Antidiuretic Factor Research Articles

The neuropeptide RhoprCCHamide2 inhibits serotonin-stimulated transcellular Na+ transport across the anterior midgut of the vector of Chagas disease, Rhodnius prolixus.

Rhodnius prolixus is a blood-feeding insect vector of Trypanosoma cruzi, a protozoan parasite that causes Chagas disease. During each blood meal, the animals ingest large volumes of blood, that may be up to 12 times the unfed body mass. These blood meals impose a significant osmotic stress for the animals due to the hyposmotic condition of the ingested blood compared with the insect's hemolymph. Thus the insect undergoes a massive postprandial diuresis that allows for the excretion of the plasma fraction of the blood in less than two hours. Diuresis is performed by the excretory system, consisting of the Malpighian tubules and gut, under the control of diuretic and anti-diuretic factors. We investigated the ion transport machinery triggered by stimulation with the diuretic factor serotonin in the anterior midgut (i.e.crop) and the effect of the diuretic modulator RhoprCCHamide2. Ussing chamber assays revealed that serotonin-stimulated increase in transepithelial short-circuit current (Isc) was more sensitive to the blockage with amiloride than 5-N-ethyl-N-isopropyl amiloride (EIPA), suggesting the involvement of Na+ channels. Incubation in Na+-free, but not Cl--free saline, blocked the effect of serotonin on Isc. Moreover, treatment with Na+-K+-2Cl- cotransporter (NKCC) and Na+-Cl- cotransporter (NCC) blockers had no effect on fluid secretion but was blocked by amiloride. Blockage of Na+/K+-ATPase with ouabain inhibited Isc but the H+-ATPase inhibitor bafilomycin had no effect. The neuropeptide RhoprCCHamide2 diminished serotonin-stimulated Isc across the crop. The results suggest that Na+ undergoes active transport via an apical amiloride-sensitive Na+ channel and a basolateral ouabain-sensitive Na+/K+-ATPase, while Cl- is transported through a passive paracellular pathway.

The role of the V‐type H + ‐ATPase in CAPA‐treated Malpighian tubules in the yellow fever mosquito, Aedes aegypti

Haematophagus insects, such as the female Aedes aegypti mosquito, face the challenge of excess ion and water intake after engorgement on a blood meal. To cope with this, adult female A. aegypti utilize their highly adapted excretory system that includes the Malpighian ‘renal’ tubules (MTs), which are under rigorous control by several hormones to regulate transepithelial movement of ions/water. Originating in the nervous system, the mosquito anti-diuretic hormone is a member of the CAPA peptide family and is a homolog of the vertebrate neuromedin U peptide. CAPA peptides inhibit fluid secretion of MTs stimulated by select diuretic factors, 5HT and DH31 through the NOS/cGMP/PKG pathway. However, the anti-diuretic signalling mechanism and downstream cellular targets, such as ion channels and transporters, remain unclear. Active ion transport in A. aegypti MTs is driven by the principal cells, whose brush-border membrane is densely packed by the V-type H+-ATPase (VA), serving as the primary energizer for transepithelial secretion of electrolytes and water. Due to its predominant role in fluid secretion, the VA is a likely target for diuretic and anti-diuretic factors. The effect of endocrine factors and the VA inhibitor, bafilomycin, on fluid secretion rate by MTs was measured using an in vitro bioassay. Bafilomycin was found to inhibit fluid secretion stimulated by serotonin (5HT) and the calcitonin-related diuretic hormone (DH31), while having no inhibitory action on MTs stimulated with the corticotropin-releasing factor (CRF)-related diuretic hormone. CAPA and bafilomycin treatment led to alkalization of the secreted fluid suggesting inhibition of the apical VA, which may lead to constrained entry of cations (Na+ and K+) across the apical membrane of MTs. VA activity was increased in adult female MTs treated with DH31, whereas CAPA treatment reduced VA activity, comparable to saline levels. Furthermore, a VA antibody was used to determine protein localization and expression in DH31 and CAPA-treated MTs. In DH31-incubated MTs, the VA was exclusively localized to the apical memrane of the MTs, whereas VA immunoreactivity was observed in both the apical membrane and cytosol of CAPA-treated tubules. These results suggest a novel mechanism for CAPA inhibition, blocking the VA to hinder fluid secretion. A. aegypti mosquitoes are vectors of a variety of pathogens leading to illnesses, such as Dengue fever, Yellow fever, and the Zika virus. The current results advance our knowledge of the anti-diuretic control of the A. aegypti MTs, suggesting that CAPA targets the VA to inhibit diuresis. Investigating the pathway of CAPA inhibition and its role in countering diuresis will help provide a deeper understanding of this critical physiological process. Studying the excretory system and discerning the underlying mechanisms behind ion and fluid transport may aid in developing novel pest management strategies to better control Aedes population and lessen its prominence as a disease vector.

Hormonal regulation and functional role of the "renal" tubules in the disease vector, Aedes aegypti.

The Aedes aegypti mosquito is a vector responsible for transmitting various arboviruses including dengue and yellow fever. Their ability to regulate the ionic and water composition of their hemolymph is a major physiological phenomenon, allowing the mosquito to adapt to a range of ecological niches. Hematophagus insects, including the female A. aegypti, face the challenge of excess salt and water intake after a blood meal. Post-prandial diuresis is under rigorous control by neuroendocrine factors, acting on the Malpighian "renal" tubules (MTs), to regulate primary urine production. The MTs are made up of two cell types; mitochondria-rich principal cells, which facilitate active transport of Na+ and K+ cations across the membrane, and thin stellate cells, which allows for transepithelial Cl- secretion. The active driving force responsible for ion transport is the apical V-type H+ ATPase, which creates a proton gradient allowing for Na+ and/or K+ cation exchange through cation/H+ antiporters. Additionally, the basolaterally localized Na+-K+-2Cl- cotransporter (NKCC) is responsible for the transport of these ions from the hemolymph into the principal cells. Numerous studies have examined hormonal regulation of the mosquito MTs and identified several diuretics including serotonin (5HT), a calcitonin-related diuretic hormone 31 (DH31), a corticotropin-related factor like diuretic peptide (DH44), a kinin-related diuretic peptide, as well as anti-diuretic factors including CAPA peptides, all of which are known to regulate fluid and ion transport by the MTs. This review therefore focuses on the control of ionic homeostasis in A. aegypti mosquitoes, emphasizing the importance of the MTs, the channels and transporters involved in maintaining hydromineral balance, and the neuroendocrine regulation of both diuresis and anti-diuresis.

Anti-diuretic action of a CAPA neuropeptide against a subset of diuretic hormones in the disease vector Aedes aegypti.

The mosquito Aedes aegypti is a vector responsible for transmitting various pathogens to humans, and their prominence as chief vectors of human disease is largely due to their anthropophilic blood feeding behaviour. Larval stage mosquitoes must deal with the potential dilution of their haemolymph in freshwater, whereas the haematophagus A. aegypti female faces the challenge of excess ion and water intake after a blood meal. The excretory system, composed of the Malpighian tubules (MTs) and hindgut, is strictly controlled by neuroendocrine factors, responsible for the regulation of diuresis across all developmental stages. The highly studied insect MTs are influenced by a variety of diuretic hormones and, in some insects, anti-diuretic factors. In the present study, we investigated the effects of AedaeCAPA-1 neuropeptide on larval and adult female A. aegypti MTs stimulated with various diuretic factors including serotonin (5-HT), a corticotropin-related factor (CRF) diuretic peptide, a calcitonin-related diuretic hormone (DH31) and a kinin-related diuretic peptide. Overall, our findings establish that AedaeCAPA-1 specifically inhibits secretion of larval and adult MTs stimulated by 5-HT and DH31, whilst having no activity on MTs stimulated by other diuretic factors. Furthermore, although AedaeCAPA-1 acts as an anti-diuretic, it does not influence the relative proportions of cations transported by adult MTs, thus maintaining the kaliuretic activity of 5-HT and natriuretic activity of DH31 In addition, we tested the effects of the second messenger cGMP in adult MTs. We established that cGMP has similar effects to AedaeCAPA-1, strongly inhibiting 5-HT- and DH31-stimulated fluid secretion, but with only minor effects on CRF-stimulated diuresis. Interestingly, although AedaeCAPA-1 has no inhibitory activity on kinin-stimulated fluid secretion, cGMP strongly inhibited fluid secretion by this diuretic hormone, which targets stellate cells specifically. Collectively, these results support that AedaeCAPA-1 inhibits select diuretic factors acting on the principal cells and this probably involves cGMP as a second messenger. Kinin-stimulated diuresis, which targets stellate cells, is also inhibited by cGMP, suggesting that another anti-diuretic factor in addition to AedaeCAPA-1 exists and may utilize cGMP as a second messenger.

Endocrine regulation of insect diuresis in the early postgenomic era1This review is part of a virtual symposium on recent advances in understanding a variety of complex regulatory processes in insect physiology and endocrinology, including development, metabolism, cold hardiness, food intake and digestion, and diuresis, through the use of omics technologies in the postgenomic era.

Diuresis, the removal of excess metabolic waste through production of primary urine while maintaining homeostasis, is an important biological process that is tightly regulated by endocrine factors. Several hormonal components that act as diuretic or antidiuretic factors in insects have been identified in the last few decades. Physiological mechanisms responsible for ion and water transport across biological membranes have been intensively studied. The large amount of data rapidly accumulating in the genomics era has led to an increased dependence on reverse genetic and physiological approaches, first identifying candidate genes and subsequently deriving functions. In many cases, the reverse approaches have been highly successful, especially in studies of the receptors for diuretic factors, which are mainly G-protein-coupled receptors. This review summarizes research on insect diuretic and antidiuretic endocrine factors, and their receptors. Emphases of the review are given to the genomics of ligands and their receptors, as well as to their implications for evolution and function.

Treatment of Urinary Incontinence in Women: A Challenge between Conservative and Surgical Therapy

Copyright: © 2011 Sanavi S. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Urinary incontinence (UI) is a socially embarrassing condition, causing withdrawal from social situations and reduced quality of life. This withdrawal from regular physical activities may be a threat to women’s general health predisposing to osteoporosis, high blood pressure, coronary heart disease, depression and anxiety [1]. UI is defined by the International Continence Society as a condition in which involuntary loss of urine is a social or hygienic problem and is objectively demonstrable [2]. UI is more common in women than in men and affects women of all ages. Prevalence rate in women between 15 and 64 years of age vary from 10% to 30% which increases with age [3-5]. The prevalence of UI is significantly higher for individuals living in nursing homes, compared to community settings, with rates ranging from 43% to 77% [5]. The severity of incontinence also increases with age [6,7]. While a specific etiologic cause of urinary incontinence is often identifiable in younger persons, a multifactorial syndrome is more likely in older persons. In the older population, UI may represent the intersection of neuro-urinary pathology, age-related factors, comorbid conditions, medications and functional and cognitive impairments [2]. Several physiologic changes occur in the lower urinary tract of both continent and incontinent older persons that predispose to urinary incontinence: detrusor overactivity, decreased bladder contractility, decreased flow rate, increased post void residual volume and change in diurnal fluid excretion (due to peripheral edema, medications, agerelated changes in the renal filtration rate and alterations in diurnal levels of anti-diuretic hormone and atrial natriuretic factor) [8-10]. In addition, low estrogen levels after menopause result in atrophy of the superficial and intermediate layers of the urethral mucosal epithelium with subsequent atrophic urethritis, diminished urethral mucosal seal, loss of compliance and possible irritation. However; these factors do not necessarily result in urinary incontinence and incontinence is not a part of “normal” aging [11-13].

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.

Genomics and Peptidomics of Neuropeptides and Protein Hormones Present in the Parasitic Wasp Nasonia vitripennis

Neuropeptides and protein hormones constitute a very important group of signaling molecules, regulating central physiological processes such as reproduction, development, and behavior. Using a bioinformatics approach, we screened the recently sequenced genome of the parasitic wasp, Nasonia vitripennis, for the presence of these signaling molecules and annotated 30 precursor genes encoding 51 different mature neuropeptides or protein hormones. Twenty-four of the predicted mature Nasonia neuropeptides could be experimentally confirmed by mass spectrometry. We also discovered a completely novel neuropeptide gene in Nasonia, coding for peptides containing the C-terminal sequence RYamide. This gene has orthologs in nearly all arthropods with a sequenced genome, and its expression in mosquitoes was confirmed by mass spectrometry. No precursor could be identified for N-terminally extended FMRFamides, even though their putative G protein coupled receptor (GPCR) is present in the Nasonia genome. Neither the precursor nor the putative receptor could be identified for allatostatin-B, capa, the glycoprotein hormones GPA2/GPB5, kinin, proctolin, sex peptide, and sulfakinin, arguing that these signaling systems are truly absent in the wasp. Also, antidiuretic factors, allatotropin, and NPLP-like precursors are missing in Nasonia, but here the receptors have not been identified in any insect, so far. Nasonia (Hymenoptera) has the lowest number of neuropeptide precursor genes compared to Drosophila melanogaster, Aedes aegypti (both Diptera), Bombyx mori (Lepidoptera), Tribolium castaneum (Coleoptera), Apis mellifera (Hymenoptera), and Acyrthosiphon pisum (Hemiptera). This lower number of neuropeptide genes might be related to Nasonia's parasitic life.

The antidiuretic neurohormone RhoprCAPA-2 downregulates fluid transport across the anterior midgut in the blood-feeding insect Rhodnius prolixus

Osmotic balance in insects is regulated by the excretory system, consisting of Malpighian tubules and the gut under the control of diuretic and antidiuretic factors. Terrestrial insects must conserve water, and antidiuresis is the norm, only interrupted by brief diuretic periods. Surprisingly, little is known about antidiuresis in insects. Two antidiuretic strategies have been described. The first antidiuretic mechanism involves the reabsorption of fluid from the primary urine in the hindgut. More recently, a second antidiuretic strategy was reported, consisting of inhibition of primary urine formation by the Malpighian tubules. Recently, we isolated, characterized, and cloned the gene encoding for the antidiuretic neurohormone (the neuropeptide RhoprCAPA-2) acting on the Malpighian tubules of Rhodnius prolixus. Here we describe a third, novel mechanism central to the antidiuretic strategy of R. prolixus, the inhibition of ion and fluid transport across the anterior midgut by RhoprCAPA-2. Our results show that RhoprCAPA-2 (1 micromol/l) reduces serotonin-stimulated fluid transport from 83 +/- 11 to 12 +/- 12 nl/min and equivalent short-circuit current from 20 +/- 4 to 5 +/- 0.7 microA/cm(2) in diuretic hormone-stimulated anterior midgut. RhoprCAPA-2 appears to function independently of intracellular cGMP or Ca(2+) in the midgut. Thus, the antidiuretic neurohormone RhoprCAPA-2 has multiple target tissues, and we hypothesize that RhoprCAPA-2 functions to coordinate the transport activity of the anterior midgut and Malpighian tubules so that the rate of fluid transport into the haemolymph by the anterior midgut matches the transport rate of Malpighian tubules to maintain the volume and ion composition of haemolymph.

Isolation, Cloning, and Expression Mapping of a Gene Encoding an Antidiuretic Hormone and Other CAPA-Related Peptides in the Disease Vector, Rhodnius prolixus

After a blood meal, Rhodnius prolixus undergoes a rapid diuresis to eliminate excess water and salts. During the voiding of this primary urine, R. prolixus acts as a vector of Chagas' disease, with the causative agent, Trypanosoma cruzi, infecting the human host via the urine. Diuresis in R. prolixus is under the neurohormonal control of serotonin and peptidergic diuretic hormones, and thus, diuretic hormones play an important role in the transmission of Chagas' disease. Although diuretic hormones may be degraded or excreted, resulting in the termination of diuresis, it would also seem appropriate, given the high rates of secretion, that a potent antidiuretic factor could be present and act to prevent excessive loss of water and salts after the postgorging diuresis. Despite the medical importance of R. prolixus, no genes for any neuropeptides have been cloned, including obviously, those that control diuresis. Here, using molecular biology in combination with matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry, we determined the sequence of the CAPA gene and CAPA-related peptides in R. prolixus, which includes a peptide with anti-diuretic activity. We have characterized the expression of mRNA encoding these peptides in various developmental stage and also examined the tissue-specific distribution in fifth-instars. The expression is localized to numerous bilaterally paired cell bodies within the central nervous system. In addition, our results show that RhoprCAPA gene expression is also associated with the testes, suggesting a novel role for this family of peptides in reproduction.

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

Acheta domesticus is reported to have an antidiuretic hormone that reduces Malpighian tubule secretion. Identified peptides known to work in this way (Tenmo-ADFa and ADFb, and Manse-CAP(2b)) were tested as candidates for the unidentified hormone, along with their second messenger, cyclic GMP. Only Tenmo-ADFb was active, but was diuretic, as was 8-bromo cyclic GMP. The activity of Tenmo-ADFb is comparable to that of the cricket kinin neuropeptide, Achdo-KII, but it is much less potent. Its activity was unaffected by deleting either the six N-terminal residues or the C-terminal phenylalanine. At high concentrations, tubule secretion is doubled by Tenmo-ADFb and Achdo-KII, but their actions are non-additive, suggesting they have a similar mode of action. Both stimulate a non-selective KCl and NaCl diuresis, which is consistent with the opening of a transepithelial Cl(-) conductance. In support of this, the diuretic response to Tenmo-ADFb and Achdo-KII is prevented by a ten-fold reduction in bathing fluid chloride concentration, and both peptides cause the lumen-positive transepithelial voltage to collapse. The Cl(-) conductance pathway appears likely to be transcellular, because the Cl(-) channel blocker DPC reduces both basal and peptide-stimulated rates of secretion. The effects of 8-bromo cyclic GMP on transepithelial voltage and composition of the secreted fluid are markedly different from those of Tenmo-ADFb. This is the first report of the antidiuretic factor Tenmo-ADFb stimulating tubule secretion. Although the actions of Tenmo-ADFb are indistinguishable from those of Achdo-KII, it is unlikely to act at a kinin receptor, because the core sequence (residues 7-12) lacks the Phe and Trp residues that are critical for kinin activity.

Distribution, activity and evidence for the release of an anti-diuretic peptide in the kissing bug Rhodnius prolixus

In the haematophagous insect Rhodnius prolixus, diuresis is accomplished through the combined actions of peptidergic diuretic hormones and 5-HT released from neurohaemal sites on the abdominal nerves. Preliminary work on anti-diuresis in this blood-feeder, previously believed to occur through a decrease in the levels of the diuretic factors, indicates that an anti-diuretic hormone, with properties similar to CAP2b (pELYAFPRVamide; recently renamed Mas-CAPA-1), might also be present in R. prolixus. Here, we present evidence from immunohistochemical analysis that suggests a PRXamide-like neuropeptide may be released from the abdominal neurohaemal sites beginning 3-4 h following feeding; a time that coincides with the cessation of diuresis. We also show evidence for an endogenous factor, isolated from the central nervous system using reversed-phase high performance liquid chromatography, which mimics the effects of Mas-CAPA-1. Specifically, this endogenous anti-diuretic factor inhibits rates of 5-HT-stimulated secretion in a dose-dependent manner and elevates intracellular cGMP levels of Malpighian tubules stimulated with 5-HT.

Body extracts from the forest ant and honeybee: chromatographic comparison of factors affecting the ant Malpighian tubule

Abstract. Crude trifluoroacetic acid extracts were prepared from different body parts of the forest ant, Formica polyctena Foerster, Hymenoptera, Formicidae, and the honeybee, Apis mellifera carnica Pollmann, Hymenoptera, Apidae. Extracts were prepurified by means of solid phase extraction over reversed‐phase cartridges. The effects of the resulting fractions on fluid secretion rates were tested on single preparations of ant Malpighian tubules. Diuretic and antidiuretic factors were shown to be present. High‐performance liquid chromatography allowed for the identification of a diuretic factor from the bee‐head extract which had a similar elution profile to a previously purified diuretic peptide from ant‐head extracts. An antidiuretic factor was also identified from the bee abdominal extract. Similar to an antidiuretic peptide, which was purified from ant abdominal extracts, this antidiuretic factor inhibited fluid secretion and depolarized the transepithelial potential of ant Malpighian tubules.

Isolation, identification and localization of a second beetle antidiuretic peptide

We isolated from head extracts of Tenebrio molitor a peptide that inhibits fluid secretion by the Malpighian tubules of this insect. This second antidiuretic factor, ADFb, like the previously published ADFa, works through cyclic GMP as a second messenger. It has primary structure Tyr–Asp–Asp–Gly–Ser–Tyr–Lys–Pro–His–Ile–Tyr–Gly–Phe–OH with an EC 50 of approximately 240 pM in a fluid secretion assay. This peptide is now the second sequenced endogenous insect ADF which inhibits Malpighian tubule fluid secretion. Immunohistochemical techniques show that the peptide is localized in the brain; it appears to be produced mainly in two pairs of bilaterally symmetrical cells in the protocerebrum.

Water and sodium in nutrition support

Severe malnutrition due to prolonged energy and protein deficiency is associated with a relative increase of the extracellular water compartment (ECW) (1). This results from several metabolic derangements: an absolute decrease of the fat-free mass, a depletion of the body fat stores and a preservation of the circulating volume and the interstitial fluid compartment. It is also possible, that during prolonged semistarvation, after an initial loss of extracellular fluid due to sodium losses, there exists an antidiuretic drive facilitating fluid retention (2). A relatively high carbohydrate intake (3) and intercurrent infections are well-known antidiuretic factors provoking the appearance of dependent oedema, a physical sign of advanced malnutrition often associated with absolute or distributional hypoalbuminaemia (4). Early works on refeeding, reported inappropriate physiological responses consisting mainly in disturbances affecting the ECW and the cardiovascular system: haemodilution, congestive heart failiure, hypertension, oedema and rapid weight gain (5–8). In addition, it has also been repeatedly reported how difficult it may be to reverse hypoalbuminaemia in patients receiving artificial nutrition support (9–11). Some of these studies suggested that high calorie intakes, particularly of carbohydrates, could be held responsible for such abnormalities that were particularly severe in patients receiving total parenteral nutrition in the ‘hyperalimentation era’, including large water volumes in addition to glucose loads over 10 g/kg/day (12).

Identification of a potent antidiuretic factor acting on beetle Malpighian tubules.

Beetles, like other insects, depend on diuretic and antidiuretic hormones to control water balance. We have isolated, using head extracts from the beetle Tenebrio molitor, a peptide that strongly inhibits fluid secretion by the Malpighian tubules of this insect. This antidiuretic factor (ADF) appears to elicit its effect via cGMP as a second messenger but does not stimulate NO production. It has primary structure: Val-Val-Asn-Thr-Pro-Gly-His-Ala-Val-Ser-Tyr-His-Val-Tyr-OH. The ADF inhibits tubule secretion with high potency: the EC(50) is around 10 fM. It bears no significant resemblance to other biologically active neuropeptides. To our knowledge this is the only endogenous insect ADF acting on Malpighian tubules to be sequenced, and the first coleopteran (beetle) antidiuretic factor fully characterized to date.

Partial purification of a novel insect antidiuretic factor from the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), which acts on Malpighian tubules

In this study, we report for the first time the presence of an antidiuretic factor in the head of the Colorado potato beetle ( Leptinotarsa decemlineata (Say)) which acts directly on Malpighian tubules. Biologically active fractions were isolated from the head and separated using molecular weight filtration and high-performance liquid chromatography (HPLC). The resulting fractions were tested for their antidiuretic activity on single isolated Malpighian tubules. Antidiuretic activity was found in the 25% acetonitrile Sep-Pak fraction and the Cn-2 (3000–10,000 MW) and Cn-3 (<3000 MW) fractions, suggesting that the antidiuretic factor was probably a peptide of 25 to 50 amino acids. The antidiuretic factor was very potent, since after five successive fractionations on two different HPLC columns, a high level of inhibition (63%) of fluid secretion by Malpighian tubules could be observed at low dose (0.14 head-equivalent/μl). The antidiuretic factor isolated from the head of the Colorado potato beetle was not affected by repeated freezing and thawing but was sensitive to heat. The differences observed between the Colorado potato beetle antidiuretic factor and other insect diuretic and antidiuretic factors may indicate the possibility of a novel family of water regulation hormones in insects.

An antidiuretic factor in the forest ant: purification and physiological effects on the Malpighian tubules

Formica polyctena antidiuretic factor (FopADF) was purified from a 15% trifluoroacetic acid (TFA) extract of the abdomens of 150,000 worker ants. After solid phase extraction of the crude extract and reversed-phase HPLC on two C 18 columns, an antidiuretic factor was isolated. Tested at a concentration of 1.0 ant-equivalents/μl (ant-eq/μl), the factor reversibly inhibited fluid secretion of isolated Malpighian tubules to 29±5% (mean±SE, n=24) of the control value. The same concentration of FopADF reversibly depolarized both the basolateral membrane potential (V bl), from −21±2 mV to −3±1 mV ( n=5), and the apical membrane potential (V ap), from −65±5 mV to −20±5 mV ( n=5). Similar effects on fluid secretion and V ap were caused by a TFA extract of the haemolymph of ants with non-secreting tubules. Unfortunately, further purification of FopADF on a C 4 column led to a loss of activity in the fluid secretion assay. This is the first time an endogenous antidiuretic factor acting directly on Malpighian tubules has been partially purified and shown to depolarize the tubule cell membranes.

Rhodnius prolixus: effects of the neolignan burchellin on in vivo and in vitro diuresis.

Supplementation of blood with the neolignan burchellin (100 microg/ml), a compound from the arboreous Lauraceae Aniba burchelli, affected the course of excretion of fourth-instar larvae of Rhodnius prolixus, especially directly after feeding, and reduced the volume of feces/urine excreted within 6 h of feeding to about 18% and, on the simultaneous addition of the diuretic hormone analogue 5-hydroxytryptamine (5-HT), about 71% of that observed in untreated bugs. In the latter, 5-HT induced a significant 60% increase in excretion. Regardless of whether Malpighian tubules originating from unfed, untreated or fed, burchellin-treated bugs were incubated in vitro in the hemolymph of these bugs or in physiological saline supplemented with 5-HT with or without burchellin or in homogenates of thoracic ganglionic masses of untreated and treated bugs, burchellin was consistently found to affect the secretion rates. Therefore, burchellin not only depresses the release of the diuretic hormone or induces the release of antidiuretic factors but also directly affects the Malpighian tubules.

PLGamide characterization and role in osmoregulation in leech brain

Neurons immunoreactive to an antiserum specifically directed against the Prolyl–Leucyl–Glycinamide peptide (PLGamide: Melanocyte Inhibiting Factor: MIF) were detected in the brain of the leech Theromyzon tessulatum. Radioimmunoassay titrations of the PLGamide-like material at different physiological stages of the life cycle indicated a maximal amount at stage 3B, which is correlated to phase of both maximal water uptake and coelomic vitellogenin accumulation. In vivo experiments demonstrate that this cerebral PLGamide-like material is an anti-diuretic factor that would act at stage 3B in order to permit a water uptake leading to water retention allowing coelomic yolk protein accumulation. In brains of the Gnatobdellid leech Hirudo medicinalis and the Pharyngobdellid leech Erpobdella octoculata, anti-PLGamide material was also detected with an amount not differing with the degree of sex maturation of the animals, confirming the link between osmoregulation and ovogenesis in rhynchobdellid leeches. Using a combination of biochemical techniques including high-pressure gel permeation chromatography followed by reversed-phase HPLC on brain extracts and Edman degradation, we demonstrated the presence of an authentic MIF-1 peptide in leech brain. Finally, since in vertebrates MIF-1 belongs to the non-classical opioid peptide family, we studied its binding displacement, in contrast to morphine, on μ-receptors and on nitric oxide (NO) release experiments in leech brain. PLGamide did not bind to μ-alkaloid opioid receptors and did not stimulate NO release.