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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 MECHANISMSCONTROLLING FLUID REABSORPTION AND ION TRANSPORT IN THELOCUST RECTUM.byLLOYD BRIAN JEFFSB.Sc., The University of British Columbia, 1990.A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTSFOR THE DEGREE OF MASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF ZOOLOGYWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAMay 1993© Lloyd Brian Jeffs, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of The University of British ColumbiaVancouver, CanadaDate 3) s-r riA\( 1(393 DE-6 (2/88)IIABSTRACTLike most insects, locusts face severe regulatory challenges associated with aridhabitats, high metabolic rates and high surface area to volume ratios. Therefore, theconservation of water, essential ions and metabolites is very important. Consequently,locusts regulate their hemolymph composition primarily by controlling epithelial transportin the excretory system. The locust excretory system is typical of many insects andconsists of the Malpighian tubules and the hindgut. The Malpighian tubules secrete aprimary isosmotic urine rich in KC1 and low in Na+ that contains most small hemolymphsolutes, waste products and toxic plant chemicals. The hindgut (ileum and rectum) isresponsible for the enormous changes in composition of the urine, by the selectivereabsorption of water, ions and metabolites. Both the Malpighian tubules and the hindgutare under endocrine control.The purpose of this study was to investigate the involvement of second messengers inthe 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 signaltransduction pathways were added to everted rectal sacs and short-circuited rectal flat-sheet bioassays. Cyclic AMP and its analogs were shown to stimulate rectal Jv and Cl-transport to the same extent as aqueous extracts of the nervous lobes of the corporacardiaca, suggesting that activation of the adenylate cyclase pathway is sufficient formaximal stimulation. It also appears that cGMP is involved, since its addition partiallystimulated both Cl- and fluid reabsorption. External Ca2+ was not required for themaintenance or stimulation of rectal transport. However, intracellular Ca2+ was shown toinfluence the control of rectal transport. The role of intracellular Ca2+ appears to be quitecomplex and may vary with its relative concentration. Finally, it was found that Proteiniiikinase C and the phosphotidylinositol cycle do not appear to be involved in the stimulationof rectal Cl- and fluid transport.2"ivTABLE OF CONTENTSPageAbstract^Table of Contents^ ivList of Tables viList of Figures viiList of Abbreviations^ ixAcknowledgments xiiiCHAPTER 1:^General Introduction^ 1Structure of the locust excretory system^ 1Mechanisms of solute and water transport in the locustrectum^ 5Solute and water transport in the ileum^ 9Endocrine control of excretion: Malpighian tubule secretion^ 11Endocrine control of excretion: Hindgut reabsorption^ 15Objectives of this study^ 19CHAPTER 2:^Effects of NCC, cAMP and Related Compounds on RectalFluid and Ion Transport^ 20Introduction^ 20Materials and Methods 23Insects 23Chemicals^ 23Salines 24Flat sheet rectal bioassay^ 24Everted rectal sac bioassay 27Preparation of NCC extracts 27Statistics^ 28Results^ 28Effect of NCC and cAMP^ 28Effect of chemicals acting on the cAMP mediatedpathway^ 33Effect of cGMP 38Discussion 43CHAPTER 3:^The Involvement of Calcium in Controlling RectalTransport^ 45Introduction 45VPageMaterials and Methods^ 49Results^ 50Effect of Ca2+ free saline^ 50Effect of agents that modulate cytosolic Calcium^ 55Discussion^ 60CHAPTER 4:^The Role of PKC and the PI Cycle in Controlling RectalTransport^ 62Introduction 62Materials and Methods^ 65Results^ 65Effect of agents acting upon protein kinase C^ 65Effects of agents acting upon the Phosphotidyl-Inositol cycle^ 66Locusta experiments 74Effect of Neuroparsins on S.gregaria recta^ 74Discussion^ 79CHAPTER 5:^General Discussion^ 81References 85viLIST OF TABLESPageTable 1.1.^A list of fully and partially characterized insect diureticpeptides. ^  12Table 1.2.^Insects shown to possess antidiuretic factorsthat increase rectal fluid reabsorption. ^ 14Table 2.1.^Effect of NCC and cAMP upon rectal electrical variables^ 29Table 2.2.^Effect of cGMP upon rectal electrical variables.^ 39Table 3.1.^Effect of Calcium-free saline upon electrical variables. ^ 51Table 4.1.^Effect of 1mM LiC1 upon electrical variables^ 67Table 4.2.^Effect of MI and DBcAMP upon electrical variables ofL. migratoria recta. ^ 75viiLIST OF FIGURESPageFigure 1.1.^Diagram of the locust excretory system. ^ 2Figure 1.2.^Comparison of ultrastructural organization and grossdimensions of locust rectal pad and ileal epithelial cells. ^ 4Figure 1.3.^Model of transport mechanisms identified in the locustrectal epithelium ^ 6Figure 1.4.^Side view of an insect showing the neuroendocrine organsimplicated in the control of fluid balance and excretion ^ 10Figure 2.1.^Diagram of the adenylate cyclase signal transductionpathway.^ 22Figure 2.2.^Standard Ussing chamber assembly used to measure Cl-dependent rectal short-circuit current (Ise).^ 25Figure 2.3.^Effect of NCC and cAMP on rectal Ise. 30Figure 2.4.^Fluid reabsorption by everted rectal sacs with time. ^ 31Figure 2.5.^Tissue swelling by the everted rectal sacs from Figure 2.4 ^ 32Figure 2.6. Effect of 2 NCC, 5 mM cAMP and 1 mM DBcAMP uponrectal Jv.^ 34Figure 2.7.^Effect of cAMP, SpcAMPS and RpcAMPS upon rectal Jv^ 35Figure 2.8.^Effect of a lh pre-incubation with 10 mM RpcAMPS onstimulation of rectal Jv by 2 NCC^ 36Figure 2.9.^Effect of ATP and Adenosine upon rectal Ise^ 37Figure 2.10. Effect of IBMX upon rectal 'Sc^ 40Figure 2.11. Effect of 5mM cGMP and 5 mM cAMP upon rectal Jv ^ 41Figure 2.12. Effect of cGMP upon rectal 'Sc. ^ 42viiiPageSchematic diagram of the cellular processes involved withthe regulation of intracellular Ca2+^ 46Fluid reabsorption by rectal sacs under Ca2+ freeconditions.^ 52Tissue swelling of rectal sacs under Ca2+ free conditions. ^ 53Effect of Ca2+ free saline (Caf) upon Jv stimulated by 2NCC and 1 mM DBcAMP ^ 54Figure3.1.Figure 3.2.Figure 3.3.Figure 3.4.Figure 3.5.^Effects of Ionomycin (IY), IY with 5.5 mM Ca2+bathing saline and IY with Ca2+ free saline upon rectal Jv.^ 56Figure 3.6.^Effect of 1 mM IY and Thapsigargin upon rectal Isc ^ 57Figure 3.7.^Effect of TMB-8 upon 5 mM cAMP stimulated Isc^ 58Figure 3.8.^Effect of TMB-8 upon 2 NCC stimulated Isc.^ 59Figure 4.1.^Diagram of the phosholipase C (PLC) mediated signaltransduction pathway. ^  63Figure 4.2.^Effect of SAG and PMA upon rectal J. ^ 68Figure 4.3.^Effect of SAG and PMA upon rectal 'Sc.  69Figure 4.4.^Effect of a 1001,tM PMXB (1000 iu/mL.) upon 2 NCCstimulated Jv.^ 70Figure 4.5.^Effect of 100 RM (1000 iu/mL.) PMXB upon rectal Ise. ^ 71Figure 4.6.^Effects of myo-inositol (MI) and LiC1 upon rectal Jv^ 72Figure 4.7.^Effects of MI and LiC1 upon rectal I.^ 73Figure 4.8.^Fluid reabsorption by Locusta migratoria everted rectalsacs ^ 76Figure 4.9.^Effect of Neuroparsins upon rectal J. ^ 77Figure 4.10. Effects of Neuroparsins upon rectal Isc 78ixLIST OF ABBREVIATIONSA Jv^- change in fluid reabsorptionClcm2 - ohms centimetre 1^- micro equivalents per centimetre squared per houruL^- microlitrepL.h- 1 - microlitres per hour[iM^- micro molarum - micron°C^- degrees Celsius5-HT - 5-hydroxytryptamineA^- Angstrom (0.1 nanometres)A/D - analog-to-digitalAA, aa^- amino acid(s)AD - antidiureticADH^- antidiuretic hormoneADP - adenosine diphosphateAG^- abdominal ganglionAP - Acheta peptideATP^- adenosine 5' triphosphateANOVA^- analysis of varianceAVG - abdominal ventral ganglionAVP^- arginine vasopressinAVP-LDH^- arginine vasopressin-like diuretic hormoneAVT^- arginine vasotocinCA - corpus allatumcAMP^- adenosine 3', 5' -cyclic monophosphoric acidxCC^- corpus cardiacumcDNA - complementary DNACGA^- colour graphics adaptercGMP - guanosine 3', 5' -cyclic monophosphoric acidcm^- centimetrecm2 - squared centimetresCRF^- corticotropin releasing factorCTSH - chloride transport stimulating hormoneD^- diureticDa - DaltonsDAG^- sn-1,2 diacylglycerolDBcAMP^- di-butyryl 3', 5' -cyclic monophosphoric acidDH - diuretic hormoneDMSO^- dimethyl sulfoxideDP - diuretic peptideEGTA^- ethyleneglycol-bis-(B-aminoethyl ether) N,N,N',N'-tetraacetic acideq. - equivalentsg^- gravity unitsGCC - glandular lobe of the corpus cardiacumGTP^- guanosine triphosphateH - hemolymph or hemocoelh^- hoursHPLC - high performance liquid chromatographyIBMX^- 3 isobutyrl- 1 -methylxanthineIns - inositolIns P^- inositol phosphateInsP3 - inositol 1,4,5, trisphosphateInsP4^- inositol 1,3,4,5 tetraldsphosphate'Sc - short-circuited currentiu^- international unitsIY - ionomycinJamm^- rate of luminal ammonia secretion- fluid reabsorptionKDa^- kilo DaltonsLCCP - locust corpus cardiacum peptide- molarM-NSC^- median neurosecretory cellsMI - myo-inositolmin^- minutesmL - millilitremm^- millimetremM - millimolarMT^- Malpighian tubule(s)MTG - meta-thoracic ganglionmV^- millivolts- numberNCC^- nervous lobe of the corpus cardiacumNpA - neuroparsin ANpB^- neuroparsin BNps - neuroparsinsP.E.^- polyethylene (tubing)PD - potential differencePdE^- phosphodiesterasePI - phosphotidylinositolxiiPtdInsP2, PIP2^- phosphotidylinositol 4,5-bisphosphatePKA^- protein Idnase APKC - protein kinase CPLC^- phospholipase CPMA - phorbol 12-mpistate-13-acetatePMXB^- polymyxin B sulfatePO - perisympathetic organsRAM^- random access memoryRpcAMPS^- adenosine 3, 5' -cyclic monophosphothioate Rp- diastereomerRt^- transepithelial resistanceS.E. - standard errorSAG^- 1-stearoy1-2-arachidonoyl-sn-glycerolScgITP - Schistocerca ion transport peptideSO-NSC^- subocellar neurosecretory cellsSOG - suboesophageal gnanglionSpcAMPS^- adenosine 3', 5' -cyclic monophosphothioate Sp- diastereomerTAG - thoracic abdominal ganglionTG^- thoracic ganglionTHAPS^- thapsigarginTMB-8 - 3,4,5 trimethoxybenzoic acid, 8-(diethylamino)-octyl esterVt^- transepthelial voltageACKNOWLEDGMENTSI would like to thank Dr. John Phillips for his support, generosity and guidancethroughout this study.I thank Drs. M. Isman, D. Jones and D. Randall for their helpful comments on themanuscript.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 andproductive.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 andfriendship.1CHAPTER 1: GENERAL INTRODUCTIONInsects inhabit a wide range of diverse and variable habitats, including deserts,freshwater, alkaline lakes and tidal pools. Insects face severe regulatory challengesassociated with high metabolic rates (e.g. when flying) and high surface area to volumeratios leading to water loss across the integument. The unusual diets of some insectsincluding toxic substances produced by plants, high ingestion rates, molting andmetamorphosis are further examples of the stresses placed on insect homeostaticmechanisms Like most insects, locusts regulate their hemolymph composition veryclosely. As well as behavioral and structural adaptations, physiological adaptationscontribute to this hemolymph homeostasis. The control of epithelial transport in theexcretory system is the principal mechanism for making rapid and major adjustments tohemolymph composition.The organization of the locust excretory system is typical of many insects, consisting ofthe Malpighian tubules and hindgut (Fig. 1.1). The Malpighian tubules actively secrete aprimary isosmotic urine, rich in KC1 and low in Nal-, that contains most small hemolymphsolutes (including amino acids), waste products (e.g. urate and ammonium) and toxic plantchemicals (reviewed by Phillips, 1981,1983). The hindgut (ileum and rectum) is thenresponsible for the enormous changes in composition of the urine by the selectivereabsorption of water, ions, amino acids and other metabolites (Phillips et al., 1986). Thisselective reabsorption can create a hyper- or hypo- osmotic urine, or even a powder dryexcreta under various environmental conditions.Structure of the locust excretory systemThere are about 250 Malpighian tubules in an adult Schistocerca gregaria that emptyinto the gut lumen at the junction of the midgut and hindgut. The hindgut (ileum, colon2Fig. 1.1. Diagram of the locust excretory system. The flow of urine is shown by the thinarrows and transfer across the epithelia is indicated by the thick arrows (from Phillips,1981; modified by Audsley, 1991).3and rectum) is lined on the luminal side with a chitinous cuticle (2-10 pm thick). Thecuticle of the ileum and rectum is permeable to small hydrophilic molecules via water-filled6 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 alsoallow the rapid diffusion of Ca2+ and Mg2+, which would otherwise be precluded becauseof their large hydrated molecular radii (Lewis, 1971).The ileum is approximately 6 mm long, has a diameter of 2.5 mm and luminal surfacearea of 0.4 cm2 (Irvine et al., 1988). The ileum consists of a simple epithelium (40 x 20pm) and is covered by a firmly attached apical cuticle (Fig. 1.2). The apical membranehas dense infoldings that are associated with mitochondria and apical junctional complexesbetween adjacent cells. The basal membrane has short narrow infoldings associated withnumerous mitochondria and may be analogous to the lateral scalariform complexes of therectum 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 unspecializedepithelial cells. In addition, the colon possesses a cuticle which makes it considerably lesspermeable than either the rectum or ileum. For these reasons the colon is not thought toplay a significant role in absorption (Maddrell and Gardiner, 1980). However, the colonhas a structural role in the separation of the gut contents into discrete fecal pellets and inthe breaking of the peritrophic membrane that surrounds the fecal material (Goodhue,1963).The rectum is immediately posterior to the colon, consists of six longitudinally arrangedthickened pads, and has a luminal surface area of 0.64 cm2 (Irvine et al., 1988). The rectalcuticle is attached at narrow junctional complexes between each rectal pad, but is freefrom the pad epithelium creating a subcuticular space above the apical border. The rectalpads 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 the14Fig. 1.2. Comparison of ultrastructural organization and gross dimensions of locust rectalpad and ileal epithelial cells (from Irvine et al., 1988).5luminal side only. The principal cells have highly folded apical and lateral membranes withclosely associated mitochondria. The lateral membranes have fmger-like projections thatare intimately associated with the lateral membranes of adjacent cells. Extensive electricalcoupling between these principal cells has been demonstrated by dye injection and cableanalysis (Hanrahan and Phillips, 1984b). The lateral membrane infolds form complexintercellular channels with three distinct regions (lateral scalariform complexes, dilatedintercellular spaces, larger intracellular sinuses with trachea) where ion recyclingassociated with the formation of hyperosmotic urine is thought to occur (Wall andOschman, 1970). The basal membrane is unfolded and devoid of mitochondria whichoccur at the apical and lateral membranes, but is externally covered by a thick basallamina. Below the basal lamina is a muscle layer, a subepithelial space and a smallersecondary epithelial cell layer. These secondary cells show some degree of membranecomplexity and possess many mitochondria, suggesting a transport function (e.g. furtherion recycling). Finally the whole structure is encased within a muscle layer through whichfluid exits at the points where trachea penetrate into the organ.Mechanisms of solute and water transport in the locust rectumWigglesworth (1931) was the first to report that insect gut contents can become verydry as they pass through the rectum and suggested that the rectum is a major site of fluidreabsorption. Active transport of ions and fluid in an insect rectum was first directlyshown by measuring changes in volume and composition of fluid injected in ligated rectaof locusts (Phillips, 1964 a-c,). It is now known that Na+, K+, Cl-, water and metabolitesare all reabsorbed by locust recta. Figure 1.3 shows the major membrane transportmechanisms demonstrated in locust rectal epithelial cells. The ileum has since been shownto have equal or greater transport capacities for ions and water than the rectum. Thedifferences 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,Ionnet(•lectro-genicpumpCI-4.3AA'aNa•Ni-1•(H•)4H••^oxidation'CO2OW-H20CELL HEMOCOELNa' 2DAA's•(WINa•N•. K^ K•ATPase4.5I cA IIon Activities (mM) and PD ( mV)Na' 75 9 75K • 7.2 70 7.282 47 82Proline 13 68 13pH 7 7.36 7mV •64 •34Net Electrochemical(Apical)PD (my)(Basolateral)Na'K •CIH•127 (favoring )12 (favoring)50 (opposing)86 (opposing)127 (opposing)20 (favoring)20 (favoring))56 (favoring)Fig. 1.3. Model of transport mechanisms identified in the locust rectal epithelium. Theneuropeptide, CTSH, acts via cAMP to stimulate or inhibit four mechanisms. Majorpumps are represented by thick arrows, carrier mediated co- or counter-transport by thinarrows through solid circles and ion channels by arrows through gaps. Steady-statevalues given for net transepithelial flux and electrochemical potential differences across thetwo borders are for stimulated recta in Ussing chambers and bathed bilaterally in controlsaline under open circuit conditions, except values for Nat and amino acids (AA) whichwere quantified under short-circuited conditions. From Phillips et al. (1988).7the urine. It can be seen from Figure 1.3 that there is a large net electrochemicaldifference opposing Cl- entry at the apical (mucosa') surface of the rectal pad cells. Thisindicates that the active step for Cl- transport is located at the apical membrane. Themodel proposes that there is a primary electrogenic Cl- pump that is stimulated by cAMPand lumina' K+. There are also basolateral Cl- channels that allow the passive exit of C1across 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 causeda 10-fold increase in net Cl- transport toward the hemocoel side and that the removal ofexternal K+ reduced net Cl- flux by 84%. They also provided evidence that Cl- transportwas not coupled to apical Nat, K+, HCO3- or H+ transport. In addition, Cl- exit throughthe basolateral membrane can be completely inhibited by Cl- channel blockers applied tothe hemocoel side of recta (Phillips et al., 1986).Potassium (K+) is the major cation in the primary urine from the Malpighian tubulesand is absorbed passively through K+ channels at both membranes (Hanrahan and Phillips,1984a). In unstimulated open-circuit recta, there are small favourable electrochemicalgradients for entry of K+ across the apical membrane (3.3 mV) and for exit across thebasolateral membrane (3.9 mV; Hanrahan and Phillips, 1983, 1984c). Addition of cAMPstimulates Cl- transport and subsequently increases apical potential (Va) by 12 mV anddecreases basolateral potential (Vb) by 13 mV. These membrane potential changesincrease the favourable apical and basolateral electrochemical gradients to 12 mV and 20mV 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 favourableelectrochemical gradient (127 mV) at the apical membrane for passive Na+ entry byseveral 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 hemocoel8against a large electrochemical gradient (127 mV) by Na+/K+ ATPase in the lateralmembranes (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 anyneuroendocrine tissue extracts tested to date (Black et al., 1987).Berridge and Gupta (1967), and Wall and Oschman (1970), have proposed a model toexplain how insect recta can absorb hypo-osmotic fluid from the lumen. The modelproposes that ions and other solutes (amino acids) are actively secreted into theintercellular spaces of the lateral scalariform complexes (Fig. 1.2), creating areas of highosmotic concentration relative to the lumen. Water then flows from the gut lumen intothese hyper-osmotic spaces (i.e. local osmosis), thereby producing a hydrostatic pressurewhich causes fluid to flow through the intercellular sinuses towards the hemocoel side.Active solute reabsorption is believed to occur in the dilated intercellular spaces andintercellular sinuses. This reabsorption of solute into the the cells results in the formationof a hypo-osmotic absorbate and concentrated excreta. Goh and Phillips (1978) haveshown that water transport in the locust rectum is indeed coupled to ion transport, andProux et al. (1984) have shown that cAMP-stimulated fluid reabsorption (Jv) is dependentupon transport. Recently Lechleitner and Phillips (1989) observed an increase in Jvwhen recta were exposed to 80 mM proline compared to incubation with 1 mM proline. Itwas also shown that a low level of Jv (2.3 liL.h-1) could be maintained for 5 hours withbathing saline containing 80 mM proline and no IC+, or Na+. Combined with theknowledge 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 thatproline is directly involved with fluid transport in the rectum.Five neutral amino acids (proline, glycine, serine, alanine and threonine) have beenshown to be actively absorbed by locust recta when Jv was prevented by making thelumen hyper-osmotic with sucrose (Balshin and Phillips, 1971; Balshin, 1973). Meredithand Phillips (1988) found that the rectum has a very high capacity for proline transport at9the 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 andis 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 ordetoxified. 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 equalmovement 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 byan apical electrogenic process (i.e. a H+ATPase), with Na+/H+ exchange playing a minorrole (Thomson and Phillips, 1992). The addition of cAMP to rectal preparations reducesactive 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 ileumMany of the Heal transport mechanisms are similar to those previously described in therectum, but there are some distinct differences. The primary difference is that the ileumcan only produce an iso-osmotic absorbate, because it lacks the complex lateral membranesystem 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 drivenlargely by proline and KC1 (Lechleitner and Phillips, 1989). Unlike the rectum, Na+ istransported at high rates in the ileum and its transport is stimulated by cAMP (Lechleitneret 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). HealNH4+ secretion rate (Jamm) is 2.5 times greater than in the rectum and is stimulated bycAMP (Lechleitner, 1988; Peach and Phillips, 1991). As previously mentioned, luminal10Fig. 1.4. Side view of an insect showing the neuroendocrine organs implicated in thecontrol of fluid balance and excretion (from Phillips 1982).11proline is the major respiratory substrate and ammonia source in the rectum, while theileum uses luminal alanine, asparagine, glutamine, serine and bilateral proline (Peach andPhillips, 1991). There is negligible proline transport in the ileum (Lechleitner and Phillips,1989).Endocrine control of excretion: Malpighian tubule secretionMalpighian tubule secretion and hindgut reabsorption are both under endocrine control.The insect neuroendocrine organs thought to be involved in the control of fluid balanceand excretion are shown in Fig. 1.4. The first demonstration of a diuretic factor thatstimulated 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 therelease of a proteinacious factor from the thoracic-abdominal nerve mass that immediatelystimulated the secretion of large amounts of urine. Various methods have been used tostudy Malpighian tubule (MT) secretion: Ramsay's (1954) in vitro fluid secretion methodwith isolated tubules and its variants; measuring changes in MT transepithelial voltage(Maddrell and Klunsuwan, 1973; Williams and Bayenbach, 1983,1984); measuringchanges in tissue cAMP levels (Morgan and Mordue, 1985; Kay et al., 1991a); and bymeasuring water loss from the whole insect (Kataoka et al., 1989). Diuretic factors havebeen reported for many types of insects, and these factors appear to be present in variouslocations throughout the neuroendocrine system (reviewed by Spring, 1990; Raabe,1991). However, only a few of these factors have been purified and their amino acidsequence determined (see Table 1.1.).High performance liquid chromatography (HPLC) has been used extensively to isolateinsect diuretic and antidiuretic factors. HPLC can rapidly separate small amounts ofmaterial with high resolution and recovery, and is far superior to older chromatographictechniques that were slower, less sensitive and required more material (Schram, 1980).12Table 1.1. A list of fully and partially characterized insect diuretic peptidesSPECIES^PEPTIDE^SOURCE^DESCRIPTIONLocusta 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 AVPand AVT respectively(Proux et al., 1987)Locusta-DP^head^46 aa amidated peptide, chemicallysimilar to DP-1, 48% and 49%homology with Acheta-DP andManduca-DH respectively(Kay et al., 1991b)Manduca sexta Manduca-DH^head^41 aa amidated peptide(tobacco^ (Kataoka et al., 1989)homworm)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, diureticpeptide/diuretic hormone; AP, Acheta peptide; AVP, arginine vasopressin; AVT, argininevasotocin; AVP-LDH, arginine vasopressin-like diuretic hormone.13Improvements to mass spectrometry, peptide sequence analysis, and the development ofautomated gas-sequencers have greatly increased the speed of peptide sequencing, andhave 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 insectdiuretic factor to be fully characterized, and was isolated using HPLC and immunologicaltechniques (Proux et al., 1987). Although AVP-LDH closely resembles mammalian AVP,its structure is unlike other purified diuretic factors. Many of the recently characterizeddiuretic peptides such as Locusta-DP, Manduca-DH, Acheta-DP (see table 1.1.),Leucophaea-DP and Periplaneta-DP (Coast, personal communication), share homologousregions in their amino acid sequences. In addition, this family of diuretic peptides hassequence 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 levelsin Acheta Malpighian tubules, mimicking the action of Acheta-DP. They have proposedthat 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 beinvolved in the regulation of fluid and solute transport, and act by elevating cAMP andperhaps by increasing intracellular Ca2+ levels.Secretion by insect Malpighian tubules is also stimulated by members of a family ofmyotropic 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 (Holmanet al., 1991). These myokinins all possess a highly conserved pentapeptide carboxyterminal 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 cAMPindependent mechanism, perhaps by elevating intracellular Ca2+.14Table 1.2. Insects shown to possess antidiuretic factors that increase rectal fluidreabsorption.SPECIES^SOURCE^REFERENCECarausius 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(migratory locust)Schistocerca gregaria(desert locust)CC^Cazal and Girardie (1968)PO De Besse and Cazal (1968)NCC, GCC^Herault et al. (1985)GCC^Mordue (1969, 1970, 1972)GCC, NCC Proux et al. (1984)AVG 5^Lechleitner and Phillips (1989)Periplan eta americana brain, MTG, CA^Wall (1967)(American cockroach)^TAG^Wall (1967), Goldbard et al. (1970)Blaberus craniifer^CC^Boureme et al. (1989)(cockroach)Leucophaea maderae^CC^Boureme et al. (1989)(cockroach)Abbreviations:GCC/NCC, glandular/nervous lobes of corpus cardiacum; CA, corpus allatum; PO,perisympathetic organs; AVG 5, fifth abdominal ventral ganglion; MTG, meta-thoracicganglion; TAG, thoracic abdominal ganglion.15Spring et al. (1988b) isolated an antidiuretic hormone (ADH) from Acheta domesticusCC that inhibited MT fluid secretion by 70%, but had no effect upon the hindgut. It wasalso shown that the release of this ADH into the hemolymph was triggered bydehydration. There is indirect evidence that this ADH may act by elevating intracellularCa2+, since the divalent ionophore A23187 also reduced tubule secretion by 85% (Springand Clark, 1990). The structure of this ADH has not yet been determined.Endocrine control of excretion: Hindgut reabsorptionVietinghoff (1966) was the first to show that insect hindgut reabsorption is underendocrine control. There have been several subsequent reports of antidiuretic (AD) anddiuretic (D) factors that control hindgut fluid reabsorption (Jv); see Table 1.2. for ADfactors (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 hindgutpreparations during an initial transitory absorptive period, when the hindgut tissue wasadjusting to the new osmotic environment (causing tissue swelling) and was possibly stillresponding to factors that were present in vivo. In addition, the earlier studies often usedsalines 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 CCthat stimulated electrogenic chloride transport and named it chloride transport stimulatinghormone (CTSH). Proux et al. (1984) later showed that CTSH also stimulates rectal fluidreabsorption. 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 isreleased into the hemolymph, presumably upon feeding (Spring et al., 1978; Spring andPhillips, 1980a,b,c; Proux et al., 1984; Hanrahan and Phillips, 1985). CTSH is thought toact by elevating intracellular cAMP levels, because exogenous cAMP stimulates both16rectal C1 transport and water reabsorption (Phillips et al., 1980; Proux et al., 1984)). Inaddition, Chamberlin and Phillips (1988) found that rectal tissue levels of cAMP increasebetween five and ten minutes after exposure to CC extracts. It has also been shown thattransport and intracellular cAMP levels increased when pharmacological agents(theophylline and forskolin) known to elevate cAMP levels in other systems, were appliedto 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 thatincrease rectal Jv over a five hour period. One factor is reported to be localized in theGCC while the other factor is exclusive to the NCC. Haault and Proux (1987) haveshown that the GCC factor elevates intracellular cAMP levels over a similar time course asCTSH. The GCC factor is an unstable peptide (H6rault et al., 1985) and has not yet beenpurified.Girardie et al. (1987b) have purified and characterized an AD factor from LocustaNCC, which they have named neuroparsins (Nps), and have shown that it is produced bythe 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). Girardieet al. (1989,1990) used anion exchange chromatography and HPLC to isolate two formsof neuroparsins, neuroparsin A (NpA) and neuroparsin B (NpB). Both forms havemolecular 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 NpBmonomer consists of 78 residues (i.e. identical to residues 6-83 of NpA). Since the NpBmonomer sequence is identical to a major part of NpA, Girardie et al. (1989) havepostulated 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 fromLocusta CC. Two of the peptides correspond to NpA and NpB, while the third peptide17possesses 81 residues (3-83 of NpA). All three peptides have been shown by massspectrometry 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 partiallysequenced 4.5 KDa, 46 residue Locusta Corpus Cardiacum peptide (LCCP) purified byMordue 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 LocustacDNA that encodes NpA. They have proposed that NpA is processed from a 107 residueprecursor in two steps; cleavage of a 22 residue signal peptide and removal of a Glu-Argdipeptide. It has also been shown that transcripts encoding NpA occur in both adult andlarval brains.Neuroparsin-like peptides have also been identified in the cockroaches Blaberuscraniifer and Leucophaea maderae (Boureme et al., 1989). Both of these peptidesincrease Locusta rectal Jv, and NpA stimulated rectal h in both cockroaches, suggestingthat these peptides are related and may belong to an intraphyletic neuroprotein family.Immunological techniques have also been used to show that neuroparsin-like factors existin brain neurosecretory cells of three other insect orders (Isoptera, Odonata andDictyoptera; Tamarelle and Girardie, 1989). In addition, NpA has 37% and 27% aminoacid sequence homology with Manduca sexta and Bombyx moni eclosion hormonesrespectively (Girardie et al., 1990). Furthermore, NpA shows some sequence identity(-30%) with some vertebrate hormones, such as hypothalamic pituitary hormones andpre-proinsulin (Girardie et al., 1990).There has also been interest in determining the second messenger systems whereby Npsstimulates rectal Jv. Fournier and Dubar (1989) have shown that Nps has no effect uponintracellular cAMP levels, while slightly increasing cGMP levels. They concluded thatboth cAMP and cGMP are not second messengers of Nps. They suggest that the slightincrease in cGMP levels is a result of Nps-stimulated elevation of cGMP in a different cell18type, such as rectal muscle cells, resulting in a small diuretic effect! Moreover, Fournier(1990b) has used a pharmacological approach to provide indirect evidence that Npsinduces phosphoinositide turnover in Locusta rectal cells. Diacylglycerol (DAG) andinositol trisphosphate (InsP3), products of phosphotidylinositol bis-phosphate (PtdInsP2)breakdown, have both been implicated as possible second messengers of Nps. DAG isknown to stimulate protein kinase C (PKC; Nishizuka, 1984), while InsP3 triggers therelease of intracellular calcium from internal stores and the entry of extracellular Ca2+(Berridge and Irvine, 1989). Fournier (1991) has since directly shown that Nps stimulatesphospholipase C (PLC) mediated hydrolysis, resulting in an increase in intracellular InsP3levels.Audsley et al. (1992) have used reverse phase (RP)-HPLC to isolate and purify a 7.7KDa factor from Schistocera gregaria CC, called Schistocerca gregaria ion transportpeptide (ScgITP). At the moment only 50 of the estimated 65 amino acid residues ofScgITP have been identified (Audsley, 1991). ScgITP is completely different from anyother known vertebrate or insect hormones, but its first 34 residues have approximately50% sequence identity with crustacean hyperglycemic and moult inhibitory hormones(Audsley et al., 1992). ScgITP has quantitatively similar effects upon ileal transport asdoes crude CC homogenates: increasing ileal Jv; stimulating C1, K+ and Na+reabsorption; and inhibiting H secretion (Audsley et al., 1992). ScgITP also sub-maximally 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 andScgITP) that control Schistocerca hindgut solute and fluid reabsorption. ScgITP isthought to act via cAMP, since exogenous cAMP mimicks most of the effects of ScgITPand of crude CC extracts. However, ScgITP mediated inhibition of ileal H+ secretion isnot mimicked by cAMP, suggesting that another second messenger may be involved.19Objectives of this studyThe purpose of this study was to investigate what second messengers are involved incontrolling rectal fluid reabsorption (Jv) and Cl- transport in the desert locust(Schistocerca gregaria). To determine which second messengers are important incontrolling these rectal transport processes, various agents known to block or activatespecific signal transduction pathways were added to everted rectal sac and short-circuitedrectal flat-sheet bioassays. Chapter 2 examines and reviews the roles of the adenylate andguanylate cyclase pathways in rectal reabsorption. In chapter 3, the following questionsare addressed: 1) Is extracellular Ca2+ necessary for rectal Jv and ion transport? 2) Doesmodulating cytosolic Ca2+ levels have any effect upon rectal transport properties?.Chapter 4 studies the role of protein kinase C (PKC) and the involvement of thephosphatidylinositol (PI) cycle in controlling rectal transport. In addition, Locusta rectalsacs and short-circuited rectal flat sheets were used to investigate the role of the PI cyclein Locusta recta. Finally, the effects of Locusta Nps, which is thought to act by stimulatingthe PI cycle in Locusta (Fournier, 1990b,1991), was tested on Schistocerca rectal Jv andIse to see if there was any cross-reactivity.20CHAPTER 2: EFFECTS OF NCC, cAMP AND RELATEDCOMPOUNDS ON RECTAL FLUID AND ION TRANSPORTINTRODUCTIONThe cyclic nucleotides cAMP and cGMP are thought to be the second messengers ofmany hormonally controlled processes in both vertebrate and insect cells (reviewed byBodnaryk, 1983). In insects, cAMP has an important role in controlling Malpighian tubulesecretion 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 secretionin various insect species (reviewed by Coast et al., 1991). These agents also stimulate fluidand 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 bythe recta of Locusta migratoria (Herault and Proux, 1987), Leucophaea maderae andBlaberus craniifer (Fournier, 1990a). Measurements of increases in intracellular cAMP inresponse to stimulants, have provided direct evidence that cAMP is a second messenger ofinsect excretory processes. Elevation of intracellular cAMP is associated with thestimulation of Malpighian tubule fluid secretion by CRF-related diuretic peptides (Coast etal., 1992). In addition, extracts of CC have been shown to simultaneously elevateintracellular cAMP levels and stimulate fluid and ion reabsorption in locust and cockroachrecta (Spring and Phillips, 1979; Chamberlin and Phillips, 1988; Herault and Proux, 1987;Fournier, 1990a). Chamberlin and Phillips (1988) have also shown that locust rectalcGMP 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 peakedbetween five and ten minutes after the addition of CC. Furthermore, this slow increase in21rectal intracellular cGMP corresponds quite well with the slow and gradual stimulatoryeffect of exogenous cGMP upon rectal^transport (Chamberlin and Phillips, 1988).The elevation of cAMP is brought about by hormonal activation of the adenylatecyclase signal transduction pathway (Fig. 2.1.). Signal transduction mechanisms translateand amplify external signals, enabling cells to respond quickly and precisely to specificstimulants/inhibitors at low concentrations. The first step in the Adenylate cyclase pathwayis the binding of a neuropeptide to its target receptor. This hormone-receptor complexthen catalyzes the binding of GTP to the a subunit of a stimulatory G protein (Gs). TheGTP-Gsa complex dissociates from the remaining f and 7 Gs subunits and binds toadenylate cyclase, which is also located on the plasma membrane (reviewed by Stryer,1988). A single hormone-receptor complex catalyzes the formation of many GTP-Gsamolecules, thus amplifying the original signal. The activated adenylate cyclase convertsATP to cAMP, resulting in another amplification of the signal. The increased amounts ofcAMP then stimulate cAMP specific protein kinases (PKA). These activated proteinkinases can now phosphorylate target proteins (e.g. enzymes, ion channels), activating orinhibiting specific cellular processes (Cohen, 1989). This protein modification results in anadditional amplification of the original, external signal. In the case of the locust hindgut, itis likely that proteins involved in transport are modified by PKA, since exogenouscAMP can stimulate C1--dependent 'sc.Increased cGMP levels are brought about by the activation of the guanylate cyclasesignal transduction pathway. The guanylate cyclase pathway is comprised of similarfunctional components as the adenylate cyclase pathway. The major differences betweenthe two pathways are that cGMP levels in animal cells are usually more than ten timeslower than cAMP levels (Bodnaryk, 1983), and there are multiple isozymes of guanylatecyclase that are found in both the cytosol and plasma membrane (Shultz et al., 1989).PKA [internal effector]22O. Neuropeptide [1st messenger]ReceptorGTP+Gsa[transducer]p [amplifier][precursor]cAMPI ATPHemocoelCytosol[2nd messenger]a cascade ofproteinmodificationsCELLULAR RESPONSESFig. 2.1. Diagram of the adenylate cyclase signal transduction pathway. Shaded circlesindicate agents that are added exogenously to affect the pathway.23In this chapter, the roles of the adenylate and guanylate cyclase signal transductionpathways in the control of rectal Jv and 'Sc were investigated. Initially, the effects of NCChomogenate, exogenous cAMP and dibutyrl cAMP upon rectal Jv and 'Sc were tested andcompared. Then the involvement of protein kinase A was tested by adding the diastereo-isomers SpcAMPS and RpcAMPS, which specifically stimulate and inhibit PKA,respectively (Rothermel and Parker-Botelho, 1988). The effects of ATP and adenosinewere tested to ensure that cAMP was not acting by stimulating purinergic cell surfacereceptors (Olsson and Pearson, 1990). The effect of inhibiting cAMP-dependantphosphodiesterase (PdE) with IBMX was also examined. The stimulatory actions ofmicromolar amounts of forskolin, which directly activates adenylate cyclase, on rectal Iscwas previously reported (Spring et al., 1978; Hanrahan and Phillips, 1985). Finally, theeffects of cGMP upon rectal Jv and Isc were tested and compared to those of cAMP.MATERIALS AND METHODSInsectsAdult female Schistocerca gregaria, 2-4 weeks past their fmal molt were used for allexperiments. The locusts were maintained on a 12 h light: 12 h dark cycle at 28°C and60% relative humidity, under crowded conditions. Animals were fed a mixture of driedgrass, bran, powdered milk and yeast; with fresh lettuce supplied daily. The nervous lobesof the corpus cardiacum (NCC) were excised from adult male and female locusts 4-6weeks past their final molt.ChemicalsMost chemicals, including those used to make the complex saline, were supplied by theSigma Chemical Company (St. Louis MO, USA). SpcAMPS and RpcAMPS were24supplied by BIOLOG Life Science Institute (La Jolla, CA, USA). Cyclic AMP, DbcAMP,SpcAMPS, RpcAMPS, ATP and cGMP were all dissolved in complex saline. IBMX andadenosine were initially dissolved in DMSO and these stock solutions were then dilutedwith complex saline (final DMSO concentration 1%).SalinesThe complex bathing saline used was based on the measured composition of locusthemolymph (Hanrahan et al. 1984) and contained (mM): 100 NaC1, 5 K2SO4, 10.9Mg504, 10 NaHCO3, 5 CaC12, 10 glucose, 100 sucrose, 2.9 alanine, 1.3 asparagine, 1.0arginine, 5 glutamine, 11.4 glycine, 1.4 histidine, 1.4 lysine, 13.1 proline, 6.5 serine, 1.0tyrosine, 1.8 valine. The saline was adjusted to pH 7.1 and continuously bubbled with a95% 02: 5% CO2 gas mixture that ensured rapid mixing. Complex saline has been shownto sustain transport activities of locust hindgut at near constant values for many hours(>8h.; reviewed by Phillips et al.,1986).Flat sheet rectal bioassayElectrogenic ion transport was measured by mounting recta between two Ussingchambers, as described by Hanrahan et al. (1984) and Audsley (1990). Recta wereremoved from animals, cut longitudinally to produce a flat sheet and immediately securedon 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 vigorousbubbling 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 throughports on the side of the chambers with leads connected to a high impedance differentialamplifier (4253, Teledyne Philbrick, Dedham, Mass. USA) which continually monitored25Fig. 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 ismounted over, 3) Neoprene 0-ring for securing rectal attachment to collar, 4) neoprenechamber seal, 5) agar bridge port for measurement of transepithelial potential (Vt), 6) gasislet 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).26Vt. Short-circuit current (Isc), a direct and continuous measure of electrogenic iontransport, was measured by maintaining Vt , at 0 mV by a second amplifier (725, NationalSemiconductor Corp., Santa Clara, CA. USA) which passed current (Isc) between twoAg-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) reportedthat locust rectal 'Sc is Cr-dependent and is therefore a direct measure of electrogenic Crtransport. Flux studies by Hanrahan (1982) have confirmed this report. Both 'Sc and Vtwere 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 ofOceanography, U.B.C.) collected and displayed the outputs from these amplifiers and allsignals were recorded at one second or greater intervals (see Jones et al., 1991). The cardand program were used with an IBM® compatible personal computer equipped with aCGA video output and 640K RAM. Corrections were made for series resistance of theexternal saline and asymmetries between voltage-sensing electrodes as described byHanrahan et al. (1984). While the tissue was under short-circuit conditions, Vt wasmonitored at intervals by stopping the voltage clamp for 30-60 seconds and using analternative circuit to measure voltage difference. Transrectal resistance (Rt) was calculatedfrom Ise and Vt using Ohm's law.The Isc and Vt decline rapidly over the first 1-2 hours after excision of the recta, butthereafter these variables of ion transport activity decline very little (if at all) over the nextseveral 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 chambersand the tissue was exposed to various substances to study their effect on rectal Isc, Vt andRt.27Everted rectal sac bioassayEverted rectal sacs were prepared as described by Goh and Phillips (1978) andHanrahan et al. (1984). A 3 cm length of PE 90 tubing with a slightly flared end wasinserted through the anus of the locust until its flared end reached the anterior boundary ofthe rectum. The hindgut was raised slightly and the anterior boundary of the rectum wasligated with surgical silk on to the flared end of the tubing. The colon and connectingtrachea were cut away and the rectum was slowly everted by pulling the PE tubingthrough the anus. The everted rectum was then cut from the animal and rinsed with 1 mLof complex saline to remove any hemolymph or fecal material and the posterior wasligated. Any remaining internal fluid was withdrawn completely with a 100 'IL. Hamiltonsyringe and the empty sac was weighed to an accuracy of ± 0.1 mg on an August Sauterbalance. Rectal sacs were filled hourly with 10pL of fresh saline, incubated at 30°C in 25mL of complex saline, and bubbled with a 95% 02: 5% CO2 gas mixture. The weight gainand the tissue volume changes were determined at hourly intervals by weighing sacsbefore and after removal of internal (Hemocoel side) fluid. The true rate of transepithelialfluid movement (iv) was determined by correcting for tissue volume changes.The rectal sacs were allowed to equilibrate for two hours and putative stimulators offluid transport were added to the hemocoel (H) side of the sac at the start of the thirdhour. The change in rate of rectal fluid reabsorption (A Jv) was calculated by subtractingthe ft, for the second hour from the third hour value. These A Jv values were thencompared to the average control values for unstimulated sacs over the same time period.Preparation of NCC extractsNCC 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 12,000g28and 4°C for 10 minutes. The pellet and the floating lipid layer were discarded and thesupernatant was kept at -20°C until use.StatisticsThe significance of differences between two means was determined by paired andindependent t-tests. Analysis of variance (ANOVA) with the Tukey test was employed todetermine differences between multiple means.RESULTSEffect of NCC and cAMPThe effects of NCC and cAMP upon rectal Isc, Vt, and Rt are shown in Table 2.1 andFig. 2.3. Large increases in 'Sc occcured within 2 minutes of addition of 5 mM cAMP, andwithin 5 minutes of adding 2NCC. (Fig. 2.3). Cyclic AMP maintained 'Sc at a constanthigh level for over an hour after initial stimulation, and NCC caused Ise to peak after 30minutes 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). Theseelectrical variables for steady-state unstimulated recta (controls) and the values after NCCstimulation were similar to those reported by Hanrahan and Phillips (1985), using extractsof whole corpora cardiacum (CC).29Table 2.1. Effect of NCC and cAMP upon rectal electrical variables'Sc^Vt^Rt(^(mV) (S2.cm2)1.44 ± 0.22a 8.48 ± 0.80a 265.41 ± 48.82a 8+ 2NCC 7.27 ± 0.92b 20.89 ± 1.77b 120.88 ± 18.85bc1.45 ±0.24a 3.98 ±0.47a 118.22 ± 18.66b 10+ 5mM cAMP 9.30 ± 1.01b 19.18^2.68b 79.27 ± 7.57cValues sharing the same letter are not significantly different from each other (Tukey test,p> 0.05).Vt values are lumen side positive30Fig. 2.3. Effect of NCC and cAMP on rectal I sc : 5mM cAMP and 2 NCC equivalentswere added to the hemocoel side of recta that had been allowed to equilibrate for 2h.Mean ± S.E. (n=6-8).1 2^3Time (h)0 4 5031Fig. 2.4. Fluid reabsorption by everted rectal sacs with time. Complex saline controlshours 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 t-test, p< 0.01).32Fig. 2.5. Tissue swelling by the everted rectal sacs from Fig. 2.4. Complex saline controlshours 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 t-test, p< 0.01).33Unstimulated rectal sacs had a mean Jv of 18.7 ± 0.8 1_11.,1-1 during the first hour afterexcision (Fig. 2.4). Jv fell steadily to 12.7 ± 0.7 during the second hour, butdecreased more slowly during the third and fourth hours. Although steady Jv was notobserved, these Jv values are much higher than those reported in earlier studies (Proux etal., 1984). Tissue swelling of 1.8 ± 0.3 [tL.h-1 was observed during the first hour, buttissue volume fell slowly over the next three hours to near the initial value (Fig. 2.5). Theaddition of 2 NCC to the hemocoel side of rectal sacs during the third hour increased Jvby 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 (Prouxet al., 1984). When rectal sacs were also exposed to NCC during the fourth hour, Jvremained elevated compared to the control value (Fig. 2.4) and there was a small butsignificant reduction in tissue swelling compared to control values (Fig. 2.5). DiButyrylcAMP (DBcAMP, 1 mM) and cAMP (5 mM) were also shown to increase Jv during thethird hour to a similar extent as NCC (Fig. 2.6). DBcAMP and cAMP caused nodifferences in tissue swelling compared to control values.Effect of chemicals acting on the cAMP mediated pathwaySpcAMPS and RpcAMPS are respectively specific stimulators and inhibitors of proteinkinase A (PKA; see Fig. 2.1). SpcAMPS (5 mM) maximally stimulated Jv, while 1-10 mMRpcAMPS had no effect upon Jv when added during the third hour (Fig. 2.7). Howeverwhen rectal sacs were pre-incubated with 10 mM RpcAMPS during the second hour andthen stimulated with NCC on the third hour, there was a significant reduction in A Jvcompared 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 beendemonstrated, it was also necessary to show that cAMP was acting intracellularly and notvia cell surface purigenic receptors. The involvement of purigenic surface receptors in** *^ -TC NCC cAMP DB cAMPFig. 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).348035Fig. 2.7. Effect of cAMP, SpcAMPS and RpcAMPS upon rectal J. Mean ± S.E. (n=6-8). Values sharing the same letter are not significantly different from each other (Tukeytest, p> 0.05).36Fig. 2.8. Effect of a lh pre-incubation with 10 mM RpcAMPS on stimulation of rectal Jvby 2 NCC. Mean ± S.E. (n=11-12). * RpcAMPS+NCC is significantly different from bothcontrol (C) and NCC values (Tukey test, p< 0.05).37 1210,NN +cAMPATP Adn.Fig. 2.9. Effect of ATP and Adenosine (Adn) upon rectal 'Sc (open bars). 5 mM ATPand 5mM Adn were added to hemocoel side at steady-state: 30 min. after testing theseagents, 5mM cAMP was added to hemocoel side of the same preparations (hatched bars).Mean ± S.E. (n=4-10).38stimulation of 'Sc was tested by adding 5 mM adenosine (preferred by Pi purigenicreceptors) and 5 mM ATP (preferred by P2 purigenic receptors) to the hemocoel side ofrecta at Steady-state. Neither adenosine nor ATP had any effect upon rectal 'Sc or on theresponse of recta to stimulation with cAMP (Fig. 2.9). The effects of these agents onrectal 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 to 4.96 ±1.33 When NCC was added Ise further increased significantly but thisIBMX + NCC value was not significantly different (Tukey test, p> 0.05) from the valuefor NCC alone.Effect of cGMPCyclic 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) alsoslowly increased Isc (Fig 2.12), increased Vt and lowered Rt (Table 2.2). Subsequentaddition of 5 mM cAMP in the presence of cGMP further increased Vt and furtherreduced Rt, and caused Ise to rise four times more rapidly (Fig 2.12 and Table 2.2).39Table 2.2. Effect of cGMP upon rectal electrical variables and the subsequent addition ofcAMP 30 minutes later (n=8).t^ 'Sc^Vt^Rt(mins) (^(mV) (cicm2)120 C 1.41 ± 0.12 6.12 ± 0.81 167.63 ± 24.06150 5mM cGMP 3.69 ± 0.26* 13.80 ± 1.77* 141.55 ± 17.42*180 5mM cAMP 7.95 ± 0.57* 20.67 ± 2.31* 97.49 ± 9.23** values are significantly different from preceding values (paired t-test, p< 0.05).t is time after mounting rectaVt is lumen side positive40141210-- ---*-_-C NCC^C IBMX IBMX+NCCFig. 2.10. Effect of 'BMX upon rectal 'sc. B3MX (0.1 mM) was added to hemocoel sideat 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).C cAMP cGMP8I0 T41Fig, 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).42Fig. 2.12. Effect of cGMP upon rectal Isc. 5 mM cGMP was added to hemocoel side atsteady-state, 30 min. later 5mM cAMP was added to hemocoel side. Mean ± S.E. (n=8).43DISCUSSIONIn this chapter, the effects of agents that influence the adenylate cyclase pathway onrectal 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 (DibutyrylcAMP, SpcAMPS) were shown to stimulate rectal Jv and 'Sc to the same extent as NCChomogenate. This suggests that activation of the adenylate cyclase signal transductionpathway 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, alsomaximally stimulated rectal Ise. While the cAMP analog SpcAMPS maximally stimulatedIv, its diastereoisomer RpcAMPS partially inhibited the stimulation of Jv by NCC. SinceRpcAMPS is known to inhibit cAMP dependant protein kinases (i.e. PKA; Rothermel andParker-Botelho, 1988), this partial inhibition of Jv could indicate that NCC factors mayutilize other signal transduction pathways to stimulate rectal reabsorption. Anotherpossibility is that RpcAMPS was unable to fully inhibit the activation of PKA by elevatedintracellular cAMP levels. It seems unlikely that cAMP and its analogs act by stimulatingcell surface purinergic receptors since addition of ATP and adenosine had no effect onrectal C1 transport. The inhibition of rectal cAMP phoshodiesterase activity by B3MX,presumably increased cAMP levels to cause a half maximal increase in rectaltransport. Chamberlin and Phillips (1988) have shown that theophylline, another PdEinhibitor, did cause an increase in rectal cAMP levels. The effects of IBMX and NCC werenot additive, since addition of both only stimulated 'Sc to the same extent as addition ofNCC alone.This study also provides evidence that the guanylate cyclase signal transductionpathway is involved with the stimulation of rectal fluid and ion reabsorption. Addition ofexogenous cGMP caused partial stimulation of rectal 'Sc and Jv (compared with NCC andcAMP). This cGMP-stimulated increase in rectal Ise is similar in magnitude to a cGMP-44stimulated increase in 'Sc previously reported by Chamberlin and Phillips (1988), and therise in 'Sc correlates with the delayed rise in rectal tissue cGMP levels observed by theseworkers. Fournier and Dubar (1989) have reported that nitroprusside, which is anactivator of soluble guanylate cyclase, and cGMP both had a diuretic effect upon Locustamigratoria recta. They also showed that neuroparsins, an antidiuretic factor isolated fromNCC of L. migratoria, does not increase cellular cAMP levels but does slightly elevatecellular cGMP. These findings suggest that S. gregaria and L. migratoria which areclosely related orthopterans, possess different antidiuretic factors that utilize differentsignal transduction mechanisms. However these differences may be of result of the unusualconditions used to measure Locusta rectal Jv. In the Locusta experiments, a- was omittedfrom the bathing saline on the first hour and added to the saline at the same time asexposure to the treatment (i.e. neuroparsins, pharmacological agents) at the onset of thesecond hour (see Fournier et al., 1987). Restoration of C1 to the tissue might be expectedto trigger tissue swelling and hence second messenger events to restore cell volume.The involvement of both the adenylate and guanylate cyclase signal transductionpathways in controlling S. gregaria rectal reabsorption, does not rule out the possibilitythat other alternative control pathways are also involved. Rectal transport mechanismsmay be controlled and fine-tuned by the interaction of multiple intracellular mechanisms(e.g. elevated intracellular cAMP might increase cellular Ca2+ levels etc.) . The followingtwo chapters will investigate the role of other signal transduction mechanisms in thecontrol of rectal transport.45CHAPTER 3: THE INVOLVEMENT OF CALCIUM INCONTROLLING RECTAL TRANSPORTINTRODUCTIONHeilbrunn and Wiercenski (1947) were the first to demonstrate that Ca2+ is involved incellular control when they injected Ca2+ into frog muscle cells and caused them tocontract. Subsequently, intracellular Ca2+ has been shown to regulate many cellularprocesses in vertebrates, but less is known about the role of Ca2-1- in controlling insectcellular 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 respondspecifically and quickly to numerous stimuli. Low resting cytosolic Ca2+ levels are crucialfor cellular control and homeostasis. Free cytosolic Ca2+ is usually less than 10-7 M,while concentrations of external Ca2+ and the Ca2+ sequestered in intracellularcompartments are normally in the millimolar range (Berridge, 1983). Therefore there is alarge 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 gradientis maintained by numerous mechanisms that include: the active pumping of Ca2+ byCa21-/ATPases into intracellular stores or outside of the cell, the expulsion of Ca2+ viaplasma membrane Na-1-/ Ca2+ ion exchangers, the entry of Ca2+ across the favourablecation gradient of the mitochondria, and binding of Ca2+ to molecules in the cytosol (seeFig. 3.1; reviewed by Alberts et al., 1989). It is important to note that entry of Ca2+ intothe cytosol is primarily across the membranes of the internal Ca2+ sequesteringcompartments, since the surface area of the plasma membrane is 10-100 times less thanthe total surface area of the Ca2+ containing organelles (Alberts et al., 1989).Calcium binding proteins, such as troponin C and calmodulin, are responsible fortranslating these Ca2+ increases into cellular responses (Berridge, 1983). These proteins46Fig.3.1. Schematic diagram of the cellular processes involved with the regulation ofintracellular Ca2+. Calcium pumps and ion exchangers are represented by shaded circlesat the plasma membrane and the membrane of the intracellular stores. Ion channels andionophores are represented by an arrow passing between parallel lines, while agents thatdecrease or increase cytosolic Ca2+ appear in solid boxes with associated -/+ signs.47possess high-affinity Ca2+ binding sites that are very specific for Ca2+ since cytosolicMg2+ levels are typically high (in the millirnolar range, Alberts et al., 1989). The bindingof 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 cellularprocesses. Cellular effects that are mediated through Ca2+ binding proteins include: Ca2+induced contraction of vertebrate skeletal muscle, pre-synaptic release ofneurotransmitters by nerve cells, and the activation of Ca2+ pumps that lower cytosolicCa2+ to create a negative feedback loop (Berridge, 1983). It must be kept in mind thatintracellular Ca2+ and cAMP can interact to control various cellular processes (reviewedby Cheung, 1982). The first of these interactions is that Ca2+ and cAMP levels canmodulate 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 increasesor decreases in intracellular Ca2+. In the second type of interaction, both Ca2+ and cAMPcan regulate the same protein. An example of this is phosphorylase kinase, which isinvolved with glycogen breakdown and can be stimulated by a cAMP-dependent kinase aswell 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 (Achetadomesticus) that was shown to inhibit Malpighian tubule secretion by 70%. Spring andClark (1990) later showed that the calcium ionophore A23187 also reduced Malpighiantubule secretion by 85%, and proposed that this antidiuretic factor inhibited fluid secretionby elevating intracellular Ca2+ (see also Kim and Spring, 1992). Since many workers havereported 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 anantagonistic manner. Fogg et al. (1990) showed that when L. migratoria Malpighian48secretion was stimulated with CC homogenate, both cAMP and inositol trisphosphate(InsP3) levels rose. Since InsP3 stimulates the release of sequestered Ca2+, it wouldappear that both Ca2+ and cAMP act cooperatively in the stimulation of fluid secretion.Coast (personal communication, 1992) has used the calcium ionophore ionomycin toincrease 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 isconflicting evidence concerning the role Ca2+ plays in this control.Calcium may also be involved with the control of rectal transport mechanisms, eventhough Hanrahan and Phillips (1985) saw no effect upon S. gregaria rectal C1 transportwhen a Ca2+ ionophore was added to the preparation. Fournier (1990b) presentedevidence to show that L. migratoria rectal fluid transport (iv) increased when Ca2+ionophores were added. He has also shown that L. migratoria neuroparsins (a potentialinsect antidiuretic peptide) increased InsP3 levels suggesting that Ca2+ is involved withthe stimulation of rectal Jv. Fournier (1990b) claims that rectal Jv is dependant uponextracellular Ca2+ levels, since removal of Ca2+ from incubating saline significantlyreduced 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 byremoving Ca2+ from the bathing medium. In addition, the ability of NCC homogenate andcAMP to stimulate rectal transport under Ca2+ free conditions was studied. The role ofintracellular Ca2+ in controlling rectal transport was explored by using agents known tomodulate cytosolic Ca2+ levels (see Fig.3.1). The calcium ionophore ionomycin (IY) inknown to increase cytosolic Ca2+ levels by inserting into both the plasma membrane andthe membranes of the intracellular organelles (Fournier, 1990b). Thapsigargin (THAPS) isalso known to increase cytosolic Ca2+ by inhibiting the Ca2+/ATPase pump that isresponsible for removing Ca2+ from the cytosol (Takemura et al, 1989; 'Thastrup et al.,1990). Trimethoxybenzoate hydochloride (TMB-8) is thought to lower cytosolic Ca2+ by49preventing its release from intracellular stores (Smith and Iden, 1979). The phosholipase C(PLC) mediated signal transduction pathway, via inositol 4,5-trisphosphate (InsP3), is alsoknown to increase intracellular Ca2+ levels. The involvement of this PLC pathway in thecontrol of rectal transport will be addressed in the next chapter.MATERIALS AND METHODSThe methods used in this study were similar to those described in Chapter 2, unlessotherwise indicated. The locust colony was maintained under similar conditions and theNCC 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 complexsaline. However, Ionomycin and Thapsigargin were initially dissolved in 100% DMSO andthese 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 salinecontrols (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 nothave 5 mM CaCl2 and contained 1 mM EGTA to ensure that any trace amounts of Ca2+would be chelated.50RESULTSEffect of Ca2+ free salineIncubation of rectal sacs in calcium-free saline (Cat) for four hours did not cause anydecrease in Jv compared to control values over the same time course (Fig.3.2). EGTA (1mM) was added to the Ca2+ free saline to ensure that any trace amounts of Ca2+ werechelated, and this saline gave similar results as Ca2+ free saline without EGTA (data notshown). When rectal sacs were incubated in saline with 10 mM EGTA, Jv was muchlower 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 foamyliquid was being collected from the hemocoel side of these sacs and the lumen sideappeared whitish instead of the usual yellow/brown. It seems that 10 mM EGTA is toxicto rectal cells and causes cell dissociation on the hemocoel side. Cell dissociation was notseen on the lumen side probably because the cuticle helped to hold the rectal tissuetogether.Both NCC and DBcAMP increased Jv to a greater extent in Ca2+ free saline than incomplex saline with Ca21- (Figs. 3.2 & 3.4). Stimulation of recta with 2 NCC in Ca2+ freesaline also caused a slight increase in rectal tissue swelling (,-,.. 1 pL.) compared to NCCcontrols (Fig. 3.3). Exposure of unstimulated recta to Ca2+ free saline had no effect uponIsc and subsequent stimulation of recta with NCC gave similar changes in electricalvariables as seen with NCC controls in normal saline (Table 3.1).51Table 3.1. Effect of Calcium free saline upon electrical variablesIsc^Vt^Rt^n(^(mV) (Ci.cm2)Control 1.44 ± 0.22a 8.48 ± 0.80a 265.41 ± 48.82a 8+ 2NCC 7.27 ± 0.92b 20.89 ± 1.77b 120.88 ± 18.85bCa2+ free 2.54 ± 0.40a 10.94 ± 2.30a 166.83 ± 22.83ab 7+ 2NCC 10.07 ± 2.10 b 20.36 ± 2.5 lb 88.55 ± 13.91bValues sharing the same letter are not significantly different (Tukey test, p> 0.05)Vt values are lumen side positive1 2^3Time (h)0 4 5052Fig. 3.2. Fluid reabsorption by rectal sacs under Ca2+ free conditions. Control complexsaline 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 NCCadded to hemocoel side on third hour (Caf+NCC). Mean ± S.E. (n=8-13). Values fromsimilar times that share the same letter are not significantly different (Tukey test, p> 0.05).53Fig. 3.3. Tissue swelling of rectal sacs under Ca2+ free conditions. 2 NCC added onhours 3+4 in presence of Ca2+ (NCC), Ca2+ free saline for hours 1-3 with 2 NCC addedon third hour (Caf+NCC). Mean ± S.E. (n=11-13). ** Caf+NCC is significantly differentfrom NCC (independent t-test, p< 0.01).1 21 0.\\1with 5mM Ca"Ca" free*-_T_54C Cal NCC DBcAMPFig. 3.4. Effect of Ca2+ free saline (Cal) upon Jv stimulated by 2 NCC and 1 mMDBcAMP. Mean ± S.E. (n=10-13). * values are significantly different from similartreatments with Ca2+ in bathing saline (Tukey test, p< 0.05).55Effect of agents that modulate cytosolic CalciumThe calcium ionphore ionomycin, an agonist of intracellular Ca2+, had a partialstimulatory 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 by10% increases the effectiveness of ionomycin; however when this procedure was triedthere was no additional stimulation of Jv by 4 iiM ionomycin (Fig. 3.5). No increase in Jvwas seen when sacs were incubated in Ca 2+ free saline and then exposed to 4 iiMionomycin.Addition of 1 1.1M ionomycin to unstimulated short-circuited recta, with calciumpresent, had no effect on Isc but reduced the stimulatory action of NCC by over 40% (4.1± 0.24 compared to 7.3 ± 0.9 for the NCC control, see Fig.3.6). Thapsigargin, another agonist of intracellular Ca2+, had no effect upon resting Isc orNCC stimulated 'Sc when used at 10-50 AM (Fig. 3.6). TMB-8, an antagonist ofintracellular Ca2+, had no effect upon rectal Ise or subsequent stimulation with cAMPwhen used at 200 and 1000 IAM concentrations. Although 1 mM TMB-8 reduced cAMPstimulated '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 Iscby 40% ( 4.3 ± 0.6 1 compared to 7.27 ± 0.92 for the NCCcontrol, see Fig. 3.8).56Fig. 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).\\N + NCC10^-*--57C^IY^10^50THAPS (MM)Fig. 3.6. Effect of 1 mM IY and Thapsigargin (THAPS) upon rectal Isc. At steady-stateagents 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 correspondingNCC control value (Tukey test, p< 0.05).1210IM + cAMP----_58C^200^1000TMB8 (AM)Fig. 3.7. Effect of TMB-8 upon 5 mM cAMP stimulated 'Sc- At steady-state TMB-8 wasadded bilaterally, 30 min. later cAMP was added. Mean ± S.E. (n=8-10).1210\\N + NCC*--59C TMB 8Fig. 3.8. Effect of TMB-8 upon 2 NCC stimulated 'Sc. At steady-state 1 mM TMB-8 wasadded bilaterally, 30 min. later 2 NCC were added. Mean ± S.E. (n=8-10). * value issignificantly different from NCC control (Tukey test, p< 0.05).60DISCUSSIONThe removal of external Ca2+ from the bathing medium did not reduce either the fluidor ion reabsorbing ability of the recta. When recta were stimulated with either NCC orcAMP under Ca2+ free conditions, there were larger increases in rectal Jv compared toincreases seen when Ca2+ was present. Calcium-free conditions did not have any effectupon the stimulation of Isc or Vt with either cAMP or NCC. Since Ca2+ free conditionsincreased stimulated Jv without changing values for stimulated electrical variables, itwould appear that the absence of Ca2+ may have an extracellular rather than anintracellular effect upon rectal cells. A possible effect of removing Ca2+ from the bathingsaline could be the loosening of the tight junctions at the apical and basal complexes thathold adjacent rectal pad cells closely together (refer to Fig. 1.2). The opening of thesecomplexes may facilitate the movement of fluid through the intercellular spaces toward thebasal membrane.Fournier (1990b,1991) has reported that bathing Ca2+ is essential for the maintenanceof resting rectal Jv in L. migratoria, since the incubation of tissues with the Ca2+ chelatorEGTA dramatically reduced Jv by 90% within the first hour. Fournier (1990b) proposesthat this apparent sensitivity of the rectum to external Ca2+ is a result of "an insufficientmobilization or lack of calcium from intracellular stores". It seems unlikely that these twoclosely related insects can possess recta that have differing dependencies upon externalCa2÷, so why are Fournier's findings so different from the results of this study? A possiblereason for Fournier's different results, is that he may have used a high concentration ofEGTA in his Ca2+ free bathing medium that proved toxic to the rectal cells. It is unclearwhat actual concentration of EGTA was used, but a Ca2+ free bathing saline containing0.8 M Ca2+ with 1.2 M EGTA has been reported in numerous papers (see Fournier1990b,1991; Fournier et al., 1992). In my study, 10 mM EGTA in Ca2+ free saline provedtoxic to the rectal cells, almost abolishing all Jv by the third hour of incubation. It also61seems unlikely that intracellular rectal Ca2+ levels, which are regulated very closely by thecell and are essential for the maintenance of cellular homeostasis, are so dependant onexternal Ca2+. In support of the fmdings of this study, Berridge (1977) has also shownthat fluid secretion by the salivary glands of the blow fly (Calliphora) was independent ofexternal bathing Ca2+.The use of agents that modulate intracellular Ca2+ levels gave mixed results. Firstly, theaddition of ionomycin to recta submaximally increased rectal Jv (-40%) compared to theincreases 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 highintracellular 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 andcAMP by 40-50%. These TMB-8 results suggest that the stimulation of transport bycAMP is dependant upon the elevation of cytosolic Ca2+ levels by release fromintracellular stores. Since thapsigargin had no effect upon resting or stimulated Ise, it mustbe concluded that either the agent did not enter the cell or that it could not elevatecytosolic 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 rectaltransport processes. However it seems that the role of Ca2+ is quite complex, and thatCa2+ 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 assuggested by Fournier (1990b,1991). Earlier evidence suggests that rectal Jv results fromtransport (Isc, Goh and Phillips, 1978). The seemingly different effects of Ca2+ onrectal Jv and 'Sc suggest that Ca2+ may control processes other than Cl transport whichinfluence Jv (e.g. movement of counter ions such as K+ and Na+, or ion recycling in thelateral spaces of the rectal epithelium).62CHAPTER 4: THE ROLE OF PKC AND THE PI CYCLE INCONTROLLING RECTAL TRANSPORTINTRODUCTIONIn the previous chapter there were some indications that intracellular Ca2+ wasinvolved with the control of rectal transport. Although the adenylate cyclase signaltransduction 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 tobe the primary method of elevating intracellular Ca2+ (Berridge and Irvine, 1984). PLC isactivated 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 ofCa2+ from intracellular stores and the entry of exogenous Ca2+, DAG stimulates theCa2+ dependant protein kinase C (PKC) which then phosphorylates various cellularproteins (see Fig. 4.1; Berridge and Irvine, 1989; Nishizuka, 1986).There is some evidence that PLC-mediated events are involved in the control secretionin insects. Berridge (1986) reported that InsP3 levels rose in Calliphora salivary glandswhen 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 cAMPand InsP3 levels occurred between 30-300 s for cAMP and 15-300 s for InsP3. Fournier(1990b), using pharmacological methods, provided evidence that L. migratorianeuroparsins (Nps) stimulated rectal Jv through PLC-mediated events. When Li+ wasused to inhibit the phosphotidylinositol (PI)IntracellularCalcium Storescascadesof proteinmodificationsNeuropeptideReceptorHemolymph11. RESPONSES63Fig. 4.1. Diagram of the phosholipase C (PLC) mediated signal transduction pathway. TheG protein activated PLC breaks down phosphatidylinositol 4,5-bisphoshate (PIP2) intodiacylglycerol (DAG) and inositol trisphophate (InsP3). InsP3 then opens Ca2+ channelsthat are represented by an arrow passing between two parallel lines. The cellular action ofagents added to the rectal preparations are shown by arrows with associated +/- signs.64cycle, the stimulatory effect of Nps was abolished. The addition of the PI cycle componentmyo-inositol was sufficient to maximally stimulate rectal Jv. Fournier (1990b) alsosuggested that PKC was involved in the stimulation of L. migratoria Jv since addition ofspecific PKC stimulators maximally stimulated Jv, while addition of the PKC inhibitorPolymyxin B (PMXB) abolished the stimulatory effect of Nps.Fournier (1991) has recently demonstrated that Nps stimulate InsP3 and inositol 1,3,4,5tetrakisphosphate (InsP4) production in L. migratoria rectal cells. In addition it wassuggested that PKC activity is necessary for the phosphorylation of enzymes involved withinositol phosphate metabolism, since the inositol phosphate cascade was mimicked usingPKC stimulators and inhibited with PMXB. It was also reported that neither the adenylatenor guanylate cyclase systems modified inositol phosphate metabolism.In this chapter the role of the PLC-mediated signal transduction pathway in controllingrectal transport was studied. Specific stimulators and inhibitors of protein kinase C wereused to explore its role in controlling rectal transport. The PKC stimulators used were 1-stearoy1-2-arachidonoyl-sn-glycerol (SAG) and phorbol 12-myristate-13-acetate (PMA),while polymyxin B sulfate was used to inhibit PKC. The involvement of thephosphatidylinositol (PI) cycle in the control of rectal transport was examined byattempting to stimulate it with myo-inositol, a component of the PI cycle. In addition, theeffects of inhibiting the PI cycle were investigated using U. Lithium is known to blockthe conversion of inositol phosphate to inositol and hence synthesis of inositoltrisphosphate (InsP3; see Fig. 4.1; Berridge and Irvine, 1989). L. migratoria rectalpreparations were used to investigate the role of the PI cycle in controlling rectal Jv andIsc, since Fournier (1990b, 1991) had previously indicated that L. migratoria neuroparsinsincreased rectal Jv by increasing InsP3 levels. Neuroparsins has also been shown to haveanti-juvenile hormone activity and increase hemolymph trehalose and lipid levels (Girardieet al., 1987; Moreau et al., 1988) Finally, neuroparsins was tested on S. gregaria rectalpreparations to test for any cross-reactivity between these species.65MATERIALS AND METHODSMost of the methods used in these experiments were similar to those described inChapter 2, unless otherwise stated. The Locusta migratoria colony was maintained aspreviously described for the Schistocerca gregaria colony. Adult 6 week old maleLocusta migratoria were used for the Locusta fluid transport experiments, while 6 weekold 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-stearoy1-2-arachidonoyl-sn-glycerol (SAG) and phorbol 12-myristate-13-acetate (PMA) were initiallydissolved in 100% dimethyl sulfoxide (DMSO) and these stock solutions were thendiluted 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).RESULTSEffect of agents acting upon protein kinase CStimulators of PKC, SAG and PMA had no stimulatory activity upon S. gregaria rectalJv and 'Sc (Figs. 4.2 & 4.3). Polymyxin B sulfate (PMXB), a PKC inhibitor, slightlyincreased NCC stimulated Jv (6.1 ± 0.6 pL.11-1 compared to 4.4 ± 0.5 pL.h-1 with NCCalone), but had no effect upon unstimulated or NCC stimulated 'Sc (Figs. 4.4 & 4.5).66Effects of agents acting upon the Phosphotidyl-Inositol cycleMyo-inositol (MI) is a component of the PI cycle and was shown to have no effect uponS. gregaria A Jv (-1.7 ± 0.6 pit.h-1) when used at 0.1-50 mM (Fig. 4.6). In addition, MIhad no effect upon rectal Isc at either low or high concentrations (Fig. 4.7). LiC1 is aninhibitor of the PI cycle, and its effects upon Jv and 'Sc were studied using a 10 minutepulse of 10 mM LiC1 applied to recta immediately before adding NCC (as described byFournier 1991). LiC1 had no effect upon NCC stimulated Jv (Fig. 4.6), did not changeunstimulated 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 uponNCC 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 Vtcompared to control values (see Table 4.1.).67Table 4.1. Effect of 1mM LiC1 upon electrical variables'Sc^Vt (mV)^Rt( (S2.em2)Control 1.4 ± 0.2 8.5 ± 0.8 265.4 ± 48.8 8+ 2NCC 7.3 ± 0.9 20.9 ± 1.8 120.9 ± 18.9LiC1 2.7 ± 0.6 20.7 ± 5.1 272.2 ± 25.9 8LiC1 + 2NCC 12.8 ± 1.5* 52.9 ± 8.4* 150.0 ± 8.6* values are significantly different from the respective control values (Tukey test, p<0.05).Vt values are lumen side positiveFig. 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).PMXB+NCCC onlyNCC708.......^6750C.)a)>'-D<20Fig. 4.4. Effect of a 100pM PMXB (1000 iu/mL.) upon 2 NCC stimulated J. Rectawere 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 NCCvalues (Tukey test, p< 0.05).*T -T--T_--8 -10-T20-I-71C NCC^C PMXB PMXB+NCCFig. 4.5. Effect of 100 pM (1000 iu/mL.) PMXB upon rectal Isc• PMXB was addedbilaterally at steady-state, and then 30 minutes later 2 NCC were added. Mean ± S.E.(n=7-8).-I -8-6TII—4420—272C^50^1^0.1^LiC1 onlyMI (mM) +NCC NCCFig. 4.6. Effects of myo-inositol (MI) and LiC1 upon rectal Jv. In the LiC1 experimentrecta were exposed to a 10 minute pulse of 10 mM LiC1 and then immediately stimulatedwith 2 NCC. Mean ± S.E. (n=6-11).-r73108201^6a)450^1^0 . 1^C NCC LiC1 LiC1MI (mM) +NCCFig. 4.7. Effects of MI and LiC1 upon rectal 'sc. In the LiC1 experiment recta wereexposed to a 10 minute pulse of 10 mM LiC1 and then immediately stimulated with 2NCC. Mean ±S.E. (n=5-17).74Locusta experimentsLocusta 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 oneand two respectively (Fig. 4.8), while Schistocerca sacs transported 18.7 ± 0.8 pL.11-1 and12.7 ± 0.7 mI..,.11-1 at the same time intervals. Locusta rectal sacs exhibited a small amountof tissue swelling (1.0 ± 0.5 tiL11-1.) during the first hour and a negligible tissue tissuevolume change over the following two hours (Fig.4.8), similar to the response observedfor Schistocerca sacs (Fig. 2.5). Addition of 50 mM MI to Locusta rectal sacs on the thirdhour had no effect upon Jv, while the addition of 1 mM DBcAMP during the fourth hoursignificantly increased Jv (Fig. 4.8).Unstimulated electrical variables for Locusta recta (Table 4.2) closely resembledequivalent Schistocerca values (Table 2.1). The addition of 50 mM MI had no effect uponLocusta electrical variables, while subsequent additon of 1 mM DBcAMP stimulated Isc,Vt and reduced Rt. Locusta electrical variables after addition of DBcAMP closelyresemble Schistocerca electrical variables after stimulation with cAMP or NCC.Unfortunately further experiments using Locusta migratoria recta could not be performedbecause the colony perished.Effect of Neuroparsins on S.gregaria rectaNeuroparsins applied at a very high concentration of 5CC gland equivalents had noeffect upon S. gregaria rectal Jv or Isc (Figs. 4.9 & 4.10). All preparations subsequentlyresponded to S. gregaria NCC. Unfortunately we were unable to test the effect of Nps onL. migratoria recta since there were no animals available.75Table 4.2. Effect of MI and DBcAMP upon electrical variables of L. migratoria recta(n=4)Isc( 1.55 ± 0.42 11.68 ± 2.73 288.56 ± 17.1050mM MI 1.44 ± 0.42 11.50 ± 3.50 289.01± 14.191mM 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 positive76Fig. 4.8. Fluid reabsorption by Locusta migratoria everted rectal sacs. During thethird hour 50 mM MI was added to the hemocoel side, then 1 mM DBcAMP wasadded to the hemocoel side during the fourth hour. Tissue swelling is representedby the dotted line. Mean ± S.E. (n=6). * fourth hour value is significantly differentfrom third hour value (p< 0.05, paired t-test).77Fig. 4.9. Effect of Neuroparsins upon rectal S. gregaria J. Neuroparsins (Nps; 5CCgland equivalents) or 2NCC were added to the hemocoel side during the third hour. Mean± S.E. (n=8-11).--810C.)rn2T078C NCC C Nps NCCFig. 4.10. Effects of Neuroparsins upon S. gregaria rectal Ise. Neuroparsins (Nps; 5CCgland equivalents) was added to the hemocoel side of recta at steady-state, 30 minuteslater 2NCC were then added to the same preparations. These results are compared tocontrol preparations to which only NCC was added. Mean ± S.E. (n=8).79DISCUSSIONIn this study, neither PKC nor the PI cycle were shown to play a stimulatory role in thecontrol of S. gregaria rectal transport. However PKC may have a slight inhibitory effectupon rectal Jv, since the PKC inhibitor PMXB slightly increased NCC-stimulated J. Italso seems that the PI cycle plays a slight inhibitory role in controlling rectal transportsince long-term Li+ exposure elevated NCC-stimulated 'Sc and Vt. These results aremarkedly different from those with L. migratoria recta, where stimulants of PKC and thePI cycle were reported to maximally increase Jv, while specific inhibitors preventedstimulation of Jv with Nps (Fournier, 1990b).Locusta migratoria rectal preparations were shown to possess similar electricalvariables as S. gregaria recta, although L. migratoria recta transported fluid at slightlylower rates than S. gregaria recta, probably because L. migratoria recta are smaller. Thepatterns of L. migratoria Jv and tissue swelling with time were similar to patternspreviously seen for S. gregaria recta. Although both L. migratoria Jv and Isc respondedto stimulation with Dibutyryl cAMP, myo-inositol had no effect. Since myo-inositol hadno 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 50mM myo-inositol can maximally increase L. migratoria rectal Jv. Finally, there was nocross-reactivity between L. migratoria neuroparsins and the S. gregaria rectalpreparations, even though the same Nps sample was later shown to have significant fluidtransporting activity on L. migratoria recta (J. Proux, personal communication). Since ourlab 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 signaltransduction pathways by Nps using our bioassays.It would initially appear that L. migratoria and S. gregaria recta are controlled bydifferent antidiuretic peptides that employ separate signal transduction mechanisms.80However, Herault and Proux (1987) showed that there is a factor in L. migratoria GCCthat increases rectal Jv by elevating intracellular cAMP. In addition, L. migratoria CChomogenates have been shown to maximally stimulate S. gregaria rectal Isc, suggestingthat L. migratoria does possess factors with S. gregaria cross-reactivity (J. Marshall,unpublished results). This present study raises an important question: Is L. migratorianeuroparsins a true antidiuretic factor, or is its reported activity an artifact of the unusualbioassay procedure used to study rectal Jv? This unusual procedure was discussed inchapter 2, and involves the omission of Cl- from the bathing saline during the first hour ofrectal incubation. On the second hour, Cl- was added back to the saline at the same timeas 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 varioustreatments, including Nps, may trigger second messenger mechanisms that are involvedwith cell volume regulation, leading to apparent increases in rectal Jv. These experimentsby Fournier's group need to be repeated using steady-state conditions, where Cl- is presentthroughout the experiment, before neuroparsins can be considered a potential antidiureticfactor.81CHAPTER 5: GENERAL DISCUSSIONThe fmdings of this study suggest that cAMP is the primary second messengerresponsible for controlling rectal C1 and fluid transport in Schistocerca gregaria, and thatother second messengers are also involved. The second messenger cGMP was also shownto have a partial stimulatory action in controlling rectal transport. The evidence for therole of Ca2+ in controlling rectal transport is interesting, suggesting that different cellularCa2+ 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 byPLC activation, were shown to have a role in stimulating rectal Jv or 'sc• Many of theresults from this study are strikingly different from those previously reported, using theclosely related species L. migratoria (Fournier, 1990b,1991). However, the unusual assayprocedures 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. Thefirst limitation was that a crude NCC aqueous extract was used to study the signaltransduction mechanisms involved in controlling rectal Jv and 'Sc. This crude extract mayhave contained multiple factors that control rectal transport. The use of individual purifiedpeptides could simplify the interpretation of the results and allow the construction of morerepresentative models. My justification for using crude NCC was the claim by Fournierand Girardie (1988) that neuroparsins is the only stimulant of rectal Jv in L. migratoriaNCC 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 yetbeen purified. Phillips et al. (1980) managed to partially purify CTSH from S. gregaria CCthat was very unstable during HPLC, while ScgITP had no effect upon rectal Jv and onlystimulated 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.82Although another attempt to purify CTSH could be undertaken, a more promisingapproach may be to concentrate on signal transduction mechanisms involved with thestimulation of ileal transport by ScgITP. The advantages of using the ileum are: 1) it is asimple epithelium comprised of fewer cell types than the rectum; 2) there is no recycling ofions in the lateral intracellular spaces between the Heal epithelial cells 3) the major CCpeptide that stimulates transport (ScgITP) has already been purified and partiallysequenced.Another limitation of this study was that the effects of NCC homogenate on secondmessenger levels were not monitored. The measurement of second messengers wouldprovide direct evidence for the involvement of specific signal transduction pathways andconfirm the proposed actions of pharmacological agents. Changes in tissue cAMP andcGMP levels could be measured using the cAMP binding protein technique and the cGMPradioimmunoassay to show that purified factors affect these pathways. These assays areboth commercially available. In fact, Chamberlin and Phillips (1988) have already shownthat crude CC homogenate elevated rectal cAMP and cGMP levels. The involvement ofPLC-mediated events, namely the activation of the PI cycle and PKC, could also bemonitored. The role of the PI cycle in the control of rectal transport could be investigatedby loading cells with tritiated myo-inositol (a precursor of the PI cycle) and examining theamounts of labeled intermediates formed (e.g. InsP3 and InsP4) using standard HPLCmethods (Kirk et al., 1990). Protrein kinase C enzyme activity could be measured using astandard assay technique described by Farese and Cooper (1990). The effect ofantidiuretic factors on cytoplasmic Ca2+ could possibly be directly measured by usingfura-2 spectrometry (see Hallett et al., 1990).Lastly, most of the experiments conducted on S. gregaria recta could not be repeatedusing 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 differentfrom Fournier's findings. For example Fournier (1990b) stated that myo-inositol maximally83stimulated rectal Jv in L. migratoria, whereas a similar experiment from this studyshowed no stimulatory effect of myo-inositol on L. migratoria rectal Jv. The onlydifference between these two experiments was the assay conditions. As previously stated,Fournier (1990b, 1991) used an unusual procedure that involved the incubation of therectal tissue in a free saline for the first hour of the experiment. Chloride was re-introduced on the second hour simultaneously with the exposure of the tissue to varioustreatments, either peptide factors or pharmacological agents. It would be interesting to seeif the same results are obtained using a conventional incubation procedure.The importance of the insect excretory system in maintaining hemolymph homeostasis iscomparable to the role of the vertebrate nephron. Both of these systems eliminate toxiccompounds and metabolic waste products, control blood volume and osmolarity, and areinvolved with acid-base regulation. The Malpighian tubules perform a function similar tothat of the glomerular capsule and are responsible for the formation of the isosmoticprimary urine. In vertebrates, the glomerular capsule and the endothelium form theendothelial capsular membrane that filters blood under pressure (60-100 mmHg) allowingthe passage of fluid and ions across the membrane but blocking the entry of largermolecules such as proteins (see Tortora and Anagnostakos, 1984). Malpighian tubulesactively secrete a primary urine that is rich in KC1 and that contains most blood solutes. Incontrast to the glomerulus, Malpighian tubule secretion is under direct endocrine control,with cAMP, InsP3 and Ca2+ being implicated as second messengers. It has been shownthat the ileum acts as the 'proximal tubule' of the locust excretory system, since it activelytransports an isosmotic absorbate, is the major site of ammonia secretion, and is involvedin acid-base regulation (Lechleitner, 1988; Peach, 1991; Thomson, 1990). In vertebrates,proximal tubule transport is stimulated by the steroidal hormone aldosterone. Aldosteroneis released from the outer cortex of the adrenal glands, it then activates the synthesis ofmore Na+ carrier proteins (Na+/K+ ATPases) and also increases the availability of ATPin the tubular epithelial cells (reviewed by Lote, 1987; Guyton, 1991). Locust ileal84transport 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 transductionpathway is not involved (Audsley, 1991). Lastly the locust rectum is responsible fordetermining the fmal ion and water composition of excreta, and can selectively createstrongly hypo- or hyper-osmotic urine. In the nephron, the loop of Henle, distal tubule andthe 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, butthis recycling is performed at different structural levels. The locust rectum can recyclesolutes in the lateral channels between the principle epithelial cells. The late distal tubuleand the collecting duct becomes permeable to water when antidiuretic hormone (ADH) isreleased by the posterior pituitary. ADH binds to the baso-lateral membrane of the targetcells, elevating cAMP levels, leading to the fusing of vesicular structures with the apicalmembrane (see Lote, 1987; Guyton, 1991). These vesicles possess proteins with largewater conducting channels. The locust rectum is also under hormonal control (e.g. CTSH)and from this study it appears that cAMP, cGMP and Ca2+ are all involved with thecontrol of rectal ion and fluid transport.Future studies should attempt to integrate the hormonal control mechanisms of theentire insect excretory system and address the following questions. Are these controllingfactors actually liberated into the hemolymph, if so, where, when, and what are thesensory pathways controlling their release? Do multiple factors control transport in thesame tissue/cell and if so, what are the combined effects of these factors? What conditionsstimulate the release/activation of these factors; feeding, dehydration, satiation?. Theanswers to these questions and the ongoing purification and characterization of peptidefactors, may eventually lead to the development of novel strategies to control insects thatare deleterious to civilization.85REFERENCESALBARWANI, S.A. (1988). 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