An antidiuretic peptide (Tenmo-ADFb) with kinin-like diuretic activity on Malpighian tubules of the house cricket, Acheta domesticus (L.

Transcription

1 3979 The Journal of Experimental iology 21, Pulished y The Company of iologists 7 doi:1.1242/je.656 n antidiureti peptide (Tenmo-DF) with kinin-like diureti ativity on Malpighian tuules of the house riket, heta domestius (L.) Geoffrey M. Coast 1, *, Ronald J. Nahman 2 and David. Shooley 3 1 Shool of iologial and Chemial Sienes, irkek, University of London, Malet Street, London WC1E 7HX, UK, 2 US Department of griulture, PMRU/SPRC, College Station, TX 77845, US and 3 iohemistry, University of Nevada, Reno, NV 89557, US *uthor for orrespondene ( g.oast@k.a.uk) epted 3 Septemer 7 heta domestius is reported to have an antidiureti hormone that redues Malpighian tuule seretion. Identified peptides known to work in this way (Tenmo- DFa and DF, and Manse-CP 2 ) were tested as andidates for the unidentified hormone, along with their seond messenger, yli GMP. Only Tenmo-DF was ative, ut was diureti, as was 8-romo yli GMP. The ativity of Tenmo-DF is omparale to that of the riket kinin neuropeptide, hdo-kii, ut it is muh less potent. Its ativity was unaffeted y deleting either the six N-terminal residues or the C-terminal phenylalanine. t high onentrations, tuule seretion is douled y Tenmo-DF and hdo-kii, ut their ations are nonadditive, suggesting they have a similar mode of ation. oth stimulate a non-seletive KCl and NaCl diuresis, whih is onsistent with the opening of a transepithelial Cl ondutane. In support of this, the diureti response to Tenmo-DF and hdo-kii is prevented y a ten-fold Summary redution in athing fluid hloride onentration, and oth peptides ause the lumen-positive transepithelial voltage to ollapse. The Cl ondutane pathway appears likely to e transellular, eause the Cl hannel loker DPC redues oth asal and peptide-stimulated rates of seretion. The effets of 8-romo yli GMP on transepithelial voltage and omposition of the sereted fluid are markedly different from those of Tenmo-DF. This is the first report of the antidiureti fator Tenmo- DF stimulating tuule seretion. lthough the ations of Tenmo-DF are indistinguishale from those of hdo- KII, it is unlikely to at at a kinin reeptor, eause the ore sequene (residues 7 12) laks the Phe and Trp residues that are ritial for kinin ativity. Key words: Malpighian tuule, fluid seretion, ion transport, eletrophysiology, diureti hormone, antidiureti fator, kinin neuropeptide. Introdution The exretory proess of insets is ontrolled y oth diureti and antidiureti hormones (Coast et al., 2a; Shooley et al., 5). In general, diureti hormones at on Malpighian tuules to inrease seretion of primary urine, whereas antidiureti hormones stimulate fluid reasorption in the hindgut (ileum and retum). There are, however, exeptions to this, with early reports of an antidiureti fator ating on Malpighian tuules to redue seretion of primary urine (Spring et al., 1988) and of a diureti fator that appears to derease fluid uptake from the hindgut (Wheelok et al., 1988). The first identified peptide shown to redue primary urine prodution was Manse-CP 2, whih is a ardioaeleratory peptide (CP) from the toao hornworm, Mandua sexta (Huesmann et al., 1995). Manse-CP 2 ats via yli GMP to redue seretion y serotonin-stimulated Malpighian tuules of the lood-suking ug Rhodnius prolixus (Quinlan et al., 1997). Surprisingly, the same peptide ats via yli GMP and Ca 2+ to stimulate primary urine prodution y Malpighian tuules of the fruit fly, Drosophila melanogaster (Dow and Davies, 3; Terhzaz et al., 6). Susequently, two other antidiureti peptides (Tenmo-DFa and Tenmo-DF) that at on Malpighian tuules have een identified from a pupal head extrat of the mealworm eetle, Tenerio molitor, ased upon their aility to inrease yli GMP prodution (Eigenheer et al., 2; Eigenheer et al., 3). oth peptides redue seretion y the free (proximal) portion of Malpighian tuules from lastinstar mealworm larvae (Eigenheer et al., 2; Eigenheer et al., 3), an effet that is mimiked y exogenous yli GMP. Tenmo-DFa is extraordinarily potent in the fluid seretion assay, with an EC 5 of 1 fmol l 1 ompared with 24 pmol l 1 for Tenmo-DF. Manse-CP 2 also stimulates yli GMP prodution y T. molitor tuules and redues fluid seretion, ut it is onsideraly less potent than either of the native peptides, with an EC 5 of 85 nmol l 1 (Wiehart et al., 2). The antidiureti ativity of Manse-CP 2 and the Tenmo- DFs appears to result from the yli GMP-dependent ativation of a yli MP-speifi phosphodiesterase, whih will lower intraellular levels of yli MP, a seond messenger that stimulates diuresis (Quinlan and O Donnell, 1998; Wiehart et al., 2). Thus, Manse-CP 2 and Tenmo- DFa antagonise the ations of diureti hormones that use

2 398 G. M. Coast, R. J. Nahman and D.. Shooley yli MP as a seond messenger, namely serotonin in R. prolixus and a ortiotropin-releasing fator (CRF)-related peptide (Tenmo-DH 37 ) in T. molitor (Quinlan and O Donnell, 1998; Quinlan et al., 1997; Wiehart et al., 2). Tenmo-DFa has also een shown to at via yli GMP in inhiiting fluid seretion and ion (Na +, K + and Cl ) transport y Malpighian tuules of the yellow fever mosquito, edes aegypti (Massaro et al., 4), possily y reduing Na + /K + /2Cl otransport aross the asal memrane, whih is known to e ativated y yli MP (Hegarty et al., 1991). The first report of an antidiureti hormone ating on Malpighian tuules ame from the oservation that haemolymph from dehydrated house rikets (heta domestius) redued primary urine prodution, whereas haemolymph from rehydrated insets had the opposite effet (Spring et al., 1988). dditionally, neuroseretory material was lost from orpora ardiaa of dehydrated rikets, whih is onsistent with the release of an antidiureti hormone. fator that redued Malpighian tuule seretion was partially purified from a methanoli extrat of the orpora ardiaa ut was not further haraterised (Spring et al., 1988). We have therefore tested those peptides that have een shown to redue primary urine prodution (Manse-CP 2, Tenmo-DFa and Tenmo- DF) for effets on riket Malpighian tuules. Of the peptides tested, only Tenmo-DF was ative, ut it had diureti rather than antidiureti ativity. Here, we desrie the ations of Tenmo-DF on riket tuules and present results from an initial struture/ativity study using N-terminal and C- terminal deleted analogues. We show that the ations of Tenmo- DF resemle those of the diureti/myotropi. domestius kinins (hdo-ks), although it most likely ats at a different reeptor. Materials and methods Insets Crikets were reared aording to methods desried previously (Clifford et al., 1977) and were maintained at 28 C under a 12 h:12 h light:dark regime. They were fed a diet of wheat germ and ground at food, with water provided ad liitum. The donor insets used in the urrent study were adult females aged etween 7 and 14 days. Fluid seretion assay The Ramsay assay used to measure fluid seretion y riket Malpighian tuules has een desried in detail elsewhere (Coast, 1988). riefly, single tuules are transferred to small (5 l) drops of saline eneath water-saturated paraffin oil. The saline used differed from that employed in earlier studies in that the K + onentration was inreased at the expense of Na + from 8.6 mmol l 1 to 25.5 mmol l 1, whih supports a higher rate of seretion y kinin-stimulated tuules (G.M.C., unpulished oservation). The omposition of the saline was as follows (in mmol l 1 ): NaCl, ; KCl, 8.6; CaCl 2, 2; MgCl 2, 8.5; NaHCO 3, 2.1; KHCO 3, 1.9; KH 2 PO 4, 4; KOH, 11; proline, 1; gluose, 24; Hepes (N-2-hydroxyethylpiperazine-N 2- ethanesulphoni aid), 25; ph adjusted to 7.2 with 1 mol l 1 NaOH. fter a 4 min equiliration period, the athing fluid was hanged and sereted droplets removed with a fine glass rod. Thereafter, sereted droplets were removed at min intervals efore and after hallenging the tuules with test ompounds dissolved in fresh saline. Droplets of sereted fluid were allowed to sink onto the non-wettale ase of the Petri dish and their diameter (d) measured using an oular mirometer. Droplet volume (piolitres; pl) was alulated as ( d 3 )/6, and the rate of seretion (pl mm 1 min 1 ) otained y dividing the volume y the olletion period (min) and y the length of tuule (mm) within the drop of athing fluid. Unless otherwise stated, results are presented either as the rate of seretion or as diureti ativity ( pl mm 1 min 1 ), whih is defined as the differene etween rates of seretion measured efore and after hallenging tuules with seretagogues. Sereted fluid analysis Sereted fluid droplets were olleted under water-saturated paraffin oil using the Ramsay assay. Samples olleted over 3 min intervals efore and after adding test ompounds to the athing fluid were transferred y miropipette to a seond Sylgard-lined Petri dish ontaining water-saturated paraffin oil for analysis using ion-seletive miroeletrodes (Coast et al., 1; Ianowski and O Donnell, 4). Separate experiments were performed for the measurement of sereted fluid ph and Cl, and for Na + and K +, whih were analysed in the same samples. Measurements of ph and Cl were made within 5 min of droplet olletion, and Na + and K + within 2 min. The ph eletrodes were prepared using hydrogen ion exhange resin (IE 1; World Preision Instruments, Sarasota, FL, US) and were akfilled with.5 mol l 1 itri aid ontaining 1 mmol l 1 NaCl adjusted to ph 6.. The referene eletrode was filled with 3 mol l 1 KCl. The K + eletrodes were ased on K + ionophore I, oktail (Fluka, uhs, Switzerland), and akfilled with 5 mmol l 1 KCl. Sodium eletrodes were ased on Na + ionophore II, oktail (Fluka), and akfilled with 5 mmol l 1 NaCl. For oth K + and Na + measurements, the referene eletrode was filled with 1 mol l 1 LiCl. Chloride-sensitive eletrodes were ased on the Corning Cl exhanger (IE-173; World Preision Instruments) and akfilled with.5 mol l 1 KCl. The tip and shank of the referene eletrode was filled with 3 mol l 1 sodium aetate, and 3 mol l 1 KCl was used to fill the shaft (Wright and O Donnell, 1992). The eletrodes were onneted via g/gcl half-ells to a high impedane eletrometer (F-223; World Preision Instruments), whih was onneted in turn to a data aquisition system (Dataan V; Sale Systems, Henderson, NV, US). Caliration solutions for ph miroeletrodes were prepared y titration of a standard referene uffer (ph 7.; Thermo Russell, Fife, Sotland, UK) with 1 mol l 1 HCl or 1 mol l 1 NaOH to give solutions enompassing the range ph 6 to ph 8. Potassium eletrodes were alirated in mixed solutions of mmol l 1 KCl and mmol l 1 NaCl, whereas Na + eletrodes were alirated in mixed solutions of mmol l 1 NaCl and mmol l 1 LiCl. Potassium is known to interfere with Na + measurements and this was orreted for as previously desried (Ianowski and O Donnell, 4). Chloride eletrodes were alirated in 2 mmol l 1 KCl. Referene and ionseletive eletrodes were positioned in sereted fluid droplets or aliration solutions eneath water-saturated paraffin oil, and the potential reorded one it had stailised (after aout 3 s). Eletrodes were deemed aeptale if the aliration urve was

3 Kinin-like ations of Tenmo-DF 3981 linear with a slope per deade hange in ion onentration of 52 mv (Na + ), 54 mv (K + ), 54 mv (Cl ) or 56 mv (H + ). Measurement of transepithelial and asolateral memrane voltages Isolated Malpighian tuules were anhored at eah end within slits ut into a small lok of Sylgard mounted in a ustom-uilt hamer. Writhing movements of the tuule were restrited y putting it under slight tension. This allowed stale reordings of oth transepithelial and intraellular voltages from the midportion of the tuule. The hamer (~25 l volume) was perfused at 1 ml min 1 with the same saline that was used in the diureti assay. Perfusion was stopped prior to the addition of test ompounds and then restarted to wash-off. Miroeletrodes (2 4 M resistane when filled with 3 mol l 1 KCl) were drawn from 1 mm o.d. filament glass tuing (GCF-75; Clark Eletromedial Instruments, Pangourne, UK) using a vertial pipette puller (PUL-; World Preision Instruments). fter akfilling with 3 mol l 1 KCl, they were onneted to a highimpedane eletrometer (M-77; World Preision Instruments) via an g/gcl half-ell, the iruit eing ompleted through a KCl referene eletrode (DRIREF-45; World Preision Instruments) plaed in the perfusion hamer. asal memrane (V ) and transepithelial voltages (V t ) were measured in the main tuule segment lose to where it was anhored into the Sylgard. Miroeletrodes were advaned at an olique angle using an hydrauli miromanipulator (MMO-23; Narishigi, Tokyo, Japan) until a sudden jump in potential indiated the asal memrane of a prinipal ell had een impaled. Reordings of V were aepted if the potential remained stale (±2 mv) for >3 s and returned to ±2 mv after withdrawal of the eletrode. Similar riteria were adopted when reording V t after the miroeletrode had een advaned through the apial memrane into the tuule lumen. Results were reorded digitally using a data aquisition system (Dataan V; Sale Systems). Reordings of V t were paused during insertion of the miroeletrode into the lumen, whih was readily seen as a positive jump in potential. Measurement of myotropi ativity Inset kinins have een shown to stimulate the ontratile ativity of the hindgut in okroahes [Leuophaea maderae (Holman et al., 1986)], houseflies [Musa domestia (Coast et al., 2),. aegypti (Veenstra et al., 1997) and R. prolixus (Te rugge and Orhard, 2)], whih may assist the exretory proess. Tenmo-DF and hdo-kii were therefore tested for myotropi ativity on riket hindgut. Insets were killed y deapitation and the adomen opened along its entire length with a mid-ventral inision. The hindgut was disseted free of traheae and Malpighian tuules and severed just anterior to the juntion of the ileum with the olon. fine thread was tied around the short portion of ileum remaining, and the terminal adominal segment with the hindgut attahed was then ut free of the remainder of the ody. The isolated hindgut (olon and retum) was transferred to a shallow hamer (volume ~1 ml) fashioned from Sylgard and seured in plae with a fine minutin pin through the utile of the terminal segment. The thread attahed to the anterior hindgut was seured to a 1 g fore transduer (WPI FORT1) oupled to a Sale Systems CP32 preamplifier. The output was reorded on a strip hart reorder or digitally using Dataan V (Sale Systems). The hamer ontaining the isolated hindgut was perfused ontinuously at 1 ml min 1 with okroah hindgut saline (Cook and Holman, 1978), whih, in ontrast to the riket saline used for the diureti assay, supported regular spontaneous ontratile ativity. Peptides dissolved in 1 ml saline were added to the preparation y swithing the perfusate etween normal saline and the test solution. The peptide was immediately washed off y perfusing with normal saline after the delivery of 1 ml of the test solution. Peptide synthesis The synthesis of Tenmo-DFa, Tenmo-DF and Manse- CP 2 has een desried elsewhere (Eigenheer et al., 2; Eigenheer et al., 3; Nahman and Coast, 7). The Tenmo- DF analogs were synthesised via Fmo methodology on Rink mide resin (Novaiohem, San Diego, C, US) using Fmoproteted amino aids (dvaned Chemteh, Louisville, KY, US) on an I 433 peptide synthesiser (pplied iosystems, Foster City, C, US) under previously desried onditions (Nahman et al., 1997). Crude produts were purified on a Waters C 18 Sep-Pak artridge and a Delta-Pak C 18 reversephase olumn (8 mm, 15 mm partile size, Å pore size) on a Waters 51 HPLC ontrolled with a Millennium 21 hromatography manager system (Waters, Milford, M, US) with detetion at 214 nm and run at amient temperature. Solvent was.1% aqueous trifluoroaeti aid (TF), and Solvent was 8% aqueous aetonitrile ontaining.1% TF. The initial solvent onsisted of 2% and was followed y the Waters linear program to % over 4 min with a flow rate of 2. ml min 1. The Delta-Pak C 18 retention times were: DF[2 13], 15. min; DF[7 13], 12.5 min; DF[8 13], 9. min; DF[1 12], 8.5 min. Most of the peptides were further purified on a Waters Protein Pak I125 olumn (7.8 3 mm) (Milligen Corp., Milford, M, US). Peptides were eluted under isorati onditions, with the solvent onsisting of 8% aetonitrile ontaining.1% TF and with a flow rate of 2. ml min 1. Retention times on the Waters Protein Pak olumn were: DF[2 13], 1.5 min; DF[7 13], 8. min; DF[1 12], 1. min. mino aid analysis was arried out under previously reported onditions (Nahman et al., 1997) and was used to quantify the peptide and to onfirm its identity. It resulted in the following analyses: DF[2 13]: sp[1.], Phe[1.], Gly[2.3], His[1.2], Ile[.8], Lys[.9], Tyr[2.]; DF[7 13]: Phe[1.], Gly[.9], His[1.], Ile[.9], Lys[.9], Pro[.9], Tyr[1.3]; DF[8 13]: Phe[1.], Gly[1.1], His[.7], Ile[.7], Pro[1.1], Tyr[.9]; DF[1 12]: sp[1.6], Gly[1.9], His[1.], Ile[.9], Lys[1.], Ser[1.], Tyr[2.]. The identities of the peptide analogues were onfirmed via MLDI-TOF-MS on a Kratos Kompat Proe MLDI- TOF MS mahine (Kratos nalytial, Ltd, Manhester, UK) with the presene of the following moleular ions (M+H + ): DF[2 13], [M+H + ]; DF[7 13], 86.9 [M+H + ]; DF[8 13], [M+H + ]; DF[1 12], [M+H + ]. Chemials The hloride hannel lokers diphenylamine-2-aroxylate (DPC) and 5-nitro-2-(3-phenylpropylamino)-enzoi aid

4 3982 G. M. Coast, R. J. Nahman and D.. Shooley (NPP) were otained from Caliohem (Merk iosienes Ltd, eeston, UK) and were prepared as stok solutions in ethanol and dimethyl sulfoxide, respetively. ll other hemials were otained from Sigma-ldrih (Poole, Dorset, UK). Calulations Net eletrohemial gradients ( /F, in mv) aross the epithelium for K +, Na +, H + and Cl were alulated as desried previously (O Donnell et al., 1996) using the equation: /F = (RT/F) ln (a L /a S ) + zv t = 59log (a L /a S ) + zv t, where z is the valeny, a L is the ativity in the lumen (mmol l 1 ), a S is the ativity in the athing fluid, R is the universal gas onstant, T is the asolute temperature (K) and F is the Faraday onstant. In pratie, ion onentrations rather than ativities were used, on the assumption that ativity oeffiients in the tuule fluid were likely to e similar to those in the athing fluid. When /F is zero, the ion is in equilirium aross the epithelium, whereas a positive value of /F means that the luminal ion onentration exeeds the equilirium value, i.e. ative transport. negative value for /F indiates that the luminal ion onentration is elow equilirium and net passive diffusion from ath to lumen is favoured. Data are presented as means ± s.e.m. for the numer of determinations indiated (N). Tests for signifiane were performed with GraphPad Instat 3.6 (GraphPad Software, San Diego, C, US) using paired and unpaired Student t-tests as appropriate. Differenes were onsidered signifiant when P<.5. Dose response urves with variale slope were fitted using Prism TM v. 4.2 (GraphPad Software). Results Effet of putative antidiureti peptides on fluid seretion In a preliminary sreen, Manse-CP 2, Tenmo-DFa and Tenmo-DF were eah tested at 1 mol l 1 for effets on fluid seretion y riket tuules. The memrane-permeant yli GMP analogue, 8-romo yli GMP (1 mmol l 1 ), was also sreened for ativity sine the yli nuleotide ats as a seond messenger for all three peptides. The riket diureti kinin neuropeptide, hdo-kii, was inluded in the assay as a positive ontrol. The results presented in Fig. 1 show that, of the three putative antidiureti peptides, only Tenmo-DF was ative, ut it stimulated rather than inhiited tuule seretion. Indeed, the diureti ativity (defined as the inrease in rate of seretion over that measured in the ontrol period; pl mm 1 min 1 ) of Tenmo-DF ( 191±32 pl mm 1 min 1 ; N=9) was not signifiantly different (P=.167; unpaired t-test) from that of hdo-kii ( 25±26 pl mm 1 min 1 ; N=13). Exogenous 8-romo yli GMP also produed a small, ut signifiant (P.1; paired t-test), inrease in fluid seretion ( 6±9 pl mm 1 min 1 ; N=1). dose response urve for the diureti ativity of Tenmo-DF is shown in Fig. 2. Data are expressed as perentages of the response to a supramaximal dose (1 nmol l 1 ) of hdo-kii Response as % maximum Diureti ativity (pl mm 1 min 1 ) (13) (13) (9) Control DFa DF CP 2 GMP (13) KII assayed alongside Tenmo-DF on tuules removed from the same inset. The apparent EC 5 is 1.5 mol l 1, with 95% onfidene limits of mol l 1, and the est fit value for the top of the urve is 91±8% of the response to hdo-kii. dose response urve for hdo-kii in the same high K + saline is shown in Fig. 2. The apparent EC 5 is 1.2 pmol l 1, with 95% onfidene limits of pmol l 1. This ompares with an EC 5 of 22 pmol l 1 for hdo-kii when assayed in riket saline ontaining 9.6 mmol l 1 K + (Coast et al., 199). Struture ativity studies Several analogues of Tenmo-DF were tested for diureti ativity and the results are summarised in Tale 1. The deletion (8) (1) Fig. 1. Peptides known to redue primary urine prodution [Tenmo- DFa (DFa), Tenmo-DF (DF) and Manse-CP 2 (CP 2 )] were tested at 1 mol l 1 for an effet on riket tuules, along with their seond messenger yli GMP [1 mmol l 1 8-romo yli GMP (GMP)]. The riket kinin hdo-kii (1 nmol l 1 ; KII) was inluded in the assay as a positive ontrol. ars represent mean values for the hange in rate of seretion ( pl mm 1 min 1 ) following addition of test ompounds, and vertial lines represent + 1 s.e.m. for the numer of repliates indiated in parentheses. Tenmo-DF and 8-romo yli GMP signifiantly inreased (P<.1) fluid seretion, and the response to the former was omparale to that of hdo-kii log Conentration (μmol l 1 ) log Conentration (pmol l 1 ) Fig. 2. Dose response urves for the diureti ativity of () Tenmo-DF and () hdo-kii. Results are expressed as a perentage of the response to a supramaximal onentration (1 nmol l 1 ) of hdo-kii. Data points are the means ± 1 s.e.m. of 7 1 repliates. Note the vast differene in poteny etween Tenmo-DF (EC mol l 1 ) and hdo-kii (EC pmol l 1 ).

5 Kinin-like ations of Tenmo-DF 3983 Tale 1. The diureti ativity and poteny of N- and C-terminal deletion analogues of Tenmo-DF EC 5 95% CL Response Sequene nalogue ( mol l 1 ) ( mol l 1 ) maximum (%) YDDGSYKPHIYGF-OH DF ±9 DDGSYKPHIYGF-OH DF[2 13] ±8 KPHIYGF-OH DF[7 13] ±8 PHIYGF-OH DF[8 13] Not ative Not ative YDDGSYKPHIYG OH DF[1 12] ±11 of six residues from the N-terminus had no effet on ativity. Indeed, Tenmo-DF[7 13] was more potent (P<.5) than the parent ompound. However, with the removal of one further amino aid, there was a omplete loss of ativity, and Tenmo- DF[8 13] had no effet on tuule seretion at 1 mol l 1, whih was the highest onentration tested. One C-terminal trunated peptide (Tenmo-DF[1 12]) was also tested and shown to retain diureti ativity with a poteny omparale to that of the parent ompound. Comparing the ativities of Tenmo-DF and hdo-kii lthough hdo-kii is onsideraly more potent than Tenmo-DF, their diureti ativity appears similar. This is further illustrated in Fig. 3, whih shows the time ourse of the response to the two peptides when tested at supramaximal onentrations (1 mol l 1 and 1 nmol l 1, respetively) first separately and then together. The data are normalised y expressing as perentages of the unstimulated rate of seretion measured at 45 min. Fluid seretion inreases y ~75% within 15 min of peptide addition, and the effets of Tenmo-DF and hdo-kii are not additive. Effet of Tenmo-DF, hdo-kii and 8-romo yli GMP on tuule fluid omposition Fig. 4 ompares the ph and the Na +, K + and Cl onentrations of tuule fluid olleted over 3 min intervals efore and after the addition of 1 mol l 1 Tenmo-DF, 1 nmol l 1 hdo-kii and 1 mmol l 1 8-romo yli GMP. Criket tuules serete K + -rih urine, and the [Na + ]:[K + ] ratio efore and after the addition of Tenmo-DF (.22±.4 and.18±.3; N=14; P=.218, paired t-test) and hdo-kii (.23±.2 and.22±.2; N=15; P=.417, paired t-test) did not hange signifiantly, although oth peptides produed small, ut signifiant, inreases in K + onentration (Fig. 4). In marked ontrast, the addition of 8-romo yli GMP aused the urine [Na + ]:[K + ] ratio to virtually doule from.23±.6 to.47±.9 (N=9; P<.1), refleting a signifiant inrease in the onentration of Na + and a orresponding derease in K + (Fig. 4). ll three seretagogues aused a small inrease in Cl onentration (Fig. 4), ut the effet of 8-romo yli GMP was not quite signifiant (P=.54). The sereted fluid was slightly more aidi after the addition of Tenmo-DF (.5±.2 ph units; N=18; P<.5, paired t-test) and hdo- KII (.8±.2 ph units; N=16; P<.1, paired t-test), whereas it was signifiantly more alkaline in tuules hallenged with 8-romo yli GMP ( +.18±.3 ph units; N=1; P<.1, paired t-test). Diureti ativity is hloride dependent Kinins stimulate tuule seretion y opening a Cl ondutane pathway, whih inreases net transport of KCl and NaCl into the lumen along with osmotially oliged water (eyenah, 3). To determine the Cl dependeny of the responses to Tenmo-DF and hdo-kii, tuules were isolated in standard saline (ontrols) and in low Cl saline (one-tenth the normal onentration, with gluonate salts replaing hloride). fter a 4 min equiliration period, fluid seretion was measured over 4 min periods efore and after the addition of either 1 mol l 1 Tenmo-DF or 1 nmol l 1 hdo-kii in standard and in low Cl saline. Finally, all tuules were transferred to standard saline ontaining the test peptides, and seretion measured over a third 4 min period. The results are presented in Fig. 5. The rate of seretion y tuules held in low Cl saline is ~25% that of tuules in normal saline and they do not respond to the addition of either Tenmo-DF or hdo-kii. When these tuules are transferred to normal saline, fluid seretion returns to levels omparale with those of the peptidestimulated ontrols. % Rate of seretion at 45 min Time (min) Fig. 3. Time ourse for the stimulation of fluid seretion y supramaximal onentrations of Tenmo-DF (1 mol l 1 ; solid symols and line) and hdo-kii (1 nmol l 1 ; open symols and dotted line). Results are normalised y eing expressed as a perentage of the unstimulated rate of seretion at 45 min. Data points are the means ± 1 s.e.m. of repliates. Fluid seretion inreases rapidly following addition of the individual peptides (downward arrow) and is not further elevated when they are applied together (upward arrow).

6 3984 G. M. Coast, R. J. Nahman and D.. Shooley Urine onentration (mmol l 1 ) Urine onentration (mmol l 1 ) Cl (17) (18) (9) ** K + (14) (15) (9) * DF KII GMP DF KII GMP Effet of hloride hannel lokers on fluid seretion Fluid seretion y unstimulated and kinin-stimulated Malpighian tuules of D. melanogaster is inhiited y DPC, whih loks hloride hannels in verterate epithelial ells (O Donnell et al., 1998). This suggests the involvement of hloride hannels in transepithelial Cl seretion, whih likely follows a transellular route. preliminary experiment showed that at high onentrations (2 mmol l 1 ), DPC almost ompletely inhiited seretion y unstimulated riket tuules, whereas.2 mmol l 1 DPC redued seretion y ~5%, and this onentration was seleted for testing the effet of the hannel ph (14) ph (1) (16) (1) ** Na + (15) (9) DF KII GMP ** ** DF KII GMP Fig. 4. Sereted fluid ph and onentrations of K +, Na + and Cl efore (open ars) and after (solid ars) the addition of 1 mol l 1 Tenmo-DF (DF), 1 nmol l 1 hdo-kii (KII) and 1 mmol l 1 8-romo yli GMP (GMP). ars represent the means + 1 s.e.m. for the numer of repliates shown in parentheses. sterisks indiate where the hange in omposition following addition of the seretagogue is signifiant in a paired t-test; *P<.5; **P<.1; P<.1. loker on the ativity of 1 mol l 1 Tenmo-DF and 1 nmol l 1 hdo-kii. Compared with ontrols (saline ontaining.1% ethanol), seretion y unstimulated tuules was redued in the presene of.2 mmol l 1 DPC ut inreased signifiantly (P<.1; paired t-test) in the presene of either Tenmo-DF or hdo-kii (Fig. 6). Rates of seretion y peptide-stimulated tuules were, however, redued signifiantly y the hloride hannel loker. We also tested another hloride hannel loker, NPP, for its effet on the response to 1 mol l 1 Tenmo-DF. t the onentration used (1 mol l 1 ), NPP had no effet on asal seretion, ut signifiantly (P<.1, unpaired t-test) redued seretion y peptidestimulated tuules from 53± 4 pl mm 1 min 1 (N=8) in ontrol saline ontaining.1% DMSO to 345±44 pl mm 1 min 1 (N=1) in the presene of the hannel loker. Tenmo-DF ats synergistially with 8-romo-yli MP Synergism etween kinins and yli MP has een desried in several insets, notaly. domestius (Coast et al., 199), Lousta migratoria (Coast, 1995) and M. domestia (Holman et al., 1999), and an e attriuted to the separate ativation of anion and ation transport proesses, respetively. To investigate possile synergism etween Tenmo- DF and 8-romo-yli MP, tuules were hallenged with eah of the seretagogues separately and together. The yli nuleotide was used at 1 mol l 1 and Tenmo-DF was tested at.3 mol l 1. The data presented in Fig. 7 show the hange in fluid seretion ( pl mm 1 min 1 ) in the 35 min period following addition of the seretagogues. The sum of the separate effets of yli MP and Tenmo-DF is signifiantly less than the response otained when they are tested together (P<.1; unpaired t- test), whih provides evidene of synergism. Synergism ould not e demonstrated etween 8-romo yli GMP and either Tenmo-DF or hdo-kii (data not shown). However, 1 mmol l 1 8-romo yli GMP signifiantly inreased seretion y tuules that were already maximally stimulated with 1 mol l 1 Tenmo-DF (N=11; P<.1, paired t- test) and 1 nmol l 1 hdo-kii (N=11; P<.1, paired t-test) (Fig. 7). Fluid seretion (pl mm 1 min 1 ) (12) (9) a a asal DF + Chloride d (12) (1) asal KII + Chloride d Fig. 5. Tenmo-DF (1 mol l 1 ; DF) and hdo-kii (1 nmol l 1 ; KII) have no effet on fluid seretion y tuules inuated in low Cl saline (one-tenth normal [Cl ]). ars represent the means + 1 s.e.m. of fluid seretion y tuules in normal (open ars) and in low Cl (solid ars) saline. The numer of repliates is shown in parentheses. In the final experimental period, all tuules were moved to normal saline (+ hloride). Note that idential letters indiate mean values that do not differ signifiantly i.e. P<.5.

7 Kinin-like ations of Tenmo-DF 3985 Effets on tuule eletrophysiology Kinins depolarise the transepithelial voltage (V t ) in Malpighian tuules of. aegypti (Hayes et al., 1989) and D. melanogaster (O Donnell et al., 1996), and we therefore determined whether Tenmo-DF and hdo-kii had a similar effet on riket tuules. The V t of unstimulated tuules, measured with an eletrode positioned in the lumen, generally osillated y aout ±1 mv (Fig. 8). Similar osillations have een reported in Malpighian tuules of. aegypti (eyenah et al., ) and D. melanogaster (lumenthal, 1) and have een attriuted to rhythmi hanges in transepithelial hloride ondutane. The mean value of V t was 42.9±3.4 mv (N=2) lumen positive, whih is sustantially higher than the value of.7 mv reported previously (Coast and Kay, 1994) using eletrodes plaed in the sereted droplet and in the athing fluid, a method that is known to e sujet to artefat (neshansley et al., 1988; Isaason and Niolson, 1989). ddition of either 1 mol l 1 Tenmo-DF or 1 nmol l 1 hdo-kii resulted in an immediate ollapse of V t, although it remained non-zero (Fig. 8,). The mean hange in V t following the addition of Tenmo-DF was 31.2±4.1 mv (N=7), whih was not signifiantly different (P=.57; unpaired t-test) from the effet of hdo-kii ( 28.2±4.1 mv; N=13). In marked ontrast to the effets of Tenmo-DF and hdo- KII, the addition of 1 mmol l 1 8-romo yli GMP resulted in a signifiant inrease in V t (Fig. 8C); the mean hange in voltage was +9.1±.9 mv (N=7; P<.1, paired t-test). Susequent addition of either 1 mol l 1 Tenmo-DF (Fig. 8C) or 1 nmol l 1 hdo-kii (data not shown) aused V t to ollapse even in the ontinued presene of 8-romo yli GMP. Fig. 9 is a representative reording that shows the effet of the hloride hannel loker DPC on V t. Following the addition of.2 mmol l 1 DPC, there is an immediate derease in V t, whih then slowly reovers even in the ontinued presene of the hannel loker, although it never returned to its initial value. The transepithelial voltage ontinued to osillate in the presene of DPC, ut the osillations were generally of redued Fluid seretion (pl mm 1 min 1 ) (23)(27) asal a a + DPC + DF (9) (9) d asal + DPC + KII Fig. 6. The hloride hannel loker DPC depresses asal seretion and redues the effet of () 1 mol l 1 Tenmo-DF (+DF) and () 1 nmol l 1 hdo-kii (+KII). ars represent the means + 1 s.e.m. for the numer of repliates shown in parentheses. Tuules were held in ontrol saline (open ars) or moved to a saline ontaining.2 mmol l 1 DPC in the first experimental period (+DPC; solid ars). Idential letters indiate signifiant differenes etween mean rates of seretion (P<.1). d amplitude. DPC did not prevent the ollapse of V t following the addition of 1 mol l 1 Tenmo-DF, ut the voltage hange was redued to 23.7±2.5 mv (N=7). However, this was not signifiantly different (P=.94, unpaired t-test) from the voltage hange produed y Tenmo-DF ( 31.7±3.6 mv; N=4) under ontrol onditions (saline ontaining.1% ethanol). Fig. 9 is a representative reording showing the effet of 1 mol l 1 Tenmo-DF and 1 nmol l 1 hdo-kii on the voltage aross the prinipal ell asal memrane (V ). The mean value of V in unstimulated tuules was 41.4±.8 mv (N=28), and the apial memrane voltage (V a =V t V ) is therefore ~84 mv lumen positive. The osillations seen in V t were not oserved in reordings of V, whih reflets what has een desried in D. melanogaster tuules (lumenthal, 1) and has een attriuted to the low resistane of the asal memrane. The hange in V following the addition of either peptide was less than ±2 mv. Transepithelial eletrohemial gradients for K +, Na +, H + and Cl Net transepithelial eletrohemial gradients ( /F) for eah of the measured ions an e alulated from their respetive onentrations in the athing medium and sereted fluid (data from Fig. 4) and from measurements of V t under asal (unstimulated) onditions and following the addition of 1 mol l 1 Tenmo-DF, 1 nmol l 1 hdo-kii or 1 mmol l 1 8-romo yli GMP (Tale 2). Values for /F are neessarily approximate, eause ion onentrations and transepithelial voltages were measured in different sets of tuules. Diureti ativity (pl mm 1 min 1 ) (9) (9) DF MP Sum oth a (11) a (11) DF (13) (11) KII (1) Fig. 7. () Evidene of synergism etween 1 mol l 1 8-romo yli MP (MP) and.3 mol l 1 Tenmo-DF (DF). ars represent the means + 1 s.e.m. for the hange in rate of seretion ( pl mm 1 min 1 ) following addition of the seretagogues individually and together. The numer of repliates is shown in parentheses, and idential letters indiate values that differ signifiantly (P<.1). The sum of the separate responses to 8-romo yli MP and Tenmo- DF (Sum) is signifiantly less than when they are tested together (oth). () 8-romo yli GMP (1 mmol l 1 ) inreases seretion y tuules that have een maximally stimulated y Tenmo-DF (1 mol l 1 ) and hdo-kii (1 nmol l 1 ; KII). ars represent the means + 1 s.e.m. for the hange in rate of seretion ( pl mm 1 min 1 ) after addition of the peptides alone (open ars) and then together with 8-romo yli GMP (grey ars). lak ars show the diureti ativity of 1 mmol l 1 8-romo yli GMP alone measured in separate groups of tuules. The numer of repliates is shown in parentheses, and idential letters indiate values that differ signifiantly (P<.1).

8 3986 G. M. Coast, R. J. Nahman and D.. Shooley C 1 mmol l 1 yli GMP 1 mv 2 min 1 mv 5 min 1 mv mv mv mv 5 min Fig. 8. Representative reordings showing the hange in transepithelial voltage (V t ) following the addition of () 1 mol l 1 Tenmo-DF, () 1 nmol l 1 hdo-kii and (C) 1 mmol l 1 8-romo yli GMP. Thik arrows show the time of insertion (downward) and withdrawal (upward) of the miroeletrode, and thin arrows show when peptides were added. The horizontal ar in C shows when 8-romo yli GMP was present in the perfusate, and the thin arrow indiates the time of addition of 1 mol l 1 Tenmo-DF. For ations (Na +, K + and H + ), /F is invarialy positive under asal onditions (Tale 2), indiating that their onentration in the sereted fluid exeeds equilirium values. The transepithelial transport of these ions must therefore e ative. Following stimulation with either Tenmo-DF or hdo-kii, the magnitude of /F dereases, refleting the ollapse of V t, ut remains positive for K + and H +, while falling to negative values for Na +. In ontrast, /F is either unhanged (H + ) or inreases (Na + and K + ) in tuules stimulated with 8- romo yli GMP. This is partiularly marked for Na +, with /F inreasing from 9.1 mv to 32.3 mv after addition of the yli nuleotide due to inreases in oth V t and the onentration of Na + in the luminal fluid. The alulated net eletrohemial potential for Cl is invarialy negative and hene favours passive movement of Cl from the ath into the lumen. The eletrohemial potential is redued after stimulation with either Tenmo-DF or hdo- KII, whih ause V t to ollapse, ut is inreased in the presene of 8-romo yli GMP. Effet of Tenmo-DF and hdo-kii on hindgut ontrations Given the similar effets of Tenmo-DF and hdo-kii on tuule seretion, oth peptides were tested for myotropi ativity on riket hindgut. The hindgut of. domestius ontrats spontaneously when athed in okroah saline, and typial reordings are presented in Fig. 1, whih shows also the effet of hallenging the same preparation with 1 mol l 1 Tenmo-DF (Fig. 1), 2 nmol l 1 hdo-kii (Fig. 1) and with saline alone (Fig. 1C). hdo-kii has a pronouned effet on the frequeny and amplitude of hindgut ontrations, whereas Tenmo-DF and saline alone were without effet. Tested on five different hindgut preparations, the perentage hange in ontration frequeny over 2 min intervals efore and after the addition of 1 mol l 1 Tenmo-DF was.4±1.6%, ompared with 1.4±1.5% after adding saline, and a 47.8±4.3% inrease with 3 nmol l 1 hdo-kii. The threshold onentration of hdo-kii needed to produe a readily oservale effet on the frequeny and/or amplitude of hindgut ontrations was 1.43±.39 nmol l 1 (N=12). Disussion tivities of identified antidiureti fators on riket tuules Spring et al. reported the presene of an antidiureti fator in. domestius that redued Malpighian tuule seretion (Spring et al., 1988). Three unrelated peptides, Manse-CP 2, Tenmo- DFa and Tenmo-DF, have sine een shown to redue tuule.2 mmol l 1 DPC mv mv 1 mv 5 min 1 mv 5 min DF KII Fig. 9. () Representative reording of the transepithelial voltage (V t ) efore and after the addition of.2 mmol l 1 diphenylamine-2-aroxylate (DPC) to the athing saline. DPC riefly depolarises V t ut does not inhiit the spontaneous voltage osillations. Nor does it prevent the redution of V t y 1 mol l 1 Tenmo-DF (thin arrow; DF). () representative reording of the asal memrane voltage (V ) showing this is not affeted y the addition of either 1 mol l 1 Tenmo-DF or 1 nmol l 1 hdo-kii (KII). Thik arrows mark the point of insertion (downward) and withdrawal (upward) of the miroeletrode, and horizontal ars show when peptides were present in the perfusate.

9 Kinin-like ations of Tenmo-DF 3987 Tale 2. Net transepithelial eletrohemial potentials ( /F) for Na +, K +, H + and Cl efore and after the addition of 1 mol l 1 Tenmo-DF, 1 nmol l 1 hdo-kii or 1 mmol l 1 8-romo yli GMP /F (mv) Tenmo-DF hdo-kii 8-romo yli GMP asal Stimulated asal Stimulated asal Stimulated Na K H Cl Calulations are ased upon measured onentrations in the sereted urine and on mean values for the transepithelial voltage (V t ). seretion y a yli GMP-dependent mehanism (Quinlan et al., 1997; Wiehart et al., 2), and it is possile that one of them is an orthologue of the unharaterised antidiureti fator from. domestius. However, when tested at 1 mol l 1, none of these peptides redued seretion y riket tuules; neither did 1 mmol l 1 exogenous 8-romo-yli GMP. On the other hand, Tenmo-DF stimulated seretion to approximately the same extent as a kinin neuropeptide, hdo-kii, although it was aout five orders of magnitude less potent in the diureti assay (EC 5 values of 1.5 mol l 1 and 1.2 pmol l 1, respetively). Comparing the ations of Tenmo-DF with those of hdo- KII and 8-romo yli GMP Data from the present study show that Tenmo-DF and hdo-kii have idential effets on riket tuules. When tested at supramaximal onentrations, they inrease seretion y aout 75% and their ativities are non-additive, whih suggests they share the same mode of ation. Consistent with this suggestion, neither peptide has any effet on the [Na + ]:[K + ] ratio of the sereted fluid, and oth ause a small ut signifiant derease in ph. Moreover, in ommon with the effet of kinins on Malpighian tuules from. aegypti and D. melanogaster (Hayes et al., 1989; O Donnell et al., 1996), oth Tenmo-DF and hdo-kii ause the lumen-positive transepithelial voltage to ollapse, although it remains non-zero. Kinins are known to at synergistially with exogenous yli MP (Coast, 1995; Coast et al., 199; Holman et al., 1999), and this has now een demonstrated with Tenmo-DF, whih further supports the kinin-like ations of this antidiureti fator from T. molitor. Tenmo-DF ats via a yli GMP-dependent mehanism in reduing seretion y T. molitor tuules (Eigenheer et al., 3), and the same seond messenger ould e impliated in its diureti ativity in rikets, where exogenous 8-romo yli GMP stimulates tuule seretion. However, this is not onsistent with the data, whih show signifiant differenes etween the effets that 8-romo yli GMP and Tenmo-DF (and hdo-kii) have on the omposition of the sereted fluid and the transepithelial voltage. Notaly, 8-romo yli GMP elevates the lumen-positive transepithelial voltage, doules the [Na + ]:[K + ] ratio of the sereted fluid and inreases its ph. Moreover, the yli nuleotide analogue aelerates seretion y tuules that are already maximally stimulated y Tenmo-DF (and hdo- KII), whih indiates it has a different mode of ation. lthough Tenmo-DF and hdo-kii have idential effets on riket tuules, only the latter stimulated ontrations y the hindgut. It is worth noting, however, that the threshold onentration for hdo-kii ativity in the hindgut assay (1.43 nmol l 1 ) is aout 14-fold higher than its EC 5 in the diureti assay. If the same differene in poteny were to apply to Tenmo-DF, then the threshold onentration for an oservale effet in the myotropi assay would e aout mol l 1, whih is 2 times higher than the maximum onentration tested. Effet on Cl ondutane The generally aepted model for the diureti ativity of inset kinins is that they open a transepithelial ondutane pathway for hloride, whih aelerates its movement into the Malpighian tuule lumen down a favourale transepithelial eletrohemial gradient. The movement of additional Cl into the lumen auses the lumen-positive transepithelial voltage to ollapse and results C 1 mg 1 min Fig. 1. representative reording of spontaneous ontrations y a riket hindgut preparation hallenged with first 1 mol l 1 Tenmo- DF (), then 2 nmol l 1 hdo-kii () and finally with saline alone (C). ars show when test sustanes were present in the perfusate.

10 3988 G. M. Coast, R. J. Nahman and D.. Shooley in a non-seletive inrease in NaCl and KCl seretion aompanied y osmotially oliged water. Evidene from the present study is onsistent with Tenmo-DF and hdo-kii ating in a similar manner. They have an insignifiant effet on the [Na + ]:[K + ] ratio of tuule fluid, their diureti ativity is hloride dependent, as evidened y a failure to stimulate seretion y tuules athed in saline ontaining one-tenth the normal hloride onentration, and oth ause V t to ollapse. Moreover, the alulated net eletrohemial driving fore for Cl ( /F Cl ) favours passive diffusion from ath to lumen oth efore and after stimulation y Tenmo-DF and hdo-kii (Tale 2) despite the fall in V t. Following peptide stimulation, /F Cl delines y ~31 mv while there is a 75% inrease in net transepithelial Cl transport (the produt of the Cl onentration in the tuule fluid and the rate of seretion). It follows that Tenmo-DF and hdo-kii must promote a sustantial inrease in the Cl permeaility (ondutane) of the epithelium. Loation of the hloride ondutane pathway The Cl ondutane pathway in the Malpighian tuules of dipteran insets lies outside of the prinipal ells, ut there is some deate as to its preise loation. In. aegypti, kinins are elieved to at on prinipal ells and to open a paraellular ondutane pathway, whih would require rapid remodelling of septate juntional omplexes (eyenah, 3a). On the other hand, in D. melanogaster, kinins at on a seond ell type, the stellate ell, to open a transellular Cl ondutane pathway (O Donnell et al., 1998; Radford et al., 2). The Malpighian tuules of. domestius lak stellate ells (Hazelton et al., 1988), and the Cl ondutane pathway must therefore e through either prinipal ells or septate juntional omplexes, as in. aegypti. Results otained with the epithelial hloride hannel lokers DPC and NPP are onsistent with Tenmo-DF and hdo- KII ating to open a transellular Cl ondutane pathway, i.e. through the prinipal ells. Thus, fluid seretion y peptidestimulated tuules was signifiantly redued y DPC (Tenmo- DF and hdo-kii) and NPP (Tenmo-DF), while DPC also dereased the extent to whih V t fell in response to Tenmo- DF, although the differene (8 mv) was not signifiant. It is worth noting, however, that DPC may have other sites of ation, eause it auses a derease in V t when added to unstimulated tuules, whih is the reverse of what would e expeted from loking a transellular Cl ondutane (lumenthal, 1). Tenmo-DF and hdo-kii do not appear to at at the prinipal ell asal memrane, eause V is unhanged despite the large derease in V t, whih suggests they target the apial memrane, ausing V a to deline from ~84 mv to ~55 mv lumen positive. Does Tenmo-DF at at a kinin reeptor? In T. molitor, the antidiureti fator Tenmo-DF has een loalized immunohistohemially to two pairs of lateral neuroseretory ells loated anteriorly in the protoererum, axons from whih projet posteriorly and enter a plexus that appears to e a neurohaemal release site (Eigenheer et al., 3). Criket heads have een examined for the presene of Tenmo- DF-like immunoreative material using the same antiserum and methods as those employed in the Eigenheer et al. study (Eigenheer et al., 3), ut without suess (L. Shoofs, personal ommuniation). Possily,. domestius has an DF-like peptide, ut it is so dissimilar from the eetle peptide that it is not reognised y the antiserum, whih would e onsistent with the low poteny of Tenmo-DF in the riket diureti assay. lternatively, rikets may lak an DF-like peptide, in whih ase the diureti ativity of Tenmo-DF ould e due to it inding and ativating a kinin reeptor, whih would aount for the similar effets of Tenmo-DF and hdo-kii on riket tuules. Considerale information is availale aout the strutural requirements for kinin ativity in riket tuules. The minimal sequene requirement for diureti ativity is a C-terminal amidated pentapeptide (Phe-Xxx 1 -Xxx 2 -Trp-Gly-NH 2 ; where X 1 is sn, His, Ser, Tyr or Phe, and X 2 is la, Pro or Ser) (Coast et al., 199). Within this ative ore, residues one (Phe), four (Trp) and five (Gly-NH 2 ) are invariant, and oth Phe and Trp are essential for ativity (Roerts et al., 1997). In the ative onformation, the two aromati residues are rought into lose proximity on one surfae of the moleule, whih adopts a type VI -turn (Nahman et al., 2). Little is known aout the struture ativity relationships of Tenmo-DF, ut the minimal sequene requirement for diureti ativity in riket tuules appears to enompass residues 7 12 (Lys-Pro-His-Ile-Tyr-Gly- OH). This sequene has virtually nothing in ommon with the kinin ative ore. Importantly, it laks the Phe and Trp residues that are ritial for diureti ativity and is non-amidated, whih suggests it is unlikely to interat with a kinin reeptor. In onlusion, Tenmo-DF has diureti rather than antidiureti ativity on riket tuules. Its effets on ion and fluid transport, and on tuule eletrophysiology, are indistinguishale from those of hdo-kii, although it is onsideraly less potent. Our data suggest that oth peptides stimulate seretion y opening a transepithelial hloride ondutane pathway ut that they most likely at at different reeptors. We gratefully aknowledge the tehnial support of lan Tyler (irkek). This work was supported in part y a NTO Collaorative Researh Grant to G.M.C. and R.J.N., and an NIH Grant to D..S. Referenes neshansley, D. J., Marler, C. E. and eyenah, K. W. (1988). Transepithelial voltage measurements in isolated Malpighian tuules of edes aegypti. J. Inset Physiol. 35, eyenah, K. W. (3a). Regulation of tight juntion permeaility with swith-like speed. 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The effets of heta diureti peptide on isolated Malpighian tuules from the house riket heta domestius. J. Exp. iol. 187,