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A pharmacological study of signal transduction mechanisms controlling fluid reabsorption and Ion transport… Jeffs, Lloyd B. 1993

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A PHARMACOLOGICAL STUDY OF SIGNAL TRANSDUCTION MECHANISMS CONTROLLING FLUID REABSORPTION AND ION TRANSPORT IN THE LOCUST RECTUM. by LLOYD BRIAN JEFFS B.Sc., The University of British Columbia, 1990. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ZOOLOGY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA May 1993 © Lloyd Brian Jeffs, 1993  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.  (Signature)  Department of The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  3) s-r riA\( 1(393  II  ABSTRACT  Like most insects, locusts face severe regulatory challenges associated with arid habitats, high metabolic rates and high surface area to volume ratios. Therefore, the conservation of water, essential ions and metabolites is very important. Consequently, locusts regulate their hemolymph composition primarily by controlling epithelial transport in the excretory system. The locust excretory system is typical of many insects and consists of the Malpighian tubules and the hindgut. The Malpighian tubules secrete a primary isosmotic urine rich in KC1 and low in Na+ that contains most small hemolymph solutes, waste products and toxic plant chemicals. The hindgut (ileum and rectum) is responsible for the enormous changes in composition of the urine, by the selective reabsorption of water, ions and metabolites. Both the Malpighian tubules and the hindgut are under endocrine control. The purpose of this study was to investigate the involvement of second messengers in the control of fluid reabsorption (iv) and Cl- transport in the rectum of the desert locust, Schistocerca gregaria.  Various agents known to block or activate specific signal  transduction pathways were added to everted rectal sacs and short-circuited rectal flatsheet bioassays. Cyclic AMP and its analogs were shown to stimulate rectal Jv and Cltransport to the same extent as aqueous extracts of the nervous lobes of the corpora cardiaca, suggesting that activation of the adenylate cyclase pathway is sufficient for maximal stimulation. It also appears that cGMP is involved, since its addition partially stimulated both Cl- and fluid reabsorption. External Ca2+ was not required for the maintenance or stimulation of rectal transport. However, intracellular Ca2+ was shown to influence the control of rectal transport. The role of intracellular Ca2+ appears to be quite complex and may vary with its relative concentration. Finally, it was found that Protein  iii  kinase C and the phosphotidylinositol cycle do not appear to be involved in the stimulation of rectal Cl- and fluid transport.  2"  iv TABLE OF CONTENTS  Page Abstract ^ Table of Contents ^ List of Tables ^ List of Figures ^ List of Abbreviations ^ Acknowledgments ^ CHAPTER 1:^General Introduction ^  iv vi vii ix xiii 1  Structure of the locust excretory system ^ 1 Mechanisms of solute and water transport in the locust rectum ^ 5 Solute and water transport in the ileum ^ 9 Endocrine control of excretion: Malpighian tubule secretion ^ 11 Endocrine control of excretion: Hindgut reabsorption ^ 15 Objectives of this study ^ 19 CHAPTER 2:^Effects of NCC, cAMP and Related Compounds on Rectal Fluid and Ion Transport^ Introduction ^ Materials and Methods ^ Insects ^ Chemicals ^ Salines ^ Flat sheet rectal bioassay ^ Everted rectal sac bioassay ^ Preparation of NCC extracts ^ Statistics ^ Results ^ Effect of NCC and cAMP ^ Effect of chemicals acting on the cAMP mediated pathway^ Effect of cGMP ^ Discussion ^  33 38 43  CHAPTER 3:^The Involvement of Calcium in Controlling Rectal Transport ^  45  Introduction ^  20 20 23 23 23 24 24 27 27 28 28 28  45  V Page Materials and Methods ^ 49 Results ^ 50 Effect of Ca2+ free saline ^ 50 Effect of agents that modulate cytosolic Calcium ^ 55 Discussion ^ 60 CHAPTER 4:^The Role of PKC and the PI Cycle in Controlling Rectal Transport ^  62  Introduction ^ 62 Materials and Methods ^ 65 Results ^ 65 Effect of agents acting upon protein kinase C ^ 65 Effects of agents acting upon the PhosphotidylInositol cycle ^ 66 Locusta experiments ^ 74 Effect of Neuroparsins on S.gregaria recta ^ 74 Discussion^ 79 CHAPTER 5:^General Discussion ^  81  References ^  85  vi LIST OF TABLES  Page Table 1.1.^A list of fully and partially characterized insect diuretic peptides. ^  12  Table 1.2.^Insects shown to possess antidiuretic factors that increase rectal fluid reabsorption. ^ 14 Table 2.1.^Effect of NCC and cAMP upon rectal electrical variables ^ 29 Table 2.2.^Effect of cGMP upon rectal electrical variables. ^ 39 Table 3.1.^Effect of Calcium-free saline upon electrical variables. ^ 51 Table 4.1.^Effect of 1mM LiC1 upon electrical variables ^ 67 Table 4.2.^Effect of MI and DBcAMP upon electrical variables of L. migratoria recta. ^  75  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 epithelial cells. ^ 4 Figure 1.3.^Model of transport mechanisms identified in the locust rectal epithelium ^  6  Figure 1.4.^Side view of an insect showing the neuroendocrine organs implicated in the control of fluid balance and excretion ^ 10 Figure 2.1.^Diagram of the adenylate cyclase signal transduction pathway. ^  22  Figure 2.2.^Standard Ussing chamber assembly used to measure Cldependent rectal short-circuit current (Ise). ^ 25 Figure 2.3.^Effect of NCC and cAMP on rectal Ise. ^ 30 Figure 2.4.^Fluid reabsorption by everted rectal sacs with time. ^ 31 Figure 2.5.^Tissue swelling by the everted rectal sacs from Figure 2.4 ^ 32 Figure 2.6. Effect of 2 NCC, 5 mM cAMP and 1 mM DBcAMP upon rectal Jv. ^  34  Figure 2.7.^Effect of cAMP, SpcAMPS and RpcAMPS upon rectal Jv ^ 35 Figure 2.8.^Effect of a lh pre-incubation with 10 mM RpcAMPS on stimulation of rectal Jv by 2 NCC ^  36  Figure 2.9.^Effect of ATP and Adenosine upon rectal Ise ^ 37 Figure 2.10. Effect of IBMX upon rectal 'Sc ^  40  Figure 2.11. Effect of 5mM cGMP and 5 mM cAMP upon rectal Jv ^ 41 Figure 2.12. Effect of cGMP upon rectal 'Sc. ^  42  viii  Figure3.1. Figure 3.2.  Schematic diagram of the cellular processes involved with the regulation of intracellular Ca2+ ^ Fluid reabsorption by rectal sacs under Ca2+ free conditions. ^  Page 46 52  Figure 3.3.  Tissue swelling of rectal sacs under Ca2+ free conditions. ^ 53  Figure 3.4.  Effect of Ca2+ free saline (Caf) upon Jv stimulated by 2 NCC and 1 mM DBcAMP ^  54  Figure 3.5.^Effects of Ionomycin (IY), IY with 5.5 mM Ca2+ bathing saline and IY with Ca2+ free saline upon rectal Jv. ^ 56 Figure 3.6.^Effect of 1 mM IY and Thapsigargin upon rectal Isc ^ 57 Figure 3.7. ^Effect of TMB-8 upon 5 mM cAMP stimulated Isc ^ 58 Figure 3.8. ^Effect of TMB-8 upon 2 NCC stimulated Isc. ^ 59 Figure 4.1. ^Diagram of the phosholipase C (PLC) mediated signal transduction pathway. ^  63  Figure 4.2. ^Effect of SAG and PMA upon rectal J. ^ 68 Figure 4.3.^Effect of SAG and PMA upon rectal 'Sc. ^ 69 Figure 4.4. ^Effect of a 1001,tM PMXB (1000 iu/mL.) upon 2 NCC stimulated Jv. ^  70  Figure 4.5.^Effect of 100 RM (1000 iu/mL.) PMXB upon rectal Ise. ^ 71 Figure 4.6. ^Effects of myo-inositol (MI) and LiC1 upon rectal Jv ^ 72 Figure 4.7. ^Effects of MI and LiC1 upon rectal I. ^ 73 Figure 4.8.^Fluid reabsorption by Locusta migratoria everted rectal sacs ^  76  Figure 4.9. ^Effect of Neuroparsins upon rectal J. ^ 77 Figure 4.10. Effects of Neuroparsins upon rectal Isc ^ 78  ix LIST OF ABBREVIATIONS  A Jv^- change in fluid reabsorption Clcm2^- ohms centimetre squared i_teq.cm-2.h- 1^- micro equivalents per centimetre squared per hour uL^- microlitre pL.h- 1^- microlitres per hour [iM^- micro molar um^- micron °C^- degrees Celsius 5-HT^- 5-hydroxytryptamine A^- Angstrom (0.1 nanometres) A/D^- analog-to-digital AA, aa^- amino acid(s) AD^- antidiuretic ADH^- antidiuretic hormone ADP^- adenosine diphosphate AG^- abdominal ganglion AP^- Acheta peptide ATP^- adenosine 5' triphosphate ANOVA^- analysis of variance AVG^- abdominal ventral ganglion AVP^- arginine vasopressin AVP-LDH^- arginine vasopressin-like diuretic hormone AVT^- arginine vasotocin CA^- corpus allatum cAMP^- adenosine 3', 5' -cyclic monophosphoric acid  x CC^- corpus cardiacum cDNA^- complementary DNA CGA^- colour graphics adapter cGMP^- guanosine 3', 5' -cyclic monophosphoric acid cm^- centimetre cm2^- squared centimetres CRF^- corticotropin releasing factor CTSH^- chloride transport stimulating hormone D^- diuretic Da^- Daltons DAG^- sn-1,2 diacylglycerol DBcAMP^- di-butyryl 3', 5' -cyclic monophosphoric acid DH^- diuretic hormone DMSO^- dimethyl sulfoxide DP^- diuretic peptide EGTA^- ethyleneglycol-bis-(B-aminoethyl ether) N,N,N',N'-tetraacetic acid eq.^- equivalents g^- gravity units GCC^- glandular lobe of the corpus cardiacum GTP^- guanosine triphosphate H^- hemolymph or hemocoel h^- hours HPLC^- high performance liquid chromatography IBMX^- 3 isobutyrl- 1 -methylxanthine Ins^- inositol Ins P^- inositol phosphate InsP3^- inositol 1,4,5, trisphosphate  InsP4^- inositol 1,3,4,5 tetraldsphosphate 'Sc^- short-circuited current iu^- international units IY^- ionomycin Jamm^- rate of luminal ammonia secretion - fluid reabsorption KDa^- kilo Daltons LCCP^- locust corpus cardiacum peptide - molar M-NSC^- median neurosecretory cells MI^- myo-inositol min^- minutes mL^- millilitre mm^- millimetre mM^- millimolar MT^- Malpighian tubule(s) MTG^- meta-thoracic ganglion mV^- millivolts - number NCC^- nervous lobe of the corpus cardiacum NpA^- neuroparsin A NpB^- neuroparsin B Nps^- neuroparsins P.E.^- polyethylene (tubing) PD^- potential difference PdE^- phosphodiesterase PI^- phosphotidylinositol  xii  PtdInsP2, PIP2^- phosphotidylinositol 4,5-bisphosphate PKA^- protein Idnase A PKC^- protein kinase C PLC^- phospholipase C PMA^- phorbol 12-mpistate-13-acetate PMXB^- polymyxin B sulfate PO^- perisympathetic organs RAM^- random access memory RpcAMPS^- adenosine 3, 5' -cyclic monophosphothioate Rp- diastereomer Rt^- transepithelial resistance S.E.^- standard error SAG^- 1-stearoy1-2-arachidonoyl-sn-glycerol ScgITP^- Schistocerca ion transport peptide SO-NSC^- subocellar neurosecretory cells SOG^- suboesophageal gnanglion SpcAMPS^- adenosine 3', 5' -cyclic monophosphothioate Sp- diastereomer TAG^- thoracic abdominal ganglion TG^- thoracic ganglion THAPS^- thapsigargin TMB-8^- 3,4,5 trimethoxybenzoic acid, 8-(diethylamino)-octyl ester Vt^- transepthelial voltage  ACKNOWLEDGMENTS  I would like to thank Dr. John Phillips for his support, generosity and guidance  throughout this study. I thank Drs. M. Isman, D. Jones and D. Randall for their helpful comments on the manuscript. I am grateful to Joan Martin for her technical assistance, advice and support.  I thank Bart Demaerschalk for his help with some of the experiments. I thank Megumi Fuse for her advice on writing this manuscript. I thank everyone in Dr Phillips lab that have made working there enjoyable and productive. I thank Brian, Eileen and Peter Jeffs for always being there when it matters. I especially want to thank Joyce Chong for her limitless encouragement, assistance and friendship.  1  CHAPTER 1: GENERAL INTRODUCTION Insects inhabit a wide range of diverse and variable habitats, including deserts, freshwater, alkaline lakes and tidal pools. Insects face severe regulatory challenges associated with high metabolic rates (e.g. when flying) and high surface area to volume ratios leading to water loss across the integument. The unusual diets of some insects including toxic substances produced by plants, high ingestion rates, molting and metamorphosis are further examples of the stresses placed on insect homeostatic mechanisms Like most insects, locusts regulate their hemolymph composition very closely. As well as behavioral and structural adaptations, physiological adaptations contribute to this hemolymph homeostasis. The control of epithelial transport in the excretory system is the principal mechanism for making rapid and major adjustments to hemolymph composition. The organization of the locust excretory system is typical of many insects, consisting of the Malpighian tubules and hindgut (Fig. 1.1). The Malpighian tubules actively secrete a primary isosmotic urine, rich in KC1 and low in Nal-, that contains most small hemolymph solutes (including amino acids), waste products (e.g. urate and ammonium) and toxic plant chemicals (reviewed by Phillips, 1981,1983). The hindgut (ileum and rectum) is then responsible for the enormous changes in composition of the urine by the selective reabsorption of water, ions, amino acids and other metabolites (Phillips et al., 1986). This selective reabsorption can create a hyper- or hypo- osmotic urine, or even a powder dry excreta under various environmental conditions. Structure of the locust excretory system There are about 250 Malpighian tubules in an adult Schistocerca gregaria that empty into the gut lumen at the junction of the midgut and hindgut. The hindgut (ileum, colon  2  Fig. 1.1. Diagram of the locust excretory system. The flow of urine is shown by the thin arrows and transfer across the epithelia is indicated by the thick arrows (from Phillips,  1981; modified by Audsley, 1991).  3 and rectum) is lined on the luminal side with a chitinous cuticle (2-10 pm thick). The cuticle of the ileum and rectum is permeable to small hydrophilic molecules via water-filled 6 A pores, but blocks the movement of larger toxic substances (Phillips and Dockrill, 1968). These water-filled pores are lined with fixed negative charges (pK — 4) that also allow the rapid diffusion of Ca2+ and Mg2+, which would otherwise be precluded because of their large hydrated molecular radii (Lewis, 1971). The ileum is approximately 6 mm long, has a diameter of 2.5 mm and luminal surface area of 0.4 cm2 (Irvine et al., 1988). The ileum consists of a simple epithelium (40 x 20 pm) and is covered by a firmly attached apical cuticle (Fig. 1.2). The apical membrane has dense infoldings that are associated with mitochondria and apical junctional complexes between adjacent cells. The basal membrane has short narrow infoldings associated with numerous mitochondria and may be analogous to the lateral scalariform complexes of the rectum that are described below (Irvine et al., 1988). The colon is an "S" shaped tube that is located between the ileum and the rectum (Fig. 1.1). It is narrower than the ileum or rectum and is comprised of small unspecialized epithelial cells. In addition, the colon possesses a cuticle which makes it considerably less permeable than either the rectum or ileum. For these reasons the colon is not thought to play a significant role in absorption (Maddrell and Gardiner, 1980). However, the colon has a structural role in the separation of the gut contents into discrete fecal pellets and in the breaking of the peritrophic membrane that surrounds the fecal material (Goodhue, 1963). The rectum is immediately posterior to the colon, consists of six longitudinally arranged thickened pads, and has a luminal surface area of 0.64 cm2 (Irvine et al., 1988). The rectal cuticle is attached at narrow junctional complexes between each rectal pad, but is free from the pad epithelium creating a subcuticular space above the apical border. The rectal pads are almost exclusively composed of large columnar principal cells (17 x 100 pm) with some small B cells. These B cells have few mitochondria and contact the  4  1  Fig. 1.2. Comparison of ultrastructural organization and gross dimensions of locust rectal  pad and ileal epithelial cells (from Irvine et al., 1988).  5  luminal side only. The principal cells have highly folded apical and lateral membranes with closely associated mitochondria. The lateral membranes have fmger-like projections that are intimately associated with the lateral membranes of adjacent cells. Extensive electrical coupling between these principal cells has been demonstrated by dye injection and cable analysis (Hanrahan and Phillips, 1984b). The lateral membrane infolds form complex intercellular channels with three distinct regions (lateral scalariform complexes, dilated intercellular spaces, larger intracellular sinuses with trachea) where ion recycling associated with the formation of hyperosmotic urine is thought to occur (Wall and Oschman, 1970). The basal membrane is unfolded and devoid of mitochondria which occur at the apical and lateral membranes, but is externally covered by a thick basal lamina. Below the basal lamina is a muscle layer, a subepithelial space and a smaller secondary epithelial cell layer. These secondary cells show some degree of membrane complexity and possess many mitochondria, suggesting a transport function (e.g. further ion recycling). Finally the whole structure is encased within a muscle layer through which fluid exits at the points where trachea penetrate into the organ. Mechanisms of solute and water transport in the locust rectum Wigglesworth (1931) was the first to report that insect gut contents can become very dry as they pass through the rectum and suggested that the rectum is a major site of fluid reabsorption. Active transport of ions and fluid in an insect rectum was first directly shown by measuring changes in volume and composition of fluid injected in ligated recta of locusts (Phillips, 1964 a-c,). It is now known that Na+, K+, Cl-, water and metabolites are all reabsorbed by locust recta. Figure 1.3 shows the major membrane transport mechanisms demonstrated in locust rectal epithelial cells. The ileum has since been shown to have equal or greater transport capacities for ions and water than the rectum. The differences between rectal and Heal epithelial transport mechanisms will be discussed later. Chloride (Cr) is the major anion that is actively reabsorbed by the locust rectum from  LUMEN  CELL  HEMOCOEL  •lectrogenic pump  ,Ion net (pEq.cm-2.h-1)  CI-  4.3  K• N•. K^ K• ATPase  (WINa• AA'a  •^ oxidation'  Na•  CO2  Ni-1•(H•) 4 H•  4.5  Na'  2D  AA's  1.5  HCO3  0.4  AcidBase  1.5  I cA I -  OW H20  Ion Activities (mM) and PD ( mV) Na' K•  75 7.2  Proline pH  82 13 7  mV  9 70 47 68 7.36  •64 Net Electrochemical (Apical)  Na'  127 (favoring )  75 7.2 82 13 7 •34 PD (my) (Basolateral) 127 (opposing)  K•  12 (favoring)  20 (favoring)  CI  50 (opposing)  20 (favoring))  H•  86 (opposing)  56 (favoring)  Fig. 1.3. Model of transport mechanisms identified in the locust rectal epithelium. The  neuropeptide, CTSH, acts via cAMP to stimulate or inhibit four mechanisms. Major pumps are represented by thick arrows, carrier mediated co- or counter-transport by thin arrows through solid circles and ion channels by arrows through gaps. Steady state -  values given for net transepithelial flux and electrochemical potential differences across the two borders are for stimulated recta in Ussing chambers and bathed bilaterally in control saline under open circuit conditions, except values for Nat and amino acids (AA) which were quantified under short-circuited conditions. From Phillips et al. (1988).  7 the urine. It can be seen from Figure 1.3 that there is a large net electrochemical difference opposing Cl- entry at the apical (mucosa') surface of the rectal pad cells. This indicates that the active step for Cl- transport is located at the apical membrane. The model proposes that there is a primary electrogenic Cl- pump that is stimulated by cAMP and lumina' K+. There are also basolateral Cl- channels that allow the passive exit of C1 across the basal (serosal) membrane. In support of this model, Hanrahan and Phillips (1983, 1984a,b) showed that addition of 1 mM cAMP to the serosal side of recta caused a 10-fold increase in net Cl- transport toward the hemocoel side and that the removal of external K+ reduced net Cl- flux by 84%. They also provided evidence that Cl- transport was not coupled to apical Nat, K+, HCO3- or H+ transport. In addition, Cl- exit through the basolateral membrane can be completely inhibited by Cl- channel blockers applied to the hemocoel side of recta (Phillips et al., 1986). Potassium (K+) is the major cation in the primary urine from the Malpighian tubules and is absorbed passively through K+ channels at both membranes (Hanrahan and Phillips, 1984a). In unstimulated open-circuit recta, there are small favourable electrochemical gradients for entry of K+ across the apical membrane (3.3 mV) and for exit across the basolateral membrane (3.9 mV; Hanrahan and Phillips, 1983, 1984c). Addition of cAMP stimulates Cl- transport and subsequently increases apical potential (Va) by 12 mV and decreases basolateral potential (Vb) by 13 mV. These membrane potential changes increase the favourable apical and basolateral electrochemical gradients to 12 mV and 20 mV respectively, resulting in increased net K+ movement to the hemocoel. Sodium (Na+) is usually present at low concentrations in the fluid entering the rectum (20-40 mM), and is almost completely reabsorbed. There is a large favourable electrochemical gradient (127 mV) at the apical membrane for passive Na+ entry by several mechanisms. These demonstated mechanisms include Na+/NH4+ and Na+/H+ counter exchange, Naliglycine cotransport, and direct entry through a Na+ channel (Phillips et al., 1986; Black et al. 1987). Na+ is actively transported into the hemocoel  8 against a large electrochemical gradient (127 mV) by Na+/K+ ATPase in the lateral membranes (reviewed by Phillips et al., 1986; Lechleitner and Phillips, 1988). Rectal Na+ transport is independent of Cl transport and is not affected by cAMP or any neuroendocrine tissue extracts tested to date (Black et al., 1987). Berridge and Gupta (1967), and Wall and Oschman (1970), have proposed a model to explain how insect recta can absorb hypo-osmotic fluid from the lumen. The model proposes that ions and other solutes (amino acids) are actively secreted into the intercellular spaces of the lateral scalariform complexes (Fig. 1.2), creating areas of high osmotic concentration relative to the lumen. Water then flows from the gut lumen into these hyper-osmotic spaces (i.e. local osmosis), thereby producing a hydrostatic pressure which causes fluid to flow through the intercellular sinuses towards the hemocoel side. Active solute reabsorption is believed to occur in the dilated intercellular spaces and intercellular sinuses. This reabsorption of solute into the the cells results in the formation of a hypo-osmotic absorbate and concentrated excreta. Goh and Phillips (1978) have shown that water transport in the locust rectum is indeed coupled to ion transport, and Proux et al. (1984) have shown that cAMP-stimulated fluid reabsorption (Jv) is dependent upon transport. Recently Lechleitner and Phillips (1989) observed an increase in Jv when recta were exposed to 80 mM proline compared to incubation with 1 mM proline. It was also shown that a low level of Jv (2.3 liL.h-1) could be maintained for 5 hours with bathing saline containing 80 mM proline and no IC+, or Na+. Combined with the knowledge that proline is actively transported apically at high rates (Lechleitner, 1988) and that 80 mM proline far exceeds the cell's metabolic needs, it has been concluded that proline is directly involved with fluid transport in the rectum. Five neutral amino acids (proline, glycine, serine, alanine and threonine) have been shown to be actively absorbed by locust recta when Jv was prevented by making the lumen hyper-osmotic with sucrose (Balshin and Phillips, 1971; Balshin, 1973). Meredith and Phillips (1988) found that the rectum has a very high capacity for proline transport at  9 the apical membrane. This proline transport system operates in the absence of lumina' Na+, Cl- or K+. The system also has an electrogenic component, probably due to H+ cotransport. In addition, proline is the major respiratory substrate in the locust rectum and is the source of the secreted ammonia (Chamberlin and Phillips, 1983; Thomson et al., 1988). Ammonia is a toxic waste product of amino acid oxidation and must be eliminated or detoxified. Ammonia is secreted into the rectal lumen via an apical Na+/NH4+ exchanger (Thomson et al., 1988). Acid (H+) secretion by the rectum is accompanied by an equal movement of base (OH-, HCO3-) equivalents to the hemocoel side (Thomson and Phillips, 1985, Phillips et al., 1988). The bulk (80-90%) of rectal acid secretion is actively driven by an apical electrogenic process (i.e. a H+ATPase), with Na+/H+ exchange playing a minor role (Thomson and Phillips, 1992). The addition of cAMP to rectal preparations reduces active rectal H+ secretion by 66% but does not change ammonia secretion (Phillips et al., 1988; Thomson et al, 1988). Solute and water transport in the ileum Many of the Heal transport mechanisms are similar to those previously described in the rectum, but there are some distinct differences. The primary difference is that the ileum can only produce an iso-osmotic absorbate, because it lacks the complex lateral membrane system for solute re-cycling found in the rectum (see Fig. 1.2.; Irvine et al., 1988). Moreover, ilea! Jv is driven by both NaC1 and KC1 transport, while rectal Jv is driven largely by proline and KC1 (Lechleitner and Phillips, 1989). Unlike the rectum, Na+ is transported at high rates in the ileum and its transport is stimulated by cAMP (Lechleitner et al., 1989b). Acid (H+) secretion in the rectum is inhibited by cAMP, while ilea! H+ secretion is inhibited by the neuropeptide ScgITP but not cAMP (Audsley, 1991). Heal NH4+ secretion rate (Jamm) is 2.5 times greater than in the rectum and is stimulated by cAMP (Lechleitner, 1988; Peach and Phillips, 1991). As previously mentioned, luminal  10  Fig. 1.4. Side view of an insect showing the neuroendocrine organs implicated in the  control of fluid balance and excretion (from Phillips 1982).  11 proline is the major respiratory substrate and ammonia source in the rectum, while the ileum uses luminal alanine, asparagine, glutamine, serine and bilateral proline (Peach and Phillips, 1991). There is negligible proline transport in the ileum (Lechleitner and Phillips, 1989). Endocrine control of excretion: Malpighian tubule secretion Malpighian tubule secretion and hindgut reabsorption are both under endocrine control. The insect neuroendocrine organs thought to be involved in the control of fluid balance and excretion are shown in Fig. 1.4. The first demonstration of a diuretic factor that stimulated Malpighian tubule secretion was in the blood feeder, Rhodnius prolixus (Maddrell 1963,1964a-b). After feeding, the body volume of Rhodnius increases ten-fold, necessitating the excretion of excess fluid. Maddrell showed that feeding triggered the release of a proteinacious factor from the thoracic-abdominal nerve mass that immediately stimulated the secretion of large amounts of urine. Various methods have been used to study Malpighian tubule (MT) secretion: Ramsay's (1954) in vitro fluid secretion method with isolated tubules and its variants; measuring changes in MT transepithelial voltage (Maddrell and Klunsuwan, 1973; Williams and Bayenbach, 1983,1984); measuring changes in tissue cAMP levels (Morgan and Mordue, 1985; Kay et al., 1991a); and by measuring water loss from the whole insect (Kataoka et al., 1989). Diuretic factors have been reported for many types of insects, and these factors appear to be present in various locations throughout the neuroendocrine system (reviewed by Spring, 1990; Raabe, 1991). However, only a few of these factors have been purified and their amino acid sequence determined (see Table 1.1.). High performance liquid chromatography (HPLC) has been used extensively to isolate insect diuretic and antidiuretic factors. HPLC can rapidly separate small amounts of material with high resolution and recovery, and is far superior to older chromatographic techniques that were slower, less sensitive and required more material (Schram, 1980).  12 Table 1.1. A list of fully and partially characterized insect diuretic peptides  SPECIES^PEPTIDE^SOURCE^DESCRIPTION Locusta migratoria^DP-1^CC^only partial amino acid sequence  (migratory locust)^  (Morgan et al., 1987)  AVP-LDH^SOG^18 aa anti parallel homodimer, 67% and 78% homology with AVP and AVT respectively (Proux et al., 1987) Locusta-DP^head^46 aa amidated peptide, chemically  similar to DP-1, 48% and 49% homology with Acheta-DP and Manduca-DH respectively (Kay et al., 1991b)  Manduca sexta Manduca-DH^head^41 aa amidated peptide  (tobacco^ homworm)  (Kataoka et al., 1989)  Acheta domesticus^AP-1^CC^18 aa peptide (Coast et al., 1990)  (house cricket)  Acheta-DP^head^46 aa amidated peptide, 41% homology to Manduca-DH  (Kay et al, 1991a)  Abbreviations: aa, amino acid; CC, corpus cardiacum; SOG, suboesophageal ganglion; DP/DH, diuretic peptide/diuretic hormone; AP, Acheta peptide; AVP, arginine vasopressin; AVT, arginine vasotocin; AVP-LDH, arginine vasopressin-like diuretic hormone.  13 Improvements to mass spectrometry, peptide sequence analysis, and the development of automated gas-sequencers have greatly increased the speed of peptide sequencing, and have reduced the amount of material required by up to 1000 fold (Holman et al., 1991). Arginine vasopressin-like diuretic hormone (AVP-LDH) was the first putative insect diuretic factor to be fully characterized, and was isolated using HPLC and immunological techniques (Proux et al., 1987). Although AVP-LDH closely resembles mammalian AVP, its structure is unlike other purified diuretic factors. Many of the recently characterized diuretic peptides such as Locusta-DP, Manduca-DH, Acheta-DP (see table 1.1.), Leucophaea-DP and Periplaneta-DP (Coast, personal communication), share homologous  regions in their amino acid sequences. In addition, this family of diuretic peptides has sequence homology with some vertebrate hormones, namely corticotropin releasing factor (CRF), sauvagine and suckerfish urotensin 1 (Coast et al.,1992). Coast et al. (1992) demonstrated that CRF, sauvagine and urotensin 1 all increased intracellular cAMP levels in Acheta Malpighian tubules, mimicking the action of Acheta-DP. They have proposed that these insect diuretic peptides belong to a superfamily of peptides that includes CRF, sauvagine and urotensin 1. The proposed members of this superfamily are known to be involved in the regulation of fluid and solute transport, and act by elevating cAMP and perhaps by increasing intracellular Ca2+ levels. Secretion by insect Malpighian tubules is also stimulated by members of a family of myotropic peptides (myoldnins), that have been isolated from Leucophaea maderae (Leucokinins) and Acheta domesticus (Achetakinins) (Hayes et al., 1989; Coast et al., 1990). Myokinins are known to stimulate contractile activity in the insect hindgut (Holman et al., 1991). These myokinins all possess a highly conserved pentapeptide carboxy terminal sequence, which is responsible for activity (Holman et al., 1990; Nachman et al., 1990). The myokinins are thought to work on Malpighian tubules via a cAMP independent mechanism, perhaps by elevating intracellular Ca2+.  14 Table 1.2. Insects shown to possess antidiuretic factors that increase rectal fluid  reabsorption.  SPECIES  ^  SOURCE^REFERENCE  Carausius morosus^brain^Vietinghoff (1966)  (stick insect)  Acheta domesticus^GCC^De Besse and Cazal (1968)  (house cricket)^CC^Albarwani (1988), Spring et al. (1988a)  Locusta migratoria  CC^Cazal and Girardie (1968) PO^De Besse and Cazal (1968) NCC, GCC^Herault et al. (1985)  Schistocerca gregaria  GCC^Mordue (1969, 1970, 1972) GCC, NCC^Proux et al. (1984) AVG 5^Lechleitner and Phillips (1989)  (migratory locust)  (desert locust)  CA^Wall (1967) Periplan eta americana brain, MTG, ^ (American cockroach)^TAG Blaberus craniifer  ^  (cockroach)  Leucophaea maderae  (cockroach)  ^  Wall (1967), Goldbard et al. (1970)  CC^Boureme et al. (1989)  CC^Boureme et al. (1989)  Abbreviations: GCC/NCC, glandular/nervous lobes of corpus cardiacum; CA, corpus allatum; PO, perisympathetic organs; AVG 5, fifth abdominal ventral ganglion; MTG, meta-thoracic ganglion; TAG, thoracic abdominal ganglion.  15 Spring et al. (1988b) isolated an antidiuretic hormone (ADH) from Acheta domesticus CC that inhibited MT fluid secretion by 70%, but had no effect upon the hindgut. It was also shown that the release of this ADH into the hemolymph was triggered by dehydration. There is indirect evidence that this ADH may act by elevating intracellular Ca2+, since the divalent ionophore A23187 also reduced tubule secretion by 85% (Spring and Clark, 1990). The structure of this ADH has not yet been determined. Endocrine control of excretion: Hindgut reabsorption  Vietinghoff (1966) was the first to show that insect hindgut reabsorption is under endocrine control. There have been several subsequent reports of antidiuretic (AD) and diuretic (D) factors that control hindgut fluid reabsorption (Jv); see Table 1.2. for AD factors (reviewed by Phillips, 1982,1983; Phillips et al., 1986; Spring, 1990). However, the validity of some of these earlier reports have been questioned (Phillips et al., 1986). Most of the earlier studies were performed on uncharacterized in vitro hindgut preparations during an initial transitory absorptive period, when the hindgut tissue was adjusting to the new osmotic environment (causing tissue swelling) and was possibly still responding to factors that were present in vivo. In addition, the earlier studies often used salines that were lacking essential metabolic substrates (e.g. proline for the locust rectum) and were not sufficiently oxygenated (see Phillips et al., 1986). Phillips et al. (1980) partially purified a neuropeptide from Schistocerca gregaria CC that stimulated electrogenic chloride transport and named it chloride transport stimulating hormone (CTSH). Proux et al. (1984) later showed that CTSH also stimulates rectal fluid reabsorption. CTSH activity is present in both the glandular (GCC) and storage lobes (NCC) of the CC but 80% of the activity is found the NCC. It appears that CTSH is released into the hemolymph, presumably upon feeding (Spring et al., 1978; Spring and Phillips, 1980a,b,c; Proux et al., 1984; Hanrahan and Phillips, 1985). CTSH is thought to act by elevating intracellular cAMP levels, because exogenous cAMP stimulates both  16 rectal C1 transport and water reabsorption (Phillips et al., 1980; Proux et al., 1984)). In addition, Chamberlin and Phillips (1988) found that rectal tissue levels of cAMP increase between five and ten minutes after exposure to CC extracts. It has also been shown that transport and intracellular cAMP levels increased when pharmacological agents (theophylline and forskolin) known to elevate cAMP levels in other systems, were applied to isolated recta (Spring et al., 1978; Hanrahan et al., 1985; Chamberlin and Phillips, 1988). H6rault et al. (1985) have isolated two AD factors from Locusta migratoria that increase rectal Jv over a five hour period. One factor is reported to be localized in the GCC while the other factor is exclusive to the NCC. Haault and Proux (1987) have shown that the GCC factor elevates intracellular cAMP levels over a similar time course as CTSH. The GCC factor is an unstable peptide (H6rault et al., 1985) and has not yet been purified. Girardie et al. (1987b) have purified and characterized an AD factor from Locusta NCC, which they have named neuroparsins (Nps), and have shown that it is produced by the Al type protocerebral median neurosecretory cells (M-NSC; Girardie et al., 1987a). As well as increasing rectal Jv, Nps inhibits the effects of juvenile hormone (Girardie et al., 1987b) and increases hemolymph trehalose and lipid levels (Moreau et al., 1988). Girardie et al. (1989,1990) used anion exchange chromatography and HPLC to isolate two forms of neuroparsins, neuroparsin A (NpA) and neuroparsin B (NpB). Both forms have molecular weights of —14 KDa, and are composed of two polypeptide chains (-7 KDa) linked by disulfide bonds. The NpA monomer has 83 amino acid residues while the NpB monomer consists of 78 residues (i.e. identical to residues 6-83 of NpA). Since the NpB monomer sequence is identical to a major part of NpA, Girardie et al. (1989) have postulated that NpB is a post-translational product of NpA. Recently, Hietter et al. (1991) used HPLC to characterize three neuroparsin-like 8-9 ICDa monomeric peptides from Locusta CC. Two of the peptides correspond to NpA and NpB, while the third peptide  17  possesses 81 residues (3-83 of NpA). All three peptides have been shown by mass spectrometry to exist as monomers containing six disulfide bridges. Hietter et al. (1991) also noticed that part of the Nps amino acid sequence is almost identical to the partially sequenced 4.5 KDa, 46 residue Locusta Corpus Cardiacum peptide (LCCP) purified by Mordue et al. (1985). Hietter et al. (1991) used a different approach for extraction, isolation and characterization of these peptides to that used by Girardie et al. (1989,1990), which may account for their differing results. Lagueux et al. (1992) have cloned a Locusta cDNA that encodes NpA. They have proposed that NpA is processed from a 107 residue precursor in two steps; cleavage of a 22 residue signal peptide and removal of a Glu-Arg dipeptide. It has also been shown that transcripts encoding NpA occur in both adult and larval brains. Neuroparsin-like peptides have also been identified in the cockroaches Blaberus craniifer and Leucophaea maderae (Boureme et al., 1989). Both of these peptides  increase Locusta rectal Jv, and NpA stimulated rectal h in both cockroaches, suggesting that these peptides are related and may belong to an intraphyletic neuroprotein family. Immunological techniques have also been used to show that neuroparsin-like factors exist in brain neurosecretory cells of three other insect orders (Isoptera, Odonata and Dictyoptera; Tamarelle and Girardie, 1989). In addition, NpA has 37% and 27% amino acid sequence homology with Manduca sexta and Bombyx moni eclosion hormones respectively (Girardie et al., 1990). Furthermore, NpA shows some sequence identity (-30%) with some vertebrate hormones, such as hypothalamic pituitary hormones and pre-proinsulin (Girardie et al., 1990). There has also been interest in determining the second messenger systems whereby Nps stimulates rectal Jv. Fournier and Dubar (1989) have shown that Nps has no effect upon intracellular cAMP levels, while slightly increasing cGMP levels. They concluded that both cAMP and cGMP are not second messengers of Nps. They suggest that the slight increase in cGMP levels is a result of Nps-stimulated elevation of cGMP in a different cell  18 type, such as rectal muscle cells, resulting in a small diuretic effect! Moreover, Fournier (1990b) has used a pharmacological approach to provide indirect evidence that Nps induces phosphoinositide turnover in Locusta rectal cells. Diacylglycerol (DAG) and inositol trisphosphate (InsP3), products of phosphotidylinositol bis-phosphate (PtdInsP2) breakdown, have both been implicated as possible second messengers of Nps. DAG is known to stimulate protein kinase C (PKC; Nishizuka, 1984), while InsP3 triggers the release of intracellular calcium from internal stores and the entry of extracellular Ca2+ (Berridge and Irvine, 1989). Fournier (1991) has since directly shown that Nps stimulates phospholipase C (PLC) mediated hydrolysis, resulting in an increase in intracellular InsP3 levels. Audsley et al. (1992) have used reverse phase (RP)-HPLC to isolate and purify a 7.7 KDa factor from Schistocera gregaria CC, called Schistocerca gregaria ion transport peptide (ScgITP). At the moment only 50 of the estimated 65 amino acid residues of ScgITP have been identified (Audsley, 1991). ScgITP is completely different from any other known vertebrate or insect hormones, but its first 34 residues have approximately 50% sequence identity with crustacean hyperglycemic and moult inhibitory hormones (Audsley et al., 1992). ScgITP has quantitatively similar effects upon ileal transport as does crude CC homogenates: increasing ileal Jv; stimulating C1, K+ and Na+ reabsorption; and inhibiting H secretion (Audsley et al., 1992). ScgITP also submaximally stimulates rectal Isc at very high doses, but has no effect upon rectal Jv or K+ permeability. This evidence indicates that there are at least two unique factors (CTSH and ScgITP) that control Schistocerca hindgut solute and fluid reabsorption. ScgITP is thought to act via cAMP, since exogenous cAMP mimicks most of the effects of ScgITP and of crude CC extracts. However, ScgITP mediated inhibition of ileal H+ secretion is not mimicked by cAMP, suggesting that another second messenger may be involved.  19 Objectives of this study  The purpose of this study was to investigate what second messengers are involved in controlling rectal fluid reabsorption (Jv) and Cl- transport in the desert locust (Schistocerca gregaria). To determine which second messengers are important in  controlling these rectal transport processes, various agents known to block or activate specific signal transduction pathways were added to everted rectal sac and short-circuited rectal flat-sheet bioassays. Chapter 2 examines and reviews the roles of the adenylate and guanylate cyclase pathways in rectal reabsorption. In chapter 3, the following questions are addressed: 1) Is extracellular Ca2+ necessary for rectal Jv and ion transport? 2) Does modulating cytosolic Ca2+ levels have any effect upon rectal transport properties?. Chapter 4 studies the role of protein kinase C (PKC) and the involvement of the phosphatidylinositol (PI) cycle in controlling rectal transport. In addition, Locusta rectal sacs and short-circuited rectal flat sheets were used to investigate the role of the PI cycle in Locusta recta. Finally, the effects of Locusta Nps, which is thought to act by stimulating the PI cycle in Locusta (Fournier, 1990b,1991), was tested on Schistocerca rectal Jv and Ise to see if there was any cross-reactivity.  20  CHAPTER 2: EFFECTS OF NCC, cAMP AND RELATED COMPOUNDS ON RECTAL FLUID AND ION TRANSPORT  INTRODUCTION  The cyclic nucleotides cAMP and cGMP are thought to be the second messengers of many hormonally controlled processes in both vertebrate and insect cells (reviewed by Bodnaryk, 1983). In insects, cAMP has an important role in controlling Malpighian tubule secretion and hindgut reabsorption (Bodnaryk, 1983; Raabe, 1989; Spring, 1990). Exogenous cAMP and pharmacological agents known to elevate intracellular cAMP (e.g. theophylline and forskolin), have been shown to increase Malpighian tubule fluid secretion in various insect species (reviewed by Coast et al., 1991). These agents also stimulate fluid and ion reabsorption in vitro by S. gregaria rectum (Spring and Phillips, 1979; Hanrahan, 1982) and ileum (Audsley and Phillips, 1990); as well as stimulating fluid reabsorption by the recta of Locusta migratoria (Herault and Proux, 1987), Leucophaea maderae and Blaberus craniifer (Fournier, 1990a). Measurements of increases in intracellular cAMP in  response to stimulants, have provided direct evidence that cAMP is a second messenger of insect excretory processes. Elevation of intracellular cAMP is associated with the stimulation of Malpighian tubule fluid secretion by CRF-related diuretic peptides (Coast et al., 1992). In addition, extracts of CC have been shown to simultaneously elevate intracellular cAMP levels and stimulate fluid and ion reabsorption in locust and cockroach recta (Spring and Phillips, 1979; Chamberlin and Phillips, 1988; Herault and Proux, 1987; Fournier, 1990a). Chamberlin and Phillips (1988) have also shown that locust rectal cGMP levels increase to a maximum value after 60 minutes of exposure to CC extract. This increase in rectal cGMP was much slower than the increase in cAMP that peaked between five and ten minutes after the addition of CC. Furthermore, this slow increase in  21 rectal intracellular cGMP corresponds quite well with the slow and gradual stimulatory effect of exogenous cGMP upon rectal ^transport (Chamberlin and Phillips, 1988). The elevation of cAMP is brought about by hormonal activation of the adenylate cyclase signal transduction pathway (Fig. 2.1.). Signal transduction mechanisms translate and amplify external signals, enabling cells to respond quickly and precisely to specific stimulants/inhibitors at low concentrations. The first step in the Adenylate cyclase pathway is the binding of a neuropeptide to its target receptor. This hormone-receptor complex then catalyzes the binding of GTP to the a subunit of a stimulatory G protein (Gs). The GTP-Gsa complex dissociates from the remaining f and 7 Gs subunits and binds to adenylate cyclase, which is also located on the plasma membrane (reviewed by Stryer, 1988). A single hormone-receptor complex catalyzes the formation of many GTP-Gsa molecules, thus amplifying the original signal. The activated adenylate cyclase converts ATP to cAMP, resulting in another amplification of the signal. The increased amounts of cAMP then stimulate cAMP specific protein kinases (PKA). These activated protein kinases can now phosphorylate target proteins (e.g. enzymes, ion channels), activating or inhibiting specific cellular processes (Cohen, 1989). This protein modification results in an additional amplification of the original, external signal. In the case of the locust hindgut, it is likely that proteins involved in transport are modified by PKA, since exogenous cAMP can stimulate C1--dependent 'sc. Increased cGMP levels are brought about by the activation of the guanylate cyclase signal transduction pathway. The guanylate cyclase pathway is comprised of similar functional components as the adenylate cyclase pathway. The major differences between the two pathways are that cGMP levels in animal cells are usually more than ten times lower than cAMP levels (Bodnaryk, 1983), and there are multiple isozymes of guanylate cyclase that are found in both the cytosol and plasma membrane (Shultz et al., 1989).  22  O. Neuropeptide [1st messenger]  Hemocoel  Receptor  Cytosol GTP+Gsa [transducer]  p [amplifier]  I  ATP  [precursor]  cAMP  [2nd messenger]  PKA  [internal effector]  a cascade of protein modifications  CELLULAR RESPONSES  Fig. 2.1. Diagram of the adenylate cyclase signal transduction pathway. Shaded circles  indicate agents that are added exogenously to affect the pathway.  23 In this chapter, the roles of the adenylate and guanylate cyclase signal transduction pathways in the control of rectal Jv and 'Sc were investigated. Initially, the effects of NCC homogenate, exogenous cAMP and dibutyrl cAMP upon rectal Jv and 'Sc were tested and compared. Then the involvement of protein kinase A was tested by adding the diastereoisomers SpcAMPS and RpcAMPS, which specifically stimulate and inhibit PKA, respectively (Rothermel and Parker-Botelho, 1988). The effects of ATP and adenosine were tested to ensure that cAMP was not acting by stimulating purinergic cell surface receptors (Olsson and Pearson, 1990). The effect of inhibiting cAMP-dependant phosphodiesterase (PdE) with IBMX was also examined. The stimulatory actions of micromolar amounts of forskolin, which directly activates adenylate cyclase, on rectal Isc was previously reported (Spring et al., 1978; Hanrahan and Phillips, 1985). Finally, the effects of cGMP upon rectal Jv and Isc were tested and compared to those of cAMP.  MATERIALS AND METHODS Insects  Adult female Schistocerca gregaria, 2-4 weeks past their fmal molt were used for all experiments. The locusts were maintained on a 12 h light: 12 h dark cycle at 28°C and 60% relative humidity, under crowded conditions. Animals were fed a mixture of dried grass, bran, powdered milk and yeast; with fresh lettuce supplied daily. The nervous lobes of the corpus cardiacum (NCC) were excised from adult male and female locusts 4-6 weeks past their final molt.  Chemicals  Most chemicals, including those used to make the complex saline, were supplied by the Sigma Chemical Company (St. Louis MO, USA). SpcAMPS and RpcAMPS were  24 supplied by BIOLOG Life Science Institute (La Jolla, CA, USA). Cyclic AMP, DbcAMP, SpcAMPS, RpcAMPS, ATP and cGMP were all dissolved in complex saline. IBMX and adenosine were initially dissolved in DMSO and these stock solutions were then diluted with complex saline (final DMSO concentration 1%).  Salines  The complex bathing saline used was based on the measured composition of locust hemolymph (Hanrahan et al. 1984) and contained (mM): 100 NaC1, 5 K2SO4, 10.9 Mg504, 10 NaHCO3, 5 CaC12, 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. The saline was adjusted to pH 7.1 and continuously bubbled with a 95% 02: 5% CO2 gas mixture that ensured rapid mixing. Complex saline has been shown to sustain transport activities of locust hindgut at near constant values for many hours (>8h.; reviewed by Phillips et al.,1986).  Flat sheet rectal bioassay  Electrogenic ion transport was measured by mounting recta between two Ussing chambers, as described by Hanrahan et al. (1984) and Audsley (1990). Recta were removed from animals, cut longitudinally to produce a flat sheet and immediately secured on tungsten pins over a 0.196cm2 opening using a neoprene 0-ring to form a seal (Fig. 2.2.). The chambers were held together with elastic bands and placed in a vice-like frame. Each chamber contained 2mL of complex saline that was continuously stirred by vigorous bubbling with 95% 02 : 5% CO2 gas mixture, at 23°C ± 2°C. Transepithelial potential (Vt) was measured by placing 3M KC1 agar bridges (size P.E. 90) near the tissue through ports on the side of the chambers with leads connected to a high impedance differential amplifier (4253, Teledyne Philbrick, Dedham, Mass. USA) which continually monitored  25  Fig. 2.2. Standard Ussing chamber assembly used to measure Cl- dependent rectal short-  circuit current (Isc). 1) rectal flat-sheet preparation, 2) Plexiglas collar that rectum is mounted over, 3) Neoprene 0-ring for securing rectal attachment to collar, 4) neoprene chamber seal, 5) agar bridge port for measurement of transepithelial potential (Vt), 6) gas islet for saline aeration and mixing, 7) current sending electrodes, 8) rear chamber seal, 9) tungsten pins for attachment of rectum to collar (taken from Hanrahan et al., 1984).  26 Vt. Short-circuit current (Isc), a direct and continuous measure of electrogenic ion transport, was measured by maintaining Vt , at 0 mV by a second amplifier (725, National Semiconductor Corp., Santa Clara, CA. USA) which passed current (Isc) between two Ag-AgC1 electrodes at either end of the chamber. A third amplifier (308, Fairchild, Mountain View CA. USA) was then used to measure Ise. Williams et al. (1978) reported that locust rectal 'Sc is Cr-dependent and is therefore a direct measure of electrogenic Cr transport. Flux studies by Hanrahan (1982) have confirmed this report. Both 'Sc and Vt were monitored by connecting the respective amplifier to an analog-to-digital (A/D) converter on a IBM® XT bus card (supplied by Brynhyfryd Consulting, Vancouver B.C.). A microcomputer logging program (supplied by David M. Jones Department of Oceanography, U.B.C.) collected and displayed the outputs from these amplifiers and all signals were recorded at one second or greater intervals (see Jones et al., 1991). The card and program were used with an IBM® compatible personal computer equipped with a CGA video output and 640K RAM. Corrections were made for series resistance of the external saline and asymmetries between voltage-sensing electrodes as described by Hanrahan et al. (1984). While the tissue was under short-circuit conditions, Vt was monitored at intervals by stopping the voltage clamp for 30-60 seconds and using an alternative circuit to measure voltage difference. Transrectal resistance (Rt) was calculated from Ise and Vt using Ohm's law. The Isc and Vt decline rapidly over the first 1-2 hours after excision of the recta, but thereafter these variables of ion transport activity decline very little (if at all) over the next several hours (i.e. steady-state phase: Williams et al. 1978; Spring and Phillips, 1980a). After the rectal tissue had reached steady-state, fresh saline was added to the chambers and the tissue was exposed to various substances to study their effect on rectal Isc, Vt and Rt.  27 Everted rectal sac bioassay  Everted rectal sacs were prepared as described by Goh and Phillips (1978) and Hanrahan et al. (1984). A 3 cm length of PE 90 tubing with a slightly flared end was inserted through the anus of the locust until its flared end reached the anterior boundary of the rectum. The hindgut was raised slightly and the anterior boundary of the rectum was ligated with surgical silk on to the flared end of the tubing. The colon and connecting trachea were cut away and the rectum was slowly everted by pulling the PE tubing through the anus. The everted rectum was then cut from the animal and rinsed with 1 mL of complex saline to remove any hemolymph or fecal material and the posterior was ligated. Any remaining internal fluid was withdrawn completely with a 100 'IL. Hamilton syringe and the empty sac was weighed to an accuracy of ± 0.1 mg on an August Sauter balance. Rectal sacs were filled hourly with 10pL of fresh saline, incubated at 30°C in 25 mL of complex saline, and bubbled with a 95% 02: 5% CO2 gas mixture. The weight gain and the tissue volume changes were determined at hourly intervals by weighing sacs before and after removal of internal (Hemocoel side) fluid. The true rate of transepithelial fluid movement (iv) was determined by correcting for tissue volume changes. The rectal sacs were allowed to equilibrate for two hours and putative stimulators of fluid transport were added to the hemocoel (H) side of the sac at the start of the third hour. The change in rate of rectal fluid reabsorption (A Jv) was calculated by subtracting the ft, for the second hour from the third hour value. These A Jv values were then compared to the average control values for unstimulated sacs over the same time period. Preparation of NCC extracts  NCC were excised from adult locusts and immediately frozen on dry ice and stored at 70°C. NCC were then homogenized in complex saline using a Tissue Tearer Homogenizer (Bartlesville OK, USA) for 2-4 minutes. The homogenate was then centrifuged at 12,000g  28 and 4°C for 10 minutes. The pellet and the floating lipid layer were discarded and the supernatant was kept at -20°C until use.  Statistics  The significance of differences between two means was determined by paired and independent t-tests. Analysis of variance (ANOVA) with the Tukey test was employed to determine differences between multiple means.  RESULTS  Effect of NCC and cAMP  The effects of NCC and cAMP upon rectal Isc, Vt, and Rt are shown in Table 2.1 and Fig. 2.3. Large increases in 'Sc occcured within 2 minutes of addition of 5 mM cAMP, and within 5 minutes of adding 2NCC. (Fig. 2.3). Cyclic AMP maintained 'Sc at a constant high level for over an hour after initial stimulation, and NCC caused Ise to peak after 30 minutes then decline slowly. NCC and cAMP caused similar (not significantly different) maximal increases in 'Sc and Vt, and both significantly reduced Rt (Table 2.1). These electrical variables for steady-state unstimulated recta (controls) and the values after NCC stimulation were similar to those reported by Hanrahan and Phillips (1985), using extracts of whole corpora cardiacum (CC).  29  Table 2.1. Effect of NCC and cAMP upon rectal electrical variables 'Sc^Vt^Rt (neq.cm-2.h-1)^(mV)^(S2.cm2)  8.48 ± 0.80a 20.89 ± 1.77b  265.41 ± 48.82a 120.88 ± 18.85bc  8  + 2NCC  1.44 ± 0.22a 7.27 ± 0.92b  + 5mM cAMP  1.45 ±0.24a 9.30 ± 1.01b  3.98 ±0.47a 19.18^2.68b  118.22 ± 18.66b 79.27 ± 7.57c  10  Values sharing the same letter are not significantly different from each other (Tukey test, p> 0.05). Vt values are lumen side positive  30  Fig. 2.3. Effect of NCC and cAMP on rectal I sc : 5mM cAMP and 2 NCC equivalents  were added to the hemocoel side of recta that had been allowed to equilibrate for 2h. Mean ± S.E. (n=6-8).  31  0  0  1  2^3  4  5  Time (h)  Fig. 2.4. Fluid reabsorption by everted rectal sacs with time. Complex saline controls hours 1-4 (C), stimulation with 2 NCC equivalents during hours 3+4 (NCC). Mean ± S.E. (n=8-11). ** values are significantly different from same hour controls (C) (independent ttest, p< 0.01).  32  Fig. 2.5. Tissue swelling by the everted rectal sacs from Fig. 2.4. Complex saline controls  hours 1-4 (C), stimulation with 2 NCC equivalents during hours 3+4 (NCC). Mean ± S.E. (n=8-11). ** value is significantly different from same hour control (C), (independent ttest, p< 0.01).  33 Unstimulated rectal sacs had a mean Jv of 18.7 ± 0.8 1_11.,1-1 during the first hour after excision (Fig. 2.4). Jv fell steadily to 12.7 ± 0.7 during the second hour, but decreased more slowly during the third and fourth hours. Although steady Jv was not observed, these Jv values are much higher than those reported in earlier studies (Proux et al., 1984). Tissue swelling of 1.8 ± 0.3 [tL.h-1 was observed during the first hour, but tissue volume fell slowly over the next three hours to near the initial value (Fig. 2.5). The addition of 2 NCC to the hemocoel side of rectal sacs during the third hour increased Jv by 4.4 ± 0.5 pL.11-1 compared to the control values for the same period (Fig. 2.4 and 2.6). This increase in Jv was similar to maximal values obtained in previous experiments (Proux et al., 1984). When rectal sacs were also exposed to NCC during the fourth hour, Jv remained elevated compared to the control value (Fig. 2.4) and there was a small but significant reduction in tissue swelling compared to control values (Fig. 2.5). DiButyryl cAMP (DBcAMP, 1 mM) and cAMP (5 mM) were also shown to increase Jv during the third hour to a similar extent as NCC (Fig. 2.6). DBcAMP and cAMP caused no differences in tissue swelling compared to control values. Effect of chemicals acting on the cAMP mediated pathway SpcAMPS and RpcAMPS are respectively specific stimulators and inhibitors of protein kinase A (PKA; see Fig. 2.1). SpcAMPS (5 mM) maximally stimulated Jv, while 1-10 mM RpcAMPS had no effect upon Jv when added during the third hour (Fig. 2.7). However when rectal sacs were pre-incubated with 10 mM RpcAMPS during the second hour and then stimulated with NCC on the third hour, there was a significant reduction in A Jv compared to the NCC control (1.8 ± 1.0 pl—h4 compared to 4.4 ± .05 AL.111; Fig. 2.8). Although the stimulatory effect of cAMP upon Jv and 'Sc had already been demonstrated, it was also necessary to show that cAMP was acting intracellularly and not via cell surface purigenic receptors. The involvement of purigenic surface receptors in  34  8  *  * *  ^  0  -  T C  NCC  cAMP DB cAMP  Fig. 2.6. Effect of 2 NCC, 5 mM cAMP and 1 mM DBcAMP upon rectal J. Mean ± S.E. (n=6-11). * values are significantly different from control (C), (Tukey test, p< 0.05).  35  Fig. 2.7. Effect of cAMP, SpcAMPS and RpcAMPS upon rectal J. Mean ± S.E. (n=68). Values sharing the same letter are not significantly different from each other (Tukey test, p> 0.05).  36  Fig. 2.8. Effect of a lh pre-incubation with 10 mM RpcAMPS on stimulation of rectal Jv  by 2 NCC. Mean ± S.E. (n=11-12). * RpcAMPS+NCC is significantly different from both control (C) and NCC values (Tukey test, p< 0.05).  37  12  ,NN +cAMP  10  ATP Adn.  Fig. 2.9. Effect of ATP and Adenosine (Adn) upon rectal 'Sc (open bars). 5 mM ATP  and 5mM Adn were added to hemocoel side at steady-state: 30 min. after testing these agents, 5mM cAMP was added to hemocoel side of the same preparations (hatched bars). Mean ± S.E. (n=4-10).  38 stimulation of 'Sc was tested by adding 5 mM adenosine (preferred by Pi purigenic receptors) and 5 mM ATP (preferred by P2 purigenic receptors) to the hemocoel side of recta at Steady-state. Neither adenosine nor ATP had any effect upon rectal 'Sc or on the response of recta to stimulation with cAMP (Fig. 2.9). The effects of these agents on rectal Jv were not tested. The effect of IBMX (a phosphodiesterase inhibitor) upon rectal 'Sc is shown in Fig. 2.10. lBMX alone significantly increased Ise from 1.44 ± 0.29 peq.cm-2.h-1 to 4.96 ± 1.33 peq.cm-2.11-1. When NCC was added Ise further increased significantly but this IBMX + NCC value was not significantly different (Tukey test, p> 0.05) from the value for NCC alone. Effect of cGMP  Cyclic GMP (5 mM) significantly increased A Jv but not to the extent of 5 mM cAMP (2.3 ± 0.6 tiL.11-1 compared to 4.5 ± 0.6 tiL.11-1, see Fig. 2.11). Cyclic GMP (5 mM) also slowly increased Isc (Fig 2.12), increased Vt and lowered Rt (Table 2.2). Subsequent addition of 5 mM cAMP in the presence of cGMP further increased Vt and further reduced Rt, and caused Ise to rise four times more rapidly (Fig 2.12 and Table 2.2).  39  Table 2.2. Effect of cGMP upon rectal electrical variables and the subsequent addition of cAMP 30 minutes later (n=8). t^ 'Sc^Vt^Rt (mins)^(peq.cm-2.h-1)^(mV)^(cicm2) 1.41 ± 0.12  6.12 ± 0.81  167.63 ± 24.06  5mM cGMP  3.69 ± 0.26*  13.80 ± 1.77*  141.55 ± 17.42*  5mM cAMP  7.95 ± 0.57*  20.67 ± 2.31*  97.49 ± 9.23*  120  C  150 180  * values are significantly different from preceding values (paired t-test, p< 0.05). t is time after mounting recta Vt is lumen side positive  40  -  14 12 -  -  10  -  *  _ -  C NCC  ^  C IBMX IBMX +NCC  Fig. 2.10. Effect of 'BMX upon rectal 'sc. B3MX (0.1 mM) was added to hemocoel side  at Steady-state: 30 min. later 2 NCC equivalents were added to hemocoel side. Mean ± S.E. (n=8). * IBMX is significantly different from its control (C), (paired t-test, p< 0.05).  41  8  I  0  T C  cAMP  cGMP  Fig, 2.11. Effect of 5mM cGMP and 5 mM cAMP upon rectal J,. Mean ± S.E. (n=6).  * cGMP is significantly different from both the Control (C) and cAMP values (Tukey test, p< 0.05).  42  Fig. 2.12. Effect of cGMP upon rectal Isc. 5 mM cGMP was added to hemocoel side at  steady-state, 30 min. later 5mM cAMP was added to hemocoel side. Mean ± S.E. (n=8).  43 DISCUSSION In this chapter, the effects of agents that influence the adenylate cyclase pathway on rectal Jv and 'Sc were found to be quantitatively similar, as expected if active transport of (Isc) drives secondary transport of fluid (Jv). Cyclic AMP and its analogs (Dibutyryl cAMP, SpcAMPS) were shown to stimulate rectal Jv and 'Sc to the same extent as NCC homogenate. This suggests that activation of the adenylate cyclase signal transduction pathway is sufficient for the maximal stimulation of rectal Jv and Ise. In support of this, Chamberlin and Phillips (1988) showed that forskolin, an adenylate cyclase activator, also maximally stimulated rectal Ise. While the cAMP analog SpcAMPS maximally stimulated Iv, its diastereoisomer RpcAMPS partially inhibited the stimulation of Jv by NCC. Since RpcAMPS is known to inhibit cAMP dependant protein kinases (i.e. PKA; Rothermel and Parker-Botelho, 1988), this partial inhibition of Jv could indicate that NCC factors may utilize other signal transduction pathways to stimulate rectal reabsorption. Another possibility is that RpcAMPS was unable to fully inhibit the activation of PKA by elevated intracellular cAMP levels. It seems unlikely that cAMP and its analogs act by stimulating cell surface purinergic receptors since addition of ATP and adenosine had no effect on rectal C1 transport. The inhibition of rectal cAMP phoshodiesterase activity by B3MX, presumably increased cAMP levels to cause a half maximal increase in rectal transport. Chamberlin and Phillips (1988) have shown that theophylline, another PdE inhibitor, did cause an increase in rectal cAMP levels. The effects of IBMX and NCC were not additive, since addition of both only stimulated 'Sc to the same extent as addition of NCC alone. This study also provides evidence that the guanylate cyclase signal transduction pathway is involved with the stimulation of rectal fluid and ion reabsorption. Addition of exogenous cGMP caused partial stimulation of rectal 'Sc and Jv (compared with NCC and cAMP). This cGMP-stimulated increase in rectal Ise is similar in magnitude to a cGMP-  44 stimulated increase in 'Sc previously reported by Chamberlin and Phillips (1988), and the rise in 'Sc correlates with the delayed rise in rectal tissue cGMP levels observed by these workers. Fournier and Dubar (1989) have reported that nitroprusside, which is an activator of soluble guanylate cyclase, and cGMP both had a diuretic effect upon Locusta migratoria recta. They also showed that neuroparsins, an antidiuretic factor isolated from  NCC of L. migratoria, does not increase cellular cAMP levels but does slightly elevate cellular cGMP. These findings suggest that S. gregaria and L. migratoria which are closely related orthopterans, possess different antidiuretic factors that utilize different signal transduction mechanisms. However these differences may be of result of the unusual conditions used to measure Locusta rectal Jv. In the Locusta experiments,  a- was omitted  from the bathing saline on the first hour and added to the saline at the same time as exposure to the treatment (i.e. neuroparsins, pharmacological agents) at the onset of the second hour (see Fournier et al., 1987). Restoration of C1 to the tissue might be expected to trigger tissue swelling and hence second messenger events to restore cell volume. The involvement of both the adenylate and guanylate cyclase signal transduction pathways in controlling S. gregaria rectal reabsorption, does not rule out the possibility that other alternative control pathways are also involved. Rectal transport mechanisms may be controlled and fine-tuned by the interaction of multiple intracellular mechanisms (e.g. elevated intracellular cAMP might increase cellular Ca2+ levels etc.) . The following two chapters will investigate the role of other signal transduction mechanisms in the control of rectal transport.  45  CHAPTER 3: THE INVOLVEMENT OF CALCIUM IN CONTROLLING RECTAL TRANSPORT INTRODUCTION  Heilbrunn and Wiercenski (1947) were the first to demonstrate that Ca2+ is involved in cellular control when they injected Ca2+ into frog muscle cells and caused them to contract. Subsequently, intracellular Ca2+ has been shown to regulate many cellular processes in vertebrates, but less is known about the role of Ca2-1- in controlling insect cellular events (see: Berridge, 1983; Berridge and Irvine, 1984). A rise in cytosolic Ca2+ concentration elicits a plethora of protein modifications that allows the cell to respond specifically and quickly to numerous stimuli. Low resting cytosolic Ca2+ levels are crucial for cellular control and homeostasis. Free cytosolic Ca2+ is usually less than 10-7 M, while concentrations of external Ca2+ and the Ca2+ sequestered in intracellular compartments are normally in the millimolar range (Berridge, 1983). Therefore there is a large gradient for the movement of Ca2+ into the cytosol, although the total Ca2+ concentration in the cell is roughly equal to the external concentration. This huge gradient is maintained by numerous mechanisms that include: the active pumping of Ca2+ by Ca21-/ATPases into intracellular stores or outside of the cell, the expulsion of Ca2+ via plasma membrane Na-1-/ Ca2+ ion exchangers, the entry of Ca2+ across the favourable cation gradient of the mitochondria, and binding of Ca2+ to molecules in the cytosol (see Fig. 3.1; reviewed by Alberts et al., 1989). It is important to note that entry of Ca2+ into the cytosol is primarily across the membranes of the internal Ca2+ sequestering compartments, since the surface area of the plasma membrane is 10-100 times less than the total surface area of the Ca2+ containing organelles (Alberts et al., 1989). Calcium binding proteins, such as troponin C and calmodulin, are responsible for translating these Ca2+ increases into cellular responses (Berridge, 1983). These proteins  46  Fig.3.1. Schematic diagram of the cellular processes involved with the regulation of intracellular Ca2+. Calcium pumps and ion exchangers are represented by shaded circles at the plasma membrane and the membrane of the intracellular stores. Ion channels and ionophores are represented by an arrow passing between parallel lines, while agents that decrease or increase cytosolic Ca2+ appear in solid boxes with associated -/+ signs.  47 possess high-affinity Ca2+ binding sites that are very specific for Ca2+ since cytosolic Mg2+ levels are typically high (in the millirnolar range, Alberts et al., 1989). The binding of Ca2+ to these high-affinity sites alters the conformation of these Ca2+ binding proteins, allowing them to associate with and activate enzymes involved with various cellular processes. Cellular effects that are mediated through Ca2+ binding proteins include: Ca2+ induced contraction of vertebrate skeletal muscle, pre-synaptic release of neurotransmitters by nerve cells, and the activation of Ca2+ pumps that lower cytosolic Ca2+ to create a negative feedback loop (Berridge, 1983). It must be kept in mind that intracellular Ca2+ and cAMP can interact to control various cellular processes (reviewed by Cheung, 1982). The first of these interactions is that Ca2+ and cAMP levels can modulate each other. For example Ca2+ levels can regulate enzymes that breakdown (e.g. phosphodiesterases) or synthesize cAMP. In addition cAMP-dependent protein kinases (e.g. protein kinase A) can phosphorylate Ca2+ channels or pumps, resulting in increases  or decreases in intracellular Ca2+. In the second type of interaction, both Ca2+ and cAMP can regulate the same protein. An example of this is phosphorylase kinase, which is involved with glycogen breakdown and can be stimulated by a cAMP-dependent kinase as well as by Ca2+ binding to calmodulin. There is some evidence that Ca2+ is involved with the control of insect excretion. Spring et al. (1988b) have isolated an antidiuretic factor from the cricket (Acheta domesticus) that was shown to inhibit Malpighian tubule secretion by 70%. Spring and  Clark (1990) later showed that the calcium ionophore A23187 also reduced Malpighian tubule secretion by 85%, and proposed that this antidiuretic factor inhibited fluid secretion by elevating intracellular Ca2+ (see also Kim and Spring, 1992). Since many workers have reported that insect diuretic factors stimulate Malpighian tubule secretion via cAMP (Aston, 1975; Morgan and Mordue, 1985; Beyenbach and Petzel, 1987; Spring and Clark, 1990; Coast et al., 1992), it would appear as though cAMP and Ca2+ may interact in an antagonistic manner. Fogg et al. (1990) showed that when L. migratoria Malpighian  48 secretion was stimulated with CC homogenate, both cAMP and inositol trisphosphate (InsP3) levels rose. Since InsP3 stimulates the release of sequestered Ca2+, it would appear that both Ca2+ and cAMP act cooperatively in the stimulation of fluid secretion. Coast (personal communication, 1992) has used the calcium ionophore ionomycin to increase Ca2+ levels and has also seen an increase in Malpighian tubule secretion. Apparently Ca2+ is important for the control of Malpighian tubule secretion, but there is conflicting evidence concerning the role Ca2+ plays in this control. Calcium may also be involved with the control of rectal transport mechanisms, even though Hanrahan and Phillips (1985) saw no effect upon S. gregaria rectal C1 transport when a Ca2+ ionophore was added to the preparation. Fournier (1990b) presented evidence to show that L. migratoria rectal fluid transport (iv) increased when Ca2+ ionophores were added. He has also shown that L. migratoria neuroparsins (a potential insect antidiuretic peptide) increased InsP3 levels suggesting that Ca2+ is involved with the stimulation of rectal Jv. Fournier (1990b) claims that rectal Jv is dependant upon extracellular Ca2+ levels, since removal of Ca2+ from incubating saline significantly reduced rectal J. This chapter deals with the role of calcium in the control of rectal Jv and 'Sc in S. gregaria.  The influence of external Ca2+ upon rectal transport was investigated by  removing Ca2+ from the bathing medium. In addition, the ability of NCC homogenate and cAMP to stimulate rectal transport under Ca2+ free conditions was studied. The role of intracellular Ca2+ in controlling rectal transport was explored by using agents known to modulate cytosolic Ca2+ levels (see Fig.3.1). The calcium ionophore ionomycin (IY) in known to increase cytosolic Ca2+ levels by inserting into both the plasma membrane and the membranes of the intracellular organelles (Fournier, 1990b). Thapsigargin (THAPS) is also known to increase cytosolic Ca2+ by inhibiting the Ca2+/ATPase pump that is responsible for removing Ca2+ from the cytosol (Takemura et al, 1989; 'Thastrup et al., 1990). Trimethoxybenzoate hydochloride (TMB-8) is thought to lower cytosolic Ca2+ by  49 preventing its release from intracellular stores (Smith and Iden, 1979). The phosholipase C (PLC) mediated signal transduction pathway, via inositol 4,5-trisphosphate (InsP3), is also known to increase intracellular Ca2+ levels. The involvement of this PLC pathway in the control of rectal transport will be addressed in the next chapter.  MATERIALS AND METHODS  The methods used in this study were similar to those described in Chapter 2, unless otherwise indicated. The locust colony was maintained under similar conditions and the NCC homogenates were prepared using the methods described in Chapter 2. Most of the chemicals were supplied by the Sigma Chemical Company (St. Louis MO, USA). Thapsigargin was supplied by CALBIOCHEM Corporation (San Diego CA, USA). Most of the tested substances were dissolved in complex saline or calcium-free complex saline. However, Ionomycin and Thapsigargin were initially dissolved in 100% DMSO and these stock solutions were then diluted with complex saline (fmal DMSO concentration 5_ 1%). DMSO (1%) alone had no effect upon rectal h or Isc compared to complex saline controls (e.g. 3rd hour rectal Jv: 10.3 ± 0.4 tiL.11-1, n=7, for the DMSO control; compared to the complex saline control of 9.8 ± 0.6 IIL.11-1, n=8). The composition and maintenance of the complex saline were described in Chapter 2. The Ca2+ free saline had a similar composition to the complex saline except that it did not have 5 mM CaCl2 and contained 1 mM EGTA to ensure that any trace amounts of Ca2+ would be chelated.  50 RESULTS  Effect of Ca2+ free saline  Incubation of rectal sacs in calcium-free saline (Cat) for four hours did not cause any decrease in Jv compared to control values over the same time course (Fig.3.2). EGTA (1 mM) was added to the Ca2+ free saline to ensure that any trace amounts of Ca2+ were chelated, and this saline gave similar results as Ca2+ free saline without EGTA (data not shown). When rectal sacs were incubated in saline with 10 mM EGTA, Jv was much lower during the first hour (5.1 ± 0.9 pL.h-1, n=6; compared to the control value of 18.7 ± 0.8 pL.h-1, n=8) and on the third hour Jv was completely abolished (0.1 ± 0.4 pL.h-1, n=6; compared to the control value of 9.8 ± 0.6 pL.h4) . By the third hour a white foamy liquid was being collected from the hemocoel side of these sacs and the lumen side appeared whitish instead of the usual yellow/brown. It seems that 10 mM EGTA is toxic to rectal cells and causes cell dissociation on the hemocoel side. Cell dissociation was not seen on the lumen side probably because the cuticle helped to hold the rectal tissue together. Both NCC and DBcAMP increased Jv to a greater extent in Ca2+ free saline than in complex saline with Ca21- (Figs. 3.2 & 3.4). Stimulation of recta with 2 NCC in Ca2+ free saline also caused a slight increase in rectal tissue swelling (,-,.. 1 pL.) compared to NCC controls (Fig. 3.3). Exposure of unstimulated recta to Ca2+ free saline had no effect upon Isc and subsequent stimulation of recta with NCC gave similar changes in electrical variables as seen with NCC controls in normal saline (Table 3.1).  51  Table 3.1. Effect of Calcium free saline upon electrical variables ^n Isc^Vt^Rt (fieq.cm-2.h-1)^(mV)^(Ci.cm2) Control + 2NCC  1.44 ± 0.22a 7.27 ± 0.92b  8.48 ± 0.80a 20.89 ± 1.77b  265.41 ± 48.82a 120.88 ± 18.85b  8  Ca2+ free + 2NCC  2.54 ± 0.40a 10.07 ± 2.10 b  10.94 ± 2.30a 20.36 ± 2.5 lb  166.83 ± 22.83ab 88.55 ± 13.91b  7  Values sharing the same letter are not significantly different (Tukey test, p> 0.05) Vt values are lumen side positive  52  0  0  1  2^3  4  5  Time (h)  Fig. 3.2. Fluid reabsorption by rectal sacs under Ca2+ free conditions. Control complex  saline hours 1-4 (C), complex saline with 2 NCC added to hemocoel side on hours 3+4 (NCC), Ca2+ free saline hours 1-4 (Cat), Ca2+ free saline for hours 1-3 with 2 NCC added to hemocoel side on third hour (Caf+NCC). Mean ± S.E. (n=8-13). Values from similar times that share the same letter are not significantly different (Tukey test, p> 0.05).  53  Fig. 3.3. Tissue swelling of rectal sacs under Ca2+ free conditions. 2 NCC added on  hours 3+4 in presence of Ca2+ (NCC), Ca2+ free saline for hours 1-3 with 2 NCC added on third hour (Caf+NCC). Mean ± S.E. (n=11-13). ** Caf+NCC is significantly different from NCC (independent t-test, p< 0.01).  54  12  10  .\\1  with 5mM Ca" Ca" free  *  _ _ T  C Cal NCC DBcAMP  Fig. 3.4. Effect of Ca2+ free saline (Cal) upon Jv stimulated by 2 NCC and 1 mM  DBcAMP. Mean ± S.E. (n=10-13). * values are significantly different from similar treatments with Ca2+ in bathing saline (Tukey test, p< 0.05).  55 Effect of agents that modulate cytosolic Calcium The calcium ionphore ionomycin, an agonist of intracellular Ca2+, had a partial stimulatory effect upon Jv (40% of maximum) when used at 4 pM, but no effect at liiM (Fig. 3.5). Fournier (1990b) claims that elevating the bathing saline Ca2+ concentration by 10% increases the effectiveness of ionomycin; however when this procedure was tried there was no additional stimulation of Jv by 4 iiM ionomycin (Fig. 3.5). No increase in Jv was seen when sacs were incubated in Ca 2+ free saline and then exposed to 4 iiM ionomycin. Addition of 1 1.1M ionomycin to unstimulated short-circuited recta, with calcium present, had no effect on Isc but reduced the stimulatory action of NCC by over 40% (4.1 ± 0.24 peq.cm-2.h--1 compared to 7.3 ± 0.9 peq.cm-2.h-1 for the NCC control, see Fig. 3.6). Thapsigargin, another agonist of intracellular Ca2+, had no effect upon resting Isc or NCC stimulated 'Sc when used at 10-50 AM (Fig. 3.6). TMB-8, an antagonist of intracellular Ca2+, had no effect upon rectal Ise or subsequent stimulation with cAMP when used at 200 and 1000 IAM concentrations. Although 1 mM TMB-8 reduced cAMP stimulated 'Sc by almost 50%, this value was not statistically different from the control (Tukey test, p> 0.05). However, 1 mM TMB-8 significantly reduced NCC stimulated Isc by 40% ( 4.3 ± 0.6 tieq.cm-2.h- 1 compared to 7.27 ± 0.92 peq.cm-2.h4 for the NCC control, see Fig. 3.8).  56  Fig. 3.5. Effect of Ionomycin (IY), IY with 5.5 mM Ca2+ (+10%) bathing saline  (Cax4IY) and IY with Ca2+ free saline (Caf4IY) upon rectal Jv. Mean ± S.E. (n=7-11). * values are significantly different from 1% DMSO controls (C), (Tukey test, p< 0.05).  57  10  \\N + NCC ^  -  * -  -  C  ^ ^ IY 10^50 THAPS (MM)  Fig. 3.6. Effect of 1 mM IY and Thapsigargin (THAPS) upon rectal Isc. At steady-state  agents were added bilaterally (open bars), and then 30 minutes later 2 NCC were added (hatched bars). Mean ± S.E. (n=5-8). * value is significantly different from corresponding  NCC control value (Tukey test, p< 0.05).  58  12  IM + cAMP -  10  _  C  ^  200^1000 TMB8 (AM)  Fig. 3.7. Effect of TMB-8 upon 5 mM cAMP stimulated 'Sc- At steady-state TMB-8 was  added bilaterally, 30 min. later cAMP was added. Mean ± S.E. (n=8-10).  59  12  \\N + NCC 10  * -  -  C TMB 8  Fig. 3.8. Effect of TMB-8 upon 2 NCC stimulated 'Sc. At steady-state 1 mM TMB-8 was  added bilaterally, 30 min. later 2 NCC were added. Mean ± S.E. (n=8-10). * value is significantly different from NCC control (Tukey test, p< 0.05).  60 DISCUSSION  The removal of external Ca2+ from the bathing medium did not reduce either the fluid or ion reabsorbing ability of the recta. When recta were stimulated with either NCC or cAMP under Ca2+ free conditions, there were larger increases in rectal Jv compared to increases seen when Ca2+ was present. Calcium-free conditions did not have any effect upon the stimulation of Isc or Vt with either cAMP or NCC. Since Ca2+ free conditions increased stimulated Jv without changing values for stimulated electrical variables, it would appear that the absence of Ca2+ may have an extracellular rather than an intracellular effect upon rectal cells. A possible effect of removing Ca2+ from the bathing saline could be the loosening of the tight junctions at the apical and basal complexes that hold adjacent rectal pad cells closely together (refer to Fig. 1.2). The opening of these complexes may facilitate the movement of fluid through the intercellular spaces toward the basal membrane. Fournier (1990b,1991) has reported that bathing Ca2+ is essential for the maintenance of resting rectal Jv in L. migratoria, since the incubation of tissues with the Ca2+ chelator EGTA dramatically reduced Jv by 90% within the first hour. Fournier (1990b) proposes that this apparent sensitivity of the rectum to external Ca2+ is a result of "an insufficient mobilization or lack of calcium from intracellular stores". It seems unlikely that these two closely related insects can possess recta that have differing dependencies upon external Ca2÷, so why are Fournier's findings so different from the results of this study? A possible reason for Fournier's different results, is that he may have used a high concentration of EGTA in his Ca2+ free bathing medium that proved toxic to the rectal cells. It is unclear what actual concentration of EGTA was used, but a Ca2+ free bathing saline containing 0.8 M Ca2+ with 1.2 M EGTA has been reported in numerous papers (see Fournier 1990b,1991; Fournier et al., 1992). In my study, 10 mM EGTA in Ca2+ free saline proved toxic to the rectal cells, almost abolishing all Jv by the third hour of incubation. It also  61 seems unlikely that intracellular rectal Ca2+ levels, which are regulated very closely by the cell and are essential for the maintenance of cellular homeostasis, are so dependant on external Ca2+. In support of the fmdings of this study, Berridge (1977) has also shown that fluid secretion by the salivary glands of the blow fly (Calliphora) was independent of external bathing Ca2+. The use of agents that modulate intracellular Ca2+ levels gave mixed results. Firstly, the addition of ionomycin to recta submaximally increased rectal Jv (-40%) compared to the increases seen with cAMP and NCC. Ionomycin had a small inhibitory effect upon resting 'Sc and reduced the stimulatory effect of NCC by 50%. This suggests that high intracellular Ca2+ partially inhibits the stimulation of transport by cAMP. In addition, TMB-8 slightly reduced resting 'Sc and reduced the stimulation of Isc by both NCC and cAMP by 40-50%. These TMB-8 results suggest that the stimulation of transport by cAMP is dependant upon the elevation of cytosolic Ca2+ levels by release from intracellular stores. Since thapsigargin had no effect upon resting or stimulated Ise, it must be concluded that either the agent did not enter the cell or that it could not elevate cytosolic Ca2+ levels to an extent where changes in 'Sc could be observed. In summary, it appears as though Ca2+ does play a role in the control of rectal transport processes. However it seems that the role of Ca2+ is quite complex, and that Ca2+ may have differing cellular effects depending upon its cytosolic concentration. Cytosolic Ca2+ alone was not able to cause a full stimulation of either Jv or Isc as suggested by Fournier (1990b,1991). Earlier evidence suggests that rectal Jv results from transport (Isc, Goh and Phillips, 1978). The seemingly different effects of Ca2+ on rectal Jv and 'Sc suggest that Ca2+ may control processes other than Cl transport which influence Jv (e.g. movement of counter ions such as K+ and Na+, or ion recycling in the lateral spaces of the rectal epithelium).  62  CHAPTER 4: THE ROLE OF PKC AND THE PI CYCLE IN CONTROLLING RECTAL TRANSPORT INTRODUCTION  In the previous chapter there were some indications that intracellular Ca2+ was involved with the control of rectal transport. Although the adenylate cyclase signal transduction pathway can alter cellular Ca2+ levels indirectly by opening Ca2+ channels, the phospholipase C (PLC) pathway, via inositol 1,4,5-trisphosphate (InsP3), is thought to be the primary method of elevating intracellular Ca2+ (Berridge and Irvine, 1984). PLC is activated by a G-protein subunit that is released from an activated receptor complex. When PLC is activated, it catalyzes the breakdown of phosphatidylinositol 4,5-bisphoshate (PIP2) into InsP3 and sn-1,2-diacylglycerol (DAG). While InsP3 activates the release of Ca2+ from intracellular stores and the entry of exogenous Ca2+, DAG stimulates the Ca2+ dependant protein kinase C (PKC) which then phosphorylates various cellular proteins (see Fig. 4.1; Berridge and Irvine, 1989; Nishizuka, 1986). There is some evidence that PLC-mediated events are involved in the control secretion in insects. Berridge (1986) reported that InsP3 levels rose in Calliphora salivary glands when serotonin (5-HT) stimulated secretion via the 5 HT]. receptor. Fogg et al. (1990) showed that there was an increase in InsP3 and cAMP levels following stimulation of L. migratoria Malpighian tubule secretion with CC homogenate. The elevation of cAMP  and InsP3 levels occurred between 30-300 s for cAMP and 15-300 s for InsP3. Fournier (1990b), using pharmacological methods, provided evidence that L. migratoria neuroparsins (Nps) stimulated rectal Jv through PLC-mediated events. When Li+ was used to inhibit the phosphotidylinositol (PI)  63  Hemolymph  Neuropeptide  Receptor 11.1.1.1.1.1...I.11  Cytosol  Intracellular  cascades  Calcium  of protein  Stores  modifications  CELLULAR RESPONSES  Fig. 4.1. Diagram of the phosholipase C (PLC) mediated signal transduction pathway. The  G protein activated PLC breaks down phosphatidylinositol 4,5-bisphoshate (PIP2) into diacylglycerol (DAG) and inositol trisphophate (InsP3). InsP3 then opens Ca2+ channels that are represented by an arrow passing between two parallel lines. The cellular action of agents added to the rectal preparations are shown by arrows with associated +/- signs.  64 cycle, the stimulatory effect of Nps was abolished. The addition of the PI cycle component myo-inositol was sufficient to maximally stimulate rectal Jv. Fournier (1990b) also suggested that PKC was involved in the stimulation of L. migratoria Jv since addition of specific PKC stimulators maximally stimulated Jv, while addition of the PKC inhibitor Polymyxin B (PMXB) abolished the stimulatory effect of Nps. Fournier (1991) has recently demonstrated that Nps stimulate InsP3 and inositol 1,3,4,5 tetrakisphosphate (InsP4) production in L. migratoria rectal cells. In addition it was suggested that PKC activity is necessary for the phosphorylation of enzymes involved with inositol phosphate metabolism, since the inositol phosphate cascade was mimicked using PKC stimulators and inhibited with PMXB. It was also reported that neither the adenylate nor guanylate cyclase systems modified inositol phosphate metabolism. In this chapter the role of the PLC-mediated signal transduction pathway in controlling rectal transport was studied. Specific stimulators and inhibitors of protein kinase C were used to explore its role in controlling rectal transport. The PKC stimulators used were 1stearoy1-2-arachidonoyl-sn-glycerol (SAG) and phorbol 12-myristate-13-acetate (PMA), while polymyxin B sulfate was used to inhibit PKC. The involvement of the phosphatidylinositol (PI) cycle in the control of rectal transport was examined by attempting to stimulate it with myo-inositol, a component of the PI cycle. In addition, the effects of inhibiting the PI cycle were investigated using U. Lithium is known to block the conversion of inositol phosphate to inositol and hence synthesis of inositol trisphosphate (InsP3; see Fig. 4.1; Berridge and Irvine, 1989). L. migratoria rectal preparations were used to investigate the role of the PI cycle in controlling rectal Jv and Isc, since Fournier (1990b, 1991) had previously indicated that L. migratoria neuroparsins increased rectal Jv by increasing InsP3 levels. Neuroparsins has also been shown to have anti-juvenile hormone activity and increase hemolymph trehalose and lipid levels (Girardie et al., 1987; Moreau et al., 1988) Finally, neuroparsins was tested on S. gregaria rectal preparations to test for any cross-reactivity between these species.  65 MATERIALS AND METHODS  Most of the methods used in these experiments were similar to those described in Chapter 2, unless otherwise stated. The Locusta migratoria colony was maintained as previously described for the Schistocerca gregaria colony. Adult 6 week old male Locusta migratoria were used for the Locusta fluid transport experiments, while 6 week  old female Locusta migratoria were used for the Locusta ion transport experiments. All chemicals were supplied by the Sigma Chemical Company (St. Louis MO, USA). Most of the tested substances were dissolved in complex saline. However, 1-stearoy1-2arachidonoyl-sn-glycerol (SAG) and phorbol 12-myristate-13-acetate (PMA) were initially dissolved in 100% dimethyl sulfoxide (DMSO) and these stock solutions were then diluted with complex saline (fmal DMSO concentration 5. 1%). Neuroparsins (Nps), purified from NCC of Locusta migratoria (Girardie et al. 1987b), was a gift from J. Proux (Laboratoire de Neuroendocrinologie, Universite de Bordeaux, Talence, France).  RESULTS  Effect of agents acting upon protein kinase C  Stimulators of PKC, SAG and PMA had no stimulatory activity upon S. gregaria rectal Jv and 'Sc (Figs. 4.2 & 4.3). Polymyxin B sulfate (PMXB), a PKC inhibitor, slightly increased NCC stimulated Jv (6.1 ± 0.6 pL.11-1 compared to 4.4 ± 0.5 pL.h-1 with NCC alone), but had no effect upon unstimulated or NCC stimulated 'Sc (Figs. 4.4 & 4.5).  66 Effects of agents acting upon the Phosphotidyl-Inositol cycle  Myo-inositol (MI) is a component of the PI cycle and was shown to have no effect upon S. gregaria A Jv (-1.7 ± 0.6 pit.h-1) when used at 0.1-50 mM (Fig. 4.6). In addition, MI  had no effect upon rectal Isc at either low or high concentrations (Fig. 4.7). LiC1 is an inhibitor of the PI cycle, and its effects upon Jv and 'Sc were studied using a 10 minute pulse of 10 mM LiC1 applied to recta immediately before adding NCC (as described by Fournier 1991). LiC1 had no effect upon NCC stimulated Jv (Fig. 4.6), did not change unstimulated Isc nor alter NCC stimulated Isc (Fig. 4.7). In addition when LiC1 (1mM) was present in the incubating saline throughout the experiment there was no effect upon NCC stimulated A Jv (3.9 ± 1.0 pL.114 compared to 4.4 ± 0.5 p.L.h-1 for NCC alone; both n=11). However, the same LiC1 treatment did increase NCC stimulated 'Sc and Vt compared to control values (see Table 4.1.).  67  Table 4.1. Effect of 1mM LiC1 upon electrical variables ^Vt (mV)^ 'Sc Rt ^ (peq.cm-2.11-1) (S2.em2) Control + 2NCC  1.4 ± 0.2 7.3 ± 0.9  8.5 ± 0.8 20.9 ± 1.8  265.4 ± 48.8 120.9 ± 18.9  8  LiC1 LiC1 + 2NCC  2.7 ± 0.6 12.8 ± 1.5*  20.7 ± 5.1 52.9 ± 8.4*  272.2 ± 25.9 150.0 ± 8.6  8  * values are significantly different from the respective control values (Tukey test, p< 0.05). Vt values are lumen side positive  Fig. 4.2. Effect of SAG and PMA upon rectal J. Mean ± S.E. (n=5-11).  Fig. 4.3. Effect of SAG and PMA upon rectal Ise. Mean ± S.E. (n=4-8).  70  8  *  .......^  7  6  T  _  -  5  0 C.)  a)  '-D  T  -  -  >  <2  -  0  -  T C  PMXB +NCC  only NCC  Fig. 4.4. Effect of a 100pM PMXB (1000 iu/mL.) upon 2 NCC stimulated J. Recta were pre-incubated with PMXB for 1 hour before stimulation with NCC. Mean + S.E. (n=11-16). * PMXB+NCC value is significantly different from both control (C) and NCC values (Tukey test, p< 0.05).  71  10  8  -  -  T  2 -I-  0  C NCC  ^  C PMXB PMXB +NCC  Fig. 4.5. Effect of 100 pM (1000 iu/mL.) PMXB upon rectal Isc• PMXB was added  bilaterally at steady-state, and then 30 minutes later 2 NCC were added. Mean ± S.E. (n=7-8).  72  8 -  6  T  4 2  -  0 —2 —4  I  I  I -  ^ C^50^1^0.1 ^ LiC1 only MI (mM) +NCC NCC  Fig. 4.6. Effects of myo-inositol (MI) and LiC1 upon rectal Jv. In the LiC1 experiment recta were exposed to a 10 minute pulse of 10 mM LiC1 and then immediately stimulated with 2 NCC. Mean ± S.E. (n=6-11).  73  10  8  ^ 1  a)  6  4  2 -r  0  ^ .1 C NCC LiC1 LiC1 50^1^0^ MI (mM) +NCC  Fig. 4.7. Effects of MI and LiC1 upon rectal 'sc. In the LiC1 experiment recta were exposed to a 10 minute pulse of 10 mM LiC1 and then immediately stimulated with 2 NCC. Mean ±S.E. (n=5-17).  74  Locusta experiments Locusta rectal sacs transported fluid at a slower rate overall than those of Schistocerca. Locusta rectal sacs transported 8.1 ± 0.9 1.iL.11-1 and 6.4 ± 0.9 1iL.11-1 during hours one  and two respectively (Fig. 4.8), while Schistocerca sacs transported 18.7 ± 0.8 pL.11-1 and 12.7 ± 0.7 mI..,.11-1 at the same time intervals. Locusta rectal sacs exhibited a small amount of tissue swelling (1.0 ± 0.5 tiL11-1.) during the first hour and a negligible tissue tissue volume change over the following two hours (Fig.4.8), similar to the response observed for Schistocerca sacs (Fig. 2.5). Addition of 50 mM MI to Locusta rectal sacs on the third hour had no effect upon Jv, while the addition of 1 mM DBcAMP during the fourth hour significantly increased Jv (Fig. 4.8). Unstimulated electrical variables for Locusta recta (Table 4.2) closely resembled equivalent Schistocerca values (Table 2.1). The addition of 50 mM MI had no effect upon Locusta electrical variables, while subsequent additon of 1 mM DBcAMP stimulated Isc,  Vt and reduced Rt. Locusta electrical variables after addition of DBcAMP closely resemble Schistocerca electrical variables after stimulation with cAMP or NCC. Unfortunately further experiments using Locusta migratoria recta could not be performed because the colony perished.  Effect of Neuroparsins on S.gregaria recta  Neuroparsins applied at a very high concentration of 5CC gland equivalents had no effect upon S. gregaria rectal Jv or Isc (Figs. 4.9 & 4.10). All preparations subsequently responded to S. gregaria NCC. Unfortunately we were unable to test the effect of Nps on L. migratoria recta since there were no animals available.  75  Table 4.2. Effect of MI and DBcAMP upon electrical variables of L. migratoria recta  (n=4)  Isc (peq.cm-2.11-1)  Vt (mV)  Rt (Q.cm2)  Control  1.55 ± 0.42  11.68 ± 2.73  288.56 ± 17.10  50mM MI  1.44 ± 0.42  11.50 ± 3.50  289.01± 14.19  1mM DBcAMP  10.27 ± 2.17*  33.75 ± 5.25*  30.25 ± 13.44*  * value is significantly different from preceding value (paired t-test, p< 0.05). Vt values are lumen side positive  76  Fig. 4.8. Fluid reabsorption by Locusta migratoria everted rectal sacs. During the  third hour 50 mM MI was added to the hemocoel side, then 1 mM DBcAMP was added to the hemocoel side during the fourth hour. Tissue swelling is represented by the dotted line. Mean ± S.E. (n=6). * fourth hour value is significantly different from third hour value (p< 0.05, paired t-test).  77  Fig. 4.9. Effect of Neuroparsins upon rectal S. gregaria J. Neuroparsins (Nps; 5CC  gland equivalents) or 2NCC were added to the hemocoel side during the third hour. Mean ± S.E. (n=8-11).  78  10  8  -  -  C.)  rn  2 T  0  C NCC C Nps NCC  Fig. 4.10. Effects of Neuroparsins upon S. gregaria rectal Ise. Neuroparsins (Nps; 5CC  gland equivalents) was added to the hemocoel side of recta at steady-state, 30 minutes later 2NCC were then added to the same preparations. These results are compared to control preparations to which only NCC was added. Mean ± S.E. (n=8).  79 DISCUSSION  In this study, neither PKC nor the PI cycle were shown to play a stimulatory role in the control of S. gregaria rectal transport. However PKC may have a slight inhibitory effect upon rectal Jv, since the PKC inhibitor PMXB slightly increased NCC-stimulated J. It also seems that the PI cycle plays a slight inhibitory role in controlling rectal transport since long-term Li+ exposure elevated NCC-stimulated 'Sc and Vt. These results are markedly different from those with L. migratoria recta, where stimulants of PKC and the PI cycle were reported to maximally increase Jv, while specific inhibitors prevented stimulation of Jv with Nps (Fournier, 1990b). Locusta migratoria rectal preparations were shown to possess similar electrical  variables as S. gregaria recta, although L. migratoria recta transported fluid at slightly lower rates than S. gregaria recta, probably because L. migratoria recta are smaller. The patterns of L. migratoria Jv and tissue swelling with time were similar to patterns previously seen for S. gregaria recta. Although both L. migratoria Jv and Isc responded to stimulation with Dibutyryl cAMP, myo-inositol had no effect. Since myo-inositol had no effect, it would seem that the PI cycle is not involved in controlling L. migratoria Jv or 'sc. This conclusion contradicts the findings of Fournier (1990b) who has reported that 50 mM myo-inositol can maximally increase L. migratoria rectal Jv. Finally, there was no cross-reactivity between L. migratoria neuroparsins and the S. gregaria rectal preparations, even though the same Nps sample was later shown to have significant fluid transporting activity on L. migratoria recta (J. Proux, personal communication). Since our lab was unable to demonstrate any transport activity with Nps on S. gregaria recta or ilea (J. Martin, unpublished results), we were unable to investigate the activation of signal transduction pathways by Nps using our bioassays. It would initially appear that L. migratoria and S. gregaria recta are controlled by different antidiuretic peptides that employ separate signal transduction mechanisms.  80 However, Herault and Proux (1987) showed that there is a factor in L. migratoria GCC that increases rectal Jv by elevating intracellular cAMP. In addition, L. migratoria CC homogenates have been shown to maximally stimulate S. gregaria rectal Isc, suggesting that L. migratoria does possess factors with S. gregaria cross-reactivity (J. Marshall, unpublished results). This present study raises an important question: Is L. migratoria neuroparsins a true antidiuretic factor, or is its reported activity an artifact of the unusual bioassay procedure used to study rectal Jv? This unusual procedure was discussed in chapter 2, and involves the omission of Cl- from the bathing saline during the first hour of rectal incubation. On the second hour, Cl- was added back to the saline at the same time as the tissue is exposed to various treatments (e.g. neuroparsins, pharmacological agents). The re-introduction of Cl- to the bathing medium combined with the action of the various treatments, including Nps, may trigger second messenger mechanisms that are involved with cell volume regulation, leading to apparent increases in rectal Jv. These experiments by Fournier's group need to be repeated using steady-state conditions, where Cl- is present throughout the experiment, before neuroparsins can be considered a potential antidiuretic factor.  81  CHAPTER 5: GENERAL DISCUSSION  The fmdings of this study suggest that cAMP is the primary second messenger responsible for controlling rectal C1 and fluid transport in Schistocerca gregaria, and that other second messengers are also involved. The second messenger cGMP was also shown to have a partial stimulatory action in controlling rectal transport. The evidence for the role of Ca2+ in controlling rectal transport is interesting, suggesting that different cellular Ca2+ levels may have varying effects (e.g. stimulatory, inhibitory/negative feedback). Another interesting finding was that neither PKC nor the PI cycle, which are stimulated by PLC activation, were shown to have a role in stimulating rectal Jv or 'sc• Many of the results from this study are strikingly different from those previously reported, using the closely related species L. migratoria (Fournier, 1990b,1991). However, the unusual assay procedures used in these previous studies may be responsible for the observed differences. There were some limitations of this study that may be overcome in future work. The first limitation was that a crude NCC aqueous extract was used to study the signal transduction mechanisms involved in controlling rectal Jv and 'Sc. This crude extract may have contained multiple factors that control rectal transport. The use of individual purified peptides could simplify the interpretation of the results and allow the construction of more representative models. My justification for using crude NCC was the claim by Fournier and Girardie (1988) that neuroparsins is the only stimulant of rectal Jv in L. migratoria NCC and acts via the PI cycle to raise cytosolic Ca2±. This is clearly not the case for S. gregaria. Unfortunately, no factor that stimulates S. gregaria rectal Jv and Isc has yet  been purified. Phillips et al. (1980) managed to partially purify CTSH from S. gregaria CC that was very unstable during HPLC, while ScgITP had no effect upon rectal Jv and only stimulated rectal 'Sc to 40% of maximum at very high doses (Audsley, 1991). In addition, this study demonstrated that neuroparsins has no effect upon S. gregaria rectal 'Sc or Jv.  82 Although another attempt to purify CTSH could be undertaken, a more promising approach may be to concentrate on signal transduction mechanisms involved with the stimulation of ileal transport by ScgITP. The advantages of using the ileum are: 1) it is a simple epithelium comprised of fewer cell types than the rectum; 2) there is no recycling of ions in the lateral intracellular spaces between the Heal epithelial cells 3) the major CC peptide that stimulates transport (ScgITP) has already been purified and partially sequenced. Another limitation of this study was that the effects of NCC homogenate on second messenger levels were not monitored. The measurement of second messengers would provide direct evidence for the involvement of specific signal transduction pathways and confirm the proposed actions of pharmacological agents. Changes in tissue cAMP and cGMP levels could be measured using the cAMP binding protein technique and the cGMP radioimmunoassay to show that purified factors affect these pathways. These assays are both commercially available. In fact, Chamberlin and Phillips (1988) have already shown that crude CC homogenate elevated rectal cAMP and cGMP levels. The involvement of PLC-mediated events, namely the activation of the PI cycle and PKC, could also be monitored. The role of the PI cycle in the control of rectal transport could be investigated by loading cells with tritiated myo-inositol (a precursor of the PI cycle) and examining the amounts of labeled intermediates formed (e.g. InsP3 and InsP4) using standard HPLC methods (Kirk et al., 1990). Protrein kinase C enzyme activity could be measured using a standard assay technique described by Farese and Cooper (1990). The effect of antidiuretic factors on cytoplasmic Ca2+ could possibly be directly measured by using fura-2 spectrometry (see Hallett et al., 1990). Lastly, most of the experiments conducted on S. gregaria recta could not be repeated using L. migratoria, because the animals were not available. A few experiments with L. migratoria recta were performed in this study but the results were completely different  from Fournier's findings. For example Fournier (1990b) stated that myo-inositol maximally  83 stimulated rectal Jv in L. migratoria, whereas a similar experiment from this study showed no stimulatory effect of myo-inositol on L. migratoria rectal Jv. The only difference between these two experiments was the assay conditions. As previously stated, Fournier (1990b, 1991) used an unusual procedure that involved the incubation of the rectal tissue in a free saline for the first hour of the experiment. Chloride was reintroduced on the second hour simultaneously with the exposure of the tissue to various treatments, either peptide factors or pharmacological agents. It would be interesting to see if the same results are obtained using a conventional incubation procedure. The importance of the insect excretory system in maintaining hemolymph homeostasis is comparable to the role of the vertebrate nephron. Both of these systems eliminate toxic compounds and metabolic waste products, control blood volume and osmolarity, and are involved with acid-base regulation. The Malpighian tubules perform a function similar to that of the glomerular capsule and are responsible for the formation of the isosmotic primary urine. In vertebrates, the glomerular capsule and the endothelium form the endothelial capsular membrane that filters blood under pressure (60-100 mmHg) allowing the passage of fluid and ions across the membrane but blocking the entry of larger molecules such as proteins (see Tortora and Anagnostakos, 1984). Malpighian tubules actively secrete a primary urine that is rich in KC1 and that contains most blood solutes. In contrast to the glomerulus, Malpighian tubule secretion is under direct endocrine control, with cAMP, InsP3 and Ca2+ being implicated as second messengers. It has been shown that the ileum acts as the 'proximal tubule' of the locust excretory system, since it actively transports an isosmotic absorbate, is the major site of ammonia secretion, and is involved in acid-base regulation (Lechleitner, 1988; Peach, 1991; Thomson, 1990). In vertebrates, proximal tubule transport is stimulated by the steroidal hormone aldosterone. Aldosterone is released from the outer cortex of the adrenal glands, it then activates the synthesis of more Na+ carrier proteins (Na+/K+ ATPases) and also increases the availability of ATP in the tubular epithelial cells (reviewed by Lote, 1987; Guyton, 1991). Locust ileal  84 transport is stimulated by a peptide factor from the insect's corpus cardiacum, ScgITP, which is thought to activate electrogenic C1 transport by elevating cellular cAMP levels. ScgITP also inhibits ileal acid secretion, but the adenylate cyclase signal transduction pathway is not involved (Audsley, 1991). Lastly the locust rectum is responsible for determining the fmal ion and water composition of excreta, and can selectively create strongly hypo- or hyper-osmotic urine. In the nephron, the loop of Henle, distal tubule and the collecting ducts are all required for the production of hypo- or hyper-osmotic urine. Both systems employ ion/solute recycling mechanisms to form hyper-osmotic urine, but this recycling is performed at different structural levels. The locust rectum can recycle solutes in the lateral channels between the principle epithelial cells. The late distal tubule and the collecting duct becomes permeable to water when antidiuretic hormone (ADH) is released by the posterior pituitary. ADH binds to the baso-lateral membrane of the target cells, elevating cAMP levels, leading to the fusing of vesicular structures with the apical membrane (see Lote, 1987; Guyton, 1991). These vesicles possess proteins with large water conducting channels. 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