Diuretic activity of C-terminal group analogues of the insect kinins in Acheta domesticus

Peptides, Vol. 16, No. 5, pp. 809-813, 1995 Copyright 0 1995 Elsevier Science Ltd printed in the USA. All tights reserved 0196-9781/95 $9.50 + .OO

Pergamon

Diuretic Activity of C-Terminal Group Analogues of the Insect Kinins in Acheta domesticus RONALD

J. NACHMAN,*’

GEOFFREY

M. COAST,_F G. MARK

HOLMAN*

AND

ROSS

C. BEIER*

*U.S. Department of Agriculture, Agricultural Research Service, Veterinary Entomology Research Unit, FAPRL, College Station, TX 77845 and fDepartment of Biology, Birkbeck College, University of London, London, UK WClE 7HX Received

21 November

1994

NACHMAN, R. J., G. M. COAST, G. M. HOLMAN AND R. C. BEIER. Diuretic activi@ of C-terminal group analogues of the insect kinins in Achefa domesticus. PEPTIDES 16(5) 809-8 13, 1995.-A series of insect kinin analogues, AFFPWG-X, modified at the C-terminal group, were evaluated in a cricket Malpighian tubule secretion bioassay. The results were compared with activity profiles observed in a cockroach hindgut myotropic bioassay for these analogues. Although the replacement of the C-terminal amide group with a negatively charged acid leads to a precipitous drop in diuretic activity, it can be partially restored with the introduction of ester groups such as methyl or benzyl. The presence of branched chain character in the C-terminal group or a Cterminal a-carbon-amide distance spanning five methylene group spacers is incompatible with the receptor interaction required for biological activity. Significant diuretic activity is retained with four or fewer methylene groups in this region. C-terminal group analogues containing -SCH3, -NHCH2CH20CH3, or -OCH2(C.&IH,) offered the greatest retention of diuretic activity while providing increased hydrophobicity and/or steric bulk. The data are of potential value in the development of mimetic analogues of this insect neuropeptide family. Mimetic analogues are potentially valuable tools to insect neuroendocrinologists studying diuresis and/or engaged in the development of future pest management strategies. Cricket

Cockroach

Hindgut

Myotropic

Peptidomimetic

Ester

nin-immunoreactive fibers innervating the perisympathetic organs form an elaborated plexus extending over most of the abdominal nerve cord. N&se1 et al. (19) suggest that if leucokinins regulate hindgut contractility in Leucophaea muderae, they probably act as neurohormones, because leucokinin-immunoreactive neurons form a substantial supply of axons to the storage lobe of the corpora cardiaca, but the hindgut is not directly innervated by leucokinin-immunoreactive fibers. The C-terminal pentapeptide sequence common to the insect kinins is all that is required to elicit a physiological response in myotropic and diuretic assays. In particular, the active core sequence Phe-Tyr-Pro-Trp-Gly-m is equipotent with the parent nonapeptide in hindgut myotropic (8) and cricket Malpighian tubule secretion (5) assays. Within the active core pentapeptide, the aromatic residues Phe’ and Trp3 are of paramount importance for activity in both bioassay systems, whereas position 2 tolerates wide variations in side-chain character ranging from acidic to basic or hydrophobic to hydrophilic (12,14,18). Aromatic residues, such as Tyr or Phe, in the variable position 2 promote the highest potencies in myotropic and Malpighian tubule fluid secretion assays (12,14,18).

THE insect kinin neuropeptide family shares the common Cterminal pentapeptide Phe-X:&a’-Xaa*-Trp-Gly-NH2 (Xaa’ = His, Asn, Phe, Ser, or Tyr; Xaa’ = Ser, Pro, or Ala) and has been isolated from such diverse sources as the cockroach Leucophaea mu&rue (S), cricket A&eta domesticus (lo), locust L.ocusra migratoria (20), as well as mosqnitos Cufex salinarius (6) and Aedes aegypti (22). Although the first members of this peptide family

were isolated on the basis of their ability to stimulate contractions of the isolated cockroach hindgut (8), they have also been associated with diuretic activity in the cricket and mosquito. The L.eucophaea cockroach hindgut preparation is extremely sensitive to these myotropic peptides, with thresholds in the range between lo-” and lo-” M (12). Hayes et al. (7) have reported that leucokinins influence transepithelial membrane potential and rate of fluid secretion in isolated Malpighian tubules from the mosquito, Aedes aegypti. Coast et al. (5) have shown that the achetakinins at 10e9 M double the rate of fluid secretion by isolated Malpighian tubules of the cricket, Acheta domesticus, and demonstrate EC& values between lo-” ;md IO-” M. Therefore, this family of peptides may regulate w.ater and ion balance in addition to hindgut motility in insects. Utilizing an antibody to cockroach leucokinin I, a group of efferent neurons was stained in abdominal ganglia of flies (2), the cockroach Leucophaea maderae (19), and in larvae of several lepidopteran species (1). In the moth Agrotis segetum, leucoki-

In previous studies, we have shown that the N-terminal region of the core pentapeptide can be modified considerably without precipitating a complete loss of activity. Modification can be in-

troduced to the extent that up to three N-terminal residues of the

’ Requests for reprints should1be addressed to Dr. Ronald J. Nachman, USDA, ARS, FAPRL, 2881 F&B Road, College Station, TX 77845. 809

NACHMAN ET AL.

pentapeptide can be replaced with simple straight-chain aryl, acyl groups that retain only the phenyl ring of the phenylalanine side chain and maintain the distance between that aromatic ring and the Trp residue. The resulting pseudopeptide analogues retain considerable activity in both myotropic and diuretic assays [( 1416,18), Coast and Nachman, unpublished data]. Experimental spectroscopy, coupled with molecular dynamic studies on an active, conformationally restricted cyclic hexapeptide analogue of the insect kinins, suggests that the peptides adopt a turn conformation involving the Pro residue in the active core region during receptor interaction (17,20). In this study, we describe the effects of modification of the C-terminal group of a superagonist analogue of the insect kinins on activity in both the cricket Malpighian tubule secretion and cockroach hindgut contractile bioassays. The superagonist AFFPWG-NH, demonstrates from fivefold to an order of magnitude greater potency in both assay systems than the most active natural insect kinins and their pentapeptide active core regions ($12). The results shed light on insect kinin receptor requirements and tolerances, and provide direction for the design of enzyme-resistant peptidomimetic analogues with the potential to penetrate the cuticle and/or gut wall of an insect. METHOD

Cricket Malpighian

Tubule Secretion Bioassays

Crickets were reared as described (3) and fed a diet of turkey starter crumbs. Water was provided ad lib. Malpighian tubules were removed from 6-12-day-old adult virgin females. Single tubules were isolated in vitro as described (4). After a 4O-min equilibration period, the bathing fluid was changed and the rate of secretion, in picoliters per millimeter length of tubule per minute (pl/mm/min), was determined over 40 min (control rate). Thereafter, the bathing fluid was exchanged for one containing the assay material and the rate of secretion was determined over an additional 40-min period (experimental rate). Diuretic activity was calculated as the difference between control and experimental rates, and results are expressed as a percentage of the response obtained with a supermaximal dose (10 nM) of achetakinin-I assayed alongside the test analogues. All experiments were performed at room temperature (21-24°C) (5). Cockroach Myotropic Biossay Leucophaea maderae cockroaches were taken from stock coionies maintained at 27°C and fed dry dog food ad lib. Cockroach hindguts, free of central nervous system (CNS) tissue, were dissected, immersed in saline, and prepared for recording of myogenie activity as previously described (9). Threshold concentrations were determined for each analogue by adding a known quantity of peptide (dissolved in 0.5 ml bioassay saline) to the bioassay chamber containing the hindgut and observing the re.sponse on a Gould 2200s oscillograph recorder. The quantity of the peptide analogues was calculated from the values obtained for Phe in the amino acid analysis. The threshold concentration was defined as the minimum concentration of peptide in the chamber required to evoke an observable change in the frequency (50%) or amplitude of contractions (5%) within 1 min and sustained for 3 min. Threshold concentrations were obtained from five cockroach hindguts on 5 consecutive days for each peptide. Quantitative data for dose-response plots were obtained as previously described (9). C-Terminal Group Peptide Analogue Synthesis The syntheses of analog,ues AFFPWG-GCH,, -NH>, -NHCH3, -N(CH&, and AFFPWG/-CH,C(O)NH, [i.e., AFFPW@Ala)-

NH*] have been previously described (18). A series of analogues with varying numbers of methylene group spacers between the (Ycarbon and amide group of the Gly in AFFPWG-NH2 was synthesized using r-Boc methodology with p-methylbenzylhydrylamine resin on a Biosearch 9600 synthesizer according to previously described procedures (13). Boc-Ala, Boc-Phe, Boc-Trp, and BocGly protected amino acids were purchased from Advanced Chemtech (Louisville, KY), and Boc-y-aminobutyric acid (Boc-Abu), Boc-e-aminocaproic acid (Boc-Aca) and Boc-7-amino heptanoic acid (Boc-Aha) were purchased from Bachem Bioscience Inc. (Philadelphia, PA). C-terminally modified insect kinin analogues were synthesized by treating 100 pg of the peptide-resin complex Ala-Phe-Phe-Pro-Trp-Gly-PAM, prepared via t-Boc methodology on a Biosearch 9600 synthesizer under previously described conditions (13) with ethanol/diisopropylethyhunine (DIEA), methanetbiol/DIEA, benzyl alcohol/DlEA, (CH,bCHCH,CH,owDIEA, CH@CH,CH,OH/DIEA, and CH,GCH,CH#H, for 2 h at room temperature. The resin was washed with anhydrous DMF and the solvent and volatile reagents were removed in vacua. The crude peptide analogues were purified on a Delta Pak Cl8 reverse-phase column on a Waters Model 5 10 HPLC controlled with a Millenium 2010 chromatography manager system (Waters-Milligen, Marlborough, MA) with detection at 214 nM at ambient temperature. Solvent A was 0.1% aqueous trifluoroacetic acid (TFA); solvent B was 50% or 80% aqueous acetonitrile containing 0.1% TFA. Conditions: initial solvent consisting of 20% B was followed immediately by Waters linear program 6 to 100% B over 40 min; flow rate: 2 ml/mm. Pure peptide samples were quantitated by analysis of the Phe concentration on a Waters Picotag amino acid analysis system (see below). The concentrations were also checked by monitoring Trp fluorescence according to previously described pro cedures (11). Peptide analogues demonstrated the following retention times (in minutes): AFFPWG-NH,, 30.1 (50% B); AFFPWGGCH3, 24.3 (80% B); AFFPWG-GCH2CH3, 44.2 (50% B); AFFPWG-NHCH3, 29.3 (80% B); AFFPWG-0C6Hs, 31.8 (80% B); AFFPWG-NHCH,CH,OCH,, 38.7 (50% B); AFFPWGGCH,cH@CH,, 42.7 (50% B); AFFPWG-GCH,CH&H(CH&, 54.8 (50% B) or 33.8 (80% B); AFFPWG\-CH2C(0)NH2 [AFFPW&4la)-NH*], 27.7 (80% B); AFFPWG\-(CH&C(0)NHZ [AFFPW(Abu)-NHJ 24.4 (50% B); AFFPWG\-(CH&C(0)NHZ ]AFFPW(Aca)-NH*], 26.0 (50% B); AFF’PWG\-(CH&C(0)NHZ [AFFPW(Aha)-NH*], 39.2 (50% B). Amino Acid Analysis

The amino acid content of the peptides purified with HPLC was determined with a Waters Picotag amino acid analysis system. Samples were hydrolyzed in vacua with gaseous HCl at 150°C for 1 h, neutralized, converted to the phenylthiocarbamyl (PTC) derivatives, and analyzed according to the instructions supplied by the manufacturer using a Waters ALC100 HPLC instrument. Quantification of the amino acids was performed by comparing sample peak areas with the peak areas determined from analyses of an amino acid standard solution supplied by the manufacturers. Synthetic peptides had the following analyses: AFFPWG-OCHICHX-A (l.O), F (2.0), G (l.l), P (1.0); AFFPWG-SCHs-A (0.9), F (2.0), G (l.l), P (1.1); AFFPWG-0C6H5--A (0.9), F (2.0), G (l.O), P (1.0); AFFPWG-GCH#ZH#ZH(CHy)2-A (0.9), F (2.0), G (l.l), P (1.0); AFFPWG-OCH~CHZOCH~-A (1 .O), F (2.0), G (l.l), P (1.0); AFFPWG-NHCH&H20CHX-A (0.9), F (2.0), G (l.O), F (1.0); AFFPW(Abu)-NHZ-A (l.O), F (2.0), P (1.1); AFFPW(Aca)-NH2-A (0.9), F (2.0), P (1.0); AFFPW(Aha)-NH,-A (l.O), F (2-O), P (1.2); AFFPWGOH-A (0.9), F (2.0), G (l.l), P (1.1).

DIURETIC ACTIVITY OF INSECT KININ C-TERMINAL

811

ANALOGUES

strated a range of ECsOvalues for the cricket diuretic assay from 5 to 140 n&f with X = -SCHs > -OCH3 > -OCH&I-I~) > -OCH$ZH, > -O(CH&,CH3 (Table 1). The analogue AF’FPWGOCHrCH,CH(CH& with a branched chain X group proved to be inactive. The diuretic maximal response ranged from a high of 115% for the analogue containing -SCH3 to a low of 54% for the analogue with -OCH2(C6H5) compared with the parent peptide (X = NH,). In the cockroach myotropic assay, threshold concentrations for this ester series ranged from a high of 1 n&f to a low of 4 @f with X = -OCHS > -SCH3 > -OCH2CH3 > -OCH2CH20CH3 > -OCH&I-&) (Table 1). The analogue AFFPWG-0CH2CH2CH(CH& with a branched chain X group was inactive. The amide analogue AFFPWG-NHCH, was observed to have an ECso value of 28 r&f, whereas the branched chain, dimethyl analogue [X = N(CH&] proved inactive (18). The extended amide analogue AFFPWG-NHCH2CH@CHS demonstrated relatively strong activity with a diuretic ECso value of 6 nM and with a full maximal response. In the myotropic assay, the analogue AFFPWG-NHCH, demonstrated a potent threshold response at a concentration of 0.7 nM, whereas the branched chain analogue AFFPWG-N(CH& proved inactive. The analogue AFFPWGNHCH2CH20CH3 demonstrated a myotropic threshold response at 100 n&f. A series of analogues containing methylene group spacers between the o-carbon of the C-terminal Gly residue and the amide group was also evaluated in both assay systems (Fig. 1). In Table 1, the analogues are identified as AFFPWG\--X where the X, preceded by a backward slash and hyphen, denotes the molecular entity following the o-carbon of Gly. The diuretic activity of analogues containing one to four methylene spacers ranged from an EC5,, of 0.4 to 2.5 n&f, whereas the analogue containing five methylene group spacers proved inactive. The order of activity

Fast Atom Bombardment Mass Spectroscopy Fast atom bombardment (FAB) mass spectra of the peptide analogues were obtained ( 16) by adding 1O-50 pg of the peptide sample in DMF (1 ml) to glycerol (1.5 ml) on stainless steel targets (6 X 1.5 mm) followed by bombardment with fast xenon atoms from a saddle-field cold cathode ion gun adjusted to 8 KV and 1 mA current using xenon gas as the partide source on a VG 70-250 EHF mass spectrometer (VG Analytical). The structural identity and a measure of the purity of the peptides was confirmed by the presence of the following (MH+) ions and the absence of contaminant ions in the mass spectra: AFFPWG-OH, 724.3 (talc. MH’: 724.34); AFFPWG-OCHrCHs, 782.5 (talc. MH+: 782.39); AFFPWG-SCH3, 754.5 (talc. MH+: 754.34); AFFPWG-O&,HS, 814.9 (talc. MH+: 814.3!)); AFFPWG-0CH2CH2CH(CH3)2, 794.3 (talc. MH’: 794.42) AFFPWG-0CH2CH20CH3, 782.5 (talc. MH’: 782.39); AFFPWG-NHCH2CH20CHs, 781.6 (talc. MH+: 781.40); AFFPW(Abu)-NH,, 751.5 (talc. MH+: 751.39); AFFPW(Aca)-NH2, 779.5 (talc. MH+: 779.42); AFFPW(Aha)NH2, 793.7 (talc. MH+: 7938.44). RESULTS

The superagonist analogue Ala-Phe-Phe-Pro-Trp-Gly-NH2 (AFFPWG-NH,) (18) was chosen as the parent peptide with which to compare modified analogues (Table 1). The table is organized to list modifications of the C-terminal X group of AFFPWG-X and the observed Et& and maximal responses in the cricket Malpighian tubule bioassay along with threshold concentrations in the cockroach hindgut myotropic bioassay. The acid analogue (X = OH) has been previously shown to be inactive in the cockroach myotropic bioassay (18), and demonstrates only a weak EC& of 1000 nM (maximal response = 70%) in the cricket diuretic assay. A series of ester analogues demon-

TABLE 1 DIURETIC AND MYOTROPIC ACTIVITY

AFFPWG-X NH2 OH WB, SCH3

OF C-TERMINAL

GROUP ANALOGUES

OF THE INSECT KININS

Cricket Mslpighian Tubule Secretion

Diuretic Maximal

Cockroach Hindgut Contractile Threshold

Stimulation ECw, (nM)

Response

Concentration (t&f)*

0.004 1000 20 5 100 50 Inactive$ 140 28 Inactive* 6

100 70 88 115 54 83 -

111

1 0.4 2.5 Inactive$

94 94 82 -

Relative Potenciest Diuretic

Myotropic

10,ooo 0.04 2 8 0.4 0.8 Inactive* 0.3 0.4 Inactive$ 7

10,000 Inactive* 70 0.4 0.1 0.02 Inactive* 0.07 100 Inactive* 2

35 88 14 Inactive*

70

(X)

GCHrCH, GCHZ’QIH, GCH,CH,CH(CH,), GCH&H@CH3 NHCH, N(C%)z NHCH2CH#BCH9 APFI’WG/-X (X) -CH,C(O)NH,

-(CB2kC(QN& -(CBz),C(O)NB, -(CB&C(O)NH,

94 70 -

0.007 (12) Inactive$ l (16) 200 490 3700

Inactive$ 1000

0.7 (16) Inactive* (16) 30

1 (16) 78 20 2100

4 0.03

* Mean for five detenninations. 7 Relative potencies are presented in comparison with the superagonist parent analogue, which is assigned a value of 10,000. The superagonist parent ana:logue AFFPWG-NH2 demonstrates five-fold to one order of magnitude greater potency in both assay systems than the most active natural insect kinins and their pentapeptide active core regions. t Inactive up to 10V6J4. 5 With this notation X is defined as the molecular entity following the o-carbon of the C-terminal Gly residue.

812

NACHMAN ET AL.

m

GIY

AFFPW - NH - CH, -

‘d-

NH,

AFFPW - NH - CHz . CHz.

C-

AFFPW - NH.

CH*. ‘d-

NH2 0

CH, . CHz.

NH, 0

AFFPW - NH - CH,

. CHz. CH* . CH,. CH2. !-

AFFPW - NH -Cl-h

- CHs . CH, . CH, . CH2. CH, . ‘d-

NH* 0 NH1

FIG. 1. A series of insect kinin analogues incorporating methylene group spacers between the a-carbon and amide group of the C-terminal Gly residue. At the top is AFFPWG-NH,, followed by AFPPW(PAla)-NH, [i.e., APFPWG\-CH2C(0)NH2], AFFPW(Abu)-NH2 [i.e., APFPWG\-(CH,)2C(0)NH.J, AFFPW(Aca)-N&-NH, [i.e., APPPWG\ -(CH,),C(O)NH,], and AFFPW(Aha)-NH2 [i.e., AFFPWG\-(CH2)5C(O)NH?] (see Table I). was X = -(CH2)$(0)NH2 zz -CH2C(0)NH, 2 -(CH&C(O)NHr >>> -(CH&C(0)NH2 (inactive) (Table 1). In the myotropic assay the threshold concentration values ranged from a high of 1 nM to a low of 2 $4. The order of potency was X = -CH#Z(O)NH, > -(CH&C(O)NH, > -(CH&C(O)NHr >> -(CH,),C(0)NHz (Table 1). DISCUSSION The very weak

activity observed for the C-terminal acid analogue AFFFWG-OH in the cricket diuretic assay is probably due to an unfavorable interaction between the negative charge of the carboxyl moiety and the receptor site rather than an absence of amide hydrogens. The methyl ester analogue AFFPWG0CH3, which lacks both a negatively charged C-terminal species and amide hydrogens, demonstrates considerably stronger retention of diuretic activity than AFFPWG-OH. The activity of these analogues in the diuretic assay mirrors that observed for the myotropic bioassay (18). The monomethyl analogue AFFPWGNHCH3, containing an amide hydrogen, is an order of magnitude less potent than the corresponding methyl ester AFFPWG-OCH, in the diuretic assay but slightly more potent in the myotropic assay. The ester analogue AFFPWG-SCH3, containing a larger and more hydrophobic sulfur instead of an oxygen, proves to be slightly more potent than AFFPWG-OCHs in the diuretic bioassay and slightly less potent in the myotropic bioassay. The analogues AFFFWG-0CH2CH3 and AFFFWG-OCH,CHrOCH,, featuring extensions of the ester chain length, demonstrate further reductions of less than one order of magnitude in diuretic activity and reductions of two and one-half and three orders of magnitude, respectively, in myotropic activity. The insect kinin receptors appear to be particularly intolerant to branched chain character at the C-terminal amide or ester group. For example, the sterically hindered N,N-dimethyl analogue AFFFWG-N(CH,), proved inactive in both bioassay systems. Movement of the position of the branched chain away from the C-terminal cr-carbon and carbonyl group, as in the ester analogue AFFFWG-OCH,CH,CH(CH,),, did not result in a restoration of biological activity. However, activity can be partially restored by incorporating branched carbons into a planar aromatic ring as in the benzyl ester analogue AFFFWG-

OCH,(C&), which demonstrates a relatively strong EC,, of 5.0 X lo-* M in the cricket diuretic assay and a weaker threshold of 3.7 X 10m6M in the cockroach myotropic assay. In the final series of analogues, the distance between the LYcarbon and amide group of the C-terminal Gly residue is modified via the incorporation of methylene group spacers (Fig. 1). The presence of one to four methylene groups leads to a drop in the diuretic EC& of about two orders of magnitude, reaching a plateau ranging from 0.4 to 2.5 r&f (Table 1). Interestingly, the most potent analogue in the diuretic assay contains two methylene groups. Maximal Malpighian tubule secretion rates were not significantly altered by these modifications as they were observed to approach that of the parent peptide for the three analogues. However, the analogue containing five methylene groups proved inactive. A precipitous drop in activity was also observed for the analogue with five methylene groups in the myotropic bioassay. Thus, both the cricket Malpighian tubule and cockroach hindgut myotropic receptors are relatively intolerant to the presence of cr-carbon-amide distances spanning five methylene groups, but can accommodate to a reasonable degree the presence of four or fewer methylene groups. In conclusion, the C-terminal amide group of the insect kinins can be modified without precipitating a complete loss of activity in either Acheta diuretic or cockroach myotropic bioassay systems. Activity lost upon replacement of the Cterminal amide with the negatively charged acid group can be partially restored with the introduction of ester moieties. This introduces the possibility of synthesizing peptide and/or pseudopeptide mimetic analogues of the insect kinins with greater hydrophobicity to penetrate the insect cuticle or gut wall and/ or greater steric bulk, which offers the potential for increased stability to degradative peptidase enzymes. However, the introduction of branched chain character or an a-carbon-amide distance encompassing five or more methylene groups is not compatible with the insect kinin-receptor interaction required for biological activity. A comparison of the activity profiles for the two bioassays indicates that, taken as a whole, the two receptors that mediate these disparate physiological responses in two different insects respond similarly to the C-terminal analogues. However, individual analogues can demonstrate different relative potencies in the two bioassay systems. The C-terminal group analogues containing -SCH3, -NHCH$I-12OCHs, and -OCH2(C6H5) moieties offered the greatest retention of diuretic activity, coupled with increased hydrophobicity and steric bulk. For the cockroach hindgut myotropic response, incorporation of -OCHj and -NHCH2CHZOCHS moieties proved to be the most promising C-terminal modifications. The C-terminally modified analogues presented here shed more light on diuretic and myotropic receptor requirements and/or tolerances for biological activity. This information, along with the results of previous studies focusing on N-terminal modifications, is valuable for the development of peptidomimetic analogues of this important insect peptide family. In addition, although the most active C-terminally modified analogues are about two orders of magnitude less potent than the analogue AFFPWG-NH*, this analogue is a superagonist and demonstrates between fivefold and one order of magnitude greater potency in both bioassay systems than the most active natural insect kinin peptides. With Et& values of between 0.4 and 5 nM in the diuretic assay, many of these analogues display quite potent responses that are well within the physiological range. Furthermore, C-terminal modifications offer the potential to introduce both greater steric bulk to increase peptidase resistance and/or greater hydrophobic character to facilitate passage through the insect cuticle and/or gut wall.

DIURETIC ACTIVITY OF INSECT KININ C-TERMINAL

Pseudopeptide and nonpeptide mimetic agonists/antagonists represent valuable tools to insect neuroendocrinologists studying diuresis mediated by the insect kinin family. Because of improved stability to peptidase degradation and hydrophobicity of mimetic analogues, they also offer the potential to disrupt the diuretic and digestive processes regulated by the insect kinins and/or modify normal insect behavior and there-

813

ANALOGUES

fore may form the basis for future pest insect management strategies. ACKNOWLEDGEMENTS

We wish to acknowledge

the capable technical

assistance

of J. Coo-

per, A. Tyler, G. Rothrock, and T. Peterson; and NATO Grant No. 90248 for financial

support (R.J.N., G.M.C., and G.M.H.).

REFERENCES 1. Cantera, R.; Hansson, B. S.; Hallberg,

2.

3.

4.

5.

6.

7.

8.

9.

10.

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E.; NLsel, D. R. Postembryonic development of leucokinin I-immunoreactive neurons innervating a neurohemal organ in the turnip moth Agcotis segetum. Cell Tissue Res. 269:65-7’7; 1992. Cantera, R.; NLsel, D. R. Segmental peptidergic innervation of abdominal targets in larval and adult dipteran insects revealed with an antiserum against leucokinin I. Cell Tissue Res. 269:459-47 1; 1992. Clifford, C. W.; Roe, R. Mr.; Woodring, J. P. Rearing methods for obtaining house crickets, A&eta domesticus, of known age, sex and instar. Ann. Entomol. Sot. Am. 70~69-74; 1977. Coast, G. M. Fluid secretion by single isolated Malpighian tubules of the house cricket, Acheto domesticus, and their response to diuretic hormone. Physiol. Enlomol. 13:381-391; 1988. Coast, G. M.; Holman, G. M.; Nachman, R. J. The diuretic activity of a series of cephalomyotropic neuropeptides, the achetakinins, on isolated Malpighian tubules of the house cricket, A&eta domesticus. J. Insect Physiol. 36(7):481-488; 1990. Hayes, T. K.; Holman. G. M.; Pannabecker, T. L.; et al. Culekinin depolarizing peptide: A mosquito leucokinin-like peptide that influences insect Malpighian tubule ion transport. Regul. Pept. 52:235248; 1994. Hayes, T. K.; Pannabecker, T. L.; Hinckley, D. J.; et al. Leucokinins, a new family of ion transport stimulators and inhibitors in insect Malpighian tubules. Life Sci. 44: 1259- 1266; 1989. Holman, G. M.; Cook, B. J.; Nachman, R. J. Isolation, primary structure, and synthesis of leucokinins VII and VIII: The final members of this new family of cephalomyotropic peptides isolated from head extracts of Leucophaea muderue. Comp. B&hem. Physiol. [C] 88(1):31-34; 1987. Holmau, G. M.; Nachman, R. J.; Schoofs, L.; Hayes, T. K.; Wright, M. S.; DeLoof, A. The Le,ucophaeu maderae hindgut preparation: A rapid and sensitive bioalrsay tool for the isolation of insect myotropins of other insect species. Insect B&hem. 21( 1): 107-I 12; 1991. Holman, G. M.; Nachman, R. J.; Wright, M. S. A strategy for the isolation and structural characterization of certain insect myotropic peptides that modify spontaneous contractions of the isolated cockroach hindgut. In: McCaffery, A. R.; Wilson, I. D., eds. Chromatography and isolation of insect hormones and pheromones. New York: Plenum Press; 1990: 195-204. Nachman, R. J.; Coast, G. M.; Holman, G. M.; Haddon, W. F. A bifunctional heterodimeric insect neuropeptide analog. Int. J. Pept. Protein Res. 40:423-428; 1992.

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