A study of signal transduction for the two diuretic peptides of Diploptera punctata.

We investigated second messengers involved in the action of the CRF-related peptide Dippu-DH46 and the calcitonin-like peptide Dippu-DH31 in Diploptera punctata. Dippu-DH46 causes a dose-dependent increase in intracellular cAMP levels, its diuretic activity is mimicked by cAMP agonists, but is attenuated by Rp-cAMPS. Dippu-DH46 acts synergistically with kinins and thapsigargin; both mobilize intracellular Ca2+. Dippu-DH46 also acts synergistically with cAMP agonists, and its effect is inhibited by a PKC inhibitor, suggesting it also activates intracellular Ca2+. Dippu-DH31 has no effect on cAMP levels and its activity is not blocked by cAMP agonists. Neither peptide stimulated cGMP levels in a dose-dependent manner, nor does cGMP have any effect on fluid secretion.

The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules.

The Drosophila melanogaster homologue of an insect calcitonin-like diuretic hormone was identified in a BLAST search of the Drosophila genome database. The predicted 31-residue amidated peptide (D. melanogaster DH31; Drome-DH31) was synthesised and tested for activity on fruit fly Malpighian tubules. It increases tubule secretion by approximately 35 % of the response obtained with a myokinin from the housefly Musca domestica (muscakinin; Musdo-K) and has an EC50 of 4.3 nmol x l(-1). The diuretic activities of Drome-DH31 and Musdo-K were additive when tested at threshold and supra-maximal concentrations, which suggests that they target different transport processes. In support of this, Drome-DH31 increased the rate of secretion by tubules held in bathing fluid with a reduced Cl- concentration, whereas Musdo-K did so only in the presence of Drome-DH31. Stimulation with Drome-DH31 increased the lumen-positive transepithelial potential in the main secretory segment of the tubule. This was attributed to activation of an apical electrogenic proton-translocating V-ATPase in principal cells, since it was associated with hyperpolarisation of the apical membrane potential and acidification of secreted urine by 0.25 pH units. Exogenous 8-bromo-cyclic AMP and cyclic GMP increased tubule secretion to the same extent as Drome-DH31 and, when tested together with the diuretic peptide, their activities were not additive. Stimulation with Drome-DH31 resulted in a dose-dependent increase in cyclic AMP production by tubules incubated in saline containing 0.5 mmol x l(-1) 3-isobutyl-1-methylxanthine, whereas cyclic GMP production was unchanged. Taken together, the data are consistent with Drome-DH31 activating an apical membrane V-ATPase via cyclic AMP. Since the K+ concentration of the secreted urine was unchanged, it is likely that Drome-DH31 has an equal effect on K+ and Na+ entry across the basolateral membrane.

Diuresis in the housefly (Musca domestica) and its control by neuropeptides.

Diuresis was studied in vivo by measuring the loss of tritiated water. The basal rate of water loss (5 nl/min) represents respiratory and cuticular losses, whereas higher rates reflect urine output, which reaches 20 nl/min after injection of 1 microl distilled water. This response to hypervolemia involves release of a diuretic hormone(s) into the hemolymph. However, housefly diuretic peptides increased urine output to a maximum of only 7 nl/min, and higher rates may require fluid reabsorption from the hindgut to be reduced. Diuresis is partially blocked by injected anti-muscakinin antibodies, providing evidence of a hormonal function for this insect myokinin.

The distribution of a kinin-like peptide and its co-localization with a CRF-like peptide in the blood-feeding bug, Rhodnius prolixus.

Rhodnius prolixus, a blood-feeding hemipteran insect, ingests large meals which are followed by rapid diuresis to eliminate excess water and salt. In Rhodnius, serotonin and an unidentified peptide(s) [33,34] have been shown to act as neurohormonal diuretic factors. In other insects, two families of diuretic peptides, the corticotropin releasing factor (CRF)-like, and kinin peptides [9], have been identified and sequenced. Recently, we demonstrated the presence of a CRF-like diuretic peptide in the CNS and digestive system of Rhodnius [47] using immunohistochemistry and bioassay. In this study, combining immunohistochemistry and radioimmunoassay (RIA) techniques, we show the presence of leucokinin-like peptide(s) in the CNS and digestive system of Rhodnius 5th instar. Additionally, double-label immunohistochemistry demonstrates that the leucokinin-like and CRF-like peptides are co-localized in the posterior lateral neurosecretory cells of the mesothoracic ganglionic mass (MTGM) and in neurohaemal areas on abdominal nerves one and two, suggesting the possibility of co-release of the peptides into the hemolymph.Partially purified extracts of the CNS and neurohaemal tissue were tested in vitro on Malpighian tubule secretion and cAMP assays. The factors eluting with increasing acetonitrile percentages from Sep-Pak cartridges were assayed in the presence or absence of ketanserin, a serotonin antagonist which blocks the effects of serotonin on Malpighian tubules. The results indicate activity of serotonin and a CRF-like diuretic peptide on Rhodnius Malpighian tubules, but fail to demonstrate activity of the leucokinin-like peptide(s). The rapid diuresis following feeding is a highly coordinated event, requiring the movement of water and salt across the epithelial cells of the crop into the hemolymph, and from the hemolymph across the cells of the Malpighian tubules. The urine then travels along the Malpighian tubules into the hindgut in order to be expelled. The presence of a leucokinin-like peptide(s) in the CNS and digestive system, which co-localizes with a CRF-like peptide(s), suggests that kinins may play a role in the rapid diuresis, although possibly not directly on the Malpighian tubules.

Ovary maturing parsin and diuretic hormone are produced by the same neuroendocrine cells in the migratory locust, Locusta migratoria.

In the migratory locust, the CRF-related diuretic hormone that stimulates fluid secretion by the Malpighian tubules, and the ovary maturing parsin, a neurohormone able to stimulate oogenesis, are produced by the same neuroendocrine cells of the pars intercerebralis in the brain.

Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects.

Insect diuretic hormones are crucial for control of water balance. We isolated from the cockroach Diploptera punctata two diuretic hormones (DH), Dippu-DH(31) and Dippu-DH(46), which increase cAMP production and fluid secretion in Malpighian tubules of several insect species. Dippu-DH(31) and -DH(46) contain 31 and 46 amino acids, respectively. Dippu-DH(46) belongs to the corticotropin-releasing factor (CRF)-like insect DH family, whereas Dippu-DH(31) has little sequence similarity to the CRF-like DH, but is similar to the calcitonin family. Dippu-DH(46) and -DH(31) have synergistic effects in D. punctata but have only additive effects in Locusta migratoria. Dippu-DH(31) represents a distinct type of insect DH with actions that differ from those of previously identified insect peptides with diuretic activity.

Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A.

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors from Drosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome allowed us to identify a putative tachykinin-related peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from a Drosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel Drosophila tachykinin-related peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predicted Drosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution.

Characterisation of multiple trypsins from the midgut of Locusta migratoria.

Three isoforms of trypsin were identified in midgut preparations from Locusta migratoria. Ammonium-sulphate-fractionated luminal contents of midguts were subjected to benzamidine affinity chromatography; proteins eluted by benzamidine were then separated by anion-exchange chromatography. Cationic (TRY 1) and anionic (TRY 2) trypsin activities were eluted from the DEAE column. TRY 1 was homogeneous, producing a single band of Mr 23,000 on SDS-PAGE. TRY 2 comprised two trypsins, TRY 2A (Mr 27,000) and TRY 2B (Mr 29,000). Following a subsequent chromatography step using a Bio-Rad UNO Q column, TRY 2A and TRY 2B were resolved to homogeneity. When homogenates of midgut caecae were the starting material for chromatography, SDS-PAGE of benzamidine-eluted proteins revealed an additional putative trypsin of Mr 17,000 (termed SERP 17) which had been absent from luminal enzyme preparations. Determination of the N-terminal 11 amino acid residues of each protein revealed unique, but similar sequences. The four sequences all began with IVGG, a motif which signifies all four proteins are serine proteases. TRY 1, TRY 2A and TRY 2B were shown to contain only trypsin activity and the preparations were sensitive to inhibition by AEBSF, PMSF, TLCK, benzamidine, leupeptin, SBTI, BPTI and E64.

The distribution of a CRF-like diuretic peptide in the blood-feeding bug Rhodnius prolixus.

The blood-feeding bug Rhodnius prolixus ingests a large blood meal, and this is followed by a rapid diuresis to eliminate excess water and salt. Previous studies have demonstrated that serotonin and an unidentified peptide act as diuretic factors. In other insects, members of the corticotropin-releasing factor (CRF)-related peptide family have been shown to play a role in post-feeding diuresis. Using fluorescence immunohistochemistry and immunogold labelling with antibodies to the Locusta CRF-like diuretic hormone (Locusta-DH) and serotonin, we have mapped the distribution of neurones displaying these phenotypes in R. prolixus. Strong Locusta-DH-like immunoreactivity was found in numerous neurones of the central nervous system (CNS) and, in particular, in medial neurosecretory cells of the brain and in posterior lateral neurosecretory cells of the mesothoracic ganglionic mass (MTGM). Positively stained neurohaemal areas were found associated with the corpus cardiacum (CC) and on abdominal nerves 1 and 2. In addition, Locusta-DH-like immunoreactive nerve processes were found over the posterior midgut and hindgut. Double-labelling studies for Locusta-DH-like and serotonin-like immunoreactivity demonstrated some co-localisation in the CNS; however, no co-localisation was found in the medial neurosecretory cells of the brain, the posterior lateral neurosecretory cells of the MTGM or neurohaemal areas. To confirm the presence of a diuretic factor in the CC and abdominal nerves, extracts were tested in Malpighian tubule secretion assays and cyclic AMP assays. Extracts of the CC and abdominal nerves caused an increase in the rate of secretion and an increase in the level of cyclic AMP in the Malpighian tubules of fifth-instar R. prolixus. The presence of the peptide in neurohaemal terminals of the CC and abdominal nerves that are distinct from serotonin-containing terminals indicates that the peptide is capable of being released into the haemolymph and that this release can be independent of the release of serotonin.

Evidence for helicity in insect diuretic peptide hormones: computational analysis, spectroscopic studies, and biological assays.

The conformation of four insect diuretic hormones has been analyzed computationally using secondary structure prediction routines and comparison with structures in the Brookhaven Protein Databank. Based on this analysis, a common seven-residue peptide fragment (DVLRQRL) had a high probability of forming an alpha-helix. Circular dichroism (CD) studies found that addition of trifluoroethanol (TFE) to an aqueous solution of the seven-residue fragment induces a change from random coil to helix. Subsequent NMR studies in water-TFE (1:1) produced nOe values and 3JalphaNH coupling constants confirming a helical conformation: 3JalphaNH coupling constants for the first five residues (D1 to Q5) were all < or = 6.0 Hz and two medium-range nOe values (dalphaN (i,i+3)) were observed between V2 and Q5, and R4 and L7. The longer fragments PLDVLRQRL in water-TFE and Lom-DH 1-26 in water alone, both containing the DVLRQRL sequence of the locust (Locusta migratoria) diuretic hormone, maintained the helicity as determined by CD analysis. However, the remaining 20 residues of the locust diuretic hormone did not maintain the same amount of helicity in water and all of the truncated fragments were not biologically active.

Isolation, characterization and biological activity of a diuretic myokinin neuropeptide from the housefly, Musca domestica.

A competitive ELISA employing a polyclonal antiserum raised against leucokinin-I was used to isolate and purify a myokinin (muscakinin) from 1.05 kg of adult houseflies (Musca domestica). Following solid-phase purification, seven HPLC column steps were used to purify 4.8 nmol of leucokinin-immunoreactive material. Sequence analysis and mass spectrometry were consistent with the structure Asn-Thr-Val-Val-Leu-Gly Lys-Lys-Gln-Arg-Phe-His-Ser-Trp-Gly NH2. This peptide was synthesized and co-eluted with the natural peptide on three different HPLC columns. The activities of natural and synthetic muscakinin were identical, with both producing a 4-5 fold increase in fluid secretion by housefly Malpighian tubules at nanomolar concentrations. The presence of a pair of basic residues (Lys-Lys) suggested muscakinin might be processed further, with the peptide pGlu-Arg-Phe-His-Ser-Trp-Gly NH2 being produced by conversion of an N-terminal glutamine to pyroglutamic acid. However, this analog was 1000-fold less active than the intact peptide, comparable to the activity of AK-V which shares the same C-terminal pentapeptide sequence. The diuretic activity of muscakinin is more than double that of a previously identified CRF-related diuretic peptide (Musca-DP) from the housefly, and the two peptides act synergistically in stimulating fluid secretion. Muscakinin also increased the frequency and amplitude of contractions by housefly hindgut which might further contribute to the excretory process.

Comparison of active conformations of the insectatachykinin/tachykinin and insect kinin/Tyr-W-MIF-1 neuropeptide family pairs.

A comparison of solution conformations of active, restricted-conformation analogues of two sequence-similar insect/vertebrate neuropeptide family pairs shed light on the potential existence of molecular evolutionary relationships. Analogues of the locustatachykinins and the mammalian tachykinin substance P, containing a sterically hindered Aib-NMePhe/Tyr residue block, share similar low-energy turn conformations incorporating a cis peptide bond. Conversely, restricted conformation analogues of the insect kinins and the mammalian opiate peptide Tyr-W-MIF-1, with near identical C-terminal tetrapeptide sequences, adopt different conformations. The insect kinins adopt a cisPro 1-4 beta-turn, in which the Phe1 is critical for bioactivity. Tyr-W-MIF-1 prefers a transPro 2-5 turn, and an additional N-terminal Phe severely inhibits mu-opiate receptor binding. Comparisons of the chemical/conformational requirements for receptor interaction are consistent with a distant evolutionary relationship between the insectatachykinins and tachykinins, but not between the insect kinins and Tyr-W-MIF-1. Therefore, analogues of the insect kinins with pest control potential can be readily designed to avoid mammalian interactions.

Immunocytochemical localisation and biological activity of diuretic peptides in the housefly, Musca domestica.

The distribution of a CRF-related diuretic peptide (Musca-DP) and the diuretic/myotropic insect myokinins in the central nervous system of larval and adult houseflies was investigated using antisera raised against Locusta-DH and leucokinin-I, respectively. Two separate, small populations of immunoreactive neurons are present in the brain and fused thoracic-abdominal ganglion mass. There is no evidence for these immunoreactivities being colocalised either within single neurons or at neurohaemal release sites. Crude extracts of tissues containing immunoreactive material increase fluid secretion by isolated Malpighian tubules from adult flies. Diuretic activity is highest in tissues containing myokinin-immunoreactive material. Consistent with this observation, myokinin analogues produce a four- to five-fold increase in fluid secretion, which is more than twice the response to Musca-DP. These effects are mimicked by treatments that increase intracellular calcium and cyclic AMP, respectively. When tested at threshold concentrations, the two classes of diuretic peptide act synergistically to accelerate tubule secretion, and their separate localisation may be important for the precise control of diuresis.

The influence of neuropeptides on Malpighian tubule writhing and its significance for excretion.

Diuretic peptides (locustakinin and Locusta-DH) increase the spontaneous contractile activity of visceral muscle fibers associated with Malpighian tubules from the migratory locust (Locusta migratoria) at concentrations that increase urine production. Muscle activity is shown to assist the flow of material in the tubule lumen, but is not essential for diuresis. Tubule writhing also serves to reduce unstirred layers (USLs) at the basolateral surface of the epithelium and thereby facilitates the excretion of solutes entering the lumen by passive diffusion.

Quantification of Locusta diuretic hormone in the central nervous system and corpora cardiaca: influence of age and feeding status, and mechanism of release.

Locusta-DH is known to have a hormonal function in the control of post-feeding diuresis in the migratory locust. This study has quantified Locusta-DH in tissues from V(th) instar nymphs and adults, and investigated the K+-induced release of the peptide from corpora cardiaca. Locusta-DH is present in thoracic and abdominal ganglia, but the amounts are small (25-200 fmol) compared with brain (approximately 1 pmol) and corpora cardiaca ( > 5 pmol) from 14-day old locusts. About 50% of the immunoreactive material in corpora cardiaca coelutes with Locusta-DH on reversed-phase HPLC. An earlier eluting fraction is also biologically active, suggesting locusts have a second, previously undetected, CRF-related peptide. The amount of peptide stored in corpora cardiaca varies with age and physiological status. Reductions on day 1 of the adult instar and immediately after feeding suggest Locusta-DH controls post-eclosion as well as post-feeding diureses. Locusta-DH is released by a Ca2+-dependent mechanism from corpora cardiaca held in salines containing > or =40 mM K+. This is blocked by verapamil, implicating L-type Ca2+ channels. Release is most rapid shortly after transfer to a high K+ saline, and more peptide is released from glands allowed to recover in normal saline between successive K+ depolarisations.

Consensus chemistry and beta-turn conformation of the active core of the insect kinin neuropeptide family.

BACKGROUND: Neuropeptides are examples of small, flexible molecules that bind to receptors and induce signal transduction, thereby eliciting biological activity. The multifunctional insect kinin neuropeptides retain full activity when reduced to only their carboxy-terminal pentapeptide (Phe1-X2-X3-Trp4-Gly5-NH2), thereby allowing extensive structure-function studies and conformational analysis. RESULTS: A combined experimental and theoretical analysis of the insect kinin carboxy-terminal pentapeptide was used to probe the role of each residue, define the bioactive conformation, and design a constrained bioactive analog. Coupling receptor-binding data with two biological activity assays allowed receptor binding and signal transduction to be differentiated. A preferred beta-turn conformation, found for residues 1-4 by molecular dynamics simulations, was tested by designing a conformationally restricted cyclic hexapeptide. This cyclic analog showed a preference for the beta-turn conformation, as shown by a conformational search and nuclear magnetic resonance spectroscopy, and it showed stronger receptor binding but decreased activity relative to highly active linear analogs. CONCLUSIONS: Each residue of the insect kinin carboxy-terminal pentapeptide has a distinct role in conformational preference, specific receptor interactions or signal transduction. The beta-turn preference of residues Phe1-X2-X3-Trp4 implicates this as the bioactive conformation. The amidated carboxyl terminus, required for activity in many neuropeptide families, may be generally important for signal transduction and its inclusion may therefore be essential for agonist design.

Circulating levels of Locusta diuretic hormone: the effects of feeding.

A radioimmunoassay was developed using 125I-labeled-[TyrO]Locusta-DH and polyclonal antibodies raised against Locusta-DH (29-46). The assay had a detection limit of 50 pM, and displayed limited cross-reactivity for other CRF-related peptides, but not for unrelated peptides. About 60% of the total immunoreactive material in locust hemolymph was attributable to Locusta-DH. The circulating level of diuretic hormone increases fivefold in fed insects, sufficient to stimulate primary urine production, and is correlated with the duration of the meal. This is consistent with the role of Locusta-DH in the control of postfeeding diuresis.

Aib-containing analogues of the insect kinin neuropeptide family demonstrate resistance to an insect angiotensin-converting enzyme and potent diuretic activity.

Analogues of the insect kinin family in which the Xaa2 residue of the C-terminal pentapeptide core sequence Phe-Xaa1-Xaa2-Trp-Gly-NH2 (Xaa1 = Asn, His, Phe, Ser, or Tyr; Xaa2 = Ala, Ser, or Pro) is replaced with sterically hindered aminoisobutyric acid (Aib) prove to be resistant to hydrolysis by housefly (Musca domestica) angiotensin-converting enzyme (ACE), an endopeptidase capable of hydrolysis and inactivation of the naturally occurring insect kinin peptides. The Aib residue is compatible with formation of turn in the active core region that is important for the biological activity of the insect kinins. One of the Aib-containing analogues, pGlu-Lys-Phe-Phe-Aib-Trp-Gly-NH2, is five- and eightfold more active than the most active endogenous insect kinins in cockroach (Leucophaea maderae) hindgut myotropic and cricket (Acheta domesticus) Malpighian tubule fluid secretion assays, respectively. As the analogue is blocked at both the amino- and the carboxyl-terminus and resistant to an endopeptidase present in insects, it is better adapted than the endogenous peptides to survive for long periods in the hemolymph. Enzyme-resistant insect kinin analogues can provide useful tools to insect researchers studying the neuroendocrine control of water and ion balance and the physiological consequences of challenging insect with diuretic factors that demonstrate enhanced resistance to peptidase attack. If these analogues, whether in isolation or in combination with other factors, can disrupt the water and/or ion balance they hold potential utility for the control of pest insect populations in the future.

Neuropeptides implicated in the control of diuresis in insects.

Primary urine production in insect Malpighian tubules is stimulated by two classes of neuropeptides, CRF-related diuretic peptides and insect kinins. The CRF-related peptide of the locust, Locusta migratoria, has a hormonal role in the control of postfeeding diuresis, but the functional role of the kinins has yet to be defined. The two classes of peptide act synergistically to stimulate tubule secretion, and the kinins may therefore have a modulatory action in the control of diuresis. The peptides differ in their effects on Malpighian tubule ion transport, and this could be important for the regulation of hemolymph volume and composition.

Synergism between diuretic peptides controlling ion and fluid transport in insect malpighian tubules.

This study provides clear evidence for synergism between diuretic peptides from Locusta migratoria, Locusta-DP (a CRF-related peptide) and locustakinin (an insect myokinin). At low concentrations, the peptides act co-operatively to stimulate Malpighian tubule fluid secretion by more than the sum of their separate responses. They work via different second messengers, and their synergistic actions can be mimicked with exogenous 8-bromo-cyclic AMP replacing Locusta-DP, and/or with thapsigargin substituting for locustakinin. Additionally, 5-HT, which had been shown previously to stimulate tubule secretion via a cyclic AMP-independent mechanism, potentiated the response to Locusta-DP. Potassium is invariably the dominant cation secreted by locust tubules, but Locusta-DP, whether in the absence or presence of locustakinin, increases JNa at the expense of JK. In contrast, JNa and JK increase in parallel with JV after stimulation by locustakinin. These findings are consistent with the known actions of cyclic AMP and leucokinin-VIII on mosquito tubules. The ability to vary the relative amounts of Na+ and K+ delivered to the hindgut may be important for the maintenance of haemolymph volume and composition in recently fed locusts.