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Ion transport in the ileum of the desert locust Schistocerca gregaria, forskal Richardson, Naomi Dinah 1993

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ION TRANSPORT IN THE ILEUM OF THE DESERT LOCUSTSCHISTOCERCA GREGARIA, FORSKAL.byNAOMI DINAH RICHARDSONB.Sc. Durham University, England, 1991.A THESIS SUBMIGGED IN PARTIAL FULFILLMENT OF THEREQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF ZOOLOGYWe accept this thesis as conformingTHE UNIVERSITY OF BRITISH COLUMBIASeptember 1993© Naomi Dinah Richardson, 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 ofBIOLOGICAL SCIENCIES , ZOOLOGYThe University of British ColumbiaVancouver, CanadaDate^16th September 1993DE-6 (2/88)ABSTRACT.Recently, Audsley (1991) purified a neuropeptide factor - scgITP from the coporacardiaca of Shistocerca gregaria. ScgITP stimulates ileal reabsorption of Cr, Kt, Na+and fluid and inhibits acid secretion. Stimulation with scgITP causes an increase in bothCr dependent short-circuit current and transepithelial voltage (Vt) and a decrease intransepithelial resistance (Rt; Irvine et al 1988). There is evidence to suggest that cAMPacts as the second messenger for scgITP (Audsley 1990).In this thesis the mechanisms underlying ileal ion transport and the effects ofcAMP were investigated. Intracellular microelectrodes were used to measure apical andbasolateral membrane potentials. Brief transepithelial current pulses (13.3 gA) and ionsubstitutions permitted the estimation of specific ion conductances.In nonstimualted ilea, Vt was slightly positive (to the lumen side) and the insideof the cell was negative with respect to both haemolymph and lumen side bathingmedia. The apical membrane potential (Va) was slightly more negative than thebasolateral membrane potential (Vb). Apical membrane resistance (Ra) was much higherthan the basolateral membrane resitance (Rb). Stimulation of ileal ion transport withcAMP resulted in an increase in Vt. There was no change in Va after cAMP stimulationbut Vb depolarized. Addition of cAMP caused a large decrease in Rt, due solely to adecrease in Ra with Rb remaining at control values.In nonstimulated ilea, a small apical Na+ conductance was measured. Voltageiimeasurements indicated an increase in apical Na+ conductance after cAMP addition.However, resistance measurements were not consistent with a cAMP stimualted apicalNa conductance. In nonstimulated ilea the conductance of the apical membrane to K+was very small. After stimulation with cAMP a large increase in apical IC -' conductancewas observed. The cAMP stimulated apical IC- conductance was not inhibited by highluminal [1(1. The basolateral membrane displayed a very high conductance to IC' andto a lesser extent Ct. There is no evidence for cAMP stimualtion of basolateral Cl-conductance. These results are formulated into a model of the mechanism of cAMPcontrol of ileal ion transport.iiiTABLE OF CONTENTS.PageAbstract ^  iiTable of Contents^  ivList of Tables  viList of Figures  viiList of Abbreviations  ixAcknowledgements ^  xiiCHAPTER 1^General Introduction  1Structure of the locust excretory system ^ 2Fluid and ion transport in the rectum  6The cAMP second messenger system  9Hormonal control of reabsorption in the rectum ^ 11Fluid and ion transport in locust ileum^ 14The effect of neuroendocrine factors on ileal transport ^ 17Towards a model of cAMP stimulation of ileal iontransport^  18CHAPTER 2^Materials and Methods ^  22Animals ^  22Solutions  22Heal preparation  23Electrical methods ^  28i) Transepithelial measurements ^ 28ii) Microelectrode measurements  30iii) Correction for saline resistance^ 31Microelectrode fabrication ^  31Statistical treatment  32Experimental protocols  32I: The effect of cAMP on ileal electrical parameters ^1.1 Control impalements ^ 32a) nonstimualted controls; the effect of salinechange ^  32b) Cyclic AMP stimulated controls ^ 331.2 Measurement of the timecourse of cAMPstimulation  33II: Investigation of apical Na+ conductance^ 34ivIII: Investigation of IC-' conductances ^ 35IV: Investigation of Cl- conductances of both apicaland basolateral membranes  36CHAPTER 3^Results ^  37I: The effect of cAMP on ileal electrical parameters ^ 37Timecourses under various conditions ^ 37Variability between cells ^ 39Effect of saline changes  41Electrical properties of ileal cells ^ 45Timecourse of cAMP stimulation  49II: Is there a cAMP stimulated increase in apical Na*conductance? ^  52Introduction  52The timecourse of the effect of Na* replacement ^ 52Comparison of electrical parameters in control andNe-free saline and the effect of amiloride ^ 54III: Investigation of IC-' conductances ^ 59Timecourse of the changes in electrical parameters ^ 59A) Is there a cAMP stimulated increase in apicalIC+ conductance ^  61B) Is there a significant basolateral rconductance?  67IV: Is there a cAMP stimulated basolateral Cl -conductance? ^  71CHAPTER 4^Discussion  75I: The effect of cAMP^  75II: Is there a cAMP stimulated increase in apical Na+conductance?  80III: A) Is there a cAMP stimulated apical 1C 4-conductance? ^  84B) Is there a significant basolateral K+conductance?  85IV: Is there a cAMP stimulated increase in basolateralCl- conductance? ^  89CHAPTER 5^General Discussion  95REFERENCE  99vLIST OF TABLES.PageTable 3.1.3 Control impalements for A: nonstimulated and B: cAMPstimulated ilea. ^  44Table 4.1.1^Comparison of electrical measurements made by Irvine etal (1988) and those made in this study for bothnonstimulated and cAMP stimulated ilea. ^ 76viLIST OF FIGURES.Pagea) Locust ailementary canal b) Locust excretory system. ^ 3Ultrastructure of the ileal epithelium and rectal pad of thelocust. ^  5Summary of ion transport processes in locust rectum. ^ 7The cAMP second messenger cascade. ^ 10The insect neuroendocrine system.  12Audsleys' model of the control of ileal ion transport. ^ 19Diagram of the perfusion chamber. ^ 24Schematic of the perfusion system.  27Schematic of the electrical set-up. ^ 29Timecourses of transepithelial measurements under variousconditions. ^  38Frequency distribution of values of basolateral membranepotential.  40The effect of saline changes in nonstimulated ilea. ^ 42The effect of saline changes in cAMP stimulated ilea. ^ 43Chart recorder trace made during the impalement of anileal cell when nonstimulated. ^  46Summary of electrical measurements for nonstimulatedand cAMP stimulated ilea.  48Timecourse of cAMP addition. ^  50Figure 1.1Figure 1.2Figure 1.3Figure 1.4Figure 1.5Figure 1.6Figure 2.1Figure 2.2Figure 2.3Figure 3.1.1Figure 3.1.2Figure 3.1.3Figure 3.1.4Figure 3.1.5Figure 3.1.6Figure 3.1.7viiFigure 3.1.8 Chart recorder trace during impalement of an ileal cell,before and 8 mins after cAMP addition. ^ 51Figure 3.2.1 Timecourse for the effect of Na+ replacement innonstimulated ilea. ^  53Figure 3.2.2 A comparison of voltage measurements before and 8 minsafter Na+ replacement or addition of amiloride. ^ 55Figure 3.2.3 A comparison of resistance measurements before and 8mins after Na+ replacement or addition of amiloride. ^ 57Figure 3.3.1 Timecourse of the effect of increasing luminal [K+]10 mM to 105 mM when ileal were cAMP stimulated. ^ 60Figure 3.3.2 The effect on Vt of varying luminal [K+] ^bothnonstimulated and cAMP stimulated ilea.  62Figure 3.3.3 The effect on Va of varying luminal [K+] ^bothnonstimulated and cAMP stimulated ilea.  63Figure 3.3.4 The effect of varying luminal [K+] ^resistances whenilea were nonstimulated. ^  65Figure 3.3.5 The effect of varying luminal [K+] ^resistances whenilea were cAMP stimulated.  66Figure 3.3.6 The effect on voltages of varying serosal [K+] ^ileawere nonstimulated. ^  68Figure 3.3.7 The effect on resistances of varying serosal [K1 when ileawere stimulated with cAMP ^  69Figure 3.4.1 A comparison of voltage measurements before and 8 minsafter Cl - replacement and the effect of longterm Cl -replacement. ^  72Figure 3.4.2 A comparison of resistance measurements before and 8mins after Cr replacement and the effect of longterm Crreplacement. ^  74Figure 5.1^Model of the control of ileal ion transport by cAMP. ^ 96viiiLIST OF ABBREVIATIONSa^ - voltage divider ratio, Va/VbA - Angstrom0 - degreesC^ - degrees Celcius[ion] c, - extracellular concentration of ion[ion] ;^- intracellular concentration of ionMsc - change in short-circuit currentAVa - apical membrane voltage deflectionAVb^ - basolateral membrane voltage deflectionAVt - transepithelial voltage deflectionp,A - micro Amperespequiv.cm-2.h-1^- micoequivalents per square centimeter per hourp1.11- '.ileum' - microlitres per hour per ileump.1.11- '.rectum'^- microlitres per hour per rectumgm^ - micronI.LM - micromolarSI^ - OhmsOcm2 - Ohms square centimetera cs - intracellular chloride activityATPase^- adenosine 5'- triphosphatasecAMP - adenosine 3': 5'- cyclic monophosphoric acidCC - copora cardiacacGMP^ - guanasine 3': 5'- cyclic monophosphoric acid- centimetercmCTSH - Chloride transport stimulating hormoneixdb^ - decibelsDPC - N- Phenylanthranil - saureG - protein^- guanasine binding proteinh^ - hoursHPLC - high performance liquid chromatographyHz - HertzID^ - inside diameterIk^- potassium currentIsc - short circuit currentJ - rate of luminal ammonia secretionJil^ - rate of luminal acidificationJv - rate of transepithelial fluid transportM - Molar (moles per litre)min^ - minuteml - millilitreml/min - millilitres per minuteMOPS^- 3 - (N - morphalino) propanesuiphonic acidmm - millimetersmM - millimolarmosmol^- milliosmolar concentrationmV - millivoltsn - numberOD^ - outside diameterP - probabilitypA - pico AmperesPDE^ - phosphodiesterasePk - potassium permeabilitypK - negative log dissociation constantxPKE^ - phosphokinasepm - picomolarpS - pico SiemensRa^ - apical membrane resistanceRb - basolateral membrane resistanceRt - transepithelial membrane resistanceRj^ - paracellular resistancescgITP - Schistocerca gregaria ion transport peptides.e. - standard errorVG5^ - 5th ventral abdominal gangliaVa - apical membrane potentialVb - baolateral membrane potentialVt^ - transepithelial potentialxiACKNOWLEDGEMENTS.I would like to thank5ohn Phillips for providing the financial support for this study and JoanMartin for her help. Here's to Lloyd 0, To&a, aigumi and Acy baby. Thanks to Enda 3 Enda 0' Sullivanfor being on the other end of the phone and for help in preparation for this thesis. Thanks to Fiwah (FionnHorgan, Fi-Fi) and Sammi-wammi (Samir Aouadi; not in any particular order) for being great and forreading the this thesis. Cheers to Shannon for being from Vlson and hackie Mt) for being herself andhelping with stats on minitab. 'Thanks to teddy and Shipra for many excellent cunys. Thanks to Grace Chofor finding everything for measuring ICt. .xiiCHAPTER 1 GENERAL INTRODUCTION.Transporting epithelia form the boundary between body fluids of different ionicand osmotic composition. Epithelia create, maintain and regulate these ionic and osmoticgradients through vectorial and selective transport processes yet, at the same time,epithelial cells must maintain their own intracellular ionic composition. In most multi-cellular animals, whole animal osmoregulation is dependent upon renal and extra-renalepithelia which are typically under hormonal control.The osmotic demands placed on insects are often extreme, especially in thefreshwater and arid terrestrial environments, due to large surface to volume ratios. Themajority of insects, however, regulate haemolymph composition within narrow limitsunder highly variable external conditions. Schistocerca gregaria displays only a 30%change in haemolymph osmotic pressure when fed either a hyperosmotic solutionequivalent to sea-water saline or tap water (Phillips 1964a). Also, during dehydration,when haemolymph volume may decrease by up to 90%, ionic composition ismaintained. Subsequent feeding on succulent material rapidly restores haemolymphvolume with little affect on ionic composition (Hanrahan 1978, Chamberlin and Phillips1979). This high capacity for haemolymph regulation is achieved through structural,behavioral and physiological adaptations (Maddrell 1971).1The excretory system is largely responsible for the rapid homeostatic control ofinsect haemolymph osmotic and ionic composition. The source of this control is the co-ordinated regulation by hormones of the membrane transport processes in the threemajor epithelia of the insect excretory system; the Malpighian tubules, the ileum and therectum. In a terrestrial phytophagous insect, such as the locust (Fig 1.1), the Malpighiantubules secrete a KC1-rich isosmotic primary urine. A small proportion of this primaryurine enters the midgut (Dow 1981). The majority, however, passes into the hindgutwhere selective reabsorption takes place. The ileum reabsorbs a near isosmoticadsorbate, playing a major role in the maintenance of haemolymph ionic composition(Irvine et al. 1988). The rectum is the main concentrating segment and control of thisprocess may lead to a very hyposmotic or hyperosmotic urine, or powder dry excreta(reviewed by Phillips 1983, Phillips et al. 1986).Structure of the Locust Excretory System.The Malpighian tubules number approximately 250 and join the alimentary canalat the junction between the midgut and ileum. The hindgut is lined with a 2-10 gm thickchitinous cuticle which acts as a molecular sieve due to the presence of water-filledpores of about 6 A, lined with a fixed negative charge, the residue having a pIC---. 4(Phillips and Dockrill 1968, Lewis 1971, Maddrell and Gardiner 1980). These poresallow small hydrophilic molecules such as ions and metabolites to pass through the2midgut Malpighian tubules cocaecaeKC1, Na and^Water and ionWater secretion reabsorptionWater, Ionand MetabolitereabsorptionStronglyhyperosmoticor hyposmoticexcretaMALPIGHIAN TUBULES ILEUM COLON RECTUM ANUSFig 1.1: a: Diagram of the locust ailementary canal. b: Detail of the locustexcretory system. Epithelial transport is denoted by the large arrows, smallarrows represent the flow of urine. Modified from Phillips et al (1981),redrawn from Audsley (1990).3cuticle but exclude large, often toxic substances. Consequently, these large moleculesaccumulate in the hindgut and are expelled in the excreta. The cuticle also protects thehindgut epithelia from mechanical abrasion by the gut contents.The locust ileum is about 6 mm long with an outside diameter of 2.5 mm. Themacroscopic surface area of the ileum is 0.4 cm 2 compared to 0.64 cm2 for the rectum.Ultrastructural observations reveal a simple epithelium of only one cell type. Cells areabout 40x20 gm in size and display the dense apical infoldings with associatedmitochondria typical of transporting epithelia (Fig 1.2; Irvine et al. 1988). The cuticleis firmly attached to the apical membrane. Apical junctional complexes between cellsare about 10 gm in length and resemble those of the rectum. The basal plasmamembrane displays numerous narrow infoldings. The basal surface of the epithelium iscovered by a thin basal lamina, overlaid by a sheet of circular muscle (Irvine et al.1988), with longitudinal muscle bands evenly spaced around the outside of the ilea(pers. obs.).The colon is much less permeable than either the ileum or the rectum (Maddrelland Gardiner 1980) and is composed of small unspecialized epithelial cells. Thus, thissegment is not thought to contribute significantly to absorption in the hindgut.The rectum consists of six, radially arranged, thick epithelial pads separated bynarrow regions of thin epithelium. The pads are composed of two types of cells,columnar epithelial cells and occasional secondary "B" cells which contact the lumenside only. The columnar cells are 17x100 gm in size and display highly folded lateralmembranes with associated mitochondria (Fig 1.2; Irvine et al. 1988). These lateral4Apical junctionalcomplexDilated intercellularspacesRECTUM ILEUM10itmBasal junctionalcomplexBasal cellsCuticleSubcuticularspaceApical membraneinfoldsMusclelayersLateral intercellular spacesFig 1.2: Comparison of the ultrastructural organization and grossdimensions of the ileal epithelium and rectal pad of the locust. Rectal "B"cells are not shown. From Irvine et al (1988).5foldings are termed lateral sclariform complexes and are thought to be the site of ionrecycling back into the rectal cells (Wall and Oschman 1970). The apical membrane, asin the ileum, is highly infolded but the cuticle is often detached from the apical surfacecreating a sub-cuticular space (Martoja and Balan-Dufrancais 1984, Chapman 1985,Irvine et al. 1988).Fluid and Ion Transport in the Rectum.The mechanisms and control of ion transport have been well characterized forthe locust rectum utilizing a variety of experimental techniques (Reviewed by Phillipset al. 1986 and 1988, Phillips 1980). Fig 1.3 summarizes the localisation of specifictransport processes in the apical and basolateral membranes of this epithelia and theircontrol by a neuroendocrine factor, CTSH acting via cAMP.The rectal epithelia transports mainly IC - and a- from the lumen to haemolymph.Na+ is also reabsorbed but to a much lesser extent due to the low levels of this cationin the primary urine. Ion substitution shows that fluid transport is secondarily coupledto ion transport, whereas inhibition by KCN, ouabain and iodoacetic acid demonstratesdependence on metabolism (Goh and Phillips 1978). In the absence of an initial osmoticgradient, the rate of net fluid reabsorption (Jv) in situ is 17 1.11.11 -1 .rectum-1 (Phillips1964a). This is higher than the rate of absorption from a NaCl-rich fluid observed forin vitro studies (Jv = 6 111.11 -1 .rectum -1 ; Goh and Phillips 1978). In vitro Jv from a KC1-6CELLLUMEN •^ HAEMOCOEL1.5A.acidsv. los= <CTSH °CEO^Net flux LEq.cm .hr011•1 1 .1 OXIDATION I•^^CO2 '''''^Cl -cAMP^••A•06 .**.•49, OH• H2O• Base 1.5RJ=800HCO3 0.4Acid-mVVa Vb-57 ---).- -67^-49 —).- -39Clcm2Ra Rb1 260 —).- 70^200 —)o- 55^I)11(apical+cAMP +cAMP *basalFig 1.3: Summary of ion transport processes in locust rectum.Upperdiagram: Proposed model for KC1 reabsorption; solid arrowsthrough circles, active transport; arrows through cylinders,channels; dotted lines, dissipative net ion movements; thickdashed lines, control by cAMP and K. Cyclic AMP stimulated netion fluxes are shown on the right hand side. Lower diagram: Apicaland basolateral membrane potentials (Va, Vb) and resistances (Ra,Rb). Rj is paracellular resistance (Redrawn from Phillips 1986 andHanrahan and Phillips 1983).7rich saline, resembling Malpighian tubule secretion is higher (10 µ1.h - '.rectum') andmore comparable with values from in vivo studies (Andrusiak et al. 1980). Initial Jv ishigher immediately after rectal excision, consistent with higher values for Isc but fallsover the first hour to a steady state (Jeffs 1993). Also, rates of fluid transport werefound to vary 2-fold depending on the hydrated state of the locust, suggesting a controlmechanism for fluid reabsorption in the rectum (Goh and Phillips 1978).The large short-circuit current in the rectum is due to the apical electrogenictransport of Cl", which represents the predominant transport process for this epithelia.C1 transport is unusual in the rectum in that it does not involve exchange for anotheranion (HCO3) or co-transport with Na - (Hanrahan and Phillips 1984c). Cr exits the cellthrough basolateral Cl - channels down a favorable electrochemical gradient (Hanrahanand Phillips 1984b,c). These channels are blockable with pharmacological agents(Phillips et al. 1986).Reabsorption of IC+ is passive through channels in both apical and basolateralmembranes and occurs by electrical coupling to transport. Low levels of luminal 1C -1-stimulate electrogenic CT transport, although KC1 co-transport has been excluded(Hanrahan and Phillips 1984a-c).Ne-ICATPase has been demonstrated in microsomal fractions isolated fromlocust rectum and identified ultrastructrally as lateral membrane fragments (Lechleitner1988). Na is pumped out of the cell basolaterally against a large electrochemicalgradient, creating a favorable gradient for passive entry across the apical membrane.Some Na+ entry is through channels but most is used to drive amino-acid (glycine)8uptake from the lumen by Nal" co-transport (Balshin 1973), and for secretion of NH,'(some W) by Na+ counter-exchange (Thompson et al. 1988b). Fr is actively secretedinto the lumen, mostly by electrogenic transport (80%), the remainder being due toNeill+ exchange. There is a concomitant reabsorption of HCO3 to the haemocoel side,derived mostly from the epithelial cells (Thompson et al. 1988a).The amino acid proline is absorbed from the lumen at high rates by the rectum.Entry is Ne-independent and may be proton linked (Meredith and Phillips 1988).Proline represents the major substrate for oxidative metabolism and ammoniagenesis inthe rectum (Chamberlin and Phillips 1983) and also drives a large component of fluidtransport (Lechleitner and Phillips 1989).The cAMP second messenger system.Neuropeptide hormones are unable to traverse the plasma membrane and thus actupon surface receptors to initiate the formation of intracellular "second messengers". Fig1.4 describes the cyclic nucleotide cAMP second messenger pathway.The ligand (eg. a hormone) binds with a receptor. This ligand/receptor complexactivates several G-proteins; the first phase of signal amplification. The G-protein a sub-unit activates adenylate cyclase which catalyses the conversion of ATP into cAMP. OneG-protein may activate several adenylate cyclase units; this is the second phase ofamplification. Adenylate cyclase may convert many ATP molecules to cAMP before it9fli IM V VI^T!.:FIIT1111g g^1 1-L-0-1 0 0 0 A UV _u(A c)++ I FORSKOLIN 1P.M.PHOSPHORYLATION)OF PROTEINS[■EFFECTOR TARGETSCELLULAR RESPONSE iFig 1.4: The cAMP second messenger cascade. See text forexplanation. PDE, phosphodiesterase; AC, adenylate cyclase;cAMP, 3':5' cyclic adenosine monophosphate; GTP, guanadinetri-phosphate; GDP, guanidine diphosphate; a, 0 and y areG-protein sub-units. R is the receptor, L is the ligand, P.M. is theplasma membrane. Adapted from Brown (1991) and Greenguard(1978).10is deactivated; the third phase of amplification. (Brown 1991).Phosphodiesterase (PDE) hydrolyses 3':5'cAMP to 5'AMP and thus down-regulates cAMP levels. Mechanisms which modulate cellular PDE activity have beenproposed but their application in insect systems has not been widely tested (Bodnaryk1983).Cyclic AMP interacts with protein kinases (PKE) to release a catalytic sub-unitwhich phosphorylates specific proteins thus altering their biological activity. The cyclicnucleotide system is reviewed in detail by Bodnaryk (1983), with special reference toinsect systems.The pharmacological agent forskolin activates adenylate cyclase and thereforemimics hormone effects (Seamon and Daly 1981). Theophylline is a stronglycompetitive cAMP-PDE inhibitor and thus acts to maintain high intracellular cAMPlevels (Butcher and Sutherland 1962). Initiation of a biological response by these agentsis considered strong evidence implicating the involvement of the cAMP secondmessenger system.Hormonal Control of Reabsorption in the Rectum.The insect neurosecretory system is shown in Fig 1.5. Aqueous extracts ofcorpora cardiaca (CC) stimulate Cl - dependent short-circuit current (Isc) by several fold(AIsc) and also cause an increase in chloride-dependent fluid transport (Spring and11Fig 1.5: Side view of an insect, showing the neuroendocrine organs. FromPhillips (1981).12Phillips 1980a-c). Transepithelial voltage (Vt) increases and transepithelial resistance(Rt) declines after CC stimulation (Spring and Phillips 1980a-c, Proux et al. 1984,Hanrahan and Phillips 1985). The actions of CC are mimicked by 1mM cAMP andintracellular measurements of this cyclic nucleotide show that maximum levels areattained at a time corresponding to the maximum increase in CC-stimulated Isc(Chamberlin and Phillips 1988). Recent pharmacological studies on stimulants of rectalJv and Isc by Jeffs (1993) provide further evidence for the involvement of the cAMPsecond messenger system in the control of rectal transport processes. However, he alsoreported a partial stimulation of rectal reabsorption by cGMP and the involvement ofCa2+ which had both stimulatory and inhibitory effects at different concentrations. Thereis no evidence for the involvement of either PKC or the PI second messenger systemin the control of rectal transport (Jeffs 1993).Using AIsc as a bioassay, Chloride Transport Stimulating Hormone (CTSH) waspartially purified from locust CC. CTSH has a molecular weight of about 8,000 Daltonsand maximally stimulates AIsc at concentrations of <7 1.1M (Phillips et al. 1980).Forskolin and theophylline both mimic the action of CTSH (Spring and Phillips 1980a),strongly implicating a CTSH receptor initiated cAMP cascade in the control of rectalreabsorption.CTSH stimulates KC1 and fluid reabsorption and inhibits fr secretion but hasno effect on Na transport (reviewed by Phillips et al. 1986). The increase in Cl -transport is due to stimulation of the apical Cl - pump. There is a 50% decline in Rt13associated with stimulation, due to an increase in conductance of the basolateralmembrane to C1 and of the apical membrane to K. Since CTSH simultaneouslyincreases both active electrogenic Cl - and passive IC' transport, this provides for a veryefficient stimulatory mechanism (Hanrahan and Phillips 1984c).In high IC+ saline, stimulation does not result in an increase in ICE permeability(Pk). This is attributed to a decline in apparent P k at high luminal [K1 due firstly tochanges in intracellular IC+ activity and membrane depolarisation and secondly to a realdecline in Pk which is [K1 0 sensitive (Hanrahan and Phillips 1984c, 1985).Neuroparsins, two neuropeptides also isolated from CC extracts have beenproposed as agents controlling rectal reabsorption in Locusta migratoria (Fournier andGirardie 1988). However, the effects of these factors on solute transport have not yetbeen investigated.Fluid and Ion Transport in Locust Ileum.The rate of fluid transport in the ilea in the absence of a transepithelial osmoticdifference and stimulants is 3 - 3.5 121.11 -1 .ileum-1 (Lechleitner et al. 1988a). Fluidtransport is inhibited by azide, KCN and iodoacetic acid and is coupled to ion transport.Fluid reabsorption can occur against osmotic gradients of up to 600 mosmol (Lechleitneret al. 1988a,b) but the reabsorbate is always near isosmotic or slightly hyperosmotic tothe luminal fluid. The inability of the ilea to produce a hyposmotic adsorbate is thought14to lie in ultrastructural differences between this segment and the rectum. As discussedpreviously, the ilea lacks the lateral sclariform complex of the rectum, thought to be thesite of solute recycling, leading to the production of a hyperosmotic excreta (Irvine etal. 1988, Wall and Oschman 1970).Measurements of Vt and Isc indicate a net active absorption of anions to thehaemocoel side. After removal of the ilea from the locust, Isc is initially large but fallsrapidly to around zero, presumably due to the removal of natural stimulants present inthe haemolymph. This, together with stimulation of ileal Isc by cAMP, suggestedhormonal control of ileal transport (Irvine et al. 1988).From a saline resembling haemolymph, everted ileal sacs reabsorb a NaCl-richfluid with substantial amounts of K. Net active absorption of Na+ was estimated by fluxstudies to be 4.2 ± 0.18 gequiv.cm2.h-1 (Irvine et al. 1988), which is twice the ratemeasured for the rectum (Black 1987).In the absence of exogenous bicarbonate and CO 2, there is an active acidsecretion into the ileal lumen at a rate of 1.56 p,equiv.cm2 .11-1 , which declines to zeroover 4 hours (Thompson et al. 1991). This is comparable to rates of acid secretionmeasured for the rectum (Thompson and Phillips 1992).Stimulation of ileal transport processes by cAMP switches absorption from lowcapacity transport of NaC1 and NaHCO3 to high capacity absorption of NaC1(Lechleitner et al. 1988a). Addition of 5mM cAMP causes a rapid increase in Vt and15Isc (AIsc) due to the stimulation of electrogenic Cr transport to the haemolymph side.Rates of fluid absorption are increased 4 fold (Irvine et al. 1988, Lechleitner et al.1988b). Fluid transport is C1 dependent with maximum rates being achieved at a Nal":1C+luminal ion ratio of 1:1 and at a concentration of 60mM (Lechleitner et al. 1988a).Glucose and 5 amino acids (alanine, asparagine, glutamine, proline and serine)are absorbed from the lumen and provide the metabolic substrate for the maintenanceof AIsc. Metabolic breakdown of these amino acids is the source of ammonia secretion(J ) to the luminal side. Ileal Jam„ is stimulated by cAMP but not CC extracts (Peach1991). In contrast to the rectum, proline sustains ileal metabolism equally well fromboth luminal and haemolymph sides and does not represent the major respiratorysubstrate (Peach and Phillips 1991).Cyclic AMP also stimulates passive IC+ and active Na+ absorption, the latterbeing the first report of control of Na+ reabsorption in any insect (Irvine et al. 1988).High levels of Na+K+ATPase have been detected in the microsomal fraction of ilealtissue (Lechleitner 1988) but the mechanism of passive Na+ entry at the apicalmembrane is unknown. Na+ substitution has no effect on Isc (i.e. Cr transport; Irvineet al. 1988) or ammonia secretion (Peach and Phillips 1991). This precludes Na/Cl co-transport or Ne/NH4+ exchange as mechanisms of apical Na+ entry into ileal cells.There is a large decrease in transepithelial resistance (Rt) after stimulation withcAMP. Ion substitution has shown this to be due to an increase in conductance primarilyto IC+ and also Cr (Irvine et al. 1988). Blocking of basolateral IC - channels with Ba+prevented cAMP induced stimulation of passive IC+ current caused by an imposed K +16gradient. Ba+ had no such effect on IC' transport at the apical membrane (Irvine et al.1988). Some preliminary intracellular electrical measurements have been made on bothnonstimulated and cAMP stimulated ilea. These will be discussed in Chapter 4.The effect of Neuroendocrine factors on ileal transport.Stimulation of ileal transport processes by the second messenger cAMP impliesregulation of ileal reabsorption by unknown hormones. Using AIsc as a bioassay, thelocust neurosecretory system (Fig 1.5) has been surveyed for agents that might act tostimulate Cl" transport, this being the dominant ion transport process in the ileum(Audsley and Phillips 1990, Audsley 1990).Extracts from most ventral ganglia cause some increase in Isc, whereashomogeanates of muscle show no stimulatory effect. Crude homogeanates of corpuscardiacum (CC) and 5th Ventral abdominal ganglia (VG5) cause the same maximalstimulation of ileal Isc as does cAMP and both act in a dose-dependent manner.However, unlike cAMP, CC and VG5 have no stimulatory effect on ileal NH 4-1" secretionbut do inhibit secretion of fr (Audsley and Phillips 1990). The stimulatory factorspresent in CC and VG5 appear to be different neuropeptides as they result in differenttime courses for AIsc and have different thermal stabilities and solvent extractionproperties (Audsley and Phillips 1990, Audsley 1990).Schistocerca gregaria ion transport peptide (scgITP) was purified from CC bya 4-step reverse phase HPLC extraction protocol (Audsley et al. 1992a-c). VG5 factor17has not yet been purified. Pure scgITP has all the actions of crude CC but requireshigher doses for a maximal response due to loss of protein during purification (Audsleyet al. 1992b). Amino acid analysis gives a molecular weight of 7700 Daltons for scgITP.The first 50 amino acids of the scgITP sequence have been determined out of a total of65 residues. Approximately 2.5 pmoles of scgITP is required for maximum stimulationof ileal Isc (Audsley et al. 1992 a-c).ScgITP has no effect on rectal Jv or IC+ permeability and elicits only a sub-maximal increase in Isc. ScgITP is therefore assumed to be a different peptide fromCTSH (Audsley et al. 1992b).Heal Isc is also stimulated by 5 mM theophylline and 50 ilM forskolin (Audsley1990). This is consistent with cAMP as the second messenger system. ScgITP anincrease in ileal cAMP levels by 1 hour after addition but the timecourse of cAMPchange remains to be studied (Audsley et al. 1992b).Towards a model of cAMP stimulation of ileal ion transport.Irvine et al. (1988) first demonstrated the importance of the ileum in hindgutreabsorption and its role in the control of the excretory process. Audsley (1990) hasshown that ileal ion transport processes are under the control of natural stimulantspresent in the locust neuroendocrine system. Based predominately on transepithelialmeasurements of ileal ion transport and the similarity of these results to those from the18Fig1.6: Model proposed by Audsley (1990) for the control of ion transportacross locust ilea. See text for details. Redrawn from Audsley (1990).19rectum, Audsley (1990) proposed a model of ion transport mechanisms in the ilea andtheir modulation by scgITP and cAMP (Fig 1.6). In essence he proposed that scgITPacts via cAMP to stimulate an apical Cl" pump and increase both apical IC' andbasolateral Cl- conductances (i.e. open ion channels). He also suggested that cAMPcontrol of Na+ transport occurs at the passive entry step at the apical membrane,possibly by opening Nal" channels.The aim of this study is to test Audsley's model using cAMP as the stimulant.ScgITP is not available in large enough quantities to warrant a direct study until thebasic features of the ion transport mechanisms are elucidated using cAMP. Intracellularrecording of cell electrical properties provides information on apical and basolateralmembrane ion transport processes, particularly ion conductances (i.e. ion channels) andtheir control by cAMP. Ion substitution permits elucidation of the specific ionsresponsible for conductance changes. Based upon Audsleys' model, this thesis utilizesthese methods to address three main questions:1) Is there a cAMP stimulated increase in apical Na + conductance?2) Is there a cAMP stimulated increase in apical IC+ conductance?3) Is there a cAMP stimulated increase in basolateral C1 conductance?Chapter 2 details the methods used in this investigation. It also describes theperfusion system developed for this study which provides a constant flow of oxygenatedsalines during the impalement of ileal cells. Chapter 3 consists of results in 4 sections.20Section I expands on the preliminary intracellular recordings made by Irvine et al.(1988) and describes the stability of electrical parameters with time and the effect ofchanging saline. This section also reports the time course of electrical changes for singleileal cells after cAMP addition. Section II tests whether apical entry of Na + occurs byion channels and if so whether cAMP opens such ion channels. The effects of amilorideinhibition and C1 removal on Na+ transport are also considered. Section III investigateswhether cAMP stimulation increases apical IC" conductance, as observed for the rectum.In section IV, Cl" conductance is investigated using bilateral replacement of this anion.Chapter 4 is a discussion in four sections. Chapter 5 is a general discussion andsummary.21CHAPTER 2 MATERIALS AND METHODS.Animals.Adult female Schistocerca gregaria Forskal, 4-5 weeks post-moult were used inall experiments. They were fed a diet of lettuce and a dry mixture of alf alfa, bran andmilk powder and were kept on a 12 h light 12 h dark cycle at 28°C, 55% relativehumidity.Solutions.The control saline used in this study is based upon measurements of locusthaemolymph composition (Hanrahan and Phillips 1983). This saline contained, in mMconcentrations; NaC1 (100), MgSO 4 (10), K2SO4 (5), Na Isethionate (10), CaC12 (5),glucose (10), MOPS (2), arginine (1), serine (6.5), proline (13.1), asparagine (1.3),glycine (11.4), valine (1.8), tyrosine (1.0), lysine (1.4), histamine (1.4), glutamine (5),alanine (2.9). Sucrose (100) balanced the osmotic contribution of haemolymph trehaloseand other solutes. The saline was bubbled vigorously with 100% oxygen for 1/2 h andthe pH was then adjusted to 7.0 with HC1 or NaOH (H 2SO4 or KOH were used for Cl-free or Na+ free salines) using a Radiometer PHM84 pH meter (Copenhagen).22Modifications to this saline are described later in this chapter. Saline containing 5 mMcAMP was made up from 100 mM cAMP stock solution in control saline and then thepH readjusted to 7.0 as above. All chemicals were obtained from the Sigma ChemicalCo. (St Louis, Mo.).Beal preparation.Locust ilea were mounted as flat sheets in a plexiglass perfusion chamber (Fig2.1). Ilea were dissected from the animal and cut longitudinally to form a flat sheet.Excreta was removed and the cleaned ilea stretched over a plexiglass collar with acentral opening of 0.196 cm 2. Eight tungsten pins embedded in this collar served tosecure the tissue and any excess below these was removed. A closely fitting neoprene0-ring was placed over an ileum thus forming a tight seal around the collar. Thistechnique has been shown to minimize edge damage (Hanrahan and Phillips 1984a). Thethree parts of the perfusion chamber were then clamped together with screws securedby wing-nuts. Any leaks were sealed on the exterior with RTV 108 silicone (GESilicones Canada, Pickering Ont.).The chamber used in this study (Fig 2.1) was developed from that used byHanrahan (1982). Each side of the chamber contained 2 ml of saline and the opendesign allowed access for microelectrodes. The preparation was illuminated from therear by fibre optics (Intralux 150H, VolpiAG, Urdorf, Switz.) and could be viewed by23Fig 2.1: Plexiglass perfusion chamber, developed from that used byHanrahan (1982).24split '0' ring ileum plexiglass collarHAEMOLYMPH SIDE OF CHAMBERLUMEN SIDE OF CHAMBERsaline outVIEWAMMO"AMMO"INK/wingnutsmeans of a horizontally mounted dissecting microscope (Zeiss, Jens, Germany) at 90-750x magnification.The preparation was continually perfused separately to both sides of the chamberat a rate of 3 ml/min. Fig 2.2 shows a schematic of the perfusion system. Varioussalines were bubbled with 100% 02 in reservoirs and selected by means of a valve. Adual-channel peristaltic pump (Microperpex, LKB, Pharmacia, Switz.) delivered salinevia PE240 tubing to the base of the chamber, in close proximity to the epithelium.Saline was removed from the chamber at the top, diagonally opposite from the point ofentry in order to maximize mixing. Saline changes took less than 3 mins (measuredvisually by dye clearance experiments). On the luminal side of the chamber (upperportion of diagram) saline removal was by means of a water vacuum pump. On thehaemocoel side, however, a peristaltic pump (Microperpex) allowed saline to be re-circulated. Saline which passed through the chamber during the first 2 hours ofexperimentation was not re-circulated. This system was designed with a view to testingthe action of scgITP directly, in the extreme allowing the re-circulation of <10 ml ofsaline. Time from dissection to the commencement of perfusion was less than 5 mins.The muscle layer was partially removed 1 h after dissection, exposingapproximately 30% of the ileal epithelium, thus enabling microelectrode impalement ofthe basal surface. This procedure had only a transitory effect on electrical parameters(Irvine et al. 1988, pers. obs.). The ilea were allowed to equilibrate for 2 h from26Fig 2.2: Schematic of the perfusion system developed for this study.Haemolymph side is towards the bottom of the diagram, lumen sidetowards the top. See text for details.27dissection before the experimental period in order to establish a steady state. Individualepithelial cells were then impaled with microelectrodes advanced at an angle of approx30° from horizontal and 45° from the plane of the epithelium using micromanipulators(Leitz, Weltzlar, Germany). Criterion for successful impalement of cells included:i) Sharp monotonic voltage deflection.ii) Steady voltage (<1 mV fluctuation) with constant voltage divider ratio.iii) Return to within ±1 mV of baseline upon microelectrode withdrawal from the cell.Electrical methods.i) Transepithelial measurements.Fig 2.3 shows a schematic diagram of the electrical set-up. Transepithelialpotential (Vt) was measured through 3M KC1 agar bridges (PE90) and calomelelectrodes connected to a high input impedance differential amplifier (10'0; 4253,Teldyne Philbrick). Vt was recorded on a pen chart recorder (Soltec 1242, San FernandoCal.). Values for transepithelial resistance (Rt) were obtained by passing transepithelialsquare-wave current pulses (13.3 pA) and monitoring the resultant voltage deflectionsin Vt (AVt). Rt could thus be calculated from Ohms' law: V= IR. Current pulses weredelivered by waveform/pulse generators (Type 160 Series, Tektronix, Beaverton, Ore.)via silver electrodes at a frequency of approximately 0.3 Hz and a 1 second duration.28Fig 2.3: Schematic of the electrical set-up, described by Hanrahan (1982).Microelectrode measurements are balanced at the microelectrode amp andswitch-box (open circles). Electrodes for microelectrode circuit, em; fortransepithelial circuit, et. See text for details.29ii) Microelectrode measurements.Apical membrane potential (Va) and basolateral membrane potential (Vb) weremeasured directly by microelectrode impalement. Microelectrodes were referenced toexternal by means of agar bridge/electrodes on either side of the epithelium, the voltagedrop across the microeletrode being balanced by variable resistors at the switch box (Vbreference) and microelectrode amp (Va reference). The head stage (Dagan probe 127,Dagan Corp. Minneapolis, Minn) was in close proximity to the microelectrode in orderto minimize signal loss. The switch box enabled either Va or Vb to be routed to themicroelectrode amp (Dagan 8800 Total clamp). The resultant signal was filtered at 3 db,5 Hz, monitored on a storage oscilloscope (D15 Tektronix) and ultimately recorded ona pen chart recorder as described by Hanrahan (1982).Values for apical (Ra) and basolateral (Rb) resistances were estimated fromvoltage deflections (AVa, AVb, respectively) due to transepithelial current pulses (asdescribed above). The relationship between AVa and AVb is normally expressed as aratio, AVa/AVb = a, where a is termed the voltage divider ratio. However, in the caseof the ileum, AVa is approximately 15-20x larger than AVb, which is typically <10 mV.Thus, small changes in AVb have a disproportionally large effect on a as they areamplified through the denominator of the ratio to render a an unacceptably inaccuratevalue. Such small changes can be a result of errors which may include;a) variance in voltage due to microelecrtode position (approximately ± lmV).b) variance in width of pen of chart recorder (approximately ± 0.1mV).30c) impalement fluctuations (± lmV).iii) Correction for saline resistance.At the end of every experiment, the ilea and 0-ring were removed from thecollar, leaving the perfusion chamber set-up as for the experiment. The deviation of themeasured transchamber (Vts) voltage from zero was recorded and values of Vt correctedfor this. This procedure was repeated for each saline used. Vts was also determinedbefore the start of each experiment and was balanced to zero by means of a variableresistor at the transepithelial amplifier.A microelectrode was placed in a similar position to where impalements of themembrane were made and current pulses were passed across the chamber (as describedabove). Thus, transchamber (Rt.), apical reference (Ra s) and basal reference (Rbs)resistances could be calculated from the resultant voltage deflections (AV....). This wasrepeated for the different salines used in each experiment. All membrane resistancevalues in this thesis are shown corrected for saline resistance which represented between15-100% of uncorrected AV, depending upon the experimental condition and whetherAV is AVt, AVa or AVb.Microelectrode fabrication.Micropipettes were fabricated from borosilicate capillary tubing (1.0 mm OD,0.5 mm ID, omega dot) using a model P-77 Brown and Flaming horizontal puller (Sutter31Inst. Co. San Franscisco, Cal.) and back-filled with 3M KC1 using PE90 tubing pulledout over a flame.Statistical treatment.Differences in electrical parameters between experimental conditions on singlecells were tested for significance using paired Students' T-tests. A P value of <0.001was considered highly significant (***), 0.005 to be strongly significant (**) and <0.05to be significant (*). Changes in electrical parameters between different experimentswere tested using independent 't'-tests for two means and ANOVAs for multiple means.Tukey Tests were applied to test for mean separation in different ANOVAs. Errors aredisplayed as ± the standard error. N is shown as the number of cells (the number ofilea).EXPERIMENTAL PROTOCOLS.I: The effect of cAMP on ileal electrical parameters.1.1 Control impalements.a) Nonstimulated controls; the effect of saline change alone.All experiments were conducted at 25°C ± 2. Heal preparations were set-up as32above. All source reservoirs contained the same control saline. Cells were impaled andthe saline source reservoir changed. The tissue was continuously perfused throughoutthe saline change. Electrical parameters were sampled immediately before the salinechange, time 0 and at 3, 8 and 13 mins after the saline change. The saline source wasthen returned to the original source reservoir with sampling at the same intervals, i.e.16, 21 and 26 mins from the start of the experiment. The same impalement wasmaintained throughout the saline change allowing direct single cell comparisons to bemade. This was repeated on different cells of the same ileal preparation and on differentpreparations.This is the basic method for all of the experiments in this study and will bereferred to as method A, modifications are referred to below.b) Cyclic AMP stimulated controls.The experiment was repeated as for nonstimulated conditions but in this case thepreparations were exposed throughout to 5 mM cAMP, starting 2 h after dissection ofthe ilea from the locust. Impalements were made between 30 mins and 6 h after cAMPstimulation. This is the basic method for cAMP stimulated preparations and will bereferred to as method B.1.2 Measurement of the timecourse of cAMP stimulation.The protocol of method A was carried out but with the following modifications.Two hours after dissection of the ilea from the locust, ileal cells were impaled and the33bathing saline on the haemocoel side changed from a control saline to a salinecontaining 5 mM cAMP. Electrical parameters were sampled immediately before salinechange (time 0), at 3 mins from saline change and then at 5 mins intervals for a totalof 23 mins. Only one experiment was performed per ileal preparation.II: Investigation of apical Nal - conductance.Method A was followed. Saline was changed on the luminal side from acomplete saline to Ne-free saline (measured as 0.054 ± 0.036 11M by flamespectrophotometer). Na+ was replaced by choline. This was repeated during cAMPstimulation using method B.In another experiment, Nal" substitution was carried out as above fornonstimulated ilea but the initial saline was C1-free (gluconate and N-methyl-D-glutamine replacing C1 and Na+, respectively). This experiment was attempted duringcAMP stimulation for 3 ileal preparations. However, these preparations never achieveda steady state and intracellular potential was only slightly negative at -10 to -20 mVmaking it difficult to judge a true impalement (i.e. one that meets the above mentionedcriterion) from baseline. Cells did not deform upon impalement and the epithelia seemed"spongy". For these reasons, no results were obtained from this experiment. J. Meredithhas also unsuccessfully attempted to impale ileal cells in Cl"-free saline (pers.comm.).Na+ channels are blocked by low concentrations (i.i.M) of amiloride (Sariban-34Sohraby and Benos 1986), higher concentrations (mM) inhibit both Na+ electroneutralexchange for H+ or NH4+ and Na+ co-transport (Sariban-Sohraby and Benos 1986, Benos1982). To test the effect of this agent on Na+ conductance, sulphate was replaced withgluconate in all salines and salines containing amiloride at 10 p.M and 1 mM were keptin darkness. Method A was followed with the following modifications; control salinewas replaced with a saline containing 10 11M amiloride and after 13 mins, instead ofreturning to control saline, the saline was changed to a saline containing 1 mMamiloride, with sampling of electrical parameters at 16, 21 and 26 mins (from start ofexperiment).III: Investigation of K+ conductances.To determine apical K+ conductance and the effect of cAMP, method A wasfollowed with the saline on the lumen side changed from a control saline (10 mM IC+)to a saline with 105 mM or 25 mM IC' or a K+-free saline (measured at 0.041 ± 0.065p,M by flame spectrophotometer). Saline [KI was varied by replacing with NaCl oradding K gluconate. This was repeated for the cAMP stimulated ilea (method B).To determine basolateral K+ conductance, [K+] varied as above but salineswere changed on the haemolymph side. This experiment was not repeated for cAMPstimulated ilea.35IV: Investigation of CI' conductances of both apical and basalateral membranes.Method A was followed but saline was changed bilaterally to C1-free saline(measured at 0.059 ± 0.082 1,LM by digital chlorideometer - DP 975M, Haake Buchler,N.J.) with gluconate replacing Cr. This was repeated for cAMP stimulated ilea (methodB).36CHAPTER 3 RESULTS.I: THE EFFECT OF cAMP ON ILEAL ELECTRICAL PARAMETERS.Timecourses under various conditions.Fig 3.1.1 shows the changes in Vt and Rt over a 10 hour period for ilealpreparations in vitro. Ten mins after dissection from the locust and 5 mins after thecommencement of perfusion (i.e. time 0), Vt was 9.3 ± 4.5 mV but fell rapidly to near0 mV, with a subsequent recovery to 5.64 ± 2.5 mV after 2 h by which time a steadystate was reached. Rt was initially 102.42 ± 8.6 acm e, rising to a steady state value of125.9 ± 6.7 fIcm 2 after 1 h. This decline in Vt from an initially high level and theincrease in resistance in the first hour are typical of previous measurements made uponthe ilea and have been attributed to the removal of ilea from natural stimulants (Irvineet al. 1988, Audsley 1990). The subsequent partial recovery reported here has not beenseen before and may be due to the rapid flushing of the epithelium at the start ofperfusion, which would remove any residual haemolymph from the ileum.There was no significant difference in Vt or Rt between preparations which wereperfused only with a control saline and those on which ion substitution experiments371a30^I^EXPERIMENTAL PERIOD206 80 102^4b15090 -70-50-I^. I•^1 •I• 1•I •^I0Time (hours)Time (hours)Fig 3.1.1: Changes with time in transepithelial voltage a: and resistance b:for control ileal preparations (U) n = (5), those on which ion substitutionexperiments were conducted (0) n = (4-13) and ilea stimulated with 5mMcAMP (A) added at 2 h, n = (6-10). Sign refers to lumen side (Vt). Values aremeans ± s.e.38were performed (Vt: P= >0.9, Rt: P= >0.05, Tukey). Thus, experimental procedures hadno deleterious effects upon the electrical properties of ilea. Addition of 5 mM cAMPcaused an increase in Vt which remained significantly higher than Vt for control orexperimental ilea throughout the experiment (P= <0.001, Tukey). For example, in cAMPstimulated ilea Vt was 19.47 ± 2.9 mV (at 3 h, 1 h after cAMP stimulation) comparedto controls at 5.86 ± 1.78 mV. Rt was significantly lower for cAMP stimulated ilea thancontrol or experimental preparations (P= <0.001, Tukey). One hour after cAMP addition,Rt was 53.31 ± 6.7 S2cm2 compared to controls at 125.56 ± 5.8 flcm 2.There was no significant change in values of either Vt (P= >0.9, ANOVA) or Rt(P= >0.9, ANOVA) for control, experimental or cAMP stimulated preparationscompared against each other over the time period of 3-10 h. There was also no changein Vt or Rt between 2 and 10 hours for control values, when a paired comparison wasmade (P= >0.09 and P= >0.5, respectively, paired 't'-test).As indicated in Fig 3.1.1, subsequent experiments were conducted within the 2to 8 h period. Ilea' preparations were monitored under control conditions for at least onehour after experimentation to detect changes in Vt or Rt which would indicate tissuedamage.Variability between cells.Fig 3.1.2 shows the frequency distribution for values of Vb for impalements onnonstimulated ilea maintained in control saline. The frequency distribution is normal,with a deviance of 2.7%, determined by normal probability plot (Ryan et al. 1985). The39Ii II hill ! hi .30 -20 -10 -0  • U-35^-40^-45^-50^-55^-60^-65^-70^-75Basolateral Voltage (mV)Fig 3.1.2: Frequency distibution of basolateral voltages (Vb). Ilea werenonstimulated, n = 284 (47). Voltage sign is for cell interior.40modal value for Vb was -63 mV (cell interior negative). Impalements which lay outsidea range of values for Vb of -50 to -70 mV were rejected for further experimentation.Effect of saline changes.Fig 3.1.3 shows the effect of changing the bathing saline without changing salinecomposition for nonstimulated ilea (control A). Fig 3.1.4 is the same experimentconducted upon ilea which had been previously stimulated with 5mM cAMP (controlB).Table 3.1.1 summarizes electrical parameters from Figs 3.1.3 and 3.1.4 for bothnonstimulated and cAMP stimulated ilea at 0 and 8 min after the saline was changed.In both cases there was no significant change in any electrical parameter associated withthe saline change (paired 't'-test). There was no significant difference in voltage orresistance measurements between all times compared against each other for eithercontrol (P= Vt, Rt, Ra, Rb >0.9; Va >0.8; Vb >0.4, ANOVA) or cAMP stimulated ilea(P= Vt, Rb >0.9; Rt, Ra >0.8; Va >0.7; Vb >0.5, ANOVA). Saline changes had,therefore, no effect on the measured electrical parameters. Although there seems to bea trend for Rt to increase with time when ilea are stimulated with cAMP, thisrepresented only a 4.7% increase in Rt at most (at 26 mins) and was not significant (P=>0.05, paired 't'-test). Since recording of electrical parameters during impalement andthrough saline changes provides relatively steady values over a 26 minute period, theeffect of cAMP addition and ion substitutions in subsequent experiments described inthis thesis are primarily paired experiments on single cells within this time period.4115 —a* *—5 —5 —25boetf"8 —45—65—850^5 10^1'5Time (mins)^• •^I20^25 30b175 —150Ng• ▪ 125 ^0—^4G100 —VCV▪ 75 —cg so -5^10^15^20^25^30Time (mins)Fig 3.1.3: The effect of saline changes (*) during impalement upon a: Vt (a),Va (A) , Vb (•), and b: Rt (0), Ra (A) and Rb (•). Ilea were nonstimulated,n = 14-17 (5). Voltage sign refers to lumen side (Vt) or cell interior (Va, Vb).Values are means ± s.e.25 -• ^0 ^042a40 —* *002000CU -20 -bAZja-40 ..............•-60 -I ai-----i^ i^-80^0^5^10^15^20^25^30Time (mins)b80 —70 -60504—...G 40 -CU2‘40 30 -.4 20-6f=4 10 11^•^•^•^9------4^^01^I 1^0^5 10^15^20^25^30Time (mins)Fig 3.1.4: The effect of saline changes (*) during impalement on; a: Vt (0),Va (A) and Vb (•). b: Rt (0), Ra (A) and Rb (•). Preparations were stimulatedwith 5mM cAMP, n = 10-21 (5). Sign refers to lumen (Vt) or cell interior(Va, Vb). Values are means ± s.e.•I43Table 3.1.1: Control impalements for both control A; nonstimulated condition, n = 17(5) and control B; cAMP stimulated condition, n = 21 (5) at 0 and 8 mins after salinewas changed from control saline to an identical saline. There is no significant differencebetween t = 0 and t = 8 mins for any parameter measured for either nonstimulated orcAMP stimulated ilea. Values are means ± s.e.CONTROL A CONTROL BTime (mins) 0 8 80Vt (mV) 9.15 ± 1.7 9.75 ± 1.65 24.66 ± 1.74 25.18 ± 1.63Va (mV) -72.97 ± 1.92 -74 ± 1.74 -67.93 ± 2.01 -68.66 ± 1.69Vb (mV) -63.83 ± 0.75 -64.25 ± 0.89 -43.28 ± 1.31 -43.48 ± 1.14Rt (C2cm2) 137.35 ± 8.23 136.95 ± 8.02 58.46 ± 3.53 59.44 ± 3.69Ra (C2cm2) 127.92 ± 7.13 127.23 ± 7.04 51.91 ± 3.6 52.72 ± 3.65Rb (0=2) 10.61 ± 1.81 11.49 ± 1.88 10.07 ± 1.09 10.64 ± 1.2444Fig 3.1.5 shows samples of an actual trace of electrical measurements madeduring impalement of an ileal cell while passing constant current pulses used to estimateresistance. Both Va and Vb displayed only minor variations and a was constant.Unfortunately, traces are too long to show in their entirety in this thesis.Electrical properties of Heal cells.Mean values for impalements made under control conditions in this study areshown in Fig 3.1.6 for both a: nonstimulated ilea and b: ilea stimulated with 5 mMcAMP, for impalements made between 30 mins and 6 hours after cAMP addition.During steady state for nonstimulated ilea, mean Vt was slightly positive (signrefers to lumen side), although the range of values of Vt included negative values. Theinside of the cell was negative with respect to both haemolymph and lumen side bathingmedia, the voltage across the apical membrane being slightly more negative at -67.53mV than across the basal membrane, -60.15 mV. Most of Rt at 132.91 fIcm2 wasaccounted for by Ra, which at 124.83 fIcm 2 was much larger than Rb at 8.3 S2cm2 .Stimulation of ileal cell transport processes with 5mM cAMP caused a 3.4-foldrise in Vt to 24.96 mV. Va did not change significantly but Vb depolarized by about 1/3to -40.9 mV.The major feature of cAMP stimulation was the large increase in the conductanceof the apical membrane, Ra decreasing to 49.54 Ocm2 , i.e. by a factor of 2.5x. Rb didnot change significantly. For 'P' values (independent 't'-test), see Table 3.1.2 which alsoprovides a comparison with measurements made by Irvine et al. (1988).45Fig 3.1.5: Actual trace of electrical measurements made during impalementof an ileal cell when nonstimulated. The deflections are caused by thepassage of constant current pulses (13.3 i.tA) used to estimate resistances.The y-axis gives the base-line potential only. The scale at right is that of thechart-recorder. Voltage deflections are uncorrected for saline resistance. Vaand Vb are shown as extracellular positive and Vt as lumen positive.4611,/Basal (Vb)1-4'-ir441^k*di*7565(3)61)O1010mVTransepithelial (Vt) 111Ji5 10 15 20 25Time (mins)1I+7.38 mV±0.49132.91 acm 2± 1.69Transepithelialhaemolymph124.83 S2CM 2^8.32 Ocm 2±1.57 ±0.36I■..^iol^lumen.11111111■■11011.-Basal-60.15 mV±0.34Apical-67.53 mV±0.57a NONSTIMULATED ILEA5 mM cAMP STIMULATED ILEAb+24.96 mV±0.6657.82 Ocm2±1.07Transepithelial01,- 41lumen 49.54 acm 2±1.077.55 Clcm 2±0.46haemolymphApical^Basal-65.86 mV -40.90 mV±0.77^±0.6Fig 3.1.6: Summary of electrical measurements made during this study fora: non-stimulated ilea n = 284 (47) and b: ilea stimulated with 5mM cAMPn = 150 (28). Values are means ± s.e.48Timecourse of cAMP stimulation.Fig 3.1.7 a and b show the changes in voltage and resistance with time afterstimulation with 5mM cAMP as measured from single cells during continuousimpalement. Over the first 8 mins after cAMP addition, Vt displayed a significantincrease from 2.86 ± 4.44 mV to 7.33 ± 2.95 mV (P= <0.05, paired 't'-test). Vbdepolarized significantly from -60.97 ± 2.32 mV at time 0 to -51.4 ±3.01 mV at 8 mins(P= <0.02, paired 't'-test). Va did not change significantly over the same time period(P= >0.5, paired 't'-test). Vt reached a new cAMP stimulated steady state value ataround 30 mins after cAMP addition (not shown).Most of the effect of cAMP stimulation upon Rt and Ra was established withinthe first 8 mins after cAMP addition. There was a rapid, significant decrease in Rt overthis time period from 128.21 ± 7.72 SIcin 2 to 71.16 ± 7.9 )cm 2 (P= <0.001, paired 't'-test). There was also a decrease in Ra which was of the same magnitude and followedthe same timecourse as the decline in Rt. Ra decreased from 113.26 ± 4.63 Ocm 2 attime 0 to 54.74 ± 7.95 Ocm2 at 8 mins after cAMP addition (P= <0.001, paired 't'-test).These results, recorded from single cells throughout cAMP stimulation were comparableto those from multiple impalements made from nonstimulated and cAMP stimulated ileapreviously reported in figs 3.1.3 and 3.1.4 and in table 3.1.1. This demonstrates that thephysical conditions of impalement before and after stimulation with cAMP were similar.Fig 3.1.8 shows actual traces of AVt, AVa and AVb caused by constant currentpulses for before and after addition of 5mM cAMP. These voltage deflections areuncorrected for saline resistance which was the same in both conditions.4920- *0 --20 -0.1fa-40 --GO-800^5^10^15^20^25Time (mins)b0 ^0 ^•^ ^•5 10^15 20 25Time (mins)Fig 3.1.7: Timecourses after the addition of 5 mM cAMP to ilea at * on a:Vt (0), Va(A), Vb(*) and b: Rt ( c), Ra (A) and Rb (•). Continuous recordingfrom single cells, n = 7 ilea (one cell per ileum). Sign refers to lumen (Vt) orcell interior (Va, Vb). Values are means ± s.e.50a: non-stimulated^b: 5mM cAMP stimulated O VtAVaAVbFig 3.1.8: Actual trace of AVt, AVa and AVb caused by the passage ofconstant (13.3 p.A) used to estimate resistances a: before and b: 8 mins afterstimulation with 5 mM cAMP. Voltage deflections are uncorrected forsaline resistance, which does not change between the two conditions. Scaleis shown at right.51II: IS THERE A cAMP STIMULATED INCREASE IN APICAL Na+CONDUCTANCE?Introduction.ScgITP and cAMP both double ileal Naf reabsorption in S. gregaria (Irvine etal. 1988). In the previous section, I demonstrated a large conductance increase in theapical membrane after the addition of 5 mM cAMP. This suggests that scgITP might actvia cAMP to open apical Nal" channels, thus increasing intracellular Na+ levels availableto a NerATPase and resulting in enhanced Na transport. In this section I test thishypothesis by replacing luminal Na+ and by the addition of the Na+ transport blockeramiloride to ilea in control saline. Since cAMP has dramatic effects upon Cr transport,the effect of Na+ replacement was studied in the presence and absence of external Cr.The timecourse of the effect of sodium replacement.Fig 3.2.1 shows the timecourse of changes in electrical parameters uponreplacement of luminal Na and subsequent return to control saline (110 mM Na+).In preliminary experiments, ilea were perfused with Nal" free saline for 23 mins,at which point the rate of change of the measured parameters slowed, indicating anapproach to a new steady-state in the Na + free condition. However, it is likely that sucha long exposure to Na free saline would alter intracellular sodium activity ([Nai l).Experiments described in section III of this chapter indicate that the apical membrane5210E^-20 -I.)bl)ft%^-40-0-60 --80 --1000I20^ 30a*^120-_1 --0Time (mins)b100 -50 -0- I I i0^ 10^ 20^ 30Time (mins)Fig 3.2.1: The effect of Na replacement for nonstimulated ilea on a: Vt 43),Va (A) and Vb (•) and b: Rt (0), Ra (A) and Rb (•). Saline is changed fromcontrol (110 mM) to Na-free at * and back to control saline at 1 n= 4-15 (6).sign refers to lumen (Vt) or cell interior (Va, Vb). Values are means ± s.e.53conductance is able to change rapidly in response to variations in luminal [K1 similarin magnitude to those of [Nal in this section. This, and the observation that the majorcAMP stimulated changes in Rt and Ra occur within 8 mins after cAMP addition (Fig3.1.7), indicates that an apical Na + conductance should be evident from changes in Rtand Ra within 8 mins after Nat substitution. For this reason, electrical parameters werecompared at time 0 and 8 mins after the ion was substituted control saline was restoredat 13 mins so that the 8 min sample time lay within an experimental window. Recoveryof electrical parameters to values similar to those recorded at time 0 upon return tocontrol saline was also monitored.Substitution of luminal Na+ resulted in an increase in Vt due to ahyperpolarization of Va. There was an increase in both Rt and Ra consistent with asmall apical Na+ conductance. Vb and Rb did not change throughout the experiment (P=>0.3 and >0.6, respectively, ANOVA. Values compared over time). Re-addition of Na+caused a return of both voltage and resistance measurements to values not significantlydifferent from those at time 0 (P= >0.05, paired 't'-test).Comparison of electrical parameters in control and Na+-free saline and the effectof amiloride.Fig 3.2.2 shows the changes in Vt, Va and Vb between time 0 and 8 mins afterluminal Na+ replacement or addition of 10 .tM and 1 mM amiloride to the haemolymphside of preparations in control saline.In nonstimulated ilea, Na l- replacement caused a highly significant increase in Vt54***^ Initial saline conditionNa free10 11M amilorideE3 1mM amiloride**********Cl-freecAMP2***3^1 1control1control^control*****controlcontrol0control cAMP Cl-free control control1, 3^1, 3cAMP2Cl-free3-100-80-60-40-200-80 —40 -30 -5 20 -10-0 ^Initial saline condition.Fig 3.2.2: A comparison of the values of a: Vt and b: Va before and 8 minsafter sodium removal or the addition of 10[tM and 1mM amiloride tocontrols (110 mM Na). Initial saline condition is complete or Cl-free whennon-stimulated. Significantly different from value in initial saline (paired't'-test) is indicated with * ; n= 59 (18). Numbers above columns indicategrouping of similar changes in Va (Tukey). Sign refers to lumen (Vt) or cellinterior (Va, Vb). Values are means ± s.e.55(P= <0.001, paired 't'-test), consistent with electrogenic transport of Ne. Vahyperpolarized significantly (by 11.5 ± 1.37 mV) as did Vb. When ilea were stimulatedwith 5 mM cAMP, there was also a significant increase in Vt (P= <0.001, paired 't'-test). Va and Vb both showed hyperpolarizations which were significantly higher thanfor nonstimulated ilea at 18.89 ± 1.85 mV and 10.65 ± 0.88, respectively, (P= <0.001,Tukey). In Cr-free saline, Vt increased as for nonstimulated and cAMP stimulated ilea.Va hyperpolarized but the change at 9.42 ± 4.44 mV (P= <0.05, paired 't'-test) wassignificantly smaller than for cAMP stimulated ilea (P= <0.001, Tukey) or ilea in controlsaline (P= <0.002).Addition of amiloride to ilea bathed in control saline resulted an increase in Vtand hyperpolarizations of Va and Vb which were not significantly different from thoseobserved after Nat replacement in nonstimulated ilea. Thus, addition of amiloride causesquantitatively the same effect as removing sodium, suggesting that amiloride is blockingan apical Na+ conductance. Since there is no difference between the results observedwith the addition of 10 j.IM amiloride and 1 mM amiloride, it is likely that the apicalconductance is a channel and not an electrogenic cotransporter.Fig 3.2.3 shows the changes in Rt, Ra and Rb between time 0 and 8 mins afterluminal Na+ replacement or addition of 10 11M and 1 mM amiloride.In nonstimulated ilea, Na+ replacement resulted in a significant increase in Rt (P=<0.001, paired 't'-test), which again supports the hypothesis of an apical Na +conductance. The increase in Rt was due solely to a significant 18% increase in Ra (P=<0.001, paired 't'-test) with Na l- replacement having no effect on Rb. When ilea were56II Initial saline conditionES] Na freeEl 10 tiM amilorideIS] 1mM amiloride2^2300 -200 -1aa. 100 -1^2***o*1*Cl-free control control2 **1 1control cAMP40 —,—,N 30 -20 -g 1 110 -If ITVcontrol^cAMP^Cl-free^control^controlInitial saline conditionFig 3.2.3: A comparison of the values of a: Rt and b: Ra before and 8 minsafter sodium removal or the addition of 1011M and 1mM amiloride (seelegend). Initial saline condition is either control or Cl-free whennon-stimulated. Significantly different from value in initial saline (paired't'-test) is indicated with * ; n=58 (18). Sign refers to lumen (Vt) or cellinterior (Va, Vb). Values are means ± s.e.57stimulated with cAMP, there was a significant increase in Rt after Nal . replacement (P=<0.05, paired 't'-test) but this was significantly smaller than that observed innonstimulated ilea (P= <0.001, Tukey). The increase in Rt was due to a significantincrease in Ra of 12.8% (P= <0.005, paired 't'-test) with Na+ replacement having nosignificant effect upon Rb. When ilea were bathed in C1 --free saline, Na+ replacementresulted in a significant increase in Rt (P= <0.005, paired 't'-test). This was due to botha significant increase in Ra (P= <0.005, paired 't'-test) and Rb (P= <0.005, paired 't'-test) which showed a 6.5 ± 1.9 S2cm2 change.The addition of amiloride to ilea in control saline did not cause a significantchange in Rt, Ra or Rb for both concentrations (10 1.iM and 1 mM) tested. This is notconsistent with the block of apical Na+ channels by this agent which should lead to anincrease apical resistance.58III: INVESTIGATION OF IC CONDUCTANCES.Timecourse of the changes in electrical parameters.The thick cuticle (10 gm) attached to the apical plasma membrane represents adiffusion barrier, which could significantly slow the response of Va and hence Vt tochanges in luminal ions. I therefore first studied the timecourse of changes in voltageand resistance to determine how long I should measure Va and Vt in other experimentsand yet minimize the time ilea were bathed in media other than control saline.Fig 3.3.1 shows the timecourse of electrical parameters when luminal [KI wasincreased from 10 to 105 mM and then returned to 10 mM. Ilea were stimulated with5 mM cAMP throughout the experiment. There was a significant decrease of Vt from+22 ± 2.85 mV at time 0 to -14.99 ± 3.32 mV at 8 mins after increasing luminal [K1(P= <0.001, paired 't'-test). This change in Vt was due mostly to a significantdepolarization of Va from -63.07 ± 3.25 mV to -19.63 ± 2.57 mV (P= <0.001, paired't'-test) over the same time period. Vb depolarized slightly but significantly from 40.69± 1.9 mV to 34.62 ± 2.02 mV over the same period (P= <0.002, paired 't'-test).There was a large, significant decrease in Rt after increasing luminal [r] from46.09 ± 1.93 S2cm2 at time 0 to 15.94 ± 1.12 Ocm 2 at 8 mins (P= <0.001, paired 't'-test). The change in Rt was due to a significant decrease in Ra from 39.92 ± 2.01 Ocm 2to 6.97 ± 1.57 S2cm 2 over the same time period (P= <0.001, paired 't'-test). Rb did notchange significantly throughout the experiment (P= >0.9, ANOVA. Values compared59a40120bTime (mins)Fig 3.3.1: Timecourse for the effect upon a: Vto, Va • and Vb. and b: Rto,Ra • and Rb • of changing luminal [KI from 10 mM (control) to 105mM at* and back to control at 1 n= 7-13 (4). Ilea were stimulated throughoutwith 5 mM cAMP. Sign refers to lumen (Vt) or cell interior (Va,Vb). Valuesare means ± s.e.60over time). There was no significant difference in either voltage or resistance values at31 mins compared to time 0 (P= >0.05, paired 't'-test). This experiment demonstratesthat changes in voltage and resistance due to varying luminal ion concentration wereessentially complete within 8 mins. While Vt and Va continued to rise slowly thereafter,this was probably due to changes in intracellular ion activities associated withexperimental perturbation. Thus, I judged 8 mins as the optimum time to estimateelectrical changes with minimal change in transmembrane ion ratios due to intracellularion changes.A) IS THERE A cAMP STIMULATED INCREASE IN APICAL IC'CONDUCTANCE?Since an increase in Na+ conductance does not account for the cAMP stimulateddecrease in Rt, I thus tested whether there is an increase in the apical conductance to1C+ after cAMP stimulation as demonstrated for the rectum (Hanrahan and Phillips,1984c)Figs 3.3.2 and 3.3.3 show the relationship between Vt and Va when luminal [r]was varied. If the apical membrane were a perfect 1C -1- electrode (i.e. only permeable toIC") then the change in Vt or Va per decade change in luminal [K1 would approach theNernstian value of 59 mV (at 25°C). For IC concentrations other than 10 mM(i.e.control saline), the electrical measurements in this section were taken 8 mins after6110^100^1000• y = 61.359 + -37.292*L0G(x)0 y = 9.0840 + -1.1625*LOG(x)Log mucosal [I<1 (mM)Fig 3.3.2: The effect on Vt (8 mins after saline change) of varying luminal[K] when ilea were nonstimulated,Q n= 29 (6) and when stimulated with 5mM cAMP, ■ n= 39 (6). Logarithmic curve best fit is shown on the graphwith dashed lines and the legend gives the equations of these lines. Vt isshown as lumen positive. Values are means ± s.e.621 10 1000100IN y = 107.23 + -41.965*LOG(x)0 y = 72.721 + -1.5050*L0G(x)Log mucosal [K+] (mM)Fig 3.3.3: The effect on Va of varying luminal [e] when ilea werenonstimulated, 0 n= 29 (6) and when stimulated with 5 mM cAMP, ■ n=39 (6). Logarithmic curve best fit is shown on the graph with dashed linesand the legend gives the equations of these lines. Sign is cell interiornegative. Values are means ± s.e.63the saline was changed.In nonstimulated ilea, both Vt and Va changed very little with changes in luminal[Kl, indicating that the apical membrane has a negligible conductance to IC4- under theseconditions. When ilea are stimulated with 5 mM cAMP there was a dramatic increasein Vt/decade [K+] 37 mV with a corresponding increase in Va/decade [K+] 42 mVover a range of 10 to 105 mM K. Thus, the ileal apical membrane displays a largecAMP stimulated increase in apical KE conductance.Fig 3.3.4 shows the effect of varying luminal [K+] resistance parameters whenilea were nonstimulated. Between 0.046 and 105 mM mucosal K", Rt decreased by only15.91 Ocm2, a change of 11% which is not significant (P= >0.1, ANOVA). The changein Rt was due to a significant decrease in Ra of 30.94 SIcm 2, a 22.5% change over thesame range of [K+] <0.02, ANOVA). Varying luminal [K+] no effect on Rb (P=>0.9, ANOVA). These results again indicate a very small apical conductance to IC+ whenilea are nonstimulated.Fig 3.3.5 shows the effect of varying luminal IC' on resistance parameters whenilea were stimulated with 5 mM cAMP. Both Rt and Ra decreased dramatically withincreasing [K1. Between 0.046 and 105 mM mucosal 1C+, Rt decreased by 47.1 5 -2cm2which represents a significant change of 74.7% (P= <0.001, Tukey). Ra also decreasedsignificantly by 47 52cm2, an change of 87% (P= <0.001, Tukey). Rb is not affected byvarying luminal [K+] >0.4, ANOVA). These results indicate that there is a verylarge increase in the IC' conductance of the ileal apical membrane when preparations arestimulated with cAMP. This is reflected in both voltage and resistance measurements.642000-• ^ •0 -^0 20Mucosal [ICJ (mM)Fig 3.3.4: The effect upon Rt 0 , Ra ♦ and Rb • of varying luminal [K + 1.Ilea were nonstimulated n= 29 (6). Values are means ± s.e.40^60^80^100^1206512020^40^60^80^100Mucosal [K +] (mM)Fig 3.3.5: The effect upon Rt ^ , Ra ♦ and Rb • of varying luminal [IC' ].Ilea were stimulated with 5 mM cAMP, n= 39 (6). Values are means ± s.e.66B) IS THERE A SIGNIFICANT BASOLATERAL IC CONDUCTANCE?Observations in section IV indicate that a Cl" conductance is not the mainconductance of the basolateral membrane. Also, it is unlikely that the passive basolateralconductance to Na+ is very high as this would increase the energy requirement of abasolateral Na+K+ATPase. I thus tested whether IC+ represents the major ion conductanceof the basolateral membrane.Fig 3.3.6 shows the relationship between voltages and varying serosal [r]. Thechange in Vb/decade [K+] 45 mV, indicating a high relative conductance of thebasolateral membrane to K. Both Va and Vb depolarized with increasing serosal [K1,consistent with a decrease in net exit of IC" from the cell, as would be expected ifbasolateral IC" exit is passive. The change in Va/decade [K+] 33.6 mV.Transepithelial conductance to IC- is low when ilea are nonstimulated, with Vt onlychanging 5 mV/decade [Kl.Fig 3.3.7 shows the relationship between resistance and serosal [K1. Rbdecreased with increasing [K1 but non-linearly, changing significantly by 48.36 ± 4.71)cm2 between 0.046 and 10 mM 1(+(P= >0.001, Tukey) but only 5.9 ± 1.3 S2cm 2between 10 and 100mM Kf (not a significant change, P= >0.4, Tukey). This indicatesthat increasing [K+] the haemocoel side increases basolateral IC+ conductance. Ra didnot change significantly with serosal [K+] >0.4, ANOVA). As expected, Rt wasmostly dependent upon changes in Rb and decreased significantly by 53.05 ± 8.55 S2cm 267^ y = 13.596 + -5.1942*LOG(x)• y = - 107.09 + 45.430*LOG(x)A y = - 103.36 + 33.610*LOG(x)Log serosal [K +] (mM)Fig 3.3.6: The effect upon VtD ,Va ♦ and Vb• of varying [K + ] on thehaemolymph side. Logarithmic curve fit is shown on the graph withdashed lines, the legend provides the equations of these lines. Ilea werenonstimulated, n= 20 (4). Sign refers to lumen (Vt) or cell interior (Va, Vb).Values are means ± s.e.6812040^60^80^100300100200Serosal [K 1 (mM)Fig 3.3.7: The effect upon RtO ,Ra À and Rb• of varying [K + ] on thehaemolymph side. Ilea were nonstimulated, n= 20 (4). Values are means ±s. e.69between 0.046 and 10 mM K* (P= <0.02, Tukey). The change in Rt between 10 and105mM K+ was not significant (P= >0.6, Tukey).70IV: IS THERE A cAMP STIMULATED BASOLATERAL C1 CONDUCTANCE?Cyclic AMP increases both apical electrogenic CT transport and basolateral Crconductance in locust rectum (Hanrahan and Phillips 1984a-c). In this section I testedif a similar situation exists in the locust ileum. Cl - was replaced bilaterally to stop Crtransport apically and thereby determine if cAMP increased basolateral conductance.Fig 4.4.1 shows the effect on voltages of bilateral C1 replacement after 8 mins(short-term) and when maintained in C1-free saline for 2-5 h, resulting in large changesin intracellular Cr activity.In nonstimulated ilea, Cr replacement (from control saline 110mM Cr) resultedin a significant 10.39 ± 0.67 mV increase in Vt (P= <0.001, paired 't'-test), due to ahyperpolarization of Va by 17.08 ± 0.96 mV (P= <0.001, paired 't'-test). This changein Va is thus in the wrong direction for the abolition of an apical Cr conductive entrymechanism alone, i.e. an apical electrogenic CT pump. Vb also hyperpolarizedsignificantly by 6.69 ± 0.89 mV (P= <0.001, paired 't'-test).When ilea were stimulated with cAMP, there was a 16.78 ± 2.41 mV (P=<0.001, paired 't'-test) decrease in Vt after Cr replacement, due mostly to a significanthyperpolarization of Vb by 14.35 ± 1.13 mV (P= <0.001, paired 't'-test) whichrepresents a change in Vb/decade [Cr]. of 4.3 mV. There was no significant change inVa after Cr replacement in cAMP stimulated ilea (P= >0.02, paired 't'-test).Longterm exposure of nonstimulated ilea to Cr free saline did not change Vt71-100 --80 --60 --40 --20 -0-80 --60 --40 --20 -0*********Initial saline conditionC1-free salineControl A from Section ILong exposure to Cl-free salinenonstimulated ilea.30 -20 -10-***•• -9;Control^cAMP^Control^Cl-freeInitial saline conditionFig 3.4.1: A comparison of voltage parameters before and 8 mins afterremoving Cl from nonstimulated and cAMP stimulated ilea n=24 (8) andthe effect of longterm (2-5 h) Cl replacement n=35 (9). Significantly differentfrom control denoted by * for paired 't'-test and • for independent 't'-test.Sign refers to lumen (Vt) ro cell interior (Va, Vb). Values are means ± s.e.72significantly from section I control A values (P= >0.5, independent 't'-test). Thus, innonstimulated ilea, Vt can be maintained in the absence of Cr. Both Va and Vb weresignificantly more positive in the absence of Cr (P= >0.001, independent 't'-test). Thisis due to the depletion of intracellular Cr levels after long exposure to Cr free saline.Fig 3.4.2 shows the effect upon ileal resistance parameters of short-term andlong-term bilateral Cl - replacement. In nonstimulated ilea, Cr replacement caused Rt toincrease significantly by 60.99 ± 4.61 S2cm2 (P= <0.001, paired 't'-test). This increasein Rt was due to both an increase in Ra and in Rb of 17.57 ± 5 C2cm2 (P= <0.002,paired 't'-test). This represents a 2.3 fold increase. These resistance changes areconsistent with Cr conductances for both membranes, as predicted.Ilea which had been stimulated with cAMP, displayed a significant increase inRt of 18.22 ± 2.41 Ocm2 after Cr replacement, due to both to a 1.6 fold increase in Rband a significant increase in Ra of 6.83 ± 1.7 C2/cm2 (P= <0.01, paired 't'-test). Thelesser effect of a replacement after cAMP addition may reflect the proportionally muchgreater apical K+ conductance after cAMP stimulation (section III) which greatly lowersRt and Ra.Longterm replacement of Cr resulted in very high values of Rt, due to increasesin both Ra and Rb.73U Initial saline conditionED CI-free salinea Control A from section IE3 Long exposure to Cl-free saline300 -^nonstimulated ilea.Initial saline conditionFig 3.4.2: A comparison of resistance parameters before and 8 mins afterremoving Cl from nonstimulated and cAMP stimulated ilea, n= 24 (8) andthe effect of longterm (2-5 h) Cl replacement n= 35 (9). Significantlydifferent from control denoted by * for paired 't'-test and • for independent't'-test. Values are means ± s.e.74CHAPTER 4 DISCUSSION.I:^THE EFFECT OF cAMP.The normal distribution observed for values of Vb confirms the findings of Irvineet al. (1988) and is consistent with a single population of cells for the ileal epithelium.The results presented here are based on a much larger sample size than in the previousstudy and represent, therefore, a stronger basis for this hypothesis which is alsosupported by electron micrograph observations of ilea (Irvine et al. 1988).Physical observations of the ileal epithelium during impalement and on removalof the muscle layer suggest the presence of another membrane between the basal laminaand muscle layer (possibly the basement membrane of the muscle layer). This membranecan be seen to deform upon impalement and during removal of the muscle layer andseems to be particularly associated with and around trachea (pers.obs., J.Meredith pers.comm.). This membrane may represent a physical barrier impeding more intimatemethods of investigation such as patch clamping.Table 4.1.1 is a comparison between results obtained in the present study andthose from the previous study by Irvine et al. (1988). There are some obviousdifferences between the two studies both for nonstimulated and cAMP stimulated ilea:Firstly, in nonstimulated ilea, Vt was much more positive at +33 mV in the75Table 4.1.1: Comparison between results of ileal impalement from Irvine et al. (1988)and those from this study. * indicates significantly different between control and cAMPstimulated ilea in the Irvine et al. study, for paired 't'test for separate impalements madeupon the same preparation before and 7-14 mins after stimulation with 5 mM cAMP.'P'-values are shown for control vs cAMP stimulated ilea for the present study(independent 't'test). Values are shown as means ± s.e. AVa and AVb are the voltagedeflections due to constant current pulses (13.3 .tA). N/A, data not available.CONTROL + CYCLIC AMP PresentstudyElectrical Irvine et al. Present Irvine et al. Present P - valuesparameter (1988) study (1988) study ('t'-test)Vt (mV) 33 ±4 7.4 ± 0.5 46 ± 4 * 25 ± 0.7 <0.001Va (mV) -95 ± 4 -67.5 ± 0.6 -87 ± 6 -65.9 ± 0.8^>0.05Vb (mV) -62 ± 1 -60.2 ± 0.3 -41 ± 4 * -40.9 ± 0.6^<0.001Rt (Ocm2) 229 ± 15 132.9 ± 1.7 98 ± 11 * 57.8 ± 1.1^<0.001AVa 14 ± 1 8.47 ± 0.11 5.6 ± 0.7 * 3.36 ± 0.07 <0.001AVb 1.0 ± 0.2 0.56 ± 0.02 0.9 ± 0.1 0.51 ± 0.03^>0.05a (range) 14 (9-23) 25.55 ± 1.6 6 (4-10) * 6.5 ± 1.08^<0.001Ra N/A 124.8 ± 1.6 N/A 49.5 ± 1.1^<0.001(S2cm2)Rb N/A 8.3 ± 0.4 N/A 7.6 ± 0.5^>0.1(C2cm2)n = 11-40 (3-5)in total284 (47) 150 (28)76previous study than reported here. This high value for Vt was attributed by Irvine et al.(1988) to either partial stimulation of the ilea, which seems unlikely due to the muchhigher values of Rt reported in that study compared to the values reported in this thesisor to the cessation of perfusion during impalement resulting in either an insufficientoxygenation of the membrane or to the changing of local ion gradients. If this is thecase, it can be seen that this effect operates at the apical membrane as values for Vbwere very similar between the two studies. Rt in the previous study was higher thanreported here. Similarly, voltage deflections due to constant current pulses (13.3 p,A) ofthe apical (AVa) and basolateral (AVb) membrane potentials (from which resistances areestimated) are higher in the previous study. This may be due to the small sample size(such high values have been recorded in this study but are uncommon), to the cessationof perfusion or to variations in the amount of stretching of ilea when mounted. It is thusimportant to compare changes on the same preparation whenever possible.Secondly, in both studies Vt was more positive in cAMP stimulated ilea than innonstimulated ilea, with a similar incremental increase of ,---13-15 mV. However, in thepresent study, this increase represents a much greater increase proportionally at 3.4xthan in the previous study at x1.5. Both studies revealed that Va was unaffected bycAMP stimulation, whereas Vb depolarized, values of Vb being again comparablebetween the two studies. Rt was shown to decrease on stimulation in both studies andthis decrease was due solely to a decrease in Ra, Rb remaining at control values. AcAMP stimulated increase in IC+ conductance has been demonstrated for the ilea (Irvineet al. 1988, Audsley 1990). The location of this increase was attributed to the apical77membrane through indirect evidence based upon a) the decrease in Ra after stimulationwith cAMP and b) the similarity of these results to those recorded from rectum wherea cAMP stimulated increase in IC+ conductance has been demonstrated (Hanrahan andPhillips 1984c). In section III exclude an increase in Na+ conductance as an explanationfor the decreases in Rt and Ra.Nonstimulated locust ileum and rectum display similar electrical properties. Inboth epithelia the inside of the cell is negative with respect to both the haemocoel andlumen side bathing media and Va is more negative than Vb (Hanrahan 1982). The majordifference between the two segments is that ileal Rb at 8.32 S2cm 2 is very much lowerthan rectal Rb at 193 Clem' (Hanrahan and Phillips 1984c). Thus, ileal a (AVa/AVb) isvery high at 25.55 ± 1.55 (n= 284 (47)) compared to 1.13 ± 0.12 for the rectum(Hanrahan and Phillips 1984c).In the ileum, cAMP caused an increase in Vt which is comparable toobservations made on the rectum. Ileal Va changed little from control values and Vbdepolarized, whereas in the rectum cAMP addition causes Va to hyperpolarize and Vbto depolarize (Hanrahan and Phillips 1984c).In the case of the rectum there is a significant decrease in Ra and Rb after cAMPstimulation, due to an increase in the conductance of the apical membrane to IC+ and thebasolateral membrane to Cl - (Hanrahan and Phillips 1984c). The ilea, however, displayedonly a decrease in Ra, with Rb remaining at control values. Since in the ilea, Rb is so78low, there may be no need during cAMP stimulation of salt reabsorption to increasebasolateral ion conductances (see section III and IV). Stimulation of ilea with cAMPtherefore causes a decrease in a to 6.5 ± 1.08 (n= 150 (28)) whereas cAMP has littleeffect upon a as measured from the rectum (Hanrahan and Phillips 1984c).79II: IS THERE A cAMP STIMULATED INCREASE IN APICAL Na+CONDUCTANCE?The rate of Na+ reabsorption in the ilea is 3-4x that measured for the rectum.(Lechleitner et al. 1988a, Black et al. 1987). In the ilea net Nal" reabsorption is increasedupon addition of 5 mM cAMP (Irvine et al. 1988), whereas in the rectum cAMP has noeffect upon Na+ transport (Black et al. 1987).Sodium entry at the apical membrane is usually passive because of the large cellnegative electrical potential and low intracellular Na+ activity caused by the activity ofthe basolateral Na+VATPase. High levels of this enzyme have been detected inmicrosomal fractions from ilea (Lechleitner and Phillips 1988). Furthermore, serosaladdition of 5 mM ouabain caused a 30% inhibition of fluid transport across everted ilealsacs (Lechleitner 1988). Similarly, ouabain partially inhibits net Na + and fluid absorptionacross rectal sacs (Irvine and Phillips 1970, Goh and Phillips 1978) and the active influxof nNa+ across short-circuited recta at 30°C (Black et al. 1987). It is thereforereasonable to assume that basolateral Na1C+ ATPase is responsible for active Na+reabsorption in both the ileum and rectum.Apical entry of Na+ can occur by exchange with fr or NH 4+, co-transport withCr or organic solutes, or by means of Na + channels. Na+ transport coupled to fr, NH 4+or Cr has been largely ruled out for the ilea (Irvine et al. 1988, Peach 1991). Amiloride(1 mM) was found to have no effect upon 22Na+ flux to the haemocoel side, suggesting80that Na+/cation exchange for I-1+ or NH 4+ is not an important pathway for apical Na+entry (Lechleitner 1988). Moveover, net acid secretion (JH) is 90% Na+-independent(Lechleitner 1988). This contrasts with the situation in the rectal epithelium where anamiloride sensitive cation exchange for both I-1+ and NH4+ accounts for 1/3 of Na+ entry(Black et al. 1987, Thompson et al. 1988a).In this section I tested whether there is an apical Na + conductive pathway innonstimulated ilea and if the observed cAMP stimulated increase in ileal Na+reabsorption (Irvine et al. 1988) is due to increased conductance of the apical membraneto Na.+When ilea were stimulated with cAMP, Irvine et al. (1988) observed similarincreases in Vt and Rt upon Na+ replacement as values reported here. In the previousstudy, the increase in Vt was attributed to the removal of a parallel Na + shunt whichwhen operative would neutralize some of the net charge transfer by the electrogenic Clpump. In this study both Va and Vb were found to hyperpolarize after Naf replacement.These changes in Va and Vb were largest in cAMP stimulated ilea, followed bynonstimulated ilea in control saline and then free saline. This could reflect arelationship between Nal" and Cl- entry. This could not be a direct coupling as Na+replacement has no effect upon ileal Isc (Irvine et al. 1988) but could be indirect withNa+ acting as a counter ion of electrogenic Cl transport. In order to draw any firmconclusions, this phenomenon would have to be investigated by the use of radiotracerNa+ flux experiments under open circuit conditions in the absence and presence of CT.Since the change in Va/decade luminal [Na} when ilea are nonstimulated is only813.4 mV, the apical membrane has a relatively low conductivity to Na. This increasesto 5 mV/decade [Na] in the presence of cAMP, indicating a small, significant increasein apical conductance (P= <0.003, Tukey).When luminal Na+ is replaced, both nonstimulated and cAMP stimulated ileadisplay a significant increase in Rt and Ra, indicating a small apical Na+ conductance.The increase is much larger in nonstimulated ilea i.e. an increase in apical Na+conductance does not contribute to the cAMP stimulated decline in Ra (section I) whichis largely due to an increase in IC+ conductance (section III). In both nonstimulated andcAMP stimulated ilea, the change in Rt is due entirely to an increase in Ra. Thisconductive element of Na+ entry could be due to either the presence of a Nal" channelor an electrogenic co-transport process. Substitution of organic solutes on the luminalside and subsequent Na+ replacement would be necessary to distinguish between thesetwo processes.The entry of neutral L-amino acids into the rectal epithelium is active and in thecase of at least glycine, is Na+ dependant (Balshin 1973). Also, a large electrochemicalpotential of 127mV favours apical Na+ entry. The uptake of amino acids in the ilea wasinvestigated by Peach (1991) who found that 5 amino acids and glucose absorbed fromthe lumen acted as respiratory substrates to support the cAMP stimulated increase in Isc.The apical entry mechanisms for Heal amino acids and glucose and their possible co-transport with Na+ have not yet been studied.Amiloride, which at 10 1.1M should exclusively inhibit apical Na+ channels, hadno effect upon Rt, Ra or Rb but did cause a significant increase in Vt and a82hyperpolarization of Va. This suggests that there is a small apical conductance to Na+,sensitive to amiloride. In the rectum, in the absence of organic solutes, 1 mM amiloridedoes not inhibit the change in iVa when Na is replaced on the luminal side withcholine (Black et al. 1987). This would suggest an insensitivity of Na+ channels toamiloride in Schistocerca. The results obtained with amiloride must, however, beregarded with some caution as the cuticular lining of the lumen may represent a barrierto the drug.In conclusion, the apical membrane of the ileal epithelium has a very low relativeconductance to Na, this conductance is probably due to the presence of Na+ channels.There is a small, significant increase in apical conductance to Na+ due to cAMPstimulation as determined from a small increase in Va/decade [Na]. An increase inapical conductance to Na* does not contribute to the decline in Rt and Ra due tostimulation with cAMP. Resistance measurements alone do not provide evidence forcAMP stimulation of apical Na+ conductance.83III: A) IS THERE A cAMP STIMULATED APICAL K+ CONDUCTANCE?Locust ilea displayed a large cAMP stimulated increase in the conductance ofthe apical membrane to K. After stimulation, Va is largely a r diffusion potential andthe increase in r conductance can account for much of the large cAMP stimulateddecrease in Ra. Irvine et al. (1988) had previously demonstrated that bilateral K±replacement abolished the decrease in Rt due to cAMP addition and subsequentrestoration of K+ reduced Rt by 40%. Since Rb does not change with cAMP stimulation,an increase in apical conductance to r is mostly responsible for the cAMP stimulateddecline in Rt (section I).The rectum also displays a large increase in apical r conductance after cAMPstimulation (Hanrahan and Phillips 1984a,c). Patch clamping of the rectal apicalmembrane revealed distinct current steps indicative of channels. Both slow 10 pA andfast 1.5 pA channel activity was observed (Phillips et al. 1986). In the ilea, the closeassociation of the cuticle with the apical membrane (Irvine et al. 1988) precludes patchclamping of the apical surface of the epithelium. The cAMP stimulated increase in ilealapical r conductance could, however, be further investigated by using noise fluctuationanalysis. This technique seems particularly appropriate in the case of the ilea where Rais much larger than Rb, even when ilea are stimulated with cAMP (see fig 3.1.6). Thiswould result in minimal attenuation of apical current noise (Lewis and Hanrahan 1988).84In the rectum, transepithelial V conductance decreases with increasing mucosal[V], with Rt remaining constant when luminal [V] was raised from 10 to 200 mM(Hanrahan and Phillips 1984a). In locust ilea, this effect was not observed. When ileaare stimulated with 5 mM cAMP, Rt and Ra decrease dramatically with increasingluminal [V] over the range 10-110 mM, indicating that luminal [V] has no modulatoryeffect upon apical V conductance. Moreover, Vt reverses to become negative at around50 mM luminal V. This is stark contrast to the situation described for cAMP stimulatedrecta where Rt remains constant for luminal [V] between 15 mM and 200 mM(Hanrahan 1982). This effect was observed whether V is varied bilaterally or on thelumen side only (Hanrahan 1982). Furthermore, there is an inverse relationship betweenapparent 'V permeability and bilateral [r] and a increases with luminal [V],indicating an increase in Ra and/or a decrease in Rb (Hanrahan 1982). The resultspresented here for the locust ileum differ in that high luminal V does not decreaseapical IC conductance over the range studied. Measurements of the relationship between42.,-+K flux across ilea and luminal [1(.1 could be made to verify these conclusions.B) IS THERE A SIGNIFICANT BASOLATERAL IC CONDUCTANCE?The Koefoed-Johnsen Ussing (1958) pump-leak model demands the presence ofa significant basolateral IC+ conductance (Koefoed-Johnsen and Ussing 1958, reviewedby Lewis et al. 1984). A V leak current allows exit of K+ from the cell down a85favorable net electrochemical gradient which is established by the active uptake of IC'by NaWATPase located at the basolateral membrane (Koefeld-Johnsen and Ussing1958). The evidence for the existence of this pump in the Heal epithelium has alreadybeen discussed in section II.Basolateral IC - channels also serve as an exit step during transepithelial transportof ICI. (Hanrahan 1982, Hanrahan and Phillips 1984a-c). I observed a high conductanceof the basolateral membrane to Ki• in nonstimulated ilea. A change in Vb/decade serosal[KI of 45 mV indicates that a IC" diffusion potential is the main source of Vb. Thedepolarization of Vb with increasing serosal [ICI was closely mirrored by adepolarization of Va. This indicates that the paracellular pathway is very "tight" to K+and that the apical membrane conductance to IC - is very low under nonstimulatedconditions as only a small amount of the change in Vb is shunted at this membrane.The change in Vb/decade serosal [K1 is only 45 mV at maximum and is non-linear, decreasing at low [Kl. This may be because the basolateral membrane isconductive to another ion (or ions), probably Cl" (see section IV) or Nal" (not tested,although unlikely, as a Na+ conductance would be in parallel with the NeK+ATPase),or due to the contribution of the NaWATPase to basolateral membrane potential.Since normal haemolymph [K1 is <10 mM, it is not surprising that between 10and 100 mM serosal [K-1, basolateral resistance changes very little and is extremely low.At [r] <10 mM, basolateral membrane resistance increases with decreasing serosal[Kl. This is probably due to the rapid depletion of intracellular K+ as basolateral K+conductance is very high and in nonstimulated ilea apical IC - conductance is very low.86Irvine et al. (1988) demonstrated that serosal addition of Ba+ eliminated a cAMPstimulated increase in the V diffusion current (Ik) due to an 80:10 mM, serosal:mucosalimposed V gradient, in the absence of Cr and with Vt clamped at 0. Mucosal additionof Ba+ had no effect on Ik under identical conditions. Thus, ileal basolateral ICconductance is sensitive to block by Be (Irvine et al. 1988). These results are similarto those obtained for the rectum (Phillips et al. 1986). Further investigation of rectalbasolateral V conductance using noise analysis (Hanrahan et al. 1986), revealed aLorentzian component of the power density spectra, consistent with the presence ofchannels. Single channel conductance was estimated at 60 pS with a mean channeldensity of 180 million/cm2. These results were comparable with the macroscopicconductance of the basolateral membrane if the probability of channels being in the openstate is 0.5 in the absence of Be.A similar investigation of basolateral V conductance in locust ilea is not feasibledue to the very low relative resistance of the basolateral membrane. However, it maybe possible to investigate ileal basolateral V conductance by patch clamp. Thistechnique cannot be applied to the basolateral membrane in the rectum due to thepresence of a secondary cell layer (Irvine et al. 1988). While some physical barriers topatch clamping are also present in the ilea, the muscle membrane can be easily removedand it may also be possible to remove the underlying basement membrane (describedin Section I), and digest away the ileal basal lamina.In summary, in nonstimulated ilea, the apical membrane has very low87conductance to IC+, the basolateral membrane, however, has a high IC+ conductance.When stimulated with cAMP the IC+ conductance of the apical membrane increasesgreatly. There is no evidence for an inhibition of apical 1C+ conductance by high mucosal[r].88IV: IS THERE A cAMP STIMULATED INCREASE IN BASOLATERAL aCONDUCTANCE?Electrogenic Cr transport is the major cAMP stimulated active transport processof both the rectum (reviewed by Phillips et al. 1986) and the ileum (Irvine et al. 1988).In the case of the rectum, the active step of Cl - reabsorption has been localisedto the apical membrane (Hanrahan and Phillips 1983, 1984c) where an electrogenic Crpump, dependent upon metabolic energy (Chamberlin and Phillips 1982) transports Cl-into the cell against a net electrochemical gradient of 50 mV which increases by 18 mVwhen recta are stimulated with cAMP (Hanrahan and Phillips 1984c).The cAMP stimulated increase in rectal Isc is due to Cl- transport at allconcentrations of Cr from 10 to 140 mM, in the presence of 10 mM IC - and absence ofbicarbonate (Hanrahan and Phillips 1984a). Similar experiments performed upon theileum revealed kinetics indistinguishable from those measured for the rectum (Irvine etal. 1988). These results suggest that both hindgut segments share the same mechanismof active Cr transport (Irvine et al. 1988). Moreover, the ultrastructural similarity of theapical membranes of both epithelia suggest a functional similarity (Irvine et al. 1988).Replacement of Cl - caused an increase in ileal Rb, consistent with the presenceof basolateral Cr channels. However, when ilea were stimulated with cAMP, althoughan increase in Vb after Cr removal was still observed, it was significantly smaller thanthat for nonstimulated ilea, thus cAMP does not result in an increase in the conductance89of the basolateral membrane to cr. In the case of the rectum, cAMP addition resultsin an 80% decrease in Rb. This decrease is abolished in Cr free saline and is due tocAMP stimulation of a basolateral Cr conductance (Hanrahan and Phillips 1984b).Chloride channel blockers 70B and 71B have been shown to completely abolishstimulated Isc in locust rectum when applied to the serosal side at concentrations of 10 4and 10' M (Phillips et al. 1986). These agents have not been tested for their effect uponilea. The effect of the Cl" channel blocker DPC upon the ilea was investigated byJ.Meredith (pers. comm.). She found that addition of 1.5 mM DPC to ilea stimulatedwith cAMP caused Vt to become more negative, as expected if Cl" exit from the cellwere prevented by Cr channel block at the basolateral membrane. However, DPC causeda significant depolarization of Vb and if DPC were blocking Cl" channels, Vb would beexpected to hyperpolarize. Furthermore, DPC caused similar increases in AVb whetherCr was present or absent in the bathing saline, suggesting that DPC was not affectinga basolateral Cr conductance (J. Meredith, pers. comm.). Blocking of basolateral cation(i.e. IC+) channels by DPC would explain both the depolarization of Vb and the increasein AVb in the absence of Cl".When ilea were stimulated with cAMP, Cr replacement resulted in adecline in Vt to values comparable to Vt as measured from nonstimulated ilea (P= >0.9,independent 't'-test, compared to control A). These results confirm those of Irvine et al.(1988) which showed that restoration of Cr to control levels fully restored the cAMPstimulated increase in Vt. Similar results were observed for cAMP stimulated Isc afterCl" replacement and subsequent restoration of this ion (Irvine et al. 1988). Thus, an90increase in active reabsorption of Cr is responsible for the cAMP stimulated increasein ileal Vt. These results are comparable to those from the rectum (Hanrahan andPhillips 1984b).Bilateral replacement of C1 in the bathing saline when ilea were nonstimulatedcaused Va to hyperpolarize. This is surprising as such a change indicates either anincreased net anion or decreased net cation entry into the cell across the apicalmembrane. In nonstimulated ilea, there is a large active secretion of fr, which is equalor even greater than C1 reabsorption (Thompson et al. 1991). The hyperpolarization ofVa may be due to an increase in ir secretion after Cl - removal possibly mediatedthrough a change in intracellular pH. When ilea are stimulated with cAMP there is nosignificant change in Va after C1 replacement. This can be explained if the highconductance of the apical membrane to IC" under these conditions (section III) providesperfect shunting of electrogenic C1 transport by passive IC' transport at the apicalmembrane.There is a significant increase in Ra for both nonstimulated and cAMP stimulatedilea when Cr is replaced, indicating an apical C1 conductance. However, the increasein Ra is much larger in nonstimulated ilea. There is a large increase in apical IC+conductance after cAMP stimulation which may mask the enhanced C1 pump activity.In both nonstimulated and cAMP stimulated ilea, C1 replacement results in ahyperpolarization of Vb. This is suprising as a reduction in serosal [Cr] would beexpected to increase the electrochemical gradient for C1 exit, thus depolarizing Vb. Thisdepolarization would be transitory as intracellular Cr levels are depleted with a91subsequent hyperpolarization of Vb as passive Cr exit from the cell ceases, revealinga larger portion of the basolateral IC÷ diffusion potential. The timecourse of the changein Vb after Cl - replacement shows no such transient depolarization (data not shown).These results may reflect the abolishment of the outward Cr current due to channelinhibition by low extracellular [Cll. The functional significance of such modulationwould be in cell volume regulation: Ilea bathed in Cl" free saline and then stimulatedwith cAMP never reached steady state and appeared to have lost cell volume, being softand nondeformable upon microelectrode impalement (pers.obs). Attempts to measure Vaand Vb were unsuccessful because very low values for these parameters madeintracellular recordings too close to baseline to be resolved reliably. In contrast ileabathed bilaterally for long periods in Cr free saline and not stimulated with cAMPmaintained a steady state and displayed high values for Va and Vb. Measurements ofintracellular [a] would be required in order to develop this hypothesis, so that theelectrochemical gradient across the apical and basolateral membranes could bedetermined during brief exposure of ilea bilaterally to Cr free saline in nonstimulatedand cAMP stimulated conditions. When recta are stimulated with cAMP and Cl" isreplaced in the bathing saline, Vb hyperpolarized to a value similar to that recordedfrom nonstimulated recta (Hanrahan and Phillips 1984b).In the ilea, after cAMP stimulation, Vb depolarizes by 27 mV (section I). If thischange in Vb were due solely to an increase in basolateral Cl" conductance and ifintracellular Cr activity (acc,) is assumed to be in the same range as for the rectum (30.7mM; Hanrahan and Phillips 1984b), then the change in Vb/decade [Cl] a would have to92increase by about 35 mV, with Vb depolarizing with decreasing [Cll.. In the case of therectum, acc, increases after cAMP stimulation to 46.6 mM (Hanrahan and Phillips1984b). This would demand a larger required increase in Vb/decade [Cll.. Since in theilea cAMP stimulation causes only a 2.3 mV increase in Vb/decade [Clio, thestimulation of basolateral Cl - conductance which occurs in the rectum (Hanrahan andPhillips 1984b) cannot be postulated for the ileal epithelium.The ileal basolateral membrane thus has a very high conductance, which consistsof both a r - and Cr conductance. Vb is due mostly to a IC diffusion potential andserosal r- replacement (from 100 mM) causes a 87 S2cm 2 increase in Vb, compared toa 6.83 f2cm2 increase when Cr is replaced (from 110 mM). Thus the basolateralmembrane has a higher conductance to r than to C1. After cAMP stimulation, Vb doesnot change. Given the high conductance of this membrane in nonstimulated ilea, it maybe possible to fully account for the exit of r and Cl- down their respectiveelectrochemical gradients without an increase in basolateral conductance. However,without measurements of intracellular Cl" activity, which would enable calculation of theCl- electrochemical potential across the basolateral membrane, this issue remainsunresolved. Thus the possibility of a nonconductive basolateral Cr exit mechanismcannot be ruled out. Such a mechanism could involve C•/HCO3 exchange or K+/Cl - co-transport. Such basolateral K+/Cl - co-transport has been postulated for epithelia wherethe basolateral conductance to Cl- is too low to account for Cl - exit (Weinstein 1992,Reuss et al. 1980, Corcia and Armstrong 1983). Ileal Cr dependent, cAMP stimulated93Isc is reduced 80% by bilateral IC - replacement (Irvine et al. 1988), although in therectum a similar effect is due to activation of the apical Cr pump by luminal IC+(Hanrahan and Phillips 1984b).In conclusion, there is evidence for a basolateral Cr conductance in locust ileumbut not for cAMP stimulation of this conductance.94CHAPTER 5 GENERAL DISCUSSION.This thesis has identified both similarities and differences between the iontransport processes of the rectum and ileum and their control by cAMP. These aresummarized below:Both epithelia display;i) a cAMP stimulated increase in apical conductance to K.ii) a small apical Na+ conductance. Amiloride has no effect on apical resistance.iii)^basolateral IC and Cl" conductances.Differences between the two epithelia are;i) There is no evidence for cAMP stimulation of basolateral Cl" conductance in theileum and basolateral Cl" conductance is smaller than that for K.ii) Apical IC+ conductance in cAMP stimulated ilea is not inhibited by high luminal[K I.iii)^There is a small increase in the conductance of the apical membrane to Na + inthe ileum after cAMP stimulation.Fig 5.1 is an updated version of Fig 1.6 (chapter 1), incorporating theconclusions of this thesis and information from the study by Peach (1991). In brief,scgITP acts through the cAMP second messenger system to stimulate an apical95=CILUMEN ^  HAEMOCOEL04.<--f-Kt  < scgITP ICl--CEEB-111C1"Na+Na+K tiaPase... 1 R^ ) ) ? INH4CELLLe"--7 ^R  <scgITP Fig 5.1: Updated model of ileal ion transport mechanisms and their controlby scgITP and the second messenger cAMP. Filled circles represent activetransport and the open circle is a carrier. Cylinders represent ion channels.R is the receptor.96electrogenic Cl - pump and cause an increase in apical IC+ conductance (i.e. open ionchannels). Na+ reabsorption is also stimulated, with cAMP causing an increase in apicalNaf conductance. Cyclic AMP also stimulates NR 4+ secretion but has no effect on 11+secretion which is inhibited by scgITP presumably through another second messengersystem.As a model to study insect epithelial transport, the ileum represents perhaps abetter candidate than the rectum for the following reasons: The ileum displays a cAMPstimulated increase in apical electrogenic C1 transport and IC+ conductance which is verysimilar to the rectum but the ileum consists of only one cell layer of one cell type. Healtransport is stimulated by scgITP, which has been purified and partially sequenced,whereas CTSH has only been partially purified and is unstable during HPLC. Finally,in the ileum, there is no recycling of ions in the intercellular spaces, and the lateralmembranes of ileal cells are simple. There are, however, two major drawbacks of theileal epithelium. Firstly, 6,Vb is very low and does not enable accurate measurement ofa. This could be improved upon somewhat by digital recording of data, eliminating thesmall errors derived from differences in chart recorder pen width and measurement fromtraces. Secondly, the cuticle is firmly attached to the apical membrane, preventing accessto the mucosal surface of the epithelium for microelectrode impalement or patch clampstudies.The main limitation of this thesis is that it does not encompass measurements ofintracellular ion activity under the various experimental conditions, leaving ambiguitiesas to the direction and magnitude of the driving forces for passive movements of ions97across the epithelial membranes. The pure peptide scgITP and CC homogeanates mustalso be assessed for their effects upon ion transport and these compared to cAMPstimulation. Audsley (1990) showed that scgITP and CC result in an inhibition of ilealW. secretion which does not involve the cAMP second messenger system.98REFERENCES.ANDRUSIAK, E.W., J.E.PHILLIPS & J.SPEIGHT: (1980). Phosphate transport bylocust rectum in vitro.Can. J. Zool 58: 1518-1523.AUDSLEY, N. (1990). Purification of a neuropeptide from the corpus cardiacum of thedesert locust which influences ileal transport.Ph.D. thesis, University of British Columbia, Vancouver.AUDSLEY, N. & J.E.PHILLIPS: (1990). Stimulants of ileal salt transport in theneuroendocrine system of the desert locust.Gen. Comp. Endocr. 80: 127-137.AUDSLEY, N., C.McINTOSH & J.E.PHILLIPS: (1992a). Isolation of a neuropeptidefrom locust corpus cardiacum which influences ileal transport.J. Exp. Biol. 173: 261-274.AUDSLEY, N., C.McINTOSH & J.E.PHILLIPS: (1992b). 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