Intracellular Na+, K+ and Cl- activities in Acheta domesticus Malpighian tubules and the response to a diuretic kinin neuropeptide.

The mechanism of primary urine production and the activity of a diuretic kinin, Achdo-KII, were investigated in malpighian tubules of Acheta domesticus by measuring intracellular Na(+), K(+) and Cl(-) activities, basolateral membrane voltage (V(b)), fluid secretion and transepithelial ion transport. Calculated electrochemical gradients for K(+) and Cl(-) across the basolateral membrane show they are actively transported into principal cells, and basolateral Ba(2+)-sensitive K(+) channels do not contribute to net transepithelial K(+) transport and fluid secretion. A basolateral Cl(-) conductance was revealed after the blockade of K(+) channels with Ba(2+), and a current carried by the passive outward movement of Cl(-) accounts for the hyperpolarization of V(b) in response to Ba(2+). Ion uptake via Na(+)/K(+)/2Cl(-) cotransport, driven by the inwardly directed Na(+) electrochemical gradient, is thermodynamically feasible, and is consistent with the actions of bumetanide, which reduces fluid secretion and both Na(+) and K(+) transport. The Na(+) gradient is maintained by its extrusion across the apical membrane and by a basolateral ouabain-sensitive Na(+)/K(+)-ATPase. Achdo-KII has no significant effect on the intracellular ion activities or V(b). Electrochemical gradients across the apical membrane were estimated from previously published values for the levels of Na(+), K(+) and Cl(-) in the secreted fluid. The electrochemical gradient for Cl(-) favours passive movement into the lumen, but falls towards zero after stimulation by Achdo-KII. This coincides with a twofold increase in Cl(-) transport, which is attributed to the opening of an apical Cl(-) conductance, which depolarises the apical membrane voltage.

Biostable agonists that match or exceed activity of native insect kinins on recombinant arthropod GPCRs.

The multifunctional arthropod 'insect kinins' share the evolutionarily conserved C-terminal pentapeptide motif Phe-X(1)-X(2)-Trp-Gly-NH(2), where X(1)=His, Asn, Ser, or Tyr and X(2)=Ser, Pro, or Ala. Insect kinins regulate diuresis in many species of insects. Compounds with similar biological activity could be exploited for the control of arthropod pest populations such as the mosquito Aedes aegypti (L.) and the southern cattle tick Rhipicephalus (Boophilus) microplus (Canestrini), vectors of human and animal pathogens, respectively. Insect kinins, however, are susceptible to fast enzymatic degradation by endogenous peptidases that severely limit their use as tools for pest control or for endocrinological studies. To enhance resistance to peptidases, analogs of the insect kinins incorporating bulky alpha,alpha-disubstituted amino acids in positions adjacent to both primary and secondary peptidase hydrolysis sites were synthesized. In comparison with a control insect kinin, several of these analogs are highly stable to hydrolysis by degradative enzymes ANCE, neprilysin and Leucine aminopeptidase. Six analogs were evaluated by calcium bioluminescence assay on recombinant receptors from mosquito and tick. Four of these analogs either matched or exceeded the potency of the control kinin peptide agonist. One of these was about 5-fold more potent than the control agonist on the tick receptor. This analog was 8-fold more potent than the control agonist on the mosquito receptor, and twice more potent than the endogenous Aedes kinin-II. The analog also demonstrated potent activity in an in vitro Aedes Malpighian tubule fluid secretion assay. Similar comparisons of analog potency cannot be made to tick kinins because no endogenous kinin has yet been identified. These potent, biostable analogs represent ideal new tools for endocrinologists studying arthropod kinin-regulated processes in vivo, particularly for ticks in which their role remains to be established.

Structure-activity relationships for in vitro diuretic activity of CAP2b in the housefly.

A series of truncated and Ala-replacement analogs of the peptide Manse-CAP2b (pELYAFPRV-NH(2)) were assayed for diuretic activity on Malpighian tubules of the housefly Musca domestica (M. domestica). The C-terminal hexapeptide proved to be the active core, the minimum sequence required to retain significant diuretic activity. However, full activity required the C-terminal heptapeptide, which was equipotent with the most active of the native housefly CAP2b peptides. Replacement of Arg(7) and Val(8) with Ala led to inactivity and a large 70-fold drop in potency, respectively, indicating that these were critical residues. The Leu(2) was semicritical, where a six-fold loss in potency was observed. Conversely, the replacement of all other residues with Ala led to much smaller effects on potency and these positions were considered to be noncritical. This structure-activity relationship data can aid in the design of mimetic agonist/antagonist analogs of this diuretic peptide family with enhanced biostability and bioavailability, as tools for arthropod endocrinologists and as potential pest management agents capable of disrupting the water balance in pest flies.

A C-terminal aldehyde analog of the insect kinins inhibits diuresis in the housefly.

The insect kinins are present in a wide variety of insects and function as potent diuretic peptides in flies. A C-terminal aldehyde insect kinin analog, Fmoc-RFFPWG-H (R-LK-CHO), demonstrates stimulation of Malpighian tubule fluid secretion in crickets, but shows inhibition of both in vitro and in vivo diuresis in the housefly. R-LK-CHO reduced the total amount of urine voided over 3 h from flies injected with 1 microL of distilled water by almost 50%. The analog not only inhibits stimulation of housefly fluid secretion by the native kinin Musdo-K, but also by thapsigargin, a SERCA inhibitor, and by ionomycin, a calcium ionophore. The activity of R-LK-CHO is selective, however, as related C-terminal aldehyde analogs do not demonstrate an inhibitory response on housefly fluid secretion. The selective inhibitory activity of R-LK-CHO on housefly tubules represents an important lead in the development of environmentally friendly insect management agents based on the insect kinins.

The effects of intracellular signalling pathway inhibitors on phagocytosis by haemocytes of Manduca sexta.

The intracellular signalling pathways activated during phagocytosis by larval haemocytes of a lepidopteran, Manduca sexta, were investigated. Using fluorescein-labelled Escherichia coli as bioparticles, a fluorescence-based assay was used to quantify phagocytosis by haemocytes in monolayers in vitro, and the intracellular signalling pathways involved in phagocytosis were examined using inhibitors. Pathways known to be involved in phagocytosis by mammalian cells were selected for the study in haemocytes, and the amino acid sequences of human isoforms of the selected protein targets were used to conduct searches of two completed databases of insect proteins, those of Drosophila melanogaster and Anopheles gambiae and EST databases of moths Bombyx mori and M. sexta. Decreases in phagocytosis produced by pathway inhibitors indicated that tyrosine phosphorlylation phosphatidylinositol 3-kinase (PI3-kinase) and mitogen-activated protein kinase/extracellular-regulated kinase (ERK/MAPK) were required for internalisation of bacteria. Inhibition of Syk, a mammalian kinase, also decreased phagocytosis. JNK/SAPK did not seem to be involved in phagocytosis. The presence of protein phosphatases probably regulates phagocytosis at the intracellular level by dephosphorlyation of serine/threonine residues.

Identification of PVK/CAP2b neuropeptides from single neurohemal organs of the stable fly and horn fly via MALDI-TOF/TOF tandem mass spectrometry.

MALDI-TOF/TOF tandem mass spectrometry has been applied to determine the complete sequences of the PVK/CAP2b neuropeptides in the stable fly Stomoxys calcitrans and horn fly Haematobia irritans, insect pests of livestock. This peptidomic analysis of single neurohemal organ preparations allows the unambiguous assignment of internal Leu/Ile positions not distinguishable by previous mass spectrometric techniques. The sequences are as follows: Stoca-PVK/CAP2b-1, AGGASGLYAFPRVa; Stoca-PVK/CAP2b-2, NAKLYPVPRVa; and Haeir-PVK/CAP2b-1, AGGASGLYAFPRVa; Haeir-PVK/CAP2b-1, NAKLYPMPRVa. Both Stoca-PVK/CAP2b-1 and -2 stimulate Malpighian tubule fluid secretion in the stable fly, with EC50 values between 3 and 11 nM. The identification of these novel neuropeptides adds to our knowledge of the peptidomes of flies, and can aid in the development of neuropeptide-based control strategies of these insect pests.

Neuropeptide control of fluid balance in insects.

This minireview considers various aspects of the control of hydromineral balance in insects with particular reference to the control of diuresis and natriuresis in mosquitoes, with new information on the diuretic peptides of Anopheles gambiae.

Mass spectrometric assignment of Leu/Ile in neuropeptides from single neurohemal organ preparations of insects.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry has been applied for the first time on an insect/arthropod target, focusing on PVK/CAP2b neuropeptides in the housefly Musca domestica and flesh fly Neobellieria bullata. The peptidomic analysis of single neurohemal organ preparations allows the unambiguous assignment of internal Leu/Ile positions not distinguishable by previous mass spectrometric techniques. The confirmation of side-chain fragments which allows assignment of Leu/Ile even from samples as small as neurohemal organs will greatly accelerate the identification of novel neuropeptides that are implicated in the regulation of critical physiological processes in insects. The unnatural Ile analog is 4.5 times more active than the native Leu sequence in a housefly Malpighian tubule fluid secretion assay, which reinforces the caveat that potency values in a biological assay cannot be relied upon to predict the native sequence.

Diuretic and myotropic activities of N-terminal truncated analogs of Musca domestica kinin neuropeptide.

Musca kinin (Musdo-K; NTVVLGKKQRFHSWG-NH(2)) and N-terminal truncated analogs of 4-14 residues in length were assayed for diuretic and myotropic activity on housefly Malpighian tubules and hindgut, respectively. The pentapeptide was the minimum sequence required for biological activity, but it was > 5 orders of magnitude less potent than the intact peptide. The pharmacological profiles of the different analogs in the two assays were very similar, suggesting the same receptor is present on both tissues. Potency was little affected by the deletion of Asn(1), but was reduced > 10-fold after the removal of Thr(2). Deletion of the next 5 residues had relatively little effect, but after the second lysyl residue (Lys(8)) was removed potency fell by one to two orders of magnitude. There was a similar drop in potency after the removal of Arg(10), and at 100 microM the pentapeptide had only 20% of the diuretic activity of the intact peptide. The importance of Arg(10) was confirmed by comparing dose-response curves for Musdo-K [6-15] and Acheta kinin-V (AFSHWG-NH(2)) in the diuretic assay; the substitution of arginine by alanine produced a significant reduction in potency and some loss of activity.

cis-peptide bond mimetic tetrazole analogs of the insect kinins identify the active conformation.

The insect kinin neuropeptides have been implicated in the regulation of water balance, digestive organ contraction, and energy mobilization in a number of insect species. A previous solution conformation study of an active, restricted-conformation cyclic analog, identified two possible turn conformations as the likely active conformation adopted by the insect kinins at the receptor site. These were a cisPro type VI beta-turn over C-terminal pentapeptide core residues 1-4 and a transPro type I-like beta-turn over core residues 2-5, present in a ratio of 60:40. Synthesis and evaluation of the diuretic activity of insect kinin analogs incorporating a tetrazole moiety, which mimics a cis peptide bond, identifies the active conformation as the former. The discovery of a receptor interaction model can lead to the development of potent agonist and antagonist analogs of the insect kinins. Indeed, in this study a tetrazole analog with D stereochemistry has been shown to demonstrate partial antagonism of the diuretic activity of natural insect kinins, providing a lead for more potent and effective antagonists of this critical neuropeptide family. The future development of mimetic agonists and antagonists of insect kinin neuropeptides will provide important tools to neuroendocrinologists studying the mechanisms by which they operate and to researchers developing new, environmentally friendly pest insect control strategies.

Enhanced in vivo activity of peptidase-resistant analogs of the insect kinin neuropeptide family.

The diuretic/myotropic insect kinin neuropeptides, which share the common C-terminal pentapeptide core FX(1)X(2)WG-NH(2), reveal primary (X(2)-W) and secondary (N-terminal to F) sites of susceptibility to peptidases bound to corn earworm (H. zea) Malpighian tubule tissue. Analogs designed to enhance resistance to tissue-bound peptidases, and pure insect neprilysin and ACE, demonstrate markedly enhanced in vivo activity in a weight gain inhibition assay in H. zea, and strong in vivo diuretic activity in the housefly (M. domestica). The peptidase-resistant insect kinin analog pQK(pQ)FF[Aib]WG-NH(2) demonstrates a longer internal residence time in the housefly than the native muscakinin (MK), and despite a difference of over 4 orders of magnitude in an in vitro Malpighian tubule fluid secretion assay, is equipotent with MK in an in vivo housefly diuretic assay. Aminohexanoic acid (Ahx) is shown to function as a surrogate for N-terminal Lys, while at the same time providing enhanced resistance to aminopeptidase attack. Peptidaese-resistant insect kinin analogs demonstrate enhanced inhibition of weight gain in larvae of the agriculturally destructive corn earworm moth. Potent peptidase resistant analogs of the insect kinins, coupled with an increased understanding of related regulatory factors, offer promise in the development of new, environmentally friendly pest insect control measures.

Occurrence of insect kinins in the flesh fly, stable fly and horn fly-mass spectrometric identification from single nerves and diuretic activity.

MALDI-TOF mass spectrometric analysis of single lateral abdominal nerves (LANs) demonstrate the presence of the insect kinin Musdo-K in the housefly Musca domestica, and identify heretofore unknown insect kinins in two other Dipteran species as Musdo-K in the stable fly Stomoxys calcitrans and horn fly Haematobia irritans. The insect kinin native to the flesh fly Neobellieria bullata is identified as Drome-K. Musdo-K and Drome-K are identical save for the conservative substitution of Ser for Thr in position 2. The sequences of the insect kinins are, therefore, remarkably conserved throughout Dipterans. The in vitro Malpighian tubule fluid secretion activity of Musdo-K in the stable fly is similar to that in the housefly, whereas that of Drome-K is 30-fold more potent in the flesh fly than in the fruit fly. Given the structural identities of the kinins and CRF-like diuretic hormones of these Dipteran species, the housefly can serve as a model insect for the study of diuretic peptides and their functions in the stable fly and horn fly, both livestock pests.

A C-terminal aldehyde insect kinin analog enhances inhibition of weight gain and induces significant mortality in Helicoverpa zea larvae.

The first reported examples of C-terminal aldehyde analogs of an insect neuropeptide are described. They are hexapeptide insect kinin analogs Boc-VFFPWG-H and Fmoc-RFFPWG-H. Activity observed for these modified analogs in an in vitro insect diuretic assay confirms that the C-terminal aldehyde group is tolerated by an insect kinin receptor. The two analogs demonstrate greatly enhanced activity over standard C-terminal amide insect kinins in a larval weight gain inhibition assay in the corn earworm Helicoverpa zea. Treatment with Boc-VFFPWG-H led to significant increases in larval mortality at doses of 500pm (45%) and 5nm (67%). Boc-VFFPWG-H represents a lead analog in the development of novel, environmentally friendly pest insect management agents based on the insect kinin class of neuropeptides.

Continuous recording of excretory water loss from Musca domestica using a flow-through humidity meter: hormonal control of diuresis.

Water loss from adult male houseflies was continuously recorded using a flow-through humidity meter, which enabled losses to be apportioned between the sum of cuticular and respiratory transpiration, salivation and excretion. Transpiration accounted for >95% of water lost from sham-injected flies, compared with excretion (3.0%) and salivation (2.4%). In contrast, excretion accounted for 40% of water lost from flies injected with > or =3 microl of saline, whereas salivary losses were unchanged. Saline injections (1-5 microl) expanded the abdomen in the dorsal-ventral plane, and this expansion was positively correlated with the magnitude of the ensuing diuresis, suggesting the signal for diuretic hormone release originates from stretch receptors in abdominal tergal-sternal muscles. The effects of decapitation, severing the ventral nerve cord within the neck or ligaturing the neck, showed the head was needed to initiate and maintain diuresis, but was neither the source of diuretic hormone nor did it control the discharge of urine from the anus. These findings indicate the head is part of the neural-endocrine pathway between abdominal stretch receptors and sites for diuretic hormone release from the thoracic-abdominal ganglion mass. Evidence is presented for Musdo-K having a hormonal role in the control of diuresis, although other neuropeptides may also be implicated.

Evaluation of insect CAP2b analogs with either an (E)-alkene, trans- or a (Z)-alkene, cis-Pro isostere identifies the Pro orientation for antidiuretic activity in the stink bug.

The CAP2b neuropeptide family plays an important role in the regulation of the processes of diuresis and/or antidiuresis in a variety of insects. While Manse-CAP2b (pELYAFPRV-NH2) and native CAP2bs elicit diuretic activity in a number of species of flies, native CAP2b sequences have been shown to elicit antidiuretic activity in the kissing bug Rhodnius prolixus and the green stink bug Acrosternum hilare, the latter being an important pest of cotton and soybean in the southern United States. Analogs of CAP2b containing either a (Z)-alkene, cis-Pro or an (E)-alkene, trans-Pro isosteric component were synthesized and evaluated in an in vitro stink bug diuretic assay, which involved measurement of fluid secretion by Malpighian tubules isolated from A. hilare. The conformationally constrained trans-Pro analog demonstrated statistically significant antidiuretic activity, whereas the cis-Pro analog failed to elicit activity. The results are consistent with the adoption of a trans orientation for the Pro in CAP2b neuropeptides during interaction with receptors associated with the antidiuretic process in the stink bug. In addition, the results are further consistent with a theory of ligand-receptor coevolution between the CAP2b and pyrokinin/PBAN neuropeptide classes, both members of the '-PRXamide' superfamily. This work further identifies a scaffold with which to design mimetic CAP2b analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting CAP2b-regulated diuretic/antidiuretic functions.

Active diuretic peptidomimetic insect kinin analogs that contain ß-turn mimetic motif 4-aminopyroglutamate and lack native peptide bonds.

The multifunctional 'insect kinins' of arthropods share the evolutionarily conserved C-terminal pentapeptide core sequence Phe-X(1)-X(2)-Trp-Gly-NH(2), where X(1)=His, Asn, Ser, or Tyr and X(2)=Ser, Pro, or Ala. Insect kinins regulate diuresis in many species of insects, including the house cricket, Acheta domesticus. Insect kinins, however, are susceptible to fast enzymatic degradation by endogenous peptidases that severely limit their potential use as tools for pest control or for endocrinological studies. To enhance resistance to peptidases, the core insect kinin sequence was structurally modified in this study to replace native peptide bonds susceptible to proteolytic degradation. These modifications include incorporation of two stereochemical variants of the ß-turn mimetic motif 4-aminogutamate in place of the X(1)-X(2) residues, insertion of a reduced peptide bond between residues Trp-Gly, and replacement of the Phe residue with a hydrocinnamyl group. The resulting biostable, peptidomimetic analogs contain no native peptide bonds and yet retain significant diuretic activity in an in vitro cricket Malpighian tubule fluid secretion assay, matching the efficacy of a native A. domesticus kinin (Achdo-KI). These novel analogs represent ideal new tools for endocrinologists studying arthropod kinin regulated processes in vivo, and provide leads in the development of novel, environmentally friendly pest insect management agents capable of disruption of the critical processes that kinins regulate.

Toward a consensus nomenclature for insect neuropeptides and peptide hormones.

The nomenclature currently in use for insect neuropeptide and peptide hormone families is reviewed and suggestions are made as to how it can be rationalized. Based upon this review, a number of conventions are advanced as a guide to a more rationale nomenclature. The scheme that is put forward builds upon the binomial nomenclature scheme proposed by Raina and Gäde in 1988, when just over 20 insect neuropeptides had been identified. Known neuropeptides and peptide hormones are assigned to 32 structurally distinct families, frequently with overlapping functions. The names given to these families are those that are currently in use, and describe a biological function, homology to known invertebrate/vertebrate peptides, or a conserved structural motif. Interspecific isoforms are identified using a five-letter code to indicate genus and species names, and intraspecific isoforms are identified by Roman or Arabic numerals, with the latter used to signify the order in which sequences are encoded on a prepropeptide. The proposed scheme is sufficiently flexible to allow the incorporation of novel peptides, and could be extended to other arthropods and non-arthropod invertebrates.

The control of Malpighian tubule secretion in a predacious hemipteran insect, the spined soldier bug Podisus maculiventris (Heteroptera, Pentatomidae).

Spined soldier bugs, Podisus maculiventris, are heteropteran insects that feed voraciously on other insects, particular the soft bodied larval forms of Lepidoptera and Coleoptera. The response of P. maculiventris Malpighian tubules (MTs) to serotonin and known diuretic and antidiuretic peptides has been investigated, and is compared with that of MT from the hematophagous and phytophagous heteropteran bugs Rhodnius prolixus and Acrosternum hilare, respectively. A CRF-related peptide diuretic hormone (DH) from the termite Zootermopsis nevadensis (Zoone-DH) stimulated MT secretion, which was reversed by a member of the CAP(2b) family of peptides from A. hilare (Acrhi-CAP(2b)-2), an antidiuretic effect. Serotonin had no effect on secretion, neither did a representative calcitonin-like DH, kinin, tachykinin-related peptide, and an antidiuretic factor from the mealworm Tenebrio molitor (Tenmo-ADFb) in both P. maculiventris or A. hilare. Serotonin is a DH in R. prolixus, and its lack of effect on MT from P. maculiventris and A. hilare suggests this is an adaptation to hematophagy. On the other hand, the antidiuretic activity of members of the CAP(2b) family in all three bugs is consistent with this being a heteropteran feature rather than a specialism for hematophagy.

Neurohormones implicated in the control of Malpighian tubule secretion in plant sucking heteropterans: The stink bugs Acrosternum hilare and Nezara viridula.

Plant sucking heteropteran bugs feed regularly on small amounts of K(+)-rich plant material, in contrast to their hematophagous relatives which imbibe large volumes of Na(+)-rich blood. It was anticipated that this would be reflected in the endocrine control of Malpighian tubule (MT) secretion. To explore this, neuroendocrine factors known to influence MT secretion were tested on MT of the pentatomid plant sucking stink bugs, Acrosternum hilare and Nezara viridula, and the results compared with previously published data from Rhodnius prolixus. Serotonin had no effect on N. viridula MT, although it stimulates secretion by R. prolixus MT >1000-fold, and initiates a rapid diuresis to remove excess salt and water from the blood meal. Kinins had no effect on stink bug MT, but secretion was increased by Zoone-DH, a CRF-like peptide, although the response was a modest 2-3-fold acceleration compared with 1000-fold in R. prolixus. Native CAPA peptides, which have diuretic activity in dipteran flies, had antidiuretic activity in MT of the stink bug (Acrhi/Nezvi-CAPA-1 and -2), as previously shown with Rhopr-CAPA-2 in R. prolixus. The antidiuretic activity of Rhopr-CAPA-2 has been linked with terminating the rapid diuresis, but results with stink bugs suggest it is a general feature of heteropteran MT.