Fluid Secretion In Tubules Research Articles

The glycoprotein hormone receptor (LGR1) influences Malpighian tubule secretion rate in Rhodnius prolixus.

In the hemipteran Rhodnius prolixus, successful post-prandial diuresis is accomplished through the synergistic actions of the peptidergic diuretic hormone RhoprCRF/DH and the biogenic amine 5-hydroxytryptamine (5-HT), and by an antidiuretic hormone RhoprCAPA-2 that terminates diuresis by inhibiting this synergy. Lateral neurosecretory cells (NSCs) in the mesothoracic ganglionic mass release RhoprCRF/DH, while midline NSCs release RhoprCAPA-2 during blood feeding. These NSCs co-express GPA2/GPB5, a conserved glycoprotein hormone involved in various physiological processes across bilaterians. This study investigated the influence of GPA2/GPB5 signaling on Malpighian tubule (MT) fluid secretion in R. prolixus. GPB5-like immunoreactivity in lateral and midline NSCs decreased following a blood meal, suggesting release and a role in diuresis. Downregulating the GPA2/GPB5 receptor LGR1 via RNA interference resulted in an increased basal fluid secretion rate in MTs, which was inhibited by the antidiuretic hormone RhoprCAPA-2. dsLGR1 treatment reduced the effects of RhoprCRF/DH and 5-HT on MT secretion and eliminated their synergism. RT-qPCR revealed that the expression of the diuretic and antidiuretic hormone receptors decreased in MTs of dsLGR1-injected insects, indicating that GPA2/GPB5 influences the expression of these other receptors. Downregulating LGR1 resulted in a smaller blood meal size and disrupted the normal time course of diuresis. As LGR1 is the most abundantly expressed G protein-coupled receptor gene in R. prolixus MTs, our results suggest that GPA2/GPB5 signaling has a critical role in regulating the timing and success of water retention in the unfed state, and in the complex processes associated with feeding and diuresis in R. prolixus.

The septate junction protein Mesh is required for epithelial morphogenesis, ion transport, and paracellular permeability in the Drosophila Malpighian tubule.

Septate junctions (SJs) are occluding cell-cell junctions that have roles in paracellular permeability and barrier function in the epithelia of invertebrates. Arthropods have two types of SJs, pleated SJs and smooth SJs (sSJs). In Drosophila melanogaster, sSJs are found in the midgut and Malpighian tubules, but the functions of sSJs and their protein components in the tubule epithelium are unknown. Here we examined the role of the previously identified integral sSJ component, Mesh, in the Malpighian tubule. We genetically manipulated mesh specifically in the principal cells of the tubule at different life stages. Tubules of flies with developmental mesh knockdown revealed defects in epithelial architecture, sSJ molecular and structural organization, and lack of urine production in basal and kinin-stimulated conditions, resulting in edema and early adult lethality. Knockdown of mesh during adulthood did not disrupt tubule epithelial and sSJ integrity but decreased the transepithelial potential, diminished transepithelial fluid and ion transport, and decreased paracellular permeability to 4-kDa dextran. Drosophila kinin decreased transepithelial potential and increased chloride permeability, and it stimulated fluid secretion in both control and adult mesh knockdown tubules but had no effect on 4-kDa dextran flux. Together, these data indicate roles for Mesh in the developmental maturation of the Drosophila Malpighian tubule and in ion and macromolecular transport in the adult tubule.

Transepithelial transport of P-glycoprotein substrate by the Malpighian tubules of the desert locust

Extrusion of xenobiotics is essential for allowing animals to remove toxic substances present in their diet or generated as a biproduct of their metabolism. By transporting a wide range of potentially noxious substrates, active transporters of the ABC transporter family play an important role in xenobiotic extrusion. One such class of transporters are the multidrug resistance P-glycoprotein transporters. Here, we investigated P-glycoprotein transport in the Malpighian tubules of the desert locust (Schistocerca gregaria), a species whose diet includes plants that contain toxic secondary metabolites. To this end, we studied transporter physiology using a modified Ramsay assay in which ex vivo Malpighian tubules are incubated in different solutions containing the P-glycoprotein substrate dye rhodamine B in combination with different concentrations of the P-glycoprotein inhibitor verapamil. To determine the quantity of the P-glycoprotein substrate extruded we developed a simple and cheap method as an alternative to liquid chromatography–mass spectrometry, radiolabelled alkaloids or confocal microscopy. Our evidence shows that: (i) the Malpighian tubules contain a P-glycoprotein; (ii) tubule surface area is positively correlated with the tubule fluid secretion rate; and (iii) as the fluid secretion rate increases so too does the net extrusion of rhodamine B. We were able to quantify precisely the relationships between the fluid secretion, surface area, and net extrusion. We interpret these results in the context of the life history and foraging ecology of desert locusts. We argue that P-glycoproteins contribute to the removal of xenobiotic substances from the haemolymph, thereby enabling gregarious desert locusts to maintain toxicity through the ingestion of toxic plants without suffering the deleterious effects themselves.

Functional plasticity of the gut and the Malpighian tubules underlies cold acclimation and mitigates cold-induced hyperkalemia in Drosophila melanogaster.

At low temperatures, Drosophila, like most insects, lose the ability to regulate ion and water balance across the gut epithelia, which can lead to a lethal increase of [K+] in the hemolymph (hyperkalemia). Cold acclimation, the physiological response to a prior low temperature exposure, can mitigate or entirely prevent these ion imbalances, but the physiological mechanisms that facilitate this process are not well understood. Here, we test whether plasticity in the ionoregulatory physiology of the gut and Malpighian tubules of Drosophila may aid in preserving ion homeostasis in the cold. Upon adult emergence, D. melanogaster females were subjected to 7days at warm (25°C) or cold (10°C) acclimation conditions. The cold-acclimated flies had a lower critical thermal minimum (CTmin), recovered from chill coma more quickly, and better maintained hemolymph K+ balance in the cold. The improvements in chill tolerance coincided with increased Malpighian tubule fluid secretion and better maintenance of K+ secretion rates in the cold, as well as reduced rectal K+ reabsorption in cold-acclimated flies. To test whether modulation of ion-motive ATPases, the main drivers of epithelial transport in the alimentary canal, mediate these changes, we measured the activities of Na+/K+-ATPase and V-type H+-ATPase at the Malpighian tubules, midgut, and hindgut. Na+/K+-ATPase and V-type H+-ATPase activities were lower in the midgut and the Malpighian tubules of cold-acclimated flies, but unchanged in the hindgut of cold-acclimated flies, and were not predictive of the observed alterations in K+ transport. Our results suggest that modification of Malpighian tubule and gut ion and water transport probably prevents cold-induced hyperkalemia in cold-acclimated flies, and that this process is not directly related to the activities of the main drivers of ion transport in these organs, Na+/K+- and V-type H+-ATPases.

FLUID AND ION SECRETION BY MALPIGHIAN TUBULES OF LARVAL CHIRONOMIDS, Chironomus riparius: EFFECTS OF REARING SALINITY, TRANSPORT INHIBITORS, AND SEROTONIN.

Larvae of Chironomus riparius respond to ion-poor and brackish water (IPW, BW) conditions by activating ion uptake mechanisms in the anal papillae and reducing ion absorption at the rectum, respectively. The role that the Malpighian tubules play in ion and osmoregulation under these conditions is not known in this species. This study examines rates of fluid secretion and major cation composition of secreted fluid from tubules of C.riparius reared in IPW, freshwater (FW) and BW. Fluid secretion of tubules from FW and BW larvae was similar but tubules from IPW larvae secrete fluid at higher rates, are more sensitive to serotonin stimulation, and the secreted fluid contains less Na(+) . Therefore in IPW, tubules work in concert with anal papillae to eliminate excess water while conserving Na(+) in the hemolymph. Tubules do not appear to play a significant role in ion/osmoregulation under BW. Serotonin immunoreactivity in the nervous system and gastrointestinal tract of larval C. riparius was similar to that seen in mosquito larvae with the exception that the hindgut was devoid of staining. Hemolymph serotonin titer was similar in FW and IPW; hence, serotonin is not responsible for the observed high rates of fluid secretion in IPW. Instead, it is suggested that serotonin may work in a synergistic manner with an unidentified hormonal factor in IPW. Ion transport mechanisms in the tubules of C. riparius are pharmacologically similar to those of other insects.

Investigations of the signaling cascade involved in diuretic hormone stimulation of Malpighian tubule fluid secretion in Rhodnius prolixus

In insects, the excretory system is comprised of the Malpighian tubules (MTs) and the hindgut, which collectively function to maintain ionic and osmotic balance of the haemolymph and rid the organism of toxic compounds or elements in excess. Secretion by the Malpighian tubules of insects is regulated by a variety of hormones including peptidergic factors as well as biogenic amines. In Rhodnius prolixus, two endogenous diuretic hormones have been identified; the biogenic amine serotonin (5-hydroxytryptamine, 5-HT) and the corticotropin releasing factor-related peptide, RhoprCRF. Both factors significantly increase secretion by MTs and are known to elevate intracellular levels of cAMP. Interestingly, applying sub-maximal doses of these two diuretic factors in combination on isolated MTs in vitro reveals synergistic effects as rates of fluid secretion are significantly higher than would be expected if rates of secretion from MTs treated with each factor alone were summed. This observed synergism suggests that different downstream targets may be activated by the two diuretic factors, but that some cellular elicitors may be shared since cAMP is elevated in response to either diuretic hormone.This study investigated the signaling cascade involved in the diuretic hormone regulation of Malpighian tubule fluid secretion. Bioassays were performed in physiological as well as modified salines (e.g. calcium-free) alone or in the presence of a variety of pharmacological compounds that interfere with prospective intracellular targets, such as the apical cation/H+ exchanger. Intriguingly, only amiloride yielded differential effects on the two diuretics with 5HT-stimulated secretion being blocked, whereas in contrast, RhoprCRF-stimulated secretion was unaffected. In addition, experiments examining the role of extracellular and intracellular calcium on fluid secretion rate showed that both diuretics are dependent on intracellular calcium availability. Finally, fluid secretion stimulated by either diuretic hormone was also sensitive to inhibition of cAMP-dependent protein kinase A. Taken together, these results suggest that each diuretic hormone activates pathways dependent upon intracellular calcium and cAMP.

Roles of PKC and phospho‑adducin in transepithelial fluid secretion by Malpighian tubules of the yellow fever mosquito

The diuretic hormone aedeskinin‑III is known to increase the paracellular Cl- conductance in Malpighian (renal) tubules of the mosquito Aedes aegypti via a G protein-coupled receptor. The increase serves the blood-meal-initiated diuresis and is associated with elevated levels of Ca2+ and phosphorylated adducin in the cytosol of tubule. In the present study we have cloned adducin in Aedes Malpighian tubules and investigated its physiological roles. Immunolabeling experiments are consistent with the association of adducin with the cortical cytoskeleton, especially near the apical brush border of the tubule. An antibody against phosphorylated adducin revealed the transient phosphorylation of adducin 2 min after stimulating tubules with aedeskinin‑III. The PKC inhibitor bisindolylmaleimide‑I blocked the phosphorylation of adducin as well as the electrophysiological and diuretic effects of aedeskinin‑III. Bisindolylmaleimide‑I also inhibited fluid secretion in control tubules. Phorbol 12‑myristate 13‑acetate increased phosphorylated adducin levels in Malpighian tubules, but it inhibited fluid secretion. Thus, the phosphorylation of adducin by PKC alone is insufficient to trigger diuretic rates of fluid secretion; elevated levels of intracellular Ca2+ may also be required. The above results suggest that the phosphorylation of adducin, which is known to destabilize the cytoskeleton, may (1) facilitate the traffic of transporters into the apical brush border supporting diuretic rates of cation secretion and (2) destabilize proteins in the septate junction thereby enabling paracellular anion (Cl‑) secretion at diuretic rates. Moreover, PKC and the phosphorylation of adducin play a central role in control and diuretic tubules, consistent with the dynamic behavior of both transcellular and paracellular transport pathways.

Active diuretic peptidomimetic insect kinin analogs that contain β-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-X1-X2-Trp-Gly-NH2, where X1=His, Asn, Ser, or Tyr and X2=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 β-turn mimetic motif 4-aminogutamate in place of the X1-X2 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.

Salt stress alters fluid and ion transport by Malpighian tubules ofDrosophila melanogaster: evidence for phenotypic plasticity

Drosophila are tolerant of high levels of dietary salt and can provide a useful model for studies of the physiology of salt stress. The effects of NaCl- and KCl-rich diets on haemolymph ionoregulation and Malpighian tubule (MT) fluid secretion, Na(+) and K(+) secretion and transepithelial potential were examined in larval and adult Drosophila melanogaster. K(+) concentrations in the haemolymph of adults reared on the KCl-rich (0.4 mol l(-1)) diet did not differ from the values for insects reared on the control diet. In the haemolymph of larvae reared on the K-rich diet, K(+) concentrations increased from 23 to 75 mmol l(-1) after 6 h, then returned to the control value within 48 h. Na(+) concentrations in the haemolymph of adults or larvae reared for 1-7 days on the NaCl-rich (0.4 mol l(-1)) diet increased by ~50% relative to values for insects reared on the control diet. Rates of secretion of fluid, Na(+) and K(+) by MTs isolated from larvae reared on the Na-rich diet for >6 h and bathed in control saline containing 20 mmol l(-1) K(+) did not differ from the values for tubules of larvae reared on the control diet. Evidence of phenotypic plasticity was seen in the response of MTs isolated from larvae reared on the K-rich diet for >6 h and bathed in saline containing 60 mmol l(-1) K(+); secretion of fluid and K(+) increased by >50% relative to the values for tubules of larvae reared on the control diet. Secretion of fluid, Na(+) and K(+) increased when tubules were bathed in haemolymph collected from larvae reared on the Na- or K-rich diets. Secretion was further increased by addition of exogenous cAMP but not by addition of thapsigargin to the haemolymph. The results show that haemolymph ionoregulation in larvae reared on salt-rich diets involves both alterations in the basal secretion rates of Na(+) and/or K(+) as well as stimulatory effects of diuretic factors present in the haemolymph. The results suggest that such factors stimulate tubule fluid and ion secretion through increases in intracellular Ca(2+) in response to salt stress.

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 α,α-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.

Beetle diuretic peptides: The response of mealworm ( Tenebrio molitor) Malpighian tubules to synthetic peptides, and cross-reactivity studies with a dung beetle ( Onthophagus gazella)

This paper reports the effects of different diuretic factors on the Malpighian tubules of beetles. Calcitonin (CT)-like peptides from silkmoth and mosquito increase fluid secretion in a dose-dependent manner in the tubules of Tenebrio molitor, but the cockroach CT-like peptide, Dippu-DH 31, has no effect. Thapsigargin induces a small but significant increase in tubule secretion rates. The interactions between different factors in mealworm tubules were explored by testing CT-like peptides, thapsigargin and the mealworm CRF-related diuretic factor Tenmo-DH 37 in various combinations, but no synergistic effects were observed. C-terminal fragments of the CRF-related diuretic peptides Locmi-DH 46 and Dippu-DH 46 fail to increase fluid secretion in mealworm tubules, unlike their corresponding whole peptides. Cross-reactivity of factors between beetle species was investigated using the scarabaeid Onthophagus gazella. Tenmo-DH 37 increases fluid secretion in isolated tubules of O. gazella in a dose-dependent manner, revealing a high degree of cross-reactivity in this distantly related beetle species. However, homogenates of O. gazella brains inhibited fluid secretion in mealworm tubules.

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 μL 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.

Secretion of Water and Ions by Malpighian Tubules of Larval Mosquitoes: Effects of Diuretic Factors, Second Messengers, and Salinity

The effects of changes in the salinity of the rearing medium on Malpighian tubule fluid secretion and ion transport were examined in larvae of the freshwater mosquito Aedes aegypti and the saltwater species Ochlerotatus taeniorhynchus. For unstimulated tubules of both species, the K(+) concentration of secreted fluid was significantly lower when larvae were reared in 30% or 100% seawater (O. taeniorhynchus only), relative to tubules from freshwater-reared larvae. The Na(+) concentration of secreted fluid from unstimulated tubules of O. taeniorhynchus reared in 30% or 100% seawater was higher relative to tubules from freshwater-reared larvae. The results suggest that changes in salinity of the larval rearing medium lead to sustained changes in ion transport mechanisms in unstimulated tubules. Furthermore, alterations of K(+) transport may be utilized to either conserve Na(+) under freshwater (Na(+)-deprived) conditions or eliminate more Na(+) in saline (Na(+)-rich) conditions. The secretagogues cyclic AMP [cAMP], cyclic GMP [cGMP], leucokinin-VIII, and thapsigargin stimulated fluid secretion by tubules of both species. Cyclic AMP increased K(+) concentration and decreased Na(+) concentration in the fluid secreted by tubules isolated from O. taeniorhynchus larvae reared in 100% seawater. Interactions between rearing salinity and cGMP actions were similar to those for cAMP. Leucokinin-VIII and thapsigargin had no effect on secreted fluid Na(+) or K(+) concentrations. Results indicate that changes in rearing medium salinity affect the nature and extent of stimulation of fluid and ion secretion by secretagogues.

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, AGGASG L YAFPRVa; Stoca-PVK/CAP2b-2, NAK L YPVPRVa; and Haeir-PVK/CAP2b-1, AGGASG L YAFPRVa; Haeir-PVK/CAP2b-1, NAK L YPMPRVa. Both Stoca-PVK/CAP2b-1 and -2 stimulate Malpighian tubule fluid secretion in the stable fly, with EC 50 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.

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.

K(+) transport in Malpighian tubules of Tenebrio molitor L: is a K(ATP) channel involved?

The presence of ATP-regulated K(+) (K(ATP)) channels in Tenebrio molitor Malpighian tubules was investigated by examining the effect of glibenclamide on both fluid secretion and basolateral membrane potentials (V(bl)). Glibenclamide, a K(ATP) channel blocker, slowed fluid secretion of Tenebrio tubules. In low bath K(+) concentration (5 mmol l(-1)), glibenclamide either hyperpolarized or depolarized V(bl), resembling the effect seen with Ba(2+). Subsequent addition of 6 mmol l(-1) Ba(2+) caused a further hyper- or depolarization of V(bl). In control Ringer (50 mmol l(-1) KCl, 90 mmol l(-1) NaCl), glibenclamide had no visible effect on V(bl). The effect of ouabain was investigated in low bath [K(+)] in the presence of Ba(2+). V(bl) responded by a small but significant hyperpolarization from -51+/-4 mV to -56+/-4 mV (n=16, P<0.001) in response to 1 mmol l(-1) ouabain. Repeating the experiments in the presence of both glibenclamide and Ba(2+) resulted in a depolarization of V(bl) when ouabain was added. In low bath [K(+)] (high Na(+)), the Na(+)/K(+)-ATPase is expected to function at a high rate. In the presence of Ba(2+), replacing Na(+) by K(+) rapidly depolarized V(bl), but this was followed by a repolarization. Repeating the experiments in the presence of glibenclamide markedly reduced the depolarizing effect and abolished the repolarization, with a gradual decrease in the sensitivity of V(bl) to the surrounding [K(+)]. These results suggest the presence of K(ATP) channels in the basolateral membrane. Glibenclamide had no visible effect on V(bl) in high K(+) or in the absence of Ba(2+), indicating that other highly conductive K(+) channels may mask the effect on K(ATP) channels. This is the first demonstration of the presence of K(ATP) channels in an insect epithelium.

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.

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.

Neurochemical fine tuning of a peripheral tissue: peptidergic and aminergic regulation of fluid secretion by Malpighian tubules in the tobacco hawkmoth M. sexta.

The actions of various peptides and other compounds on fluid secretion by Malpighian tubules in the tobacco hawkmoth Manduca sexta sexta are investigated in this study. Using a newly developed pharate adult Malpighian tubule bioassay, we show that three tachykinin-related peptides (TRPs), leucokinin I, serotonin (5-HT), octopamine, the cardioacceleratory peptides 1a, 1b and 2c, cGMP and cAMP each cause an increase in the rate of fluid secretion in pharate adult tubules. Whereas the possible hormonal sources of biogenic amines and some of the peptides are known, the distribution of TRPs has not been investigated previously in M. sexta. Thus we performed immunocytochemistry using an anti-TRP antiserum. We show the presence of TRP-like material in a small subset of cells in the M. sexta central nervous system (CNS). The larval brain contains approximately 60 TRP-immunopositive cells and there are approximately 100 such cells in the adult brain including the optic lobes. Every ganglion of the ventral nerve cord also contains TRP-like immunoreactive cells. No TRP-containing neurosecretory cells were seen in the CNS, but endocrine cells of the midgut reacted with the antiserum. We propose the hypothesis that the control in insects of physiological systems by hormones may not always involve tissue-specific hormones that force stereotypical responses in their target systems. Instead, there may exist in the extracellular fluid a continuous broadcast of information in the form of a chemical language to which some or all parts of the body continuously respond on a moment-to-moment basis, and which ensures a more effective and efficient coordination of function than could be achieved otherwise.

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 1X 2WG-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.