Cross reactivity studies of CRF-related peptides on insect Malpighian tubules

Camp. Biochem. f’hysiol.Vol. I lOA, No. I, pp. 87-93. 1995 Copyright 0 1995 Elsevier Science Ltd

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Cross reactivity studies of CRF-related insect Malpighian tubules Neil Audsley,* Iain Kay,? Timothy and Geoffrey M. Coast*

peptides

on

K. Hayes1

*Department of Biology, Birkbeck College, University of London, Malet Street, London WClE 7HX, U.K.; tSt John’s Institute of Dermatology, St Thomas’s Hospital, London SE1 7EH, U.K.; and IDepartment of Entomology, Texas A & M University, College Station, TX 77843, U.S.A. Manduca sexta diuretic peptide II (Mas-DPII) stimulates fluid secretion by adult Malpighian tubules and cyclic AMP production by larval proximal and adult tubules of M. sexta in a dose-dependent manner. Mas-DPII has no effect on fluid transport across the larval cryptonephric complex. M. serta diuretic hormone (Mas-DH) and CRF-related insect diuretic peptides from A&eta domesticus, Locusta migratoria, and Periplaneta americana also cause similar increases in the production of cyclic AMP by the Malpighian tubules of both larval and adult M. sexta. Insect CRF-related diuretic peptides exhibit varying degrees of potency when assayed on Malpighian tubules from L. migratoria and A. domesticus. Sauvagine, bovine-CRF, and human-CRF have only a small, but significant, effect on cyclic AMP production by M. sexta Malpighian tubules. However, sauvagine, bovine-CRF, and sucker fish urotensin-1 have no effect on L. migratoria tubules. Stimulation of cyclic AMP production by M. sexta Malpighian tubules could potentially be used as a screening assay to identify other insect CRF-related diuretic peptides. Key words: Munduca se.utn; A&zeta domesticus; Locusta migratoria; Diuretic tubules; Fluid transport; Cyclic AMP; Corticotropin releasing factor. Corp.

Bioclzem. Phj2siol. 1 lOA, 87-93,

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Malpighian

1995.

Introduction There are five reported members of the corticotropin-releasing factor (CRF)-related insect diuretic peptide (DP) family from Manduca sexta (Kataoka et al., 1989; Blackburn et al., 1991) Acheta domesticus (Kay et al., 1991a), Locusta nzigratoriu (Kay et al., 1991 b; Lehmberg et al., 1991). and Peripluneta americana (Kay et al., 1992). The primary effect of insect diuretic peptides is considered to be stimulation of fluid secretion by Malpighian tubules. This increase in fluid secretion is preceded or paralleled by cyclic AMP production when tubules are stimulated in zlitro Correspondence 10: N. Audsley. Dept. of Biology, Birkbeck College, University of London, Malet Street, London WClE 7HX. U.K. Received 31 March 1994; revised IO July 1994; accepted 18 July 1994.

by CRF-related diuretic peptides (Coast and Kay, 1994). Maddrell (1980) and Petzel et al. (1987) have also shown similar responses to crude extracts of neurohaemal tissue (containing diuretic factors) by Malpighian tubules of Rhodnius prolixus and Aedes aegypti, respectively. Thus, an increase in cyclic AMP production by isolated Malpighian tubules in vitro is assumed to be a diuretic response to CRF-related peptides. All insect CRF-related diuretic peptides, except for M. sexta diuretic peptide II (Mas-DPII), have been shown to stimulate Malpighian tubule fluid secretion via a cyclic AMP dependent mechanism (Kay et al., 1991b, 1992; Lehmberg et al., 1991; Audsley et al., 1993). Mas-DPII was isolated using an in vivo bioassay which measured fluid excretion from adult insects (Blackburn et al., 1991). They

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suggested that Mas-DPII acts on either the Malpighian tubules or the rectum, but presented no evidence to support either of these possibilities (Blackburn et al., 1991). However, the receptor for M. sexta diuretic hormone (MasDH) has recently been cloned from mRNA extracted from 5th instar larval M. se.uta tubules and expressed in COS-7 cells (Reagan, 1994). Mas-DPII binds to COS-7 cells expressing this receptor and stimulates cyclic AMP production, indicating that it is likely to have a direct effect on Malpighian tubules. The purpose of this study was to demonstrate the activity of Mas-DPII on M. sexta Malpighian tubules, and to determine the cross-reactivities of CRF-related peptides on larval and adult Malpighian tubules from M. sexta, A. domesticus, and L. migratoria.

Materials and Methods Insects M. sexta eggs and pupae were supplied by Prof. S. E. Reynolds, University of Bath. Larvae were reared as described by Yamamoto (1969). Larval experimental animals were 3rd day, 5th instars. and adults were 12-48 hr post emergence. A. domesticus and L. migratoria were reared as described by Coast (1988) and Goldsworthy et al.. (1972). Crickets were 6-12 day old adult virgin females and locusts were IO-14 day old adult females. Bioassavs 1. Fluid secretion by Malpighian tubules. The effect of Mas-DPII on adult M. sexta Malpighian tubule fluid secretion was determined using the bioassay described in detail by Audsley et al. (1993). Briefly, short segments (6-12 mm) of M. sexta Malpighian tubules are bathed in small drops (5 ~1) of saline beneath water-saturated liquid paraffin. The ends of the tubules are drawn into the paraffin, and secreted fluid collected. After a 30 min equilibration period, the bathing fluid was replaced, and secreted fluid was collected over the following 60 min in order to determine the basal (unstimulated) rate of fluid secretion. The bathing fluid was then substituted for fresh saline (controls) or saline containing Mas-DPII, and the rate of secretion (experimental rate) determined over a second period of 60 min. Rates of secretion were determined from the volume of the droplets secreted (assuming them to be spherical). Results are expressed as the difference in the rate of secretion between the experimental and control periods (A nl min-‘) so that each tubule segment served as its own control. 2. Cyclic AMP assa_v. Cyclic AMP production in Malpighian tubules was measured as described

et al.

previously for L. migratoria and A. domesticus by Kay et al., (1991) and Coast et al., (1991) and for M. sexta by Audsley et al. (1993). The amount of cyclic AMP produced is compared with control preparations (IBMX saline addition) over the same period, and to that produced when tubules are stimulated with a supra-maximal dose of the diuretic peptide (in IBMX saline) from the same insect. Cyclic AMP determinations were made using a modification of the competitive binding assay of Gilman (1972) and as described by Coast et al. (1991). Results are represented as cyclic AMP produced (pmol) per tubule or tubule segment. IBMX was prepared as a 200 mmol 1-l stock in dimethyl sulphoxide (DMSO) and diluted in physiological saline for tissue incubation (final concentration of IBMX = 5 x 10-j mol 1-l. and DMSO = 0.25%). 3. Fluid absorption by the cryptonephric complex of M. sexta larvae. Methods for studying fluid transport across the larval cryptonephric complex using everted sacs from 5’h instar M. sexta larvae were described in detail by Audsley et al. (1993). Briefly, at hourly intervals, over 5 hr, weight gain and tissue volume changes were determined by weighing the sacs (to within 0.25 mg) before and after removal of fluid in the sac. Transepithelial fluid movement was then determined by correcting for tissue volume changes. Physiological saline (10 ~1) was added to the haemolymph side of the sacs for the first three hours, and the sacs were placed in 50 ml of oxygenated physiological saline at 3O’C to obtain control rates. At the end of the third hour, Mas-DPII or physiological saline (control) were added to the sacs. The rate of fluid transport was then measured for the next one or two hours and compared with control preparations over the same period and with rates during the previous control period for the same preparation. Results are expressed as /tl hr- ’ tissue-‘. Salines The saline used for both M. se.vta Malpighian tubule fluid secretion and cyclic AMP measurements is described by Audsley et al. (1993). A saline based on insect haemolymph (Coast, 1988) was used to determine cyclic AMP production by Malpighian tubules of both A. domesticus and L. migratoria Malpighian tubules. CRF-related

peptides

Mas-DPII and Acheta-DP were synthesized at Birkbeck using methods described by Kay et al. (1991b). Locusta-DP, Mas-DH and the norleucine analogues of AchetaDP ((Nle20.2s)Achet;~~ ,;Ed mey;;zL (Wk., [Was-DW, residues at the positions indicated were replaced

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with norleucine, were synthesized in Texas as previously described (Coast et al., 1992). Synthetic peptides were purified by reversedphase high performance liquid chromatography as described by Kay et al. (1991 b). PeriplanetaDP was the native peptide (Kay et al., 1992). Mas-DH-acid, where the C-terminal amide is replaced by the free acid, was a gift from Prof. D.A. Schooley (Reno, NV). Sauvagine, bovine-CRF, human-CRF, and Sucker fish urotensin-I, were purchased from Sigma. All peptides were made up as stock solutions in 80% methanol, and diluted in saline for assay, so that the methanol content was less than l%, which has no effect on fluid secretion or cyclic AMP production by Malpighian tubules. Osmolarity

a

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I

/

I

-11

-10

Log

-9

Dose

(moles

-8

-7

I-‘)

Fig. 1. Effect of increasing doses of Mas-DPII on fluid secretion by isolated adult Malpighian tubules, shown as a semi-log plot (means + SE, n = 7-9).

of salines

Changesinsalineosmolarityduetotheaddition ofpeptidesweremonitoredwithaWestcorvapour pressure osmometer (Model 5500; Logan, and were found to be not significant. Statistical

peptides

Utah)

The efSects of Mas-DPII the larval cryptonephric

treatment

Results are presented as the error (SE). Differences between considered significant when indicated a P value < 0.05. The determined from a generalised using the computer programme

mean f standard treatments were Student’s t-test EC& values were logistic equation FigP (Biosoft).

Results Eflect of Mas-DPII fluid secretion

on adult Malpighian

tubule

Under control conditions (when no peptide was present) rates of fluid secretion by short segments of adult Malpighian tubules were approximately 0.25 nl min-‘. On the addition of Mas-DPII to the bathing saline, fluid secretion was stimulated in a dose-dependent manner as shown in Fig. 1. The response to the peptide is shown here as the difference between the basal and stimulated rates of secretion measured over 60 min periods. As little as 0.05 nmol ll’ peptide caused a significant increase in tubule secretion, and a maximal response was observed at a dose of 1.5 nmol ll’. The it+, is 0.35 f0.003 nmol 1-l. Eflect of Mas-DPII on adult Malpighian cyclic AMP production

manner to approximately 40 pmol tubule segment-’ on the addition of Mas-DPII. The EC,, values are shown in Table 1. on fluid transport across complex

Control rates of fluid transport across the cryptonephric complex after 3 hr were 3.34 + 1.03 ~1 tissuee’hr-’ (N= 6). No significant increase in fluid transport was observed on the addition of 1 x lo-* mol 1-l Mas-DPII over the fourth hour (3.65+ 1.32; N=6). However, the same preparations could be stimulated to a maximum of 11.04 + 2.10 ~1 tissue-’ hr-’ (N= 6) when 1 x lo-* mol 1-l Mas-DH was added over the fifth hour. The relative eflects of insect CRF-related diuretic peptides on cyclic AMP produced by M. sexta Malpighian tubules All the insect CRF-related diuretic peptides that were tested caused dose-dependent increases

tubule

Cyclic AMP production by larval and adult tubules due to Mas-DPII is shown in Fig. 2. Under control conditions (when no peptide was present) the amount of cyclic AMP produced by both M. sexta adult and larval proximal tubules over a 1 hr period was less than 1.0 pmol tubule-‘. This was increased in a dose-dependent

-11

-10

Log Fig. 2. Effect of increasing production by isolated (solid circles) M. sextu semi-log plot

-9

Dose

(moles

-8

-7

I-‘)

doses of Mas-DPII on cyclic AMP larval (solid triangles) and adult Malpighian tubules, shown as a (means + SE, n = 410).

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Table I. The relative effects of CRF-related insect diuretic peptides on cyclic AMP production by Malpighian tubules of . . adult and larval M. sex& (N=4-12). Peptide Mas-DH (Nle, ,,)Mas-DH Mas-DH-acid Mas-DPii Achefa-DP Locusra-DP Periplaneta-DP

Apparent

ECjo

(nmol 1-l) Adult 6.92 3.46 5120 5.01 6.03 9.33 6.46

Larva 9.55 4.51 Not tested 2.04 0.87 4.47 2.34

in cyclic AMP production by larval and adult tubules to a maximal response of > 35 pmol tubule-’ (results not shown), similar to the response obtained with Mas-DPII (Fig. 2). From the dose-response data, the concentration of peptides required to elicit a half maximal response (apparent ~c~a values) was determined and is presented in Table 1. All these peptides appeared to have very similar potencies, and ~c~a values ranged from 5 to 9.3 nmol l-‘, except for A&eta-DP on larval tubules, which was 2-10 times more potent, and Mas-DH-acid on adult tubules which was approximately lOOOfold less potent than the other diuretic peptides (Table 1). EfSects of insect CRF-related diuretic peptides on cyclic AMP produced by A. domesticus and L. migratoria Malpighian tubules The effects of CRF-related insect diuretic peptides on A. domesticus and L. migratoria Malpighian tubules are shown in Table 2 All peptides apparent as EGO values. which produced a response did so in a dose-dependent manner. When assayed on A. domesticus tubules, the order of potency was Acheta-DP > Locusta-DP > Periplaneta-DP > Mas-DH (Table 2). However, Mas-DH could only stimulate cyclic AMP production to 60% of a maximum response observed with Acheta-DP (results not shown), and Mas-DPII had no significant effect, even at high doses (lo-’ mol 1-l). On L. migratoria tubules, the order of potency was Locusta-DP > Acheta-DP > Periplaneta-DP. No significant increases in cyclic

Table 2. The relative effects of CRF-related insect diuretic peptides on cyclic AMP production by Malpighian tubules of L. migraroria and A. domesticus (N=410) Treatment Acheta-DP Locusta-DP Periplaneta-DP Mas-DH Mas-DPII

Apparent L. migratoria 6820 1.9 671 No effect No effect

Ec,,(nmol

I-‘)

A. domesticus 2.5 140 194 380 No effect

et al. Table 3. The effects of vertebrate CRF-related peptides (10-l mollk’) on cyclic AMP production by Malpighian tubules (mean k SE, N= 48) Treatment Control (no peptide) Bovine-CRF Human-CRF Sauvagine Urotensin-I Means + SE, N = 5. Significant difference

Cyclic AMP (pmol tissue-‘) M. sexta 0.036 * 0.02 0.89+0.14** 0.82+0.21* 1.94*0.30** Not tested from controls

**Pi

L. migratoria 0.16&0.07 0.14*0.09 0.21*0.11 Not tested 0.13t0.09 0.005. *P
AMP production were observed due to Mas-DH or Mas-DPII (lo-’ mol 1-l). Of the Malpighian tubules used in this study, those from L. migratoria were the least responsive to CRF-related peptides from other insects. The effect of (Nle2,&Acheta-DP on cyclic AMP production by A. domesticus and L. migratoria tubules was identical to that of Acheta-DP (results not shown). EfjGects of zlertebrate CRF-related peptides on cyclic AMP secreted by the Malpighian tubules At a concentration of 1O-5 mall-‘, sauvagine, human-CRF and bovine-CRF had a small but significant effect (PcO.05) on cyclic AMP production by adult M. sesta tubules compared with control values (Table 3). Sauvigine was the most potent, but when compared with the maximal response caused by 100 nmol 1-l Mas-DH, this was only a 7% increase in cyclic AMP production. None of the vertebrate CRF-related peptides had any significant effect on cyclic AMP production when assayed on L. migratoria tubules (Table 3).

Discussion This study has established the site of action of Mas-DPII and described the relative potencies of CRF-related peptides on Malpighian tubules from M. sexta, A. domesticus and L. migratoria. During the isolation of Mas-DPII, biological activity was followed using an in rive bioassay which measured weight changes in decapitated adult female M. se.uta (Blackburn et al., 1991) so its site of action was not established. Here, Mas-DPII in common with other CRF-related insect diuretic peptides is shown to act by stimulating Malpighian tubule fluid secretion via a cyclic AMP dependent mechanism. This peptide was isolated from the CC-CA complex and from type IIb cells of the medial protocerebrum. As type IIb cells differentiate postembryonically during adult development, Blackburn et al. (1991) proposed that Mas-DPII may have an adult specific function. However,

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this is almost certainly not the case; when extracts of larval M. sexta corpora cardiaca are subjected to reversed-phase high performance liquid chromatography, two separate fractions stimulate cyclic AMP production by larval proximal tubules. These fractions have the same retention times as Mas-DPII and Mas-DH when these peptides were run under identical conditions, suggesting that both Mas-DPII and Mas-DH are present in larval heads (N. Audsley, unpublished observations). More significantly, the results described in this study show that Mas-DPII acts not only on adult Malpighian tubules, but also stimulates cyclic AMP production by larval proximal tubules. The latter is taken here to be a diuretic response, because although it has not been possible to develop a reliable bioassay for fluid secretion by larval Malpighian tubules (see Audsley et al., 1993), it is now well established that CRF-related diuretic peptides use cyclic AMP as a second messenger (Coast and Kay, 1994). Indeed, in A. domesticus tubules, dose-response curves for the stimulation of fluid secretion and cyclic AMP production are identical (Coast and Kay, 1994), and during the isolation of Ache&-DP, Locusta-DP and Peripluneta-DP, fractions which increased cyclic AMP production by Malpighian tubules were shown subsequently to stimulate fluid secretion (Kay et al.. 1991a,b, 1992). Audsley et al. (1993) showed that Mas-DH has both diuretic and antidiuretic activity; it stimulates fluid secretion and cyclic AMP production by Malpighian tubules of adult and larval M. sexta, and increases fluid absorption across the larval cryptonephric complex. The latter effect is thought to be due to stimulation of fluid secretion by the cryptonephric tubules which results in water being drawn osmotically from the rectal lumen. The net effect would be to increase fluid recycling between the haemolymph and excretory system rather than water loss from the insect (Audsley et al., 1993). In contrast, Mas-DPII has no effect on fluid uptake across the cryptonephric complex. This suggests that only the proximal tubules in M. sexta larvae possess receptors for Mas-DPII, whereas both the Table 4. Sequences Achera-DP Loctrsta-DP Periplaneta-DP Mas-DH Mas-DPII Human-CRF Bovine-CRF Sauvagine Urotensin-I

on insect CRF-related TGA.QSLSIV MGMGPSLSIV TGSGPSLSIV ..RMPSLSID . . . ..SFSVN .SEEPPISLD .SQEPPISLD ..QGPPISID .NDDPPISID

peptides

proximal and cryptonephric tubules have receptors for Mas-DH. However, Reagan (1994) has recently shown that Mas-DPII binds to and activates Mas-DH receptors expressed in COS-7 cells. The reason why Mas-DPII does not appear to activate Mas-DH receptors on the cryptonephric tubules in vitro to promote fluid transport is unclear, but it suggests that Mas-DH receptors on the distal (cryptonephric) tubules differ from those on the proximal tubules. In any event, it is likely that Mas-DH and Mas-DPII have different roles in larval M. sexta. for Mas-DH (Audsley The ECUS values ef al., 1993) and Mas-DPII (this study) on fluid secretion by adult tubules are around 0.3 nmol l-‘, whereas for cyclic AMP production, the values are 5-30-fold greater (Table 1). The greater potency for the fluid secretion response may be due to enzymatic cascades within Malpighian tubule cells which amplify the initial signal. In both rat and human adrenocortical cells, there is about a 40-fold lower adrenocorticotropic hormone concentration required for half-maximal steroidogenesis than for half-maximal cyclic AMP production (Ramachandran er al., 1988). This is somewhat different from the situation described in A. domesticus, where the two dose-response curves are identical (see above). All CRF-related insect diuretic peptides that were assayed (Mas-DH, Mas-DPII, Acheta DP, Locusta-DP and Periplaneta-DP) cause maximal stimulation of cyclic AMP production by both adult and larval M. sexta Malpighian tubules, and their ECU,, values are very similar (Table 1). Reagan (1994) also reports that these same peptides bind to Mas-DH receptors and stimulate cyclic AMP production by COS-7 cells expressing Mas-DH-receptors equally well. These results are remarkable in view of the limited sequence identity between insect CRF-related diuretic peptides which (excluding DPII) range from 44 to 63% (Table 4). However, most of this similarity is over the N-terminal half of the molecule, which has been shown to be involved in both signal transduction and receptor binding. Thus, in Ache&-DP, the first six N-terminal

diuretic peptides, urotensin

APLDVLRQRL NPMDVLRQRL NPLDVLRQRL LPMSVLRQKL PAVDILQH.. LTFHLLRE.. LTFHLLRE.. LSLELLRK.. LTFHLLRN..

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bovine CRF, sauvagine

MNELNRRRMR LLEIARRRLR LLEIARRRMR SLS.KERKVH .RYMEKV... VLEMARAEQL VLEMTKADQL MIEIEKQEKE MIEMARIENE

ELQGSRIQQN DAE.EQIKAN QSQ.DQIQAN AL...RAAAN . . . . ..AQNN A...QQAHSN A . ..QQAHNN K...QQAANN R...EQAGLN

and sucker fish RQLLTSI-NH, KDFLQQI-NH, REILQTI-NH, RNFLNDI-NH, RNFLNRV-NH, RKLMEII-NH, RKLLDIA-NH, RLLLDTI-NH, RKYLDEV-NH,

The sequenceswerealigned usingacomputer program, MALIGN (Mark Johnson, Birkbeck College). The gaps (.) in the sequences are inserted by MALIGN so as to optimize alignment scores.

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residues can be deleted without any loss of activity in the fluid secretion assay, but deletion of the next five amino acids causes a substantial loss of activity, and A&eta-DP(23-46) is inactive (Coast et al., 1994). Similarly, binding studies with plasma membrane preparations of 5th instar M. sexta larvae have revealed that N-terminal truncation of Mas-DH beyond residue 12 resulted in a progressive reduction in receptor affinity, and Mas-DH(31-41) showed no binding activity at all (Reagan et al., 1993). These results are consistent with those obtained with mammalian CRF, where residues 4-8 are necessary for biological activity, and binding affinity is greatly reduced in analogues truncated beyond residue 9 (Kornreich et at., 1992; Rivier et al., 1984). In contrast with their actions on M. sextu tubules, the potencies of insect CRF-related peptides vary considerably when assayed on Malpighian tubules from A. domesticus and L. migrutoriu (Table 2). These results are similar to those observed by Coast et al. (1992,1994) for the effects of CRF-related peptides on fluid secretion by Malpighian tubules of the latter insects. Clearly, there are major differences between receptors on M. sexfu Malpighian tubules and those on tubules from A. domesricus and L. migrutoriu. Those on M. sextu appear to be far more liberal in their requirements for receptorligand interaction, both in terms of binding (potency) and signal transduction (activity). Interestingly, Reagan (1994) reports that although A&eta-DP is a potent Mas-DH receptor agonist, the A&eta-DP receptor does not appear to have high overall sequence identity with the Mas-DP receptor. This may account for the reduced activity and substantially lower potency of Mas-DH on A. domesticus tubules compared with the native peptide. An analogue of Mas-DH, where the methionine residues at positions 2 and 11 were substituted with norleucine ((Nle,,,,)Mas-DH), produces a maximal response on A. domesticus Malpighian tubule fluid secretion (Coast et al., 1992), whereas Mas-DH produces only a 60% response in this assay and on cyclic AMP production compared with A&eta-DP. The difference between Mas-DH and (Nlez.,,)Mas-DH was attributed to methionine oxidation which has been demonstrated in Locustu-DP when stored under identical conditions, i.e. in methanol at 4°C without an antioxidant (N. Audsley and I. Kay, unpublished observations). However, there is no significant difference between the potencies of Mas-DH and (Nle,.,,)Mas-DH, in stimulating cyclic AMP production by larval and adult Malpighian tubules. Thus, if methionine residues had oxidized in Mas-DH, then this does not affect biological activity when assayed on

M. sextu tubules. The effects of A&eta-DP and )Achetu-DP on cyclic AMP production (NleZO,,, by A. domesticus and L. migratoria tubules are also identical, but these peptides had been newly synthesized and stored dry at - 20°C which may prevent or minimize oxidation. All CRF-related peptides are amidated, and this appears to be important for biological activity. Thus, replacing the C-terminal amide of Mas-DH with a free acid (Mas-DH-acid) causes a substantial reduction in potency, although this analogue still gives a maximum response in the cyclic AMP assay (Table 1). Similarly, in an in vivo assay, Kataoka et al. (1989) observed a 1OOO-fold reduction in potency with Mas-DHacid compared with the amide. Reagan et al. (1993) report that receptor binding studies with Mas-DH-acid were unsuccessful, and suggest that this was due to the peptide being rapidly degraded by the Malpighian tubule membrane preparation. It is possible that the amide gives some protection to the C-terminal from exopeptidases. Alternatively, the presence of a negative charge at the C-terminus may reduce the affinity of the receptor for the free acid. Ovine-CRF-acid and ovine-CRF( I-39)-amide also have less than 0.1% potency of ovineCRF( 1-41) (Kornreich et al., 1992). Mas-DPII is considerably shorter (30 and only has 26-33% sequence residues), identity to the other insect CRF-related diuretic peptides, but is still considered to be a member of this family (Coast et al., 1994). Most of this sequence identity is over the C-terminal region of this molecule (last 11 residues). However, over the N-terminal half of these peptides, which is important for signal transduction and receptor binding (see above), the sequence similarities are minimal. It is remarkable, therefore, that Mas-DPII binds to and fully activates Mas-DH receptors (Reagan, 1994). Although Mas-DPII has similar potency/activity to Mas-DH on M. sextu tubules, it has no effect on Malpighian tubules from A. domesticus or L. migrutoriu. This is hardly surprising in view of the low ( < 27%) similarity scores of Mas-DPII with the native peptides from the latter insects. The absence of biological activity could be due to failure of the peptide to bind to or activate receptors on A. domesticus or L. migratoriu tubules. In the absence of receptor-binding data, it is not possible to distinguish between these two alternatives, but it is significant that no second CRF-related peptide was identified during the isolation of Locusta-DP or A&eta-DP. The corollary to this is that receptors for a DPII-like peptide will be lacking in these insects. There is even less sequence identity between insect and vertebrate CRF-related peptides (approximately 17%). which would account for

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the relatively small effects of sauvigine, humanCRF, bovine-CRF, and urotensin-I (Table 3) on cyclic AMP production by L. migratoria and M. sexta Malpighian tubules. Similarly, when assayed on A. domesticus tubules, urotensin-I, sauvigine, and human-CRF at a concentration of low6 mol I-’ caused only small increases in fluid secretion and cyclic AMP production (Coast et al., 1992). Kataoka et al. (1989) also reported that ovine CRF, urotensin-I, and sauvigine (500 pmol) had no effect on excretion of fluid when injected into post-eclosion Pieris rapae adults. In conclusion, Mas-DPII has been shown to have a diuretic effect in both adult and larval M. sexta tubules, stimulating fluid secretion and/or cyclic AMP production. However, unlike Mas-DH, Mas-DPII has no effect on the cryptonephric complex. All other CRF-related insect diuretic peptides that were tested promote cyclic AMP production by M. sexta Malpighian tubules at doses similar to the native peptide, and can evoke maximal responses. In contrast, the activities and potencies of diuretic peptides on Malpighian tubules from A. domesticus and L. migratoria vary considerably. These differences between the responses of Malpighian tubules from M. sexta, L. migratoria, and A. domestic-us to insect diuretic peptides suggest that cyclic AMP production by the tubules of M. sexta could be used as a heterologous screening assay for the identification of CRF-related diuretic peptides from other insects. Indeed, in collaboration with Mark Holman (USDA, College Station, Texas), a CRF-like diuretic peptide has been identified from Musca domestica using as a screening assay the ability of HPLC fractions to stimulate cyclic AMP production by adult Manduca tubules (Clottens et al., 1994). This peptide was subsequently shown to stimulate fluid secretion by M. domestica tubules. Acknowledgements--We wish to acknowledge Prof. SE. Reynolds, School of Biological Sciences, University of Bath, for his generous supply of M. se.yta eggs and pupae. This work was supported by a grant from the Science and Engineering Research Council.

References Audsley N., Coast G.M and Schooley D.A. (1993) the Effects of Manduca Sexra Diuretic Hormone on Fluid Transport by The Malpiahian Tubules and Crvntonenhric Complex I of Manduca’ S&a. J. Exp. Biol. 178; 231-243. Blackburn M.B., Kingan T.G., Bodnar W., Shabanowitz J.. Hunt D.F.. Kempe T., Wagner. R.M.. Raina A.K., Schee M.E. and Ma M.C. (1991) Isolation and Identification of A New Diuretic Peptide From the Tobacco Hornworm, Manduca Serta. Biochem. Biophys. Res. Commun. 181, 927-932. Clottens F.L.. Holman G.M., Coast G.M.. Totty N.F., Hayes T.K., Kay I.. Mallet AI., Wright M.S., Chung J-S., Trung 0. and Bull D.L. (1994) Isolation and Characterization of a Diuretic Peptide Common to the House Fly and Stable Fly. Peptides 15, 971-979.

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