Science, Faculty of
Zoology, Department of
DSpace
UBCV
Audsley, Neil
Doctor of Philosophy - PhD
University of British Columbia
Previous studies on the regulation of salt and water reabsorption in the insect excretory system have concentrated on the rectum, while regulation of the ileum has received little attention. Cl⁻ transport is the predominant ion transport process in both the ileum and rectum of the desert locust and drives fluid absorption. The central nervous system (CNS) was surveyed for factors which stimulate Cl⁻-dependent short-circuit current (I[formula omitted]) using in vitro flat sheet preparations of locust ileum as a bioassay. All ganglia extracts tested (except the corpora allata) caused significant increases in ileal I[formula omitted]. Extracts of muscle tissue, used as a control, had no effect on ileal I[formula omitted] indicating that stimulants were not general metabolites present in locust tissue. Crude extracts of the corpus cardiacum (CC) and fifth ventral ganglion (VG5) stimulated ileal I[formula omitted] in a dose-dependent manner and both caused an increase in K⁺ and Na⁺ absorption as previously observed with cAMP. CC and VG5 had no effect on ileal NH₄⁺ secretion but both inhibited ileal H⁺ secretion. Most of the stimulatory effects of CC and VG were largely abolished by treatment with trypsin and chymotrypsin, suggesting that the stimulants were peptides. CC and VG5 factors were apparently separate compounds because they differed in the time courses of ileal I[formula omitted] response, thermal stability, and extraction properties. Reversed-phase high performance liquid chromatography (RP-HPLC) of water extracts of CC identified two distinct factors (fractions D and F) which stimulated ileal I[formula omitted] and a third factor (fraction G) which had little effect on I[formula omitted], but which caused a five-fold increase in ileal fluid transport (J[formula omitted]). None of these fractions increased rectal J[formula omitted]; moreover, fraction D stimulated rectal I[formula omitted] at higher doses. These results provided the first indication that separate stimulants act on locust rectum and ileum. The most potent factor in CC acting on ileal I[formula omitted] was isolated using a four-step purification procedure, utilizing C₈ and phenyl RP-columns for separation. Amino acid analysis of this purified peptide indicated a molecular weight of 7700 daltons and a near complete amino acid sequence (50 out of 65) was determined. The purified factor (S. gregaria ion transport peptide; ScgITP) was assayed on all ileal ion transport processes influenced by crude CC extracts. ScgITP caused quantitatively the same range of effects as crude CC extracts, in that it stimulated Cl⁻, K⁺, and Na⁺ reabsorption and inhibited H⁺ secretion. High doses of ScgITP (5 CC equiv.ml⁻¹) caused the same maximum response on all these systems as crude CC extracts (0.25 CC equiv.ml⁻¹). ScglTP is unlikely to be chloride transport stimulating hormone, previously reported to act on the rectum, because a maximum rectal I[formula omitted] response was not achieved and there was no effect on rectal J[formula omitted], which is Cl⁻-dependent. It appears that ScgITP acts through cAMP as the second messenger to stimulate reabsorptive processes because this cyclic nucleotide mimicked the actions of ScgITP and crude CC extracts. In support of this view, ileal I[formula omitted] was also stimulated to maximum levels by 5mM theophylline and 50μM forskolin. The inhibition of H⁺ secretion by ScgITP must occur through a different intracellular pathway because this action was not mimicked by cAMP.
PURIFICATION OF A NEUROPEPTIDE FROM THE CORPUS CARDIACUM OF THE DESERT LOCUST WHICH INFLUENCES ILEAL TRANSPORT by NEIL AUDSLEY B.Sc.(HONS), The University of Newcastle Upon Tyne, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ZOOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA NOVEMBER, 1990 © Neil Audsley, 1990 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 The University of British Columbia Vancouver, Canada Date DE-6 (2/88) i Abstract Previous studies on the regulation of salt and water reabsorption in the insect excretory system have concentrated on the rectum, while regulation of the ileum has received little attention. CI" transport is the predominant ion transport process in both the ileum and rectum of the desert locust and drives fluid absorption. The central nervous system (CNS) was surveyed for factors which stimulate Cl'-dependent short-circuit current (Isc) using in vitro flat sheet preparations of locust ileum as a bioassay. All ganglia extracts tested (except the corpora allata) caused significant increases in ileal L^ .. Extracts of muscle tissue, used as a control, had no effect on ileal L. indicating that stimulants were not general metabolites present in locust tissue. Crude extracts of the corpus cardiacum (CC) and fifth ventral ganglion (VG5) stimulated ileal la. in a dose-dependent manner and both caused an increase in K+ and Na+ absorption as previously observed with cAMP. CC and VG5 had no effect on ileal NIL,* secretion but both inhibited ileal H+ secretion. Most of the stimulatory effects of CC and VG were largely abolished by treatment with trypsin and chymotrypsin, suggesting that the stimulants were peptides. CC and VG5 factors were apparently separate compounds because they differed in the time courses of ileal I,,, response, thermal stability, and extraction properties. Reversed-phase high performance liquid chromatography (RP-HPLC) of water extracts of CC identified two distinct factors (fractions D and F) which stimulated ileal I„, and a third factor (fraction G) which had little effect on I„., but which caused a five-fold increase in ileal fluid transport (Jv). None of these fractions increased rectal Jv; moreover, fraction D stimulated rectal I«. at higher doses. These results provided the first indication that separate stimulants act on ii locust rectum and ileum. The most potent factor in CC acting on ileal I,,. was isolated using a four-step purification procedure, utilizing C„ and phenyl RP-columns for separation. Amino acid analysis of this purified peptide indicated a molecular weight of 7700 daltons and a near complete amino acid sequence (50 out of 65) was determined. The purified factor (S. gregaria ion transport peptide; ScglTP) was assayed on all ileal ion transport processes influenced by crude CC extracts. ScglTP caused quantitatively the same range of effects as crude CC extracts, in that it stimulated CI", K+, and Na+ reabsorption and inhibited H+ secretion. High doses of ScglTP (5 CC equiv.ml'1) caused the same maximum response on all these systems as crude CC extracts (0.25 CC equiv.ml"1). ScglTP is unlikely to be chloride transport stimulating hormone, previously reported to act on the rectum, because a maximum rectal I«. response was not achieved and there was no effect on rectal JVi which is Cl'-dependent. It appears that ScglTP acts through cAMP as the second messenger to stimulate reabsorptive processes because this cyclic nucleotide mimicked the actions of ScglTP and crude CC extracts. In support of this view, ileal ^ was also stimulated to maximum levels by 5mM theophylline and 50u,M forskolin. The inhibition of H + secretion by ScglTP must occur through a different intracellular pathway because this action was not mimicked by cAMP. iii Table of Contents Page Abstract i Table of Contents iii List of Tables vi List of Figures vii List of Abbreviations x Acknowledgements xiv Chapter 1: General Introduction 1 Structure of the locust excretory system 4 Fluid and ion transport in the hindgut 6 Mechanisms of solute and water transport across the locust ileum. 7 Insect hormones 10 The endocrine control of excretion 11 Regulation of reabsorption in the hindgut 16 Source, distribution and release of neuropeptides 19 Second messengers of insect peptides 21 Isolation of insect neuropeptides 24 Chapter 2: Actions of Corpus Cardiacum and Ventral Ganglia on Ileal Salt Transport 32 Introduction 32 Materials and Methods 35 Electrical measurements 35 Salines 37 Preparation of tissue extracts 37 Assay procedures 39 Estimation of potassium permeability 39 Ammonia secretion 40 Acid secretion 41 Extraction of stimulants in CC or VG with different solvents 42 Effect of proteases on CC and VG stimulants 42 Stability of CC and VG factors 43 Statistical treatment 43 Results 44 Effect of cAMP on ileal I« 44 Survey of the central nervous system for stimulants . . . . 44 Time course of stimulation and relative activity in separated lobes of CC 50 Dose-response relationship for CC homogenate 50 Time course of response to VG5 homogenate 52 iv Dose-response relationship for VG5 homogenate . . . . . . . 54 Effect of CC and VG5 on potassium permeability 54 Effect of cAMP, CC and VG5 on ammonia secretion . . . 54 Effect of cAMP, CC and VG5 on acid secretion 57 Effect of CC and VG on rectal I„ 57 Effect of CC and VG5 homogenates on rectal Ik 57 Effect of temperature on CC and VG stimulants 63 Effect of proteolytic enzymes on CC and VG stimulants . 63 Stimulatory activity extracted with different solvents . . . . 65 Effect of known or putative insect neurotransmitters on ileal k 66 of exogenous stimulants on ileal 66 Discussion 72 3: Isolation and characterization of a factor from the CC which influences ileal transport 77 Introduction 77 Materials and Methods 80 Bioassays 80 Electrogenic chloride transport 80 Fluid absorption 80 Extraction and HPLC 81 Preparation of tissue extracts 81 Separation of CC extracts 82 Preparative step 82 Reversed-phase HPLC - C8 step 82 Reversed-phase phenyl step 83 Electrophoresis 84 SDS-PAGE 84 DBF 85 Effect of proteolytic enzymes on purified CC 85 Amino acid analysis and sequencing 85 Results 86 C4 Separation 86 Cg Separation 86 Phenyl separation of fraction D 94 Estimation of purity of fraction Diii 97 SDS-PAGE 97 Sequence analysis 97 Dose-response relationship of fraction Diii (PI + P2) . . . 97 Phenyl separation of fraction Diii 101 Effect of proteolytic enzymes on fraction Diii3 106 Estimation of purity of fraction Diii3 106 SDS-PAGE 106 Isoelectric focusing 110 Amino acid analysis and sequence data 110 Discussion 113 V Chapter 4: Actions of Ion Transport Peptide from the Corpus Cardiacum on Ileal Ion Transport 120 Introduction . 120 Materials and Methods 122 Sodium flux measurements 122 Statistical treatment 123 Results 124 Dose-response relationship and time course of ileal 1^ with ITP 124 Effect of ITP+P2 and ITP on ileal IK 124 Effect of ITP+P2 and ITP on ileal Na+ reabsorption . . . . 127 Effect of ITP-I-P2 and ITP on ileal acid secretion 132 Discussion 135 Chapter 5: General Discussion 138 References 152 vi List of Tables Page Table 1.1 Factors which increase (diuretic) fluid secretion by the Malpighian tubules of insects and their sources 13 Table 1.2 Factors which increase (antidiuretic) fluid reabsorption in the rectum of insects and their sources 14 Table 2.1 Effect of saline extracts of various parts of the nervous system on transepithelial difference and transepithelial resistance across short-circuited ilea 48 Table 2.2 Effect of cAMP, CC and VG on ileal ammonia secretion under open circuit conditions 58 Table 2.3 Effect of cAMP, CC and VG on ileal acid secretion under open circuit conditions 59 Table 2.4 Effect of different temperature pre-treatments on ileal I« following addition of CC or VG extracts 64 Table 2.5 Effect of protease pre-treatments of CC and VG homogenates on stimulation of ileal L. 67 Table 2.6 Effects of exogenous stimulants on ileal I,,. 70 Table 3.1 Effect of 60% CFLCN fractions from C4 cartridge on rectal and ileal IK 87 Table 3.2 The twenty common protein amino acids 99 Table 3.3 Effect of protease pre-treatments of purified factor Diii3 on stimulation of ileal L. 108 Table 4.1a Effect of ITP+P2 on ileal acid secretion under open circuit conditions 134 Table 4.1b Effect of ITP on ileal acid secretion under open circuit conditions 134 Table 5.1 A comparison between the transport parameters across the locust ileum between unstimulated (control) conditions, and due to crude CC and ScglTP stimulation 139 vii List of Figures Page Figure 1.1 Diagram of the locust excretory system 2 Figure 1.2 Comparison of ultrastructural organization and gross dimensions of locust rectal pad and ileal epithelium 6 Figure 1.3 Model of transport mechanisms identified in locust rectal epithelium 8 Figure 2.1 Standard Ussing chamber assembly used for measuring I*., V M JH+. h> JAmm and J N a + 36 Figure 2.2 Change in ileal 1K with time under unstimulated and cAMP stimulated conditions 45 Figure 2.3 Effect of saline extracts of various parts of the nervous and endocrine systems, and muscle (control) on electrical parameters across short-circuited locust ilea 46 Figure 2.4 Effect of different doses of CC extract on the time course of ileal response 49 Figure 2.5 Increase in I« one hour after adding various doses of CC homogenate to the haemocoel side of ilea 51 Figure 2.6 The time course of ileal 1^ in response to addition of fifth ventral ganglia (VG5) extracts to the haemocoel side at different doses 53 Figure 2.7 Dose-response curve for maximum stimulation of ileal 1^ by V G 5 extracts 55 Figure 2.8 Effect of CC and V G extracts on ileal IK 56 Figure 2.9 Increases in rectal 1^ . one hour after adding various doses of crude CC to the haemocoel side 60 Figure 2.10 Dose-response curve for maximum stimulation of rectal 1^ to V G extracts 61 Figure 2.11 Effect of CC and V G homogenates on rectal IK 62 Figure 2.12 Maximum stimulation of ileal I,,, following extraction of whole CC (A) and V G 5 (B) by different solvents 68 Figure 2.13 A dose-response curve for forskolin stimulation of ileal !„. . . . 71 vi i i Figure 3.1 Absorption profile of extracts from 300 whole CC from C8 reversed-phase-HPLC 88 Figure 3.2 Absorption profile of 50 NCC from C8 reversed-phase HPLC 89 Figure 3.3 The effect of whole CC HPLC fractions on ileal I«. one hour after addition of fractions 90 Figure 3.4 The effect of HPLC fractions from whole CC on rectal I,,. one hour after addition of fractions 92 Figure 3.5 The effect of whole CC HPLC fractions on ileal fluid transport in the absence of any initial osmotic concentration difference 93 Figure 3.6 Dose-response relationship of HPLC fraction G on ileal fluid absorption 95 Figure 3.7 Absorption profile of fraction D from phenyl RP-HPLC separation 96 Figure 3.8 SDS-PAGE of fraction Diii 98 Figure 3.9 Partial amino acid sequences of peptides from fraction Diii . . . 100 Figure 3.10 Increase in ileal I« one hour after adding increasing amounts of fraction Diii to the haemocoel side 102 Figure 3.11 Increases in rectal I« one hour after adding various amounts of fraction Diii to the haemocoel side 103 Figure 3.12 Dose-response relationship for fraction Diii on the increase in ileal fluid absorption after one hour of exposure to the stimulant on the haemocoel side 104 Figure 3.13 Absorption profile of fraction Diii from phenyl RP-HPLC separation 105 Figure 3.14 SDS-PAGE of fraction Diii3 107 Figure 3.15 Plot of log of molecular weight of known peptides against their relative mobilities by SDS-PAGE 109 Figure 3.16a Comparison of amino acid and sequence analyses of pure peptide from fraction Diii3 112 ix Figure 3.16b Partial amino acid sequence of pure peptide from fraction Diii3 112 Figure 4.1 Increase in I*. one hour after adding various doses of ITP to the haemolymph side of ilea . 125 Figure 4.2 Time course of ileal I,,, response to various doses of ITP added to the haemocoel side 126 Figure 4.3 Effect of ITP+P2 on ileal IK 128 Figure 4.4 (a) The time course of unidirectional Na+ fluxes across short-circuited ilea under control and ITP+P2 stimulated conditions; (b) The net flux to the haemolymph side; (c) The short-circuit current across the tissue during flux measurements 129 Figure 4.5 The time course of unidirectional forward Na+ fluxes across short-circuited ileal under control and ITP stimulated conditions 133 Figure 5.1 Proposed model for control of ion transport across the locust ileum 141 Figure 5.2a Comparison of the amino acid sequences of diuretic peptides from L.migratoria, A. domesticus, and M. sexta 149 Figure 5.2b Comparison of the amino acid sequences of neuroparsins B and ScglTP 149 X List of Abbreviations Clem2 - ohms centimetre squared |j,A - microamps u,equiv.cm2.hl - microequivalents per square centimetre per hour |xl - microlitre uLbAileum"1 - microlitres per hour per ileum (j.m - micron uM - micromolar A - Angstrom AD - antidiuretic ADH - antidiuretic hormone ADP - antidiuretic peptide AG - abdominal ganglion AKH - adipokinetic hormone AMP - adenosine 5'-monophosphate ATP - adenosine 5'-triphosphate AVP - arginine vasopressin B - brain BSA - bovine serum albumin CA - corpus allatum cAMP - adenosine 3':5'-cyclic monophosphoric acid CC - corpus cardiacum cGMP - guanosine 3':5'-cyclic monophosphoric acid CNS - central nervous system xi CTSH - chloride transport stimulating hormone D - diuretic Da - Daltons DAG - diacylglycerol DH - diuretic hormone DP - diuretic peptide GCC - glandular lobe of the corpus cardiacum GLDH - glutamate dehydrogenase h - hours H - haemocoel or haemolymph HPLC - high performance liquid chromatography IE - ion exchange EEF - isoelectric focussing IK - potassium current n>3 - inositol 1,4,5, trisphosphate Isc - short circuit current J A M M - rate of luminal ammonia secretion JH - juvenile hormone V - rate of luminal acidification T + ">Na - rate of sodium flux Iv - transepithelial fluid transport L - lumen 1 - litre LCCP - locust corpus cardiacum peptide LDH - lactate dehydrogenase xii M - muscle min - minutes ml - millilitre mM - millimolar MOPS - 3-(N-morpholino)propanesulphonic acid mV - millivolts MW - molecular weight n - number N - normal NADH - B-nicotinamide adenine dinucleotide, reduced form NCC - storage (nervous) lobe of the corpus cardiacum nm - nanometre Nps - neuroparsins NSC - neurosecretory cells PI - pars intercerebralis PIP2 - phosphatidylinositol 4,5 bisphosphate PMA - phorbol-12-myristate 13 acetate pmoles - picomoles PO - perisympathetic organs PTTH - prothoracicotropic hormone RP - reversed-phase RPCH - red pigment concentrating hormone R t - transepithelial resistance s - seconds SAG - l-stearoyl-2-arachidonoyl-sn-glycerol XUl ScglTP - Schistocerca gregaria ion transport peptide SDS-PAGE - sodium dodecyl polyacrylamide gel electroph SE - standard error SEC - size exclusion chromatography SOG - suboesophageal ganglion TFA - trifluroacetic acid TG - thoracic ganglion UV - ultra-violet VG - ventral ganglion Vt - transepithelial potential xiv Acknowledgements I wish to thank Dr. John PhilUps for his support, guidance and generosity throughout this study. I also thank Dr. Chris Mcintosh for his advice, generous use of equipment and laboratory space, and comments on the manuscript. I thank Joan Martin for her help, advice, and keeping me "sane" throughout the countless hours we spent dissecting locusts. I am also grateful to all the undergraduate students who assisted in dissecting ganglia from locusts. I thank Dr. Richard Lechlietner for testing the effects of HPLC fractions on fluid transport. I thank Drs. J. Gosline, D. Randall, and A. Perks for comments on the manuscript. I especially thank Rebecca for her support over the past years, and for her help in preparing this manuscript. 1 CHAPTER ONE: General introduction Insects inhabit a wide range of environments and exhibit considerable variation in their feeding habits and are, therefore, subject to different osmotic problems. However, insects can regulate their haemolymph osmotic pressure and composition over a wide range of conditions. In Schistocerca gregaria Forskal there is only a 30% difference in haemolymph osmotic pressure between animals kept in air at 100% relative humidity and fed tap water and those at 70% relative humidity and fed concentrated saline (Phillips, 1964a). This homeostasis in insects results from structural, behavioural and physiological adaptations (Maddrell, 1971). In terrestrial insects, water is lost (i) by evaporation across the integument, which is reduced considerably by a wax layer in the epicuticle, (ii) by respiration through the spiracles, and (iii) by excretion. The excretory system of insects can make rapid adjustments in haemolymph composition (PhiUips et ai, 1986) to solve the problems of salt and water balance. The process in most insects involves the same basic mechanisms, namely, production of a primary isosmotic urine by the Malpighian tubules followed by differential reabsorption from, or secretion into, this fluid during subsequent passage through the hindgut (Stobbart and Shaw, 1974; Phillips, 1981, 1983; PhiUips et a/., 1978, 1986). The locust excretory system (Fig. 1.1) is typical of most terrestrial phytophagous insects, consisting of the Malpighian tubules and the hindgut (ileum, colon and rectum). The Malpighian tubules produce a primary, isosmotic urine, rich in KC1 and low in Na+, which contains most haemolymph solutes at lower concentrations than* in the haemolymph. Fluid secretion by the Malpighian tubules 2 MIDGUT MALPIGHIAN _ T _ \ TUBULES ILEUM COLON RECTUM KC1, Na+ and Water secretion ANUS Water and ion reabsorption Water, ion and metabolite reabsorption Strongly hyperosmotic or hyposmotic excreta Fig. 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). 3 is driven by active transport of K+ and CI". The tubules also actively secrete toxic molecules which would otherwise diffuse slowly across the tubule wall (reviewed by Phillips, 1981, 1983). This fluid then passes into the gut, where some moves forward into the midgut (Dow, 1981), but the majority passes posteriorly into the hindgut where selective reabsorption of water, ions and metabolites occur (Fig. 1.1) to produce a hyposmotic or hyperosmotic urine, or powder dry faeces in the rectum (reviewed by Phillips, 1983; Phillips et al., 1986). The usual problem for terrestrial insects is the removal of unwanted ions and solutes present in the diet, excretion of nitrogen and the retention of water. When locusts are dehydrated, nearly all the fluid secreted by the Malpighian tubules is reabsorbed in the hindgut so that dry faeces are produced. Haemolymph volume at such times can be reduced by 50-90%, and H + ions are secreted into the hindgut to counteract the haemolymph acidosis. Haemolymph volume and pH can be restored within a few hours of feeding on succulent plant materials (Phillips 1981). The food often contains ions in concentrations which are vastly different from those in the haemolymph, i.e. rich in K + and low in NaCl. These ions and solutes enter the haemolymph (passively) by absorption across the midgut wall in the same proportions as they occur in the diet, and excesses are subsequently expelled via the excretory system. There is therefore generally a need to retain NaCl and eliminate excess K+, and the rate of water loss can increase 10-fold when locusts feed on a succulent diet (reviewed by Phillips et al., 1986). 4 Structure of the locust excretory system The excretory system of the desert locust consists of approximately 250 Malpighian tubules which join the gut at the junction between the mid and hind sections (Fig. 1.1). The hindgut is divided into the ileum, colon and rectum. The ileum is about 6mm long with an outside diameter of 2.5mm and a macroscopic surface area of 0.4 cm2 (Irvine et ai, 1988). The rectum has a surface area of 0.64 cm2. The hindgut is lined with a chitinous cuticle (2-lOu.m thick) which, in the ileum and rectum is permeable to small hydrophilic molecules due to the presence of water filled pores of 6A radius (negative charge, pK about 4, Lewis 1971; Phillips and Dockrill, 1968; Maddrell and Gardiner, 1980), These cuticular pores allow reabsorption of major ions and metabolites from the primary urine whilst excluding larger, often toxic substances, thereby leading to their accumulation in the final excreta as a result of rectal fluid reabsorption. In contrast, the colon consists of smaller unspecialized epithelial cells and is much less permeable. For this reason, the colon is not thought to play any significant role in absorption (Maddrell and Gardiner, 1980). The locust rectum consists of six radially arranged pads where the cuticle is often detached to create a sub-cuticular space (Martoja and Balan-Dufrancais, 1984; Chapman, 1985; Irvine et al., 1988; Fig. 1.2). The pads are composed of columnar epithelial cells (17 x 100 urn), and also occasional secondary type "B" cells which have few mitochondria and contact the lumen side only. The columnar cells have highly folded lateral membranes with closely associated mitochondria. These membranes form complex intercellular channels with three distinct regions, 5 Fig. 1.2. Comparison of ultrastructural organization and gross dimensions of locust rectal pad and ileal epithelium. (From Irvine et al, 1988). 6 where ion recycling is believed to take place (Wall, 1970). In contrast, the ileum consists of a single layer and one type of epithelial cell (40 x 20 urn) which is covered by a firmly attached apical cuticle (Fig. 1.2). The basal surface is covered by a thin basal lamina, and there are no elaborate intercellular lateral membrane systems or secondary epithelial cell layer, there is only one type of basolateral membrane evident. Deal cells have elaborate narrow infoldings of the basal membrane, associated with numerous mitochondria, which may be analogous to lateral scalariform complexes of rectal pad cells (Irvine et al., 1988). Fluid and ion transport in the hindgut Wigglesworth (1931) first suggested that the rectum was the main site of water reabsorption in many terrestrial insects, although it was not until the early 1960's that active transport of fluid and the major monovalent ions across the locust rectum was demonstrated (Phillips, 1961, 1964a, b, c). It has since been demonstrated that both the ileum and the rectum play major roles in solute and water transport to determine the final composition of the excreta. Insect epithelia are unusual in that they commonly secrete or reabsorb KCl-rich fluids of low Na+ content (Maddrell, 1978; Phillips, 1981). This is true of secretion by Malpighian tubules, lepidopteran midgut, dipteran salivary glands and the integument during moulting, and of reabsorption in the locust midgut, rectum, reabsorptive segments of some Malpighian tubules and salivary glands. Of the reabsorptive epithelial transport processes which have been studied, their cellular organisation and control in the locust rectum is best understood, as recently reviewed by Phillips et al. (1986, 1988). 7 Mechanisms of solute and water transport across the locust rectum In the rectum, Na\ K+, CI", water and basic metabolites are all reabsorbed from the fluid entering the hindgut (Fig. 1.3). CI', the major anion in the fluid, is actively transported at the apical membrane by an electrogenic process stimulated by cAMP and by low levels of luminal K+. CI" exits passively at the basolateral membrane by a conductive mechanism, blockable by typical anion channel blockers (Hanrahan and Phillips, 1983, 1984a, b). CI" transport is not coupled to movement of other ions (Hanrahan and Phillips, 1983, 1984a, b), although relation to H + recycling is still under investigation. K+, the major cation, is reabsorbed passively through K+ channels with different properties at the apical and basolateral membranes due to the favourable electropotential (haemocoel negative) generated by CI" transport (Hanrahan and Phillips, 1983, 1984a, b; Hanrahan et al., 1986). Na+ levels in fluid entering the hindgut are low (20mM) relative to K+. Active reabsorption of Na+ occurs by a Na+-K+-ATPase at the basolateral membrane (Hanrahan and Phillips, 1982; Black et al., 1987). Passive Na+ entry at the apical membrane occurs by cotransport with some neutral amino acids (e.g. glycine), by exchange for NIL* (some H+) and by a conductive pathway which is possibly a channel (Hanrahan and PhiUips, 1982; Black et al, 1987). The rectum can actively reabsorb fluid from the lumen against very large osmotic gradients (1000 mOsm) to produce a hyperosmotic urine. A model was proposed by Berridge and Gupta (1967) and Wall and Oschman (1970) to explain the production of a hyposmotic absorbate in the rectum. Ions and other solutes (eg. amino acids) are actively secreted into the intercellular space between the 8 L U M E N •Itctro-C«niC pump c r ^ f C E L L H E M O C O E L .-CICTSMI •ton Jnt> ( > / E Q cm'^h" 1 ) G-^?—<V ci* 43 4.5 £0 A At U HCO, Acid-Bat* 0.4 15 Ion Activitiaa (RIM) and PD (mv) N a * 75 S 75 K» 7.2 70 7.2 C I * 52 47 62 Prolin* 13 ee 13 PH 7 7.36 7 mV *64 0 *34 Nat Eltetroeharnieal PC (Apical) ( ("<v) Baaolataral) Na* K* cr H * 127 (favoring) 12 (favoring) 60 (oppoaing) •6 (oppoaing) 127 (oppoaing) 20 (favoring) 20 (favoring) 56 (favoring) Fig. 1.3. Model of transport mechanisms identified in locust rectal epithelium. The neuropeptide, CTSH, acts via cAMP to stimulate or inhibit four mechanisms: thick arrows, major pumps; thin arrows through solid circles, carrier-mediated co-or counter-transport; arrows through gaps, ion channels. Steady-state values given for net transepithelial flux and eletrochemical potential differences across the two borders are for stimulated recta in Ussing chambers and bathed bilaterally in control saline under open-circuit conditions, except short-circuited state for Na+ and amino acids (AA). From PhiLlips et al. (1988). 9 highly convoluted plasma membranes of adjacent epithelial cells so that local pockets of high solute concentration are formed Thus, an osmotic gradient is developed down which water flows from the lumen into these apical intercellular spaces. This produces a hydrostatic pressure which causes fluid flow towards the haemolymph. As the fluid moves through the larger inner intracellular spaces and subepithelial sinus, active reabsorption of ions is believed to occur across the epithelial cell membrane without much water, leading to an absorbed fluid (absorbate) which is hyposmotic to the lumen. Water reabsorption in the rectum is linked to both CI" and proline transport, but net transepithelial proline transport is absent in the ileum, which cannot concentrate its contents like the rectum (Lechleitner and Phillips, 1989). Several neutral amino acids are actively reabsorbed (reviewed by Phillips et al, 1986). Balshin and Phillips, (1971) and Balshin (1973) demonstrated active uptake of proline, glycine, serine, alanine and threonine. Proline transport is largely NaMndependent and may be proton-linked (Meredith and Phillips, 1988). Five amino acids entering the hindgut from the Malpighian tubules act as metabolic substrates in the ileum (Peach and Phillips, 1990), whereas luminal proline is the major metabolic substrate in the rectum (Chamberlain and Phillips, 1983). Ammonia produced by amino acid oxidation is secreted into the rectal lumen via an apical NaTNTL,* exchange mechanism (Thomson et al, 1988). The rectum also actively secretes H + by an electrogenic process (80%) and to a lesser extent (20%) also by Na+/H+ electroneutral exchange. This is accompanied by HC03" reabsorption. The mechanisms for similar secretion of H + and NH/ in the ileum have not yet been studied. 10 The ileum possesses most of the ion transport processes found in the rectum, but there are some notable differences (reviewed by Lechleitner, 1988). The ileum is only able to produce an isosmotic absorbate as it lacks the complex membrane system responsible for solute recycling found in the locust rectum. Moreover, fluid uptake in the ileum is primarily driven by both NaCl and KC1, whereas that in the rectum is driven largely by KC1 and proline (Lechleitner and Philhps, 1989). Finally Na+ is actively reabsorbed at much higher rates in the ileum as compared to the rectum and can be stimulated by cAMP, corpus cardiacum (CC) and ventral ganglia (VG) extracts (Lechleitner et ah, 1989a). Ileal NFL,* secretion is also much greater than in the rectum (Lechleitner, 1988). Insect hormones Ecdysteroids, the juvenile hormones (JHs) and neuropeptides constitute the three main classes of known insect "hormones". In addition, prostaglandins (Brady, 1983), biogenic amines (Orchard 1982) and oestrogens (Mechoulan et al., 1984; Ohnishi et al., 1985) also act as insect hormones under certain circumstances. Until recently, insect endocrinologists have concentrated on the ecdysteroids and JHs, which are relatively simple molecules that regulate moulting and metamorphosis. Neurohormones are generally peptides and as such have greater diversity than the JHs and ecdysteroids. Insect neuropeptides have been shown to regulate a wide range of physiological, biochemical and developmental functions, mcluding osmoregulation, lipid and carbohydrate metabolism, reproduction, growth and development, and also regulate the release of the JHs and ecdysteroids. Prothoracicotropic hormones (PTTHs), which are produced by the brain and stimulate moulting by initiating ecdysone synthesis and release by the prothoracic 11 glands (Bollenbacher and Granger, 1985), were first demonstrated by Kopec in the early 1920s in the gypsy moth, Lymantria dispar. This was the first insect hormone to be discovered, and one of the first neurohormones to be described in any animal. Early preoccupation with the control of insect development has meant that significant advances in the discovery of other insect hormones has progressed rapidly only in the last 20-25 years. There are now around twenty-five physiological processes which are known to be influenced by neuroendocrine tissue, and therefore presumably by neuropeptides. The endocrine control of excretion Maddrell (1963, 1964a, b) provided the first comprehensive study of the control of diuresis using isolated Malpighian tubules of Rhodnius prolixus. He demonstrated, by ligation, that a diuretic (D) factor was released into the haemolymph from the mesothoracic ganglia, and its release can be promoted by high (K+ + Ca2+) salines (Maddrell and Gee, 1974). Since then the excretory process has been shown to be regulated and controlled by factors distributed throughout the central nervous system (CNS). These diuretic (D) and antidiuretic (AD) factors, and Chloride Transport Stimulating Hormone (CTSH) influence secretion of fluid by the Malpighian tubules and fluid and ion reabsorption in the rectum (reviewed by Gee, 1977; Phillips, 1982, 1983; Phillips et al., 1986; Raabe, 1982; Spring, 1990; See table 1.1 and 1.2). However, the often confusing and contradictory evidence as to the source, distribution and actions of neuroendocrine factors acting on diuresis may be reflected by the number of different methods used for their detection. Phillips (1983) and Phillips et al. (1986) have questioned some of the assay methods used. 12 Key for Tables 1.1 and 1.2 Br = Brain CC = Corpora cardiacum (whole) NCC = Storage lobe of CC GCC = Glandular lobe of CC CA = Corpora allatum SOG = Suboesophageal ganglia TG = Thoracic ganglia AG = Abdominal ganglia PO = Perisympathetic organs (neurohaemal areas of thoracic and abdominal ganglia) 13 Table 1.1. Factors which increase (diuretic) fluid secretion by the Malpighian tubules of insects and their sources. SPECIES SOURCE REFERENCE Acheta domesticus Aedes aegypti Aedes taeniorhynchus Anopheles freeboni Calliphora erythrocephala Carausius morosus Cenocorixa blaisdelli Corethra Dysdercus fasciatus Glossina austeni Locusta migratoria Onymacris plana Periplaneta americana Pieris brassica Rhodnius prolixus Schistocerca gregaria CC Head Head Head, TG TG B, CC Neurohormone C of brain Head AG B, CC TG CC PI SOG, TG B, CC, SOG, AG Terminal AG B, CC TG CC Coast (1988, 1989) Spring and Hazleton (1987) Petzel et al. (1985, 1986, 1987) Beyenbach & Petzel (1987) Maddrell and Phillips (1978) Nijhout and Carrow (1978) Schwarts and Reynolds (1979) Pilcher (1970a, b) Viennghoff (1966, 1967) Cooper et al. (1987) Gersch (1967) Berridge (1966) Gee (1975a, b) Mordue (1969, 1970, 1972) Stone & Mordue (1980) Cazal & Girardie (1968) Proux et al. (1982) Nicolson & Hanrahan (1986) Mills (1967) Nicolson (1976, 1980) Maddrell 1963, 1964a,b) Mordue (1969, 1970, 1972) Mordue and Goldsworthy (1969) Phillips et al. (1980) 14 Table 1.2. Factors which increase (antidiuretic) fluid reabsorption in the rectum of insects and their sources. SPECIES SOURCE REFERENCE Carausius morosus Gryllus domesticus Locusta migratoria Periplaneta americana Schistocerca gregaria Neurohormone D of brain GCC CC PO NCC, GCC NCC B, Meta-TG Terminal-AG CA Terminal-AG CC, CA GCC GCC, NCC Vietinghoff (1966) De Besse & Cazal (1968) Cazal & Girardie (1968) De Besse & Cazal (1968) Herault et al. (1985) Founder & Girardie (1987) Wall (1967) Goldbard et al. (1970) Steele & Tolman (1980) Mordue (1969, 1970, 1972) Proux et al (1984) 15 In general, D factors in insects have usually been identified by their stimulatory effect on Malpighian tubule function in isolated preparations rather than by diuresis in the whole animal. This has been achieved by direct measurement of fluid secretions using the method of Ramsay (1954), or modifications thereof. Other in vitro bioassay methods include measurement of dye secretion by the tubules (Mordue, 1969; Mordue and Goldsworthy, 1969), changes in Malpighian tubule transepithelial potential (Petzel et al., 1985, 1986, 1987), or changes in intracellular cAMP concentrations (Morgan and Mordue, 1985). In addition, water loss from the whole insect has also been used (Highman et al., 1965; Dores et al., 1979; Kataoka et al, 1989). There are reports of AD factors affecting tubule secretion (Altmann, 1956; Wall and Ralph, 1964; Vietinghoff, 1967; Cazal and Girardie, 1968; deBesse and Cazal, 1968) but this AD action has not be confirmed (Mordue, 1969, 1970; Gee, 1977). It has since been suggested that AD effects on Malpighian tubules were due to enzymatic destruction of D factors, or by a reduction in the amount of D factors released (Maddrell, 1964b; Berridge, 1966; Pilcher, 1970a, b; Mordue, 1972; Gee, 1975a, b; Phillips, 1983). However, Spring et al (1988) have provided evidence of an AD factor which inhibits fluid secretion by the Malpighian tubules of Acheta domesticus. Both D and AD factors were detected in methanolic extracts of haemolymph depending on the hydration state of this insect. Reversed-phase HPLC identified a single peak of AD activity which caused a 70% reduction in tubule secretion (Spring et al. 1988), but had no effect on rectal reabsorption (Spring, 1989). A possibility exists that AD activity was due to action on proximal reabsorptive areas of the tubules. 16 Regulation of reabsorption in the hindgut Regulation of Malpighian tubule secretion by D factors is now well established, but the control of fluid and ion reabsorption in the hindgut, particularly the ileum, is not as extensive. AD factors generally act on fluid reabsorption in the rectum and can be determined using the methods of Goh and PhilUps (1978). There are several reports of putative D and AD factors which inhibit or stimulate hindgut fluid reabsorption respectively (reviewed by Phillips, 1982, 1983; PhilUps et al, 1982, 1986; Spring, 1990; Table 2). PhilUps et al, (1986) questioned some of the earUer reports, as most studies were carried out on uncharacterized in vitro preparations usually during the initial transient phase when the tissue was adjusting to new osmotic conditions (sweUing) and still subject to stimulatory factors which may initiaUy be present in situ. In addition, the salines used often lacked essential metabolic substrates (such as proUne for locust rectum) and were not sufficiently oxygenated. This may account for some reports of putative D factors which subsequently have not been confirmed. The first report of regulation of rectal fluid reabsorption was in Carausius morosus by Vietinghoff (1966), who demonstrated that Neurohormone D and Neurohormone C of the brain had AD and D activities respectively. Since then most reports have shown that the retrocerebral complex (CC) and pars intercerebralis (PI) of the brain contain AD and D factors. WaU and Ralph (1964) and Wall (1967) reported AD activity in the brain, metathoracic and terminal abdominal ganglia, and the corpus aUatum. Goldbard et al. (1970) observed both D and AD activity in the terminal abdominal ganglia of the same insect, whereas Steele and Tolman (1980) reported only AD activity in the corpus cardiacum and allatum. De Besse and Cazal (1968) found only AD activity in the CC and 17 perisympathetic organs of Gryllus domesticus. Mordue (1969, 1970, 1972) reported a D factor in the storage lobe of the CC (NCC) and an AD factor in the glandular lobe (GCC) of both L. migratoria and S. gregaria, whereas only AD activity was reported by Cazal and Girardie (1968) and Herault et al. (1985) in L. migratoria and by Proux et al. (1985) in S. gregaria. Two reasons for these contradictory results have been suggested (Fournier et ah, 1987; Phillips et al, 1986). The putative D factor could be very unstable and therefore its activity lost; alternatively, fluid reabsorption (Jv) was measured during the first hour of assay during the transient fall in Jv, before the tissue had attained a near steady-state. Herault et al. (1985) demonstrated that there are two AD factors in the CC of the migratory locust, Locusta migratoria. These factors are located in the NCC and GCC, and differ in their thermal stabilities and extraction properties. In addition, the actions of the GCC factor appear to be mediated by cAMP (Herault and Proux, 1987; Herault et al., 1988), whilst the NCC factor may act via a phosphoinositol system (Fournier, 1990). Fournier and Girardie (1988) claim that neuroparsins A and B (NpA, NpB), which are two sulphur containing proteins produced by the Al-type median neurosecretory cells of the migratory locust (Girardie et al., 1987), are the only factors in the NCC with AD activity. These factors are multifunctional, in that they also inhibit the effects of juvenile hormone (Girardie et al., 1987) and induce an increase in haemolymph lipid and trehalose levels (Moreau et al., 1988). They have recently been isolated from NCC of L. migratoria by HPLC, and the sequence of NpB determined. None of the above studies of D and AD activity looked at the effects on solute movements in the insect hindgut. 18 Spring et al, (1978) first reported a factor from the CC of the desert locust, S. gregaria, which stimulates chloride transport across voltage-clamped rectal preparations as indicated by a 2-3 fold increase in short-circuit current (LJ and increases in transepithelial potential (V,; Spring and Phillips, 1980a, b). This factor, called Chloride Transport Stimulating Hormone (CTSH), apparently acts via cAMP, as its actions are mimicked by this cyclic nucleotide and uM levels of forskolin. Moreover, when crude CC extracts are added to rectal tissue, intracellular levels of cAMP increase to a peak at the time of maximum increase in L. (Spring and Phillips, 1980a; Chamberlain and Phillips, 1988). Haemolymph from fed locusts mimics the action of cAMP and CC extracts and is twice as effective as haemolymph from starved locusts, suggesting that CTSH is released into the haemolymph. Cardiatectomy also almost completely abolished stimulation by haemolymph (Spring and Phillips, 1980c). Spring (1986) also demonstrated that rectal transport of CI" in Romalea could be stimulated by cAMP and CC extracts. Crude extracts of CC from P. americana also stimulated locust rectal I*. (Phillips et al, 1982). Partial purification by gel filtration chromatography indicated that CTSH had a molecular weight of about 8,000 (Phillips et al, 1980). Since fluid transport is secondarily driven by the transport of salt, CTSH may be responsible for AD activity in locust CC. Proux et a\ (1985) provided evidence that CTSH has AD activity; fluid reabsorption across locust rectal sacs is stimulated by CC homogenates. This stimulation is abolished when recta are bathed in CI' free saline, and restored when CI" is added back, suggesting that water movement is linked to CI" transport. Similar results indicating dependence on CI' have been reported for AD action on everted rectal sacs of L. migratoria (Fournier et al, 1987). 19 In the vertebrate kidney, salt and fluid reabsorption is controlled by AD hormone and aldosterone, the former increasing water permeability and both stimulating Na+ transport. Two factors may therefore be necessary to control volume and osmolality of the hindgut absorbate in insects. Lechleitner et al. (1989) have demonstrated that crude extracts of CC and ventral ganglia (VG) increase both fluid transport and the osmotic permeability of ileal epithelia in 5. gregaria. However, in the absence of a favourable osmotic difference for passive water absorption, an increase in water permeability alone cannot increase water transport without a concomittent stimulation of solute transport. Source, distribution and release of neuropeptides Factors which can influence excretory processes are widespread throughout the CNS (Philhps, 1983; PhiUips et al, 1986; Tables 1 and 2). Most insect neuropeptides are synthesized in neurosecretory cells (NSC) and then transported down axons to neurohaemal areas for storage, thereby enabling the products to be released close to target organs. Histochemical studies have shown that these NSC are located in the brain, particularly the pars intercerebralis (Pp, in all of the ventral ganglia (VG) and in ganglia of the sympathetic nervous system (Raabe, 1984; Delphin, 1963, 1965; Jarial and Scudder, 1981; Girardie and Girardie, 1972; Maddrell, 1974; Remy and Girardie, 1980). The neurohaemal organ for the brain neurosecretory cells is the NCC, while those of the VG are the perisympathetic bodies found in segmental nerves near the CNS. Neurosecretions are released from these storage areas into the haemolymph and transported to their site of action (Raabe, 1983; 1984). Spring and Phillips (1980a) and Proux et al, (1985) found significant CTSH activity only in the PI of the brain and in the CC of the 20 desert locust. Girardie and Girardie (1972) demonstrated by 35S- labelling that a D factor in the migratory locust is synthesized in A cells of the PI. Maddrell and Gee (1974) showed that a D factor produced in the mesothoracic ganglia is stored in neurohaemal organs close to the Malpighian tubules before release. The widespread distribution of vasopressin (VP)-like diuretic hormone (DH) throughout the CNS of L. migratoria (Proux and Rougon-Rapuzzi, 1980; Proux et a/., 1982) was demonstrated by Remy and Girardie (1980) by radioimmunoassay. They showed that two NSC in the sub-oesophageal ganglia (SOG), which produce VP-like DH, send axon branches through the whole CNS, from optic lobes to terminal abdominal ganglia. Therefore widespread distribution of stimulants in the insect nervous system is perhaps not surprising. Mordue (1969) suggested that neurohaemal areas have greater potency than synthesis sites. There is much less D (Mordue, 1969) or CTSH (Spring and Phillips, 1980a) activity in the brain, compared to the considerable amounts of these peptides in the NCC. As an explanation it has been suggested that precursors synthesized in the brain are metabolized to more active forms in the NCC. Release of neuropeptides occurs in response to nerve impulses at neurosecretory endings (Gee, 1977; Raabe, 1983, 1984). As in mammals, this release is by exocytosis (Maddrell, 1974). Using electron microscopy, Maddrell (1966) observed omega-shaped profiles characteristic of exocytosis during periods of D activity release in Rhodnius prolixus. High (K+ + Ca2+) salines can initiate release of D activity in R. prolixus (Maddrell and Gee, 1974), Glossinia austeni (Gee, 1975a, b) and Calliphora erythrocephala (Schwartz and Reynolds, 1979). A neurophysin-like protein in CC can be released from L. migratoria by high K+ or 21 by electrical stimulation of the nerve (NCC1) from the PI to the CC (Orchard et al, 1981). Release of an AD factor from the GCC of L. migratoria is initiated by octopamine and by extracts of lateral NSC of the brain (Herault and Proux, 1987). Second messengers of insect peptides Neuropeptides are generally hydrophilic and therefore cannot readily penetrate cell membranes. Instead they utilize second messengers to bring about their effects (Alberts et al., 1989). Several common second messengers have been identified, mcluding cyclic AMP, cyclic GMP, Ca2\ inositol 1, 4, 5, trisphosphate (1P3) and diacylglycerol (DAG; reviewed by Berridge, 1983, 1986; Bodnaryk, 1983; Nathanson, 1983; Putney 1987; Rasmussen et al, 1985). Peptide hormones acting via cAMP bind to a specific receptor protein on the cell surface to form a receptor-hormone complex. This complex then binds to and activates a GTP-binding protein (either stimulatory or inhibitory). Binding of GTP to this protein changes its conformation so that it activates or inhibits adenylate cyclase to convert ATP to cAMP. Cyclic AMP exerts its effects by activating cAMP-dependent protein kinases (A-kinases) which phosphorylate target proteins so as to regulate their activity. Thus if the target protein is an ion channel or carrier protein, the rate of ion transport will be altered. Cyclic AMP levels are also regulated by phosphodiesterase, which break it down to 5'AMP. This resulting fall in cAMP levels dephosphorylates the target proteins, thus regulating the duration of the response (Alberts et al, 1989). Ca2+ has been implicated as a second messenger in the control of ion permeability across several cell membranes (reviewed by Berridge, 1983). The source of Ca2+ can be external (i.e. the opening of channels in the plasma 22 membrane) or internal, In the latter case IP3, production at the plasma membrane initiates Ca2* mobilization from the endoplasmic reticulum (Berridge and Irvine, 1984; Putney, 1987). In the IP3 system, hormone binding to cell-surface receptors activates a GTP-binding protein which regulates phospholipase C. This molecule cleaves phosphatidylinositol 4, 5, bisphosphate (PIP2) to (1, 4, 5) IP3 and diacylglycerol (DAG) or other inositol phosphates (Putney, 1987; Majerus et al, 1988). DAG in turn stimulates protein kinase C in the plasma membrane (Nishizuka, 1984) and IP3 stimulates Ca2+ release from intracellular stores (Streb et al, 1983; Berridge, 1986). Ca2+ commonly acts through receptor proteins such as calmodulin, or sometimes binds directly to the target enzyme to change its activity. In this way, Ca2+ can alter the permeability of the plasma membrane (Berridge, 1983). Recent evidence suggests that cAMP and Ca2+ nearly always act as synarchic messengers (reviewed by Rasmussen et al, 1985), and both regulate similar types of cellular processes, such as secretion (Berridge, 1983). Insect neuropeptides commonly act via the cAMP system (reviewed by Bodnaryk, 1983). However, in some cases the only evidence in support of this view is that exogenous cAMP mimics peptide action. This is true of D action on Malpighian tubules of G. austeni (Gee and Whitehead, 1977) and R, prolixus (Aston, 1975), and CTSH action on the rectum of S. gregaria (Spring and PhilUps, 1980b,) using CC extracts for stimulation. Hanrahan and PhUUps (1983) proposed that CTSH stimulates CI' transport across the locust rectum by promoting the synthesis of cAMP. Spring and PhiUips (1980b), Hanrahan et al (1985), and Hanrahan and PhUUps (1985), aU found that ImM theophyUine and u.M levels of forskolin stimulate electrogenic ion transport across rectal preparations of S. 23 gregaria. More recently, Chamberlain and Phillips (1988) showed that crude CC extracts, forskolin and theophylline all increase cellular levels of cAMP in this same epithelium, with a peak between 5 and 10 minutes when the I*. increase is the greatest. A rise in cGMP only occurs after 60 minutes of exposure to CC extracts when I„ has already been at maximum for 30 minutes. Herault and Proux (1987) observed that an AD factor located in the GCC also increases intracellular cAMP levels. Neuroparsin (Nps) A and B which are AD factors present in L. migratoria NCC do not act via cAMP or cGMP, and therefore they may act via the phosphoinositol system (Fournier and Dubar, 1989). In C. erythrocephala salivary glands, Ca2+ mediates the action of 5HT on chloride permeability at the basolateral membrane (Berridge et al., 1975). 5HT also increases cAMP production, thereby activating K+ transport (Berridge and Prince, 1972). Therefore KCl-rich fluid secretion is stimulated by 5HT using both these second messengers. Despite the progress in the knowledge of phosphoinositides in vertebrates (reviewed by Berridge, 1986; Irvine, 1986; Majerus et al., 1988; Putney, 1988), little is known of their role in insects. Recently, using indirect methods, Fournier (1990) has reported that Nps AD action may be mediated by phosphoinositide turnover and calcium. By measuring Jv across everted Locusta rectal sacs, he demonstrated that stimulators of protein kinase C, l-stearoyl-2-arachidonoyl-sn-glycerol (SAG) and phorbol-12-myristate 13 acetate (PMA), stimulated fluid reabsorption. In contrast, polymoxin B, a protein kinase C inhibitor, abolished the response to Nps, SAG or PMA. In addition, mysinositol and Ca2+ used in association with Ca2* ionophores mimicked the Nps response. Using rectal I« and Jv in S. gregaria as bioassays Demarachuk and Jeffs (pers. comm.) were unable to 24 observe any of the stimulatory or inhibitory effects reported by Founder, even though preparations responded to crude NCC or cAMP. Therefore, rP3 or Ca2+ may not be involved as second messengers in this insect. Whether these conflicting results reflect species differences (which seems unlikely) or methodological differences remains unclear. It remains to be seen whether Nps stimulates rectal 1^ and Jv in S. gregaria. Isolation of Insect neuropeptides Isolation of insect neuropeptides has, in the past, been limited by insensitive, slow, and low-resolution chromatographic and detection techniques. In addition, this problem has been compounded by lack of starting materials, lability of factors which are isolated, and poor bioassays (Mordue and Morgan, 1985). The characterization of the first two insect neuropeptides, proctolin and adipokinetic hormone (AKH) was therefore a significant achievement. Proctolin is a myotropic substance which acts on the longitudinal muscles of the proctodeum. It was isolated from 125 kg of whole cockroaches by an eleven-step isolation procedure (Brown and Starratt, 1975) to produced 180u.g of pure peptide. Its structure was determined to be a pentapeptide, Arg-Tyr-Leu-Pro-Thr (Starratt and Brown, 1975). A year later, AKH, which mobilizes lipid during prolonged flight, was isolated and its structure determined from S. gregaria and L. migratoria (Stone et al., 1976). Only two chromatographic steps were required, because of the high content of AKH per locust (100-250 pmole), and corpora cardiaca (the source of the peptide) were dissected out of locust heads. AKH is a decapeptide, with the structure pGlu-Leu-Asp-Phe-Thr-Pro-Asp-Trp-Gly-ThrNH2 (Stone et al, 1976). 25 These workers did not have the advantage of modern liquid chromatographic methods and AKH is now routinely isolated using a one-stage (HPLC) procedure (reviewed by Gade, 1990). High performance liquid chromatography was developed in the late 1970's, and has greatly facilitated the isolation of insect peptides. Three types of HPLC are now being routinely used, namely size exclusion (HPSEC), reversed phase (RP-HPLC) and ion exchange (T£-HPLC). The development of HPLC has revolutionised the field of peptide chemistry (reviewed by Regnier, 1983a, b). HPLC permits rapid separation of small amounts of material with high resolution and recovery. This coupled to advances in gas-phase sequencing and fast-atom bombardment mass spectrometry has meant that the number of insect peptides fully characterised has risen dramatically over the last few years. Several laboratories are currently attempting isolation of insect D and AD factors by HPLC, particularly using RP-HPLC. Like all chromatography, RP-HPLC depends on a differential distribution of solutes between moving and stationary phases to achieve separation. RP-HPLC separates compounds according to their hydrophobicity, which is responsible for absorption to the stationary phase, so that hydrophilic compounds move through the column faster than hydrophobic ones. RP-HPLC has improved resolution, detection sensitivity, recovery and separation speed over other chromatographic techniques, making it an ideal method for insect neuropeptide purification (Hayes and Keeley, 1983). The separation of D peptides has been hampered by their lability (Aston and White, 1974; Gee, 1977; Nijhant and Carrow, 1978; Schwartz and Reynolds, 1979; Morgan and Mordue, 1983, 1985). Progress has been made, however, in the purification of D factors from R. prolixus, L. migratoria and P. americana. Aston 26 and White (1974) detected two active fractions from R. prolixus thoracic ganglia by gel filtration and gel electrophoresis. Using Bio-gel P-30, P-60 and P-2 columns, most D activity was found in the void volume, indicating a molecular weight greater than 60,000 daltons (Da). A second area of D activity had an estimated weight of less than 2,000 Da. Hughes (1979) suggested the larger compound was possibly a prohormone, though prohormones do not usually possess biological activity. Hormones are generally synthesized as parts of larger precursors (prohormones) which have no activity, and specific enzymes then cleave the active hormone from its precursor (Schwyzer, 1982). Hughes (1979) also suggested that possibly the high molecular weight form of D peptide is inactive, but is converted to the physiologically active form during the assay procedures. In P. americana, a D peptide of greater than 30,000 Daltons eluted in the void volume of a Bio-Gel P-30 column (Goldbard et al., 1970). This D factor may be similar to the higher molecular weight form in R. prolixus, but no low molecular weight form was detected in P. americana. Morgan and Mordue (1983) separated a peptide of approximately 2,000 Da from NCC of L. migratoria on a Bio Gel P-6 column, and reported no evidence of a higher molecular weight form. Using RP-HPLC of methanol extracted NCC, two fractions (DHI and DHJ.I) were obtained with estimated molecular weights between 1,000 and 2,000 Da. A year-later, Morgan and Mordue (1984b) reported two D peptides (DPI and DPII) which act at different receptors. DPI, has an estimated MW of 6,000 - 7,000 Da, acts via cAMP, whereas DPII (ca. 1,000 Da) acts via an undetermined transduction pathway (Morgan and Mordue, 1984a). DPI was purified by HPSEC and RP-HPLC and a partial sequence determined. A second peptide, which was arbitrarily named locust corpus cardiacum peptide (LCCP), chromatographed with DPI by 27 HPSEC, occurred in relatively large amounts and was also partially sequenced (Morgan et al, 1987). It has been suggested the D peptide of L. migratoria is a vasopressin-like molecule (Proux and Rougon-Rapuzzi, 1980; Proux et al, 1982), because the D peptide is immunoreactive to arginine vasopressin radioimmunoassay. This peptide was isolated from a total of 51,000 sub-oesophageal and thoracic ganglia, using RP-HPLC. Two factors (Fl and F2) were immunoreactive, with the bulk of the immunoreactivity being in Fl and the bioactivity mostly in F2. Amino acid analysis of both factors were identical, but size exclusion chromatography indicated that Fl had an apparent molecular weight of 700 Da, and F2 of 1,470 Da, suggesting F2 was a dimer of Fl. It was determined that the AVP-Like D factor is an antiparallel homodimer of fourteen amino acids (Proux et al, 1987; Schooley et al, 1987). Rafaeli et al. (1986) presented evidence that D activity in Locusta CC is related to vertebrate adrenocorticotropin. Crude CC and 10'5 M adrenocorticotropin both gave similar rates of Malpighian tubule fluid secretion. There is, therefore, evidence from various workers that D activity in L. migratoria CNS is widespread, and these factors have different properties. There are also conflicting reports on D distribution. Proux et al (1982) found no D activity in the NCC of L. migratoria, contradicting the well established findings of Mordue (1969, 1970, 1972), Mordue and Goldsworthy (1969), and Morgan and Mordue (1983). In addition, no AVP-like material was found in L. migratoria CC (Proux and Rougen-Rapuzzi, 1980) indicating that AVP-like hormone is chemically distinct from D factors in the NCC. The partial sequence of DP-1 (Morgan et al 1987) bears no relation to the AVP-like D factor sequenced by Proux et al (1987). It appears therefore that there are a number of D peptides in L. migratoria which 28 affect fluid secretion by isolated Malpighian tubules, but the relationship between these factors and their involvement in regulation in vivo is not yet known. Full sequence data of other D peptides will aid in answering this question. A similar pattern has emerged in Acheta domestica, where D activity is widespread throughout the CNS (Coast and Wheeler, 1990). In the CC, there are four active peaks from RP-HPLC separation, and purification of the largest ultra violet absorbing peak has been accomplished using phenyl columns, HP-SEC and HP-IEC (Coast et al, 1990). This factor (API) eluted as a single peak and was sequenced, and a molecular weight of 1,925 Da was determined. However, synthetic API had no D activity on isolated Malpighian tubules, and Coast et al. (1990) discuss some of the possible reasons for this result. In this same insect two peaks with D activity were observed by Spring and Hazelton (1987) and Spring et al (1988) using methanolic extracts of CC and RP-HPLC. These two factors have opposite effects on intracellular cAMP: DH1 increased cAMP, whilst DH2 reduced intracellular cAMP by 50% (Spring and Clark, 1990). A diuretic factor was isolated from pharate adult heads of Manduca sexta by a riine-step purification procedure (Kataoka et al, 1989), utilizing a combination of LE-HPLC and RP-HPLC. The complete structure of a 41-residue peptide with no disulphide bonds was determined. This peptide was synthesized and shown to have chromatographic and biological properties identical to those of the natural peptide. All the above studies have been mainly concerned with control of Malpighian tubule secretion. Much less attention has been given recently to control of hindgut reabsorption. 29 Goldbard et al (1970) partially purified a diuretic and an antidiuretic factor from the terminal abdominal ganglia of the American cockroach, both of which acted on Jv across isolated rectal preparations. The diuretic factor had an estimated molecular weight greater than 30,000 Da, and the antidiuretic factor was about 8,000 Da. CTSH was partially purified from saline and methanol-water extracts of locust CC by gel filtration (Phillips et al, 1980). Using Bio-Gel P-30 columns, two areas of activity acting on electrogenic ion transport across short-circuited rectal preparations were observed. The major activity was determined to have an estimated molecular weight of 8,000 Da (CTSH), whilst traces of activity were observed in the void volume (> 30,000 Da, which may be a prohormone). It was estimated that as little as 7nmoles of partially purified CTSH could cause a maximum increase in when diluted in 5ml of saline (20 times locust blood volume; Phillips et al, 1980). CTSH is most stable at pH 7-10, at which value it has an excess negative charge, as determined by electrophoresis. By reciprocal bioassay, PhilUps et al (1980) determined that CTSH activity was not due to AKH and DH present in the CC of this insect. The AD factor partially purified by Goldbard et al. (1970) from the American cockroach may be similar to CTSH. In support of this, Phillips et al. (1980) report that crude homogenates of P. americana CC stimulate across locust recta. Neuroparsins A (NpA) and B (NpB) are reported to be the only AD factors from the NCC acting on rectal fluid transport in the migratory locust (Fournier and Girardie, 1988). NpA and NpB isolated from NCC by a two-step purification procedure utilizing anion exchange and reversed-phase HPLC (Girardie et al, 1989). Both consist of two polypeptide chains linked by disulphide bridges. NpB 30 is a homodimer: a complete sequence (78 residues) of its monomer (8,188 Da) was determined (Girardie et al, 1989). The sequence of NpB is homologous to that of locust corpora cardiaca peptide partially sequenced by Morgan et al. (1987). The goal of this study was to identify and characterize the natural factors which regulate ion transport in the hindgut of the desert locust. Spring et al. (1978) first reported the presence of CTSH, a peptide from the CC which stimulates ion transport across the rectum. Since the initial partial purification of this factor in 1980 (Phillips et al., 1980), no further attempt has been made to characterize CTSH, and crude homogenates have since been routinely used as a stimulant. Recently, Irvine et al. (1988) demonstrated a major role for the ileum in the excretory process, and obtained the first direct evidence that ileal salt and water transport in insects may be under hormonal control. As there are large cAMP-induced changes in ion transport across locust ilea, questions arise as to the distribution of natural stimulants of this hindgut segment throughout the central nervous system, and whether these factors are identical to those acting on the rectum. The voltage-clamped preparation developed by Williams et al. (1978) and improved by Hanrahan et al. (1984) offers a rapid, convenient and reliable bioassay to identify factors which may regulate CI" transport (the principal active ion transport process) across locust hindgut. In vitro preparations of locust hindgut have been shown to remain viable in a near steady-state condition for at least 8 hours (Williams et al, 1978; Irvine et al, 1988). Chapter two reports the identification of factors from the CNS which stimulate salt transport across locust hindgut, as indicated by changes in CI-31 dependent short-circuit current. Previous studies (Spring and Phillips, 1980b; Proux et al., 1985) have shown that CTSH is located in the PI of the brain and the CC. A wider distribution of stimulants acting on both hindgut segments is reported and evidence presented for the existence of more than one factor. The most potent factor acting on ileal I„ was located in the CC and was therefore presumed initially to be CTSH. Preliminary studies on its stability and extraction properties suggested that this factor was stable enough to attempt its isolation. Chapter three reports the purification of this factor using reversed-phase high performance liquid chromatography and a near complete amino acid sequence. Crude extracts of CC influence several transport processes in locust ileum and rectum. While stimulation of these several processes by exogenous cAMP is consistent with a common control by a single peptide, this hypothesis required confirmation. The peptide purified from CC (Chapter 3) was tested on most of these hindgut transport processes, as described in Chapter 4. The purified factor does affect most ileal transport processes influenced by cAMP but has limited or no action in the rectum. In the final chapter (5) the results in chapters 2 to 4 are discussed in relation to ionic and acid-base regulation and nitrogen excretion in whole locusts. 32 CHAPTER TWO: Actions of Corpus Cardiacum and Ventral Ganglia on Heal Salt Transport INTRODUCTION Haemolymph composition in insects is ultimately regulated by the excretory process, involving selective reabsorption of ions, water and metabolites in the hindgut from a primary urine secreted by Malpighian tubules (reviewed by Phillips et al, 1986). Most previous work has concentrated on specific transport processes and their control in the Malpighian tubules and the rectum of insects while the role of the intermediate segment in the excretory system, namely the ileum, has been largely neglected (reviewed by Gee, 1977; Bradley, 1985; PhilUps, 1983; PhiUips et al, 1986; 1988). Recently, however, Irvine et al (1988) have demonstrated that ion transport processes across short-circuited Uea of the desert locust, Schistocerca gregaria Forskal, are remarkably similar to those reported earlier for the rectum. In both segments cyclic AMP causes a several-fold increase in electrogenic CI" transport from the lumen as indicated by increases in Cl-dependent short-circuit current (LJ, and in transepithelial potential (V,), while transepithelial resistance (RJ decreases. In addition, this cyclic nucleotide also increases K+ permeabiUty, water reabsorption and ammonia secretion in both segments, whereas a large increase in active absorption of Na+ is only stimulated in the ileum and is largely electroneutral, and a decrease in acid secretion is observed in the rectum. Evidence for control of rectal fluid absorption by putative antidiuretic (AD) and diuretic (D) factors in several insects has recently been reviewed by Phillips et al (1986). Recent studies have reported only AD activity in the corpus cardiacum (CC) using fluid absorption by rectal sacs as a bioassay. Proux et al (1984) 33 observed AD activity in both the storage (NCC) and glandular (GCC) lobes of the CC from Schistocerca gregaria, and this was subsequently confirmed for Locusta migratoria (Herault et al., 1985). In the latter species, the active factors from the NCC and GCC differ in size and extraction properties. The factors in the NCC were found to be neuroparsins (Nps) A and B (Fournier and Girardie, 1987) and NpB was recently fully characterized (Girardie et al., 1989). As active absorption of fluid across epithelia against (or in the absence of) osmotic differences is invariably driven by solute transport, AD activity in insects may be the result of factors which first stimulate salt transport. Chloride Transport Stimulating Hormone (CTSH) which was partially purified from CC of S. gregaria (Phillips et al., 1980) and stimulates CI" dependent !« across locust recta, also has AD activity (Proux et al., 1984). CTSH was first reported by Spring and Phillips (1978) to be present in both NCC and GCC. Partial purification indicated that CTSH is a peptide of about 8,000 Daltons. Irvine et al. (1988) demonstrated large cAMP-induced changes in ion transport across locust ilea are comparable to those observed in the rectum, so questions arise as to the distribution of potential natural stimulants of ileal ion transport throughout the locust central nervous system (CNS) and whether these agents are identical to those acting on the rectum (i.e. CTSH). In this chapter, the distribution of stimulants in the CNS using Cl"-dependent I*, across flat sheet preparations of ilea in Ussing-type chambers as the bioassay is reported. The effects of these factors on other ion transport processes are determined and compared to the actions of cAMP. Some of the chemical and physical properties of these factors are also determined. These observations lay the groundwork for the purification of the 34 major stimulant in CC as reported in the next chapter. The results show that natural stimulants from the CNS have the same broad range of effects on the ileum, as does cAMP. Both the CC and VG contain proteinaceous stimulants with different properties. 35 MATERIALS AND METHODS The experimental animals were adult Schistocerca gregaria, 2-3 weeks past their final moult. They were reared at 28°C and 55% relative humidity under a 12:12 light:dark cycle, and fed a diet of lettuce and a mixture of dried grass, bran and milk powder. Ilea from females were used because of their larger size. Electrical measurements To measure electrogenic ion transport, ilea or recta were mounted as flat sheets between two modified Ussing chambers and voltage-clamped at zero mV, as described by Hanrahan et al. (1984) for locust rectum. Each chamber contained 2ml of saline which was stirred by vigorously bubbling with 100% 02, or a mixture of 95% Oj/5% C02 at 22°±2°C. Tissues were removed from animals, cut longitudinally to produce a flat sheet and immediately (within 5 min from start of dissection) secured over a 0.196 cm2 opening by means of tungsten pins and an overlaying neoprene O-ring (Fig. 2.1). Edge damage was negligible with this technique (Hanrahan and Phillips, 1984a). The chambers were clamped together in a vice-like frame. To measure the transepithelial potential (V,) 3M KC1 agar bridges (size PE 90) were placed near the tissue through ports on the side of the chambers and connected to a high input impedance differential amplifier (4253, Teledyne Philbrick, Dedham, Mass.) which continuously monitored Vt. Short-circuit current (LJ* a direct continuous measure of electrogenic ion transport, was measured by maintaining V t at 0 mV by a second amplifier (725, National Semi-conductor Corp. Santa Clara, Calif.) which passed current (LJ between two 36 Fig. 2.1. Standard Ussing chamber assembly used for measuring I,,., V t , JH+, IK, J A M M and J N . + - (1) flat-sheet hindgut preparation, (2) plexiglass collar over which rectum or ileum is mounted, (3) neoprene O-ring for securing hindgut attachment to collar, (4) neoprene chamber seal, (5) agar bridge port for measurement of V„ (6) gas inlet for saline aeration and mixing, (7) current sending electrodes, (8) rear chamber seal, (9) tungsten pins for attachment of hindgut to collar (taken from Hanrahan et al, 1984). 37 Ag-AgCl electrodes at either end of the chamber. A third amplifier (308, Fairchild, Mountain View, Calif.) was used to measure I*.. Both 1^. and V, were monitored on a strip chart recorder (Soltec 1242, Soltec Corp., Sun Valley, Calif.). Corrections were made for series resistance of the external saline and asymmetries between voltage-sensing electrodes (Hanrahan et ai, 1984). Under short-circuit current conditions, transepithelial potential was monitored at intervals by stopping the voltage-clamp for 20-30 seconds and using an alternative circuit to measure voltage difference. The locust rectal and ileal I„. were reported previously to be Cl-dependent and a measure of electrogenic active transport of this anion (Williams et al, 1978; Irvine et al, 1988). Tissue resistance (R,) was calculated from L, and Vt using Ohms law. Salines The complex saline was based on the composition of locust haemolymph (Hanrahan and PhilUps, 1983) and contained (mM): 100 NaCl, 5 K2S04, 10 MgS04, 10 NaHC03, 5 CaCl2, 10 glucose, 100 sucrose, 2.9 alanine, 1.3 asparagine, 1.0 arginine, 5 glutamine, 11.4 glycine, 1.4 histidine, 1.4 lysine, 13.1 proline, 6.5 serine, 1.0 tyrosine, 1.8 valine, and was bubbled with 95% 0 2 / 5% C02 and adjusted to pH 7.2. This saline was used in aU experiments unless otherwise stated. Preparation of tissue extracts Brain (B)» corpora cardiaca (CC), corpora aUata (CA), suboesophageal ganglia (SOG), and aU eight ventral gangUa (VG) were removed from adult male locusts, 2-6 weeks past their final moult. Male locusts were used to avoid cycUc 38 changes associated with female reproduction. EHssecting the glands involved the following procedure: the head was separated from the prothorax at the neck region and the corpus cardiacum was removed from the head as described by Stone and Mordue (1980). The head was placed face up and a single cut made slightly to one side of the midline to expose the CC and most of the brain in the larger portion. The CC and brain were removed with watchmakers forceps and fine scissors, and the rest of the brain was taken from the smaller section of the head. Separate storage (NCC) and glandular (GCC) lobes were dissected from different animals, and the connecting stalk sections were discarded to avoid contamination of each lobe with the products of the other. As the stalks from the NCC project part way into the GCC (Highnam, 1961), the anterior third of the GCC was removed in some experiments to avoid potential contamination from NCC products. To remove the 3 thoracic and 5 abdominal ganglia (i.e. 8 ventral ganglia), the ventral portion of the thorax and abdomen was removed from the dorsal part by cutting laterally along the body with scissors. All internal organs and fat body were removed from the ventral surface to expose the ventral nerve and ganglia. Individual ganglia were dissected by cutting the ventral nerve cord on either side of the ganglia and removing the ganglia with forceps. Glands were immediately frozen on dry ice and stored at -70°C. Extracts were prepared by mechanically homogenizing these tissues in saline on ice (stock concentration 50 ganglia per ml) using a Tissue Tearer homogenizer (Bartlesville, OK), and then centrifuging for 5 min at 12,000 g and at 4°C. The supernatant was removed and stored at -20°C prior to use. The relative weights of different ganglia of S. gregaria were reported earlier (Proux et al., 1984). Pieces of flight muscle, several times the weight of VG, were treated in the same manner and used as a control. 3 9 Assay procedures Small volumes (1-lOOu.l) of saline extracts were added to the haemocoel side of ilea or recta when these tissues had reached a low steady-state level 60-120 min after dissection, as reported by PhilUps et al. (1986) and Irvine et al., (1988). The resulting increases in ileal and rectal I,,, were followed until a maximum value was attained. At the end of each experiment 5mM cAMP was added to the haemocoel side to determine that ilea or recta still gave a normal response when stimulated. The rare preparations which failed to respond to cAMP were discarded. This test was not necessary with CC extracts due to the sustained increase in L, caused by CC factors. Cyclic AMP, cGMP, theophylUne, forskoUn and various neurotransmitters were all made up as lOOmM stock solutions in saline, and small amounts were added to the haemocoel side of the tissue to give the desired final concentration. Neurotransmitters were tested at 10"6 to 10 3 M final concentration. Each preparation was only used for one assay, because a substantial variation in response to a second stimulation (often a potentiation) was found. Estimation of potassium permeability The procedures described by Hanrahan et al. (1986) in their study of basolateral K + channels in locust rectum were followed to determine the effects of CC and V G homogenates on Ueal and rectal K + conductance. A simple saline was used which contained (mM): 70 NaCl, 10 KC1, 10 MgCl 26H 20, 10 glucose, 100 sucrose, 10 proline, 5 glutamine, 5 CaCl 2, 3-(N-morpholino)propanesulphonic acid, 10 (MOPS), adjusted manually to pH 7.0 with concentrated HN0 3 or NaOH using a Radiometer PHM 84 pH meter (Copenhagen), and bubbled with 100% 0 2 . 40 Irvine et al., (1988) showed that cAMP increases ileal K+ conductance in a similar manner to that reported by Hanrahan et al. (1986) for the rectum. Ilea and recta were first allowed to attain a steady-state. All external CI" was then replaced by gluconate on both sides to abolish electrogenic CI" absorption and basolateral CI" conductance. Haemocoel K+ was then raised to 80mM to create a K+ concentration difference of 80:10mM across the epithelium, thereby inducing a transcellular K+ diffusion current (IK) to the lumen side. This reverse gradient avoided the known effects of high luminal K+ on apical K+ channels in locust rectum. CC or VG homogenates were then tested for their ability to stimulate IK. Ammonia secretion To measure ammonia secretion, ilea were mounted as flat sheets in Ussing-type chambers (as previously described) under open-circuit conditions and allowed to come to steady-state (60 min) under bilateral perfusion (5-8 ml min1) with saline, and open-circuit conditions. The saline on both sides of the chamber was always mixed by bubbling with 100% 02. Salines were C02-HC03 free for comparisons with previous work on the rectum (Thomson et al., 1988) and the ileum (Lechleitner, 1988), and to remove the effects of volatile buffer components other than NH3. Salines were based on the composition of locust haemolymph, except they were C02-HC03 free and contained (in mM) 100 NaCl, 5 K2S04, 10 MgS04, 10 Na-isethionate, 10 glucose, 100 sucrose, 5 CaCl2, 2mM MOPS, and amino acids as described for complex saline. All salines were also initially ammonia free (<20 u.M measured) so any ammonia measured was endogenously produced. Salines were aerated with 100% Oz for 2-3 h before use, and perfusion reservoirs were continuously aerated 41 throughout the experiments. The salines were filtered, and pH manually titrated to 7 with concentrated HN03 or NaOH, using a Radiometer PHM 84 pH meter (Copenhagen) before each experiment. Bilateral perfusion (but not mixing) was stopped, and 5mM cAMP, 1.0 VG5, 0.5 CC (experimental), or nothing (control) was added to the haemocoel side of the tissue. After one hour (experimental period) 1ml samples of saline were collected from the lumen side for assay of ammonia levels. The ammonia concentrations of samples were determined by enzymatic assay using an assay kit from Sigma (Procedure No. 170-UV), which utilizes the reductive amination of 2-oxoglutarate by glutamate dehydrogenase to bring about a change in extinction (at 340nm) proportional to the ammonia content of the sample: NADH + NFL, + 2-oxoglutarate + glutamate dehydrogenase -> L-glutamate + NAD+ + H20 + glutamate dehydrogenase. The rate of ammonia secretion was determined as (total final ammonia) - (total initial ammonia), expressed as a flux rate cm'2.h'2. Thomson et al. (1988) established that increase in ammonia concentration was linear under the same conditions for at least 4 hours for the rectum and J. Peach (personal communication) obtained the same results for locust ileum. Acid secretion Ilea were mounted and perfused in salines as described to measure ammonia secretion, and measurements made under the same experimental conditions. Rates of luminal acidification (JH+) were determined with a pH-stat technique (PHM 84 research pH meter, TTT 80 titrator, ABU 80 autoburette; Radiometer, Copenhagen). J„+ was calculated as the rate of titrant addition (0.01N NaOH) 42 required to maintain the initial pH, and is expressed as uequiv.cm-.h"1 over the second hour after mounting the tissue. Extraction of stimulants in CC or V G with different solvents Twelve pooled CC or VG were homogenized in different solvents (physiological saline, distilled water, 70% methanol, or 0.2M acetic acid) using the same technique as described earlier. The supernatant was removed after centrifuging the homogenates, and the pellet was resuspended in physiological saline. After the initial extraction, two or three subsequent extractions of the pellet in saline were carried out to determine how efficient the original solvents were in recovering activity from these tissues. Because acetate is actively transported by the locust hindgut, the supernatant from the acidic extraction was dried down by a stream of N 2 gas at ,room temperature and then lyophilized. None of the solvents used alone had any effect on ileal I*. when added in amounts of up to lOOul. Small amounts (less than 100|j.l) of the supernatant from solvent extractions of CC or VG were added to saline on the haemocoel side of ilea to achieve final doses of 0.5CC ml'1 and 1VG ml"1. Subsequent saline extracts of the pellets from solvent extractions were assayed in the same manner. Effect of proteases on CC and V G stimulants Saline extracts (12 ganglia per ml) of CC and VG5 in 0.1M TRIS buffer were treated with trypsin at pH 7.6 or chymotrypsin at pH 7.8 (Sigma Chemical Co.), at a concentration of lmg ml1 for 2 hours at 25°C before testing extracts on short-circuited ilea. As controls, ileal preparations were exposed to proteases alone, and to CC and VG5 extracts which were sirnilarly incubated without 43 proteases. Stability of CC and VG factors Homogenates of CC and VG5 at a concentration of 12 ganglia per ml saline were maintained in a water bath at various temperatures and were then tested after various periods of time for activity on ileal IK. As a control, freshly prepared homogenates were also tested. Statistical treatment Differences between treatments were considered significantly different when Student's T-test indicated a P value less than 0.05. 44 RESULTS Effect of cAMP on ileal I« The effect of cAMP on ileal 1^. is shown in figure 2.2. The initial large 1^ . (7.6 ± 0.3 |iequiv.cm"2.h"') falls rapidly over the first 1-2 h to a low steady-state level close to zero (-0.6 ±1 .0 Ltequiv.cm'Mi'1), which persists for at least 7 h. There is a concomitant fall in Vt, from 8.7 ± 0.4 to -1.0 ± 2.8 mV and an increase in R< from 44 ± 16 to 102 ± 52 Qcm2 (n = 74-121). Addition of 5mM cAMP to the haemocoel side of the tissue after 2 or 6 h causes a 10-fold (9.82 ± 0.88) increase in I*. within 30 min, which persists for at least 6 h, confirming the viability of the tissue over this time period. This stimulated l^. can be rapidly inhibited by at least 90% within 5 min by the addition of 5mM azide, indicating a dependence on aerobic metabolism. Survey of the central nervous system for stimulants The effects of saline extracts from different parts of the central nervous system, added to the haemocoel side, on ileal IK are shown in Fig. 2.3 and Table 2.1. The brain (B), suboesophageal ganglia (SOG), all ventral ganglia at a dose of 1 ganglia ml"1 saline, and CC at 0.5 gland pair ml"1 all caused some stimulation of ion transport, as reflected by increases in ileal I*, and Vt, and decreases in Rt. CC homogenates were by far the most potent source of stimulants, resulting in an average increase in per mg wet gland weight of 10080 Liequiv.cm h^"1. In contrast, the fifth VG caused a 600-fold smaller increase of 16 Ltequiv.cm'^ .mg"1 wet gland weight. Corpora allata and muscle tissue (control) had no effect on I« across any ileal preparation. 45 Time (hours) Fig. 2.2. Change in ileal IK with time under unstimulated (control) conditions (solid circles). Cyclic AMP (5mM) was added to the haemocoel side of the tissue (solid arrow) after 2h (solid squares) or 6h (open circles). (Means ± SE, n = 6, except where stated otherwise.) 46 Fig. 2.3. Effect of saline extracts of various parts of the nervous and endocrine systems, and muscle (control) on electrical parameters across short-circuited locust ilea (mean ± SE, n = 6-10). The dosage was 1 gland equivalentml'1, except 0.5 glandml'1 for CC. (A) maximum increase in short-circuit current ( A L J from steady-state levels indicating CI' transport. Baseline before stimulation was always close to O uA, as first reported by Irvine et al. (1988). (B) As for (A) but expressed as AL^. per mg wet weight of ganglia or other tissue. Abbreviations: B, whole brain; CC, whole corpus cardiacum; SOG, suboesophageal ganglion; CA, corpus allatum; M, flight muscle; 1-8, ventral ganglia. 47 14 12-10-8 6 4-2 0 I I I I — • — T -2 3 4 5 6 B CC CA SOG 1 8 M Ventral ganglia 10080-j 16 12 8-AAA -i—1—1—r 2 3 —T— 7 - i — 8 B CC SOG 1 4 5 6 Ventral ganglia 48 Table 2.1. Effect of saline extracts of various parts of the nervous system on transepithelial potential difference (V t, sign refers to lumen side) and transepithelial resistance ( R J across short-circuited ilea (mean ± SE, n= 6-10). R t (ohms.cm2) V, (mV) unstimulated stimulated unstimulated stimulated B 94.6±9.5 62.3±4.5 -0.511.1 10.812.2 CC 88.1±7.9 47.8+3.6 -1.910.9 14.811.8 SOG 99.2±11.6 50.8±3.8 1.311.3 5.310.9 VG1 95.3±9.3 56.8±7.1 1.811.3 7.011.3 VG2 122.5±10.4 82.8+9.3 -1.310.9 7.712.8 VG3 97.5±8.4 57.2±3.4 -2.011.3 11.511.9 VG4 85.8±9.8 45.6±4.6 1.611.0 6.311.3 VG5 91.6±10.2 43.8±4.6 -0.610.8 16.111.7 VG6 101.0±8.5 44.2±6.1 -2.811.8 6.511.8 VG7 93.4±8.2 46.9±5.2 -0.910.9 8.311.2 VG8 82.3±6.1 58.116.8 -0.310.9 2.911.0 Al l stimulated values are significantly different from unstimulated values (P <0.05). The dosage was 1 gland equivalent ml"1, except 0.5 glands ml"1 for CC. Abbreviations: B , whole brain; CC, whole corpus cardiacum; SOG, suboesophageal ganglion; VG, ventral ganglia. 49 1 2 n Time (hours) Fig. 2.4. Effect of different dosages of CC extract ( • , 0.025; 0 , 0.1; • , 0.25 glands.ml1) added to the haemocoel at t=0 hours on the time course of ileal L*. (Mean ± SE, n = 6-8). 50 Time course of stimulation and relative activity in separated lobes of C C The time course of ileal response to CC homogenates is long lasting (i.e. sustained for at least 8 h), and the time taken to reach a maximum stimulation varies with dose (Fig. 2.4). At low doses (0.025 gland pair ml"1) there is a gradual increase in I*. to a maximum after 6 h. At high doses (0.25 gland pair ml1) there is a rapid initial increase in I,,, to a maximum after 0.5 h, followed by a gradual partial decline in I,,, over the next 5-6 h. The time lag before any change in I*. is apparent after addition of CC homogenate is also dose-dependent, ranging from 30 s at 0.5 gland pair ml'1 to 4 min at 0.025 gland pair ml"1 (data not shown). Separated storage lobes (NCC) and glandular lobes (GCC) of corpora cardiaca were tested for their relative effects on ileal IK. As described by Phillips et al. (1980), the intermediate region of CC was discarded during dissection to ensure that only one lobe was tested At a concentration of 0.1 lobe ml"1, the storage lobe ( AlK of 7.8+1.1 uequiv.cm-.h"1) had 3.5 times the activity of the glandular lobe ( A I K of 2.34±0.63 li-equiv.cm^.h"1). In other experiments where only the anterior third of the GCC was dissected (to eliminate any NCC projections into the GCC), activity in the saline extract was observed at higher doses. At a concentration of 1.0 GCC ml"1 an increase in 1^ of 3.13±0.34 uequiv.cm-.h"1. was observed (n = 4). Al l these increases were highly significant (paired t-test; P < 0.05). Dose-response relationship for C C homogenate As reported above, time courses of ileal I« response to CC homogenates varied with dosage so that the dose-response curve of maximum change in was 51 Fig. 2.5. Increase in I,,, one hour after adding various dosages of CC homogenate to the haemocoel side (Mean ± SE, n = 6-8); solid regression line fitted by least squares method is expressed by log y = log 1.4 x + 1.7; r = 0.99. 5 2 atypical and biphasic (not shown). However, a typical dose-response relationship was observed when the AI*. after 1 h of stimulation was used (Fig. 2.5). There is a good linear relationship between logarithm of I,,. (Liequiv.cm^ .h"1) and logarithm of dose over the range of 0.005 to 0.5 glandml"1. The increase in I,,, caused by 0.05 gland pair.ml'1 was significant and a maximum response was produced by 0.5 gland pair.ml'1 (12 u.equiv.cm'2.h'1). For comparison, the total haemolymph volume of S. gregaria is about 250 ul, suggesting that release of about 10% of the CC contents would cause maximum stimulation in situ. Time course of response to VG5 homogenate The effects of VG5 homogenates on ileal I,,, are shown in Fig. 2.6. VG5 was chosen as a representative of the ventral ganglia because all ganglia gave similar time courses of I,,. response and VG5 was found to be the most active of the VG (see Fig. 2.3). Unlike ileal response to the CC, response to VG5 reached a maximum value within 0.5 h regardless of dosage and fell back to original levels within approximately 3 h. The greater the dose, the longer lasting was this decline in I^ , presumably reflecting greater time required for enzymatic destruction of larger amounts of VG factor or of intracellular second messengers. This interpretation requires near saturation of a step in the destructive pathway. The lag time before a response to VG5 homogenates was evident was dose-dependent, ranging from 3 min (0.01 ganglia-ml"1) to 30 s (2.5 ganglia.ml"1; results not shown). 53 18n Time (hours) Fig. 2.6. The time course of increases in ileal IK (Mean ± SE, n = 6-10) in response to addition of fifth ventral ganglia (VG5) extracts to the haemocoel side at different doses (gland equivalents ml'1): • , 0.10; O, 0.25; • , 0.5; • , 0.75; A , 1.5; A , 2.5. 54 Dose-response relationship for VG5 homogenate Fig. 2.7 shows the maximum change in ileal IK (u^ equiv.cnr-.h"1) with increasing amounts of VG5. There is a good linear relationship between log dosage and A I,,, over the range 0.1 to 1.5 ganglia .ml"1. As little as 0.05 ganglia per ml is required to give a significant response (0.82±0.2 u.equiv.cm"-.h"1) and a maximum response was observed with 1.5 ganglia .ml"1 (13.9+ 1.1 uequiv.cm-.h1). The lag time, the rate of rise in !„, and the maximum I,,, attained were all similar for maximum doses of CC and VG. Effect of CC and VG5 on potassium permeability The effects of CC and VG5 on ileal potassium permeability, as indicated by increases in IK, are shown in Fig. 2.8. In the presence of control saline (i.e. with CI" present) I,,. fell to a steady level of 0.9+0.2 (lequiv.cm .^h'1 after 1 h. There was only a slight change in a residual when all CI" was replaced by gluconate (0.6 (lequiv.cm'-.h'1), and when IK was then initiated by raising haemocoel K+ to 80 mM (1.0510.1 uequiv.cm--.h-1). Both 0.5CC ml1 and 1.0 VG5 ml"1 subsequently caused significant several-fold increases in potassium diffusion current (IK). These increases in IK caused by CC and VG5 (Fig. 2.8) were quantitatively similar to those caused by cAMP stimulation of this tissue (Irvine et al., 1988). Effect of cAMP, CC and VG5 on ammonia secretion. Table 2.2 shows the effects of 1^ . stimulants on ammonia secretion in the ileum, over a 1 h period. Under control conditions the rate of ammonia secretion (JAMM) to the lumen is 1.5 umole cm-.h1, which increased significantly (P < 0.05) by two-fold on the addition of cAMP. CC and VG5 extracts have no 55 Fig. 2.7. Dose-response curve for maximum stimulation of ileal I,,, by VG5 extracts (Mean ± SE, n = 4-12). The solid regression line fitted by least square method is described by y = log 10.3 x + 11.6; r = 0.95. 56 10 8-5 6 I A CM* I 6 o i—< o o CO [ K + ] haemocoel: lumen (mM) 10:10 [Cl~] bi lateral (mM) 110 Stimulant (ganglia m l - * ) ~ 10:10 80:10 80:10 80:10 0 0 0 0 0.5 CC 1 VG 5 Fig. 2.8. Effect of CC and V G extracts on current (Mean ± SE, n = 6-12) required to clamp ileal V t at OmV under different sequential external conditions. The bar starting on the left represents normal unstimulated I,,., which was slightly reduced by bilateral replacement of all CI" (second bar). Imposition of a haemocoel-to-lumen K + gradient has little effect in causing a potassium diffusion current (IK, third bar), until CC or VG5 extracts were added (fourth and fifth bar). Mean increases are significant at * (P<0.05). 57 significant effect on J ^ . Effect of cAMP, CC and VG5 on acid secretion Table 2.3 shows the effect of stimulants on ileal acid secretion over a one hour period. The rate of acid secretion to the lumen was 1.4 u.equiv.cm'-.h"1 under control conditions, a similar rate to that observed for locust rectum (1.55±0.10 equiv.cm'Mi'1) under similar conditions (Thomson, 1990). Both CC and VG extracts reduced lumen acidification, but cAMP had no significant effect, unlike the situation in the rectum where Thomson (1990) observed a 66% reduction in JH caused by cAMP under open-circuit conditions. Effect of CC and VG on rectal I« Dose-response relationships of CC and VG5 saline extracts on rectal I*. are shown in Figs. 2.9 and 2.10 respectively. As little as 0.03 CC was required to produce a significant response, whereas a similar response to VG5 was observed at a doses of 0.1 gland. Both sources gave maximum stimulation at 0.5 gland ml'1 of 7.2±0.9 for VG5 and 8.6i0.66 for CC. The large response to VG extracts contradicts earlier reports (Spring and Phillips, 1980a; Proux et al., 1985) which only observed substantial stimulation of rectal I*. by extracts of the brain and CC. Effect of CC and VG5 homogenates on rectal IK. Both CC and VG5 cause a significant increase in potassium diffusion in the rectum (Fig. 2.11). As observed with ileal I*, rectal I«, fell to a steady-state level of 1.3±0.25 uequiv.cm-.h'1 after 2 h. There was a slight decrease in residual I*. when all CI' was replaced by gluconate on both sides, which returned to the 58 Table 2.2. Effect of cAMP, CC and V G on ileal ammonia secretion under open circuit conditions (Mean ± SE, n = 4-6) J A m m (uMxm-2.!!-1) V t (mV) Control 5mM cAMP 1.0CC ml"1 2.5VG ml"1 1.51±0.32 3.55±0.32* 1.39±0.09 2.00±0.49 2.57±0.89 23.5±4.37* 12.67±1.67* 13.5±4.03* * significant difference from control, P < 0.005. Measured over lh starting lh after dissection Ammonia refers to total of ammonia plus ammonium ions 59 Table 2.3. Effect of cAMP, CC and V G on ileal acid secretion (JH+) under open circuit conditions (Mean ± SE, n = 4-6) JH+ (u.equiv.cm'2.h'1) V, (mV) Control 5mM cAMP 0.5CC ml"1 1.0CC ml'1 2.5VG ml"1 1.40±0.08 1.31±0.34 0.7410.14* 0.0910.89* 0.5710.14* 3.5010.96 25.013.44* 18.011.08* 17.313.97* 16.314.06* * significant difference from control, P < 0.005. 60 CQ' I 6 o .a & CD O W 10-, 8 44 ~ 2 T -o 1 0.00 0.25 0.50 0.75 CC Dose (gland pair m l - 1 ) 1.00 Fig. 2.9. Increases in rectal I« one hour after adding various doses of crude C C to the haemocoel side (Mean ± SE, n = 6-8). 61 Fig. 2.10. Dose-response curve for maximum stimulation of rectal 1« to VG5 homogenates (Mean ± SE, n = 6-8). 62 CM' I H o i—i o o w 8 6 * T [K + ] haemocoel-.lumen (mM) 10:10 [Cl~] bi lateral (mM) 110 Stimulant (ganglia m l - - - ) -10:10 80:10 80:10 80:10 0 0 0 0 0.5 CC 1 VG 5 Fig. 2.11. Effect of CC and V G extracts on current (Mean ± SE, n = 6-12) required to clamp rectal V t at OmV under different sequential external conditions. The bar starting on the left represents normal unstimulated IK, which was slightiy reduced by bilateral replacement of all CI' (second bar). Imposition of a haemocoel-to-lumen K + gradient has little effect on causing a potassium diffusion current (IK, third bar), until CC or VG5 extracts were added (fourth and fifth bar). Mean increases are significant at * (P<0.05). 6 3 steady-state value when IK was induced by raising haemocoel K+ to 80 mM. The significant increases (P < 0.05) in IK due to CC and VG were quantitatively similar to those caused by cAMP stimulation (Hanrahan et al., 1986). VG5 caused a greater increase in rectal IK (7.13±0.28 uequiv.cm .^h1) than that observed with CC (5.09±0.31 uequiv.cm-Mi-1). Effect of temperature on CC and VG stimulants The effects of different temperature pre-treatments on CC and VG stimulatory activity are compared in Table 2.4. When left at 4°C for 24 h, CC extracts lost 17% of its activity, whereas there was no measurable loss in VG activity. CC homogenates also lost more activity at room temperature over a 24 hour period, namely a 77% reduction compared to only 32% for VG5. Boiling caused rapid loss of all VG5 stimulatory activity within one min, whereas CC stimulants remained stable. Indeed CC homogenates lost only 50% of their activity after boiling for 10 min, indicating significant heat stability of a major stimulant in CC as compared to VG. Both CC and VG stored as intact glands at -70°C retain their full stimulatory activity for at least one year. As homogenates at -20°C they show no discernable loss in activity after 3 months (data not shown), indicating that all major stimulants of ileal 1^ are stable when stored frozen and unpurified. Effect of proteolytic enzymes on CC and VG stimulants The effects of pre-treatment with proteolytic enzymes on stimulatory activity in CC and VG homogenates are shown in Table 2.5. Both CC and VG factors were almost completely destroyed by trypsin digestion (99 and 98% reduction from 64 Table 2.4 Effect of different temperature pre-treatments of CC or V G extracts at a dose of 1 gland .ml'1 on ileal !„. (Mean ± SE, n=6). AL*. (uequiv.cm'Mi"1) PRE-TREATMENT CC V G Control 8.310.52 7.38+1.04 4°C, 24h 6.9210.96 7.6010.26 Room temperature, 24h 2.2210.62** 4.8710.51* boiling for 1 min 8.4810.73 0.00** 10 min 4.0710.64** 0.00** 1 Freshly thawed glands used within lh. * Significant difference (P < 0.05) and ** highly significant difference (P < 0.005) from controls. 65 control values respectively). Chymotrypsin was less effective in that it destroyed 80% of CC activity and 89% of VG activity. These results suggest that both CC and VG stimulants are proteinaceous. Stimulatory activity extracted with different solvents As shown in Fig. 2.12, stimulants in whole CC were extracted equally well in physiological saline and distilled water, with nearly all activity retained in the first supernatant. With saline extractions, the third extract caused a small negative response in I*, (i.e. a reversal of current direction) which lasts longer than 2 h. However, in other experiments using a very small dose of CC homogenate, a similar response was observed but IK gradually rose to a positive value over a 6-7 h period (results not shown). This suggests that a separate less water-soluble stimulant of cation (i.e. Na+) absorption might be present in CC, but this possibility has not been pursued. CC stimulants showed some solubility in acetic acid (0.2M), but no activity was recovered in the pellet by subsequent saline extraction. This suggests that acid conditions destroyed activity. In contrast, 70% methanol was a poor solvent, but stimulatory activity was largely recovered by three subsequent saline extractions of the pellet. VG stimulants were best extracted in physiological saline, but required three extractions to recover all activity. Solubility in distilled water was poor compared to CC stimulants and activity was completely destroyed by 0.2M acetic acid. Stimulants in VG also exhibited low solubility in 70% methanol and, like CC factors, required three subsequent saline extractions to recover all of the original activity. 66 Effect of known or putative insect neurotransmitters on ileal L,c. Of the known or putative neurotransmitter substances tested (acetylcholine, adrenalin, dopamine, octopamine, glycine, glutamate, serotonin), only dopamine at excessively high concentrations (ImM) had any effect on ileal 1^ ., ( I*, of 1.1±0.5 liequiv.cm'-.h'1-, n=8), but the response was several times slower than that initiated by CC or VG homogenates and was then only equivalent in magnitude to that induced by very small quantities of CC or VG stimulants. No evidence was obtained that these common neurotransmitter substances could account for stimulation by locust neural tissue homogenates, especially considering that dilution of extracts was 103 to 105 fold during our assays. For example, one would have to postulate dopamine concentrations in excess of one molar in locust neural tissues to explain the slight stimulation of ileal IK observed. Effect of exogenous stimulants on ileal I8C The effect of cyclic nucleotides (cAMP and cGMP), theophylline and forskolin on ileal !« are shown in Table 2.6. Cyclic AMP, cGMP, and theophylline (all at 5mM) caused similar maximum increases in ileal I*, to values previously observed with CC and VG stimulants. Fig. 2.13 shows a dose-response relationship for forskolin over the range of 10-100 |iM. 67 Table 2.5 Effect of protease pre-treatments of CC and VG homogenates (1 gland equiv.ml1) on stimulation of ileal I*. (uequiv.cm'2.h'"; Mean ± SE, n = 6). CC VG CONTROL (no protease) 6.95+0.49 7.38±1.04 TRYPSIN 0.11±0.04** 0.21±0.06** CHYMOTRYPSIN 1.18±0.42** 0.76±0.23** Note: Protease alone at the concentration and over the time period used in this experiment did not affect ileal I,,.. ** Highly significant from controls (P < 0.005). 68 Fig. 2.12. Maximum stimulation of ileal I*, following extraction of whole CC (A) and VG5 (B) by different solvents, tested at a dosage of 0.5 (A) or 1 (B) gland equivalentml'1 bathing saline (open bars). None of the solvent controls alone had any effect on IK in the amounts (100u.l) added to the saline bath. Subsequent sequential extraction of stimulatory activity remaining in the original pellet with physiological saline is indicated by PI, P2, P3 (hatched bars) for each extraction solvent (Mean ± SE, n = 6-8). A I s c (/xequiv.cm .h l ) •—^ i—^ »—»• o w ^ o j c o o r o t f ^ i i i i i i i i_ SALINE PI P2 WATER PI P2 METHANOL PI P2 P3 7ZZZZ2r^ 7ZZZZZZZZZZZZ. ZZZZZZZZZZZZZZZZZZZ2r^ >///////////?r ACETIC ACID PI A I g c ()uequiv.cm .h •*) I i—»• i—^ i—^ r o o r o ^ c o c D o r o ^ i i i i i i i i i_ SALINE PI P 2 WATER PI P 2 METHANOL PI P 2 ACETIC ACID PI P 2 7777777777777777^ 77777?^ 22< 7k 70 Table 2.6. Effect of exogenous stimulants on ileal I,,. OJ.equiv.cm"2.h*1; Mean ± SE, n = 6-8) Unstimulated Stimulated 5mM cAMP -0.6±0.10 12.9±0.70 5mM cGMP -0.5±0.21 9.3±0.92 5mM Theophylline -0.710.53 9.711.12 lOOuM Forskolin -0.210.30 8.911.3 71 Fig. 2.13. A dose-response curve for forskolin stimulation of ileal I*. (Mean ± SE, n = 6-8). 72 DISCUSSION It has long been known from in vivo studies that desert locusts must regulate salt and water reabsorption in their hindgut in order to regulate the large changes in volume and ionic concentrations of the excreta observed under different extremes of osmotic stress (Phillips, 1964 a-c). The major solute transport mechanisms in both the locust ileum (Irvine et al., 1988; Lechleitner et al, 1989 a,b) and rectum (reviewed by Phillips et al, 1986, 1988) have since been identified, localized to apical or basolateral membranes, and partially characterized. Both these segments of locust hindgut possess qualitatively similar ion transport mechanisms for CI", Na+ and K+. These can be stimulated in a similar manner by exogenous cAMP or agents (forskolin, theophylline) which typically increase intracellular levels of this second messenger (Chamberlain and Phillips, 1988; Table 2.4). Moreover, Chamberlain and Phillips (1988) showed that locust CC homogenates and all other forms of stimulation tested caused transitory elevation of cAMP levels in rectal tissue at the time that CI" dependent I*. increased rapidly. While exogenous cGMP also causes some stimulation of rectal I,,. (Chamberlain and Phillips, 1988) and ileal I„ (Table 2.4), increases in cGMP levels in rectal tissue following CC stimulation only occurred long after the initial rise in L*.. All the attempts to implicate intracellular Ca2+ in the response of rectal I,,. to CC homogenate have been negative to date (Hanrahan and Phillips, 1985; Phillips et al., 1986). In summary, there is good evidence that an intracellular cAMP-mediated control system for NaCl and KC1 absorption is present in locust hindgut epithelia. The questions remain as to (a) which specific substances normally control hindgut salt transport in situ and (b) whether these factors act largely via this cAMP control system. 73 Potential sources of natural factors in neurosecretory tissues, which are capable of controlling salt transport in locust ileum have been identified and these stimulants were found to be stable enough under some extraction conditions to attempt their purification by HPLC. The dose-response curves obtained for saline extracts of CC and VG provide the basis for assessing recovery of stimulants during purification as reported in Chapter 3. These results indicate that both corpus cardiacum and the ventral ganglia contain proteinaceous factors which are capable of causing large dose-dependent increases in ileal IK and Vt, and decreases in Rt, similar to those caused by exogenous cAMP. In addition, results also confirmed that CC extracts (presumably CTSH) have similar effects on KC1 transport in the locust rectum, and demonstrated a similar rectal KC1 stimulation due to VG5 extracts. Previous reports found that only the brain and CC caused significant increases in rectal 1^ ., (Spring and Phillips, 1980a; Proux et al., 1985). Although activity was observed in both the NCC and GCC, it is unclear whether the GCC contains factors stimulating ileal Ire or if this is due to contamination with products in projection of the NCC into GCC. The latter would appear the more likely explanation. The NCC store and secrete neuropeptides which are produced in the brain, and GCC produce their own neurosecretions (Raabe 1982). Histochemical staining with chrome-haematoxylin-phloxin shows that the NCC contains large amounts of A-material (acidic neurosecretory material). This A-material is also found in the stalks of the NCC which project into the GCC. Moreover, other than these extensions, the GCC contain no A-material (Highman, 1961). The electrical changes in the ileum in response to cAMP and CC are similar to those previously observed for locust rectum. For both locust hindgut segments, 74 36CT flux measurements, electrical studies, and ion substitutions all indicate that increases in !«. initiated by stimulants are due to increases in active electrogenic transport of CI" from the lumen (reviewed by Phillips et al., 1986, 1988). Lechleitner and PhilUps (1989) and Lechleitner et al. (1989 a,b) found that locust CC and VG5 extracts increased both CI" (JC]) and fluid (Jv) absorption across everted ileal sacs (i.e. open-circuit state) by several-fold over a similar dose-response range as observed for CC homogenates in the rectum (Proux et ah, 1984), and this stimulation of ileal Jv was also entirely Cl-dependent. In other words, crude extract stimulants of CI" transport (i.e. LJ have an antidiuretic action on both locust hindgut segments. Likewise, both VG and CC homogenates also enhance salt absorption by increasing passive permeability to a major counter-cation for CI" transport, namely K+, as judged by increases in IK across both ileum and rectum; All of these common results are therefore entirely consistent with the initial hypothesis that the same locust hormones (including CTSH partially purified by Phillips et al., 1980) act through cAMP to increase salt and hence water absorption in both ileum and rectum. This hypothesis does not exclude the possibility that other second messengers may also play a role in association with cAMP, or that one of several natural stimulants in CC or VG may not act via cAMP at all. These results demonstrate that factors in the CC and VG5 inhibit acid secretion into the lumen of the ileum, but have no significant effect on ammonia secretion. Acid secretion in the rectum is partially inhibited by cAMP (Thomson, 1990), but this cyclic nucleotide had no significant effect on ileal JH+ (Thomson et al. in preparation; this chapter). The inhibition of JH+ caused by CC and VG (Tables 2.2 and 2.3) must therefore proceed by another transduction pathway (Ca2+, diacylglycerol or inositol trisphosphate). Cyclic AMP causes a large increase in 75 ammonia secretion in the ileum (Lechleitner, 1988; this chapter), but neither CC nor VG5 have any significant effect, suggesting that there may be factors in other ganglia which regulate ammonia secretion in this tissue, or that control of might be neural. Rectal ammonia secretion (0.6 |iM. cmMr1.; Thomson, 1988) is less than half of that observed for unstimulated ileum, and rectal JA M M is unaffected by cAMP. In contrast, cAMP causes a two-fold increase in ammonia secretion in the ileum to values four times those of the rectum. The effects of CC and VG on acid and ammonia secretion in the rectum have not been studied. Lechleitner (1988) demonstrated that cAMP, CC and VG5 all stimulated active Na+ flux to the haemocoel in the ileum. Some of this Na absorption may occur in exchange for secreted ammonia. In contrast, rectal J N A is not similarly controlled (Black et al., 1987). These results and those reported by Lechleitner (1988) using ileal sacs demonstrate that factors from the central nervous system regulate all major ion transport processes (except ammonia secretion) described to date in the ileum, leading to increased ileal fluid transport (reviewed by Phillips et al., 1988). The same tissue sources also increase fluid and KC1 transport in the locust rectum. The widespread distribution of stimulatory activity which was observed throughout the ventral nerve cord was not unexpected. Remy and Girardie (1980) showed that two neuroendocrine cells in the locust suboesophageal ganglion send axons throughout the central nervous systems from brain to the last abdominal ganglion. Likewise, stimulants of insect Malpighian tubule secretion have a wide distribution, at least at low concentrations, in many insects (reviewed by PhilUps, 1983). 76 While CC and VG factors stimulated an identical pattern of absorptive events in both locust hindgut segments, the major stimulants from these two sources must be somewhat different substances. For example, the time course of the decline in ileal I,,, is much faster following VG as compared to CC stimulation. Only the VG stimulant was destroyed by boiling for one min. While major stimulants from CC and VG are probably proteins (i.e. destroyed by proteolytic enzymes or prolonged boiling) and are saline-soluble (i.e. not steroids), they differ in solubility properties. For example, about 80% of CC activity could be extracted with 0.2 M acetic acid, whereas this acidic treatment completely destroyed all VG stimulation of ileal Ire. Ileal stimulants from both CC and VG could only be partially extracted with 70% methanol even after vigorous sonication, but most of the residual stimulatory activity could be recovered by subsequent saline extraction of the pellet. In contrast, CTSH (assayed by rectal LJ is readily soluble in 80% methanol according to Phillips et al. (1980). This difference provides the first suggestion that a stimulant of ileal L^ . which may differ from CTSH is present, particularly in VG. This question will only be resolved by comparison of stimulants purified by HPLC and tested on both hindgut segments. 77 CHAPTER T H R E E : Isolation and characterization of a factor from the C C which influences ileal transport. INTRODUCTION Results of chapter 2 and previous studies (reviewed by PhilUps et al., 1986) demonstrate that ion and fluid transport in the locust hindgut can be controlled by proteinaceous stimulants from the central nervous system. It was demonstrated that ileal KC1 reabsorption is stimulated by CC and VG extracts, which also inhibit H + secretion in the ileum, whUst no significant effect is observed on ammonia secretion. Lechleitner et al. (1989a, b) and Lechleitoer (1988), also report that the same CC and VG extracts regulate fluid and Na+ absorption in the ileum. In addition, the stimulatory action of crude CC on rectal CI- reabsorption was confirmed and the stimulatory effect of VG5 homogenates rectal 1K was demonstrated. Moreover, both CC and VG5 stimulate IK in the rectum. Effects of crude extracts on rectal acidification and ammonia secretion were not studied. While all these hindgut transport processes are influenced by crude extracts, it is possible that there are a number of different factors with actions on specific transport processes which serve to regulate excretion in the insect hindgut. To answer this question factors from the CC of the desert locust acting on ileal L, were separated for three reasons: (1) a partial purification of a factor (CTSH) which stimulates rectal I*. was reported by Phillips et al. (1980), but no further attempts have been made since to purify it; (2) crude CC were the most potent source of rectal and Ueal I«. stimulants and therefore the best source of starting material; (3) I„ is the most convenient, rapid and sensitive bioassay to test HPLC 78 fractions. A purified factor could then be tested on all other bioassays. I,,, measures the major active transport process in the hindgut, namely CI- absorption, which drives fluid recovery. Crucial information on stability of the ileal I,,, stimulant in CC to physical and chemical treatments and solubility in solvents useful for purification were reported in chapter 2. Thus, the results reported in chapter 2 provided the groundwork necessary to attempt purification of the CC factor. CTSH was found to be a proteinaceous compound of approximately 8,000 Da, using a Bio-Gel P-30 column. (Phillips et al., 1980). These workers also report that CTSH extracts well into 80% methanol and is unstable below pH 5. Results reported in chapter two show different results using ileal L. as a bioassay, in that most activity remains in the pellet after a methanol extraction of CC and the active factor(s) extract well in 0.2N acetic acid. As these two reports used different tissue for the assay (rectum and ileum respectively) it remained possible that the stimulation of rectal and ileal I«. were due to different factors in CC. This question can be resolved by isolating factor(s) from the CC, and comparing effects of specific fractions on both hindgut segments. The factor(s) from the CC which stimulate ileal I„. are stable in strong acidic solutions, so reversed-phase HPLC could be used for separation. A number of groups that are isolating insect peptides now use this method (Fournier and Girardie, 1988; Coast, 1988; reviewed by Hayes and Keeley, 1983), particularly utilizing wide-pore columns (300 A) which are recommended for peptides of greater than 30 amino acids (Pearson et al, 1982). Only one factor which might control excretion in an insect hindgut (rectum) has been fully characterized to date (Girardie et al., 1989). The NCC of L. 79 migratoria stimulates fluid reabsorption in the rectum of this insect (Herault et al, 1985). Fournier and Girardie (1988) reported that neuroparsins (Nps), produced by the median neurosecretory cells (Girardie et al,1987), are the only antidiuretic factors in the NCC of this insect acting on rectal Jv. Neuroparsins A (NpA) and B (NpB) were isolated by anion-exchange and RP-HPLC, (Fournier and Girardie, 1988; Girardie et al, 1989). Neuroparsins B was found to be a homodimer, and the sequence of its monomer (molecular weight 8,188 Da) was determined. In this chapter the characterization of a factor named S. gregaria ion transport peptide (ScgLTP) from the CC of S. gregaria, using RP-HPLC for separation and voltage-clamped locust ilea as the main bioassay is described. A comparison between Neuroparsins, CTSH, and this factor is made, which indicates that ScglTP is a new peptide. The distribution of active factors from the initial chromatographic separation of locust CC and their influence on Cl-dependent L, and water absorption (antidiuretic activity) using isolated ileal and rectal preparations is also described. Finally an amino acid analysis and near complete sequence for ScglTP is reported. 80 MATERIALS AND METHODS BIOASSAYS 1. Electrogenic chloride transport To test activity of extracts and HPLC fractions, recta and ilea were mounted as flat sheets between two modified Ussing chambers and voltage-clamped at zero mV, as described in chapter 2. Each chamber contained 2 ml of saline which was stirred by vigorously bubbling with a mixture of 95% 0J5% C02 at 22°±2°C. Short-circuit current (J*), a direct continuous measurement of electronic CI" transport in this tissue (Irvine et al., 1988), was recorded continuously on a strip chart recorder (Soltec 1242, Soltec Corp., Sun Valley, Calif.). As this assay is sensitive to acetonitrile, aliquots of fractions to be assayed were dried in polypropylene microcentrifuge tubes (Robbins Scientific, CA.; rinsed with a 0.5% bovine serum albumin (BSA) solution) by centrifugal evaporation (Speed-vac, Emerston Instruments Inc., Ont.). BSA was found to be necessary to prevent loss of activity due to non-specific binding to the surface of the tubes: if active peptide was dried alone it became insoluble. Fractions were resuspended in small volumes (10-100 |iL) of physiological saline (chapter 2) and aliquots added to the haemolymph side of ilea or recta once a steady-state level had been reached (1-2 h after dissection) to give the desired concentration. BSA, which does not affect hindgut IK, was also added to the bathing saline to reduce non-specific binding of active peptide to the walls of the Ussing chamber. 2. Fluid absorption Methods for studying ileal and rectal fluid transport using everted hindgut sacs were similar to those previously described for locust rectum (Hanrahan et al., 81 1984) and ileum (Lechleitner et al., 1989a). At hourly intervals weight gain and tissue volume changes were determined by weighing ilea or recta (to within 0.1 mg) before and after removal of fluid in the sac. The true rate of transepithelial fluid movement was determined by correcting for tissue volume changes. Fluid transport (Jv) was measured hourly over 5h as described by Goh and PhilUps (1978). Usually rates are at near steady-state after the first hour. HPLC fractions were prepared as described for assay on CT transport. Effects of tissue homogenates and HPLC fractions in saline were determined by adding small aliquots (1-3 to the inside of the sacs (haemolymph side). Changes in saline osmolarity due to the addition of these fractions were monitored with a Westcor vapour pressure osmometer (Model 5500; Logan, Utah) and were found not to be significant. In a typical experiment, physiological saline (3 to 5ul) was added to the haemolymph side of the Ueal or rectal sacs for the first two hours, and the sacs were placed in 50 ml of physiological saline to obtain control rates. At the end of the second hour, crude CC, HPLC fractions, or physiological saline (control) were added to the sacs. The rate of fluid transport was then measured for the next two hours and compared to control preparations over the same period and to rates during the previous control period for the same preparation. EXTRACTION AND HPLC 1. Preparation of tissue extracts Whole corpora cardiaca (CC), storage (NCC) or glandular (GCC) lobes of CC were removed from adult male and female locusts, 2-6 weeks past their final moult, as described in chapter 2. Extracts were prepared by mechanically homogenizing 1,000 whole CC (50 NCC or GCC) in 5 ml HPLC water using a 82 Tissue Tearer homogenizer (Bartlesville, OK), and then centrifuging for 20 min at 12,000 g and at 4°C. The supernatant was removed and after two further extractions of the pellet, the supernatants were combined. 2. Separation of CC extracts a) Preparative step Supernatants were applied to a custom-made reversed phase C4 cartridge, 0.5 g, 300 A, 10|im packing, (Hypersil, Phenomenex, CA.) in a 3ml polypropylene filtration column (Supelco, Ont.), equilibrated with 0.1% trifluoroacetic acid (TFA). The cartridge was eluted stepwise with 2 ml each of 0.1% TFA, 30% CH3CN/0.1% TFA, 60% CH3CN/0.1% TFA and 100% CH3CN/0.1% TFA. These fractions were concentrated by centrifugal evaporation (Speed-Vac) to dryness for bioassay, or until volatile solvents were removed for further separation. Commercially available cartridges were found to be unsuitable for sample preparation, as recovery of biological activity from these cartridges was < 50% that from custom-made C4 cartridges. b) Reversed-phase HPLC - C8 step The concentrated active fraction (1-2 ml) from the preparative step (a) was injected via a Waters 2ml loop injector, onto a Nucleosil C8 300 A, 10um, reversed-phase column (224 x 4.4 mm: Phenomenex, CA.) fitted with a guard column (30 x 4.6 mm) of similar packing material. Chromatography was performed using two Beckman pumps, a Beckman 421A system controller (Beckman 114 M Solvent Delivery Module) and a Waters variable wavelength UV detector, set at 225 nm. The column was eluted with a gradient of 30 - 45% 83 CH3CN/0.1% TFA for 10 min, then 45 - 55% for 5 min at a flow rate of 1 ml/min. Nineteen fractions were collected manually, according to the peaks observed. Aliquots of these fractions were dried in Eppendorf tubes containing BSA (as described earlier) by centrifugal evaporation (Speed-Vac) and assayed for activity on both voltage-clamped and everted sac preparations of both ilea and recta. c) Reversed-phase phenyl step. After concentrating by centrifugal evaporation, the most potent fraction (D) from step (b) was injected on to an Aquapore 300 A, 7fi RP-phenyl column (250 x 4.6 mm) fitted with a guard column of the same type (15 x 4.6 mm; Chromatographic Specialties, Ont.), using the same injector, pumps, controller and detector as in step (b). The column was eluted with a 28 - 38% CH3CN / 0.1% TFA gradient for 10 min at a flow rate of 1 ml min1. Nine fractions were manually collected, and prepared for assay as described above (b). The active fraction was tested for purity by SDS-PAGE and contaminants were considered low enough to attempt amino acid sequencing. However, two N-termini were detected by the sequencer (Fig. 3.9) so larger amounts of active peptide were re-chromatographed on the RP-phenyl column isocratically at 29% CH3CN for 20 min. The major peak and surrounding fractions were collected and bioassayed on ileal I,,, The major peak was tested for purity by electrophoresis. 84 ELECTROPHORESIS Electrophoresis was performed on a PhastSystem utilizing PhastGels and PhastGel buffer strips (Pharmacia, Sweden). Two separation methods were utilized: sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE), and isoelectric focusing (LEF). 1. SDS-PAGE A high density homogenous polyacrylamide gel designed for the separation of peptides and small proteins (1000 - 10000 MW), was used with PhastGel SDS buffer strips. The gel (0.45 mm thick) has a 13mm stacking zone (7.5% total acrylamide (T), 2% cross Unking (C)) and a 32 mm continuous separating gel zone (20% T, 2% C) and contains 30% ethylene glycol. The buffer system consists of 0.2 M tricine, 0.2 M TRIS, 0.55% SDS, pH 8.1. The buffer strips are made of 3% agarose LEF. The sample must contain 1-5 ng.u.1'1 of each peptide for silver staining. Sample peptides equivalent to 10 - 20 CC in water were diluted (1:1) with SDS (non-reducing) or heated in the presence of SDS + B-mercaptoethanol (reducing conditions) before applying to the gel. The running conditions were; 400V, 10mA, 3.0 W for 25 min at 15°C. Molecular weight (MW) of the peptide was estimated by running calibration peptides on the same gel. The standard peptides used and their apparent molecular weights (kDa) are insulin, 3.035; bovine trypsin inhibitor, 5.835; lysozyme, 14.4; a lactoglobulin, 18.4; carbonic anhydrase, 29.1. There is a linear relationship between log MW and relative mobility of a protein/peptide. SDS-PAGE separates compounds according to their size, the distance moved is inversely proportional to 85 molecular weight so that small peptides move further than large ones under similar conditions. 2. IEF For IEF, a PhastGel LEF 3-9 (pH 3-9) was used which is a homogenous polyacrylamide gel (5% T, 3% C). The running conditions were 2000 V, 2.5 mA, 3.5 W for 25 min at 15°C. Peptides migrate under an electric field to a point in the pH gradient which corresponds to their isoelectric point. Sample peptides (10 CC equivalents) in water were applied to the gel. The gels from the methods above were stained with silver as described by Heukeshoven and Dernick (1985). Effect of proteolytic enzymes on purified CC The purified fraction was resuspended in 0.1M TRIS buffer and treated with trypsin at pH 7.6 or chymotrypsin at pH 7.8, at a concentration of lmg ml1 for 2 h at 25°C before testing extracts on short-circuited ilea. As controls, ileal preparations were exposed to proteases alone, and to the purified fraction which were similarly incubated without proteases. Amino acid analysis and sequencing Both amino acid analysis and sequencing were conducted by Dr. S. Kieland at the University of Victoria using an Applied Biosystems model 420A derivatizer-analyser system and sequence analysis using an Applied Biosystems 470A gas phase sequencer with on-line PTH-analyser and 900A system controller and data analyser. 86 RESULTS C 4 cartridge separation Stimulation of CI- transport across both hindgut segments was only observed in the 60% CFLCN fraction. The effects of this fraction are shown in Table 3.1. At a concentration of 1 CC equivalent ml1, this fraction caused a similar maximum stimulation of ileal 1^ as observed with 0.5 crude CC ml"1 (chapter 2). No activity was observed in the 0.1% TFA, 30% CH3CN or 100% CH3CN fractions when assayed on either hindgut segment. In addition, the ileum was more sensitive to the active factor(s) in this fraction than the rectum, at both concentrations assayed. C 8 separation An absorption profile for HPLC separation of whole CC is shown in Fig. 3.1. The fractions collected are indicated by letters, and the effects on ileal and rectal 1^ are represented in Figs. 3.3 and 3.4. Fig. 3.2 shows absorption profiles for separation of NCC extracts, and fractions which stimulate ileal I^ (D and F) are indicated by the solid bars. Nineteen fractions were collected from each separation and these were labelled using a similar coding. Fig. 3.3 shows activity of whole CC fractions on voltage-clamped locust ilea Activity was associated with two main peaks, D and F, which eluted from the column at CH3CN concentrations of 38 and 40% respectively. Some activity was also observed in adjacent fractions E,H and G. All other fractions gave no significant increase in ileal I«. Fraction D was the most potent source of the stimulant with a maximum response (9.9710.84 uequiv.cm .^h'1) observed at a concentration of ICC equivalents ml1, a value equal to the maximum obtainable 87 Table 3.1. Effect of 60% CH3CN fractions from C4 cartridge on rectal and ileal I«c, represented as a change in I,,. (ALJ from steady-state levels (Mean ± SE, N = 4-8) DOSE RESPONSE I, (CC equiv.ml1) (u.equiv.cnr2.lrI) ILEUM 0.5 4.4±0.42** 1.0 9.1910.61** RECTUM 0.5 2.9911.44* 1.0 5.5611.62** All increases are significantly different at * (P < 0.05) or ** (P < 0.01). 88 6 a in 02 CV2 Fraction Retention 0 Time (min) Fig. 3.1. Absorption profile of extracts fom 300 whole CC from reversed-phase Nucleosil Cg HPLC. Chromatographic conditions are described in materials and methods. The solid horizontal line represents the concentration of CH3CN in 0.1% TFA. The letters indicate the 19 fractions collected. 89 Fig. 3.2. Absorption profile of 50 NCC from reversed-phase Nucleosil Cg HPLC. Chromatographic conditions are described in the materials and methods. The horizontal line represents the concentration of CH3CN in 0.1% TFA. A solid bar indicates ileal I*, activity, and the letters represent the nineteen fractions collected. 90 I C\2* I S o > •I—t cr CD o CO 1 2 T 1 0 8 6 4 2 0 ** _ L A - C D E F G H I J - S FRACTION Fig. 3.3. The effect of whole CC HPLC fractions (ICC equivalent ml1) on ileal I«.. AIK is the change from steady-state levels, one hour after addition of fractions. (Means ± SE., n = 4-10). Means increases were significantly different at * (P < 0.05) or ** (P < 0.01). 91 with crude CC. Fraction F, at the same concentration, evoked only 46% of this response (4.61±0.49 uequiv.cm'-.h"1). Stimulatory activity on voltage-clamped recta is shown in Fig. 3.4. As observed in the ileum, most activity is associated with fraction D, but a far greater dose (5CC equivalents ml'1) is required to evoke a maximum response of 7.5±0.73 uequiv.cm'-.h"1. At a dose of 1.0 CC ml'1, which causes maximum increase in ileal I K , the increase in rectal I K is only 20% of maximum (data not shown). Fraction F gave no response at all, but fractions G , H and I all showed some effect, but only at very high dosages (5CC ml'1). When assayed on ileal I*., separated NCC had two significant areas of activity (Fig. 3.2) which correspond to active fractions (D and F) of whole CC. At a concentration of 1 CC equivalents.ml'1, fractions D and F from NCC separation increased ileal I K by 8.9+.1.02 and 2.7±0.87 |i.equiv.cm'2.h'1 respectively (n = 6). Presumably the active factors in whole CC are stored in the NCC. The effect of HPLC fractions on ileal fluid transport are shown in Fig. 3.5. Values represent the increase in fluid transport between the control period (second hour) and one or two hours after fractions were added (i.e. over the third and fourth hour). There was no significant change in control values over the second to fourth hour with fluid transport rates averaging 3 - 3.5 uL hour"1 ileum1, as previously reported by Lechleitner et al. (1989). During the first hour of exposure to HPLC fractions significant increases in fluid transport were observed with fractions D to G inclusive, but fractions H to S were completely inactive. As observed for ileal L^ , fraction D had the greatest stimulatory activity, causing a 5-fold increase in Jv (the maximum) at a dose of 0.05 CC equivalents nl'1. Crude CC causes a similar increase in Jv at the same dosage. The increase in Jv caused 92 I C\2 O > • r-t cr cu o CO 8 6-0 A - C ! D E i H I - S FRACTION Fig. 3.4. The effect of HPLC fractions from whole CC on rectal L^ . (open bars, 2.5 CC equivalents ml'1; stippled bars 5.0 CC equivalents ml'1). A L^ . is the change from steady-state levels one hour after addition of fractions. (Mean ± SE., n = 4-6). Mean increases are significant at * (P < 0.05) or ** (P < 0.01). 93 _ 3 J A B C D E F G H I - S F r a c t i o n Fig. 3.5. The effect of whole CC HPLC fractions (0.05 CC equivalents uT1) on ileal fluid transport in the absence of any initial osmotic concentration difference. AAbsorption is the difference in fluid transport between hour 2 and 3 (open bars) and between hours 3 and 4 (stipled bars). The fractions were added at the start of the second hour and renewed at the third hour (Mean ± SE., n = 6). Mean increases are significant at * (P < 0.05) or ** (P < 0.01). 94 by fraction F was 30% of maximum and in proportion to its lesser effect on ileal I„ as compared to fraction D. There is therefore a good correlation between ileal !« and Jv stimulation by both fractions D and F. Longer exposure of ilea to fractions D,E and F (i.e. for 2h) caused no further significant increase in Jv, however there was a large and significant delayed increase in fluid transport due to fraction G. As this fraction had little effect on ileal I«., both at high doses (5CC equivalents ml1; 1.12±0.31 uequiv.cm-.h1, n = 4) and following 2 h of exposure (1.52±0.27 uequiv.cnrMr1, n = 4), its effects on fluid transport were investigated further. Fig. 3.6 shows dose-response relationship for fraction G on ileal Jv after 1 and 2 hours of exposure. At the lower dose there was a significant increase in fluid absorption only in the second hour, but at higher doses of 0.10 CC equivalents ul1, the increase in Jv is significant in both the first and second hour. The 4-fold increase in Jv due to fraction G is similar to the maximum caused by crude CC, but the time course is slower. Clearly the factor in fraction G must act on ileal Jv by stimulating a solute transport other than CI- (i.e. U RP-phenyl separation of fraction D An absorption profile of RP-phenyl separation of fraction D is shown in Fig. 3.7. The fractions collected are indicated by Roman numerals. The activity from this separation was only associated with fraction iii (Diii), using ileal L^ as the bioassay. At a dose of ICC equivalent ml-1, ileal I„ was increased by 11.2 ± 1.09 |iequiv.cm-.h4., (n = 8), while all other fractions gave no response (n = 4). 95 Fig. 3.6. Dose-response relationship for HPLC fraction G on ileal fluid absorption (i.e. increase over control values) after 1 h (open symbols) and 2 h (solid symbols) of exposure. (Mean ± SE., n = 6). 96 0.2 -Retention 0 5 10 15 20 Time (min) Fig. 3.7. Absorption profile of fraction D from Phenyl RP-HPLC separation. Conditions are described in the materials and methods. Solid horizontal line represents the acetonitrile concentration, and the Roman numerals indicate the fractions collected. 97 Estimation of purity of fraction Diii 1) SDS-PAGE Fraction Diii eluted as a single band on a SDS-PAGE under both reducing and non-reducing conditions, with a preceding "shadow" (Fig. 3.8), but was considered "pure" enough to attempt sequence analysis. The peptide moved approximately the same distance as bovine trypsin inhibitor and therefore has a similar size (5835 Da). A more accurate estimation of molecular weight from electrophoresis is considered later. 2) Sequence analysis Sequence analysis revealed that fraction Diii consisted of two peptides with unblocked N-termini (Fig. 3.9), and these were arbitrarily termed peptide 1 (PI) and 2 (P2). From the sequence data peptide 1 was estimated to be present at 4-5 times the amount of peptide 2. These two peptides also have no homology with each other. From this run, 21 amino acids of PI and 20 residues of P2 were determined. As the fraction Diii contained an impurity, RP-HPLC was again utilized to separate the two peptides as discussed later. However, fraction Diii (PI and P2) was tested on a number of transport processes, as reported below. Dose-response relationship of fraction Diii (PI + P2) Fig. 3.10 shows the relationship observed between the increase in ileal after one hour of stimulation and increasing doses of fraction Diii. There is a good linear relationship between L^ . (u,equiv.cnr2.h"1) and dose over the range of 0.075 to 0.5 gland-ml1. The increase in !« caused by 0.075 CC equiv.ml1 was 98 Fig. 3.8. SDS-PAGE of fraction Diii (2 and 3) and proteins of known molecular weight (1; listed in materials and methods), (i) indicates the major peptide and (ii) the preceding "shadow" from fraction Diii. (a) is bovine trysin inhibitor (MW: 5835 Da.). 99 Table 3.2. The twenty common protein amino acids Amino acid 3-letter 1-letter abbreviation abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D (Asparagine or aspartic acid) Asx B Cysteine Cys C Glutamic acid Glu E Glutamine Gin Q (Glutamic acid or glutamine) Glx z Glycine Gly G Histidine His H Isoleucine He I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine - Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V 100 Fig. 3.9. Partial amino acid sequences of peptides from fraction Diii. Peptide 1: S F F D I Q ? ? G V Y D ? S I F A R L D R I I E Peptide 2: D A A D F G D P Y S F L D/Y R L I E R G D 101 significant, and a maximum response was produced by 1.0 CC equiv.ml-1 (11.2+.1.09 iiequiv.cm'-.h1). With crude extracts a significant response was observed with 0.005 CC ml'1, and a similar maximum response required 0.25 CC ml'1. Thus there was a four-fold difference in the concentrations of crude and purified extracts required for a maximum response, due to losses and perhaps removal of a co-stimulant. Fig. 3.11 shows the effect of fraction Diii on rectal I*.. A maximum response of 3.63±0.29 (O-equiv-cm^ .h'1 is observed with 2CC equiv.ml1, and I*. could not be increased further by doubling the dose. This is only 41% of the maximum rectal response to crude CC using a 4-fold greater equivalent concentration of Diii. It appeared that a maximum rectal 1K response could not be produced with the active factor in fraction Diii, suggesting that neither peptide in this fraction is CTSH. Fraction Diii factor stimulated ileal Jv by up to 4-fold in a dose-dependent manner over the range 0.025 - 0.1 CC equiv. ul"1 (Fig. 3.12), compared to 0.005 to 0.2 CC for crude extracts (Lechleitner et al, 1989a). The ileal Jv of 13.9+1.2 ulh"1 achieved with fraction Diii was quantitatively similar to the maximum response to crude CC. Phenyl RP-HPLC separation of Fraction Diii Utilizing the same phenyl column as used in the previous chromatography step, but an isocratic elution of 29% acetonitrile:0.1% TFA, it was possible to remove the contaminating peptide. The absorption profile is represented in Fig. 3.13, where numbers (1-4) represent the fractions collected. The active fraction eluted as a large single peak (3), and a small preceding 1 5 - , CC equivalents ml Fig. 3.10. Increase in ileal L. one hour after adding increasing amounts of fraction Diii to the haemocoel side (Mean ± SE, n = 6-8). 103 Fig. 3.11. Increases in rectal I« one hour after adding various amounts of fraction Diii to the haemocoel side (Mean ± SE, n = 6-8). 104 U H 1 1 1 1 — 0.00 0.05 0.10 0.15 0.20 CC equivalents jA Fig. 3.12. Dose-response relationship for fraction Diii on the increase in ileal fluid absorption after one hour of exposure to the stimulant on the haemocoel side (Mean ± SE, n = 6, except at 0.05 CC equiv.uT1 where n = 19). 105 0.1-s LO C\2 Fraction Retention 0 Time (mins) - r -5 10 2 3 4 15 20 Fig. 3.13. Absorption profile of fraction Diii from phenyl RP-HPLC separation, at an isocratic 29% acetonitrile concentration. The numbers represent the four fractions collected. The only major peak (3) stimulated ileal L.. 106 shoulder (2). The peak and fractions around the peak were collected and assayed. Heal I*. activity was only associated with the major peak, which caused an increase in ileal !« of 9.96±0.66 iiequiv.cm'-.h"1 (n = 8) at a dose of 5 CC equivalents ml1. This peptide (fraction Diii3) was again tested for purity by electrophoresis and amino acid and sequence analyses were conducted. Effect of proteolytic enzymes on fraction Diii3 The effects of pre-treatment with proteolytic enzymes on stimulatory activity in purified CC are shown in Table 3.3. Activity was reduced 65% by chymotrypsin and 78% by trypsin from control values, confirming the peptidic nature of Diii3. These results also suggested that the active peptide may contain the amino acids lysine or arginine (trypsin) and phenylalanine, tryptophan or tyrosine (chymotrypsin). Estimation of purity of fraction Diii3 1. SDS-PAGE Figure 3.14 shows electrophoretic separation by SDS-PAGE of fraction Diii3 from HPLC separation (non-reducing conditions), and calibrating peptides used to estimate molecular weight. The active factor from HPLC ran as a single band, suggesting this factor was pure. The estimated molecular weight of Diii3 is approximately 5750 Daltons from a plot of log molecular weight against relative mobility (fig. 3.15). 107 Fig. 3.14. SDS-PAGE of fraction Diii3 (2) and calibrating peptides (1; i = insulin, ii = bovine trypsin inhibitor, iii = lysozyme, iv = a lactoglobulin, v = carbonic anhydrase). 108 Table 3.3. Effect of protease pre-treatments of purified factor Diii3 (2.5 gland equiv.ml'1) on stimulation of ileal (^ .equiv.cm'Mi"1; Mean ±SE, n=4). Treatment Control (no protease) 9.2±0.75 Chymotrypsin 3.2±1.00** Trypsin 2.0±0.70** Note: Protease alone at the concentration and over the time period in this experiment did not affect ileal I*.. ** Highly significant difference from controls (P < 0.005). 109 Relative Mobility Fig. 3.15. Plot of log of molecular weight of known peptides (open circles) against their relative mobilities by SDS-PAGE. The relative mobility of the peptide from fraction Diii3 is 0.69, and is indicated by the dashed line. The molecular weight calculated from the corresponding position on the ordinate. The regression line is expressed as y = 4.71 - 1.37x. 110 2) Isoelectric focusing No bands from fraction Diii3 were observed by isoelectric focusing. These negative results could be due to problems with the staining techniques used or the amount of peptide required, however, this method was repeated three times, and the standards were clearly visible. 3) Amino acid analysis and sequence data The amino acid analysis of Diii3 is shown in fig. 3.16a This indicates a molecular weight of 7700 Daltons, which is greater than predicted by SDS-PAGE (5750 Da). Sequence data is represented in Fig. 3.16a, b. A 55 amino acid sequence was obtained including 5 modified amino acids in positions 7, 23, 26, 48 and 50. These modified amino acids are possibly glycosilated, phosporylated or cysteine residues. The first 24 amino acids of the pure peptide (Diii3) correspond to the partial sequence of peptide 1 from fraction Diii, confirming that the most abundant peptide is active. From the sequence data (Fig. 3.16a, b) there are 50 identified amino acids of which 23 are non-polar and 27 are polar. Of the polar residues, 8 are negatively charged (acidic) and 10 are positively charged (basic) at pH 7.0. There appears to be a relatively random distribution of these classes of amino acids throughout the sequence. Amino acid analysis identified 65 residues. The additional amino acids (from Fig 3.16a) are glutamine or glutamic acid (4), cysteine (3), leucine (3), methionine (2), lysine (2), asparagine or aspartic acid (1), alanine (1), isoleucine I l l (1) and valine (1). The total amount of peptide used for amino acid analysis was estimated as 146 pmoles from 300 CC equivalents. From this estimate, 1 CC equivalent is equal to 0.487 pmoles (approximately) and 4.87 pmoles of pure peptide is required for maximum stimulation of ileal 112 Fig.3.16a. Comparison of amino acid and sequence analyses of pure peptide from fraction Diii3. Number of residues Name 1. Amino acid 2. Sequence 1 - 2 analysis analysis B (D+N) 8 7 1 Z(E+Q) 9 5 4 H 1 1 -K 5 3 2 R 4 6 -2 S 5 5 -T 0 0 -Y 2 2 -A 2 1 1 C 3 0 3 F 6 7 -1 G 2 2 -I 4 3 1 L 8 5 3 M 2 0 2 P 2 2 -V 2 1 1 TOTAL 65 50 Fig 3.16b. Partial amino acid sequence of pure peptide from fraction Diii3. 1 10 20 S F F D I Q ? K G V Y D K S I F A R L D 30 40 R l ? E D ? Y N L F R E P Q L H S L R R 50 S D D F K F P ? F ? R G L Q S 113 DISCUSSION Chapter 2 demonstrated that the corpus cardiacum contains stimulants of ileal KC1 transport. Activity is found in both the NCC and GCC with the former having four times the stimulatory activity of the latter. However it is unclear whether activity in the GCC is not due to contamination by NCC products, as discussed in chapter 2. As proposed for CTSH, it is probable that factors influencing ileal transport processes are produced in the brain and transported to the NCC for storage, and from there they are released into the haemolymph. Lechleitner et al. (1989a,b) demonstrated that CC extracts also have an antidiuretic action on the locust ileum, but there was an equal effect of both NCC and GCC on Jv. These results demonstrate that the CC and more specifically the NCC of the desert locust contains two distinct factors (fraction D and F) which differ in their retention times on a RP-HPLC column, both of which stimulate ileal CI- and also fluid reabsorption. A third CC fraction (G) contains a stimulant of ileal Jv, which has little effect on ileal CI" transport. Physiological functions in insects are commonly regulated by more than one peptide. Lipid mobilization during flight is regulated by adipokinetic hormones (AKHI and AKHTI), which apparently have the same function (reviewed by Gade 1990). Partial purification of diuretic peptides has revealed several compounds which differ in size, but which stimulate fluid secretion by isolated Malpighian tubule preparations (reviewed by Mordue and Morgan, 1983; Phillips, 1983). Fluid reabsorption in the rectum of L. migratoria is stimulated by two factors which also differ in size and extraction properties (Herault et ah, 1985) and anion exchange HPLC of NCC identified six fractions of twenty-one which had antidiuretic activity (Fournier and Girardie, 1988) in this same insect. Three of these fractions 114 react with neuroparsin immune serum, and three have properties similar to that of a factor in the GCC described by Herault et al. (1985). Neuroparsin immune serum destroys all fluid transport activity of crude NCC extracts, so Fournier and Girardie (1988) concluded (a) that fractions which do not react with this antibody must be due to contamination of NCC extracts with GCC, and (b) neuroparsin is the only antidiuretic factor in the NCC of L. migratoria. In all cases involving the insect hindgut, it remains to be shown that these factors are normally released into the haemolymph to control hindgut reabsorption in situ. It is unlikely that the major stimulant of ileal salt and fluid transport from 5. gregaria CC is neuroparsin, as their extraction properties differ. Fournier et al. (1987) report that AD activity from the NCC of L. migratoria extracts equally well in saline and 70% methanol, whereas factors in S. gregaria CC affecting ileal I^ remain mainly in the pellet after a methanol extraction (Chapter 2). Sequence data confirms that the peptide in fraction Diii3 and neuroparsin are different compounds (discussed later). PhilUps et al., (1980) partiaUy purified a proteinaceous factor from 5. gregaria CC by gel filtration using a Bio-gel P-30 column and rectal I,,. as a bioassay. This factor, chloride transport stimulating hormone (CTSH) is located in both lobes of the CC, with 80% of the activity in the NCC (Spring and PhiUips, 1980a). There was no apparent difference in the estimated molecular weights of the NCC and GCC factors (8000 Da.) suggesting both lobes contain CTSH (PhiUips et al, 1980) My results demonstrate that the factor in fraction D is the most potent stimulant of rectal I,,., but much greater doses were required to attain maximum stimulation of rectalcompared to Ueal L,. AU other fractions showed Uttle or no 115 effect on the rectum, even at high concentrations (5CC ml'1). In addition, no fraction from the HPLC separation or preparative C4 cartridge had any effect on rectal Jv (data not shown). This lack of stimulation of rectal fluid reabsorption is surprising because Proux et al. (1984) determined that rectal Jv was stimulated 2-fold by crude CC extracts, and the distribution of AD activity in the locust nervous system is similar to that of CTSH activity in that they are both located in the NCC and GCC. In addition, stimulation of rectal fluid transport ceases in CI' free saline, suggesting that AD activity is due to rectal stimulation of active CI" transport. Fraction D is around twenty times less potent than crude CC in stimulating rectal I*., but there is only a four-fold difference when assayed on ileal 1^ . In contrast similar doses of crude CC cause similar increases in IK across both recta and ilea (chapter 2). These results suggest that the purified factor in fraction Diii3 is different from CTSH. Earlier attempts to isolate CTSH by RP-HPLC, using rectal 1^ as the bioassay, also resulted in substantial loss of rectal I^ activity (Meredith, pers. comm.). This loss of rectal Inactivity in CC may be due to the solvent mixture used for elution, as TFA is strongly acidic (pH < 3), and Phillips et al. (1980) report that CTSH is unstable below pH 5. CTSH activity may also be reduced by enzymatic breakdown during homogenization: a peptidase inhibitor may be necessary to prevent this, however, inclusion of such an inhibitor had no significant effect on recovery of factors in CC which stimulate ileal I„. (results not shown). Lability and stability of peptides has in the past been a major problem in their isolation. This is especially true of the diuretic peptides (reviewed by Mordue and Morgan, 1985). As fluid absorption against or in the absence of osmotic differences is 116 invariably driven by solute transport (Spring, 1983), the stimulation of fluid by fraction G may be due to increases in transport of some other ion. Lechleitner (1988) observed that crude CC extracts increase active Na+ reabsorption in locust ileum. It is therefore possible that fraction G may cause increased fluid absorption by stimulating Na+ transport. This can be investigated by measuring "Na* fluxes across ileal flat sheet preparations. In the rectum, Meredith and Phillips (1988) observed a net proline flux under steady-state conditions, which drives active fluid absorption in the absence of luminal Na+, K+ and CI'. There in no net proline flux under unstimulated conditions across the locust ileum (Lechleitner and Phillips, 1989), but if ileal absorption of proline (or other amino acids) was activated by fraction G, this also could account for the stimulation of fluid movement. By varying the osmotic concentration differences across ileal sacs, Lechleitner et al. (1989) showed that CC and VG extracts increased the osmotic permeability of the ileal epithelium at low osmotic concentration differences. This effect of crude CC on osmotic permeability may be due to one of the factors partially purified. This possibility could be explored by stimulating fluid transport across everted ileal sacs at different osmotic concentrations, as described by Lechleitner et al. (1989a). The separation of CC extracts has revealed a number of different factors potentially capable of regulating salt and fluid transport in the locust hindgut. As cAMP and CC extracts cause large increases in active CI' transport, K+ permeability, fluid reabsorption, inhibition of acid secretion in both hindgut segments and active Na+ reabsorption in the ileum (reviewed by Phillips et al., 1988), it is unclear whether one or several different peptides stimulate these different transport processes via cAMP or other second messengers. This question 117 will only be fully resolved by purification of all these factors and determining their range of physiological actions on different processes. The major active peptide from the CC was purified using a four step procedure, utilizing RP-HPLC. Purity of a peptide should be confirmed by at least two different separation methods. After two HPLC separations the active factor was considered "pure" enough to attempt sequencing. This factor eluted as a single peak on HPLC and ran as a single band on SDS-PAGE (albeit with a faint preceding "shadow"). For the purpose of amino-acid sequence analysis, a peptide is considered pure if impurities do not interfere with the correct interpretation of the structural data. For peptides with more than 20 residues, the concentration of impurities must be present in a molar ratio of less than 5% (Allen, 1986). The final test for purity is the observation of a single sequence. Sequence analysis of the factor(s) in fraction Diii (third step) produced two N-terminal and two partial sequences. Peptide 1 was present in greater amounts and therefore presumed to be the active species. To attempt removal of the impurity from this fraction, the the gradient programme, the ion pairing agent (to heptafluorobutyric acid), and the wavelength of detection (to 214 nm; not reported in results) was altered. The active factor eluted as a single peak under all these conditions. Finally, an isocratic programme was used, and by injecting large amounts of peptide onto the column (500 CC equivalents), a large peak (active) with a preceding shoulder which could be separated was observed. From 1200 CC equivalents, amino acid sequence analysis revealed a single peptide which corresponded to peptide 1 from the previous analysis. Fifty residues were determined, and 5 undetermined positions, which are possibly modified amino acids or cysteine residues. A computer search for similarity to a reported protein sequence indicated that this 118 peptide was novel. This confirms the prediction that this peptide is not neuroparsins, however its molecular weight (7700 Da) is similar to that estimated for CTSH (8000 Da, Phillips et al, 1980), and until CTSH is characterized, the relation between the two will remain unclear. As the sequence analysis procedure used can normally only determine up to approximately 50 residues at high sensitivity (Jones, 1986), a complete sequence cannot be obtained by a single analysis. This peptide must now be fragmented, the fragments purified, and subjected to sequence analysis to establish the remaining structure. The predicted weight of this peptide is 7700 from the 65 residues detected by amino acid analysis. By subtraction, 10 - 15 residues were not identified by sequence analysis. These unidentified residues will include the additional amino acids identified by amino acid analysis and listed in the results. The recovery of active peptide from fraction Diii (third step) acting on ileal IK is quite high after two HPLC runs, requiring ICC equivalent ml"1 at this step for maximum stimulation, compared to 0.25CC ml'1 of crude extract. This recovery is even greater when one considers that around 30% of activity is contained in another fraction after the first HPLC step. However, the loss of activity on the final run is far greater, requiring 2.5CC equivalents ml"1 to attain a maximum response. As a maximum ileal IK response is observed, presumably activity is lost due to non-specific binding of the active peptide to the column and vessels rather than its separation from the second peptide (P2) in fraction Diii. In support, one of the fractions 1,2 and 4 from the final separation step presumably contained P2, but these fractions were completely inactive when assayed on ileal L ,^ at high doses. However, it cannot at present be excluded that P2, while itself not a stimulant, might somehow enhance stimulation by ScgLTP. 119 Now that a factor from the CC in the desert locust has been purified, it will be possible to determine its role in the regulation of salt transport in the hindgut. The various physiological effects of this purified peptide on in vitro preparations of locust ilea is the topic of the next chapter. As the major role of the pure peptide is to influence ion transport across the locust ileum, the proposed nomenclature for this peptide is S. gregaria ion transport peptide (ScglTP). 120 CHAPTER FOUR: Actions of Ion Transport Peptide from the corpus cardiacum on ileal ion transport INTRODUCTION Despite our knowledge of the presence and distribution of factors regulating the excretory process, little is known of their biochemical nature and as such, even less is known of the physiological roles of individual peptides. Only three peptides regulating excretion have been fully characterized to date. Two are diuretic peptides (which stimulate fluid secretion by Malpighian tubules) from Acheta domestica CC (Coast et al., 1990) and L. migratoria suboesophageal and thoracic ganglia (Proux et al, 1986; Schooley et al., 1987). The third peptide (neuroparsins B) has antidiuretic activity on the rectum of L. migratoria (Girardie et al., 1989). Using in vivo preparations, Kataoka et al. (1989) have isolated a diuretic peptide from Manduca sexta CC which stimulates fluid excretion when injected into larval M. sexta and newly emerged Pieris rapae. However, whether this factor is causing diuresis by stimulating Malpighian tubule secretion, (which is generally assumed), has not been established. Using crude extracts of neuroendocrine tissues, some of the transport processes in the insect excretory system have been demonstrated to be under the control of natural peptidic stimulants. Fluid secretion by most insect Malpighian tubules is driven by active transport of K+ and CI" ions (Coast, 1969). There is limited information on how diuretic factors increase Malpighian tubule fluid secretion (reviewed by Phillips et al., 1982). In L. migratoria, the tubules continue to secrete a KCl-rich fluid , unaltered in ionic or osmotic composition after 121 stimulation but at a greater rate (Phillips, 1983). Most work on the effects of diuretic factors centre around the second messengers involved in their actions, rather than the specific transport processes which are influenced (reviewed by Raabe, 1989). Fluid reabsorption in the rectum of L. migratoria and S. gregaria is dependent on CI" (Proux et al., 1985; Fournier et al, 1987). As active solute transport is the driving force behind the secondary active transport of fluid, the bioassays (i.e. Jv) used to isolate these peptides do not demonstrate the primary physiological actions of these factors. The transport processes across the locust rectum are now well characterized (reviewed by PhilUps et al., 1986), and those in the ileum have recently been elucidated (reviewed by PhUUps et ah, 1988). The control of these processes by crude extracts of CC and VG have been reported by Spring and PhUUps (1980a, b), Lechleitner et al (1989a, b), and Lechleitner and PhUUps (1989). These studies and those outlined in chapter 2 demonstrate that crude extracts stimulate reabsorption of CI", K+, Na+ and fluid, and inhibit H+ secretion in the Ueum, and regulate KC1 and fluid reabsorption in the rectum. In chapter three the isolation of a novel peptide, named Ion Transport Peptide (TTP), from the CC which stimulates ileal I,,,, was described. Bioassays of FTP on locust recta and ilea suggest that ITP is different from CTSH reported by PhiUips et al, (1980). For example, maximum stimulation of rectal I*, cannot be produced by fraction DiU, which is largely ITP. The question remains as to whether FTP causes the same range of effects on salt and fluid transport in the ileum as does crude CC extracts. By repeating experiments demonstrating the actions of crude CC, results in this chapter demonstrate that ITP does indeed have the same range of functions on ileal transport processes as do crude CC. In chapter 3, the dose-response relationship 122 between the active fraction (Diii) from the penultimate purification, step which contains ITP+peptide 2 (P2), and ileal was described. In this chapter the effects of ITP and ITP+P2 on all the major ileal transport processes are compared. MATERIALS AND METHODS To measure ion transport across locust ilea, the methods described in chapter 2 for CT, K+, and H + were repeated, using similar experimental animals, the same electrical measurements, and the same salines. ITP and ITP+P2 were prepared for assay by drying down aliquots of these fractions by centrifugal evaporation (Speed-Vac,. Emerston Instruments Ltd., Ont.) The polypropylene centrifuge tubes were rinsed with a 0.5% BSA solution. The purified peptide sticks to the walls of vessels and becomes insoluble if dried without BSA. Sodium flux measurements 22Na+ fluxes across ilea were determined by mounting the tissue as flat sheets in Ussing type chambers containing physiological saline, which was stirred by vigorously bubbling with 95% 0 2 : 5% C02 at 26°C - 28°C (TTP+P2) or at 20° C (ITP). The tissue was voltage-clamped at zero and allowed to come to steady-state under short-circuit current conditions and L^ . was monitered continuously throughout the experiment. N^a* was purchased from New England Corporation as NaCl. Aliquots of stock solution were added to the lumen (for forward fluxes; L -H) or haemocoel (back fluxes; H - L) sides of the tissue, thirty minutes before the experimental period. A 50u,l sample was taken from the "hot" side (in duplicate) and added to 500ul of cold saline in polypropylene scintillation vials. Flux of N^a* across ilea was determined at 15 min intervals, 2 h before and after 123 stimulation with ICC equiv.ml'1 of JTP+P2 or 5 CC equiv.ml'1 of LTP added on the haemolymph side of the tissue. Samples of saline (0.5 ml) were taken from the "cold" side of the chambers every 15 min for determination of increase in radioactivity and replaced with 0.5ml of cold saline. If taken from the haemolymph side after stimulation, additional extract was added back to keep the concentration the same throughout the experiment. Samples (0.5ml) were counted with an automatic well-type gamma counter (Nuclear Chicago Model 1058). Unidirectional flux was calculated using the following formula (Williams et al, 1978). Ji.2 = asVC/a/TA Where Ji . 2 is the unidirectional flux ( equiv.cm'^ h"1.), aj is the radioactivity of the "hot" side (cpm.ml1) ^ is the increase in radioactivity of the "cold" side (cpm.ml"1) C is the concentration of the unlabelled Na+ in solution (mM) V is the volume of solution in the chambers (2ml) A is the tissue area (0.196 cm2) T is the time interval between samples (h). Statistical treatment Differences between treatments were considered significantly different when Student's T-test indicated a P value less than 0.05. 124 RESULTS Dose-response relationship and time course of ileal lK with ITP The time course of ileal I,,, response to LTP is similar at the four concentrations tested (Fig.4.1), rising to a maximum for each dose between 0.5 and 1.0 h, and remaining at this level for the next hour. This differs from the time course of response to crude extracts (chapter 2). As reported in chapter 2, time courses of ileal I*, response to CC homogenates varied with dosage so that the dose-response curve of maximum 1^. was atypical and biphasic. However, a typical dose-response relationship was observed when the I«. after 1 h of stimulation was used. The latter procedure was used to determine the dose-response relationship of ITP on ileal I«, (Fig.4.2). A linear dose-response relationship over the range of 1.0 to 5.0 CC equivalents.ml'1 was observed, which corresponds to 0.48 to 2.4 pmoles.ml1 of pure peptide as determined from the amino acid analysis data (chapter 3). At 5 CC equiv.ml1 of ITP, the increase in I*. response of 9.96±0.66 uequiv.cm-.h-1 agrees well with the maximum response of 10.32±1.29 caused by 0.25 crude CC ml1 (chapter 2, Fig 2.5) or of 11.2±1.09 uequiv.cm--.h-1 produced by 1.0 CC equiv.ml-1 of ITP+P2 (chapter 3, Fig. 3.10) There is therefore a twenty-fold difference in the concentrations of crude and pure extracts required for a maximum response, and most of this activity is lost during the final purification step. Effect of ITP+P2 and ITP on ileal IK. The effects of ITP+P2 and ITP on increases in ileal IK, are shown in Fig. 4.3. In the presence of control saline (i.e. with CI" present) I„. fell to a steady level of 0.8210.24 uequiv.cm-.h1 after one hour. There was only a slight change 125 12i ° 0 15 30 45 60 75 90 105 120 TIME (min) Fig. 4.1. Time course of ileal I« response to various doses of ITP added to the haemocoel side (Mean ± SE, n = 4-8): O, 1 CC; • , 2 CC; • , 2.5 CC; • , 5 CC equiv.ml1 of ITP. 126 Fig. 4.2. Increase in I« one hour after adding various doses of ITP to the haemolymph side of ilea (Mean ± SE, n = 4-8). 127 in residual I,,. when all CI" was replaced bilaterally by gluconate (0.35 nequiv.cm" 2.h"1), and when IK was then initiated by raising haemocoel K+ to 80 mM (1.67±0.22 l^equiv.cm .^h"1). Subsequently 1.0 CC equiv.ml1 of ITP + P2 or 5.0 CC equiv.ml"1 of ITP cause significant several-fold increases in potassium diffusion current to 5.41±0.32 and 4.6±0.93 uequiv.cm .^h'1 respectively, which is qualitatively similar to that caused by crude CC stimulation (Chapter 2). Addition of lOmM Ba2+ completely abolished the IK due to ITP when added to the haemolymph side of the tissue, in a similar manner to that previously described for cAMP-induced IKin both ileum and rectum (Irvine et ai, 1988; Hanrahan et ai, 1986). In contrast to its effects on ileal IK, ITP+P2 had no significant effect on rectal IK, whereas crude CC caused an increase in IK of 3.58 (lequiv-cm .^h1 in the rectum (chapter 2). This provides further evidence that the factor in crude CC (CTSH) which stimulates rectal salt transport is not the same as ITP which stimulates ileal salt transport. Effect of ITP+P2 and ITP on ileal Na+ reabsorption The effect of ITP + P2 on unidirectional and net Na+ fluxes is shown in Fig. 4.4a, b. Short-circuit current values in Fig 4.4c indicate an anion (CI) movement from the lumen to the haemolymph side of the tissue of 1.12±0.05 ixequiv.cm^ .h"1 (unstimulated) which increased to 11.4910.09 on the addition of ITP+P2. Before stimulation there is a mean back flux of 2.6110.14, a forward flux of 4.5010.13 and a net flux of 1.8910.22 nequiv.cm-Mi"1 of Na+ to the haemolymph side over the first two hours. After the addition of ITP+P2 the back flux falls slightly to 2.1810.7 and the forward flux increases significantly to 128 6 43 I o CO o o CO 1 [K+] haemocoehlumen (mM) 10:10 10:10 80:10 80:10 80:10 80:10 [CI-] bilateral (mM) 110 - - - - -Stimulant - - - ITP+P2 ITP ITP 5mM B a 2 + (haemocoel) _ _ _ - - + Fig. 4.3. Effect of LTP+P2 (ICC equiv.ml1) and ITP (5CC equiv.ml'1) on current (Mean ± SE, n = 6-12) required to clamp ileal Vt at OmV under different sequential external conditions. The bar starting on the left represents normal unstimulated L^ , which was slightly reduced by bilateral replacement of all CI" (second bar). Imposition of a haemocoel-to-lumen K+ gradient has little effect on causing a potassium diffusion current (IK, third bar), until stimulants were added (fourth and fifth bar). The sixth bar shows the effect of adding barium to the haemocoel. Means which are significantly difference from the control value are represented by * (P < 0.05). 129 Fig. 4,4. A) The time course of unidirectional ^ Na* fluxes across short-circuited ilea under control and ITP+P2 stimulated conditions. Time zero is 90 min after dissection. (L-H represents the forward flux from lumen to haemolymph side, and H-L the back flux of haemolymph to lumen side). Control preparations (solid circles, solid squares) were bathed in normal saline throughout and ICC equiv.ml-1 of ITP+P2 was added to the haemolymph side of experimental preparations (open circles, open squares) two hours after the start of the experiment. B) The net flux to the haemolymph side was calculated by subtraction from unidirectional fluxes in (a). The differences are significantly different (P < 0.05). C) Represents the short-circuit current across the tissue during flux measurements. (Means ± SE, n = 4-8). 130 A) 12i 9 6 3 I B o .1-1 cr 3 + cd !z; CV2 CN2 0 -3 -6 ITP+P2 T T / B) 8i 6^ 4 2H 0 -2 O o. ITP+P2 / o x / o o \ I 1 0 30 60 90 120 150 180 210 240~ TIME (min) 131 I A CV2* I s o > • H cr1 a> o CO c) 14 12 10 8 64 44 24 A T I I 1 I 1 ^o-o-o-o-o-o-o ° 1 1 1 1 1 I ITP+P2 -2 0 30 60 90 120 150 180 210 240 TIME (min) 132 8.08±0.24 e^quiv.cm'2.*!"1, resulting in a significant increase (P < 0.05) in net flux to 5.65±0.26 iiequiv.cnr2.!!'1 to the haemolymph. This increase in net Na+ flux caused by ITP+P2 is equivalent to the maximum attained with crude CC (Lechleitner et al, 1988) or cAMP (Irvine et al, 1988). . ITP action on the active forward flux of "Na* is shown in Fig. 4.5. Before stimulation there is a mean forward flux of 1.25±0.28 l^equiv.cmlh1 to the haemolymph side over the first 2 h. This flux falls slightly over the third and fourth hour to 0.9310.42 iiequiv.cm'Mi'1 (control) and increases to 3.5510.19 (lequiv.cnrMr1 upon the addition of 5 CC equiv.ml1 FTP over this time period. The differences in the control flux measurements and stimulated values between these two sets of experiments (Fig. 4.4 and 4.5) is probably due to the temperature differences during the experiments (27°C versus 20°C) Regardless, ITP causes a large increase in active Na+ influx, similar to that caused by crude CC (Lechleitner, 1988). Effect of ITP+P2 and ITP on ileal acid secretion Table 4.1a shows the effect of ITP+P2 on ileal acid secretion. The rate of acid secretion to the lumen is 1.4 |iequiv.cm"2.h_1. under control conditions. Addition of ITP+P2 significantly (P < 0.005) reduced lumen acidification by 64 and 84% at doses of 1.0 and 2.0 CC equiv.ml1 respectively. The effect of ITP on acid secretion is shown in Table 4.1b. Under control conditions the rate of acid secretion to the lumen is 0.93 |j.equiv.cm'2.h_1 and this is significantly (P < 0.005) reduced by 75% when 5 CC equiv.ml-1 ITP is present. Both ITP+P2 and ITP reduce acid secretion to the same extent as crude extracts of CC (chapter 2). 133 6-1 43 cV I a o & CD 0 + CQ 4-0 1 1 i , / l \ T T 30 60 90 120 150 180 210 240 TIME (min) Fig. 4.5. The time course of umdirectional forward (lumen to haemolymph) N^a" fluxes across short-circuited ilea under control (solid circles) and ITP stimulated (open circles) conditions. Time zero is 90 min after dissection. Control preparations were bathed in normal saline throughout and 5 CC equiv.ml-1 ITP was added to the haemolymph side of experimental preparations two hours after the start of the experiment. The increase caused by ITP is significant (P < 0.05; mean ± SE, n = 4-8). 134 Table 4.1a. Effect of ITP+P2 on ileal acid secretion (JH+) under open circuit conditions (Mean ± SE, n = 4-6) TREATMENT JH+ V t (mV) (p-equiv.cm'2.!!1) Control (no treatment) 1.40±0.08 1 CC equiv.ml1 ITP+P2 0.6310.11** 2 CC equiv.ml-1 ITP+P2 0.22±0.09** ** significant difference from control, P < 0.005. Table 4.1b. Effect of ITP on ileal acid secretion (JH+) under open circuit conditions (Mean ± SE, n = 4-6) TREATMENT JH+ V, (mV) (Hequiv.cnrMi-1) Control (no treatment) 0.93±0.021 5.25±1.31 ITP (5 CC equiv.ml1) 0.23310.043** 10.011.96* 3.50±0.96 11.7511.10** 18.2513.64** Significant difference from control, * (P < 0.05) and ** (P < 0.005) Measured over the second hour after mounting the tissue. 135 DISCUSSION In this chapter the physiological actions of ITP, a factor isolated from the CC of the desert locust, on ileal salt transport are described. This factor qualitatively has the same effects on all the transport processes in the ileum regulated by crude extracts (chapter 2, Lechleitner et al., 1989a, b). While higher doses of ITP are required, the same maximum stimulation of all these processes is achieved as with crude CC. ITP does not maximally stimulate rectal 1^ (chapter three), and has no effect on rectal fluid (chapter 3) or potassium reabsorption (this chapter). The small effects of ITP on rectal I,,. may therefore be pharmacological rather than a normal physiological response or ITP may augment actions of CTSH on the rectum. Presumably CTSH activity is destroyed during one of the purification steps. A comparison between the effects of the active fraction from the penultimate purification step, which contains ITP+P2, and ITP shows that the stimulation of ileal salt transport is due to ITP. Both these fractions cause the same maximum increase in ileal I*., IK and Na+ reabsorption and inhibition of H+ secretion. These two fractions have the same range of effects on ileal transport processes. In addition, the impurity (P2) removed by the final purification has no effect on ileal I« at a concentration of 5 CC equivalents.ml1, as reported in chapter 3. The time course of ileal I„. response to low doses of ITP is different to that observed with crude CC extracts. The reason is unknown, but crude CC does contain a number of active factors, including ITP, which stimulate ileal I,,.. There may also be factors which act to inhibit ileal response to ITP or modify receptor affinity for ITP, which could explain the discrepancy. 136 The differences in control values for Na+ absorption and acid secretion are probably due to the effects of different room temperatures recorded and variations in the response of different populations of locusts at different times of the year. Such differences have been commonly noted in our laboratory. It is to be expected that the greater the purity of a peptide, the greater will be the relative losses due to non-specific binding to vessels. This is the most obvious explanation for the five-fold loss of total activity in the last purification step. This loss can be reduced by adding BSA to vessels used for concentrating the active factor and to the Ussing chambers. Experience has proved that without BSA, the peptide becomes insoluble if dried and sticks to polypropylene vials. This has since been found to be a common problem with other insect peptides (Coast, Schooley, pers.comm.). ScglTP probably acts via cAMP to regulate reabsorption of CI", K+ and Na+, as these processes are also stimulated by cAMP (reviewed by Irvine et al., 1988, Phillips et al., 1988). Moreover, in some preliminary experiments, ITP was observed to increase intracellular cAMP after one hour of exposure (work in progress). However cAMP levels must be measured during the initial rise in ileal IK to confirm these preliminary results. As H + secretion is inhibited by both crude CC extracts and pure ITP, but not cAMP, ITP must act via a different intracellular messenger to mediate this specific response. Intracellular levels of Ca2*, cGMP, arachidonic acid, and inositol phosphates will have to be measured before and after stimulation with ITP to determine their possible roles as second messengers controlling ileal transport functions. Fournier (1990) has provided some evidence that phosphoinositols act as the second messengers during neuroparsin B stimulation of fluid reabsorption in the rectum of L. migratoria. 137 If several reabsorptive processes in the ileum can all be stimulated by cAMP, it is to be expected that a factor (eg. ITP) which elevates intracellular cAMP concentration is also going to regulate those same processes. Since other stimulants of ileal transport were detected in the CC (chapter 2), the action of ITP may be enhanced and modified by these agents, which could act through other second messengers. The ileum consists of a single layer of epithelial cells of apparently one cell type (Irvine et al., 1988), so all regulatory factors presumably must act on several processes in these cells. This suggests therefore that different factors may act via different receptors and possibly different second messengers to assert their effects on all reabsorptive processes within this one cell type, rather than on different processes restricted to different cells. The same may also be true of factors acting on rectal transport. It still remains to be shown that ITP and other CC factors are normally released into the haemolymph to influence ileal transport activity in situ. CTSH and other factors in the CC and VG must also be isolated to determine their physiological functions and how these factors interact to regulate excretion. Some of the possibilities are discussed in the next chapter. 138 CHAPTER FIVE: General discussion The goal of this thesis was to identify and isolate natural factors from the central nervous system which may normally regulate ion and fluid transport across the hindgut of Schistocerca gregaria. This thesis, and a previous study by Lechleitner (1988), determined that factors in crude extracts of CC and VG, which have different physical and chemical properties, have the same broad range of effects on the locust ileum as does cAMP. Cyclic AMP, CC and VG all increase K\ Na+, CI" and water reabsorption, and CC and VG inhibit H+ secretion. Likewise cAMP, CC, and VG were all found to increase KC1 and fluid reabsorption in the rectum (reviewed by Phillips et al, 1986; Lechleitner and Phillips, 1989; chapter 2) Partial purification of aqueous extracts of CC (chapter 3) revealed two distinct factors which stimulate CI- transport in the ileum. A third factor has little effect on ileal CI" transport, but causes a significant increase in fluid movement, presumably by first stimulating the transport of another ion (Na+?) or amino acids. The specific solute involved in this transport has yet to be identified. ScglTP, the factor in the CC which has the greatest stimulatory effect on ileal ^ has been isolated and characterized. LTP has a molecular weight of 7700 Da by amino acid analysis and a partial sequence was determined (chapter 3). The specific major actions of LTP on the ileum were determined by repeating those experiments previously carried out using crude extracts. Its actions include all those influenced by crude CC in the ileum (table 5.1), in that it stimulates the active absorption of CI", Na\ passive absorption of K+ and inhibits active acid secretion (chapter 4). Hanrahan and Phillips (1984a) developed a model for the 139 Table 5.1. A comparison between the stimulation of transport processes in the locust ileum by crude CC (0.25 CC equivalents ml1) and pure ScglTP (5 CC equivalents ml'1). PARAMETERS (liequiv.cm .^h'1) CONTROL CRUDE CC ScglTP 1^ (CI" absorption) -0.87+0.63 10.4±0.53 11.211.09 K + absorption 1.1010.10 9.03±1.30 4.6010.93 Na+ absorption * 3.60±0.10 5.70+0.11 Na+ absorption 1.67±0.22 3.5510.19 H + secretion * 1.40+0.08 0.65±0.09 H + secretion 0.92±0.83 0.2310.13 All experiments were conducted at 2212°C except * (26-28°C) 140 transport processes in the locust rectum and identified specific sites of control by CTSH acting through cAMP. They proposed that CTSH elevates intracellular cAMP to stimulate active CI' entry, apical membrane K+ conductance and basolateral CI' conductance. While intracellular recording of ion activities has not yet been done on locust ilea, all other observations on this segment are consistent with this rectal model with regard to transport mechanisms and their location and control (Irvine et al, 1988). A a similar model for the regulation of ion transport across the locust ileum is proposed (Fig. 5.1). ScglTP may also act via cAMP to stimulate active CI", Na\ and passive K+ absorption, because these processes are also stimulated by cAMP in a similar manner (Irvine et al, 1988). In support of this view, forskolin (10-50 |iM), which stimulates adenylate cyclase, and the phosphodiesterase inhibitor, theophylline (5 mM), also stimulate Cl'-dependent I„. (chapter 2). Intracellular levels of cAMP must be monitored in parallel with the rise in ileal I*. to confirm this prediction. Preliminary observations confirm that intracellular cAMP does increase one hour after adding ITP (work in progress). Two notable differences between the actions of cAMP, crude tissue extracts and ITP on ileal transport were observed: (1) cAMP had no significant effect on H + secretion, but the latter could be inhibited by ITP, CC and VG; and (2) ammonia secretion is stimulated by cAMP, but not by ITP, crude CC nor VG. This suggests firstly that ITP and possibly other factors in the CC and VG which inhibit acid secretion must act via second messengers other than cAMP, and secondly a separate control process for ammonia secretion exists (possibly neural). In the mammalian salivary gland, adrenaline acts through a- and B-adrenergic receptors to increase intracellular levels of Ca2+ and cAMP respectively, leading to 141 LUMEN CELL HAEMOLYMPH Fig. 5.1. Proposed model for control of ion transport across the locust ileum. I propose that ScglTP acts via cAMP to stimulate CI" , Na+, and K+ entry at the apical membrane and basolateral membrane CI" conductance. Active secretion of H+ is also controlled by ScglTP via an unknown second messenger pathway. 142 fluid and amylase secretion by different second messengers (Eckert et al., 1988). It is therefore feasible that LTP binds to two different receptors, one to increase cAMP (thereby promoting reabsorption of salt and water) and the second to increase an unidentified second messenger which inhibits H + secretion. Cyclic GMP has been shown to stimulate ileal IK (chapter 2), and the effects of inositol trisphosphate and diacylglycerol can be mimicked with pharmacological agents (Ca2+ ionophores, derivatives of diacylglycerol or phorbol esters). The effects of these agents on H+ secretion must be studied and intracellular levels determined to confirm which of these second messengers are involved. If cAMP stimulation of ammonia secretion is a physiological rather than pharmacological effect, it is surprising that neither CC nor VG5 extracts have any effect on this process, especially if they are acting via this second messenger to promote reabsorption of salt and water. A factor acting specifically on may be present in a gland other than the CC or VG5, so the whole CNS must be surveyed to identify possible sources of activity. If the regulation of ammonia secretion is neural, known or putative neurotransmitters could be tested for their effects on J^. It is still uncertain whether LTP or other factors from the CNS are normally released to control ileal function in situ. Nethertheless, it is a common occurrence for physiological processes in insects to be regulated by more than one neuropeptide. Adipokinetic hormones (AKH) have been isolated in a number of insects in two forms, whose effects are qualitatively but not quantitatively the same (reviewed by Gade, 1990). It has been suggested that the control of diuresis in L. migratoria Malpighian tubules is due to two peptides which act at different 143 receptors via different second messengers (Morgan and Mordue, 1985). Likewise, two antidiuretic factors capable of acting on reabsorption of fluid in the rectum of this same insect were reported by Herault et al. (1985), although again control in situ remains to be investigated. In vertebrates, renal salt and water reabsorption is regulated by at least three different hormones, antidiuretic hormone (arginine vasopressin), aldosterone, and atrial natriuretic peptides (Ganong, 1989). These presumably permit integration of relevant information from several sources. In starved dehydrated locusts, an alkaline fluid (pH 7.8) rich in bicarbonate is absorbed in the ileum (Lechleitner, 1988). This may serve to counteract the acidosis associated with periods of starvation between meals . Using in vitro preparations it was observed that the ileum also secretes FT ions into the lumen under control conditions (i.e. when no natural stimulant is present; Thomson et al., 1990; chapter 2). When starved, the haemolymph volume is low and rich in NaCl but not K+ (PhilUps et al., 1982). Most plant material is K+-rich, low in NaCl and of near neutral pH. On feeding, haemolymph volume and K+ concentrations increase as ions and water are absorbed across the midgut wall from this plant material. This leads to a relative shortage of CI' which is further compounded by increased secretion of KCl-rich fluid in the Malpighian tubules due to diuretic factors released after feeding (Gee, 1977; PhUUps et al, 1982). The insect thus faces the problem of maintaining NaCl levels in the blood whilst excreting other harmful substances. Increased recycling of fluid through the excretory system is beUeved to enhance accumulation of waste products from the meal in the hindgut (PhUUps et al, 1982). Spring and PhiUips, (1980c) present evidence that CTSH is released into the haemolymph to increase rectal reabsorption, when starved insects are fed. I 144 hypothesize that ITP is also released at this time to enhance ileal transport processes. This must be confirmed by measuring the presence of ITP in the haemolymph after feeding. Lechleitner (1988) showed that after stimulation, the ileum switches from absorbing an alkaline fluid to one which more closely resembles ion ratios (Na+, K+, CI" and HCGy) and pH (7.1) in the haemolymph, helping to increase haemolymph volume without changing pH. The pH of the faeces of starved insects is acidic (4.7), and becomes more alkaline (6.2) and moist on feeding (Harrison pers. comm.). These observations are consistent with the hypothesis on ITP function in situ. In summary, I propose that CTSH and ITP are both released into the haemolymph immediately on feeding to begin rapid reabsorption of ions and fluid in the hindgut to balance the increased secretion of the primary urine by the Malpighian tubules. In addition, ITP inhibits H+ secretion and concomitant HCCy reabsorption in the ileum, as there is no longer a need to counteract the acidosis associated with starvation. I also propose that other factors in the CC and VG are also released to modulate the ITP response. The factor in fraction G (G-factor; chapter 3) may act via Na+ transport to change the Na+:K+ ratio of the absorbate while simultaneously increasing Jv. The ratio of ITP to G-factor may be adjusted according to ion ratios in the food source to achieve the correct K+:Na+:Cl" ratio in the haemolymph. As the time course of ileal response to VG is rapid and short-lasting compared to the prolonged CC response, possibly this factor(s) is released immediately to regulate the initial diuresis. The need to conserve water as feeding progresses is reduced, as haemolymph volume is restored. Excess fluid must be eliminated, and moist faecal pellets are observed a short time after the ingestion of a meal (Bernays and Chapman, 1974; Loveridge, 1975; Mordue, 1972). The effects of ITP may persist to maintain homeostasis 145 after the need of VG stimulation has ceased. Factor(s) in the CC and VG may be broken down by receptor-mediated endocytosis or by enzymes released into the haemolymph as observed with diuretic factors acting on the Malpighian tubules. In R. prolixus (Maddrell, 1964b), Dysdercus (Berridge, 1966), Carausius (Pilcher, 1970a) and G. austeni (Gee, 1975a, b), diuretic activity is destroyed by the Malpighian tubules, and in G. austeni, an enzyme which is presumed to be released into the haemolymph with diuretic factors, also destroys diuretic activity (Maddrell and Gee, 1974; Gee, 1975a, b). Our knowledge of events in the whole animal is very limited and virtually nothing is known of the release of peptides controlling excretion. In only a few cases to date have neuropeptide factors capable of influencing excretory epithelia been shown to act as natural hormones in vivo. For this reason Wheeler and Coast (1990) suggest that until such a status is confirmed these "hormones" should be referred to as "factors" or "peptides". One criterion to characterize a factor as a hormone is its release and transport into the circulatory system of an animal at the time that target organ activity is modified in situ. The release of diuretic factors into the haemolymph after feeding has been demonstrated in several species (Nijhout and Carrow, 1978; Pilcher, 1970a, b; Maddrell, 1963; Berridge, 1966; Maddrell and Gardiner, 1976). Spring and Phillips (1980c) observed that haemolymph of fed locusts could stimulate rectal !«, and the factor in the haemolymph had the same retention time on a Sephadex G75 column as the active factor in crude CC (Phillips et al., 1980). Cardiatectomy reduced haemolymph CTSH activity indicating the CC was the source of 146 haemolymph activity. This indicates CTSH is released into the blood after feeding, however, CTSH must be first isolated and antibodies raised to detect its presence in the haemolymph and changes after feeding. Likewise, the presence of LTP in the haemolymph has still to be shown, to confirm that it is indeed a hormone. Haemolymph levels of LTP will provide information on its release and open the possibility of studying the sensory pathways involved. Release of neurohormones from neurohaemal sites (eg. CC) is probably initiated by nerve impulses in the neurosecretory axons. In Rhodnius prolixus this depolarization can be initiated by high K+ solutions in the presence of Ca2+ (Maddrell and Gee, 1974). Electrical stimulation of NCCI (the nerve from the pars intercerebralis of brain to the storage lobe of the CC), and high K+ treatment both initiate release of a diuretic factor from the CC in L. migratoria (Orchard et ah, 1981). Similar treatments of CC and cardiatectomy in S. gregaria coupled with detection of LTP in the haemolymph with antibodies would confirm its role as a natural hormone and identify the site of release. In R. prolixus, stretcMng of the abdominal wall triggers hormone release (Maddrell, 1974), and it has been suggested that stretching of the foregut causes the release of hormones in insects, such as S. gregaria, that feed more or less continuously (Berridge, 1966; Mordue, 1969). Presumably there is a direct relationship between the amount of food consumed, the amount of hormone released and the quantity of fluid secreted by the Malpighian tubules (Gillott 1980). Such a simple mechanism may work for R. prolixus, whose food has a constant water content and ionic composition, but the physical stimulus of stretching alone is not a precise enough mechanism for water regulation in insects whose food varies in water and ionic content. It is also difficult to envisage such a simple 147 arrangement for the regulation of release of all the different factors which have been shown to regulate fluid secretion by the Malpighian tubules and reabsorption of fluid and salt in the hindgut. Some other sensory control system must operate. The pars intercerebralis-corpus cardiacum-allatum system of insects has been compared to the hypothalamus-neurohypophysial system of vertebrates (Scharrer and Scharrer, 1954; Gee, 1977; Phillips, 1982). Release of arginine vasopressin results from changes in blood volume and osmolality. Osmoreceptors in the hypothalamus detect the osmolality of the extracellular fluid which stimulates the release of AVP from the neurohypophysis (Verney, 1947). Stretch receptors in the left atrium detect blood volumes, resulting in increased or a reduction in AVP release, controlling urine output. Insects may be able to monitor the water and ionic content of their food directly. Alternatively, the nervous system may monitor changes in the ionic and osmotic concentration of the haemolymph in order to regulate the reabsorption of fluid and ions in the hindgut. Phillips (1964b) showed that the rate of Na+ and K+ reabsorption in the rectum is correlated with the ionic content of the haemolymph. In P. americana, the proventriculus, which regulates food movement, has been shown to respond to osmotic pressure of the ingested fluid (Davey and Treherne, 1963). They suggested that the osmotic concentration of ingested fluid is monitored by osmoreceptors in the pharynx, and this information is relayed to the ingluvial ganglia (which controls the proventriculus) via the frontal ganglia. There is no evidence concerning the stimuli which control the release of diuretic and antidiuretic factors in S. gregaria. Peptide hormones belong to "families" whose members have similar amino acid sequences and/or common precursors (Goldsworthy and Wheeler, 1985). One 148 family of invertebrate peptides is the adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) family, first reported by Stone et al. (1976). These authors found that locust AKH had an amino acid sequence similar to that of the previously characterized RPCH of prawns (Fernhund and Joseffson, 1972). There are now fourteen peptides of this family sequenced, one of the largest families known (reviewed by Gade, 1990). More sequence data for peptides regulating insect excretory processes will ultimately determine whether a similar family exists in this group. To date, diuretic peptides appear to be very diverse (reviewed by Wheeler and Coast, 1990). There are reports of arginine vasopressin-like (Proux et al., 1987), and adrenocorticotropin-like (Rafaeli et al, 1986) factors, based on the observation that extracts of CC react with antibodies raised to these vertebrate hormones. The sequence of the AVP-like diuretic peptide from SOG and TG of L. migratoria (Proux et al, 1987) bears considerable homology to arginine vasopressin. However, this peptide is unlike diuretic peptides isolated by Morgan et al, (1987), Coast et al (1990) or Kataoka et al (1989), from the CC of L. migratoria, A. domesticus and M. sexta respectively (Fig. 5.2). There is no similarity between the structure or size of neuroparsins B (Girardie et al, 1989) and ScglTP (Fig. 5.3). NpB is a homodimer, and its monomer has a MW of 8166 Da. Whether these two peptides have a role in regulating diuresis in vivo remains to be determined. Once synthetic peptides and antibodies are available, it will be possible to carry out reciprocal bioassays and show whether these factors have a common function in the control of excretion in insects. ITP, or other stimulants which I partially purified from the CC, are also 149 Fig. 5.2a. Comparison of the amino acid sequences of diuretic peptides from L. migratoria, A. domesticus, and M. sexta. L. migratoria DP-1 ? G ? G I Q A ? V/M Y K L. migratoria C L I T N C P R G NH2 AVP-like C L I T N C P R G NH2 A. domesticus AP-1 p E R D I F H A Q T D I F Q V P K OH M. sexta R M P S L S I D L P M S V L R Q L S L E K E R K V H A L R A A A N R N F L N D I NH2 Fig. 5.2b. Comparison of the amino acid sequences of neuroparsins B and ScglTP. Neuroparsins B S C E G A N C V V D L TR. C E Y G D V T D L. migratoria F F G R K V C A K G P G D L C G G P Y E L H G K C G V G M D C R C G L C S G C S L H N L Q C F F F E G G L P S S C 5. gregaria-TTP S F F D I Q 7 K G V Y D K S I F A R L D R I 7 E D 7 Y N L F R E P Q L H S L R R S D D F K F P ? F ? R G L Q S 150 unlikely to be CTSH, which was partially purified by Phillips et al. (1980), as they have a reduced effect on rectal I^ , and no significant effect on rectal potassium and fluid reabsorption. In addition, their extraction properties differ (chapter 2), and the purified factor (TTP) cannot mimic maximum rectal stimulation produced by crude extracts, even at high doses (chapter 4). If CTSH activity is destroyed during HPLC separation, different methods must be used for its purification using stimulation of rectal I*. as the bioassay. As peptides are lipid insoluble, they cannot penetrate membranes and therefore must bind to surface receptors to initiate a cellular response. There are two aspects of the interaction of peptides with target cells (Hruby, 1981). The hormone must first bind to the receptor and then activate the receptor complex to initiate a cellular response. The binding and activation steps can involve separate or overlapping portions of the amino acid sequence. A change in a peptide hormone that alters its conformation will prevent it from binding to its receptor and prevent its action. Neuroendocrine manipulation has been suggested as a means for insect pest control, and the general endocrine process (synthesis-secretion-transport-action-degradation) has numerous steps which would be sensitive to disruption. Disruption of hormone-receptor interactions could prove a useful means of insect pest control, by suppressing key physiological responses (Keeley and Hayes, 1987). Isolation and characterization of peptide hormones is a prerequisite for developing pest management programmes by this method in order to provide necessary information for structure/activity studies. A number of such investigations have been carried out with the AKH/RPCH family (reviewed by Gade, 1990) and the myotropic peptides (Holman et al., 1987). They have been 151 able to determine, by amino acid substitution, which amino acids are necessary for activity by synthesizing analogues to the native peptides. Such analogues to diuretics and/or antidiuretic peptides in insects could result in the excretion of copious quantities of salt and fluid resulting in dehydration of the insect, and subsequent debilitation or death. AKH and myoactive peptides are relatively short, (less than 10 amino acids) so synthetic peptides are relatively easy and cheap to produce. With the larger diuretic and antidiuretic peptides, it may be necessary to first cleave the peptide, isolate the fragments, and determine which section of the peptide is necessary for activity. The active portion can then be synthesized and amino acid substitutions made to determine its active and /or binding sites. This study and that conducted by Lechleitner (1988) has identified a number of factors capable of regulating the excretory process. Such peptides could form the basis of insect control studies even if they are not normally released into the haemolymph, because they clearly are potent activators of hindgut receptors. In conclusion, I have isolated one of these factors, ITP, a novel peptide, which influences salt and water transport across in vitro preparations of locust ilea. 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Purification of a neuropeptide from the corpus cardiacum of the desert locust which influences ileal transport
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