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Substrate utilization and ammonia secretion in the locust ileum Peach, Jacqueline Lenore 1991

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SUBSTRATE UTILIZATION AND AMMONIA SECRETION IN THE LOCUST ILEUM by JACQUELINE LENORE PEACH B.Sc, The University of British Columbia, 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1991 © Jacqueline Lenore Peach, 1991 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 it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Zoology  The University of British Columbia Vancouver, Canada D a t e October 11, 1991 DE-6 (2/88) ii Abstract Increases in chloride-dependent short-circuit current (AL^ .; an indicator of the major energy-requiring membrane transport process) were used to determine which external substrates support aerobic respiration in locust ileum stimulated by cAMP and theophylline in vitro. At high bilateral concentrations, only glucose and 5 amino acids (alanine, asparagine, glutamine, proline and serine) sustained ileal AL^ ., while trehalose, lipid (diolein) and all other amino acids normally found in locust haemolymph were not utilized. When individual substrates were provided unilaterally at physiological concentrations, the predominant source of substrates supporting ileal I,,, was the luminal fluid, which is mostly derived in situ from Malpighian tubule secretion. Only proline was used almost equally well from both the lumen or haemolymph sides. This situation contrasts with locust rectum where luminal proline is by far the predominant source of respiratory substrate. An inhibitor of amino transferases, amino-oxyacetate, largely abolished ileal AL^ sustained by alanine, but not that sustained by either proline or glutamine. The 5 amino acids that caused an increase in chloride-dependent J^. were assayed for their effect on luminal secretion of ammonia (J^), with and without cAMP. A saline with alanine, asparagine, glutamine, proline and serine supported ileal J,,,,,,, not significantly different from with complete saline. Each of these 5 amino acids, when applied individually, resulted in a significantly above a substrate-free control. The remaining amino acids found in a complete physiological saline (arginine, glycine, histidine, lysine, and valine) that do not stimulate ileal were combined in one saline and gave a J,,,,,,,, value insignificant from the substrate-free control. iii Asparagine and glutamine have the greatest effect on J,,,,,,, with both bilateral and luminal presentation. The addition of cAMP caused an increase in when either the complete saline or the 5 amino acid saline were present bilaterally, but no significant effect on caused by a luminal addition of individual amino acids. J,,,,,,, was unaffected by changes in luminal pH from 4.5 to 7.5, Na+ substitution and amiloride addition, indicating that luminal J,,,,,,, is primarily occurring by NH 4 + transport and not by diffusion trapping (NH3). iv Table of Contents Page Abstract ii Table of Contents iv List of Tables vi List of Figures • vii List of Abbreviations ix Acknowledgements xii Chapter 1: General Introduction 1 Ultrastructure of the locust hindgut 2 Ion and fluid transport 4 Amino acid transport and metabolism 5 Ammonia secretion 7 Chapter 2: Metabolic Support for Chloride-Dependent Short-Circuit Current 12 Introduction 12 Materials and Methods ; 14 Animals 14 Short-circuit current 14 Solutions 16 Statistics 16 Results 17 The substrate assay protocol 17 Effect of individual substrates applied bilaterally at high concentrations 17 Effect of individual substrates added bilaterally at haemolymph levels 21 Sidedness of amino acid utilization 23 Substrate utilization of amino acids at physiological concentrations 25 Transaminase inhibitor effects 25 Discussion 27 V Chapter 3: Amino Acid Support for Ammonia Secretion 31 Introduction 31 Materials and Methods 33 Animals 33 Ammonia secretion 33 Acid secretion 34 Solutions 34 Statistics 35 Results 36 The characterization of ammonia secretion 36 Amino acids as possible precursors of ammonia 40 The nature of ileal ammonia secretion 42 Discussion 48 Identification of the specific amino acids responsible for ammonia secretion 48 Sidedness of amino acid uptake and ammonia secretion 49 The effect of cAMP on 49 Mechanism of ammonia secretion 50 Chapter 4: General Discussion 52 References 56 vi List of Tables Page Table 2.1 Substrates with negligible effect on short-circuit current across ilea previously depleted of substrate 22 Table 2.2 Reduction in ileal 1^ . caused by 10 mM amino-oxyacetate across preparations bathed bilaterally in a single substrate 28 Table 3.1 Effect of luminal Na+ substitution on ammonia secretion rates . . . . 46 Table 3.2 Effect of amiloride on luminal ammonia secretion rates 47 S i Vll List of Figures Page Diagram of the locust excretory system 3 Diagram of rectal metabolism 8 L^ . with time for ilea bathed bilaterally and continously in complete physiological saline and in substrate-free saline with 5mM cAMP and 5mM theophylline 15 Restoration of L^ . in substrate-depleted ilea after the addition of complete saline 18 A typical experiment to test single substrates: L^ . with time for substrate-depleted ilea 19 Effect of bilateral addition of a single substrate on change in short-circuit current (ALJ across ilea 20 Effect of unilateral addition of a single substrate at physiological concentrations on ileal L^ . 24 Effect of a unilateral addition of a single substrate at the concentration typically observed on either haemocoel side or on lumen side on ileal he 26 Ammonia concentration with time for ilea bathed bilaterally in complete physiological saline with 5 mM cAMP and 5 mM theophylline 37 viii Figure 3.2 Effect of the source of amino acids on ammonia secretion rates for ilea bathed in a complete saline 38 Figure 3.3 Effect of bilateral addition of amino acids on ammonia secretion rates to the lumen 39 Figure 3.4 Effect of saline ammonia concentration on the rate of ileal ammonia secretion to the lumen 41 Figure 3.5 Effect of luminal addition of amino acids at the concentration typically observed on the lumen side, on the rate of ammonia secretion 43 Figure 3.6 Effect of pH gradients on luminal ammonia secretion rates and luminal acid secretion rates 45 ix List of Abbreviations ockg - a-ketoglutarate Al^ . - change in short-circuit current e^quiv.cm"2.h", - microequivalents per square centimeter per hour ul - mircoliter(s) uM - micromolar umoles.cm^ .h'1 - micromoles per square centimeter per hour acetyl CoA - S acetyl Coenzyme A ADP - adenosine 5'-diphosphate ala - alanine arg - arginine asn - asparagine ATP - adenosine 5'-triphosphate ATPase - adenosine 5'-triphosphatase cAMP - adenosine 3':5'-cyclic monophosphoric acid Da - Daltons GDH - glutamate dehydrogenase gin - glutamine gly - glycine GTP - glutamate-pyruvate transaminase h H his a^mm J« kg 1 L lys M MDH ME min ml mM mmol MOPS mosmol mV n N - hours - haemocoel or haemolymph - histidine - short-circuit current - rate of ammonia secretion - rate of acidification - kilogram - liter(s) - lumen - lysine - moles per liter (molar) - malate dehydrogenase - malic enzyme - minute - milliliter(s) - millimolar - millimole(s) - 3-(N-morpholino)propanesulfonic acid - milliosmolar concentration - millivolt(s) - number - normal xi NAD - B-nicotinamide adenine dinucleotide, oxidized form NADH - 6-nicotinamide adenine dinucleotide, reduced form nm - nanometer oaa - oxaloacetate pro - proline ScglTP - Schistocerca gregaria ion transport peptide SE - standard error ser - serine val - valine xii Acknowledgements I wish to thank Dr. John Phillips for his encouragement, support and generosity throughout this study. I thank Drs. W. Milsom, P. Hochachka and J. Gosline for comments on the manuscript. I thank Joan Martin for her help, advice, morning tea and especially for measuring ileal acid secretion. I am grateful to Dr. Jon Harrison for many enjoyable conversations, comments on the manuscript, and for his abundant enthusiaum for life, insects and science. I thank Andrew Stagg for his friendship, support and many enjoyable times both inside and outside of the lab. I thank Donna, Trevor and Lisa Peach for being a mountain of support. I especially wish to thank Kevin Harris for teaching me to be computer literate, for his patience over the years, and for being a wonderful friend. 1 CHAPTER ONE General Introduction Insects live in a wide variety of environments where they are subject to fluctuating regulatory challenges, such as severe dehydration-hydration and starvation-feeding cycles. In the face of such fluctuating conditions, the desert locust (Schistocerca gregaria) is able to regulate its haemolymph osmotic concentration and ionic composition extremely well (Phillips, 1964). The regulation is primarily accomplished by the homeostatic functions of the excretory system, which includes the Malpighian tubules and hindgut (i.e. ileum, colon and rectum). The structure and function of the locust hindgut has been reviewed by Phillips (1977, 1981; also Wall and Oschman, 1975). The excretory process involves the production of an isosmotic primary urine in the Malpighian tubules. This fluid contains high levels of K+, and low levels of Na+. Secretion of this fluid is driven by the active transport of K+, possibly as a result of IT recycling and K +/H + exchange (Wieczorek et al, 1989). Most amino acids and organic solutes enter the tubules passively, and are found in the tubule fluid of Rhodnius (Maddrell and Gardiner, 1980), Schistocerca and other insects at lower levels than in the haemolymph (Maddrell and Gardiner, 1974). Some organic anions and cations are actively secreted by insect tubules, as is also proline in Schistocerca gregaria (Chamberlin and Phillips, 1982b). 2 The 250 Malpighian tubules of the desert locust empty into the gut at the junction of the midgut and hindgut. The tubule fluid flows mostly into the hindgut where selective reabsorption of major ions, amino acids and water occurs to produce a hyposmotic or hyperosmotic excreta (Fig. 1.1). Ultrastructure of the locust hindgut The rectum, with a gross surface area of 0.64 cm2, consists of 6 longitudinal pads covered apically by a permeable cuticle. Rectal pads are made up of 2 cell types, with large columnar cells predominating. Both apical and lateral membranes of these cells are highly folded and associated with many mitochondria. These folded membranes form complex intercellular channels where ion recycling may take place to drive water extraction from the luminal contents (Wall, 1970). The locust ileum has a surface area of 0.40 cm2 and is made of a single layer of epithelial cells covered by a tough apical cuticle. The basal surface is covered by a thin basal lamina. As in the rectum, the apical membrane of the ileum is highly folded and associated with mitochondria. Unlike rectal epithelial cells, ileal cells have elaborate narrow infoldings of the basal plasma membrane and relatively straight lateral cell membranes (Irvine et al, 1988). 3 MIDGUT MALPIGHIAN TUBULES ILEUM RECTUM KC1, Na+ and Water secretion COLON 4--, ANUS Water and ion reabsorption Water, ion and metabolite reabsorption Strongly hyperosmotic or hyposmotic excreta Figure 1.1 Diagram of the locust excretory system. The flow of urine is indicated by the thin arrows and transfer across the epithelia is indicated by the thick arrows (Modified from Phillips, 1981). 4 Ion and fluid transport The ileum and rectum are the major organs for water and solute reabsorption in the excretory system. The ileum reabsorbs the bulk of the water and solutes (Lechleitner et al,1989a), and the rectum adjusts the final osmolality of the faeces (Phillips, 1964). The locust ileum and rectum both actively reabsorb Na+, CI", K+ and HC03" to the haemocoel side and actively secrete ammonia and H + to the lumen (Audsley, 1991; Lechleitner and Phillips, 1989; Thomson et al, 1988; Irvine et al, 1988; Williams et al, 1978). CI" is the predominant anion in the luminal fluid and is actively transported into the cell apically via an electrogenic pump which is stimulated by cAMP and by low levels of luminal K+ (Spring and Phillips 1980a, b). CI" exits passively at the basolateral cell border by a conductive mechanism (Hanrahan and Phillips, 1983, 1984a, b). K + is the predominant cation in the luminal fluid and is reabsorbed passively through apical K+ channels and exits at the basolateral membrane by barium-sensitive channels (Phillips et al, 1986) according to the electropotential generated by CI" transport (Hanrahan and Phillips, 1983, 1984a, b). Na+ levels in the hindgut fluid are frequently low. Na+ entry at the apical membrane occurs passively and coupled with transport of other ions (eg.by exchange for NH4+), with neutral amino acids, or by a putative channel (Hanrahan and Phillips, 1982; Black et al, 1987). Na+ is actively pumped out of the cell basolaterally by the ubiquitous Na+,K+-ATPase (reviewed by Phillips et al, 1986). 5 Cyclic AMP, and homogenates of corpora cardiaca and ventral ganglia, all increase K+, CI" and water reabsorption in locust ileum and rectum, and also Na+ in the ileum only (Lechleitner et al, 1989a, b). Recently, ScglTP, a 7700 Da peptide purified from corpora cardiaca and partially sequenced, has been shown to have a large stimulatory effect on ileal CI", Na+, K + transport, water absorption and an inhibitory effect on H+ secretion (Audsley et al, 1990; Audsley, 1991). The ileum appears to possess ion transport processes similar to the rectum, but there are some differences (review by Lechleitner, 1988). The rectum actively absorbs water from the lumen to produce a hyperosmotic urine, whereas the ileum is only able to transport an isosmotic absorbate because it lacks the lateral scalariform complexes found in the rectal cells and responsible for solute recycling. Fluid absorption in the ileum is driven by NaCl and KC1 absorption, whereas in the rectum it is driven largely by KC1 and proline (Lechleitner and Phillips, 1989). Finally, in the ileum, Na+ is actively reabsorbed at much higher rates than in the rectum and appears to be under hormonal control (Lechleitner and Phillips, 1989b). Amino acid transport and metabolism Insect haemolymph contains high levels of amino acids (50 mmol/1) and^ the levels of haemolymph amino acids are closely regulated even when blood volume drops significantly (Rutherford and Webster, 1978; Chamberlin and Phillips, 1979). Ramsay (1958) and Maddrell and Gardiner (1974) observed low levels of some amino acids in Malpighian tubule fluid compared to those in the haemolymph. They showed that several amino acids are passively 6 transported by the Malpighian tubules. Chamberlin and Phillips (1982b) demonstrated that locust Malpighian tubules actively secrete proline, and that it constitutes 80% of the total amino acids in Malpighian tubule fluid. Reabsorption and metabolism of amino acids in the hindgut are believed to contribute to regulation of haemolymph amino acid levels (Chamberlin and Phillips, 1982a). Luminal amino acids also serve other important roles. They have been shown to be a major source of respiratory substrates required to sustain ion and water reabsorption in rectal epithelia (Chamberlin and Phillips, 1982b), and the major source of ammonia in the rectum (Thomson etal, 1988). Little is known about the metabolic support of ion transport in insect epithelia. Berridge (1966) showed that exogenous alanine, glutamine, pyruvate and other sugars supported fluid secretion by the Malpighian tubules of Calliphora. Giordana and Sacchi (1978) reported that pyruvate and alanine supported a transepithelial potential across the midgut of Bombyx. Active absorption of amino acids in insects was first demonstrated in the locust rectum. Balshin and Phillips (1971) and Balshin (1973) showed that 5 major neutral amino acids (proline, glycine, serine, alanine and threonine) in the haemolymph were all actively absorbed against large concentration gradients. Only small fractions of the total proline, alanine, serine and threonine which are transported across the locust rectum are metabolized by this tissue (Balshin 1973, Phillips et al, 1986). While glutamate enters the rectal cells from the^ umen and acts as a metabolic substrate, it is not transported transepithelially (Chamberlin and Phillips, 1983; Balshin, 1973). The 2 major amino acids of the haemolymph and primary urine, proline and glycine, are transported at a high rate and against large concentration gradients in the locust rectum. Meredith and Phillips (1988) observed that 85% of 14C-activity appearing on the haemocoel side was proline when 2 or 80 mmol/1 proline was added to the lumen. Only a small fraction of 1 4C-proline was oxidized to 1 4C0 2 during net transport across short-circuited locust recta (Spring and Phillips, 1984). The addition of cAMP caused a large increase in the oxidation of 14C-proline and a 40% decrease in the net flux of proline across the tissue (Spring and Phillips, 1984). To maintain cAMP-stimulated CI" transport, only 1 mmol/1 luminal proline was required as a metabolic substrate (Meredith and Phillips, 1988), which raises the question as to why the Malpighian tubules secrete fluid containing 38 mmol/1 proline. The locust rectum actively transports Cf and this transport can be stimulated by cAMP in vitro (Spring and Phillips, 1980a, b). CI' transport is the major energy-dependent process in rectal epithelial cells (Chamberlin and Phillips, 1982a). Both the ileum and rectum are highly aerobic tissues in which active transport of CI" is rapidly abolished by azide, cyanide or anoxia (Baumeister et al, 1981) Studies by Chamberlin and Phillips (1982a) indicate that isolated rectal mitochondria of locusts oxidize proline preferentially over other amino acids, carbohydrates and lipids found to occur in locust haemolymph. The locust rectum has high activities of the enzymes glutamate dehydrogenase, glutamate-pyruvate transaminase and glutamate-oxalacetate transaminase which are associated with amino acid metabolism and NH3 production. A metabolic pathway for amino acid catabolism has been proposed for the locust rectum by Chamberlin and Phillips (1982c) based on these and other observations (Fig. 1.2). 8 active secretion of prol ine proline Figure 1.2 Rectal metabolism and J^ . are sustained by the main respiratory substrate luminal proline, supplied from Malpighian tubules. Proline actively enters the apical membrane infoldings to fuel mitochondria, thereby providing ATP for the apical CI" pump (LJ and hence fluid transport, while ammonia from proline oxidation is secreted into the lumen. The metabolic pathway for rectum elucidated by enzyme analyses, substrate utilization and inhibitor studies. Based on data in Chamberlin and Phillips (1982a, b; 1983) and Thomson et al (1988). 9 Ammonia Secretion Ammonia is known to be the most important nitrogenous excretory product of several aquatic insects (Cochran, 1985; Wigglesworth, 1972). Terrestrial insects were thought to excrete nitrogen primarily as uric acid (Cochran, 1985), although ammonia has been shown to be the major excretory product in Sarcophaga bullata (Prusch, 1972), Periplaneta americana (Mullins and Cochran, 1972; 1976) and recently Schistocerca gregaria (Harrison and Phillips, submitted). Early studies by Razet (1966) reported that S. gregaria primarily secretes urate and trace amounts of ammonia. Stagg et al. (1991) found that the Malpighian tubules secrete small amounts of ammonia and urate in an 1:1 ratio. In contrast, the ileum and rectum secrete ammonia at high rates and account for over 70% of the ammonia excreted in the faeces. There is no evidence to date for urate secretion in the locust hindgut. In starved locusts, final urate and ammonia concentrations are 68 and 278 mM/kg H20 respectively (Harrison and Phillips, submitted). Loss of ammonia by evaporation before analysis of faecal pellets probably explains why high levels went undetected in earlier studies. Harrison and Phillips (submitted) report and explain that NH 4 + excretion is consistent with water conservation in desert species as the NH 4 + is present primarily as a precipitate. Chamberlin and Phillips (1982b; 1983) demonstrated that amino acids secreted by the Malpighian tubules were actively absorbed in the rectum and oxidatively deaminated, leading to ammonia loss from the tissue. Hanrahan (1982) and Hanrahan and Phillips (1982) also reported transport of ammonia, especially to the lumen side of short-circuited locust recta. Thomson et 10 al. (1988) showed that some amino acids (alanine, glutamine, proline and serine) supplied luminally caused an increase in ammonia secretion to the lumen side of the rectum. The locust rectum produces ammonia and transports it in a similar manner to the hindgut of Sarcophaga bullata (Prusch, 1972) and to the proximal tubules of the mammalian kidney, i.e. by amiloride-sensitive, Na7NH4+ exchange (Thomson et al., 1988). Pitts (1973) demonstrated that NH3 synthesized in renal cells of vertebrates diffuses into acid tubular fluid, becomes trapped and excreted as an ammonium ion (diffusion trapping ). Good and Knepper (1985) found that there may be diffusion trapping in the proximal tubules however, the preferential addition of ammonia to the lumen suggested NH 4 + transport to the lumen also occurs. Studies by Nagami and Kurokawa (1985) on mouse proximal tubules have demonstrated that ammonium produced by the cells is preferentially secreted into the lumen. The lumen-to-bath distribution of ammonium exceeds that predicted by the diffusion trapping model by nearly 2 orders of magnitude. Nagami and Kurokawa have proposed that preferential ammonium secretion into the proximal tubule lumen may occur by direct transport of ammonium ions resulting from substitution of ammonium for protons on the apical membrane Na+/H+ exchanger. The excretion of ammonium by the kidney plays a critical role in the regulation of whole-body acid-base balance. Studies have demonstrated that renal net acid excretion (NH4+ excretion + titratable acid - bicarbonate excretion) can change to excrete quantitatively either an acid or alkali load (Knepper et al., 1989). Ammonium excretion increases during a metabolic acidosis and decreases during metabolic alkalosis. It is likely that the regulation of ammonium production 11 by the proximal tubule is in large part responsible for the changes in ammonium excretion in altered acid-base states. Virtually all the ammonium excreted by the vertebrate kidney is produced by the renal cells from amino acids, chiefly glutamine (Pitts, 1973). Studies by Vinay etal. (1982) and Good and Burg (1984) have demonstrated that most of the glutamine-dependent ammonium production in the kidney occurs in the proximal tubules. Glutamine is metabolized to form ammonium and a-ketoglutarate. The ammonium formed is secreted into the tubule lumen and eventually in the urine. The a-ketoglutarate formed from glutamine is further metabolized to bicarbonate, which exits the cell across the basolateral membrane and, is therefore returned to the extracellular fluid. The return of this new bicarbonate to the extracellular fluid restores bicarbonate that was lost due the intake or generation of acid in the body and therefore plays an important homeostatic role (Knepper, 1988). Since the role of the ileum in the insect excretory process has only recently been appreciated (Irvine et ai, 1988), little is known of the metabolic substrates supporting membrane transport and ammonia secretion in this tissue. In this thesis, I examined the role of amino acids as metabolic substrates supporting absorption and as a source of ammonia secreted into the ileal lumen. In chapter 2,1 investigate which respiratory substrates provide the metabolic energy to drive CI" transport (the major active process) using short-circuited ilea. In chapter 3,1 determine which of the respiratory substrates found in chapter 2 lead to ammonia secretion in the ileum and whether secretion occurs by Na+/NH4+ exchange, as shown for the rectum. In both chapters I also reported on the sidedness of substrate usage. 12 CHAPTER TWO Metabolic Support for Chloride-Dependent Short-Circuit Current Introduction The role of Malpighian tubules and recta of insects in the excretory process have long been studied (reviewed by Phillips et al, 1986) but the contribution of the ileum to this process has only recently received attention. Irvine et al (1988) showed that the ileum of the desert locust, Schistocerca gregaria, possesses absorptive mechanisms for CT, Na+ and K +, similar to those of the rectum. These can all be stimulated by cAMP, and also by extracts of corpus cardiacum and ventral abdominal ganglia. The concurrent 4-fold stimulation of ileal fluid transport against osmotic gradients exceeds that of the rectum (Lechleitner et al, 1989a, b; Lechleitner and Phillips, 1989). In essence, the locust ileum appears to be functionally analogous to the vertebrate proximal tubule in achieving substantial bulk reabsorption of primary excretory fluid without large changes in luminal osmolality. Like vertebrate proximal tubules and locust rectum, the locust ileum also actively secretes H + (Thomson et al, 1991) and ammonia (lechleitner, 1988), the latter at twice the rate (per unit surface area) for locust rectum. In locust rectum, secreted NH4 + and the ATP to drive ion transport are both produced largely by oxidation of amino acids (predominantly proline) which are actively reabsorbed from luminal (i.e. Malpighian tubule) fluid. The cellular ammonia so produced, is exchanged as NH4 + for luminal Na+. This process achieves the multiple functions of energizing the major solute 13 transport process of this epithelium (i.e. CI"), eliminating both nitrogenous waste and also excess H + (i.e. rT + NH3 —> NH4+), while conserving Na+ which is generally in short supply in the ingested plant material. Proline transport in the rectum is an order of magnitude greater than that required to sustain fully aerobic respiration and this large solute movement drives a substantial fraction of fluid reabsorption, leading to water conservation (Chamberlin and Phillips, 1982a,b, 1983; Thomson et al, 1988; Lechleitner and Phillips, 1989). In contrast, active transepithelial reabsorption of proline and associated fluid movements do not occur in locust ileum (Lechleitner and Phillips, 1989) suggesting some metabolic differences between the two hindgut segments. Both ileum and rectum are highly aerobic tissues in which active transport of CI" (the predominant process) is rapidly abolished by azide, cyanide or anoxia. In this chapter I investigate for the first time which respiratory substrates provide the metabolic energy to drive salt transport and hence water reabsorption in locust ileum. I also determine whether these substrates are obtained preferentially from haemolymph or the luminal (i.e. Malpighian tubule) fluid. The method used is essentially similar to that first employed with the locust rectum (Chamberlin and Phillips, 1982a). That is, I monitored a major energy-requiring process in this epithelium, CI" transport, which gives rise to a large short-circuit current (LJ upon stimulation with cAMP (Irvine et al, 1988). Metabolism of specific respiratory substances can therefore be assessed from their ability to restore J .^ across ilea previously deprived of exogenous and endogenous (i.e. intracellular) substrates. A positive response of 1^  also implies the existence of selective membrane transfer mechanisms into the cells for the effective substrates, nearly all of which are strongly hydrophilic. 14 M e t h o d s a n d M a t e r i a l s Animals Adult female Schistocerca gregaria 2-4 weeks beyond their final moult were used in all experiments because of the larger size of their ilea. They were reared as previously described (Chamberlin and Phillips, 1982 a,b). Short-Circuit Current Locust ilea were mounted as flat sheets between two Ussing-type chambers and bathed bilaterally with identical salines, which were stirred by bubbling with 95% 0 2 and 5% C02, as described by Irvine et al. (1988). The applied short-circuit current (I*.) required to maintain transepithelial potential at 0 mV was continuously monitored with a Soltec 220 recorder (see Hanrahan et al, 1984 for electrical circuitry and other details). Irvine et al. (1988) demonstrated that the large increase in ileal ^  from near zero at lh after dissection to about 10 uequiv.cm2!."1 following addition of cAMP was due to an increase in electrogenic transport of CI" from the lumen. Throughout all of the present experiments, locust ilea were continuously and maximally stimulated by 5 mmol/1 cAMP with 5 mmol/1 theophylline. This combination of stimulants was found to sustain 1^ . at near constant rates for more than 8h in preliminary experiments using a complete saline resembling locust haemolymph (Fig. 2.1). 15 Time (hrs.) Figure.2.1 1^  with time for ilea bathed bilaterally and continuously in complete physiological saline (•) and in substrate-free saline (o) with 5mM c A M P and 5mM theophylline. Mean ± SE. (n=8). 16 Solutions The saline used in the assay contained (in mmol/1): NaCl, 100; K2S04, 5; MgS04, 10; NaHC03, 10; CaCl2, 5; glucose, 10; sucrose, 100, alanine, 2.9; asparagine, 1.3; arginine, 1.0; glutamine, 5; glycine, 11.4; histidine, 1.4; lysine, 1.4; proline 13.1; serine, 1.5; valine, 1.8; with pH adjusted to 7.0. Haemolymph osmotic pressure is 420 mosmol/1, so the final osmotic concentration of the salines are adjusted with sucrose (100 mmol/1), mostly to replace trehalose (about 50 mmol/1 in locust blood). Salines containing a single potential metabolic substrate were prepared by replacing all other organic substrates listed above with osmotically equivalent amounts of sucrose, which is not metabolized. The same procedure was used to test other potential substrates which are present in haemolymph, but not included in the complete saline listed above (i.e. diolein, trehalose, glutamate, and aspartate). All chemicals were reagent grade and obtained from Sigma Co. To deplete tissues of endogenous substrates, stimulated ilea were bathed with saline lacking all organic components except sucrose. All experiments were conducted at 22-24°C. Statistics Data are presented as means ± standard error of mean (SE), with n indicating number of ilea. Significance of change in measured immediately before and 0.5h after adding specific substrates to substrate-depleted ilea were determined by paired Mests. Comparisons between mean increases in I,,. caused by different substrates (or mixtures) was by Student's f-test. A P-value of <0.05 was considered indication of statistical difference. 17 Results The Substrate Assay Protocol In the absence of any substrates in the bathing saline, ileal 1^. falls rapidly by about 90% within lh and thereafter remains stable at these low values for at least 8h (Fig. 2.1). Between the first and second hour, ileal L^ . could be fully restored to near control values by re-exposing preparations to complete saline. Even after 4h without external substrates, about 80% of L^ . could be restored by re-adding complete saline. The changes in 1^ . caused by substrate additions were completed within 0.5h (Fig. 2.2). At 1.5h, the ability of individual substrates to increase ^ across substrate-depleted ilea was compared with the AI^ . caused by complete saline in control preparations. As a second control, we checked that all ilea exposed to single substrates subsequently responded fully to complete saline at the end of experiments. Effect of Individual Substrates Applied Bilaterally at High Concentrations Considering that some substrates might enter the ileal epithelium slowly, we first tested individual substrates applied bilaterally at a high concentration of 20 mmol/1, i.e. at two to twenty times normal levels of most substrates in locust haemolymph or Malpighian tubule fluid. The time course of a typical experiment testing alanine is shown in Fig. 2.3. This single amino acid was almost as effective in restoring ileal as was a complete saline. A subsequent addition of complete saline caused a slight additional increase in 1^ . The results for individual substrates tested in this manner are summarized in Fig. 2.4. Alanine, asparagine, glutamine, proline and serine at 20 mmol/1 and glucose at 2 mmol/1 all quickly restored ileal J^. to levels which were 18 Figure 2.2 Restoration of 1^. in substrate-depleted ilea after the addition of complete saline at lh (•) and 3.5h (o). Mean ± SE. (n=8). 19 XI w I a o & g. S o 0 0 20 n 15 4 10 0 20mM Alanine Complete Saline T X 0.0 1.0 2.0 3.0 4.0 5.0 Time (hrs.) Figure 2.3 A typical experiment to test single substrates: I,,, with time for substrate-depleted ilea after the bilateral addition of 20 mM alanine at Th and complete saline at 3.5 h. Mean ± SE. (n=8). 20 Bilateral external cone. - 2 J Substrate None All Ala Asn Gin Glucose Pro Ser *Conc.(mM) - - 2.9 1.3 5.0 10.0 13.1 1.5 Figure 2.4 Effect of bilateral addition of a single substrate on change in short-circuit current (ALJ across ilea previously depleted of substrate (none) compared to complete saline (all). Hatched bars are for substrate concentrations in complete saline as indicated below figure. 1^ . measurements were made 1-1.5 h post-tissue mount. Mean ± SE. (n=8). 8 Not significantly different from substrate-free control by paired f-test (P <0.05). 21 50% or more of maximum caused by a later addition of complete saline. Depriving ilea of all substrates apparently causes compensatory metabolic adjustments, since addition of complete saline at the second hour caused a larger AL^ . (10.5 uequiv.cm .^h"1) than observed for preparations maintained continuously from dissection in the complete medium during this series of experiments (AI^  of 5.6 uequiv.cm.2!."1). In contrast, (Table 2.1) several other amino acids present in locust haemolymph (arginine, aspartate, glycine, histidine, lysine, valine, all at 20 mmol/1, and glutamate at a physiological level of 1 mmol/1) had no significant stimulatory effect on ileal 1^ . Indeed ileal 1^  actually declined slightly over the test period in most cases, as did the 1^ . for substrate-depleted controls (-0.69 uequiv.cnrlh"1). Likewise, the major carbohydrate in locust haemolymph, trehalose at 20 mmol/1, and a lipid substrate (diolein) at high concentration had no effect on ileal 1^ (Table 2.1). This is in agreement with earlier results for locust rectum (Chamberlin and Phillips, 1989a). When I compared 1^ . caused by the five effective amino acids, there was no significant difference between alanine and glutamine, asparagine and proline, or glutamine and proline, amongst all possible paired comparisons. In summary, locust ileum is capable of using five amino acids and glucose from amongst many potential substrates available in situ (or in complete saline) to sustain aerobic respiration and hence I*.. Effect of Individual Substrates Added Bilaterally at Haemolymph Levels Having determined which of the available respiratory substrates can be used at high concentrations by locust ileum, I tested their relative importance at concentrations present in complete saline, which approximates the levels in locust haemolymph. Since proline and 22 Table 2.1 Substrates with negligible effect on short-circuit current across ilea previ-ously depleted of substrate. Substrate Concentration A Isc (mM) (uEquiv*cm2*h1) Arginine 20 -1.28 ± 0.2 Aspartate 0.9* 0.29 ±0.1 Diolein 10 mg/ml++ -0.21 ±0.1 Substrate Free - -0.69 ±0.1 Glutamate 1.0 * 0.52 ± 0.2 a Glycine 20 -1.14 ±0.1 Histidine 20 -1.29 ± 0.2 Lysine 20 -0.73 ± 0.2 Trehalose 20* -0.43 ± 0.2 a Valine 20 -0.66 ±0.1 A Isc, change in short-circuit current. Isc measurements were made 1-1.5 hours post-tissue mount. Mean ± SE (n=8). * Physiological levels in haemolymph. + + Lipid concentration in haemolymph of Locusta migratoria (Jutsum and Goldsworthy,1976) a Not significantly different from control by paired t -test(P < 0.05). 23 glutamine are exceptional in that they are apparently secreted actively by locust Malpighian tubules (Chamberlin and Phillips, 1982b), I also tested these two amino acids at the higher concentrations normally occurring in the hindgut lumen (50 and 20 mmol/1 respectively). Again, identical substrate concentrations were used on both the haemolymph and luminal sides of the ileal preparation in this series of experiments (Fig. 2.4 and Table 2.1). At physiological concentrations, alanine (2.9 mmol/1) and glutamine (5 mmol/1), are as effective in restoring ileal I,,, as they are at 20 mmol/1, suggesting firstly saturation at low concentrations and secondly that these amino acids are major substrates in situ. In contrast, asparagine (1.3 mmol/1) and proline (13.1 mmol/1) were significantly less effective at haemolymph concentrations than at 20 mmol/1; i.e. I^ . was more concentration dependent Raising proline levels to those typical of luminal fluid (50 mmol/1) only increased 1^ . slightly beyond that observed at 20 mmol/1, suggesting saturation over this high concentration range. Sidedness of Amino Acid Utilization Under physiological conditions, the predominant metabolic substrate used by locust rectum is proline which is almost exclusively obtained from the lumen side (i.e. from Malpighian tubule fluid; Chamberlin and Phillips, 1982b). I therefore investigated the relative importance of haemolymph and hindgut luminal fluid as a source of metabolic substrates to sustain ileal 1^ . I compared 1^ . caused by substrates added to either the lumen or haemolymph side only of ilea. The concentrations of substrate used were those present in complete saline (Fig. 2.5). Complete saline was equally effective at restoring ileal 1^ . from either haemocoel or lumen sides (i.e. difference not significant). Individual amino acids were then tested. Alanine and serine only acted 24 Figure 2.5 Effect of unilateral addition of a single substrate at physiological (i.e. complete saline) concentrations on ileal L^ . compared to the effect of complete saline. Tissues were bathed in substrate-free saline for one hour before the addition to either haemocoel or lumen side. L^ . measurements were made 1-1.5 h after tissue mount. Mean ± SE. (n=5). ' Not significantly different from substrate-free control by paired f-test. (P <0.05). * Not significantly different by independent /-test. (P <0.05). 25 from the lumen side. Asparagine, glutamine and glucose were several times more effective from the lumen side and only proline caused somewhat greater AI^ . from the haemocoel side (unlike the situation for locust rectum). There was a strong correlation (r = 0.979) between AI^ . caused by various substrates added at physiological concentration on the lumen side only with that caused by bilateral addition. In summary, given equal (i.e. haemolymph) concentrations, most individual substrates apparently are preferentially obtained by locust ileum from the lumen side. Substrate Utilization of Amino Acids at Physiological Concentrations To obtain a more precise estimate of the relative importance of the haemolymph and hindgut fluid as a source of metabolic substrates in situ, I also tested the principal substrates at hindgut fluid concentrations on the lumen side only, using substrate values for these body fluids reported by Chamberlin and Phillips (1982b; Fig. 2.6). Allowing for reported concentration differences on the two sides of ilea in situ, the most effective substrates are luminal alanine, glutamine and glucose, even though their concentrations are relatively low in hindgut fluid, and also proline from both sides. Transaminase Inhibator Effects Chamberlin and Phillips (1983) demonstrated two pathways for proline oxidation in locust rectum: (1) deamination to a-ketoglutaric acid via glutamate dehydrogenase, and (2) transamination via glutamate-pyruvate transaminase to form a-ketoglutarate and alanine. Oxidation of proline via the first pathway clearly predominates in intact locust recta because amino-oxyacetate, an inhibitor of transaminases, does not reduce stimulated rectal 1^. sustained 26 Figure 2.6 Effect of a unilateral addition of a single substrate at the concentration typically observed on either haemocoel side (complete saline values) or on lumen side (luminal fluid values) on ileal 1^ : mM values indicated on figure. Mean ± SE. (n=5). * The effect of a single substrate on haemocoel vs lumen side at physiological concentrations are not significantly different by independent f-test (P <0.05). All increases in 1^  are significantly different from substrate-free control by paired r-test. (P <0.05). 27 by only proline. In contrast, this inhibitor largely abolished rectal 1^ . sustained by alanine (Chamberlin and Phillips, 1982a). I therefore conducted similar experiments with amino-oxyacetate on ileal I,,, supported by individual substrates applied bilaterally at physiological levels (Table 2.2). This inhibitor had no significant effect on ileal oxidation of proline or glutamine as judged by AI^ ., whereas alanine-supported 1^ . was largely abolished. Thus results with amino-oxyacetate for locust ileum and rectum are essentially similar except that oxidation of alanine (by the transaminase pathway) is quantitatively much more important in the ileal segment. Discussion The results of this study show that five of the exogenous amino acids (alanine, asparagine, glutamine, proline and serine) and glucose supplied at physiological concentrations support stimulated ileal 1^ ., while the main haemolymph sugar (trehalose) and lipid (diolein) do not. Amino acids and glucose entering the lumen from the Malpighian tubule fluid are apparently the predominant source of metabolic substrates in the ileum as judged by CI* dependent 1^ . These results are qualitatively similar to those observed by Chamberlin and Phillips (1982a, 1983) for locust rectum. The latter gut segment uses the same substrates at high concentrations and almost exclusively from the lumen side. However at physiological concentrations, the predominant rectal substrate is luminal proline. In contrast, other luminal amino acids are preferred by the ileum under physiological conditions, and proline is equally well used from both sides. This difference may ensure that proline, which is actively secreted by locust Malpighian tubules at high levels (40 mmol/1), is largely allowed to pass through the ileum into the rectum where it has several 28 Table 2.2 Reduction in ileal Isc caused by 10 mM amino-oxyacetate across preparations bathed bilaterally in a single substrate. Substrate Concentration (mM) Inhibitor A Isc (uEquiv*cm2*h1) Alanine Glutamine Proline 2.9 5.0 13.1 Amino-oxyacetate Amino-oxyacetate Amino-oxyacetate -6.42 ± 1.4 a 0.0 ± 0.2 -0.86 ± 0.3 A Isc, change in short-circuit current caused by adding inhibitor to ilea exposed bilaterally to a single substrate at concentration indicated. Tissues were bathed in substrate 1 hour before addition of the inhibitor. A Isc is the difference between measurements immediately before and 1.0 hour after adding the inhibitor. Mean ± SE (n=5). a Significantly different from previous control values by paired t -test (P < 0.05). 29 important functions (See Introduction). Why are amino acids the preferred hindgut substrates, when locusts as a whole largely burn lipids? It has recently been observed that ammonia concentrations in excreta of starved desert locusts (O^M.kg'HjO) substantially exceeds that of urates, when care is taken to prevent ammonia loss which occurs by evaporation from eliminated faecal pellets (Wong et al, 1990). Comparison of ammonia secretion rates by locust Malpighian tubules (Stagg et al, 1991) in situ with those of ileum (Lechleitner, 1988) and rectum (Thomson et al, 1988) in vitro suggests that ileal secretion is responsible for more than half of all excreted ammonia, whereas Malpighian tubules account for less than 10%. Thus the ileum apparently makes the major contribution to total nitrogen and acid (i.e. H + trapped by NH3 as NH4+) excretion in locusts. It has also recently been established that the ileum is the major site of Na+ reabsorption in locusts and that this recovery process is under hormonal control (Irvine et al, 1988; Lechleitner et al, 1989b; Lechleitner, 1988). Since ileal Na+ reabsorption is largely electroneutral (Irvine et al, 1988), possibly Na+/NH4+ exchange may occur in the ileum, as has been demonstrated for the rectum (Thomson et al, 1988). In summary, recent observations suggest that ammonia production and secretion in locust ileum probably serves several important homeostatic functions. I therefore predicted that amino acids are likely to be major respiratory substrates and sources of secreted ammonia in locust ileum, as they are in locust rectum. The results of this chapter support this hypothesis, although I must still determine relative ammonia production from individual amino acids. My results to date on locust hindgut are reminiscent of mammalian proximal tubules. In mammals, a majority of the urinary ammonia is derived from the metabolism of specific amino acids in the epithelial cells of the proximal tubules (Good and Knepper, 1985). This seems to be 30 the predominant site for adaptive changes in ammonia production and hence acid elimination in response to acute acidosis in whole animals (Brosnan et al, 1987). The increase in ammonia secretion supported by particular amino acids in acidotic dogs has been correlated with their rate of oxidative deamination in vitro (Pitts, 1973). Under metabolic acidosis, L-glutamine, L-asparagine, L- and D-alanine and L-histidine were found to be highly effective in their capacity to enhance ammonia excretion. Interestingly, three of these amino acids (L-glutamine, L-asparagine, L-alanine), also support oxidative metabolism in the locust ileum. Lechleitner (1988) has demonstrated that the ileum has the highest rate of ammonia secretion in the locust hindgut. Therefore, as for the locust rectum and vertebrate proximal tubule, the oxidation of exogenous amino acids is most likely the main source of ammonia secreted in the locust ileum. 31 CHAPTER THREE Amino Acid Support for Ammonia Secretion Introduction The renal mechanisms by which nitrogenous wastes are excreted in insects are poorly understood (Cochran, 1985). Ammonia secretion plays a major role in total nitrogen and acid secretion in the locust, S. gregaria (Harrison et al, submitted). The major site of ammonia secretion is in the ileum (Lechleitner, 1988; Stagg et al, 1991), where the amino acids alanine, asparagine, glutamine, proline and serine, absorbed from the lumen, are the predominant source of metabolic substrates (chapter 2). The goal of this chapter is to examine the role of exogenous amino acids in the support of ammonia secretion by the ileum. Specifically, this study determined: (1) the relative production of NH3 from individual amino acids, (2) sidedness of substrate uptake and subsequent ammonia release, (3) the effect of cAMP stimulation on ^m a a , and (4) the nature of the species of ammonia1 which crosses the apical side of the ileal epithelium. The amino acids which support short-circuit current and NH 4 + production in the ileum and rectum are primarily neutral amino acids, sited above (Chamberlin and Phillips, 1982a; Thomson et a/.,1988; chapter 2) and are similar to amino acids (alanine, glutamine and serine) utilized by The terms, ammonia, total ammonia, and refer to the sum of NH3 and NH4*. The terms NH3 and NH4 + (ammonium) refer to the non-ionic and ionic forms of ammonia, respectively. 32 the vertebrate kidney (Pitts, 1973). Amino acid catabolism in mammalian kidneys involves the hydrolytic deamination of glutamine to glutamate and ammonia and the subsequent deamination of glutamate (via glutamate dehydrogenase) to a-ketoglutarate and ammonia. Oxidation of amino acids in the locust rectum involves essentially the same pathway, but it is not known whether the catabolism is complete. However, enzymes such as glutamate dehydrogenase which are necessary for complete oxidation are abundant in the rectum (Chamberlin and Phillips, 1983). In the rectum and kidney, the ammonium formed from the amino acid oxidation is secreted into the tubule lumen. Studies by Lechleitner (1988) on the rectum show that oxidation of amino acids, especially proline, supports ammonia secretion and further studies by Thomson et al. (1988) showed that ammonium ions were pumped preferentially to the lumen side in exchange for Na+. Ammonia may be secreted in 2 forms: The non-ionic form (NH3) diffuses across the cell membrane rapidly, according to external pH and total ammonia concentration. Ammonia secretion via non-ionic diffusion has been reported in the rabbit cortical collecting ducts (Knepper et al, 1984) and the turtle urinary bladder (Schwartz et al, 1983). The ionic form (NH4+) may be exchanged for Na+ by an amiloride-sensitive mechanism (Evans and Cameron, 1986) or by an H7Na+ exchanger (Kinsella and Aronson, 1981). Alternatively, NH 4 + may be secreted via hydrophilic channels, pumps or other exchangers. Active transport of ammonia by a Na+/NH4+ exchange has been demonstrated in the hindgut of the locust rectum (Thomson et al, 1988), the hindgut of Sacrophaga bullata (Prusch, 1972) and recently in the proximal tubules of the mammalian kidney (Kinsella and Aronson, 1981; Nagami and Kurokawa, 1985; Nagami, 1988). 33 Methods and Materials Animals Locusts (Schistocerca gregaria) were reared as previously described (Hanrahan et al, 1984). Adult female locusts, 2-4 weeks beyond their final moult, were used in all experiments because of the larger size of their ilea. Ammonia Secretion Ilea were mounted as flat sheets between Ussing-type chambers each holding 2 mis of solution (Hanrahan et al, 1984). Epithelial preparations were bathed bilaterally with identical salines, which were vigorously aerated and mixed by bubbling with 100% oxygen. Using a complete physiological saline, ilea were brought to steady-state under open-circuit conditions within one hour, as reported earlier by Irvine et al (1988). The tissue preparations were bathed for one hour in salines containing a single amino acid. Then 5 mmol/1 cAMP was added to the saline and observations were made for a further 1.25 hour period. Saline samples (500ul) were collected from the lumen side after each period and assayed for total ammonia content. The rate of ammonia secretion OamJ was determined as (total final ammonia) - (total initial ammonia) in the external saline and expressed as a flux rate per square centimetre of tissue per hour. This rate is measured as a net accumulation in the bathing saline without specification as to the species transported (NH3/NH/). Ammonia concentrations were determined by the enzymatic assay of Kun and Kearny (1974), which uses the reductive animation of 2-oxoglutarate catalysed by the enzyme glutamate dehydrogenase to bring about a change in extinction (at 340 nm) 34 proportional to the ammonia concentration of the sample: NADH + NH 4 + + 2-oxoglutarate + glutamate dehydrogenase » L-glutamate +NAD+ + H 20 + glutamate dehydrogenase. Acid Secretion Locust ilea were mounted as above and bathed in a complete physiological saline stirred and oxygenated with 100% oxygen. Ilea were brought to steady state under short-circuit conditions using agar salt bridges as described by Thomson et al. (1991). The bathing saline pH was maintained at pH 7.0 throughout the experiment by continuous perfusion of both the haemocoel (2ml/min) and luminal (5ml/min) sides of the tissue, except for 3 experimental periods of 45 minutes, during which luminal perfusion was stopped (but mixing continued by bubbling 100% oxygen). Acid secretion (calculated as flux rates per square centimetre of tissue per hour) to the lumen side was measured by a pH-stat technique (PHM 84 research pH meter, TTT 80 titrator, ABU 80 autoburette; Radiometer, Copenhagen, Denmark) as the amount of titrant (0.010 N NaOH or KOH) required to maintain the luminal pH at 7.00. 1^  (uequiv.cm"2.!.'1) was continuously monitored on a dual channel strip-chart recorder (1242 Soltec, Sun Valley Ca.) Solutions Salines were C0 2 and HC03" free to facilitate comparisons with previous work on the rectum (Thomson et al, 1988), and to remove the effects of volatile buffer components other than NH3. The composition of the complete physiological saline was based on locust haemolymph (Hanrahan and Phillips, 1983), and contained (in mmol/1): NaCl, 100; K2S04, 5; MgSQ4, 10; CaCl2, 5; 3-(N-morpholino) propanesulfonic acid (MOPS; pK„ 7.2 at 25°C), 10; 35 glucose, 10; sucrose, 100; alanine, 2.9; asparagine, 1.3; arginine, 1.0; glutamine, 5; glycine, 11.4; histidine, 1.4; lysine, 1.4; proline, 13.1; serine, 1.5; valine, 1.8; with pH adjusted to 7.0 . For experiments measuring ammonia secretion, sucrose was adjusted to 110 mmol/1 to replace NaHC03. For the acid secretion experiments, 10 mmol/1 sodium-isethionate and 1.0 mmol/1 tyrosine were added, and 2 mmol/1 MOPS was used so that haemolymph osmotic concentration remained constant at 420 mosmol/1. Experimental salines containing one or a few amino acids were prepared by replacing all other organic substrates listed above with isosmotic amounts of sucrose, which is not metabolized (chapter 2, Fig. 2.1). Sodium was replaced with gluconate salt to prepare a Na+-free saline. Amiloride was made up in sulfate-free salines just prior to use and added to the luminal side of the tissue 30 minutes before and during the experimental period. All salines were also initially ammonia free (<40uM measured) to confine the scope of the investigation to endogenously produced ammonia. All experiments were conducted at 23-25°C. All chemicals were reagent grade and obtained from Sigma Co. (St. Louis, Mo.). Statistics Data are reported as means ± standard error of mean (SE), with n indicating number of ilea. A P value of < 0.05 was considered an indication of statistical significance using paired and independent Mests. 36 Results The characterization of ammonia secretion During long-term flux studies when the chambers were bathed in a complete physiological saline containing 5 mmol/1 cAMP and 5 mmol/1 theophylline, ilea maintained a constant rate of ammonia secretion for at least 4 hours, during which time the ammonia concentration of the bath rose linearly from nearly zero to 800 umol/1 on the lumen and to 150 umol/1 on the haemocoel sides (Fig. 3.1). Ammonia was secreted preferentially to the lumen side at rates of 1.40 ± 0.15 umoles.cm'2.!."1 compared to 0.37 ± 0.10 umoles. cm"2.!.'1 to the haemocoel side. In the sections which follow, the ammonia secretion rates presented are to the lumen unless otherwise stated. All subsequent experiments were completed within 3.25 hours and before bath ammonia exceeded 800 umol/1, ensuring that was measured over time periods during which was constant The rise in chamber ammonia was not due to absorption from air. When Ussing chambers without ilea present were bubbled with oxygen and bathed with a complete saline no increase in ammonia concentration was observed over 2 hours. There is evidence for uptake and metabolism of amino acids in the ileum (chapter 2) and in the rectum (Chamberlin and Phillips, 1982a). In the ileum (chapter 2), five amino acids supplied luminally are the major metabolic substrates used to support CI" transport. Similarly, a luminal addition of a complete saline supports ileal ammonia secretion rates as high as bilateral exposure (Fig. 3.2). Amino acids supplied serosally supported a about 50% of that present with bilateral amino acids (Fig. 3.2). An addition of cAMP has an effect on ammonia secretion with only a bilateral source of substrate (Fig. 3.2). 37 Figure 3.1 Ammonia concentration with time for ilea bathed bilaterally in complete physiological saline with 5 mM cAMP and 5 mM theophylline. Saline pH was maintained at 7. Mean ± SE. (n=8). 38 Figure 3.2 Effect of the source of amino acids on ammonia secretion rates for ilea bathed in a complete saline. Mean ± SE. (n=4). * 8 No significant difference from each other by independent f-test (P < 0.05). 39 Substrate Free Complete AA Ala Asn Gin Pro Ser Other Conc.(mM) - 2.9 1.3 5.0 13.1 1.5 Figure 3.3 Effect of bilateral addition of amino acids on ammonia secretion rates to the lumen. AA, an amino acid saline including alanine, asparagine, glutamine, proline and serine at concentrations shown. Other, a saline including the other amino acids in the complete saline (arginine, glycine, histidine, lysine and valine) at concentrations found in complete saline. Mean ± SE. (n=4-8). •Significantly different by paired f-test (P <0.05) "treatment without cAMP, btreatment with cAMP, significantly different from complete saline control by independent r-test (P <0.05) 40 Trace levels of ammonia (up to 40 umol/1) were present in the amino acid saline. To determine the contribution of exogenous ammonia to J ^ , bilateral external concentrations of ammonia in substrate-free saline were varied from 0.01 - 0.09 mmol/1. Under unstimulated conditions, it was found that increased 4 times with a 9 times rise in external ammonia levels (Fig- 3.4). to the haemocoel under these conditions were 0-0.03 umoles.cm"2.!."1 (data not shown) and unchanged by external ammonia concentrations. In contrast, with cAMP present, was unaffected by variation in external ammonia (Fig. 3.4). In subsequent experiments, J,,,,,,,, due to saline ammonia was subtracted from experimental rates to determine secretion due to addition of individual amino acids per se. Amino acids as possible precursors of ammonia Individual amino acids were assayed for their effect on luminal secretion of ammonia, with and without cAMP (Fig. 3.3). Amino acids were applied bilaterally at typical concentrations in the haemolymph. The 5 amino acids studied (ala, asn, gin, pro and ser) were selected because of their ability to support CI' dependent short-circuit current in the previous chapter. A saline with these 5 principal amino acids (AA saline) supported not significantly different from the Jamm with complete saline. Each of these 5 amino acids, when applied individually, resulted in a significantly above the control value. The remaining amino acids found in the complete saline (arg, gly, his, lys, and val) that do not stimulate ileal L^ . were combined in one saline (other; Fig. 3.3); which gave a value insignificantly different from that with substrate-free saline. 41 Figure 3.4 Effect of saline ammonia concentration on the rate of ileal ammonia secretion to the lumen. Salines were substrate-free and provided bilaterally. Mean ± SE. (n=4-8). 42 The addition of cAMP when complete saline or the 5 amino acid saline were present causes an increase in (Fig. 3.3). However, cAMP does not lead to significantly increased rates of ammonia secretion when individual amino acid salines are assayed (Fig. 3.3). For alanine and serine salines, significantly decreases after stimulation with cAMP. I next studied the ability of amino acids applied unilaterally to support (Fig. 3.5). The principal substrates applied on the lumen side only were assayed at concentrations reported for Malpighian tubule fluid by Chamberlin and Phillips (1982b). Substrate-free saline was present on the serosal side during these experiments. Under stimulated and unstimulated conditions, all 5 amino acids which support ileal L^ . bilaterally stimulated J ^ . Asparagine and glutamine have the greatest effect on with both bilateral and luminal presentations (Figs. 3.3, 3.5). The addition of cAMP does not have a significant effect on the caused by any luminal amino acid. The nature of ileal ammonia secretion Non-ionic diffusion of NH3 occurs if there is a transmembrane pH or ammonia concentration gradient. If the apical and basolateral membranes are equally permeable to NH3, the free base will passively distribute itself according to the direction of these transmembrane gradients. If the pH on one side is lowered, the free base (NH3) will diffuse into the acid medium and bind to the hydrogen ions to form non-diffusible ammonium ions (NH4+). If the species transferred across the apical membrane is NH3, rather than NH 4 +, then should vary with a pH gradient and rate of acid secretion (JH), but should not be affected by ion substitutions (eg. Na+) or the inhibitor of cation exchange (i.e. amiloride). 43 Figure 3.5 Effect of luminal addition of amino acids at the concentration typically observed on the lumen side, on the rate of ammonia secretion. AA, an amino acid saline including alanine, asparagine, glutamine, proline and serine at concentrations shown. Mean ± SE. (n=4). All values not significantly different by paired /-test (P <0.05) "treatment without cAMP, Veatment with cAMP, significantly different from amino acid (AA) saline control by independent Mest (P <0.05) 44 When ilea were exposed to a luminal pH of 4.5 to 7.5 maintained by pH stat, (haemolymph pH=7), mucosal acid secretion varied but ammonia secretion did not change significantly (Fig. 3.6). Even when the pH was the same on both sides of the tissue (Fig. 3.1) the rates of secretion to the mucosa were significantly higher than to the serosa. These results suggest that NH3 is not the primary species transferred across the epithelial membrane. A NH4+/Na+ exchange mechanism has been demonstrated in the locust rectum (Thomson et al, 1988); therefore, two experiments were conducted to determine if the ileal occurs by the same mechanism. The first experiment involved a luminal Na+ substitution. Hea were exposed to a complete saline with bilateral Na+ (110 mmol/1) for one hour, and then with Na+-free saline ( <0.5mmol/l) on the lumen side for one hour, and finally with bilateral high Na+ restored, for one hour. The same procedure was followed with serosal addition of 5mmol/l cAMP. The removal of Na+ does not affect (Table 3.1) with or without cAMP present, suggesting that NH 4 + is not exchanged for luminal Na+. The second experiment involved a luminal addition of 0.10 mmol/1 amiloride, a Na7NH4+ exchange inhibitor in other cell types tfivans and Cameron, 1986). The protocol was the same as the Na+ substitution experiments. The data presented in table 3.2 shows that amiloride does not inhibit J ^ , suggesting again that NH 4 + is not exchanged for Na+. 45 -2.0 L ' ' • 1 4.0 5.0 6.0 7.0 8.0 Luminal pH Figure 3.6 Effect of pH gradients on luminal ammonia secretion rates and luminal acid secretion rates (no cAMP). Haemocoel pH maintained at 7. Mean ± SE. (n=6-23). 46 Table 3.1 Effect of luminal Na+ substitution on luminal ammonia secretion rates Ammonia Secretion Rate /imoles .cm2 .h'1 without c AMP with c AMP Complete Saline with Na+ 0.83 ± 0.22 0.70 ± 0.20 without Na+ 0.75 ± 0.12 0.98 ± 0.08 with Na+ 0.85±0.10 0.91 ±0.11 Mean ± SE (n=4 - 5). pH l u m e n = pH, ^ =7. Within treatment groups all values not significantly different by paired t -test (P < 0.05) 47 Table 3. 2 Effect of amiloride on luminal ammonia secretion rates Ammonia Secretion Rate //moles .cm2 .h 1 Control Saline 1.50 ±0.13 +0.10 mM Amiloride 1.61 ± 0.63* Mean ± SE (n=6). Amiloride added to luminal bath only. pH l u m e n = pH h a e m o c o e l =7. * No significant difference from pooled control by paired t -test {P < 0.05) ) 48 Discussion Identification of the specific amino acids responsible for ammonia secretion Several amino acids, when supplied bilaterally at haemolymph concentrations, are capable of supporting high rates of ileal J ^ . The five amino acids (ala, asn, gin, pro and ser) that cause significant increases in CI" dependent 1^ . (chapter 2) are the same 5 amino acids that support ileal J ^ . Although the tissue uses these 5 amino acids, asparagine and glutamine are preferentially utilized for ammonia secretion. The 5 major amino acids clearly serve a dual function in the ileum as respiratory substrates and sources of secreted ammonia. In the mammalian kidney (Nissim et al., 1986) and in the locust ileum, glutamine is a major source of ammonia. However, other sources of amino acids other than glutamine may contribute significantly to ammoniagenesis. The rectum, on the other hand, utilizes a number of amino acids to support J ^ , but cannot be supported by just one amino acid (Thomson et al., 1988). Some ammonia secretion due to endogenous production (in substrate-free saline) does occur, especially at high external NH4 + and without cAMP (Fig. 3.4). Similar results have been reported for the rectum of S. gregaria (Thomson et al, 1988) and S. bullata (Prusch, 1972) and the mammalian kidney (Lowe et al, 1985). Substantial (0.9 umoles.cm .^h"1) occurs at external ammonia concentrations similar to that reported in locust haemolymph (Stagg et al, 1991 and Harrison, 1988) suggesting that endogenous sources of NH/ (i.e. cellular protein) can make some contribution in vivo. The rise in NH4 + excretion with increase in haemolymph and luminal NH 4 + concentration can provide a mechanism for increasing ileal NH 4 + secretion when 49 organismal production rises. In the mammalian kidney, Lowe et al. (1985) find that the external concentration of NH 4 + may act on an external modifier site to stimulate Na+/H+ or Na+/NH4+ exchange. Sidedness of amino acid uptake and ammonia secretion Generally, luminal presentations of substrates supported as well as bilateral applications, indicating that luminal uptake of substrates is predominant (Figs. 3.2, 3.3, 3.5). Similar conclusions were reached when the effect of sided amino acid presentation was tested on transepithelial 1^ . (chapter 2). Whether the substrates were added to the lumen or haemocoel side of the tissue, is predominantly to the lumen side, allowing the epithelium to use the amino acids as respiratory substrates and to contribute to acid-base regulation by releasing nitrogenous waste to the lumen. The effect ofcAMP on In the ileum, cAMP significantly increases by 30% (similar increases reported in rectum, lechleitner, 1988; Audsley, 1991) when a complete physiological saline is available bilaterally (Figs. 3.1, 3.3). In the rectum, cAMP increases by 20% when the only luminal amino acid is proline, but not when a complete physiological saline is provided (Phillips et al, 1986). In the locust rectum, given that exogenous amino acids appear to be the primary fuel for oxidative metabolism in the locust hindgut (Chamberlin, 1981; chapter 2), why does (with a bilateral addition of complete saline) not increase with I,,. after cAMP ? The pathway for the production of ammonia involves the hydrolytic deamination of glutamine to glutamate and 50 ammonia (Chamberlin and Phillips, 1983). A further oxidation of glutamate produces oc-ketoglutarate and ammonia. When cAMP increases ATP production, amino acids may be transaminated and their carbon skeletons oxidized, but the NH 4 + groups may be trapped in the form of glutamate, and concentrations of glutamate may increase in the cell. Alternatively, cAMP may stimulate the oxidation of endogenous glycogen or lipid stores. Finally, cAMP may increase the catabolism of metabolic intermediates to C0 2 without stimulating further deamination of amino acids or of glutamate. Mechanism of ammonia secretion The rate of ammonia transport in the locust ileum is independent of transmembrane pH gradients and JH. By these criteria, luminal in the locust ileum must be occurring primarily by NH 4 + transport and not by diffusion trapping. In the presence of luminal amino acids, is unchanged in Na-free saline, and is also unchanged with the addition of 0.10 mmol/1 amiloride. This suggests that NH 4 + transport does not occur via a Na+ exchange as documented for the locust rectum (Thomson et al, 1988). The large luminal might be due to the deamination of amino acids by apically bound enzymes. Lechleitner (1988) has found y-glutamyl transferase, an enzyme that cleaves amino acids, in an apical membrane fraction. This enzyme could account for the sidedness of luminal Jamm» m e unchanged with changes in pH and the lack of effect of Na+ substitution or amiloride. To date, there is no direct evidence of deamination of luminal amino acids in the hindgut. This is an interesting area for future studies. 51 In conclusion, the preferential secretion of NH 4 + in the locust ileum is supported by the mucosal addition of alanine, asparagine, glutamine, proline and serine. Ammonia secretion is particularly favoured by the addition of asparagine and glutamine. does not occur by non-ionic diffusion trapping, or by Na+/NH4+ exchange as in the locust rectum. M i l 52 CHAPTER FOUR General Discussion The overall objectives of this thesis were to determine, 1) which external substrates support aerobic respiration, 2) the role of exogenous amino acids in the support of ammonia secretion and, 3) the magnitude of these processes relative to the locust rectum and mammalian kidney. The results presented in previous chapters indicate that amino acid metabolism is an important source of energy for ion and water transport and a source of secreted ammonia in the locust ileum. This conclusion is supported by the following observations. 1) The "k. of substrate-depleted ilea is maximally stimulated by physiological concentrations of ala, asn, gin, pro, ser and glucose and this stimulation can be achieved by adding these amino acids individually to the luminal side of the tissue (chapter 2). 2) The ileum preferentially secretes ammonia in the form of NH 4 + into the lumen when ala, asn, gin, pro and ser are present on the luminal side (chapter 3). Although there are high levels of amino acids in insect haemolymph (Chamberlin and Phillips, 1979; Rutherford and Webster, 1978), the metabolically demanding flight muscle oxidizes carbohydrates and lipids (Weis-Fogh, 1952) for extended periods of flight (Brosemer and Veerabhdrappa, 1965). The rectum, normally exposed to metabolic substrates on both the luminal and haemocoel sides of the tissue, preferentially takes up luminal amino acids, primarily proline as its main respiratory substrate (Chamberlin and Phillips, 1982a). The Malpighian tubule 53 fluid supplies the lumen with amino acids and glucose. Tested at high concentrations, the ileum and rectum both utilize alanine, proline, glutamine and glucose. However, at physiological concentrations, a luminal presentation of glucose causes a very small stimulation of J^ ., whereas proline maximally stimulates I*.. Preferential uptake of proline occurs on the luminal side of the tissue. Basolateral uptake is very low, due to a muscle layer, the presence of secondary cells, and an unfavorable outflow of absorbed fluid (Phillips, 1964). Both tissues are capable of oxidizing some carbohydrates and a wide variety of amino acids; however, neither of the 2 hindgut segments use the main haemolymph sugar (trehalose) or lipid (diolein). Chamberlin (1981) proposed that since flight muscles readily oxidize lipid (Weis-Fogh, 1952) and carbohydrates such as glucose and trehalose, these muscles may out compete the hindgut epithelia for these substrates, making it advantageous to oxidize amino acids which the flight muscles only moderately oxidize (Brosemer and Veerabhdrappa, 1965). From recent observations (Thomson et al, 1988; Harrison and Phillips, submitted; chapter 3) it seems more likely that the hindgut, a transporting epithelium, metabolizes amino acids to provide NH, to aid in acid-base regulation. Amino acid metabolism is a source of ATP in the rectum. The complete oxidation of proline yields 32 ATP which is comparable to the 36 ATP produced by glucose oxidation (Chamberlin, 1981). The ATP is used to drive ion transport (Chamberlin, 1981), and the cellular ammonium produced is secreted to the lumen (Thomson et al, 1988). Chamberlin and Phillips (1983) demonstrated that the rectum has the enzyme glutamate dehydrogenase, which is associated with amino acid metabolism. Proline carbons enter the TCA cycle via glutamate dehydrogenase with pyruvate serving as an amino accepter, forming alanine. Alanine may then 54 be metabolized via a transaminase. The complete oxidation of proline leads to the production of ammonium rather than ammonia, since ammonium is one of the products of glutamate dehydrogenase (Lehninger, 1975; Chamberlin and Phillips, 1983). While biochemical pathways have not been investigated for the ileum, presumably amino acids are catabolized via similar pathways as in the rectum. These predictions require future experimental confirmation. Lechleitner (1988) compared the locust ileum and the mammalian kidney proximal tubules. The ileum has a large capacity for reabsorption of Na+, K+, CI", HC03" and fluid from the primary urine: this reabsorbed fluid is isosmotic to the original urine and haemolymph. The proximal tubules remove a large portion of glomerular filtrate while having no effect on urine osmolality. In terms of ammonia excretion, both the ileum (Lechleitner, 1988; Audsley, 1991; chapter 3) and vertebrate proximal tubules (Brosnan et al, 1985) are the predominant sites of ammonia production in their respective renal systems. There is reason therefore to propose that the locust ileum is functionally analogous to proximal tubules of the vertebrate kidney. Renal ammonium excretion plays a role in whole-body acid-base balance. NH 4 + secretion is equivalent to acid secretion if, (1) neutral amino acids are oxidized by the transport epithelia, (2) glutamate generated is fully oxidized to bicarbonate and ammonia, and (3) bicarbonate is moved to the haemocoel in equimolar quantities to ammonia secretion to the lumen (Harrison and Phillips, submitted). The locust ileum (chapter 2, 3) and rectum (Balshin and Phillips, 1971; Balshin, 1973) both catabolize primarily neutral amino acids, as does the proximal tubules. If the glutamate derived from neutral amino acids is oxidized, bicarbonate and ammonia will be produced in equal quantities (Walser, 1986). Chamberlin and Phillips (1983) have shown that rectal mitochondria have a large capacity for glutamate oxidation. Since bicarbonate 55 concentrations in the faeces are less than 0.5% of ammonia concentrations (Harrison and Phillips, submitted), and bicarbonate has been shown to move to the haemocoel side at high rates (Lechleitner et al, 1989a), ammonium excretion could aid in acid-base regulation. This may explain why the locust hindgut has evolved to utilize amino acids as metabolic substrates. 56 REFERENCES A L T M A N , P.L. (1961). Blood and other fluids, (ed. D.S. Dittmer), Federation of the American Society for Experimental Biology, Washington, D.C, 540. AUDSLEY, N. (1991). Purification of a neuropeptide from the corpus cardiacum of the desert locust which influences ileal transport. Ph. D. thesis. University of British Columbia, Vancouver, Canada. AUDSLEY, N., AND PHILLIPS, J.E. (1990). Stimulants of ileal salt transport in neuroendocrine system of the desert locust. Gen. Comp. Endocrin. 80, 127-137. BALSHIN, M. (1973). 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