation. To appreciate these preliminary observat-ions, a brief review of the excretory process in insects is in order (reviewed by Maddrell, 1971; Phillips, 1970;Stobbart and Shaw, 1964). Excretion in insects occurs mainly through the activity of the Malpighian tubules and the rectum (reviewed by Maddrell, 1971; Phillips, 1970; Stobbart and Shaw, 1964). The Malphighian tubules produce a primary excretory fluid by active secretion of K+ and in some cases Na+ and -4-possibly phosphate ions into the tubule lumen (Ramsay, 1958, 1961; Berridge, 1968, 1969; Maddrell, 1969). Water flow is probably passively coupled to ion transport by local osmosis. As a consequence, electro-chemical gradients are set up which favor the passive movement of other solutes, such as ions, amino acids, sugars and urea into the lumen. The fluid produced by the tubules is isosmotic. or slightly hyposmotic to the hemolymph and the concentrations of the individual solutes are a direct function of their individual levels in the hemolymph. As Ramsay (1956) first pointed out, the activity of the Malphigian tubules would alone radically upset the hemolymph composition, were i t not for selective reabsorption of this fluid in the rectum. For example, high concentrations of useful metabolites are present in Malpighian tubule fluid. The Malpighian tubule fluid to hemolymph concentration ratios(U/P) range between 0.2 and 0.65 for amino acids and 0.3 to 0.9 for sugars in the stick insect. These ratios and the kinetics of excretion are consistent with passive movement of amino acids (Ramsay, 1958). Since most insect Malpighian tubules secrete a considerable fraction of the total body water per day (20% for the desert locust), most insects would be rapidly depleted of their metabolites, ions and water i f re-absorption did not occur in the rectum. Indeed, i t is usually difficult to detect amino acids in insect excreta unless excess amounts of these substances are artificially introduced by injection into the hemolymph (stick insect; Ramsay, 1958), or are normally ingested (honeydew of aphids and excreta of bees; Craig, 1960). Mechanisms for active reabsorp-tion of Na+, K+, Cl~ and water have been demonstrated in the locust rectum. This reabsorption is regulated in accordance with the state of -5-hydration of the insect. Under dehydrated conditions a limiting rate of ion absorption is observed ( i.e. saturation kinetics; Phillips, 1964b). Such kinetics represent an intrinsic mechanism for regulation which is generally characteristic of excretory systems throughout the animal kingdom (Pitts, 1968). By analogy, the regulation of hemolymph amino acids might be accomplished by active reabsorption in the rectum of insects. Transport of amino acid from rectal lumen back to the hemolymph would account for the low levels of amino acids in excreta compared with the relatively high concentrations in the hemolymph and Malphighian tubule fluid. A carrier-mediated process would provide saturation kinetics and hence a potential mechanism of regulation. Evidence for or against active absorption of amino acids in the insect rectum, or indeed in any insect tissue, is sparse and inconclusive. Ramsay (1958) observed a unidirectional flux of labelled amino acids across in Vitro rectal sacs of the stick insect, indicating permeability of the epithelia to these substances. Treherne (1959) did not observe substantial absorption of glycine from the hindgut of the desert locust. This is possibly because his experiments were concerned with establishing the main site of absorption of ingested amino acids and were of relatively short duration. Wall and Oschman (1970) measured the concentration of ninhydrin-positive substances in various compartments of the cockroach, Periplaneta  americana. These were lower in the rectum than in the hindgut and hemolymph. Moreover, the total concentration in fluid obtained from the subepithelial space (absorbate) of the rectal epithelium was -6-significantly higher (2x) than in the rectal lumen. They proposed that amino acids were actively reabsorbed. However, their measurements were made at only one point in time, and from one tissue compartment, so that net movement of amino, acids was not rigorously established; moreover, the contribution of solvent drag and the electropotential gradient to movement across the rectal wall absorption were not considered. Since the concentrations of individual amino acids were not determined, and since the contribution from rectal tissue (by metabolic conversion or synthesis) to the levels in the absorbate were not investigated, their conclusion as to the active nature of the process was tentative. Indeed, Berridge (1969) suggested that amino acid absorption in the cockroach rectum might be explained by entrainment in fluid flow within intra-cellular spaces (solvent drag). A demonstration of active transport of amino acids in insects is of wider evolutionary interest. Schultz and Curran (1970) predict such mechanisms in most phyla capable of Na+ transport. From the comparative point of view, the lack of data on insects represents a serious handicap. In 1971, Balshin and Phillips presented evidence for active trans-port of glycine by in vitro rectal sacs of the desert locust. Since then Nedergaard (1972) has demonstrated active absorption of a non-metabolized amino acid analogue, Y - aminobutyric acid, by the isolated midgut of Hyalophora cecropia. This is of considerable comparative interest because, unlike most other amino acid transport systems, the uptake in Cecropia is not coupled to Na+ absorption. Sridhara and Chitra (1972) have also recently reported evidence for active transport of glycine by the silk glands of Bombyx mori This uptake is only slightly sensitive to Na+ -7-and K in the bathing medium. From the comparative point of view, amino acid transport has been most extensively studied in vertebrate systems. Most published results in the literature until 1951 suggested that amino acids moved across the cell membrane by diffusion down their gradient. Gibson and Wiseman (1951), using enzymatic techniques which enabled them to measure and distinguish one amino acid isomer in the presence of the other, first showed that L-isomers of amino acids leave the lumen of the rat small intestine more rapidly than the corresponding D-forms. The same phenomenon was found in the small intestine of dogs (Clarke e_t al_., 1951) , humans (Kuroda & Gimbel, 1954) and chickens (Paine et a l . , 1959). That L-amino acids entered the blood faster than did the D-isomers was later shown by Matthews and Smyth (1954). Wiseman (1951, 1955)> employing everted sacs of rat small intestine in vitro, showed clearly that the L-isomers of amino acids were absorbed against chemical concentration gradients, while D-isomers were not. Sodium dependency for amino acid movement across animal cell membranes was first clearly recognized by Csaky (1961, 1963), using in vitro preparations of toad small intestine. Christensen and his co-workers suggested that asymmetrical distribution of either K+ or Na+ across the cell membrane has an influence on amino acid uptake by duck erythrocytes (1952c), and Ehrlich ascite tumor cells (1952 a,b). However, they favored the hypothesis of an interaction between amino acid uptake and potassium rather than sodium movement (Christensen, 1960, Oxander & Christensen, 1963, Riggs e_t a l . , 1958). These workers did not exclude the possibility that amino acid uptake might be coupled to the downhill - 8 -movement of sodium into the cell ( Riggs et a l . , 1958). However, i t was clearly demonstrated by Kromphardt e_t a l . , (1963) that glycine uptake depended on the extracellular sodium concentration. This has since been confirmed for uptake of other amino acids in various types of cells (reviewed Schultz and Curran, 1970). It has been known since the work of Cori (1926) that different amino acids may compete for the absorptive site in rat intestine. Since then many workers have reported the same phenomenon. Only those amino acids actively absorbed when present alone compete for the absorptive site. Amino acids which move across the intestine wall slowly when present alone, show a greater inhibitory effect on the absorption of other amino acids than do those which move rapidly when present alone in the lumen. It was suggested, by Wiseman (1956) that L-methionine and other slowly absorbed neutral L-amino acids formed more stable complexes with some part of the transport system and thereby exerted a stronger inhibitory effect. Chapter III of this thesis will present evidence for the accumula-tion of amino acids across in vitro rectal sacs of the locust against large electrochemical gradients and in the absence of a net flow of water. The extent of metabolic conversion of these compounds during transport will be evaluated and the effect of metabolic inhibitors will be explored. Once having established active transport in the classical sense (i.e. an energy requiring process ultimately driven by metabolism of the cell), the next section of the thesis (Chapter IV) will be concerned with the site and nature of the transport mechanism. Evidence will be presented for a carrier-mediated process controlling the entry of amino acid into - 9 -the rectal tissue from the lumen and the passive movement (downhill) of amino acid from the former compartment to the hemocoel. Finally, in Chapter V, the ion dependence of glycine transport will be investigated to determine whether the energy for transport might be provided by ionic gradients across the rectal epithelium as proposed by the Na+ gradient hypothesis. CHAPTER II MATERIALS AND METHODS A. Experimental animals Adult male locusts, Schistocerca gregaria Forksal, reared at 28 C° and 60% relative humidity on a diet of bran and lettuce, were used in a l l experiments. Animals were taken 4-7 days after the final molt and were given only tap water for 1-2 days in order to remove the faeces from the rectum before the start of experiments. B. Preparation of everted rectal sacs and incubation procedure The animals were anaesthetized with a mixture of carbon dioxide and ether under a dissecting microscope. Preparation of the everted rectal sac, as described below, followed the method of Goh(1971). The animal was secured to a plasticine block and a dorso-lateral U-shaped cut was made in the cuticle of the fifth to the seventh abdominal segments. The resulting flap of cuticle was held back and secured on the block with dissecting pins. A 3 cm. length of polyethylene tubing (P.E. 160) with the end slightly flared was inserted through the anus into the rectum until the flared end passed the anterior end of the rectal pads. The hindgut was raised and the fat body and tracheae were removed from the rectum, using fine forceps. A ligature of human hair was tied between the anterior end of the rectal pads and the end of the polyethylene tubing. The hindgut was then severed anterior to the ligature and the cannula slowly withdrawn posteriorly to evert the rectum. The cannulated sac was then cut away from the anus. The hemocoel (H) side (inside) of the everted sac was rinsed with 1 ml of Ringer (Mordue, 1969; Table I) using a -10--11-Table I Chemical composition of modified Ringers used in various experiments Mordue Na+-free K+-free CI -free Choline Compound Ringer Ringer Ringer Ringer Ringer (mM) (mM) (mM) (mM) (mM) NaCl NaN03 Choline CI KC1 KN03 MgCl2 Mg(NG3)2 CaCl0 Ca(N0^>2 NaH2P04 KH2P04 NaHCOo 168 KHCO, Glucose 6.4 3.6 2.2 6.1 2.1 16.6 174.4 3.6 2.2 6.1 2.1 16.6 174.4 3.6 2.2 6.1 2.1 16.6 168 6.4 3.6 2.2 6.1 2.1 16.6 168 6.4 3.6 2.2 6.1 2.1 16.6 Mordue, 1969 -12-tuberculin syringe inserted into the cannula. All excess fluid was removed using the syringe and the posterior end of the rectum closed with a second ligature. The entire procedure was completed within about 8 minutes. A 10 Pi aliquot of the desired experimental medium (Table I) was inserted into the sac with a "Hamilton" syringe and after mixing, a 2 ul aliquot of this internal fluid was removed within 1 minute for an i n i t i a l analysis. The upper end of the polyethylene cannula was blocked with a wax plug to prevent evaporation and a copper-wire hook was attached to the tubing to facilitate weighing. The everted sacs were then placed in perspex test tubes containing 2 ml of external media. In most experiments, the out-side bathing medium was identical to the internal solution except that sucrose was often added externally to minimize fluid movement. Care was taken to match the height of internal fluid with the external meniscus to avoid any hydrostatic pressure difference. The perspex test tubes were placed in a constant temperature bath maintained at 30.0 - 0.1°C. Previous experiments (Goh, 1971) indicated that vigorous oxygen-ation of the outside solution was required to sustain transport activity. There was no difference in the viability of the preparation when both the inside and outside solutions were oxygenated; consequently, a mixture of 95% oxygen and 5% carbon dioxide, delivered through polyethylene tubing (P.E. 10), was bubbled through the external medium (Fig. 1.). The effect of different ions on the amino acid transport system was studied by replacing Na+, K"1" and CI with isosmotic amounts of K+ or choline, Na+, and NOo respectively. The composition of these Ringer The experimental set-up for measuring absorption by jLn vitro rectal sacs (Schematic) (details are given in the text) COPPER HO_OK_ n WAX PLUG PERSPEX TEST TUBE CONSTANT TEMPERATURE BATH (30°C) P E. CANNULA RINGER + xmM 1 4 C-A.A. 1 4 / 420mosm' SUCROSE (2m l ) r B U B B L E 9 5 % 0 2 5 % C 0 2 EVERTED RECTAL SAC -14-solutions is given in Table I. Since the pH of the hemolymph in vivo is about 7.1 and that of rectal fluid approximately 1.0 - 2.5 pH units lower (Phillips, 1961), the Ringer was adjusted to an intermediate pH of 6.4 with KOH in sodium-free and choline Ringers, and with the NH^ OH in Chloride -free Ringer. The pH measurements were made using a 'Radiometer Model 25' pH meter. After 6 hours of oxygenation, the pH of the Ringer increased by less than 0.3 pH units. C. Experiments with radioactive tracers Net movement of amino acids across the rectal wall was measured by placing the same Ringer containing x^C-labelled amino acids (New England Nuclear; Boston) at constant specific activity (40 uC/mmole) on both sides of the preparation. If not otherwise stated, the same specific activity was used in various experiments. Unlabelled amino acids were usually obtained from Sigma Chemical Co. (St. Louis); however, ultra-pure grade reagents which were used in various ion-free experiments were obtained from British Drug House (England). The preparations were usually pre-incubated for 1 hour to permit exchange of x^C-labelled amino acids with the tissue pool of these substances. Media were subsequently removed and replaced with fresh labelled media of the same composition as the former. After different periods of incubation, 2 ul aliquots of internal and external media were withdrawn using 'Drummond Microcap' pipets and placed in 10 ml of Bray's (1960) liquid scintillation fluid. These samples were counted using a 'Nuclear Chicago Mark I* liquid scintillation counter and the channels ratio method of quench correction. In some later experi-ments the same procedure was followed, but the labelled compound was initially present in the external medium only. In these experiments, the - 15 -radioisotope was absent during the pre-incubation period. D. Measurements of net water movement and ion concentrations Net water movement was measured during a l l experiments by weighing the rectal sacs on a Torsion balance ( reproducibility ± 0.25 mg.) after fluid had been removed with filter paper (Whatman No. 1). Rectal sacs were weighted immediately after the i n i t i a l , and before the final samples of media were taken (1 minute and 1 to 6 hour samples, respectively). In most experiments net water movement was prevented or minimized by adding sucrose (420 mosm/1) to the external media. (The required amount of sucrose was determined empirically in preliminary experiments). This excluded the possibility of amino acid movement across the rectal wall due to solvent drag. Sucrose was used to adjust the osmotic gradient since the rectal cuticle is not permeable to this molecule (Phillips, 1964a; Phillips & Dockrill, 1968; Wall, 1967). Thus, a net flux of sucrose, which might cause net movement of amino acids by solute-solute drag (Franz, et a l . , 1968), was avoided. Sodium concentrations of media were determined with a 'Techtron AA 120 flame spectrophotometer'. One yl samples of media were diluted in 10 ml of distilled water in plastic vials prior to analysis. E. Measurements of electro-potential difference across the rectal wall ; The electro-potential difference across the rectal wall was measured with a 'Keithly' electrometer (Model 602) or a 'Radiometer Model 25' pH meter. Salt-bridges (3% agar-Ringer in P.E. 10 tubing) connected the inside and outside compartments (containing Ringer) to two beakers, each containing a saturated KC1 solution and a calomel -16-electrode. The calomel electrodes were connected to the electrometer to complete the circuit. The assymetry potential was determined by placing the tips of both salt-bridges in a common dish of Ringer. F. Amino acid analysis The method used was that of Mitchell and Simmons (1962). Isolated recta (individual or pooled) were weighed and homogenized in 95% methanol (1:40 W/V). The homogenate was centrifuged, the supernatant collected and the precipitate washed with 95% and 50% methanol. These operations extract the free amino acids and small peptides. The whole procedure was carried out at -20°C to reduce possible changes caused by enzyme reactions during extraction. The extract was then concentrated in a vacuum flask rotary evaporator at 30°C. The resulting sample was further purified by shaking with chloroform and decanting the upper fraction. Finally i t was diluted in sodium citrate buffer (pH2) to a known volume. Amino acid composition was determined using an amino acid analyser (Bio-cal 200 or Beckman 120C). Aliquots (10 ul to 2 ml ) of internal and external media were treated in a similar manner. The known concentration of the test amino acid in the external medium served as a control for recovery during these procedures. In one case a fraction collector was connected to the amino acid analyser column, and the collected fractions were counted in the liquid scintillation counter. G. Metabolism of amino acids by rectal tissue To determine if amino acids were metabolized during transport, the characteristics of the transported species were checked by ascending two-dimensional thin-layer chromatography and high-voltage paper -17-electrophoresis. In a typical experiment, everted rectal sacs were incubated with Mordue Ringer as the internal solution and Mordue Ringer containing 10 mM of "*"4C- label led amino acid and 420 mosm/1 of sucrose as the external solution. After 6 hours incubation, 2 $1 aliquots were taken from the inside solution and placed at the origin of the plate. Twelve unlabelled amino acids, as well as l^rj-iabelled a m i n o acid from the external solution (before incubation started),were used as standards for the chromatograms and electrophoretograms. The amino acids chosen as standards were those known to constitute the free pool of the rectal tissue on the basis of previous results observed with an amino acid analyser. 1. Thin layer chromatography The stationary phase was silica gel G on glass plates. Ethanol: water (7:3) was used as the first irrigation solvent and n-propanol: water (7:3) as the second solvent (Brenner, et a l . , 1965). Two Ml aliquots of bathing media were placed on the origin and the plates were run at a temperature of 23°C for 2.5 hours in solvent A and 2.1 hours in solvent B. Amino acids were identified by spraying the plates with cadmium ninhydrin reagent (Heilmann, et a l . , 1957). To localize the radioactivity on the chromatogram, 'Kodak' film sheets (No. N-5-54T) were pressed against the chromatography plates and exposed for 7 days at room temperature and then developed according to conventional film developing methods. 2. High-voltage paper electrophoresis Following a 6 hour incubation period, 10 ul aliquots were with-drawn from inner compartment of the rectal sacs (initially no amino -18-acid or radioactive material was present in the sacs). The inside samples were mixed with 12 unlabelled amino acids naturally occurring in the rectal tissue. Samples (100 ul) of this mixture were spotted, one in each corner of the paper strip. Equal volumes of pure glycine standard solutions and the external Ringer containing X 4 C-glycine were spotted in between the mixture spots along the origin. The paper strips (Whatman No. 3) were subjected to high-voltage paper electrophoresis in an apparatus similar to that .described by Michl (1951). The pH 1.8 buffer system (formic acid: acetic acid: water, 1:4:45 by volume) was that of Amber (1963). Three thousand volts at 100 mA were applied across the paper for 30 minutes. After the paper strips were dry, they were sprayed with ninhydrin solution to identify amino acids. The electrophoretograms were then subjected to radio-autography as previously described. 3. Incorporation of into COQ Isolated recta were incubated in 2 ml of Ringer containing 10 mM of X ^C-labelled glycine in sealed Warburg flasks. After shaking the flasks for 6 hours, a CO2 absorbant (0.3 ml of hyamine hydroxide) was injected into the central well and 0.5 ml of a 60% perchloric acid solution was injected through a rubber stopper into the outside space to drive the soluble CO2 from the Ringer. After 30 minutes the hyamine hydroxide was transferred to scintillation vials containing 10 ml of Bray's solution for determination of X 4 C-activity as previously described. Other flasks without rectal sacs were treated in an identical manner and served as controls. -19-H. The accumulation of C-labelled amino acids In the rectal tissue The everted rectal sacs were pre-incubated for 1 hour in Mordue Ringer solution lacking amino acids to permit stabilization of the intra-cellular ion concentrations. Any net movement of water across the rectal wall was abolished by the end of this hour (i.e. 420 mosm/1 sucrose gradient was present).. After pre-incubation the rectal sacs were placed 14 in Ringer containing various concentrations of a C-labelled amino acid at constant specific activity. At various times after the addition of amino acids to the external solution,rectal sacs were removed from the incubation medium and their external (apical) surface rinsed with ice-cold isosraotic mannitol solution for 0.5 minutes to remove surface activity. The sacs were then gently blotted on Whatman No. 1 fi l t e r paper. The ends beyond the two ligatures were cut away, and the remaining central part of each rectal sac was cut into two portions of approximately equal size, each of which was blotted and weighed on a 'precision' Torsion balance. The entire weighing procedure was completed within 1 minute and the water loss due to evapor-ation was shown to be negligible. One portion of the tissue was dried at 80°C for 48 hours to determine the dry weight and, hence by subtraction, the tissue content of water. This portion was then put in a platinum boat and placed in an oven at 460°C for 48 hours. The ash was placed in plastic vials containing 10 ml of distilled water for sodium determination by flame photometry. The second portion of rectal tissue was extracted for 24 hours in 0.1 N nitric acid (1:40 W/V). Aliquots (25 or 100 yl) of this extract were transferred to vials containing 10 ml Sray's solution for determination of radioactivity as previously described. This is - 2 0 -similar to the method employed by Schultz, et a l . , (1967) to measure the accumulation of -^C-labelled amino acids in rabbit ileum. Influx of radiotracers from the external media into rectal tissue was estimated from the i n i t i a l linear rate of accumulation of -^C-activity in rectal tissue, before measurable amounts of radioactivity appeared on the hemocoel side (see Results). The specific activity of ^C-glycine in these experiments was 100 yC/mmole. I. The effect of respiratory inhibitors on amino acid transport To test the effect of KCN on glycine and water transfer by everted rectal sacs, in vitro preparations were prepared as described in Sections B-D. Since KCN increases the pH of the Ringer, a 5 mM solution of PIPES buffer [piperazine-N, N-bis (2-ethane sulfonic acid) monosodium monohydrate] was used to adjust the Ringer to pH 6.6. The preparations were preincubated in Mordue Ringer containing 10 M KCN for 1 hour to allow time for the inhibitor to reach its site of action. No sucrose was present externally to create an osmotic gradient. After pre-incubation the preparations were transferred to fresh Mordue Ringer 14 -3 containing 10 mM of C-glycine and 10 M KCN. The composition of the bathing medium was initially the same on both sides of the rectal wall. The radioactivity of the internal media and rectal tissue were determined as previously described in Sections C and H. 14 J. Efflux of C-labelled amino acids: The l^c-giycj-ne efflux from rectal tissue was determined directly 14 by loading the cell with C-labelled glycine during a pre-incubation period (see Section C) and following the appearance of radioactivity in -21-the external solution after transferring to an unlabelled media of the same chemical composition. The preparations wereipre-incubated in Mordue Ringer containing -^C-glycine (10 mM) for 1 hour. Four preparations 14 were extracted to determine the C-glycine level in the c e l l . Another group of preparations were rinsed externally and transferred to test tubes containing 1 ml solution of unlabelled glycine (10 mM or 50 mM) media of identical composition and 25 ul aliquots of external media were collected at 2, 4, 8 and 20 minute intervals for radioactivity determinations. CHAPTER III TRANSPORT OF AMINO ACIDS AGAINST  CONCENTRATION GRADIENTS A. Introduction In this chapter, I consider whether amino acids are transported across the rectal wall in vitro against electrochemical gradients and in the absence of external driving forces (e.g. solvent drag). Metabolic conversion of amino acids during transport across the rectal epithelium and the effects of respiratory inhibition by KCN on the net transport process are considered. B. Viability of ±a_ vitro preparations Goh (1971) reported that everted rectal sacs of the desert locust prepared in the same manner as in this study actively transported sodium, potassium and chloride ions at rates comparable to those in vivo (Phillips, 1964b). The rates of transport and electro-potential difference across the rectal wall decreased only slightly between the first and sixth hour after setting up the everted sacs. Likewise, water was transported at a constant rate of 7 yl/hr./rectum in the absence of an osmotic gradient*, and there was no movement of water for several hours when the lumen was made hyperosmotic to the hemocoel side by 420 mosm/1. The absorbate was hyposmotic to the bathing media. These experiments demonstrated that the in vitro preparation retained the transport activities characteristic of the intact rectum (in vivo) and remained a viable, steady-state system for several hours. -22--23-The in vitro preparations used in the present study behaved as reported by Goh (1971). Water is absorbed at a constant rate of 7.2 ± 0.6 yl/hr./rectum (mean ± S.E.) during the first six hours in the absence of a concentration gradient, but no movement of water occurs over the same period of time when an osmotic gradient of 420 mOsm/1 is applied across the rectal wall (Fig. 2, Table H). In the absence of net water movement, the transepithelial potential difference (P.D.) of 28 preparations was initially 89 ± 1.6 mV, hemocoel ^  side negative (Fig. 3). This P.D. f e l l to a constant value of 50-60 mV after the first hour. This is further evidence that ion transport processes and membrane permeabilities change relatively l i t t l e during the experimental period ( 1 - 6 hours). These potentials are of the same sign, but larger than those observed in vivo (i.e. 15-32 mV; Phillips, 1964b) under different experimental conditions, and in vitro by Vietingholf e£ al.,(1969). The P.D. observed by the latter authors f e l l to 0 by the second hour. C. The accumulation of ^ C-glycine against an electrochemical gradient A widely accepted method of demonstrating an active transport process is to place identical concentrations of the molecule in question on both sides of the membrane. If during the experiment the concentration of the substance increases in one compartment, and the possibility of an external driving force, (e.g. solvent or solute drag) is eliminated, i t is safe to assume that a primary or secondary active transport has taken place. Such experiments were carried out using four different glycine concentrations in the bathing medium (Fig. 2). In a l l cases the R e -activity inside the sacs increased with tims so that a 10-fold concentration Fig. 2. The C-glycine ratio (H/L) and the net movement of water across rectal sacs with time. The H and L bathing media of in vitro preparations contained initially either 1 ( O ) , 5 ( • ) , 10 ( • ), or 50 ( A ) mM 14C-glycine (same specific activity). Preparations were pre-incubated for 1 hour in the experimental media. Sucrose (420 mosm/1 ) was present in the external medium to prevent net water movement. Vertical lines represent ± S.E. of the mean of at least 4 preparations. -24-VOLUME CHANGE ( u l / r e c t u m ) C-GLYCINE RATIO (H/Lside) I 4-Fig. 3. The potential difference (H side negative) everted rectal sacs with time across Both compartments ( H & L) contained Mordue Ringer. Sucrose (420 mosm/1) was present in the external solution to prevent net water movement. Vertical lines represent ± S.E. of the mean of 4 preparations. -25-- 2 6 -gradient was reached after 6 hours across sacs incubated in Ringer 14 initially containing 10 mM of glycine. The C-activity ratios developed at other glycine concentrations were lower. The pH of both the external and internal media remained between 6.4 and 6.7 over the experimental period. Over this pH range, 99% of glycine molecules possess no net charge and the remaining 1% possesses a net negative charge (Edsall,1965). Therefore, the observed accumulation of "^C-glycine must occur against an electrical gradient" During these experiments, a sucrose gradient prevented significant water absorption throughout the 6 hour experimental period (Fig. 2.). These results indicate that net transport of ^C-glycine from the L to the H side occurs against an electrochemical gradient, and in the absence of net absorption of water. According to the criteria of Ussing (1954) the absorption of glycine i s , therefore, an active process. 14 If either the maximum or the average rate (Table II) of C-glycine accumulation in the sacs is used to calculate net rates of glycine movement, the uptake exhibits saturation type kinetics as expected for an active transport mechanism. This is more clearly demonstrated by the influx of "^C-glycine into the rectal tissue (see Chapter IV, Section F). Addition of 10 M KCN to the bathing media on both sides of the sac decreased •^C-glycine and water absorption by 80% after pre-incubation for 1 hour (Table II). Higher concentrations of KCN (10~2'M) completely inhibits absorption (Balshin & Phillips, 1971). This observation supports the 14 view that absorption of C-glycine by the rectal sacs involves an energy requiring process. Fig. 4. The C-glycine ratio (H/L) and the net rate of water movement across the rectal wall in the absence of an osmotic gradient. The H and L bathing media of in vitro preparations contained initially 10 (^^) mM. The incubation was performed under isosmotic conditions (no sucrose was.present in the external medium). Net rate of water movement ( • ) was determined every 20 minutes. Vertical lines represent ± S.E. of the mean of 4 preparations. - 27 --28-Table II Net rates of glycine and water absorption by in vitro recta in media of various composition Composition of Media Net Rates of Absorption C-glycine Ratio (6 hr.) Glycine (mM) External sucrose (mosm/1) Glycine nmoles/hr./rectum Water ul/hr./rectum H/L 1 420 9.0 ± 2.0 -1.0 ± 0.5 4.1 ± 0.3 5 420 50.5 ± 6.7 -0.3 ± 0.2 7.5 ± 1.2 10 420 106.0 ±11.0 0.4 ± 0.9 10.0 ± 0.5. 50 420 202.0 ±31.0 -1.0 ± 1.1 3.8 ± 0.1 10 0 171.0 ±14.0 7.2 ± 0.6 1.6 ± 0.2 10 with M KCN 10"3 0 9.7 ± 3.8 0.5 ± 0.3 1.1 ± 0.04 10 control* 420 65.5 ± 5.0 0.3 ± 0.1 2.3 ± 0.1*** Taken from Table VI, i.e. incubated without bubbling 02 through media. This probably accounts for the lower rate of transport by the controls as compared to the rates at comparable glycine concentrations, using the standard method. ** Values were taken from Fig. 2 (mean ± S.E.), using the increase in internal activity; i.e. average rate over 6 hours. 'Ratio determined after 2 hours (Fig. 13). -29-In the absence of an osmotic gradient (i.e. no sucrose on the apical side) fluid absorption averaged 7.2 ul/hr./rectum (Fig. 4). This rate is 18 times greater than the average rate observed when a 420 mosm/1 sucrose gradient is present across the rectal wall (Table II). Under isosmotic conditions the ^C-glycine activity ratio reached a maximum of only 1.6 due to dilution by absorbed water and the rate of glycine transfer across the rectal wall was increased to 171 nmoles/hr./rectum (Table II) . Thus the rate of ^C-glycine accumulation increases by only 70%,while the rate of water absorption increases at least 18-fold. These results indicate that glycine movement might be increased due to solvent drag, but this is not the only or principal driving force for the glycine transport. D. Tissue content and metabolism of glycine The conclusions in Section C regarding glycine transfer were based on the assumption that the specific activity of 14rj_giyCin e w a s n ot significantly changed during the above experiments and that most of the 14 C-activity remained in the form of glycine. These assumptions are considered in this section. 1. Tissue content The tissue pool of free amino acids might contribute to changes in the specific activity of ^C-glycine even though pre-incubation probably eliminates this possibility (Chapter IV). Table III shows the free amino acid content of rectal tissue immediately after the extirpation from the locust, :.as determined by an amino acid analyser. The concentration of free glycine naturally occuring in rectal tissue -30-Table III Concentration of free amino acids and urea in the rectal  tissue using an amino acid analyser Amino Acid nmoles/mg. Concentration (mM) T i s s u e * ** Tissue water Hemolymph Alanine 3.82 4.5 3.7 Aspartic Acid 0.57 0.7 -Glutamic Acid 1.90 2.3 5.1 Glycine 10.65 13.0 33.0 Histidine 0.64 0.8 1.0 Isoleucine 0.25 0.3 -Leucine 0.38 0.5 2.6 Lysine 0.57 0.7 -Ornithine 0.26 0.3 -Proline 65.70 79.0 4.0 Serine 3.04 3.6 35.0 Threonine 0.63 0.8 2.3 Tyrosine - - 2.5 Valine 1.31 1.6 5.0 Amides asparagine) glutamine ) 11.80 14.0 11.0 Pho spho ethanolamine 1.03 1.0 -Phosphoserine 1.03 1.0 -Urea 0.75 0.9 — Total Organic 125.0 105.2 The relative concentrations of amino acids in rectal tissues were .confirmed by two additional amino acid analyser runs on pooled samples, but absolute concentrations were not obtained from these preliminary runs. ** Treherne, 1959 -31-is 13 mmoles/kg.tissue water. The i n i t i a l content of free glycine in a typical 10 mg.: rectum was, therefore, less than 1% of the external glycine pool and approximately 10-20% of the total glycine transported (at an external concentration of .10mM;See Table II) over a 6 hour period, 14 taking into account the i n i t i a l specific activity of C-glycine in the 14 media. Dilution of transported C-glycine with unlabelled tissue glycine would lead to an under-estimation of the true rate by 10-20% at the very most. Later observations (Chapter IV) on tissue specific 14 activities of C-glycine after 1 hour pre-incubation indicate no difference in specific activity compared to bathing media. The total concentration of the 14 free amino acids observed in rectal tissue (Table III) was 125 mM/kg. tissue water, which is only slightly greater than that of the locust hemolymph (reported by Treherne, 1959). Proline is responsible for 60-70% of the total tissue content, while glycine and amines (asparagine and/or glutamine) are the next most important. The concentration of urea is very low. 2* Metabolism Solutions from the inside and the outside compartments of the rectal sacs were removed after 6 hours of incubation in Mordue Ringer initially containing 10 mM -^C-glycine on the outside only. By this 14 time C-activity was approximately 10 times greater on the H than on the L side. The possibility that the x^C-radioactivity accumulated in rectal sacs was the result of metabolic conversion of glycine to other molecules, was tested by three methods: i) thin layer chromatography, ii ) high voltage paper electrophoresis, and i i i ) incorporation of ±4C into C O 2 . -32-i) Thin layer chromatography Selected chromatographs and autoradiographs of external and internal media, either alone or mixed with unlabelled amino acids standards, are presented in Fig. 5-9. The values are summarized in Table IV. Good separation of amino acid standards was obtained in a l l cases. Only one amino acid was detectable in both the external and internal media, and this had R^  values corresponding to those of glycine. In a l l cases only one radioactive spot was detected in the internal media by autoradiography of these chromatographs. This corresponded with the position of glycine. These results indicated 14 that most, if not a l l , of the C-species transported across the rectal wall to the H side remained in the form of glycine. The intensity of the black spots on autoradiographs was greater for the internal than the external media. This provides an independent semi-quantitative demonstration of accumulation of glycine against a chemical gradient. i i ) High voltage paper electrophoresis A high voltage paper electrophoretic separation of 12 unlabelled amino acid standards mixed with internal media (after 6 hour incubation) is shown in Fig.lOj (column 1). External medium and a glycine standard were run simultaneously on the same paper strip (columns 2 and 3 respect-ively) . Glycine is clearly separated from other amino acids. An autogadiograph (Fig. 10-Q) °f t n e electrophoretograph indicates only one spot of radioactivity in the external and internal media correspond-ing to the position of glycine. These results confirm the chromatographic results. Fig. 5. Two-dimensional thin layer chromatography-of samples withdrawn from the inside medium  of a rectal sac incubated with -*-4C-glycine. At the end of 6 hours incubation in a media initially containing 14C-glycine (10 mM) in the external side only, 2 yl samples from the inside were co-chromatographed with 5 amino acids as standards (ascending technique). The inside Ringer was placed at the origin (bottom left corner). From right to l e f t , proline, serine, threonine, aspartic acid, glycine and external medium were put in order and from top down. Fig. 5j» Autoradiography of the above thin layer chromatogram. ' -33-Fig. 6. Two-dimensional thin layer chromatography of samples withdrawn from the inside medium of the rectal sac, incubated with -^C-glycine. At the end of 6 hours incubation in a medium initially containing x^C-glycine (10 mM) in the external side only, 2 ul samples from the inside were co-chromatographed with 5 amino acids as standards (ascending technique); The inside Ringer was placed at the origin (bottom left corner). From right to le f t , methionine, valine, alanine, glutamic acid, glycine and external medium were placed in order and from top down. Fig. 6-j-. Autoradiography of the above thin layer  chromatogram. -34-Fig. 7. Two-dimentional thin layer chromatography of samples withdrawn from the inside of the rectal sac after incubation. At the end of 6 hours incubation in a media initially containing l^C-glycine (10 mM) in the external side only, 2 ul samples from the inside were co-chromatographed with 5 amino acids as standards (ascending technique). Inside Ringer was placed at the origin (bottom left corner). From right to left, arginine, tyro-sine, phenyl alanine, leucine, glycine and external medium were placed in order and from top down. Fig. 7j_. Autoradiograph of the above thin layer chromatogram. - 3 5 -Fig. 8. Two-dimensional thin layer chromatogram of a media withdrawn from the inside of the rectal sac after incubation. At the end of 6 hours incubation in a media initially containing x^C-glycine ( 1 0 mM) in the external side only, inside Ringer was placed at the origin, mixed with the standards (bottom left corner). From right to l e f t , proline, lysine, isoleucine, histidine, glycine and external medium were placed in order and from top down. Fig. 8 . Autoradiograph of the above thin layer  chromatogram. -3.6-Fig. 9. Two-dimensional thin layer chromatogram of a media withdrawn from the inside of the rectal sac after incubation. At the end of 6 hours incubation in a media initially containing 14C-glycine (10 mM) in the external side only, inside Ringer was placed at the origin, mixed with the standard (bottom left corner). From right to le f t , phenyl alanine, tyrosine,threonine, valine, leucine, glycine, and external medium were placed in order and from top down. Fig. 9-r' Autoradiograph of the above thin layer chromatogram -37-- 3 8 -Table IV R^  values for unlabelled amino acid standards, ninhydrin  positive spots in external (E.M.) and internal (I.M.)  media after 6 hr. incubation, and radioactive spots  identified by autoradiography. Amino acids in brackets indicate identification of unknowns. Amino acid Solvent A Solvent B Rf X10"2 R f xio-2 1. (Fig. 5) Migration: solvent A, 11 cm., solvent B, 9.8 cm. Standards Proline 34 19 Serine 46 28 Threonine 47 29 Aspartic acid 31 14 Glycine 41 24 I. M. (Glycine) 42 24 E. M. (Glycine) 41 24 Radioactive spots I. M. 41 24 E. M. 42 24 -39-Amino acid Solvent A Solvent B Rf X10-2 Rf X10"2 2. (Fig. 6) Migration: solvent A, 11.4 cm., solvent B, 8.3 cm; Standards Methionine 65 48 Valine 61 42 Alanine 54 34 Glutamic acid 53 24 Glycine 50 33 I.M. (Glycine) 51 33 E.M. (Glycine) 50 33 Radioactive spots I.M. 50 33 E.M. 49 33 3. (Fig. 7) Migration: solvent A, 10.6 cm., solvent B, 8.6 cm. Standards Arginine 7 3 Isoleucine 57 42 Phenyl alanine 61 .46 Leucine 60 45 Glycine 49 28 I.M. (Glycine) 49 28 E.M. (Glycine) 49 28 Radioactive spots I.M. 48 28 E.M. 49 28 -40-Amino acid Solvent A Rf X10"2 Solvent B Rf X10"2 4. (Fig. 8) Migration: solvent A, 14.2 cm., solvent B, 14.0 cm. Standards E.M. I.M. + standards spot no. 1 2 3 4 5 Radioactive spot Proline Lysine Isoleucine Histidine Glycine (Glycine) (Lysine) (Histidine) (Proline) (Glycine) (Isoleucine) 36 2 59 37 50 50 2 38 40 51 60 49 21 2 42 20 25 25 2 20 21 23 41 23 5. (Fig. 9) Standards Migration: solvent A, 12.4 cm., solvent B , . l l . Phenylalanine 57 48 Tyrosine 60 49 Threonine 48 34 Valine 53 39 Leucine 61 48 6 cm. -41-Amino acid Solvent A Solvent B, % xicr2 Rf xio-? Glycine 33 28 E.M. (Glycine) 32 28 I.M. + standards spot no. 1 (Phenylalanine 53 51 2 (Valine) 47 40 3 (Threonine) 45 33 4 {Glycine) 26 28 Radioactive spot 27 28 Fig. 10 . High voltage paper electrophoresis of samples from the Internal medium ot sacs exposed  externally to ^4C-glycine. At the end of 6 hours incubation in a media initially containing 14C-glycine (10 mM) in the external side only, samples from the inside were mixed with 12 unlabelled amino acid standards (naturally occurring in rectal tissue). One sample of this mixture was placed at each corner (column 1). The same volume (100 ul) of unlabelled glycine standard was placed in the center (column 3) and samples of external media (column 2) were placed on the origin between the mixture and the glycine standard. The small dots in column 3 are from the radioactive marker ink used for comparing I and II. Fig. 10-j.j- Autoradiograph of the above electrophoretogram -4-2-% * « • I t C\J CO CM C\J CO CM o - -fx*.--43-i i i ) Incorporation of ^ C-activity into CO^ 14 14 The amount of C-glycine converted to CO^ during the 6 hour incubation period in 10 mM of glycine (specific activity identical to that in previous experiments, Section C) was 13.5 ± 3.8 . nmoles/ 6 hr./rectum (mean ± S.E. of 4 experiments). This amount of l^C-activity was not significantly different from that collected in control flasks lacking rectal sacs (10.3 ± 2.4 nmoles/6 hr./ rectum). The minute amount of ^ C-activity in control flasks might be due to bacterial degradation, impurities in the radioactive glycine obtained commercially, or splashing during.incubation. Even if a l l of the activity in the experimental flasks represents ^CG^ produced by the rectal sacs, this amount is only 2% of the total quantity of glycine transported across a rectal sac during the same period (Table II). These observations indicate that the glycine which moves across the rectal wall is not an important energy 14 source for respiration in this tissue. Moreover, no C was found in the TCA precipitate of a tissue-homogenate from preparations 14 which were incubated for 1 hour in a medium containing 10 mM C-14 glycine. This excludes incorporation of C-glycine into proteins. In summary, there is no evidence for considerable metabolic conversion of ^C-glycine during transfer across the rectal wall in vitro (Balshin and Phillips, 1971). Murdock and Koidl (1972) reached the same conclusion for transfer of glycine across the midgut of Locusta  migratoria. - 4 4 -E. Transport of other amino acids Chromatographic analysis of the locust's hemolymph (Schistocerca gregaria), performed by Treherne (1959), revealed the presence of 10 amino acids ( Table III). Of these, glycine and serine are present in relatively high concentrations (32.2 and 34.6 mM respectively) compared to the other eight (range 1-11 mM). Are a l l of these amino acids actively transported by everted rectal sacs, and i f so, is there any relationship between their rates of accumulation and their natural concentrations in the hemolymph; that i s , might relative rates of rectal re-absorption be in some measure responsible for the relative concentrations of amino acids in the hemolymph? With these questions in mind, the accumulation of 8 14 C-labelled amino acids was investigated in the same manner as previously described for glycine (Section C). In a l l experiments Mordue Ringer 14 solution containing 10 mM of C-labelled amino acid was placed on both sides of a rectal sac and water movement prevented by the presence of 420 mosm/1 sucrose in the external solution. Following pre-incubation, 14 the H/L ratio of C-activity was determined after 2 and 6 hours (Fig. 11). Rates of transport were estimated from the net increase in activity at these intervals of time. Results are presented in Table V. All four of the neutral L-amino acids normally present in the locust hemolymph (glycine, serine, alanine, threonine) and L-proline were accumulated against substantial concentration gradients (Fig. 11). On the other hand, the acidic (L-glutamic acid),basic (L-histidine) and aromatic (L-tyrosine) amino acids naturally occurring in the hemolymph were not accumulated in measurable amounts. The amino acid analogue, y-aminobutyric acid was not transported. With the exception of histidine, Fig. 11. The i^C-amino acid ratios (H/L) and the net movement of water across the rectal sacs wi't;h time of incubation. Both bathing media (H &L) of in vitro preparations initially contained 10 mM of the test amino acid: L-alanine ( A ) , y-aminobutyric acid (•), L-serine (x ), L-proline ( O ) , L-threonine ( • ), glycine ( 0 ) , L-glutamic acid ( A ) , L-histidine (•), L-tyrosine ( + ), (constant specific activity). Sucrose (420 mosm/1) was present in the external medium. Vertical lines represent S.E. of the mean of 3 or 4 preparations. No lines were drawn when the S.E. values were comparable to the size of the symbols. -45-VOLUME C H A N G E pi /rectum) [ U C - A m i n o Acid] RATIO ( H / L ) — o — O CO O T~1 I o x o 1 -46-Table V Estimated net rates of amino acid transfer and water  absorption across the wall"of in vitro recta Rectal sacs were initially bathed on both sides with 10 mM of a single C-labelled L-amino acid. The values were calculated from data presented in Fig. 11.(means ± S.E. ) Test amino acid Incubation period (hr.) Net Rates Amino acid Water nmoles/hr./rectum u 1/hr.'/rectum Serine Alanine Threonine Y-aminobutyric acid Proline Glutamic acid Histidine 2 6 2 6 2 6 2 6 2 6 2 6 2 6 77.00 ± 1.70 44.00 ± 5.70 62.05 ±19.-20 37.77 ±10.90 11.30 ± 0.60 21.40 ±-2.8 0.90 ± 0.06 1.10i± 0.05 79.70 ± 7.08 93.40 ±22.30 0.55 ± 0.08 1.14 ± 0.15 1.05 ±. 0.05 1.00 ± 0.00 0.66 ± 0.41 0.64 ± 0.19 -0.37 ± 0.52 0.30 ± 0.09 0.40 ± 0.50 0.83 ± 0.16 0.31 ± 0.48 0.12 ± 0.70 0.66 ± 0.41 0.27 ± 0.19 0.06 ± 0.35 -0.45 ± 0.48 0.44 ± 0.35 -0.47 ± 0.07 Tyrosine 2 6 0.64 ± 0.12 0.98 ± 0.17 -0.42 ± 0.60 -0.49 ± 0.34 -47-a l l these amino acids have isoelectric points close to or below that of test solution pH (6.4). These amino acids would be in a neutral or negatively charged form. Therefore, the electrical gradient existing across the rectal wall could not contribute positively to the accumulation of neutral amino acids in the rectal sacs. The iso-electric point of histidine is 7.58. Under the experimental conditions, most of the histidine molecules would be positively charged. Although the electrical gradient across the rectal wall should favor movement of positively charged molecules from the L to the H side, the Rc-activity ratio did not exceed unity during the incubation with L-histidine for 6 hours. Amongst the neutral amino acids studied, there is some correlation between the rate of transport under standard conditions and the natural level of amino acid in the hemolymph (see Chapter v i ) . A better correlation can be found between the rates of accumulation on the H side and concentration in the rectal tissue. Proline is the predominant amino acid in the rectal tissue and then glycine, alanine and serine, in decreas-ing order of concentration (Table III). All 4 of these amino acids have relatively high rates of accumulation in the rectal sacs. A l l the amino acids, in the present work, which were accumulated against concentration gradient, are monoaminocarboxylic acids. All but proline are aliphatic. There is a negative relationship between the length of the side chain of the carbon skeleton and the rates of accumulation. Addition of an hydroxyl group enhances absorption rate at a given side chain length (e.g. serine vs. alanine). Proline, on the other hand, has a pyrolidine ring. Koser and Christensen (1971) found that proline was transported extremely rapidly by a neutral amino acid pump; they attribute this to the ring Fig. 1 2 H i g h voltage paper electrophoresis separation  of internal media bathing rectal sacs exposed to different x^C-amino acids. At the end of 6 hours incubation in a media initially containing a single C-amino acid (10 mM) in the external side only, one sample of internal media was placed at the origin on the right side of the paper strip, and a sample from the external media on the left side. Each paper strip contained a different amino acid. 1. Serine, 2. proline, 3. threonine, 4. alanine, 5. glutamic acid, 6 hisitidine, 7. tyrosine, 8. y-aminobutyric acid. Fig. 12JT. Autoradiograph of the above elctrophoretograms - 48 --49-structure. 14 The possibility that the C-activity in rectal sacs was the result of metabolic conversion of the present test amino acid to other form was tested by high voltage paper electrophoresis. The procedure was basically the same as in Section D, except that the internal solutions were not mixed with the standards before carrying out separation (Fig. 12). An autoradiograph of the electrophoretograph indicates only one spot of radioactivity in the external and internal media corresponding to the position of the labelled amino acid initially present on the lumen side only. F. Summary (1) Monoaminocarboxylic acids, naturally found in locust blood and rectal tissue are accumulated in rectal sacs against large concentration gradients. (2) The transport process occurs in the absence of net fluid movement (i.e. solvent drag) .and i t is not caused by the electro-potential difference existing across the rectal wall. (3) Significant metabolic conversion of the amino acids which are transported against concentration gradients does not occur during the transport process. (4) Glycine transport exhibits saturation kinetics, and is inhibited by KCN. -50-G. Discussion Many attempts have been made to distinguish between transport phenomena that can be explained in terms of external physical and chemical forces and those that appear to involve some coupling to metabolic energy sources. In the previous chapter I have used the term active transport in the classical thermodynamic sense, i.e. an energy requiring process of accumulation in which energy is provided directly or indirectly by cell metabolism. This is done without any consideration of specific mechanisms or mode of energy coupling. More recent definitions (reviewed, Curran and Schultz, 1968), using concepts of non-equilibrium thermo-dynamics, have been proposed which also consider the mode of coupling of transport to chemical reactions, either directly (primary transport) or indirectly (secondary transport). The three main criteria I have adopted to determine whether the absorption of amino acids by isolated rectum of the locust i s , in fact, an active transport process, are: 1. The transport of amino acids takes place against a concentration gradient. 2. The transport of amino acids is not driven by an electrical potential difference between the hemolymph and the lumen. 3. The transport is a. process which is suppressed by metabolic inhibitors. The present results show that the isolated rectum has the capability of transporting L-monoaminocarboxylic acids against large concentration differences. Under conditions of no net water uptake, only glycine, -51-L-serine, L-proline, L-alanlne and L-threonine, which are naturally found in locust blood and rectal tissue in high concentrations, are vectorially translocated from the lumen to the hemolymph side. The rate of glycine transfer across the rectal wall shows typical saturation properties and inhibition by KCN. The electrical profile of the rectum in vivo (Phillips, 1964b) and in vitro (Vietingholf et a l . , 1969)cannot account for translocation of amino acids. Both the intracellular compartment and the blood side were shown to be electro-negative to the lumen side. Such electro-potential differences cannot facilitate accumulation of the transported amino acids, which are in either a neutral or negative form at the experimental pH. I used four independent methods in order to determine the import-ance of metabolic conversion of the amino acids in the translocation process. Chromatography or high voltage paper electrophoresis of aliquots taken from hemolymph and lumen compartments after different incubation periods revealed a single radioactive spot corresponding to the test amino acid initially present on the lumen side only. Significant amounts of l^CC^ are not generated from i4C-glycine by everted sacs during the 14 incubation. A final proof that C increase in the hemolymph or tissue compartment truly represents the accumulation of the glycine, was performed with amino acid analyser (see Chapter IV). Under the conditions of the previous experiment there was no net volume flow through the rectal wall. However, since a primary function of the rectum is the reabsorption of water from the lumen (Phillips, 1964a), a net flow normally occurs through the rectal wall. Such a flow could have an influence on solute flow. Ussing and Anderson(1955) first demonstrated this phenomenon in toad skin. The two unidirectional -52-fluxes of thiourea were equal in the absence of significant volume flow. When net water flow was induced, a net flow of thiourea was observed in the same direction as the water flow. In such a case, the rate of solute drag should be directly proportional to the rate of solvent flow (Ussing,1954). In order to determine the relative importance of solvent drag in amino acid absorption from locust rectum, I compared the glycine absorption in the presence and absence of net solvent flow. An 18 fold increase in water absorption is followed by, at most, a 1.7 fold increase in glycine absorption. These results suggest that solvent flow enhances amino acid movement to some extent. This factor might be especially important in dehydrated animals when water absorption from the lumen is greatest. The increase in amino acid accumulation inside the sacs when net water uptake takes place might be explained by a local gradient of amino acids produced by the high rate of solvent flow. This flow might flush out lateral intercellular spaces, as proposed by Berridge (1969). This possibility is discussed in detail in Chapter IV. Another possibility is that solvent flow might facilitate glycine movement across the intima to a transport site on the apical membrane (see Chapt. IV). Extrapolation of results obtained with in vitro preparations to the in vivo situation might be questioned. Obviously confirmation in vivo is desirable. However, I have observed that the present preparation exhibits most characteristics of the organ in vivo. For instance, the potential difference across the rectal wall remains constant during the experimental period and is completely abolished when a metabolic inhibitor is introduced, or when oxygenation is not provided (repeatedly observed, but not reported in results section). In addition, the absorption of water and solutes against concentration gradients by the rn vitro preparations (Goh, 1971) is probably comparable to that observed in  vivo (Phillips, 1964 a,b). The rates of water absorption measured in the present work are in excellent agreement with the rates reported by Goh (1971). Moreover, the ability of the rectal sac preparation to produce hyposmotic absorbate again indicates the viability of in vitro prepara-tions. The occurrence,of active transport mechanisms for amino acids in insect tissues has been a controversial subject for years. Treherne (1959) could not show the presence of an active transport of amino acids in the locust rectum or midgut. The apparent lack of such a mechanism in his experiments on the rectum was possibly a consequence of the relatively short duration of observation, since his main concern was to establish the main site for absorption of ingested amino acids. Moreover, active trans-port mechanisms could not be excluded on the basis of his experiments, since he did not attempt to determine whether net movement could take place against an amino acid concentration gradient. Shyamala and Bath (1966) confirm the conclusion of Treherne. In the midgut of Bombyx mori larva, amino acid absorption is not sensitive to DNP or KCN. On the other hand, Wall and Oschman (1970) have suggested an active absorption of amino acids in the cockroach rectum. The results reported in this chapter and in a previous publication (Balshin and Phillips, 1971) provides the first clear evidence for an active transport mechanism for amino acids in insects. This has since been confirmed for other insect tissues. -54-Nedergaard (1972) has conclusively demonstrated that y amino-isobutyric acid is actively transported by the midgut of Cecropia. Also, active glycine uptake by the integument and silk gland of the silkworm Bombyx mori was reported by Ravi Rao and Bhat (1971) and Sridhara & Chitra (1972) respectively. CHAPTER IV LOCATION AND CHARACTERISTICS OF THE TRANSPORT SYSTEM A. Introduction The specific site controlling absorption of amino acids across vertebrate epithelia has been assigned to the mucosal border (reviewed by Schultz and Curran, 1970), since tissue concentrations usually greatly exceed those of the bathing media. Thus the entry of amino acids into the epithelial cells is probably an energy-requiring process, whereas exit to the serosal side could be a passive process by simple or facilitated diffusion. In this chapter I consider whether the same situation holds for the rectal sacs of the desert locust. In the Discussion, a number of processes (i.e. models suggested in the literature), that might lead to accumulation of amino acids across an epithelial membrane are considered. Without elaborating on these processes at this point, they can be conveniently divided into two groups: non-specific and specific. For example, recycling of water and ions might occur within lateral intercellular channels of the rectal epithelium (Berridge, 1970; Phillips, 1970; Wall, 1971). Amino acids might become entrained in such a flow by frictional drag. Thus, net movement of amino acids could occur even if net transport of water or ions across the whole rectal wall was not observed. If so, accumulation should be relatively non-specific since any small water-soluble molecule entering such channels should be entrained and accumulated. Furthermore, perturbations which do not affect-water and ion transport in the system should not alter amino acid accumulation. -55--56-If, on the other hand, transport of amino acids involves an 'enzyme-substrate type' combination with a 'carrier molecule' or a binding site, movement of organic molecules across rectal sacs should be much more selective. Moreover, one might anticipate those properties generally attributed to a 'carrier'; i.e. stereo-specificity, saturation (Michealis-Menton) kinetics, and competitive inhibition between molecules sharing the 'carrier'. The characteristis of amino acid movement into rectal sacs are considered in this chapter with a view to deciding whether the mechanism is of the specific or non-specific type. 14 B. Tissue accumulation of C-labelled amino acids 1) Glycine The x"*C-activity ratios, T/L .and H/L, developed after incubating rectal sacs for 2 hours in 10 mM of l4C-glycine (water-movement prevented) are presented in Fig. 13 and Table VI. The T/L (4/1) is twice the H/L (2/1) ratio suggesting an uphill gradient for entry of glycine into the tissue, but a downhill gradient for exit across the basal (serosal) border. When this experiment was repeated in the presence of 10-3M KCN ( no osmotic gradient and no net water movement; Table VI), significant accumulation, both in tissue and 14 inside the sac, was abolished. The increase of C-glycine activity in rectal tissue with time is also shown in Fig. 24. The T/L ratio exceeds 11/l after 3 hours. 2) Serine 14 A second experiment was conducted in which 10 mM of C-labelled L-serine was placed only on the outside of sacs bathed in Mordue Ringer (water movement prevented with sucrose). The i 4C-activity Fig: 13. The 14C-glycine ratios (H/L and T/L) developed across rectal sacs at two hours of incubation. Rectal sacs were incujjated in media initially containing 10 mM of C-glycine on both sides. Water movement was prevented with sucrose (420 mosm/1). Other preparations were exposed to 10 M KCN during the same period of time in which case no sucrose was added externally. Net absorp-tion rates of glycine and water, as well as % dry weight of rectal tissue at the end of experiments, are given in Table VI. Vertical lines represent S.E. of the mean of 4 preparations. - 5 7 -Table VI l^c-glycine ratios (H/L and T/L), net rates of glycine and water absorption, and  % dry weight of rectal tissue after a two hour incubation in the presence or  absence of 10"% KCN. (Means ± S.E. of 4 preparations). H-compartment T-compartment i oo Incubation 14c-glycine Net absorption rate i 4 C-glycine Accumulation of co dition ratio(H/L glycine water ratio (T/L glycine in the % D.W. con x xon a f c 2 h r ^  (n m oles/hr. / . (ul/hr./ at 2 hr.) tissue,,nmoles/hr. / rectum) rectum) 10 mg. rectum 10-3 M KCN i.i5 ± o.04 9.74 ± 3.8 -0.53 ± 0.32 1.2 ± 0.07 23.45 ± 8.45 27.5 ± 1.3 10 mM glycine 420 mosm/1 2.31 ± 0.10 65.5 ± 5.0 -0.13 ± 0.15 4.17 ± 0.37 417.60 ± 37.9 26.25 ±1.7 sucrose 10 mM glycine -59-in rectal tissue increased linearly with time (Fig. 14). The T/L ratio exceeded unity after 15 minutes and reached 7/1 after 2 hours. During this experiment the internal bathing medium was replaced with fresh Mordue Ringer every 20 minutes. The flux rate of x4C-serine from the tissue to the hemocoel side was estimated from the radio-activity of internal media removed every 20 minutes. No,x4C-serine appeared inside the sac during the first 20 minutes. After this lag (while x^C-serine accumulated in • the tissue) the flux rate was directly proportional to the tissue concentration of x4C-serine. During the whole experiment tissue concentrations of x4C-serine exceeded those in the internal media. These results are consistent with simple diffusion of serine from the tissue to the hemocoel side down a concentration gradient. It is not possible to exclude a second 'carrier' mediated process on the basal side since saturation of such a mechanism might only occur at very high concentrations. 3) Comparative survey 14 The tissue accumulation of C-amino acids normally found in the hemolymph of the desert locust were determined in two types of experi-ment.? 1. Rectal preparations were extracted after 3 hours of incubation in media initially containing 10 mM of a single test x**C-amino acid on the L side only (Table VII). 2. Data on tissue accumulation was also obtained during experiments concerned with amino acid transfer across the whole rectal wall (Chapter III, Section D). In these experiments the -^C-labelled Fig. 14. The C L-serine content in rectal tissue and the flux rate of this amino acid into the  internal bathing media during incubation. The flux rate ( A ) into the H compartment and the tissue content ( • ) were measured at various times. The rectal sacs were initially incubated in Mordue Ringer containing L-serine (10 mM) and sucrose (420 mosm/l) in the external compartment only. The internal bathing solution (which initially consisted of Mordue Ringer) was replaced with fresh media every 20 minutes. Vertical lines represent ± S.E. of the mean of 4 preparations. Since the internal volume was 10 Vl, the total content of the hemocoel compartment can be read off the left hand legend. An approximation of the concentrations in the tissue and hemocoel compartments can, therefore, be read directly off the graph. -60-20 40 60 80 T I M E (rtfn.) 100 120 CO > o -61-Table VII Uptake of different L-amino acids by rectal tissue A, single-^C-labelled. amino acid(10 mM) was initially present on the L side only ( means ± S.E. of 4 prep-arations. Test amino acid 14C ratio (T/L after 3 hr.) Accumulation in the tissue nmoles/3 hr./ 10 mg. rectum Structural formula Glycine Serine Threonine Y-amino butyric acid Proline Glutamic acid Histidine 11.2* ± 0.25 7.2**± 0.80 2.0 + 0.14 1.0 ± 0.05 10.0 ± 0.78 0.16 + 0.04 0.19 ± 0.01 H2N-CH2-C02H Tyrosine 0.08 ± 0.05 CH20H JsHCH-H2N-" H-C02H CH3CH0HCH(NH )COOH H NCH (CH ) COOH 2 2 2 2 8 1120 ± 25 720 ± 75 200.9 + 13.6 110.2 ± 5.4 1068.5 ± 77.5 16.2 ± 3.9 19.0 ± 1.1 CH CH(NH )C00H // \ 2 2 8.0 ± 4.5 H0<\' > CH2CH(NH?)C00H COOH H00C(CH ) CH(NH )C00H 2 2 2 Taken from Fig. 24. After 2 hours of incubation (taken from Fig. 14) -62-test amino acid ( 10 mM) was initially present in both bathing media (Table VIII). After 3 hours of incubation, the T/L ratios for the amino acids which were previously shown to be transported across the rectal wall reached 2.1 to 11.1 in the first type of experiments (Table VII). In the second type, after 3 and 7 hours of incubation, the ratios were 2.5 to 7.6 and 3.3 to 11 respective-ly (Table VIII). On the other hand, T/L ratios for the other 4 amino acids (Table VIII) did not exceed unity. Those amino acids which are not transported (acidic, basic and aromatic amino acids) entered the rectal wall from the L side very slowly so that the T/L ratios were very low (;0.08:1 to 0.19:1) after 3 hours incub-ation (Table VII). This suggests a permeability barrier to these amino acids on the apical, but not the basal, side. On the other hand, Y-aminpbutyric acid, which is not present in the locust's blood, penetrated into the rectal wall, but in spite of its low molecular weight, the final T/L ratio did not exceed unity. These observations again suggest that the absorptive site for the amino acids accumulated in the sacs is probably located in the apical membrane of the rectal wall. Figure 15 indicates that the relative rate of amino acid transport across rectal wall (indicated by H/L ratios) is positively correlated with accumul-ation of a given amino acid in rectal tissue (i.e. T/L ratio). C. The amino acid composition of the rectal tissue and the bathing  media during incubation Changes in the concentration of the free amino acids both in rectal tissue and bathing media were followed during glycine uptake by -63-Table VIII The T/L ratios of C-activity and the % dry weight of rectal  sacs exposed to various L-amino acids. Various 14C amino acids (initially at a concentration of 10 mM) were present on both sides of rectal sacs. Net water movement was prevented by adding sucrose (420 mosm/1) on the L side. Values represent means ± S.E. of 3-4 preparations. Preparations used in this experiment were also employed in the determinations of H/Lratio shown on Fig. 11 and Table V. The T/L ratios and % D.W. were measured after 3 and 7 hour incubation periods. 3 hr. •7 hr. Test Solution 14 C ratio (T/L) % D.W. 14C ratio (T/L) % D.W. Serine 6.90 + 0. 44 30. 3 + 0. 66 11. 05 + 0.88 28. 7 + 0. 94 Alanine 5.40 + 0. 89 23. 3 + 1. 90 7. 40 + 0.59 30. 6 + 1. 40 Threonine 2.50 + 0. 07 27. 1 + 1. 06 3. 30 + 0.35 27. 8 + 2. 00 Y-amino 1.20 + 0. 14 31. 0 + 0. 48 1. 08 + 0.11 22. 0 + 0. 70 butyric acid Proline 7.60 + 1. 70 28. 0 + 1. 50 10. 00 + 1.60 24. 9 + 2. 80 Glutamic acid 0.60 + 0. 15 27. 9 + 1. 25 0. 83 + 0.1© 27. 1 + 1. 10 Histidine 1.05 0. 05 27. 0 + 1. 00 1. 07 + 0.07 27. 2 + 1. 10 Tyrosine .0.65 + 0. 06 27. 6 + 0. 88 0. 88 + 0.09 33. 0 + 1. 10 These times include the 1 hour pre-incubation period. The corresponding time for H/L accumulation (Fig. 11 and Table V) is 1 hour less. Fig. 15. Comparison of accumulation ratios for eight -^ C-amino acids in tissue (T/L) and in internal  bathing media (H/L) relative to the external media. Values were taken from Tables VII, VIII and Fig. 11 for 2 (0) and 6 (#) hours of incubation (excluding pre-incubation time of 1 hour) in 10 mM of amino acid. Net water movement was prevented. The broken line indicates equal values for T/L and H/L ratios. Note that in a l l cases the T/L exceeds the H/L ratio and that there is a direct correlation between these two ratios; i.e. between the rate of accumulation of an amino acid in the rectal tissue and its rate of active transport across the rectal wall. Vertical and horizontal lines represent ± S.E. of the mean. (GABA = y-aminobutyric acid ). -6.4;-\ -65-the rectal tissue (Table IX) using an amino acid analyser. The objectives were: (a) To obtain direct chemical confirmation for amino acid accumul-ation previously demonstrated with radiotracers. (b) To measure loss of indigenous amino acids from tissue to bathing media. The T/L ratio of total glycine obtained after 1 hour pre-incubation was 5:1 (Table IX), a value similar to that obtained with x4C-glyince as shown on Fig. 24 and Table D of the Appendix. Another proof that the x^C-increase in the tissue compartment truly represents the accumulation of the glycine was obtained by collecting the buffer solution drained from the amino acid analyser column with a fraction collector. All the radioactivity was obtained in a single fraction corresponding to the glycine peak. The glycine concentration calculated in the two different methods were similar (approximately 400 nmoles/hr./10 mg. rectum). This shows that by the end 14 of the pre-incubation period, the specific activity of the C-glycine in the rectal tissue had largely equilibrated with that of the bathing media. This supports the view that the net movement of x 4C-activity after the pre-incubation period truly reflects the movement of glycine. The main trend was for amino acids naturally present in the tissue to flow toward the H side. The total loss was 77.6 nmoles/7 hr./10 mg. rectum (Table IXa) and 172.7 nmoles/7 hr./.10 mg. rectum (Table IXb) . In the two experiments the accumulation of glycine in the tissue during the incubation period (7-8 fold increase over 7 hours) was in large measure balanced by a loss of other amino acids, so that the total amino acid concentration in the tissue changed very l i t t l e . The principal amino acids lost to the internal bathing media were those previously shown to be transported. The total quantity of a l l amino acids that had - 6 6 -Table IX The composition of free amino acids in the rectal tissue  and in the incubation media when rectal sacs are exposed  to different amino acids (a) Rectal sacs were bathed with 10 mM of glycine on the L-side-Sucrose (420 mosm/1) was added to prevent a net water move-ment. Determination of amino acids was performed with an amino acid analyser. The very low levels of amino acids in the external medium at 7 hours were measured by running the total volume (Lml) through the analyser. Amino Rectal- Rectal Rectal Internal Internal External Acid tissue tissue tissue media media medium before pre-- after 1 hr. after 7 after after after incubation of pre- hrs. of 1 hr. 7 hrs. 7 hrs. incubation incubation x 100 (nmoles/10 mg. rectum) (nmoles/10 ul aliquot) Aspartic acid 11.5 14.5 6.1 5.5 1.7 1.7 Threonine 7.7 5.0 2.0 10.5 2.5 Serine 96.9 47.2 44.9 10.9 11.87 6.7 Amides 192.3 66.0 28.9 35.7 23.8 Proline 646.1 222.0 28.9 96.4 17.8 47.9 Glutamic 66.1 98.6 60.0 2.0 1.2 3.3 acid Glycine 140.7 512.4 982.3 145.7 669.9 105.0 Alanine, 53.8 60.2 74.0 22.1 33.2 Cysteine 27.3 2.2 Isoleucine 0.8 Leucine 1.4 Tyrosine 18.5 5.2 1.3 1.1 0.8 100.0 34.8 76.5 41.8 37.9 65.5 Arginine 26.9 28.8 10.0 7.1 0.5 1.8 Valine 4.0 -67-(b) Experimental protocol was similar to (a), except that a different group of animals and different amino acid analyser were used. Amino Acid Internal media after 7 hrs. of incubation (nmoles/10 yl) Aspartic acid 0.7 8.0 0.9 Threonine 7.3 8.5 4.5 Serine + Amides 30.0 33.5 27.5 Proline 16.3 2.9 9.4 Glutamic acid - - 12.5 Alanine 108.0 66.2 68.0 Glycine 720.0 1050.0 * _ Valine 5.2 - -Metionine trace 5.6 trace Isoleucine 3.5 7.3 -Leucine trace - trace Tyrosine - 11.2 -Phenyl alanine 2.4 - -Lysine - 8.7 7.3 Histidine - 3.2 8.2 Arginine trace trace -Rectal tissue after 7 hrs. of incubation (nmoles/10 mg. rectum) External media after 7 hrs. of incubation x 100 " (nmoles/10 yl) not determined. -68-(c) Experiment l.x^C-serine (1.0 mM) was initially present only on the L-side. Experiment 2.i"*C L-alanine (10 mM) was initially present only on the L-side. Rectal tissue Amino acid concentration after. .. 6 hrs. of incubation (nmoles/lO mg; rectum) Concentration of internal media after 6 hrs. of incubation (nmoles/10' ul) Lysine 46.0 not determined Histidine 15.4 II Ammonia 15.5 it Aspartic acid 8.8 it Serine 1981.0 826 Glutamic acid 57.6 not determined Proline 92.8 II Glycine 207.0 ii Alanine 51.4 ii Arginine trace 2. Ammonia 157.0 not determined Aspartic acid 48.0 " Serine 82.6 " Glumatic acid 264.0 " Proline 182.9 Glycine 46.9 " Alanine 1284.6 Arginine 33.0 -69-leaked out from the tissue into the external medium during the incubation period (14.6 nmoles/hr./10 mg. rectum, Table IX ; and 23 . nmoles/hr/10 mg. a rectum, Table IX^) is approximately 15% of the total amount of glycine transported. This observation excludes the possibility that glycine is concentrated in the rectal tissue by exchange for other amino acids which move across the apical membrane out of the cell (i.e. transport by counter-flux; Rosenberg and Wilbrant,1957). 14 Similar experiments in which C-labelled L-serine or alanine were placed initially on the L-side of rectal sacs provided direct chemical evidence for the accumulation of these amino acids against large concentr-ation gradients (Table IX^). D. Specificity of absorption The possibility that a non-specific process (e.g. drag effect) was responsible for amino acid accumulation was tested by studying the net movement of amino acid stereo-isomers and other solutes across the rectal wall. The experimental protocol was identical to that of earlier experi-14 ments conducted with C-glycine (Chapter III, Section B), except that 14 10 mM of other test molecules labelled with C were placed on both sides of rectal sacs (net water movement prevented). The ratios of ^C-activity across the rectal wall after 6 hours of incubation and the corresponding net rates of solute and water movement are presented in Table X. L-alanine, was accumulated across the rectal wall, but D-alanine, urea and 3-0-methylglucose were not. The rectal sac was shown to be permeable to urea and to a lesser extent to 3-0-methylglucose. If the molecule is added initially to the external bathing media, substantial quantities of Table X Net rates of organic solute • and water absorption by in vitro recta The bathing media contains 10 mM of test molecule. 420 mosm/1 sucrose is present in the external media. Values are given as means ± S.E. of 4 preparations Test Solute u ** C ratio after M.W. 6 hr.(H/L) Net absorption rates Solute Water nmd.les/hr. /rectum ul/hr. /rectum Glycine 75 10.0 + 0.5 106 + 11 0.4 + 0.9 L-alanine 89 2.5 + 0.4 25 + 5 0.1 + 0.1 D-alanine 89 1.0 + 0.1 0.2 + 0.1 Urea 60 1.0 0.1 0.6 + 0.4 0.0 + 0.1 *( 0.5 + 0.04) 3-0-methyl- 194 0.9 + 0.02 -0.1 0.02 0.3 + 0.1 glucose *( 0.3 + 0.03) The test solute is added to the.outside compartment only. Initial ratios in a l l cases were close to 1 (range 0.94 - 1.19), - 71 -radioactivity appear in the internal bathing media after 6 hour incubation period (Table X). Thus, some permeating molecules ( e.g. urea) are not accumulated by rectal sacs. It is interesting that 3-0-methylglucose is not accumulated by rectal sacs. This compound is actively transported by glucose transport systems of many cells and epithelial tissues (Schultz and Curran, 1970). In the present study, the ratio of 3-0-methylglucose found after a 6 hour incubation period was only 0.3:1. This low ratio may be due to low permeability of the intima to this molecule. To suggest, only on the basis of this single observation, that in vitro rectal sacs lack a glucose transport sytem would be premature, since no information is available on the specificity properties of such a system. Preliminary observations indicate that ±4C-glucose accumulation occurs against a concentration gradient. L-alanine crossed the rectal wall at least 10 times as quickly as D-alanine (Table X),.even though these isomers have identical solubility properties, rates of free diffusion, and molecular size. This is a clear indication that transport of L-alanine at some stage involves combination with a stereo-specific binding site or 'carrier' molecule. Experiments with stereo-isomers of serine support this conclusion (Table XI). When rectal sacs were exposed to '^C-labelled isomers in the external bathing media for 30 minutes, L-serine accumulated against a concentration gradient in rectal tissue, but only a small amount of D-serine could be detected. The rate of accumulation of the L form was again about 10 times that of the D form. This again supports the view -72-Table XI The uptake of l^c-serine stereo-isomers by the rectal tissue. 14C-serine (10 mM) was added to the external Ringer solution only. Water movement was prevented by 420 mosm/1 of sucrose present in the external media. (Mean + S.E. of 4 preparations). T/L *Rate of % D.W. Test l^C-ratio accumulation of rectal „ . , after 30 min. in tissue tissue Molecule , , , _ . (nmoles/hr./rectum) L-serine 2.2 ± 0.27 . 444.5 ± 55.7* 22.6 ± 2.6 D-serine 0.27 ± 0.04 55.9 ± 8.7 25.3 ± 1.9 * L-serine uptake is significantly higher than D-serine uptake, (P .<0.001, T test). Dry weight under the two conditions is not different ( P>0.4). - 7 3 -that the apical membrane is the selective absorption site. Since some small permeating molecules are not accumulated and the transport of neutral amino acids exhibits stereo-specificity, the trans-port mechanism for amino acids in rectal sacs is clearly a highly specific process. 14 E. Kinetics of C-glycine influx 14 Unidirectional influx of C-glycine from the external media into rectal tissue was estimated within 2-10 minutes of exposing in vitro sacs to various external concentrations of this amino acid (no net fluid movement). Since the amount of x^C-activity incorporated in tissue over 14 such short incubation periods is small, C-glycine in superficial layers of fluid might represent a serious source of error. It was, therefore, important to assess the rinse procedure used to remove superficial activity (see Methods Section). 14 It is known that rectal cuticle is impermeable to C-inulin (Phillips and Dockrill, 1968). The success of the rinse procedure could, therefore, be estimated by incubating rectal sacs in external media containing this isotope and using the standard rinse procedure to estimate 14 C-inulin activity in rectal tissue. The inulin space (Table XII) is negligible (0.13 ul/10 mg. tissue; i.e. 1% of rectal tissue) and does not change between 30 and 60 minutes of incubation. This indicates that the rinse procedure removes virtually a l l external surface activity. 14 Since C-labelled amino acids only appear on the hemocoel side after 20 minutes of placing these radioisotopes in the external medium (Fig. 14), the radio-activity found in the internal surface layers of fluid (H side) is not a source of error in these experiments. (The difficulties of - 7 4 -Table XII The external inulin space of the rectal sacs Measurements were made after 30 and 60 minutes of incubation with ^C-inulin in the external medium. The rectal tissue was extracted for radioactivity measurements. Values are given as means of 2 recta. Duration of Inulin space % D.W. of T_ . , / n / n n ^ - N rectal sacs incubation (ul/10 mg. tissue) (minutes) 30 60 0.125 0.130 25.3 26.8 - 7 5 -obtaining a meaningful estimate of total extra-cellular space in the rectal wall are considered in the Discussion). The uptake of x^C-glycine by rectal tissue at various external concentrations is shown in Figs. 16 and 17. (Actual values are given in Table A, Appendix). In a l l cases the accumulation is a linear function of time; indeed, this is so for the first 1.5 hours (Fig. 24). The average influx rate calculated over the first 2, 4 and 6 minutes (575 nmoles/hr«/ 10 mg. rectum; Fig. 17) is in reasonable agreement with that calculated over 1.5 hours (473 nmoles/hr/10 mg. rectum; Fig. 24) at an external concentration of 10 mM glycine. The negligible 1 4C-activity in rectal tissue after 10 seconds of incubation (Fig. 16) also indicates the effectiveness of the rinse procedure. 14 The rate of C-glycine accumulation in rectal tissue over these short time intervals was judged to be a direct measure of unidirectional influx because: (a) No loss of x^C-activity occurred to the H side; i.e. a two compart-ment system is approximated. (b) The linearity of the uptake suggests that no significant backflux of radioisotopes to the external medium takes place over this time. (c) The total activity (and specific activity) in the rectal tissue was low compared to the external media so that any back diffusion probably involved primarily unlabelled glycine. A double reciprocal plot of influx rate against external concentr-ation of glycine (Lineweaver-Burke plot) yields a straight line (Fig. 18) as expected for an enzymic reaction. The K and V for glycine influx T max are 22 mM and 2000 nmoles/hr./10 mg. rectum respectively. Initial Fig. 16. The entry of C-glycine into the rectal epithelium. In vitro sacs were exposed to Ringer containing TO mM l^C-glycine and 420 mosm/1 sucrose on the external side for different periods of time. Vertical lines represent ± S.E. ef the mean of 4 preparations. -76--76a-Fig. 17. The entry of C-glycine into the rectal epithelium at different concentrations of  glycine. In vitro sacs were exposed to Ringer containing different concentrations of glycine and 420 mosm/1 sucrose on the external side for different periods of time. Glycine concentrations were: 2.5 mM (O); 5 mM (A); 10 mM W ; 20 mM (•) ; 40 mM (T) ; 80 mM (•) . (Each point represents the mean of 4 preparations; vertical lines represent ± S.E. of the mean of 4 preparations. -77--77a-Fig. 18. Double reciprocal plots of i n i t i a l rates of glycine influx into the rectal tissue as a function of external glycine concentration. The Ringer solution contained different concenctration of C-glycine, either with (•) , or without (O) Na+. Choline was used to substitute for Na+. Net movement of water was prevented as previously described. (Each point represents the average of 12 experiments; vertical lines represent ± S."E. of the mean; data taken from Figs. 17 & 25). Values for glycine influx and net water movement can be found in Table A of the Appendix. -78-1/GLYCINE INFLUX -78a. -79-unidirectional influx rates into rectal tissue (Table A, Appendix) are approximately 5-6 times the average rates of net glycine transfer across the whole rectal wall over a 6 hour period (Table II). This kinetic study provides further evidence that amino acid transport by rectal sacs involves a saturable carrier molecule or binding site, probably located on the apical membrane of the rectal epithelium. F. Competition . ' It has been widely observed that the transport of one amino acid may be inhibited by another ( Fridhander and Quastel,1955; Hagihira,et a l . , 1960). I hoped to find out i f mutual inhibition occurred amongst the amino acids transported across the rectal wall. Such inhibition has commonly been accepted as evidence that amino acids share the same carrier mechanism (Wiseman, 1968). In this experiment, 5 or 10 mM of l^C-glycine was added to the external medium only (no net water movement), and the 14 level of C-activity in rectal tissue was determined after 30 minutes (during linear uptake phase; Fig. 24). The effect of adding increasing concentrations of a second amino acid (L-serine, L-proline) to the external media on this influx of glycine is shown in Fig. 19 and Table B, Appendix. When the serine concentration equalled that of glycine in the external media (5 mM), the influx of ^C-glycine was inhibited by 40%. This inhibition increased to 85% as the serine concentration was raised to 50 mM. Serine and glycine were previously shown to be transported at about the same rate at equal external concentrations (Table VII). These two observations suggest a common carrier or binding site having similar affinities for glycine and serine. It is quite possible that Fig. 19. The,rate of C-glycine uptake by rectal  tissue when different concentrations of  L-serine or proline are present in the test solution. Various concentrations of L-serine (•) or proline (O)were initially present in the external bathing media. Net water movement was prevented. The % dry weight of rectal tissue at the end of experiments remained between 24.6 ± 2.2 to 28.5 + 1.2 (see Table B, Appendix). Each point is the average of 4 to 7 experiments; vertical lines indicate ± S.E. Influxes under different concentrations of the second amino acid differ significantly(P < 0.001). -80--81-a l l 4 neutral amino acids which are transported across the rectal wall share a common'carrier' mechanism. A significantly lower inhibition was caused by proline. When the external concentration of proline was 5 times that of glycine, the influx of the latter was inhibited by only 50%. Yet proline is trans-ported as quickly as serine and glycine. While proline could share the same 'carrier' with the neutral amino acids, an alternative suggestion is that two carrier systems compete for a common energy source. In order to determine i f the inhibition of glycine transport by serine was of the competitive or non-competitive type,the serine concentr-ation was held constant in the external media and the concentration of ^C-glycine was varied. The uptake of ^ C-glycine by rectal tissue was estimated after 10 minutes of incubation. Other details of protocol were the same as in the first experiment. Figure 20 shows a double reciprocal plot of ^ C-glycine uptake rate as a function of the in i t i a l concentration of glycine in the test solution, in the presence and absence (from Fig. 18) of 5 mM serine. At a l l glycine concentrations the uptake was lower in the presence of serine. Fig. 20 shows that the lines obtained in the absence and presence of the inhibitor intersect at the same point on the ordinate; this indicates that the transport of glycine is competitive with serine, and if the concentration of glycine is increased sufficiently, the inhibition by serine can be overcome. These experiments provide further evidence that a specific 'carrier' process is involved in amino acid transport across rectal sacs since there is no evidence that amino acids inhibit the general activity of rectal tissue. For example, rectal sacs maintain the same osmotic Fig". 20.' Double reciprocal plot of glycine influx into rectal tissue in the presence or absence of L-serine. Different concentrations of C-glycine were added to the external medium, either in the presence ( • ) or absence ( O ) of L-serine (5 mM). Each point represents the average of 4 experiments; vertical lines indicate ± S.E. The values for glycine influx in the absence of L-serine were taken from Fig. 18. -82--83-gradients in the presence or the absence of these amino acids. G. Summary 1) Only those amino acids transported across the rectal wall are accumul-ated in the rectal tissue against large concentration gradients. The T/L ratios for 5 monoaminocarboxylic acids present in the locust's blood are much greater than the corresponding H/L ratio. This suggests that entry of these amino acids into the rectal epithelium is an energy requiring process, whereas exit could occur by passive diffusion. 2) Alanine and serine transport are stereo-specific processes. Glycine influx is characterized; by Michaelis-Menton kinetics and is competit-ively inhibited by serine. All these properties suggest that the amino acid transport across the rectal wall involves an i n i t i a l combin-ation of the amino acid with a specific 'carrier' molecule or binding site. 3) Transport rates decrease as the length of the hydrocarbon side chain increases amongst the 4 aliphatic amino acids present in locust hemo-lymph. The rates are further enhanced by the presence of an hydroxyl group in the side chain of the molecule. H. Discussion In Chapter III the translocation of amino acids across the rectal wall was found to be vectorial and active in nature. The present chapter explores several properties of this absorptive process (in particular, tissue localization, specificity and kinetics) in the hope that such studies might suggest a mechanism for the active transport. Several fundamentally different types of active transport mechanism -84-for organic solutes have been proposed in the general literature. Their properties are summarized in Fig. 21. It is possible to exclude some of ... these models on the basis of experimental observations presented in this chapter. Ussing (1966) has observed accumulation of sucrose across the frog skin against an electrochemical gradient and in the absence of net fluid movement. Although he initially reasoned that an active transport coupled to cell metabolism was probably involved, i t was subsequently shown that this apparently 'active' accumulation was coupled to (driven by) a net passive diffusion of urea. Thus, the accumulation was not dependent on cell metabolism, but was driven by an external energy source (net urea diffusion) by a process of solute-solute drag (Franz et a l . , 1968; Galey and Van Bruggen, 1970; Biber & Curran, 1968). Ussing has recently suggested that solute-solute drag may be due to fluid recycling and hence represent a form of solvent drag (Ussing, 1969). Such processes would exhibit the following general characteristics (Fig. 21; Mechanism A): (1) Accumulation should proceed when cellular respiration is inhibited ( i.e. insensitive to metabolic inhibitors). (2) A net movement of a passively permeating solute must occur across the membrane • (3) The frictional drag effect might be expected to be non-specific and cause accumulation of most rapidly permeat-ing hydrophilic molecules (i.e. should not distinguish between L- and D- isomers). The absorption of amino acids by rectal sacs does not f u l f i l any of these requirements. In the present experiments, the accumulation of Fig. 21. A 'key' (binary classification) to the principal types of mechanisms which have been proposed to  account for net transfer of amino acids across  epithelial membranes against electrochemical  gradients. Mechanisms sharing a similar type of energy source (external or internal) are linked by heavy lines. A l l of the mechanisms on the left hand side have, in common the property of being non-specific ( e.g. unable to distinguish between L and D stereo-isomers). Those on the right hand side exhibit enzyme-like properties (saturation kinetics, competitive inhibition and stereo-specificity). -85-POSSIBLE MECHANISMS OF AMINO ACID ACCUMULATION A,EXTERNAL ENERGY SOURCE (solute or solvent drag) Characteristics: (1) insensitive to respiratory inhibitors passively permeating molecule required to drive accumulation non-specific (2) (3) B. NON-SPECIFIC ion transport followed by frictional drag (e.g.) solute-solute solvent-solute INTERNAL ENERGY SOURCE (i.e. respiration) P PRIMARY TRANSPORT direct energy coupling to specific carrier SECONDARY TRANSPORT indirect energy coupling SPECIFIC carrier shared by a.a. and ion: i.e. CO-TRANSPORT -85a--86-glycine has been shown to be completely inhibited by KCN. While a concentration gradient of sucrose was present across the rectal sacs in most experiments and potentially could cause solute-solute drag of amino acids, the rectal intima ( the first barrier to molecular penetration) which is virtually impermeable to this molecule (Phillips, 1965; Phillips and Dockrill, 1968; Wall, 1967) would have prevented such an effect. Finally, small permeating molecules, such as urea, are not accumulated across rectal sacs and, in addition, the latter are highly selective for the L-isomers of amino acids. On the basis of a l l three criteria, solute-solute drag driven by an external energy source can be eliminated as a possible mechanism. A variation on the above mechanism should be considered (Fig. 21; Mechanism B). While no transepithelial concentration difference existed initially for solutes other than sucrose, net active transport of Na+, K+, and CI ions are known to occur across rectal sacs (Phillips, 1964b; Goh, 1971). These active fluxes of ions might cause solute-solute drag of amino acids or solvent drag by fluid-recycling (in the absence of a significant net fluid movement). On ultrastructural grounds, such a model has been proposed by Berridge (1970; Fig. 22) to account for the observat-ions of Wall and Oschman (1970). This represents a form of secondary active transport (i.e. amino acid movement indirectly coupled to cell metabolism), sensitive to respiratory inhibitors, but relatively non-specific. I can rule out such a process on the basis of stereo-specificity observed for amino acid absorption by rectal sacs of the locust. However, one might modify Berridge's model to explain stereo-specificity of amino acid absorption by postulating a passive carrier-mediated entry Fig. 22 A model illustrating the possible role  of an extracellular compartment in the  absorption of amino acid across insect rectal wall. The water transport into;, the extracellular compartment (EC), or recycling from the blood, creates a favorable gradient for the passive diffusion of amino acids (X) from the lumen into the extracellular compartment. The water flow drags the amino acid from the (EC) out to the blood through the restricted openings in the basal membrane. (From Berridge, 1970). Numbers refer to relative concentrations of amino acids. - 8 7 -Lumen Cell Blood Apical Basal membrane membrane -87a--88-step (i.e. facilitative diffusion mechanism) prior to a solute drag step. Such a mechanism seems highly unlikely for three reasons: a) One might expect such a process to accumulate a l l small rapidly permeating ions, assuming that a l l solutes move by the same route through the tissue. However, accumulation of urea, 3-0-methylglucose and several amino acids, has not been observed in the present experiments. b) It is difficult to imagine a friction drag (i.e. within a fluid f i l l e d lateral space, as proposed by Berridge (1970), + + which is specific enough to distinguish between Na and K . It will be shown in Chapter' V that amino acid absorption is abolished after 2 hours when Na+ is not present in the external bathing media ( Na+ replaced by K+). Under such conditions Na+ transport is abolished by the same time, but this is compensated for by increased transport of K+" so that the rate of fluid transport under isosmotic conditions (Goh, 1971) and the maximum gradient against which a net fluid movement will occur (Fig. 23, Chapter V) are not decreased. It should be pointed out that high '••'• K+ transport rates are the normal situation in vivo (Phillips, 1964, b,c; reviewed by Maddrell, 1971). Thus, under the above experimental condition of Na+ deficiency, when total ion transport and presumably any fluid recycling in the epithelium are not decreased, glycine transport is abolished. c) Ultrastructural studies indicate that the columnar epithelial cells of rectal pads with their fluid fill e d lateral intercellular spaces constitute most of the volume of the rectum (Gupta and - 8 9 -Berridge, 1966; Oschman and Wall, 1969; Phillips, 1970). For passive net diffusion of amino acids to lateral spaces where drag or sweeping away effects might occur, a downhill concentration gradient of amino acid would be required; i.e. [Lumen]> [/Intracellular] > CUpper lateral space]. Thus, the whole tissue concentration of transported amino acids should be less than the lumen concentration. This is not the case for the locust rectum. During a l l experiments the tissue concentrations of a transported amino acid exceeds the lumen concentration by factors of 2 to 11 times. (The problems and uncertainties of equating tissue content with concentr-ation are discussed below). However one might postulate water reabsorp-tion in the basal part of the lateral channels as a mechanism of con-centrating amino acids ( i.e. recycling), but this compartment would represent an extremely small fraction of tissue volume and would require reabsorption of most of the secreted water in the lateral channel (as opposed to fluid recycling by net diffusion at other sites). The concentration in this small compartment would have to be at least 100 times that of the c e l l . This accumulation might also have to occur across a membrane relatively permeable to amino acids (discussed below). Such a process would, therefore, be energetically very wasteful and would not make much sense for a system concerned with producing hypo-osmotic absorbate by ion recycling (Phillips, 1970; Wall, 1971). In summary, I conclude that the mechanism of facilitated diffusion in series with secondary transport by frictional drag in lateral extra-cellular spaces (modified Berridge's model) is highly unlikely. However, this model may explain the additional amino acid transport component previously demonstrated when net fluid absorption occurs (Table II). -90-Amino acid absorption across the locust rectum is a highly specific process exhibiting a l l the properties typical of an enzyme-substrate interaction; saturation kinetics, stereo-specificity and competitive inhibition. These observations suggest the presence of a 'carrier' molecule as proposed by either the primary transport, or co-transport concepts ( e.g. Na+-gradient hypothesis, Fig. 29, Chapter V). Attempts to distinguish between these two models ( Fig. 21; Mechanisms C and D) are considered in Chapter V. It is also important to decide whether accumulation against a concentration gradient occurs at the apical or basal boundaries of epithelial layers. It is not possible to draw a rigorous conclusion concerning this question on the basis of the present data because only the content of -^ C-amino acid was measured.rather than concentrations within specific tissue compartments. Measurement of, and correction for, extracellular spaces in rectal tissue of the locust were not considered feasible because the cuticle and tissue layers on the hemocoel are barriers to the entry of large molecules such as inulin, which might consequently be restricted to the intercellular space. Moreover, the composition of fluid in the intercellular (lateral) spaces is known to vary drastically in different areas of the rectal pads (Wall, 1971). It seems unlikely that the high concentrations of transported amino acids in the tissue are due to binding of specific sites within the cytoplasm because the T/L ratio for glycine is reduced from approx-imately 10/1 to 1/1 when respiration is inhibited with KCN. Moreover, when various amino acids and other organic molecules which are not transported across the rectal wall are present on the H side for several hours -91-( L-tyrosine, L-histidine, L-glutamate, D-alanine, urea, 3-0-methylglucose), the T/L ratios are a l l close to one (range 0.6 to 1.1). This is consistent with a relatively free passive distribution of these molecules in the tissue fluid, or at least in the major tissue compartments. On the other hand, the T/L ratios abtained by non-transported amino acids (L-histidine, L-tyrosine, L-glutamate, D-serine), when they were placed on the L side only, are relatively low (ratios of 0.1 to 0.2). This suggests that permeability for entry of external amino acids across the basal surface into the rectal tissue is 3 to 8 times greater than across the apical border. The amino acid analyser estimations of relative amino acid loss to the lumen and hemocoel side, when the bathing media'.have initially no amino acids, also support this conclusion. The location of a carrier mechanism at the basal or lateral borders seems unlikely for the following reasons: a) It would be difficult to account for the high T/L x4C ratio observed for transported amino acid, unless extremely high concentrations (100 fold) were postulated for lateral or sub-epithelial intercellular spaces. b) Such high concentration ratios would be difficult to reconcile with the relatively high permeability of the basal border which is observed. Moreover, i t would be energetically wasteful to have a transport mechanism in parallel with a large passive leak. On the other hand, high permeability of basal border would facilitate movement to the hemocoel of amino acids accumulated in the tissue by a mechanism located at the apical border. Indeed, passive exit by diffusion -92-is supported by the observation that the rate of C-serine efflux from the tissue to the homocoel side is a linear 14 function of the C-serine content in the tissue measured over a wide range of concentration, c) There is no evidence for any amino acid transport across the serosal borders of epithelial membranes in other animal groups. On the other hand, there is considerable evidence for such a mechanism on the apical side (reviewed by Curran and Schultz, 1968). In summary, a l l the experimental observations presented in this chapter are consistent with the hypothesis that the main barrier to the penetration of organic molecules by simple diffusion is at the apical side. The properties of the short-time (10 minutes) influx of ^C-glycine from lumen into rectal tissue are consistent with a selective entry process at the apical side. The very large T/L ratios observed for trans-ported amino acids are most easily reconciled with accumulation across the apical border where back diffusion would be less due to relatively low passive permeability. It is interesting that the predominant free amino acids in rectal tissue in vivo at the time of dissection are also the most rapidly transported by in vitro sacs, and are the ones most rapidly lost across the basal border when external amino acids are absent. Finally, the observations on amino acid content of rectal tissue and transport rates observed with rectal sacs may be of some relevance.to the question of water transport by the locust rectum - 9 3 -(reviewed by Phillips, 1970). The most promising hypothesis to account for hyposmotic fluid transport is one of local osmosis and solute recovery (recycling) within the lateral extracellular spaces (Wall,1971). This theory requires a high osmotic pressure within the cell to permit flow into the latter from the lumen. The present experiments suggest that amino acids or urea are not likely to create the required high osmotic pressure. Indeed, the estimated concentration of tissue ions and amino acids, together was rather similar to that of the hemolymph, even though determinations were made on recta maintaining a 420 mosm/1 gradient. Furthermore, i t seems unlikely that the hypothesized solute driving water movement is an amino acid, although the latter might make some contribution. This tentative conclusion is made because water transport continues for up to 6 hours at a rate of 6-7 yl/hr./rectum when no amino acids are present in the two bathing media (Goh,1971). The results presented in Section C indicate that, under such conditions, most of the tissue amino acids are rapidly lost, but water absorption does not disappear. CHAPTER V SODIUM DEPENDENCE OF GLYCINE TRANSPORT A. Introduction With a few notable exceptions, amino acid transport systems which have been demonstrated in various animal cells and tissues are Na+ dependent processes. In general Na+ is. required for uphill transport, but not passive exchange (i.e. facilitated diffusion) of amino acids and sugars. The dependence on Na+ is quite specific in a number of intensively studied systems, but there have been suggestions of dependence on other ions in other cells and tissues. Certainly K+ has been shown to have stimulatory and inhibitory effects at low and high concentrations respectively on Na+ dependent amino acid transport (reviewed by Schultz t and Curran, 1970). The first objective of the experiments described in this chapter was to establish whether the amino acid transport across the rectal sacs is a Na+-dependent process. There are at least three possible explanations for the Na+ dependence of metabolite transport: 1. Sodium is required as an activator of a carrier mechanism, by influencing the membrane structure to favor amino acid transport. More specifically, i t has been suggested (Matthews, 1972) that alkali metal ions affect amino acid transport through inter-action with the polar groups in the lipid layer of the membrane. 2. Sodium is required for direct activation of the energy output process (Kimmich, 1970). According to this hypothesis, metabolite transport might be driven by (i.e. directly coupled to) an -94--95-energy-yielding metabolic reaction. This would then be a case of primary transport. 3. More widely held is the Na+ gradient hypothesis. According to this model, metabolic energy is used directly to transport Na+ out of the c e l l , thereby maintaining a Na+ activity gradient across the plasma membrane. If amino acids and Na+ share a common transfer mechanism for entry into the c e l l , then the passive influx of Na+ down its activity gradient could provide the energy for accumulation of an amino acid in the cell against its activity gradient. Thus, metabolic energy would not be directly invested in amino acid move-ment, but in a primary Na+ pump. This model, therefore, represents a case of secondary transport (see Stein, 1967, Chapter 6, for definitions of primary and secondary transport). The Na+ gradient hypothesis makes the following predictions (Schultz and Curran, 1970): 1) The entry of glycine into the rectal tissue should be increased in the presence of external Na+. 2) There should be a mutual effect of external glycine on Na+ influx and possibly accumulation in rectal tissue. Thus, a stoichiometric relationship should exist between Na+ and amino acid transfer. 3) Reversal of the Na+ activity gradient should cause reversal of amino acid transport. 4) Agents inhibiting the active Na+ extrusion mechanism (e.g. ouabain) should, in time, result in disappearance of the Na+ -96-gradient and active transport of amino acids should no longer be possible, even though external Na+ is present. The first three of these predictions were tested in the case of glycine transport across rectal sacs of the locust. B. The influence of monovalent ions on the net transport of glycine.. across the rectal wall The effect of Ions on glycine transport was studied by incubating rectal sacs in various ion-free Ringers (Table I) containing 10 mM of x^C-glycine (constant specific activity). Identical media were placed on both sides of the sacs and net water movement was prevented by adding 420 mosm/1 sucrose to the external medium. The H/L ratios of x"*C-activity and the changes in volume that develop with time (after pre-incubation) are shown in Fig. 23. Average rates.calculated from Fig. 23 are given in Table XIII. When a l l external Na+ was replaced by K+, the H/L ratio of glycine (maximum of 1.8/1) was greatly reduced compared to the control (10/l) and did not change after the second hour. In other words:, net glycine transport -did—'not occur between the second and sixth hour of incubation when Na+ was absent. Loss of tissue Na+ to the bathing medium might contribute some trace amounts of this ion which might account for the slight i n i t i a l accumulation of glycine observed. However, in other systems which have been studied, the amino acid transport is completely inhibited in the absence of external Na+, even when a tissue pool of sodium is present (Schultz and Curran, 1970). Therefore, a small Na+-independent component F ig- 2 3- T h e C-glycine ratio (H/L) and the net movement of water across the rectal wall in media of various, composition'. The different Ringer solutions used as bathing media contained initially 10 mM of x4C-glycine (same specific activity). They are listed as: Na+-free (high K4") Ringer (A); K+-free (high Na+) Ringer (•); CI. .-free (high N03) Ringer (O); and control (Mordue Ringer) (•). Sucrose (420 mosm/1) was present in the external (L) medium to prevent net water absorption. Each point represents an average value of 4 experiments. Vertical lines indicate ± S.E., if the latter was not included within the symbol. -97-VOLCHANGE (ul/hr/rect.) 1 4 C - G L Y C I N E ACTIVITY RATIO (H/L) I + — * —» O —» O N D C O 4 S ( _ n C D - v j C D C D O -98-Table XIII Net rates of glycine and water absorption by In vitro  recta in media of various composition. Values were calculated from data shown in Fig. 23. Average net rate (Mean ± S.E.) RC-glycine Medium + for 6 hrs. concentration Glycine (nmoles/hr./rectum) Water (pl/hr./rectum) ratio at 6 hr. (H/L) Sodium-^ f ree Ringer Potassium-free Ringer Chloride-free Ringer Control 1122 ± 1.3 0.15 ± 0.07 36.36 ± 6.3 0.33 ± 0.14 44.5 ± 11.8 -0.06 ± 0.09 1.8 ± 0.12 3.7 ± 0.50 5.4 ± 0.87 106.0 ± 11.0 0.40 ± 0.90 10.0 ± 0.50 * - + + Na replaced by K **K+ replaced by Na+ ***C1~ replaced by NO^  +Composition of various media are shown in Table I. -99-of glycine transport (20%) cannot be excluded in the case of rectal sacs. Only a few other epithelial membranes have been reported to contain a Na+-independent amino acid transport component (Nedergaard, 1972; Munk and Schultz, 1969; Thier, 1968; Fox et a l . , 1964). Replacing the small amount of K* (6.4 mM) in the control Ringer with Na+ (Na+-Ringer) caused a 60% reduction in the rate of glycine absorption. However, this rate of glycine transport was maintained for 6 hours, unlike the situation in Na+-free Ringer. There is obviously not an absolute requirement for external K+, but low levels of external K+ appear to be essential for maximum stimulation of glycine transport. This has been widely observed in other systems (reviewed by Schultz and Curran, 1970). The accumulation of glycine after 6 hours was reduced by 50% when a l l external Cl~ was replaced with NO^  , although a steady rate of transport was maintained over the 6 hour period. The influence of Cl~ on amino acid transport has not been extensively investigated in other systems. Recently, Imber & Vidaver (1972) found that replacement of external CI by N0~;ions reduced glycine uptake by pigeon red blood cell by 35%. The substitution of NO" for Cl~ is known to reduce the P.D. across in vitro rectal sacs (Goh, 1971). If this reduction occurs largely across the apical plasma membrane, as proposed by Goh (1971), the electrochemical gradient for Na+ across this border would be greatly decreased. Thus, the resulting decrease in Na+ influx might be directly responsible for inhibition of glycine transport rather than the absence of external CI-. -100-Another explanation for the inhibitory effects of the ions substitutions described above is that general metabolic activity or viability of the rectal epithelium is not maintained. There are positive reasons to believe this is not the case. Under a l l of the above conditions, no significant net water movement occurred (Fig. 23), indicating the ability of a l l preparations to maintain a large osmotic gradient for 6 hours. Such gradients decrease rapidly when rectal sacs are exposed to 10-3 M KCN. Goh(1971) studied rates of water and ion transport across in vitro rectal sacs under isosmotic conditions. The same ions substit-utions as those used in the present study did not cause any significant change in the rate of water movement over a 6 hour period. In fact, the absorbate was most'^ hyposmotic to the bathing media in Na+-free (high K+)Ringer. The decreases in Na+ transport were compensated for by an increase in K1" transport. Thus, ion substitutions which do not appear to alter significantly either the total ion or the water transport across rectal sacs do in fact inhibit glycine transport. This further proves that the glycine transport is not due entirely to a non-specific frictional drag associated with water and ion flow, or to a re-cycling within the rectal wall. The effect of ion replacements on glycine transfer would seem to be more direct. ' '- • C. The effect of Na"1" on the tissue accumulation of glycine 1. Replacement with K"*" According to the Na+ gradient hypothesis, the influx and the accumulation of glycine in the rectal tissue should increase with increasing external Na+ concentration. Consequently, the entry step -101-should be the Na dependent one (Crane, 1965; Csaky, 1963). So far, no evidence has been obtained for a. Na"1" dependent efflux of amino acids across the serosal border of any epithelia is Na+ dependent (Schultz and Curran, 1970). In my experiments, the time dependent accumulation of ±4C-activity 14 in rectal tissue was measured after adding 10 mM of C-glycine to the external bathing medium. Net movement of water was prevented with sucrose. The effect of replacing a l l external Na+ with K+ is shown in Fig. 24. In normal Ringer, the x^C-glycine accumulation increases linearly with time during the first 2 hours, reaching a T/L ratio, of 11/1 by the third hour of incubation. In the absence of external Na+ (i.e. KT^-Ringer), the ini t i a l linear rate of glycine accumulation (30 minutes) was only 50% of the control rate. Moreover, accumulation f e l l off quickly and reached a constant T/L ratio of only 1.8/1 after the first hour of incubation. This Na+-dependent increase in influx is relatively small compared to the 10 to 30 fold increase reported for several other systems' (Schultz and Curran, 1970). At times sufficient for the establishment of a steady-state or equilibrium, the T/L ratio of 1.8/1 (Fig. 24) is identical to the H/L ratio (Fig. 23) of -^C-glycine in the absence of external Na+. This is consistent with a small Na+ independent component for glycine entry into the rectal tissue and a passive movement to the H compartment. Other evidence that -^C-glycine concentrations are comparable in tissue water and internal medium at equilibrium is also provided by studies with inhibitors ( Fig. 13). Fig. 24. .The effect of Na+ on the 14C-glycine 'accummulation by rectal tissue. Rectal sacs were .exposed to normal (•), or Na+-free (high K+) Ringer (A) containing 10 mM of C-glycine and 420 mosm/1 of sucrose externally. Each point is the average of at least 4 experiments; vertical lines indicate ± S.E. The broken line indicates the external level of ^C-glycine. -102-0 60 120 180 T I M E (min) -102a--103-The % dry weight of rectal tissue incubated in K+ -Ringer was 20.9± 0.6 (Mean ± S.E., 24 preparations) compared to 23.8 ± 0.5 (Mean ± S.E., 19 preparations) for the controls. These results indicate a slight swelling of the tissue in high K+ media, as previously reported by Goh (1971). Nevertheless, an increase of 3% in the tissue water content cannot account for the large decrease in glycine accumulation in Na+-free media. 2. Replacement with Choline In order to demonstrate that the inhibition,of glycine uptake observed in KT^ -Ringer was due to the lack of Na+ and not to the high concentrations of K+, Na+ was replaced by choline in the test solution. Fig. 25 shows the tissue uptake of "^C-glycine with time when rectal sacs were incubated in choline-Ringer containing 2.5, 10 or 20 mM of •*-4C-glycine and 200 mosm/1 of sucrose in the external media. The uptake of ^C-glycine is linear over the first 15 minutes at a l l 3 glycine concentrations tested. These i n i t i a l rates of accumulation are considered unidirectional influxes under these conditions (see Chapter IV, Section E). Previous experiments with Mordue Ringer (Fig. 17), which were performed at the same time and on the same batch of animals, served as controls. Pre-incubation under both conditions was in normal Ringer. A double reciprocal plot of glycine influx rate versus external glycine concentration is shown in Fig. 18. The glycine influx fits the Michaelis-Menten Kinet ics, either in the presence or absence of external sodium. At 10 mM of external glycine, the influx of the latter is twice as large as in choline-Ringer (Table C, Appendix). Fig. 25. The effect of Na+ on the 14Crglycine influx into the rectal tissue. Rectal sacs were incubating in Ringer containing different concentrations of x^C-glycine added to the external compartment. Na+ was replaced by choline in the Ringer solution. Sucrose (200 mosm/1) was present in the external Ringer to prevent significant net water movement. The uptake of glycine was estimated from 14c-activity, using the specific activity of the external medium. External glycine concentrations were 2.5 mM (T) ; 10 mM (•); and 20 mM (O). (Each point represents the mean of 4 preparations; vertical lines indicate the ± S.E.). -104-C-GLYCINE UPTAKE (n moles/10mg. rectum) -104a--105-This is quantitatively similar to the effect of replacing external Na with K (Fig. 24). ' Na replacement (Fig. 18) causes a two fold increase in apparent for glycine entry without changing the Vmax. In other systems, Na replacement causes an increase in Kj, alone, a decrease in V only, or changes both the Km and the V max J ° J- max of the amino acid transport.(Schultz and Curran, 1970) D. The effect of external Na+ concentration on glycine influx According to the Na+ gradient hypothesis, both the amino acid and the Na+ combine with the same 'carrier' to cross the apical plasma membrane. Thus, unidirectional influx of glycine should be a saturable function of both external glycine (Fig. 18) and Na+. In some vertebrate tissues, the amino acid flux is a hyperbolic function of external Na+ concentration; i.e. a double reciprocal plot of external Na+ versus amino acid influx is linear. This suggests that one Na ion participates as a co-substrate for the transfer of a single amino acid molecule. Other systems, however, exhibit a sigmoid relationship, i.e. a double reciprocal plot of the Na+ concentration raised to a higher power versus the amino acid influx would yield a straight line. This . suggests that two or more Na+ ions are co-substrates for each amino acid molecule transferred (reviewed by Schultz and Curran, 1970. Christensen, 1971). The relationship between external Na+ concentration and the rate of 14 C-glycine influx into rectal tissue was studied while keeping the external concentration of glycine constant at 5 mM. Na+ was replaced with choline (Fig. 26 and Table C, Appendix). 14 The accumulation of C-glycine in the rectal tissue was approximately Fig. 26. The effect of different concentrations of Na+ on the glycine influx. Rectal sacs were incubated in media initially containing l^C-glycine ( 5mM) and different concentrations of Na+ (replaced with choline) in the external compartment. Na+ concentrations were: 0 (•); 22 (O); 44 (•); 87 (•); and 174 (•) mM. In a l l cases the water movement was prevented with sucrose (Table C, Appendix). Influxes were calculated from tissue x^C-activities using the specific activity of the external medium. Vertical lines indicate the ± S.E. of the mean of 4 preparations. -106-[Na](mM/U 1 2 3 4 5 6 TIME (min.) -106a--107-linear during the first 6 minutes of incubation at most Na concentrations (Fig. 26). The estimated rates of glycine influx are plotted against the external Na+ concentrations in Fig. 27. The relationship is sigmoid or possibly exhibits a maximum at approximately 40 mM Na+. The increase in glycine influx occurs as the external Na"1" is increased from 20 to 40 mM (P •> 0.01). This suggests that more than one sodium might accompany each glycine into the cell and that saturation of the transfer system with Na+ is achieved at relatively low external Na+ concentrations. Of course, this would apply only to the Na+-dependent fraction of influx. E. Efflux of glycine Removal of external sodium had a considerably greater effect on the T/L ratio ( 6-fold) than on the i n i t i a l rate of ^C-glycine influx (2-fold) into the rectal tissue (Fig. 24). This suggests that the external Na+ might enhance the tissue accumulation of glycine by inhibiting the back flux (efflux) of this amino acid from the tissue to the external medium, although this has not been widely observed in other systems (Schultz and Curran, 1970). However, such an effect has been reported by Vidaver and Shepard (1968) for glycine efflux from pigeon erythrocytes, but this inhibition is relieved by increasing external glycine concentr-ations. To estimate the efflux, a l l rectal sacs were first pre-incubated on both sides with Mordue Ringer containing 10 mM of ^C-glycine for one hour. By this time, the glycine content of the rectal tissue had increased to approximately 50 mM/kg. tissue water and the radioisotope in the tissue had largely equilibrated with that in the external medium (Fig. 24 and Fig. 27. The influence of the external Na"1" concentration on the in i t i a l rate of -"-^C-glycine influx into the rectal tissue. The external concentration of glycine was 5 mM. Choline was used to replace Na in normal Ringer. Each point is the average of 12 determinations, taken from Table C (O) and from Table A (•), Appendix, for two groups of locusts. The influxes were estimated from tissue ±^C-activities, using the specific activity of the external medium. (Vertical lines indicate ± S.E. of the mean) -108-400 44 87 [Na] (mM) 174 -108a--109-Table IX). The sacs were then rinsed with an isosmotic mannitol solution (< 1 minute) to remove surface radioactivity. Control sacs (Na+ present) were filled with fresh labelled media and placed in 1 ml. of Mordue Ringer containing 10 mM of unlabelled glycine. Experimental sacs (Na+-free) were treated in the same manner except that K+ replaced a l l Na+ in the fresh media added to the external side of the rinsed sacs. (Net water movement was prevented at a l l stages). Aliquots ( 25 ul) of 14 external medium were then removed periodically for estimations of C-activity. The increase in the external radioactivity, as i t develops with time, is shown in Fig. 28. Since unlabelled glycine from the external medium is rapidly absorbed, the specific activity of labelled glycine in the rectal tissue undoubtedly falls rapidly following transfer. This probably accounts 14 for the hyperbolic nature of C-efflux in Fig. 28. Thus, unidirectional efflux can only be accurately estimated over the i n i t i a l linear, phase of the efflux curve ( first 2-4 minutes) before there is time for large changes in the tissue's specific activity. Although this method gives only an approximate value of the unidirectional efflux, i t can be consid-ered adequate for the comparison of different experimental treatments. The replacement of external Na+ with K+ causes the glycine efflux to increase approximately 2-fold from 105 ± 17 to 200 ± 40 nmoles/hr./10 mg. rectum (calculated over the first 4 minutes). When the external concentration of unlabelled glycine was raised in the presence of Na+ to 50 mM, which is equivalent to the level in the 14 tissue water after pre-incubation, the efflux of C-glycine was not significantly increased over that of the control (10 mM of external Fig. 28. The efflux of C-glycine from rectal sacs. The rectal sacs were loaded with this isotope during 1 hour pre-incubation in normal Ringer containing 10 mM of x^C-glycine and 420 mosm/1 sucrose externally. After rinsing off the external activity with isosmotic mannitol, the sacs were transferred at time .0. to a .1 ml solution of normal (•sO) or Na*-free (A) Ringer containing either 10(solid symbols; mean ± S.E.) or 50 (individual observations, open circles ) mM of unlabelled glycine. In a l l cases net water movement was prevented by external sucrose (420 mosm/1). The efflux was estimated from the i n i t i a l rate of appearance of 14C -activity in the external media, using the specific activity value in the tissue following the pre-incubation step. -110-- 1 1 1 -glycine (Fig. 28). Although more experiments are required, this preliminary observation suggests that the glycine efflux rate is not greatly influenced by external glycine concentrations which would indicate that there is l i t t l e or no transconcentration effect ("'. V-idaver and Shepard, 1968). F. The effect of external glycine on the accumulation of 22Na+ by  the rectal tissue If glycine and sodium share a common entry mechanism (as proposed by the Na+ gradient hypothesis), adding glycine to the external medium should 22 + increase the unidirectional influx of Na into the rectal tissue. This 22 + might be expected to result in a greater accumulation of Na in the tissue, unless the efflux to the inner or outer bathing media are also accelerated by this treatment. Thus, a positive demonstration of an increase in tissue Na may be taken as an evidence in favor of the Na gradient hypothesis. A negative result, however, would not exclude this hypothesis (Schultz and Curran, 1970). To test this prediction, rectal sacs were first pre-incubated for 1 hour in Mordue Ringer with no glucose or amino acids. Net movement of water was prevented with sucrose. The sacs were then transferred to the 22 + same external media (containing 174 mM of Na ;0.6uC/mraole,with or without 10 mM.-glycine. . The glucose also was omittted ( replaced with additional isosmotic amount of sucrose) from the incubation medium because, in some cases, glucose is reported to have an inhibitory or stimulatory effect on the amino acid transport systems (Schultz and Curran, 1970). The tissue 2 2 + contents of water and Na were determined after 1 hour (Table XIV). No 77 + significant difference in the Na concentration in the tissue is observed -112-Table XIV The effect of external glycine on /<;Na*i" levels in rectal tissue Rectal sacs were pre-incubated for 1 hour in Mordue Ringer with no amino acids or glucose before they were+transferred to a similar Ringer containing Na and 420 mosm/1 sucrose (externally). Glycine (10 mM) was present in the external medium, except in the control. After 1 hour incubation, the external 22Na+ was rinsed off the rectal tissue with an ice-cold isosmotic mannitol solution and the isotope concentration of this tissue was then determined. The Na+ concentration in the tissue was estimated from the external specific activity of 22a^+ (m e a n + S.E.). Treatment (incubation solution) No. of exp. Rectum D.W. (mg.) Tissue H20 content (% W.W.) Na+ (mequiv. concentr- Na+/Kg. ation tissue (nmoles/ R^ O) 10 mg.rectum Without glycine 4 2.5 ± .06 74.9 ±0.7 826 ± 151 110 ± 21 (control) With glycine (experimental) 4 2.7 ± .18 74.2 ± 1.1 879 ± 105 119 ± 16 -113-in the two treatments. A more direct test of this prediction would be to study the uni-77' + directional influxes of Na into the tissue over short periods of time. However, the net rate of Na+ transport across the rectal sacs in media lacking amino acids ( 600 nmoles/hr./rectum; Goh, 1971) is approximately six times the net rate of glycine transport at 10 mM external glycine (106 nmoles/hr./rectum; Table II). If only half the influx of glycine is Na~*"-dependent (Fig. 27) , but two Nations accompany each of these glycine molecules (Section D), then on the average, one Na+ should enter the tissue with each glycine molecule. Then the rate of Na+ influx into the rectal tissue might be increased by only 15% or less upon addition of 10 mM of external glycine. Considering the variability observedcin this study, changes of this magnitude would be extremely difficult to detect. Experiments employing lower Na+ (40 mM; Fig. 27) and higher glycine concentrations externally might be more successful. G. The net transfer of -*-^C-glycine upon the reversal of the Na+ gradient Perhaps the most direct and compelling evidence for the Na+ gradient hypothesis in several vertebrate systems is that the direction of amino acid transport against an activity difference can be reversed upon reversal of the Na+ activity gradient between intracellular and external compart-ments. Such experiments involve the inhibition of the active Na+ transport with ouabain or KCN (Hajjar e_t _al. , 1970). However, Schultz and Curran (1970) emphasize that "unfortunately, studies of efflux kinetics are fraught with difficulties" because i t is difficult to determine with certainty the concentration, let alone the activity of Na+ and glycine in various compartments of the ce l l . This problem has been convincingly -114-surmounted in experiments with erythrocyte ghosts (Vidaver, 1964). There have been other studies, however, where amino acid movement has been reversed when the concentration of Na+ in the tissue exceeds that of the external medium. It is at this level of uncertainty that the preliminary experiments were conducted on rectal sacs of the locust. Rectal sacs were pre-incubated on both sides for 1 hour with Mordue Ringer containing 10 mM of ^ C-glycine and 10-3M KCN. Under these conditions the active abosrption of Na"r, glycine and water across the rectal wall are completely inhibited ( Fig. 13; Goh, 1971). By the end- of the pre-14 incubation, the concentration of C-glycine in the tissue is only slightly greater ( 1.2X; Fig. A, Appendix), whereas Na+ slightly lower (151 ± 9 mM/ kg. tissue water; Table E) than in the external medium. No significant concentration differences exist between the internal and the external bathing media (Fig. A, Table E, Appendix). At this point, 10 y l . of fresh radioactive media (Mordue Ringer) containing KCN was placed inside the sacs. Half of the preparations were exposed externally to fresh labelled Mordue Ringer. The other half of the preparations were placed in Na+-free (high K+) Ringer containing 10 mM of "^C-glycine at the same specific activity as in the controls. Thus, in the experimental preparations, a downhill gradient was estab-lished favoring net Na+ movement from H to L side (also from T to L). If glycine movement is coupled to passive Na+ flux by a reversible mechanism, the internal concentration in the small H compartment, and hence the H/L ratio of C-glycine,might be expected to f a l l significantly below 1/1. This would indicate a reversal of net glycine movement against a concentration gradient. -115-The results are presented in Table XV. After 3 hours of incubation the H/L ratio of 14c_giyCin e na& n ot changed significantly from the ini t i a l value for either experimental or control preparations. These results do not confirm the prediction of the Na+ gradient hypothesis; however, i t would be premature to consider this negative result as rigorous evidence against the hypothesis. A further series of experiments is first required in order to determine (a) whether the membrane permea-bility and the integrity of the entry mechanism for glycine are maintained during the KCN inhibition, (b) whether the transfer of glycine might have been reversed across the apical, but not the basal border, (Some prelim-inary observations suggest that when external Na+ is removed, the T/L ratios f a l l below 1/1 (Fig. A and Table E, Appendix), but this f a l l might have been caused by the tissue swelling under high K"1". When the comparison is done on the basis of dry weight, there is no difference between controls and experiments), (c) finally, i t is necessary to ascertain whether any significant net Na+ efflux occurs^under the experi-mental conditions employed. Studies by Goh(1971) indicate that a sub-stantial net efflux of Na+ (110 nmoles/hr./rectum) should have occurred in the present experiment. H. Summary 1. Glycine transport across the rectal wall may involve a small Na+ independent component. External high Na+ and low K+ concentrations are essential for prolonged glycine transport at maximum rates. Sub-stituting NO^  for Cl~ also causes partial inhibition of glycine transport. -116-Table XV The H/L ratio of l^C-glycine upon reversal of the Na"1" gradient Rectal sacs, pre-incubated in normal Ringer containing C-glycine (10 mM) and 10_3M KCN, were transferred to fresh normal Ringer (control), or K"t" -Ringer (experimental) , both containing 14C -glycine and KCN at the same concentrations and specific activities. Under both conditions, normal radioactive Ringer (10 ul) was left inside the sacs. Values are given as mean ± S.E. of 4 preparations. H/L ratio H_0 Treatment ^ absorption 0 hr. 3 hr. (ul/hr/rectum) Control (no Na+ gradient) 1.07 ± 0.02 1.14 ± 0.02 Experimental (Na+ gradient reversed) 1.01 ± 0.02 1.07 ± 0.05 0.62 ± 0.26 0.25 ± 0.32 -117-2. Na dependence of C-glycine accumulation in rectal tissue is a consequence of both an increased influx (K_, but not V ) and a decreased efflux of glycine between the external media and the tissue. The enhancement of glycine influx behaves as a quasi-sigmoidal function of the external Na+ concentration. 3. The tissue concentration of Na+ was not affected by changes in external glycine levels. The net transfer of glycine across KCN-poisoned rectal sacs was not reversed upon reversal of the-Na+ activity gradient. The Na+ gradient hypothesis could not be rigorously excluded on the basis of these observations alone. I. Discussion In most groups of animals in which intestinal amino acid absorption has been studied, the naturally occurring L-isomers of neutral amino acids are absorbed against a concentration gradient by a process involving at least one carrier. This absorptive process shows a general specificity for the monoaminocarbbxylic acids. Such a process has been frequently described in the literature for the mammalian intestine (reviewed by Wiseman, 1968). More recent studies with in vitro intestinal preparations of marine invertebrates have also demonstrated the presence of such a neutral carrier, as in the mollusc,Cryptochiton stelleri (Greer & Lawrence, 1967), the echinoderms,Stichopus;parerimensis and Echinus esculentus (Bamford and James, 1972), and in the annelid, Arenicola marina (Bamford and Stewart, 1973). • Ion-dependence of the amino acid transport is a - wide spread phenomena ?in various animal tissues (Schultz and Curran, 1970) . Among the monovalent ions Na+ and K+ are the pair of ions more -118-thoroughly studied (Christensen & Riggs, 1952; Krompardt et al . , 1963; Csaky et al . , 1961, 1963). However, relatively few studies have been carried out to determine whether the same type of phenomena occur in invertebrates in general and in insects in particular. The main question which the present chapter attempts to answer is whether the carrier mediated process is a secondary (Na+ gradient hypothesis) or a primary transport mechanism. The results of the present chapter have shown that: (a) when NO^  is substituted for Cl~, glycine accumulation is reduced by 40%, (b) when K is replaced by Na , 50% inhibition is obtained,and (c) when Na+ is replaced by K1", an 82% inhibition is obtained. In (a) and (b) glycine continues to accumulate slowly throughout the experimental period. However, in (c) the inhibition is completed after the first hour and there is no accumulation of glycine. These results show that the amino acid transport mechanism in locust rectum is sensitive to the ion content of the external medium, particularly to Na+. The latter ion increases the influx and decreases the efflux across the apical border. I tried next to see i f my findings f i t the "Na+ gradient hypothesis" first suggested by Crane (1965). According to this model (Fig. 29) there is a coupled entry of Na+ and organic solutes into the cell across the brush border side. The Na+ level in the cell is kept low by an energy dependent active extrusion pump located at the serosal border. Na+ might facilitate the amino acid accumulation in the c e l l , either by sharing a common carrier with the amino acid, or else Na+ might act to decrease the ICj, for amino acid binding to the carrier and hence facilitate the entry. In either case, low internal concentration of -119-Fig. 29 Schematic diagram of the co-transport model (Na"1" gradient hypothesis) . The model was first proposed by Crane for the mammalian intestine. E-r is a facilitated diffusion carrier, co-transporting sodium and glucose, or amino acid. E2 is a sodium pump sensitive to ouabain and requiring ATP. The height of the blocks below the main diagram indicate the prevailing levels of amino acid and sodium within the epithelial cell (i) and the intestinal lumen (e). Phlorizin' selectively inhibits the sugar transport. Serosa Epithelia Mucosal surface SB N a e A e -120-Na results in more amino acid bound to the carrier moving from the lumen side and amino acids are released into the cell interior and hence, net amino acid transport occurs. In this case, a direct link of the transport process to a metabolic energy supply is not necessary. There is an indirect link, though, since ATP is utilized by the Na+ pump to keep the internal Na+ concentrations.' low. Phillips (1964b) and Goh(L971) have shown, in vivo and in vitro respectively, that there is an asymmetrical distribution of alkali ions across the apical membrane of the locust rectum. These observations, together with the present evidence for carrier mediated entry at the apical border, suggest that a co-transport model may be operative in the rectal epithelium. An important question for consideration in vivo is whether a downhill Na+ gradient normally exists across the rectal wall. The Na+ concentration in rectal tissue of locusts kept 3 days on tap water ( similar to the present treatment) was found to be 55 mequiv./ Kg. tissue water. The hemolymph of locusts fed on hypertonic saline contained 160 mequiv/l Na"*and that of water fed animals contained 90 mequiv./l Na+(Phillips, 1964b). The U/P ratio for Na+ excreted by locusts Malpighian tubules is approximately 0.2 (Phillips, 1964, b,c). However, Na"1" concentrations increase or decrease in the rectal lumen, depending on the environmental conditions. The Na+ concentrations in the rectal lumen of dehydrated locusts can become very high and there is no doubt that a downhill gradient for Na+ entry into the tissue exists-. The situation in hydrated, ion-depleted locusts is less certain. Further studies of N a + activity across the apical membrane under the latter condition would -121-provide a crucial test of the Na gradient hypothesis. It is questionable whether a downhill Na+ gradient from L to T side has existed in the present experiments. The glycine influx increased markedly when Na+ in the external media was raised from 20 to 40 mM. Since the pre-incubation was done in normal Ringer and the influx was determined within 1-6 minutes, I assumed that the Na+ concentration of the rectal tissue did not change significantly in such a short time.If this assumption is correct, then the glycine accumulation might have occurred in the absence of a favorable Na+ downhill gradient, since Goh(1971)reported that the Na+ concentration in the tissue, under similar incubation condit-ions, is about 80 mequiv./Kg: tissue water. A major difficulty in any conclusive evaluation of this hypothesis is the mncertainty about the exact distribution and activity of intra-cellular Na+. A direct determination of Na+ gradient across apical border would require the use of Na+ selective glass microelectrode (Lee and Armstrong, 1972) to determine the Na+ activity in the cellular compartment. One of the predictions made by the-Na+ gradient hypothesis (Schultz and Curran, 1970) was confirmed with rectal sacs of the locust: the entry of the amino acid into the tissue is dependent on the presence of Na+ in the external solution. Positive confirmation of two Other predictions, however, was not obtained, since the external glycine did not increase the Na+ content of rectal tissue and the reversal of the electrochemical gradient for Na+ did n°t cause a reversal of the amino acid transport. If there is a common entry step for glycine and Na+, adding the glycine to the external medium should increase the unidirectional influx of Na. I have found that after 1 hour of incubation, the level of Na - 1 2 2 -was not significantly affected by the presence, or absence, of glycine (Table XIV). However, this does not necessarily contradict the prediction, because it is not the net Na+ accumulation that is c r i t i c a l , but rather the rate of Na+ entry. It is possible that extra Na+ entry is matched by additional extrusion of Na+ from the tissue resulting in no net accumu-lation. Since the net Na+ transport across the rectal wall without glycine is 6 times higher than the glycine flux, one could expect to observe at most, a 15% increase in the net influx of Na+. However, such changes would be difficult to detect. Probably the most direct evidence for the Na+ gradient hypothesis is the demonstration that the direction of amino acid transport against a concentration gradient can be reversed when the gradient of Na+ activity between the intracellular and external compartment is reversed. The present results do. not seem to be in line with this view, since i t is quite clear that under this experimental condition, a substantial Na+ efflux to the L side should have taken place (Goh, 1971). However, i t is possible that KCN may have affected the integrity of the glycine transport system, in addition to inhibiting the energy sources of the ce l l . The more specific inhibitor, of Na+ transport, ouabain (584 M.W.) could not be used because this compound does not penetrate through the intima and thus could not reach any Na+, K+-ATPases. In summary, the present results demonstrate that Na+,and other ions to a lesser extent, have an important role in the active transport of glycine across the locust rectal epithelium. However, a number of findings do not support the Na+ gradient hypothesis: 1) the reverse of the Na" gradient did not drive glycine back into the lumen against its own -123-concentration gradient; 2) there is a small amount of transport against a concentration gradient in the absence of Na+ in the external medium; 3) the presence of glycine on the L side did not increase the Na concentr-ation gradient in the tissue. Although these three observations do not support the co-transport model, i t would be premature to make a clear choice between the Na+ gradient hypothesis and a primary transport mechanism on the basis of the present results. However, observations in this chapter indicate the presence of at least a small Na+ independent component. A Na+ independent amino acid transport phenomena was recently reported in Cecropia (Nedergaard, 1972). In recent years considerable evidence has been presented which argues against the classical Na+ gradient hypothesis. On the basis of their studies of amino acid uptake by 53-S ascites tumor cells, Matthews, et a l . , (1971) have suggested an alternate hypothesis regarding the effect of ions on sugar and neutral amino acid transport. This hypothesis postulates an interaction between the polar groups of the lipids and the transported solutes. They suggest that various ions facilitate transport of amino acids by screening the strong field sites associated with the polar lipids on the surface of the membrane. An alternate model for a Na+ dependent, metabolite transport, which is directly driven by a metabolic reaction,was recently proposed by Kimmich (1970). In a very elegant series of experiments he showed that rates of accumulation of sugar and amino acids are independent of the Na+ gradient and the process is inhibited instantly by ouabain and oligomycin before the Na+ gradient is dissipated. Kimmich proposed that energy for a -124-number of energy-dependent transport processes may be derived from a common intermediate which is generated by the Na+ + K+ activated ATPase. Although in the present work there is some evidence which is not consistent with the Na+ gradient hypothesis, there is no doubt that Na+ plays an important role in the transport process of glycine across the locust rectal epithelium. Further work is required in order to reveal the exact mechanism by which Na+ influences the amino acid transport system. CHAPTER VI GENERAL DISCUSSION The results and arguments presented in previous sections of this thesis support the view that the uptake of amino acids by the rectum is an active and saturable process which might play an important role in controlling the levels of amino acids in the hemolymph. Since the present results were obtained with a viable in vitro preparation, caution obviously must be used in the extrapolation of these results to the in vivo situation. Nevertheless, some lines of evidence support such an extrapolation.cFor example,the:-rates of ion and water transport in vitro are in agreement with the rates observed in vivo, as previously discussed (Chapter III. ). The first question one has to answer is whether the observed reabsorption rates of in vitro rectal sacs can possibly account for the complete recovery of amino acids normally secreted by the Malpighian tubules. The latter situation is the case when blood levels of amino acids are near normal and no net excretion of the latter compounds from the rectum takes place. The rate at which an amino acid is delivered to the rectum of an adult locust in vivo can be represented by: R= VMJ/P.C where R. is the rate of Malpighian tubule secretion of an amino acid in nmoles/hr., y_ is the volume secretion rate by the tubules in ul/hr.., U/P is the concentration ratio of the amino acid in the tubule secretion over that in the hemolymph, and (] is the concentration of the amino acid (mmoles/1) in the hemolymph. —1-25--126-The rate of fluid delivery into the rectum of hydrated or partially hydrated locusts is approximately 8 ul/hr. This rate is comparable to the rate of fluid reabsorption found in the present in vitro preparations (7.2 yl/hr./rectum, Table II; also Goh, 1971). Although the hemolymph levels of amino acids in adult locusts have been reported by Treherne (1959), i t is not clear whether these values are constant under various environmental and physiological conditions. Nevertheless, these values might be used as a first approximation. So far, U/P ratios are available only for the Malpighian tubules of •• Rhodnius prolixus (Ramsay, 1958). The values observed range from 0.19 for glycine, 0.25 for alanine, to 0.56 for proline. For the present consideration, various U/P values from 0.1 to 1.0 have been used to estimate the possible in vivo rates of glycine secretion by the Malpighian tubules of the desert locust. These secretion rates are compared with the expected i n i t i a l rates of reabsorption in the rectum (Table XVI) using values obtained with rectal sacs (Table II) and using the calculated amino acid concentration in the Malpighian secretion resulting from an assumed U/P value. From Table XVI, one learns that the observed rates of reabsorption in vitro are sufficient to recover a l l the glycine secreted on x±n& as long as the U/P ratio is lower than 0.5 (which is the case for Rhodnius at least). However, the glycine uptake in the presence of net water absorption (which is the normal case in vivo) may be up to 1.7 times greater (Table II). Under such iconditions, a complete recovery of glycine in the rectum might occur,even when the U/P ratio approaches 0.7. Normally this ratio should be less than unity. -127-Table XVI Calculated secretion rates (R) of glycine by Malpighian tubules of the desert locust in,vivo  Tor various U/P ratios"] These values are compared to the reabsorption rates (A) observed with rectal sacs in the absence of fluid movement (Table II). The hemolymph concentration of glycine is assumed to be 33 mM (Treherne, 1959), and the volume secretion rate 8 yl/hr. (Phillips, 1964c). The calculated concentration of glycine in Malpighian tubule secretion is given by C. U/P C R A ratio (mM/1) nmoles/hr. (nmoles/hr.) 0.1 3.3 26 32 0.2 6.6 53 67 0.5 16.5: 132 126 0.7 23.1 184 144 1.0 33.0 264 172 -128-The above conclusions might be complicated by the fact that rectal absorption rates depend on the concentrations of amino acids in the rectum, rather than the in i t i a l concentrations of these substances in the fluid entering the rectum. If the i n i t i a l ratio of water to glycine reabsorption is greater than that in the Malpighian secretion, then the concentration of glycine in the rectum will increase during the absorption (or be higher under steady state conditions). Ultimately, this would place the concentration of amino acids near the saturation level of the reabsorption process. High luminal concentrations might also increase the importance of any passive diffusion component of transfer. Evidence was presented (Chapter IV) for a low rate of diffusion of amino acids from the rectal lumen due to the low permeability of the apical border. Thus, even when the concentration of amino acids in the lumen is high, a net diffusion component should not become significant. A final factor which complicates the picture, presented in Table XVI, is the competitive inhibition between amino acids (Chapter IV) for a common transport system. This can drastically reduce the reabsorption rates. It is possible to make some estimate of this effect by using data in Chapter IV, assuming that the predominant blood amino acids . (serine and glycine) have the same affinity for a common carrier (see Section F, Chapter IV), and a l l other transported amino acids together have an equivalent inhibitory effect. This is a reasonable assumption since the other rapidly transported amino acid, proline, has a relatively low competitive effect on glycine uptake (Fig. 19). If one assumes a low concentration of glycine in the rectal fluid (e.g. 10 mM, equivalent to -129-a U/P ratio of approximately 0.3) and an i n i t i a l reabsorption which occurs in the presence of water flow (Malpighian tubule secretion being isosmotic to blood; reviewed by Maddrell, 1971), then, the reabsorption rate of glycine should be 1/3 of 170 nmoles/hr. (Table II), or 57 nmoles/hr. This is in good agreement with the estimated in vivo secretion rate of 53 nmoles/hr. by the Malpighian tubules under the same conditions. These calculations are not intended to give an accurate quantitative picture, but rather they are presented here in order to indicate that within the limits of available knowledge, the estimated reabsorption rates in the rectum at normal hemolymph levels of amino acids are probably adequate to explain the recovery of most of the amino acids secreted by the Malpighian tubules. Moreover, the rates of secretion and reabsorption are close enough that saturation of the reabsorption mechanism (and hence excretion of amino acids) might be predicted if the hemolymph levels of amino acids increases far above those observed by Treherne (1959). Another possible source of control which should^be investigated is that U/P ratios might increase as the level of blood amino acids increases. A second consideration from the point of view of regulation is whether the relative concentrations of different amino acids in the hemolymph of the locust reflect and are related to their relative absorption rates in the rectum. This comparison is made in Table XVII. The comparison in Table XVII is based on relatively few values (4 at each time, total of 8), so that a better quantitative picture is s t i l l to be obtained. For instance, the accumulation of serine and alanine were not linear with time (Fig. 11). This may be a -130-Table XVII The relative average absorption rates of amino acids by _in_ vitro recta*, compared to relative  concentrations of amino acids in the hemolymph in vivo** Relative Relative Amino Acid Hemolymph Reabsorption Concentration Rate Glycine 1.00 1.00 Serine 1.00 0.72 Alanine 0.11 0.60 Threonine 0.07 0.18 Proline 0.12 1.03 Histidine 0.03 ca. 0.00 Glutamate 0.15 ca. 0.00 Tyrosine 0.07 ca. 0.00 5 Taken from Table V (average over 2 and 6 hour incubation). fe*Taken from Treherne (1959). Glycine = 1. -131-fact, or a result of small sample size. Despite these reservations, the predominant hemolymph amino acids, serine and glycine (70% of total), are amongst the most rapidly absorbed. These two amino acids are present in the hemolymph in equal concentrations and are also reabsorbed at comparable rates. The concentrations of the other amino acids in the hemolymph are about 10% or less than that of glycine and serine. The rate of threonine reabsorption is about 10 to 20% of the value for glycine uptake. Alanine reabsorption may also be lower, but the present data is not sufficient to fully support this view. However, proline is clearly an exception to this relationship. Proline is the predominant amino acid within the rectal tissue (66% of total). Possibly, the low concentration of proline in the hemolymph is not due to a slow reabsorption, but rather to its rapid removal from this fluid by general tissue uptake. Although proline has been shown to be the principal respiratory substrate used by flight muscles in some insects (Bursell, 1963), i t is not that clear whether this also holds for the desert locust. There is a second reason why deviations from the exact relationship between blood concentration and reabsorptive rate might be expected. The U/P ratios for secretion of various amino acids by the Malpighian tubules probably differ. For example, the U/P ratio for proline is 3 times that for glycine in Rhodnius (Ramsay, 1958). If the.same situation holds for Schistocerca gregaria, this would help to reconcile the high rate of proline absorption in the rectum with the low hemolymph level, since proline would simply be recycled more quickly. Obviously, the possible regulatory function of the rectum with regard to amino acids -132-cannot be fully demonstrated until more is known about the relative rates of amino acid secretion by the Malpighian tubules and the relative rates of net exchange between the hemolymph and other body tissues. These topics should be fruitful areas for further investigations. APPENDIX -13i--134-Fig. A. The movement of glycine across rectal sacs upon reversal of the Na+ gradient Rectal sacs were incubated in Mordue Ringer containing IO-3 M KCN + 10 mM 14C-glycine for 1 hour. They were subsequently transferred to a K+ Ringer (Na+ free) containing 10 mM l^C-glycine and KCN (solid line). Control tissues remained in the in i t i a l Ringer (broken line). Vertical lines indicate ± S.E. of the mean of 4 preparations. 7 6 5 4 3 • 2 1 60 50 40 14 13 12 11 10 9 - 1 3 4 a -Na+both sides Ext. Na* replaced by K* 2 3 T I M E (min ) -135-Table A -f- & Effect of Na on glycine influx No. of Pre-incubation Test Exp. solution solution Glycine influx (nmoles/hr./ 10 mg. rectum) 9 Na+ Ringer (Mordue) Na+Ringer (Mordue) 9 +2.5 mM Glycine 168.7 ± 4.3 12 +5 mM " 349.0 ±16.6 12 +10 mM " 575.5 ±34.3 11 +20 mM " 868.7 ±64.7 10 +40 mM " 1162.2 ±80.0 9 +80 mM " 2004.3 ±126.7 9 Choline Ringer + 2.5 mM Glycine 90.4 ± 5.4 12 Choline Ringer + 10 mM Glycine 311.3 ±14.1 12 Choline Ringer + 20 mM Glycine 522.3 ±30.0 % D.W. 24.8 ± 0.4 22.4 ± 0.7 23.3 ± 0.6 21.6 ± 0.6 19.9 ± 0.2 Data graphed in Figs. 17 and 25. -136-Table B Rates of -^C-glycine uptake by rectal tissue when different concentrations of serine or proline are  present in the test solution Net water movement was prevented. The % dry weight of a l l rectal sacs used in these studies was determined at the end of the experiment. No. of Glycine uptake « IT m - . . / , # , # - , „ /°D.W. Test solution exp. (nmoles/hr./ 10 mg. rectum) Glycine (5 mM) 7* 287.7 ± 22.9 26.2 ± 1.7 Glycine + L-serine(5 mM) 4 170.9 ± 30.9 25.3 ± 2.0 + L-serine(15 mM) 4 52.15 ±11.1 28.5 ± 1.2 11 + L-serine(50 mM) 4 39.8 ± 4.7 27.6 ± 2.4 Glycine (10 mM) 4 298.4 ± 16.3 Glycine + L-proline(10 mM} 4 228.3+10.8 + L-proline(50 mM) 4 138.6 ± 7.5 24.6 ± 2.2 * Average values for 4 preparations were taken from Table A, together with another 3 independent observations. -137-Table C Effect of different concentrations of Na on the influx of glycine (measured between O^ and 6 min. after exposure to radioactive glycine ) Test No. of Pre-incubation Glycine influx % D.W. solution exp. solution (nmoles/hr./ (mM Na+) 1 0 m g' tissue> 0 9 Na"1" free Choline 165.2 ± 12.0 24.2 ± 3.7 Ringer 22 12 " 169.9 ± 12.9 27.4 ± 2.7 44 12 " 319.2 ± 18.9 27.5 ± 3.1 87 12 " 223.7 ± 16.2 26.8 ± 4.3 174 11 11 251.0 ± 16.6 26.1 ± 4.1 Calculated from Fig. 26, using the value of the specific activity measured in the external medium. (Glycine influxes are also presented in Fig. 27.). Table D Cell water and x4c_giyC:Lne content and cell glycine concentration after different periods of time in the presence or absence of external sodium in the incubation media. (No. of experiments indicated in brackets; values are given as mean ± S.E.) Cell H20 Cell HO Cell Glycine Cell Glycine Cell Glycine Cell Glycine Condition after 1 hr. after 3 hr. content after concentration concentration concentration incubation incubation 1 hr. incub. after 3 hr. after 1 hr. after 3 hr. (ul/mg.D.W.) (pl/mg.D.W.) (nmoles/mg.D.W.) incub. incub. (mM) incub.(mM) (nmoles/mg.D.W.) Mordue Ringer (174 mM Na*) 4.5 ± 0.5 50.4 ± 3 11.5 14C-glyc ine + 10~3 KCN= (4) 2nd and 3rd hour transferred to the same Ringer as above (4) 4.2 ± 0.4 52.7 ± 7.6 12.2 ± 0.9 2nd and 3rd hour transferred to Na"*7 free Ringer (K4" replaced Na+) (4) 5.9 ± 0.3 52.5 ± 3.3 8.98 ± 0.4 Table E Na+ content of rectal tissue after different periods of incubation In various media (No. of experiments indicated in brackets; values are given as means ± S.E.) Incubation media D.W. (mg.) Water content (% W.W.) Na+ content (yequiv./lO mg. rectal tissue) Na+concentration (mequiv./Kg. tissue H2O) Mordue Ringer (1 hr.) 2.45 (6) 75.5 .911 120.9 Mordue Ringer 174.4 mM Na+ 10 mM 14C-glycine + IO-3 M KCN 2.20 (4) 78 1118 ± 0.07 151.2 ± 9.2 2nd and 3rd hours transferred to the same Ringer as above 2.26 (4) 77.4 1.74 ± 0.05 224.5 ± 6.5 2nd and 3rd hours transferred to Na+ free Ringer (K"1" replaced Na+) + 10~3M KCN 1.74 (4) 82.6 .54 ± 0.04 65.77 ± 4.9 -140-BIBLIOGRAPHY Amber, R. P.,1963. 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