Anti-diuresis in the blood-feeding insect Rhodnius prolixus Stål: antagonistic actions of cAMP and cGMP and the role of organic acid transport

Secretion of primary urine by upper Malpighian tubules of the blood-sucking insect Rhodnius prolixus has recently been shown to be inhibited by cyclic GMP (cGMP). In the present work, we have demonstrated that cGMP has effects antagonistic to those of cAMP in Rhodnius tubules and have further characterized the effects of cGMP on tubular secretion. Cyclic GMP inhibited secretion at all concentrations from 5x10(-6) to 10(-3)M, though this inhibition was partially or wholly reversed by large (2mM) doses of cAMP. While sub-maximal concentrations of cGMP did not significantly alter [K(+)] and [Na(+)] of secreted fluid, high external [cGMP] reduced secretion to minimal levels and caused [K(+)] and [Na(+)] to approach pre-stimulation levels. Cyclic GMP does not appear to affect the permeability of the lower Malpighian tubule to water. Both cAMP and cGMP likely enter tubule cells by way of an organic acid transporter whose activity is induced by feeding. Sensitivity of the tubules to exogenous cGMP and cAMP, which is assumed to be a function of transport activity, reaches a peak approximately 5 days after the blood meal and declines rapidly thereafter. Transport of anions into upper tubules involves at least two different transporters: one for acylamides (e.g., p-aminohippuric acid) and another for sulphonates (e.g., amaranth, phenol red). Amaranth and phenol red blocked the actions of both cGMP and cAMP, whereas p-aminohippuric acid was without effect. This suggests that cyclic nucleotides enter by way of the sulphonate transporter.     

Characterization of tetraethylammonium uptake across the basolateral membrane of the Drosophila Malpighian (renal) tubule

Basolateral transport of the prototypical type I organic cation tetraethylammonium (TEA) by the Malpighian tubules of Drosophila melanogaster was studied using measurements of basolateral membrane potential (V(bl)) and uptake of [(14)C]-labeled TEA. TEA uptake was metabolically dependent and saturable (maximal rate of mediated TEA uptake by all potential transport processes, reflecting the total transport capacity of the membrane, 0.87 pmol.tubule(-1).min(-1); concentration of TEA at 0.5 of the maximal rate of TEA uptake value, 24 muM). TEA uptake in Malpighian tubules was inhibited by a number of type I (e.g., cimetidine, quinine, and TEA) and type II (e.g., verapamil) organic cations and was dependent on V(bl). TEA uptake was reduced in response to conditions that depolarized V(bl) (high-K(+) saline, Na(+)-free saline, NaCN) and increased in conditions that hyperpolarized V(bl) (low-K(+) saline). Addition of TEA to the saline bathing Malpighian tubules rapidly depolarized the V(bl), indicating that TEA uptake was electrogenic. Blockade of K(+) channels with Ba(2+) did not block effects of TEA on V(bl) or TEA uptake indicating that TEA uptake does not occur through K(+) channels. This is the first study to provide physiological evidence for an electrogenic carrier-mediated basolateral organic cation transport mechanism in insect Malpighian tubules. Our results also suggest that the mechanism of basolateral TEA uptake by Malpighian tubules is distinct from that found in vertebrate renal tubules.     

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.     

AedesCAPA-PVK-1 displays diuretic and dose dependent antidiuretic potential in the larval mosquito Aedes aegypti (Liverpool)

This study reveals that AedesCAPA-PVK-1 (GPTVGLFAFPRV-NH(2)) inhibits basal and serotonin stimulated fluid secretion in the Malpighian tubules of larval Aedes aegypti at femtomolar concentrations. Conversely 10(-4)moll(-1) of the peptide stimulated fluid secretion rates. The diuretic effects of 10(-4)moll(-1)AedesCAPA-PVK-1 and antidiuretic effects of 10(-15)moll(-1)AedesCAPA-PVK-1 were abolished by protein kinase A (PKA) and protein kinase G (PKG) inhibition, respectively. Similar to the peptide, 10(-3)moll(-1) cGMP stimulated fluid secretion but doses in the micromolar to nanomolar range inhibited fluid secretion of the Malpighian tubules. Stimulatory effects of cGMP were abolished by PKA inhibition and inhibitory effects of cGMP were abolished by PKG inhibition. Furthermore, the nitric oxide synthase inhibitor l-NAME attenuated the inhibitory effects of AedesCAPA-PVK-1 but did not affect inhibition by cGMP. Based on the results we propose that AedesCAPA-PVK-1 inhibits fluid secretion rates of larval Malpighian tubules via the NOS/cGMP/PKG pathway and that high doses of the peptide lead to diuresis through the cGMP mediated activation of PKA.     

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.     

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/microl). 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.     

Immunocytochemical localization of a diuretic hormone of the beetle Tenebrio molitor,Tenmo-DH(37), in nervous system and midgut

Although the mealworm Tenebrio molitor inhabits very dry environments, it has at least two diuretic peptides, which increase fluid secretion by the free portions of the Malpighian tubules. Unlike other insect corticotropin-releasing factor (CRF)-related peptides isolated to date, these are non-amidated peptides. The immunocytochemical localization of Tenmo-DH(37) was investigated using antisera raised against this hormone. Immunoreactive neurosecretory cells were found in the brain and abdominal ganglia with immunoreactive processes projecting to the peripheral nervous system. Intense staining of the neurohaemal release site, the corpora cardiaca, was observed. In addition, neurosecretory cells immunoreactive to Tenmo-DH(37) were found in the posterior midgut and a network of immunoreactive nerve processes extended over the surface of the midgut. Tenmo-DH(37) is widely distributed and its staining pattern resembles that found for other, amidated CRF-related diuretic peptides.     

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.     

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.     

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.     

Dopamine-induced epithelial K(+) and Na(+) movements in the salivary ducts of Periplaneta americana

K(+)- and Na(+)-selective double-barrelled microelectrodes were used for intracellular and luminal measurements in salivary ducts of Periplaneta americana. The salivary ducts were stimulated with dopamine (10(-6) mol l(-1)). Dopamine decreased intracellular [K(+)] from 112+/-17 mmol l(-1) to 40+/-13 mmol l(-1) (n=6) and increased intracellular [Na(+)] from 22+/-19 mmol l(-1) to 92+/-4 mmol l(-1) (n=6). Luminal [K(+)] was 15+/-3 mmol l(-1) in the unstimulated salivary ducts and increased to 26+/-11 mmol l(-1) upon stimulation with dopamine (n=10). Luminal [Na(+)] was insignificantly increased from 105+/-25 mmol l(-1) to 116+/-22 mmol l(-1) (n=12) by stimulation with dopamine. The potential difference across the basolateral membrane (PD(b)) was depolarized from -65+/-6 mV to -31+/-13 mV (n=12) and the transepithelial potential difference (PD(t)) was hyperpolarized from -13+/-6 mV to -22+/-7 mV (n=22, lumen negative) upon stimulation with dopamine. The re-establishment of prestimulus values of intracellular [K(+)] and [Na(+)] and PD(b) was inhibited by basolateral addition of ouabain (10(-4) mol l(-1)). Furosemide (10(-4) mol l(-1)) in the bath inhibited the dopamine-induced increase in intracellular [Na(+)], the decrease in intracellular [K(+)] and the depolarization of PD(b). We propose a model for dopamine-stimulated ion transport in the salivary ducts involving basolateral Na(+)-K(+)-2Cl(-) cotransport and active extrusion of K(+) via the apical membrane.     

Isolation, characterization and biological activity of a CRF-related diuretic peptide from Periplaneta americana L.

A diuretic peptide (Periplaneta-DP) has been isolated from extracts of whole heads of the cockroach, Periplaneta americana. The purified peptide increases cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro. In the fluid secretion assay, the response to native Periplaneta-DP is comparable to that obtained with crude extracts of cockroach corpora cardiaca, and the EC50 lies between 10(-8) and 10(-9) M. The primary structure of Periplaneta-DP was established as a 46-residue amidated peptide: T G S G P S L S I V N P L D V L R Q R L L L E I A R R R M R Q S Q D Q I Q A N R E I L Q T I-NH2. Periplaneta-DP is a further member of the recently established family of CRF-related insect diuretic peptides.     

Intracellular and luminal pH measurements of Malpighian tubules of the mosquito Aedes aegypti: the effects of cAMP

In order to understand the critical role that hydrogen ions play in fluid secretion in Malpighian tubules, intracellular and luminal pH and K+ measurements were performed in isolated Malpighian tubules of the yellow fever mosquito (Aedes aegypti). The intracellular pH was 7.03+/-0.05 (n=15 Malpighian tubules (MT)) and the luminal pH was 7.19+/-0.09 (n=99 MT) when bathed in saline at a pH of 7.0. The lumen potential is positive, thus net proton secretion into the lumen is active. The intracellular and the luminal K+ concentrations were 75+/- 9 mM (n=15) and 102+/-13 mM (n=9 MT) respectively. Cyclic AMP analogues accelerated fluid secretion and at the same time acidified the cell without affecting the luminal pH. Both effects were abolished by an isomer of adenosine-3',5' cyclic monophosphothioate (cAMPS), the Rp-cAMPS, known to inhibit protein kinase A. The results suggest that in the presence of cAMP the properties of the cation/H+ exchanger are affected and that this may be a result of phosphorylation of a Na+/2H+ antiporter located on the apical membrane.     

The synthesis and biological activity of linear and cyclic analogs of the two diuretic peptides of Diploptera punctata

We have investigated the effect of analogs of the two Dippu diuretic hormones, Dippu-DH(46) and Dippu-DH(31), on fluid secretion by Malpighian tubules of male Diploptera punctata. We synthesized analogs containing the amino acid methyl-homoserine, to replace methionine residues, to render these modified peptides less subject to oxidation. We have also synthesized C-terminal fragments and their corresponding cyclic analogs to determine their effect on fluid secretion in D. punctata. Our results indicate that the modified peptides retain significant activity in the Ramsay secretion assay. The linear fragments displayed no activity or some inhibitory activity whereas the cyclic analog fragments showed stimulatory activity, in the case of DH(46), or slight inhibitory activity, in the case of DH(31).     

Unusual physiology of scale-less carp,Gymnocypris przewalskii,in Lake Qinghai: a high altitude alkaline saline lake

The scale-less carp (Gymnocypris przewalskii) inhabits Lake Qinghai located on the Qinghai-Tibet plateau (elevation, 3200 m) in western China. The lake waters are alkaline (pH 9.4, titratable alkalinity=30 mmol l(-1)), Mg(2+)-rich (18.7 mmol l(-1)), Ca(2+)-poor (0.30 mmol l(-1)) and saline (9 per thousand ). These fish make annual spawning migrations into freshwater rivers. We investigated the physiology of nitrogen excretion and ionoregulation of fish from the lake and river. Fish from both waters were ammonotelic, although ammonia-N excretion rates were lower in lake fish (175 vs. 344 micromol kg(-1) h(-1), P<0.05) resulting in unusually high levels of ammonia in blood plasma (2.23 vs. 0.32 mmol l(-1)), bile, liver, muscle and brain. Exposure to 0.4 mmol l(-1) total ammonia in lake water ([NH(3)]=0.16 mmol l(-1)) killed fish within 8 h. River fish survived exposure to 1.0 mmol l(-1) total ammonia in river water at pH 8.0 ([NH(3)]=0.023 mmol l(-1)) for 24 h suggesting high ammonia tolerance in lake fish. High glutamate dehydrogenase and glutamine synthetase activities in tissues probably allow the fish to alleviate ammonia toxicity by amino acid accumulation. Neither lake nor river fish relied on urea excretion to remove excess N. Urea-N excretion rates were below 20 micromol kg(-1) h(-1) for both groups, and levels of urea in plasma and tissues were moderate. When exposed to elevated ammonia, urea-N excretion increased slightly (approximately 50 micromol kg(-1) h(-1)) and liver and muscle urea levels increased in the river fish. Plasma ion levels were within the range typical of cyprinids, but river fish had significantly higher plasma [Na(+)] and [Cl(-)] and lower [K(+)] than fish from the lake. During 48-h lake-to-river water transfer, plasma Na(+) and Cl(-) levels rose significantly. Significantly higher Na(+)/K(+)-ATPase activity in the gills of river fish may be related to the higher plasma ion levels. Plasma [Mg(2+)] and [Ca(2+)] were tightly regulated despite the great differences in the lake and river water levels.     

Presence and activity of a Dippu-DH31-like peptide in the blood-feeding bug, Rhodnius prolixus

The blood-feeding bug, Rhodnius prolixus, ingests large blood meals, then undergoes a period of rapid diuresis which is under neurohormonal control. In both cockroach (Diploptera punctata) and fruit fly (Drosophila melanogaster) a calcitonin-like DH31 neuropeptide has been identified [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795-804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469-74] and demonstrated to be active on Malpighian tubule secretion [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795-804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469-74]. Using an antibody raised against D. punctata (Dippu) DH31, we demonstrate the presence of Dippu-DH31-like immunoreactivity in the CNS, salivary glands, hindgut and neurohemal sites of 5th instar Rhodnius. Double-label immunohistochemistry for Dippu-DH31-like and serotonin-like immunoreactivity demonstrates some co-localization of these factors in cells of the mesothoracic ganglionic mass (MTGM) and in neurohemal sites on the abdominal nerves. When tested on Rhodnius 5th instar Malpighian tubules, Dippu-DH31 stimulated minor increases in rate of secretion. Dippu-DH31 tested in combination with serotonin resulted in increases in the rate of secretion which were at least additive.     

Characterization of salicylate uptake across the basolateral membrane of the malpighian tubules of Drosophila melanogaster

The organic anion salicylate is a plant secondary metabolite that can protect plants against herbivores. Transport of salicylate across the basolateral membrane of the Malpighian tubules of Drosophila melanogaster was studied using a radioisotope tracer technique. The uptake of [(14)C]salicylate by the Malpighian tubules was active, saturable and Na(+)-dependent; the maximum uptake rate (J(max)) and the half saturation concentration (K(t)) were 12.6 pmoltubule(-1)min(-1) and 30.7micromoll(-1), respectively. In contrast to organic anion transport by vertebrate renal tissues, salicylate uptake was not trans-stimulated by glutarate (0.01-1.0 mmoll(-1)) or cis-inhibited by high concentrations (5 mmoll(-1)) of various alpha-keto acids (glutaric acid, alpha-ketoglutaric acid, succinic acid, and citric acid). Changes in basolateral membrane potential or physiologically relevant changes in bathing saline pH did not affect the rate of [(14)C]salicylate uptake. Ring-structure monocarboxylic acids (benzoic acid, nicotinic acid, gentisic acid, unlabelled salicylic acid, alpha-cyano-4-hydroxycinnamic acid, probenecid, fluorescein, and P-aminohippuric acid) strongly inhibited [(14)C]salicylate uptake rate. In contrast, short-chain monocarboxylic acids had little (butyric acid) or no effect (lactic acid, pyruvic acid, and propionic acid). Our results suggest that salicylate uptake across the basolateral membrane of D. melanogaster Malpighian tubules is mediated by a non-electrogenic, alpha-cyano-4-hydroxycinnamic acid-sensitive, Na(+):salicylate cotransport system.     

Invitro effect of Canavalia ensiformis urease and the derived peptide Jaburetox-2Ec on Rhodnius prolixus Malpighian tubules

Ureases are metalloenzymes that are widespread among plants, fungi and bacteria. Urease isoforms (jack bean urease-JBU and canatoxin) from Canavalia ensiformis seeds are toxic to insects and fungi, suggesting a role in plant defense. The entomotoxic effect is due to the release of a 10-kDa peptide by cathepsin-like enzymes in the insect's midgut. Urease causes a decrease in post-feeding weight loss in Rhodnius prolixus, suggesting an effect on water balance. To investigate how this impairment occurs, we have evaluated the action of JBU and the urease-derivated peptide Jaburetox-2Ec on R. prolixus Malpighian tubules and also investigated the involvement of second messengers. JBU and Jaburetox-2Ec affect serotonin-induced secretion from Malpighian tubules. This effect is not cAMP-dependent, but the Jaburetox-2Ec effect is cGMP-dependent. Eicosanoid metabolites and calcium ions appear to be involved in JBU effect on diuresis, but are not involved in the action of Jaburetox-2Ec. Jaburetox-2Ec, but not JBU, causes a change in the transepithelial potential of the tubules. Canatoxin has a similar effect on tubules secretion, decreasing the secretion rate, but the urease from Helicobacter pylori has no significant effect. These data are helpful in our understanding of the actions of ureases and derived peptides on insects, and also reinforces the potential use of these proteins as biopesticides.     

The biological activity of diuretic factors in Rhodnius prolixus

The rapid post-feeding diuresis of Rhodnius prolixus is under neurohormonal control and involves the integrated activity of the crop, Malpighian tubules and hindgut. One of the factors which is involved in this rapid diuresis is serotonin, however a peptide(s) is also considered to be involved. In other insects, corticotropin releasing factor (CRF)-like and kinin-like, calcitonin-like peptides and CAP(2b) have been demonstrated to be diuretic factors/hormones.In the present study, serotonin and CRF-like peptides increased secretion rate and cAMP content of Rhodnius Malpighian tubules, while the kinin-like peptides tested did not increase secretion rate or cAMP content of the tubules. Extracts of the CNS were processed and several HPLC fractions revealed kinin-like immunoreactivity but these fractions did not increase secretion rate when tested on Malpighian tubules. However, these same fractions did possess activity when tested on the hindgut contraction assay. In addition, material eluting at higher acetonitrile concentrations from the HPLC increased secretion and cAMP content of Rhodnius Malpighian tubules. This material eluted at concentrations of acetonitrile consistent with the elution time of CRF-like peptide standards.Synergism was demonstrated using the pharmacological agent forskolin and serotonin, tested on the rate of secretion of Rhodnius Malpighian tubules, in agreement with data of Maddrell et al. As well, synergism could be demonstrated using mesothoracic ganglionic mass (MTGM) homogenates and serotonin at some concentrations of serotonin. However, combinations of CRF-like material and serotonin increased secretion additively, not synergistically. Kinin-like peptides, tested along with CRF-like material and serotonin, at low concentrations, did not increase secretion above that of those factors tested alone.     

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.