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The biological activity of diuretic factors in Rhodnius prolixus
Peptides 23 (2002) 671– 681
The biological activity of diuretic factors in Rhodnius prolixus V.A. Te Bruggea,*, D.A. Schooleyb, I. Orcharda a
Department of Zoology, University of Toronto, 25 Harbord Street, Toronto, Ontario, Canada M5S-3G5 b Department of Biochemistry, University of Nevada, Reno, Nevada 89557, USA Received 1 May 2001; received in revised form 16 October 2001; accepted 16 November 2001
Abstract 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 [25], 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 CAP2b 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. [24]. 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. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Malpighian tubule; Diuresis; cAMP; Hindgut contraction; Neuropeptide
1. Introduction Rhodnius prolixus 5th instars ingest a blood meal which is up to10 times their initial body weight. During gorging the insect initiates a period of rapid diuresis which results in the loss of 40% of the weight of the bloodmeal. This diuresis 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 [25]. Serotonin is released into the hemolymph at the beginning of feeding reaching a peak titer of 10⫺7 M 5 min after the start of feeding. The hemolymph titer of serotonin decreases over the next 15 min to between 10⫺8 and 2 ⫻ 10⫺8 M and remains at this level for the next few hours [20]. A peptide(s) is also considered to be involved in this rapid diuresis [1], and in other insects, cor* Corresponding author. Tel.: ⫹1-416-978-4602; fax: ⫹1-416-9783522. E-mail address: [email protected] (V.A. Te Brugge).
ticotropin releasing factor (CRF)-like, kinin-like (see review [7]), calcitonin-like [9,11] and cardioactive peptide 2b (CAP2b) [10] peptides, have been demonstrated to be diuretic factors/hormones. Interestingly, while CAP2b has been shown to have diuretic effects on Drosophila tubule secretion, studies done on Rhodnius Malpighian tubules with CAP2b have suggested an antidiuretic effect [31]. Previously, we have demonstrated the presence of CRFlike [34] and kinin-like peptides [35], and serotonin [21] in the CNS of Rhodnius prolixus. These factors are found in neurohemal terminals of the abdominal nerves and corpus cardiacum (CC). CRF-like and kinin-like peptides, but not serotonin, are co-localized in the neurohemal terminals of the abdominal nerves [34,35]. These results suggest that all three factors are released into the hemolymph and that the release of serotonin can be independent of the release of the peptides. The CRF-like peptide, Locusta-DH, has previously been shown to increase the secretion of Rhodnius Malpighian tubules with an EC50 of 0.14 M [7], although in other
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insects CRF-like peptides increase secretion at low nanomolar concentrations [6,7]. CRF-like peptides are considered to work through a cAMP dependent pathway [6,7]. Kinin peptides were originally isolated from the cockroach, Leucophaea maderae, based on their ability to induce contraction of the hindgut muscle [14]. Subsequently, hindgut assays, in combination with high-performance liquid chromatography (HPLC), were used to isolate kinin peptides from several other insect species [5,12,16,30,33] or to confirm bioactivity [15,37]. As well as having activity on hindgut, kinin-like peptides have been shown to increase the rate of secretion of isolated Malpighian tubules of several species of insects, including Acheta domesticus [8], Locusta migratoria [6], Drosophila melanogaster [36], Musca domestica [15] and Culex salinarus [12]. However, only very small increases in secretion were observed in Aedes aegypti [37] and no stimulation by native kinin-like peptides was obtained in Helicoverpa zea [4]. The EC50 of the Acheta-kinins for Malpighian tubule secretion in Acheta is in the order of 0.3– 0.02 nM [7]. Similar values were found for Musca and Locusta-kinins when tested on their respective tubules [6,15]. Insect kinins have been shown to stimulate changes in transepithial potentials of the Malpighian tubules [36] and to increase the Cl⫺ permeability [38] of the Malpighian tubules. This increased permeability to Cl⫺ is mediated by the release of intracellular Ca2⫹ [3,28]. In Drosophila Malpighian tubules, kinin-like peptides have been shown to increase intracellular Ca2⫹ specifically in the stellate cells [32,36]. Immunohistochemical experiments on fed 5th instar Rhodnius suggest a reduction in staining intensities of CRFlike and kinin-like immunoreactive material in the mesothoracic ganglionic mass (MTGM) within 30 min of feeding (Te Brugge, unpublished). In addition, serotonin is known to be released into the hemolymph at the start of feeding, with the titer peaking at 5 min and remaining at a lower level for the next few hours [20]. Preliminary experiments suggest the presence of material(s) in the hemolymph at 5 min and 2 h, from the start of feeding, which increases the rate of secretion of Malpighian tubules but is not blocked by the serotonin antagonist ketanserin (Te Brugge unpublished). These pieces of evidence suggest that serotonin, CRF-like and kinin-like peptides may all be present in the hemolymph during and following feeding. The control of the post-feeding diuresis in Rhodnius involves the crop, Malpighian tubules and hindgut [23]. All of these tissues must have a rapid initiation and cessation of activity associated with diuresis such that the insect does not retain excess water or become desiccated. Mixtures of small amounts of diuretic factors have been shown to interact synergistically to increase the rate of secretion of Malpighian tubules by an amount greater than the sum of the rates of secretion induced by the individual factors [6]. Thus, serotonin and forskolin or tissue extracts have been shown to synergistically increase the secretion rate of Rhodnius Malpighian tubules [24]. Moreover, the synergistic
interactions of diuretic factors have been shown to result in a steeper dose-response curve. This is shown clearly in the synergism between Locusta CRF-like and kinin-like peptides on Locusta tubules [6]. Synergism between CRF-like and kinin-like peptides has also been shown in Musca [15]. This study focuses on serotonin, CRF-like and kinin-like analogues and tissue extracts tested on Malpighian tubule secretion rate and cAMP content assays in Rhodnius prolixus to more fully characterize the range of putative diuretic factors present in the nervous system. Rhodnius CNS extracts were partially purified by HPLC. These fractions were tested for diuretic activity and kinin-like immunoreactivity using RIA; as well a hindgut contraction assay was established to test for myotropic activity.
2. Materials and methods 2.1. Animals Fifth instar larvae of Rhodnius prolixus were taken from a long standing colony maintained at 25°C under high humidity. The insects were unfed, 6 – 8 weeks post emergence, and previously fed on rabbit’s blood as 4th instars. 2.2. Malpighian tubule secretion assay The Malpighian tubules of 5th instars were dissected under physiological saline [18], and the tubules were freed from trachea and fat with the aid of fine glass probes. The secretion assays were conducted as described in Te Brugge et al. [34]. In brief, the upper portions of the tubules were transferred to a 20 l drop of physiological saline under water-saturated heavy mineral oil. The open end of the tubule was pulled out and wrapped around a minutin pin. Saline containing serotonin (Sigma Chemical Co., St Louis, Missouri, USA), Diploptera punctata (Dippu)-DH46 [11] or Zootermopsis nevadensis (Zoone)-DH [2], kinin-like peptides (obtained from Sigma) or tissue extracts was exchanged for the equilibrating saline. The tubules were allowed to secrete for a further 20 –30 min. Droplets of urine from the cut end of the tubule were collected every 5 min and their diameter measured under mineral oil using an eyepiece micrometer and the volume calculated. In experiments testing the CRF-like peptides, a maximal rate of secretion was determined using 10⫺6 M serotonin, with each tubule acting as its own control. The percent relative to the maximal rate of secretion was calculated for each test substance. In some experiments rates were expressed as the actual rate of secretion. Mean ⫾ SEM were calculated and compared using an unpaired Student’s t test. 2.3. Malpighian tubule cAMP assay The cAMP content of the Malpighian tubules was assayed as previously described [34]. In experiments testing
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tissue extracts, Malpighian tubules from 5th instars were dissected and then transferred to a microfuge tube containing 0.5 mM of the phosphodiesterase inhibitor, 3-isobutyl1-methyl-xanthine (IBMX) in 50 l of physiological saline, along with tissue extract, serotonin, CRF-like or kinin-like peptides. Tubules were incubated for 10 min at room temperature and the reaction stopped with 500 l of boiling 0.05 M sodium acetate. Samples were placed in a boiling water bath for a further five minutes, then frozen at -20°C until assayed. The samples were thawed, sonicated and centrifuged at 8800 g for 10 min and the supernatant decanted. The cAMP content of the supernatant was measured using an RIA kit (Mandel/NEN, Guelph, Ontario) with modifications as previously described [19]. The mean ⫾ SE were calculated and compared using an unpaired Student’s t test. 2.4. Radioimmunoassay The radioimmunoassay (RIA) for kinin-like material was conducted as previously described in Te Brugge et al. [35]. The assay utilizes the same anti-leucokinin 1 antiserum that was used in immunohistochemistry studies [35] at a final concentration of 1:100,000 in RIA buffer. Leucokinin I with a tyrosine residue on the N terminus was custom synthesised (Research Genetics, Huntsville, AL, USA) and the peptide iodinated using the Chloramine T method. The standard curve for the RIA used leucokinin I (Sigma) with concentrations ranging from 5–1000 fmol/100 l RIA buffer. 125I-Y leucokinin I working tracer, leucokinin I antiserum and either standard or sample (100 l of each) were combined in 1.5 ml polypropylene microfuge tubes for 18 h at 4°C. Standards and samples were run in duplicate. Unbound peptide was precipitated by the addition of 250 l of dextran-coated charcoal. The charcoal suspension was added to the microfuge tubes and incubated for three minutes, the tubes were then centrifuged for eight minutes at 8800 g; 400 l of supernatant was removed to glass test tubes and counted for 1 min in a Beckman 4000 gamma counter. 2.5. Isolation of CNS CRF-like and kinin-like peptides One hundred 5th instar CNSs were dissected under physiological saline and collected in acidified methanol (methanol: acetic acid: water; 90:9:1) on ice, then stored at -20°C. The tissues were then sonicated and centrifuged for 10 min at 8800 g. The supernatant was decanted and dried in a Speed-Vac concentrator (Savant, Farmingdale, NY, USA). The dried extract was dissolved in water with 0.01% TFA and then applied to a C18 Sep-Pak cartridge (Waters Associates, Mississauga, Ontario, Canada) which had been previously equilibrated as described by Miggiani et al. [27]. The cartridge was then washed sequentially with 3.0 ml each of water, 30% and 60% acetonitrile (Burdick and Jackson, Muskegon, MI, USA) with 0.1% TFA (BDH,
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Toronto, Ontario, Canada) and the eluents collected. The collected extracts were dried in the Speed-Vac and stored at -20°C until use. The 30 and 60% acetonitrile eluents were then applied to a reversed-phase HPLC system which utilized a Brownlee C18 column (Mandel/Alltech, Guelph, Ontario, Canada) with a 1 ml injection loop. The HPLC gradient was 9% to 60% acetonitrile with 0.01% TFA over 40 min. The fractions were collected, subsampled, dried and stored at -20°C until use. The samples were then taken up in physiological saline prior to bioassay. 2.6. Rhodnius hindgut assay Rhodnius hindgut assays were conducted on isolated 5th instar hindguts maintained under physiological saline. The preparation consisted of a small piece of ventral cuticle surrounding the anus to secure the hindgut to a Sylgard (Dow Corning, Midland Michigan, USA; Paisley Products, Scarborough, Ontario) coated dish, while the anterior end of the hindgut and a small portion of the posterior midgut was tied by a fine thread to a miniature force transducer (Aksjeselskapet Mikro-elektronikk, Horten, Norway). The output from the transducer was connected to a data acquisition system (Biopac MP100 workstation, Biopac systems Inc, Santa Barbara, CA, USA). Tissues were equilibrated in 20 l of saline for 20 min, then the saline removed and replaced with either saline or test solutions containing peptides or tissue extracts. Basal tonus and phasic contractions were recorded and measured using the Biopac system.
3. Results 3.1. The effects of synthetic factors on Malpighian tubules Serotonin and both of the CRF-like peptides increased the rate of secretion of Rhodnius Malpighian tubules. Serotonin increased the rate of secretion in a dose-dependent manner, with an EC50 of 4.2 ⫻ 10⫺8 M (see Fig. 1A). The CRF-like peptides, Zoone-DH and Dippu-DH46, also increased the rate of secretion of Rhodnius Malpighian tubules in a dose-dependent manner, with both CRF-like peptides having a similar dose-response curve. The EC50 values were 6.7 ⫻ 10⫺7 M and 5.5 ⫻ 10⫺7 M for Zoone (Fig. 1B) and Dippu-DH46 (not shown) respectively, threshold (lowest concentration to produce a significant increase in secretion) at 2 ⫻ 10⫺7 M, and maxima (lowest concentration to produce the highest (maximum) rate of secretion, relative to serotonin) at approximately 10⫺6 M. Leucokinin 1, LK8 or Locusta-kinin each failed to increase the rate of secretion significantly over saline controls (Table 1) and at some concentrations may even have decreased the rate of secretion. Both CRF-like peptides, as well as serotonin, increased the cAMP content of Rhodnius Malpighian tubules in the presence or absence of IBMX. Zoone-DH, in the presence
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Fig. 2. CRF-like peptide, Zoone-DH, dose-response curve on cAMP content of Rhodnius 5th instar Malpighian tubules. Points are mean ⫾ SE n ⫽ 4. Regression curve was fitted by SigmaPlot version 5 curve fitting program. The EC50 is 7.5 ⫻ 10⫺7 M.
3.2. The effects of CNS factors on Malpighian tubules
Fig. 1. (A) Serotonin dose-response curve on the rate of secretion of Rhodnius 5th instar Malpighian tubules. Points are mean ⫾ SE, n ⫽ 4 – 8. Regression curve fitted by Sigmaplot version 5 curve fitting program. The EC50 is 4.2 ⫻ 10⫺8 M. (B) CRF-like peptide, Zoone-DH, dose-response curve on the rate of secretion of Rhodnius 5th instar Malpighian tubules. Points are mean ⫾ SE, n ⫽ 4 – 8. Regression curve fitted by SigmaPlot version 5 curve fitting program. The EC50 is 6.7 ⫻ 10⫺7 M.
of IBMX, increased the cAMP content in a dose-dependent manner (Fig. 2) with an EC50 of 7.5 ⫻ 10⫺7 M, threshold at 5 ⫻ 10⫺8 M and maxima at approximately 2 ⫻ 10⫺6 M. None of the kinin-like peptides increased the cAMP content of Malpighian tubules.
Table 1 The effects of kinin-like peptides on rate of secretion from Rhodnius Malpighian tubules. Mean ⫾ SE n ⫽ 4 –9. Compound Rhodnius saline Leucokinin I
Leucokinin VIII Locusta-kinin
Concentration [M]
10⫺9 10⫺8 ⫺7 10 10⫺6 5 ⫻ 10⫺6 5 ⫻ 10⫺6
Rate of secretion ⫾ SE (nl/min) 0.13 ⫾ 0.09 0.01 ⫾ 0.01 0.18 ⫾ 0.18 0.2 ⫾ 0.2 0.2 ⫾ 0.2 0.23 ⫾ 0.11 0.04 ⫾ 0.03
3.2.1. Kinin RIA Material eluting from the Sep-Pak with 30% acetonitrile and 0.1% TFA was injected into the RP-HPLC system and 1 ml fractions were collected. These fractions were subsampled and tested using the RIA for kinins at 2 CNS equivalents per sample. Kinin-like material was found in fractions 18, 22, 23 and 25–30 (the remaining fractions only revealing the non-specific background levels). The greatest amount of kinin-like material was seen in fractions 25 and 26 (Fig. 3A). 3.2.2. Effects on tubules Subsamples (2, 4, 12.5 CNS equivalents/20 l) of all of the RP- HPLC fractions were tested in secretion assays and expressed as percent maximum secretion relative to10⫺6 M serotonin. As well, subsamples from all fractions were tested in Malpighian tubule cAMP assays. In bioassays of fractions where kinin-like material was found (fractions 18, 22, 23 and 25–30), there was no increase in the rate of secretion above baseline rates of secretion. Malpighian tubule rate of secretion was increased using fractions 6, 7 and 12 tested at 4 CNS equivalents (Fig. 3B) and these same fractions were also capable of increasing the cAMP content of Malpighian tubules to 10.3, 12.1 and 8.3 pmol/tissue respectively (versus 3.6 ⫾ 0.5 pmol/tissue for control values) when tested in the presence of IBMX. For comparison, a maximum dose of serotonin (10⫺6 M) resulted in Malpighian tubule cAMP content increasing to 11.2 ⫾ 1.7 pmol/tissue. Material from the Sep-Pak cartridge that eluted with 60% acetonitrile was run on RP-HPLC and 1 ml fractions were collected. The fractions were subsampled and these subsamples were tested in Malpighian tubule secretion and
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Fig. 3. (A) Kinin-like immunoreactivity in fractions of the RP-HPLC run of the 30% acetonitrile eluent from C18 Sep-Pak. Sub-samples, 2 CNS equivalents, of the 1 ml fractions were tested in the leucokinin 1 RIA. Note that background levels within the RIA are 27.1 0.95 ⫾ fmol. Fractions 1–5 were pooled resulting in elevated background levels due to additional non-specific binding. Leucokinin 1-like material was detected in fractions 18, 22, 23 and 25–30. A single example is shown, but this pattern of elution is consistent between 4 individual HPLC runs of CNS material. The elution times for LK1 and serotonin are indicated by arrows. (B) Rhodnius Malpighian tubule secretion assay of sub-samples of the RP- HPLC fractions tested in secretion assays at 4 CNS equivalents, expressed as percent maximum secretion with respect to serotonin at 10⫺6 M.
cAMP assays at 2 and 4 CNS equivalents. When tested at 4 CNS equivalents, fractions 43, 44 and 45 increased the rate of secretion of Rhodnius Malpighian tubules (Fig. 4), with fraction 43 eliciting 45% of the maximum rate of secretion induced by 10⫺6 M serotonin. Subsamples of these fractions were also tested in Malpighian tubule cAMP assays in the presence of IBMX. Fractions 43, 44 and 45 resulted in an increase of cAMP content of Malpighian tubules to 11.9, 12
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Fig. 5. Fraction 44 of the RP-HPLC run of the 60% acetonitrile eluent from C18 Sep-Pak was tested in secretion assays. Fraction 44 increased the rate of secretion of 5th instar Malpighian tubules in a dose-dependent manner. Points are mean ⫾ SE n ⫽ 4.
and 8.6 pmol/tissue respectively, relative to baseline levels of 4.8 ⫾ 0.5 pmol/tissue. The material that stimulated both secretion and increases in cAMP content eluted at an acetonitrile concentration of approximately 57% (Fig. 4). Fraction 44 (since fraction 43 was used for other assays) was further tested and shown to stimulate dose-dependent increases in the rate of secretion from Malpighian tubules (Fig. 5). 3.2.3. Hindgut assays In order to provide a positive test for bioactivity of the kinin-like material, subsamples of fractions 17–35 from the 30% Sep-Pak fractionated RP-HPLC run were tested in the hindgut contraction assay. Kinin RIA positive fractions stimulated an increase in the frequency of contraction and an increase in basal tonus (Table 2). The samples with the greatest activity resulted in large changes in basal tonus, which masked the superimposed phasic contractions. Other fractions were also myostimulatory, and two fractions were myoinhibitory (Table 2). 3.3. Additivity and synergism studies
Fig. 4. Rhodnius Malpighian tubule secretion assays of fraction subsamples from the RP-HPLC run of the 60% acetonitrile eluent from C18 Sep-Pak. HPLC fractions tested in secretion assays at 4 CNS equivalents, expressed as percent maximum secretion with respect to serotonin at 10⫺6 M. Consistent elution profiles were obtained from 3 HPLC runs of CNS material. The elution time for Dippu DH46 is indicated by arrow.
Homogenates of the MTGM contain serotonin, CRF-like and kinin-like peptides as well as many other compounds. Previously Maddrell et al. [24] had tested MTGM homogenates (0.05 MTGM/100 l) and serotonin (4 ⫻ 10⫺8 M) on secretion rates, using a single concentration of the two factors. In order to look more fully at the interactions of serotonin and tissue homogenates on the resultant doseresponse curve characteristics, we tested a range of concen-
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Table 2 The effects of fractions 18 –35 from the RP-HPLC run of the 30% SepPak eluent of Rhodnius 5th instar CNS extracts on Rhodnius hindgut contractions Fraction #
Hindgut stimulation/inhibition
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
0 0 0 ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹⫹ ⫹ ⫺ ⫺ ⫹ ⫹ 0
0 no change ⫹ increase in frequency of contraction and basal tonus ⫹⫹ strong increase in basal tonus, masking the superimposed phasic contractions ⫺ decrease in frequency of contraction
trations of serotonin with the same fixed concentration of MTGM homogenate (0.05 MTGM/100 l). Fig. 6 shows the dose-response curve for serotonin with an EC50 of 4.2 ⫻ 10⫺8 M. In addition Fig. 6 illustrates the dose-response curves for a combination of increasing concentrations of serotonin and a fixed dose of MTGM extract. The sum of
Fig. 6. Synergism studies. Serotonin dose-response curve on secretion rate from Malpighian tubule (solid line). The EC50 for the serotonin doseresponse curve is 4.2 ⫻ 10⫺8 M. The dose-response curve for serotonin tested in the presence of MTGM extracts (0.05 equivalents/100 l) is shown by the dashed line. The EC50 of the dose-response curve for the combination of serotonin and MTGM extract is 1.6 ⫻ 10⫺8 M. The dotted line represents the calculated rate of secretion of the individual factors added together. Points are mean ⫾ SE n ⫽ 4 – 8.
the data for the two factors tested separately is shown by the dotted line, and the observed rate of secretion with the two factors tested in combination is shown by the dashed line. The curve for the rate of secretion with the two factors tested in combination is shifted to the left with an EC50 of 1.6 ⫻ 10⫺8 M for serotonin. The rate of secretion measured when the two factors are combined is only significantly greater (P ⬍ 0.05) at two low concentrations of serotonin (10⫺8 M and 2 ⫻ 10⫺8 M). Serotonin and CRF-like peptides were also tested in combination. Low concentrations of serotonin (3 ⫻ 10⫺8 M) and Zoone-DH (2.5 ⫻ 10⫺7 M) each resulted in only small increases in the rate of secretion. When applied in combination, these factors resulted in an increase in the rate of secretion which was not significantly greater than the sum of the two factors tested alone (Fig. 7A). In a similar manner serotonin (3 ⫻ 10⫺8 M) and fraction 44 (2 CNS equivalents) both increased the rate of secretion. When tested in combination, the two factors resulted in an increased rate of secretion which was not significantly greater than the sum of the two factors (Fig. 7B). In order to verify our technique for analysis of synergism, we tested serotonin and forskolin individually and in combination on Malpighian tubule secretion, since this combination has previously been shown to increase the rate of secretion of Rhodnius tubules synergistically [24]. In these assays, the forskolin was dissolved in DMSO. A concentration of 0.1% DMSO had no measurable effect on secretion. Forskolin, tested at a concentration of 1 ⫻ 10⫺5 M, increased the rate of secretion, as did serotonin tested at 3 ⫻ 10⫺8 M. When the two factors were tested together the resulting rate of secretion was significantly greater (3.9 fold) than the sum of the values of the two factors tested individually; a synergistic response. This rate of secretion stimulated by low concentrations of the two factors in combination resulted in rates of secretion which were close to maximal rates of secretion induced by 10⫺6 M serotonin (Fig. 8). Malpighian tubule secretion was tested with kinin-like peptides both singly, and in combination with serotonin and CRF-like peptides at low concentrations, to look for any effects on secretion by the kinins when other diuretic factors are present. Various kinin-like peptides were tested and none increased the rate of secretion significantly from rates of secretion stimulated by the factors tested alone. The results of secretion assays using Locusta-kinin in combination with serotonin and the CRF-like peptide Zoone-DH are shown in Fig. 9. No synergism was observed with these combinations. Rhodnius 5th instar Malpighian tubules were incubated for 10 min in the presence of IBMX with serotonin (3 ⫻ 10⫺8 M) and Zoone-DH (2.5 ⫻ 10⫺7 M) individually and in combination. Both serotonin and Zoone-DH each increased the cAMP content of the Malpighian tubules significantly over saline controls. The mixture of the two factors resulted
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Fig. 8. Forskolin and serotonin were tested separately and together at low concentrations (1 ⫻ 10⫺5 M and 3 ⫻ 10⫺8 M respectively) to look at their combined effect on Malpighian tubule rate of secretion. At these concentrations both factors increased rate of secretion over saline controls. The calculated value of the addition of the individual rates of secretion is shown as well as the actual rate of secretion. The final column is the maximum rate of secretion obtained with 10⫺6 M serotonin. The calculated value of secretion (addition of the individual rates of secretion) was significantly different than the observed rate of secretion. Points are mean ⫾ SE n ⫽ 4 – 6. Fig. 7. (A) CRF-like peptide (Zoone-DH) and serotonin were tested separately and together at low concentrations (2.5 ⫻ 10⫺7 M and 3 ⫻ 10⫺8 M respectively) to look at their combined effect on Malpighian tubule rate of secretion. At these concentrations both factors increased rate of secretion over saline controls. The calculated value of the addition of the individual rates of secretion is shown as well as the actual rate of secretion. The final column is the maximum rate of secretion obtained with 10⫺6 M serotonin. The calculated value of secretion (addition of the individual rates of secretion) was not significantly different than the observed rate of secretion. Points are mean ⫾ SE n ⫽ 4 – 6. (B) Fraction 44 and serotonin were tested separately and together at low concentrations (2 CNS eq and 3 ⫻ 10⫺8 M respectively) to look at their combined effect on Malpighian tubule rate of secretion. At these concentrations both factors increased rate of secretion over saline controls. The calculated value of the addition of the individual rates of secretion is shown as well as the actual rate of secretion. The final column is the maximum rate of secretion obtained with 10⫺6 M serotonin. The calculated value of secretion (addition of the individual rates of secretion) was not significantly different than the observed rate of secretion. Points are mean ⫾ SE n ⫽ 4 – 6.
in a small but not significant increase in cAMP content over that found for the individual factors (Fig. 10A). Rhodnius 5th instar Malpighian tubules were incubated for 10 min in the presence of IBMX with serotonin (3 ⫻ 10⫺8 M) and forskolin (1 ⫻ 10⫺5 M) individually and in
combination. DMSO (0.01%) had no measurable effect on cAMP content of the Malpighian tubules. Serotonin and forskolin, at these concentrations, increased cAMP content of the Malpighian tubules significantly (P ⬍ 0.1 and P ⬍ 0.05 respectively) above the saline controls. In contrast to the earlier result where these two factors acted synergistically on the rate of secretion of Malpighian tubules, the mixture of the two factors resulted in a small but not significant increase in cAMP content over that found for the individual factors (Fig. 10B). That is to say, no synergism was observed.
4. Discussion Previously, we have used immunohistochemisty to demonstrate the presence of CRF-like peptides [34], kinin-like peptides [35] and serotonin [21] in the CNS of 5th instar Rhodnius. These factors are found in neurohemal terminals of the abdominal nerves and CC and so may be released into the hemolymph from both sites. CRF-like and kinin-like
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Fig. 9. Locusta-kinin, Zoone-DH and serotonin were tested separately and together at low concentrations (3 ⫻ 10⫺8 M, 2.5 ⫻ 10⫺7 M and 3 ⫻ 10⫺8 M, respectively) to look at their combined effect on Malpighian tubule rate of secretion. At these concentrations Zoone-DH and serotonin increased rate of secretion. The kinin-like peptide did not increase secretion. The individual rates of secretion stimulated by serotonin or Zoone-DH were not significantly different than the rate of secretion of serotonin or Zoone-DH combined with Locusta-kinin. The combination of all three factors resulted in increases in secretion that were not significantly different than the additive rates of serotonin or Zoone-DH. Points are mean ⫾ SE n ⫽ 4 – 6.
peptides, but not serotonin, are co-localized in the neurohemal terminals on the abdominal nerves. Serotonin and CRF-like peptides increase secretion rate and cAMP content of Rhodnius Malpighian tubules in a dose-dependent manner. The dose-response curve of Malpighian tubule secretion stimulated by serotonin was consistent with that previously shown by Maddrell et al. [26]. The curves are relatively steep, have similar EC50, threshold and maxima. This is in contrast to a later serotonin doseresponse curve [24] which was shifted to the right (higher concentrations) and was less steep. All of the CRF-like peptides tested to date on Rhodnius tubules (Lom-DH [7], Dippu-DH and Zoone-DH (present study)) have a moderate degree of sequence identity and have similar EC50’s on Rhodnius Malpighian tubules. However, the concentrations of CRF-like peptide required for secretion are quite high relative to native CRF-like peptides tested on their conspecific tubules, which are active in the nanomolar range [6,7]. The kinin-like peptides failed to increase rate of secre-
Fig. 10. (A) CRF-like peptide (Zoone-DH) and serotonin were tested in Malpighian tubule cAMP assays in the presence of IBMX. Serotonin (3 ⫻ 10⫺8 M) and Zoone-DH (2.5 ⫻ 10⫺7 M) were tested individually and in combination. Both serotonin and Zoone-DH increased the cAMP content of the Malpighian tubules significantly over saline controls. The mixture of the two factors did not significantly increase cAMP over that seen when testing them individually. Points are mean ⫾ SE n ⫽ 4. (B) Forskolin (1 ⫻ 10⫺5 M) and serotonin (3 ⫻ 10⫺8 M) were tested in Malpighian tubule cAMP assays in the presence of IBMX. DMSO (0.01%) had no measurable effect on cAMP content of the Malpighian tubules. Serotonin and forskolin increased cAMP content of the Malpighian tubules above the saline controls. The cAMP content of the Malpighian tubules incubated with both factors was not significantly different from the cAMP content of Malpighian tubules incubated with the individual factors. Points are mean ⫾ SE n ⫽ 4.
tion or cAMP content of Rhodnius Malpighian tubules. Kinins tested in other insects have been active on Malpighian tubule secretion, including those in Acheta [8], Culex [13], Locusta [7], Drosophila [36] and Musca [15, 17]. In contrast the kinins isolated from Helicoverpa zea are inactive in Helicoverpa [4], but are active on Manduca sexta tubules. Aedes kinins resulted in only small increases in the
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rate of secretion from Malpighian tubules of Aedes, and Aedes kinin II did not increase secretion at all [37]. Although the kinin-like peptides did not increase secretion of Rhodnius tubules, they were biologically active when tested on Rhodnius hindgut muscles, increasing basal tonus and frequency of phasic contractions. As such the Rhodnius hindgut responds to kinins in a similar fashion as other insect hindguts [5,14,15,16,30,33,37]. Extraction and separation of the 5th instar Rhodnius CNS indicates the presence of serotonin and kinin-like peptides in the 30% eluent. Previously, we have shown that over 90% of the secretion caused by the 30% eluate is blocked by the serotonin antagonist, ketanserin [35]. The RP-HPLC separation of the 30% C18 Sep-Pak eluate indicated several fractions that were positive for kinin-like material tested using RIA. However, those fractions which contained kininlike material did not increase secretion rate of Rhodnius Malpighian tubules. Several RP-HPLC fractions collected at the beginning of the RP-HPLC run (fractions 6,7) increased secretion and cAMP content of the Malpighian tubules and likely reflect the activity of serotonin, which elutes from the C18 column at a time consistent with these active fractions (Fig. 3A). The nature and identity of the activity seen in fraction 12, which increased both secretion and cAMP content of Malpighian tubules, is not known. The 60% eluent from C18 Sep-Pak was run on RP- HPLC and three fractions, 43, 44 and 45, increased secretion and cAMP content of Rhodnius 5th instar Malpighian tubules. Fraction 44 gave a dose-dependent increase in the rate of secretion of Malpighian tubules. The active fractions elute at concentrations of acetonitrile consistent with that found in other studies looking at CRF-like peptides [29] and CRFlike standards (Fig. 4). The CRF-like peptides, Locusta-DH ([7], Te Brugge unpublished), Dippu-DH46 and Zoone-DH each increased secretion and cAMP content of Malpighian tubules. Taken together, the evidence suggests that there is a CRF-like peptide present in Rhodnius with diuretic activity, which may be similar to other members of the CRF-like family of peptides. The CRF-like and kinin-like peptides which are co-localized in the neurohemal terminals of the abdominal nerves have the potential to be released together and independent of serotonin. Serotonin concentrations reach a peak of 10⫺7 M at 5 min after the commencement of feeding. The serotonin concentration decreases to a level close to 10⫺8 M over the next 15 min, remaining at low levels for the next few hours [20]. Preliminary results suggest the presence of a CRF-like peptide in the hemolymph at 5 min and 2 h after feeding (unpublished data). It is possible then, that several potential diuretic factors could be present in the hemolymph at the same time. Coast [6] has demonstrated that the interaction of Locusta CRF-like and kinin-like peptides on Locusta tubules results in a synergistic response in tubule secretion. These factors together caused the dose-response curve to be steeper and shifted to the left. Maddrell et al. [24] found that serotonin and either MTGM extracts or
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forskolin also had a synergistic interaction on the rate of Malpighian tubule secretion. In the present study we found some synergism between MTGM homogenates or forskolin and serotonin on the rate of secretion. This was strongest using the pharmacological agent forskolin in combination with serotonin (3.9 fold greater rate of secretion than the sum of the rates of secretion of the two factors). This confirms the results of Maddrell et al. [24] and verifies our technique for looking at synergism on Malpighian tubule secretion. Forskolin was used since it increases cAMP content of the Malpighian tubules, as do both serotonin and the ‘diuretic peptide’ known to be involved in diuresis [24]. The combination of serotonin and forskolin was used to test whether two agents that increase cAMP could have synergistic effects on secretion. However, this synergism between serotonin and forskolin on Malpighian tubule secretion was not evident in experiments looking at the cAMP content of Malpighian tubules. CRF-like analogues and RP- HPLC fraction 44 tested in the presence of serotonin did not elicit a diuretic response significantly greater than an additive response, when tested for rate of secretion or cAMP content of Malpighian tubules. Why there is such strong synergism between serotonin and forskolin on Malpighian tubule secretion but not cAMP content is unclear. It may indicate that the synergism is caused by other cAMP- independent pharmacological effects of forskolin [22], the interaction of different second messenger systems or pathways downstream of cAMP formation. Tissue extracts tested in the presence of serotonin demonstrated synergism at 10⫺8 M serotonin (2 fold greater response than the sum of two diuretic factors) and at 2 ⫻ 10⫺8 M serotonin (1.7 fold greater response than the sum of the two diuretic factors), and the dose-response curve for serotonin was shifted to the left. However, the results obtained are not as dramatic as those seen with serotonin and forskolin. The other “diuretic” factor tested, kinin-like peptides, did not cause any change in the rate of secretion when tested in combination with either serotonin and/or CRF-like peptides at low concentrations. As there was some evidence of synergism between the tissue homogenates and serotonin, there may be other diuretic factors that play a role in the rapid post-feeding diuresis. The interactions of various diuretic factors on Malpighian tubules may also affect the various characteristics of diuresis, such as ion composition, speed of response and duration of the response, in addition to the rate of secretion. Serotonin, CRF-like and kinin-like peptides have the potential to be released from neurohemal sites into the hemolymph. Thus, these factors are possibly exposed to many tissues such as salivary gland, cuticle, crop and hindgut, as well as Malpighian tubules. The present work indicates that there are multiple diuretic factors present in Rhodnius CNS, but the kinins, which have diuretic activity in other insects, do not appear to be active on Rhodnius Malpighian tubule secretion. The kinins do, however, have an action on hind-
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gut muscle contraction; the hindgut being involved with the voiding of fluid following diuresis.
Acknowledgments This work was funded through an NIH grant (GM 48172) to D.A.S. and I.O. and through an NSERC grant to I.O.
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[37] Veenstra JA, Pattillo JM, Petzel DH. A single cDNA encodes all three Aedes leucokinins, which stimulate both fluid secretion by the Malpighian tubules, and hindgut contractions. J Biol Chem 1997;272: 10402–7. [38] Wang S, Rubenfeld AB, Hayes TK, Beyenbach KW. Leucokinin increases paracellular permeability in insect Malpighian tubules. J Exp Biol 1996;199:2537– 42.
The biological activity of diuretic factors in Rhodnius prolixus V.A. Te Bruggea,*, D.A. Schooleyb, I. Orcharda a
Department of Zoology, University of Toronto, 25 Harbord Street, Toronto, Ontario, Canada M5S-3G5 b Department of Biochemistry, University of Nevada, Reno, Nevada 89557, USA Received 1 May 2001; received in revised form 16 October 2001; accepted 16 November 2001
Abstract 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 [25], 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 CAP2b 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. [24]. 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. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Malpighian tubule; Diuresis; cAMP; Hindgut contraction; Neuropeptide
1. Introduction Rhodnius prolixus 5th instars ingest a blood meal which is up to10 times their initial body weight. During gorging the insect initiates a period of rapid diuresis which results in the loss of 40% of the weight of the bloodmeal. This diuresis 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 [25]. Serotonin is released into the hemolymph at the beginning of feeding reaching a peak titer of 10⫺7 M 5 min after the start of feeding. The hemolymph titer of serotonin decreases over the next 15 min to between 10⫺8 and 2 ⫻ 10⫺8 M and remains at this level for the next few hours [20]. A peptide(s) is also considered to be involved in this rapid diuresis [1], and in other insects, cor* Corresponding author. Tel.: ⫹1-416-978-4602; fax: ⫹1-416-9783522. E-mail address: [email protected] (V.A. Te Brugge).
ticotropin releasing factor (CRF)-like, kinin-like (see review [7]), calcitonin-like [9,11] and cardioactive peptide 2b (CAP2b) [10] peptides, have been demonstrated to be diuretic factors/hormones. Interestingly, while CAP2b has been shown to have diuretic effects on Drosophila tubule secretion, studies done on Rhodnius Malpighian tubules with CAP2b have suggested an antidiuretic effect [31]. Previously, we have demonstrated the presence of CRFlike [34] and kinin-like peptides [35], and serotonin [21] in the CNS of Rhodnius prolixus. These factors are found in neurohemal terminals of the abdominal nerves and corpus cardiacum (CC). CRF-like and kinin-like peptides, but not serotonin, are co-localized in the neurohemal terminals of the abdominal nerves [34,35]. These results suggest that all three factors are released into the hemolymph and that the release of serotonin can be independent of the release of the peptides. The CRF-like peptide, Locusta-DH, has previously been shown to increase the secretion of Rhodnius Malpighian tubules with an EC50 of 0.14 M [7], although in other
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insects CRF-like peptides increase secretion at low nanomolar concentrations [6,7]. CRF-like peptides are considered to work through a cAMP dependent pathway [6,7]. Kinin peptides were originally isolated from the cockroach, Leucophaea maderae, based on their ability to induce contraction of the hindgut muscle [14]. Subsequently, hindgut assays, in combination with high-performance liquid chromatography (HPLC), were used to isolate kinin peptides from several other insect species [5,12,16,30,33] or to confirm bioactivity [15,37]. As well as having activity on hindgut, kinin-like peptides have been shown to increase the rate of secretion of isolated Malpighian tubules of several species of insects, including Acheta domesticus [8], Locusta migratoria [6], Drosophila melanogaster [36], Musca domestica [15] and Culex salinarus [12]. However, only very small increases in secretion were observed in Aedes aegypti [37] and no stimulation by native kinin-like peptides was obtained in Helicoverpa zea [4]. The EC50 of the Acheta-kinins for Malpighian tubule secretion in Acheta is in the order of 0.3– 0.02 nM [7]. Similar values were found for Musca and Locusta-kinins when tested on their respective tubules [6,15]. Insect kinins have been shown to stimulate changes in transepithial potentials of the Malpighian tubules [36] and to increase the Cl⫺ permeability [38] of the Malpighian tubules. This increased permeability to Cl⫺ is mediated by the release of intracellular Ca2⫹ [3,28]. In Drosophila Malpighian tubules, kinin-like peptides have been shown to increase intracellular Ca2⫹ specifically in the stellate cells [32,36]. Immunohistochemical experiments on fed 5th instar Rhodnius suggest a reduction in staining intensities of CRFlike and kinin-like immunoreactive material in the mesothoracic ganglionic mass (MTGM) within 30 min of feeding (Te Brugge, unpublished). In addition, serotonin is known to be released into the hemolymph at the start of feeding, with the titer peaking at 5 min and remaining at a lower level for the next few hours [20]. Preliminary experiments suggest the presence of material(s) in the hemolymph at 5 min and 2 h, from the start of feeding, which increases the rate of secretion of Malpighian tubules but is not blocked by the serotonin antagonist ketanserin (Te Brugge unpublished). These pieces of evidence suggest that serotonin, CRF-like and kinin-like peptides may all be present in the hemolymph during and following feeding. The control of the post-feeding diuresis in Rhodnius involves the crop, Malpighian tubules and hindgut [23]. All of these tissues must have a rapid initiation and cessation of activity associated with diuresis such that the insect does not retain excess water or become desiccated. Mixtures of small amounts of diuretic factors have been shown to interact synergistically to increase the rate of secretion of Malpighian tubules by an amount greater than the sum of the rates of secretion induced by the individual factors [6]. Thus, serotonin and forskolin or tissue extracts have been shown to synergistically increase the secretion rate of Rhodnius Malpighian tubules [24]. Moreover, the synergistic
interactions of diuretic factors have been shown to result in a steeper dose-response curve. This is shown clearly in the synergism between Locusta CRF-like and kinin-like peptides on Locusta tubules [6]. Synergism between CRF-like and kinin-like peptides has also been shown in Musca [15]. This study focuses on serotonin, CRF-like and kinin-like analogues and tissue extracts tested on Malpighian tubule secretion rate and cAMP content assays in Rhodnius prolixus to more fully characterize the range of putative diuretic factors present in the nervous system. Rhodnius CNS extracts were partially purified by HPLC. These fractions were tested for diuretic activity and kinin-like immunoreactivity using RIA; as well a hindgut contraction assay was established to test for myotropic activity.
2. Materials and methods 2.1. Animals Fifth instar larvae of Rhodnius prolixus were taken from a long standing colony maintained at 25°C under high humidity. The insects were unfed, 6 – 8 weeks post emergence, and previously fed on rabbit’s blood as 4th instars. 2.2. Malpighian tubule secretion assay The Malpighian tubules of 5th instars were dissected under physiological saline [18], and the tubules were freed from trachea and fat with the aid of fine glass probes. The secretion assays were conducted as described in Te Brugge et al. [34]. In brief, the upper portions of the tubules were transferred to a 20 l drop of physiological saline under water-saturated heavy mineral oil. The open end of the tubule was pulled out and wrapped around a minutin pin. Saline containing serotonin (Sigma Chemical Co., St Louis, Missouri, USA), Diploptera punctata (Dippu)-DH46 [11] or Zootermopsis nevadensis (Zoone)-DH [2], kinin-like peptides (obtained from Sigma) or tissue extracts was exchanged for the equilibrating saline. The tubules were allowed to secrete for a further 20 –30 min. Droplets of urine from the cut end of the tubule were collected every 5 min and their diameter measured under mineral oil using an eyepiece micrometer and the volume calculated. In experiments testing the CRF-like peptides, a maximal rate of secretion was determined using 10⫺6 M serotonin, with each tubule acting as its own control. The percent relative to the maximal rate of secretion was calculated for each test substance. In some experiments rates were expressed as the actual rate of secretion. Mean ⫾ SEM were calculated and compared using an unpaired Student’s t test. 2.3. Malpighian tubule cAMP assay The cAMP content of the Malpighian tubules was assayed as previously described [34]. In experiments testing
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tissue extracts, Malpighian tubules from 5th instars were dissected and then transferred to a microfuge tube containing 0.5 mM of the phosphodiesterase inhibitor, 3-isobutyl1-methyl-xanthine (IBMX) in 50 l of physiological saline, along with tissue extract, serotonin, CRF-like or kinin-like peptides. Tubules were incubated for 10 min at room temperature and the reaction stopped with 500 l of boiling 0.05 M sodium acetate. Samples were placed in a boiling water bath for a further five minutes, then frozen at -20°C until assayed. The samples were thawed, sonicated and centrifuged at 8800 g for 10 min and the supernatant decanted. The cAMP content of the supernatant was measured using an RIA kit (Mandel/NEN, Guelph, Ontario) with modifications as previously described [19]. The mean ⫾ SE were calculated and compared using an unpaired Student’s t test. 2.4. Radioimmunoassay The radioimmunoassay (RIA) for kinin-like material was conducted as previously described in Te Brugge et al. [35]. The assay utilizes the same anti-leucokinin 1 antiserum that was used in immunohistochemistry studies [35] at a final concentration of 1:100,000 in RIA buffer. Leucokinin I with a tyrosine residue on the N terminus was custom synthesised (Research Genetics, Huntsville, AL, USA) and the peptide iodinated using the Chloramine T method. The standard curve for the RIA used leucokinin I (Sigma) with concentrations ranging from 5–1000 fmol/100 l RIA buffer. 125I-Y leucokinin I working tracer, leucokinin I antiserum and either standard or sample (100 l of each) were combined in 1.5 ml polypropylene microfuge tubes for 18 h at 4°C. Standards and samples were run in duplicate. Unbound peptide was precipitated by the addition of 250 l of dextran-coated charcoal. The charcoal suspension was added to the microfuge tubes and incubated for three minutes, the tubes were then centrifuged for eight minutes at 8800 g; 400 l of supernatant was removed to glass test tubes and counted for 1 min in a Beckman 4000 gamma counter. 2.5. Isolation of CNS CRF-like and kinin-like peptides One hundred 5th instar CNSs were dissected under physiological saline and collected in acidified methanol (methanol: acetic acid: water; 90:9:1) on ice, then stored at -20°C. The tissues were then sonicated and centrifuged for 10 min at 8800 g. The supernatant was decanted and dried in a Speed-Vac concentrator (Savant, Farmingdale, NY, USA). The dried extract was dissolved in water with 0.01% TFA and then applied to a C18 Sep-Pak cartridge (Waters Associates, Mississauga, Ontario, Canada) which had been previously equilibrated as described by Miggiani et al. [27]. The cartridge was then washed sequentially with 3.0 ml each of water, 30% and 60% acetonitrile (Burdick and Jackson, Muskegon, MI, USA) with 0.1% TFA (BDH,
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Toronto, Ontario, Canada) and the eluents collected. The collected extracts were dried in the Speed-Vac and stored at -20°C until use. The 30 and 60% acetonitrile eluents were then applied to a reversed-phase HPLC system which utilized a Brownlee C18 column (Mandel/Alltech, Guelph, Ontario, Canada) with a 1 ml injection loop. The HPLC gradient was 9% to 60% acetonitrile with 0.01% TFA over 40 min. The fractions were collected, subsampled, dried and stored at -20°C until use. The samples were then taken up in physiological saline prior to bioassay. 2.6. Rhodnius hindgut assay Rhodnius hindgut assays were conducted on isolated 5th instar hindguts maintained under physiological saline. The preparation consisted of a small piece of ventral cuticle surrounding the anus to secure the hindgut to a Sylgard (Dow Corning, Midland Michigan, USA; Paisley Products, Scarborough, Ontario) coated dish, while the anterior end of the hindgut and a small portion of the posterior midgut was tied by a fine thread to a miniature force transducer (Aksjeselskapet Mikro-elektronikk, Horten, Norway). The output from the transducer was connected to a data acquisition system (Biopac MP100 workstation, Biopac systems Inc, Santa Barbara, CA, USA). Tissues were equilibrated in 20 l of saline for 20 min, then the saline removed and replaced with either saline or test solutions containing peptides or tissue extracts. Basal tonus and phasic contractions were recorded and measured using the Biopac system.
3. Results 3.1. The effects of synthetic factors on Malpighian tubules Serotonin and both of the CRF-like peptides increased the rate of secretion of Rhodnius Malpighian tubules. Serotonin increased the rate of secretion in a dose-dependent manner, with an EC50 of 4.2 ⫻ 10⫺8 M (see Fig. 1A). The CRF-like peptides, Zoone-DH and Dippu-DH46, also increased the rate of secretion of Rhodnius Malpighian tubules in a dose-dependent manner, with both CRF-like peptides having a similar dose-response curve. The EC50 values were 6.7 ⫻ 10⫺7 M and 5.5 ⫻ 10⫺7 M for Zoone (Fig. 1B) and Dippu-DH46 (not shown) respectively, threshold (lowest concentration to produce a significant increase in secretion) at 2 ⫻ 10⫺7 M, and maxima (lowest concentration to produce the highest (maximum) rate of secretion, relative to serotonin) at approximately 10⫺6 M. Leucokinin 1, LK8 or Locusta-kinin each failed to increase the rate of secretion significantly over saline controls (Table 1) and at some concentrations may even have decreased the rate of secretion. Both CRF-like peptides, as well as serotonin, increased the cAMP content of Rhodnius Malpighian tubules in the presence or absence of IBMX. Zoone-DH, in the presence
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Fig. 2. CRF-like peptide, Zoone-DH, dose-response curve on cAMP content of Rhodnius 5th instar Malpighian tubules. Points are mean ⫾ SE n ⫽ 4. Regression curve was fitted by SigmaPlot version 5 curve fitting program. The EC50 is 7.5 ⫻ 10⫺7 M.
3.2. The effects of CNS factors on Malpighian tubules
Fig. 1. (A) Serotonin dose-response curve on the rate of secretion of Rhodnius 5th instar Malpighian tubules. Points are mean ⫾ SE, n ⫽ 4 – 8. Regression curve fitted by Sigmaplot version 5 curve fitting program. The EC50 is 4.2 ⫻ 10⫺8 M. (B) CRF-like peptide, Zoone-DH, dose-response curve on the rate of secretion of Rhodnius 5th instar Malpighian tubules. Points are mean ⫾ SE, n ⫽ 4 – 8. Regression curve fitted by SigmaPlot version 5 curve fitting program. The EC50 is 6.7 ⫻ 10⫺7 M.
of IBMX, increased the cAMP content in a dose-dependent manner (Fig. 2) with an EC50 of 7.5 ⫻ 10⫺7 M, threshold at 5 ⫻ 10⫺8 M and maxima at approximately 2 ⫻ 10⫺6 M. None of the kinin-like peptides increased the cAMP content of Malpighian tubules.
Table 1 The effects of kinin-like peptides on rate of secretion from Rhodnius Malpighian tubules. Mean ⫾ SE n ⫽ 4 –9. Compound Rhodnius saline Leucokinin I
Leucokinin VIII Locusta-kinin
Concentration [M]
10⫺9 10⫺8 ⫺7 10 10⫺6 5 ⫻ 10⫺6 5 ⫻ 10⫺6
Rate of secretion ⫾ SE (nl/min) 0.13 ⫾ 0.09 0.01 ⫾ 0.01 0.18 ⫾ 0.18 0.2 ⫾ 0.2 0.2 ⫾ 0.2 0.23 ⫾ 0.11 0.04 ⫾ 0.03
3.2.1. Kinin RIA Material eluting from the Sep-Pak with 30% acetonitrile and 0.1% TFA was injected into the RP-HPLC system and 1 ml fractions were collected. These fractions were subsampled and tested using the RIA for kinins at 2 CNS equivalents per sample. Kinin-like material was found in fractions 18, 22, 23 and 25–30 (the remaining fractions only revealing the non-specific background levels). The greatest amount of kinin-like material was seen in fractions 25 and 26 (Fig. 3A). 3.2.2. Effects on tubules Subsamples (2, 4, 12.5 CNS equivalents/20 l) of all of the RP- HPLC fractions were tested in secretion assays and expressed as percent maximum secretion relative to10⫺6 M serotonin. As well, subsamples from all fractions were tested in Malpighian tubule cAMP assays. In bioassays of fractions where kinin-like material was found (fractions 18, 22, 23 and 25–30), there was no increase in the rate of secretion above baseline rates of secretion. Malpighian tubule rate of secretion was increased using fractions 6, 7 and 12 tested at 4 CNS equivalents (Fig. 3B) and these same fractions were also capable of increasing the cAMP content of Malpighian tubules to 10.3, 12.1 and 8.3 pmol/tissue respectively (versus 3.6 ⫾ 0.5 pmol/tissue for control values) when tested in the presence of IBMX. For comparison, a maximum dose of serotonin (10⫺6 M) resulted in Malpighian tubule cAMP content increasing to 11.2 ⫾ 1.7 pmol/tissue. Material from the Sep-Pak cartridge that eluted with 60% acetonitrile was run on RP-HPLC and 1 ml fractions were collected. The fractions were subsampled and these subsamples were tested in Malpighian tubule secretion and
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Fig. 3. (A) Kinin-like immunoreactivity in fractions of the RP-HPLC run of the 30% acetonitrile eluent from C18 Sep-Pak. Sub-samples, 2 CNS equivalents, of the 1 ml fractions were tested in the leucokinin 1 RIA. Note that background levels within the RIA are 27.1 0.95 ⫾ fmol. Fractions 1–5 were pooled resulting in elevated background levels due to additional non-specific binding. Leucokinin 1-like material was detected in fractions 18, 22, 23 and 25–30. A single example is shown, but this pattern of elution is consistent between 4 individual HPLC runs of CNS material. The elution times for LK1 and serotonin are indicated by arrows. (B) Rhodnius Malpighian tubule secretion assay of sub-samples of the RP- HPLC fractions tested in secretion assays at 4 CNS equivalents, expressed as percent maximum secretion with respect to serotonin at 10⫺6 M.
cAMP assays at 2 and 4 CNS equivalents. When tested at 4 CNS equivalents, fractions 43, 44 and 45 increased the rate of secretion of Rhodnius Malpighian tubules (Fig. 4), with fraction 43 eliciting 45% of the maximum rate of secretion induced by 10⫺6 M serotonin. Subsamples of these fractions were also tested in Malpighian tubule cAMP assays in the presence of IBMX. Fractions 43, 44 and 45 resulted in an increase of cAMP content of Malpighian tubules to 11.9, 12
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Fig. 5. Fraction 44 of the RP-HPLC run of the 60% acetonitrile eluent from C18 Sep-Pak was tested in secretion assays. Fraction 44 increased the rate of secretion of 5th instar Malpighian tubules in a dose-dependent manner. Points are mean ⫾ SE n ⫽ 4.
and 8.6 pmol/tissue respectively, relative to baseline levels of 4.8 ⫾ 0.5 pmol/tissue. The material that stimulated both secretion and increases in cAMP content eluted at an acetonitrile concentration of approximately 57% (Fig. 4). Fraction 44 (since fraction 43 was used for other assays) was further tested and shown to stimulate dose-dependent increases in the rate of secretion from Malpighian tubules (Fig. 5). 3.2.3. Hindgut assays In order to provide a positive test for bioactivity of the kinin-like material, subsamples of fractions 17–35 from the 30% Sep-Pak fractionated RP-HPLC run were tested in the hindgut contraction assay. Kinin RIA positive fractions stimulated an increase in the frequency of contraction and an increase in basal tonus (Table 2). The samples with the greatest activity resulted in large changes in basal tonus, which masked the superimposed phasic contractions. Other fractions were also myostimulatory, and two fractions were myoinhibitory (Table 2). 3.3. Additivity and synergism studies
Fig. 4. Rhodnius Malpighian tubule secretion assays of fraction subsamples from the RP-HPLC run of the 60% acetonitrile eluent from C18 Sep-Pak. HPLC fractions tested in secretion assays at 4 CNS equivalents, expressed as percent maximum secretion with respect to serotonin at 10⫺6 M. Consistent elution profiles were obtained from 3 HPLC runs of CNS material. The elution time for Dippu DH46 is indicated by arrow.
Homogenates of the MTGM contain serotonin, CRF-like and kinin-like peptides as well as many other compounds. Previously Maddrell et al. [24] had tested MTGM homogenates (0.05 MTGM/100 l) and serotonin (4 ⫻ 10⫺8 M) on secretion rates, using a single concentration of the two factors. In order to look more fully at the interactions of serotonin and tissue homogenates on the resultant doseresponse curve characteristics, we tested a range of concen-
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Table 2 The effects of fractions 18 –35 from the RP-HPLC run of the 30% SepPak eluent of Rhodnius 5th instar CNS extracts on Rhodnius hindgut contractions Fraction #
Hindgut stimulation/inhibition
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
0 0 0 ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹⫹ ⫹ ⫺ ⫺ ⫹ ⫹ 0
0 no change ⫹ increase in frequency of contraction and basal tonus ⫹⫹ strong increase in basal tonus, masking the superimposed phasic contractions ⫺ decrease in frequency of contraction
trations of serotonin with the same fixed concentration of MTGM homogenate (0.05 MTGM/100 l). Fig. 6 shows the dose-response curve for serotonin with an EC50 of 4.2 ⫻ 10⫺8 M. In addition Fig. 6 illustrates the dose-response curves for a combination of increasing concentrations of serotonin and a fixed dose of MTGM extract. The sum of
Fig. 6. Synergism studies. Serotonin dose-response curve on secretion rate from Malpighian tubule (solid line). The EC50 for the serotonin doseresponse curve is 4.2 ⫻ 10⫺8 M. The dose-response curve for serotonin tested in the presence of MTGM extracts (0.05 equivalents/100 l) is shown by the dashed line. The EC50 of the dose-response curve for the combination of serotonin and MTGM extract is 1.6 ⫻ 10⫺8 M. The dotted line represents the calculated rate of secretion of the individual factors added together. Points are mean ⫾ SE n ⫽ 4 – 8.
the data for the two factors tested separately is shown by the dotted line, and the observed rate of secretion with the two factors tested in combination is shown by the dashed line. The curve for the rate of secretion with the two factors tested in combination is shifted to the left with an EC50 of 1.6 ⫻ 10⫺8 M for serotonin. The rate of secretion measured when the two factors are combined is only significantly greater (P ⬍ 0.05) at two low concentrations of serotonin (10⫺8 M and 2 ⫻ 10⫺8 M). Serotonin and CRF-like peptides were also tested in combination. Low concentrations of serotonin (3 ⫻ 10⫺8 M) and Zoone-DH (2.5 ⫻ 10⫺7 M) each resulted in only small increases in the rate of secretion. When applied in combination, these factors resulted in an increase in the rate of secretion which was not significantly greater than the sum of the two factors tested alone (Fig. 7A). In a similar manner serotonin (3 ⫻ 10⫺8 M) and fraction 44 (2 CNS equivalents) both increased the rate of secretion. When tested in combination, the two factors resulted in an increased rate of secretion which was not significantly greater than the sum of the two factors (Fig. 7B). In order to verify our technique for analysis of synergism, we tested serotonin and forskolin individually and in combination on Malpighian tubule secretion, since this combination has previously been shown to increase the rate of secretion of Rhodnius tubules synergistically [24]. In these assays, the forskolin was dissolved in DMSO. A concentration of 0.1% DMSO had no measurable effect on secretion. Forskolin, tested at a concentration of 1 ⫻ 10⫺5 M, increased the rate of secretion, as did serotonin tested at 3 ⫻ 10⫺8 M. When the two factors were tested together the resulting rate of secretion was significantly greater (3.9 fold) than the sum of the values of the two factors tested individually; a synergistic response. This rate of secretion stimulated by low concentrations of the two factors in combination resulted in rates of secretion which were close to maximal rates of secretion induced by 10⫺6 M serotonin (Fig. 8). Malpighian tubule secretion was tested with kinin-like peptides both singly, and in combination with serotonin and CRF-like peptides at low concentrations, to look for any effects on secretion by the kinins when other diuretic factors are present. Various kinin-like peptides were tested and none increased the rate of secretion significantly from rates of secretion stimulated by the factors tested alone. The results of secretion assays using Locusta-kinin in combination with serotonin and the CRF-like peptide Zoone-DH are shown in Fig. 9. No synergism was observed with these combinations. Rhodnius 5th instar Malpighian tubules were incubated for 10 min in the presence of IBMX with serotonin (3 ⫻ 10⫺8 M) and Zoone-DH (2.5 ⫻ 10⫺7 M) individually and in combination. Both serotonin and Zoone-DH each increased the cAMP content of the Malpighian tubules significantly over saline controls. The mixture of the two factors resulted
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Fig. 8. Forskolin and serotonin were tested separately and together at low concentrations (1 ⫻ 10⫺5 M and 3 ⫻ 10⫺8 M respectively) to look at their combined effect on Malpighian tubule rate of secretion. At these concentrations both factors increased rate of secretion over saline controls. The calculated value of the addition of the individual rates of secretion is shown as well as the actual rate of secretion. The final column is the maximum rate of secretion obtained with 10⫺6 M serotonin. The calculated value of secretion (addition of the individual rates of secretion) was significantly different than the observed rate of secretion. Points are mean ⫾ SE n ⫽ 4 – 6. Fig. 7. (A) CRF-like peptide (Zoone-DH) and serotonin were tested separately and together at low concentrations (2.5 ⫻ 10⫺7 M and 3 ⫻ 10⫺8 M respectively) to look at their combined effect on Malpighian tubule rate of secretion. At these concentrations both factors increased rate of secretion over saline controls. The calculated value of the addition of the individual rates of secretion is shown as well as the actual rate of secretion. The final column is the maximum rate of secretion obtained with 10⫺6 M serotonin. The calculated value of secretion (addition of the individual rates of secretion) was not significantly different than the observed rate of secretion. Points are mean ⫾ SE n ⫽ 4 – 6. (B) Fraction 44 and serotonin were tested separately and together at low concentrations (2 CNS eq and 3 ⫻ 10⫺8 M respectively) to look at their combined effect on Malpighian tubule rate of secretion. At these concentrations both factors increased rate of secretion over saline controls. The calculated value of the addition of the individual rates of secretion is shown as well as the actual rate of secretion. The final column is the maximum rate of secretion obtained with 10⫺6 M serotonin. The calculated value of secretion (addition of the individual rates of secretion) was not significantly different than the observed rate of secretion. Points are mean ⫾ SE n ⫽ 4 – 6.
in a small but not significant increase in cAMP content over that found for the individual factors (Fig. 10A). Rhodnius 5th instar Malpighian tubules were incubated for 10 min in the presence of IBMX with serotonin (3 ⫻ 10⫺8 M) and forskolin (1 ⫻ 10⫺5 M) individually and in
combination. DMSO (0.01%) had no measurable effect on cAMP content of the Malpighian tubules. Serotonin and forskolin, at these concentrations, increased cAMP content of the Malpighian tubules significantly (P ⬍ 0.1 and P ⬍ 0.05 respectively) above the saline controls. In contrast to the earlier result where these two factors acted synergistically on the rate of secretion of Malpighian tubules, the mixture of the two factors resulted in a small but not significant increase in cAMP content over that found for the individual factors (Fig. 10B). That is to say, no synergism was observed.
4. Discussion Previously, we have used immunohistochemisty to demonstrate the presence of CRF-like peptides [34], kinin-like peptides [35] and serotonin [21] in the CNS of 5th instar Rhodnius. These factors are found in neurohemal terminals of the abdominal nerves and CC and so may be released into the hemolymph from both sites. CRF-like and kinin-like
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Fig. 9. Locusta-kinin, Zoone-DH and serotonin were tested separately and together at low concentrations (3 ⫻ 10⫺8 M, 2.5 ⫻ 10⫺7 M and 3 ⫻ 10⫺8 M, respectively) to look at their combined effect on Malpighian tubule rate of secretion. At these concentrations Zoone-DH and serotonin increased rate of secretion. The kinin-like peptide did not increase secretion. The individual rates of secretion stimulated by serotonin or Zoone-DH were not significantly different than the rate of secretion of serotonin or Zoone-DH combined with Locusta-kinin. The combination of all three factors resulted in increases in secretion that were not significantly different than the additive rates of serotonin or Zoone-DH. Points are mean ⫾ SE n ⫽ 4 – 6.
peptides, but not serotonin, are co-localized in the neurohemal terminals on the abdominal nerves. Serotonin and CRF-like peptides increase secretion rate and cAMP content of Rhodnius Malpighian tubules in a dose-dependent manner. The dose-response curve of Malpighian tubule secretion stimulated by serotonin was consistent with that previously shown by Maddrell et al. [26]. The curves are relatively steep, have similar EC50, threshold and maxima. This is in contrast to a later serotonin doseresponse curve [24] which was shifted to the right (higher concentrations) and was less steep. All of the CRF-like peptides tested to date on Rhodnius tubules (Lom-DH [7], Dippu-DH and Zoone-DH (present study)) have a moderate degree of sequence identity and have similar EC50’s on Rhodnius Malpighian tubules. However, the concentrations of CRF-like peptide required for secretion are quite high relative to native CRF-like peptides tested on their conspecific tubules, which are active in the nanomolar range [6,7]. The kinin-like peptides failed to increase rate of secre-
Fig. 10. (A) CRF-like peptide (Zoone-DH) and serotonin were tested in Malpighian tubule cAMP assays in the presence of IBMX. Serotonin (3 ⫻ 10⫺8 M) and Zoone-DH (2.5 ⫻ 10⫺7 M) were tested individually and in combination. Both serotonin and Zoone-DH increased the cAMP content of the Malpighian tubules significantly over saline controls. The mixture of the two factors did not significantly increase cAMP over that seen when testing them individually. Points are mean ⫾ SE n ⫽ 4. (B) Forskolin (1 ⫻ 10⫺5 M) and serotonin (3 ⫻ 10⫺8 M) were tested in Malpighian tubule cAMP assays in the presence of IBMX. DMSO (0.01%) had no measurable effect on cAMP content of the Malpighian tubules. Serotonin and forskolin increased cAMP content of the Malpighian tubules above the saline controls. The cAMP content of the Malpighian tubules incubated with both factors was not significantly different from the cAMP content of Malpighian tubules incubated with the individual factors. Points are mean ⫾ SE n ⫽ 4.
tion or cAMP content of Rhodnius Malpighian tubules. Kinins tested in other insects have been active on Malpighian tubule secretion, including those in Acheta [8], Culex [13], Locusta [7], Drosophila [36] and Musca [15, 17]. In contrast the kinins isolated from Helicoverpa zea are inactive in Helicoverpa [4], but are active on Manduca sexta tubules. Aedes kinins resulted in only small increases in the
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rate of secretion from Malpighian tubules of Aedes, and Aedes kinin II did not increase secretion at all [37]. Although the kinin-like peptides did not increase secretion of Rhodnius tubules, they were biologically active when tested on Rhodnius hindgut muscles, increasing basal tonus and frequency of phasic contractions. As such the Rhodnius hindgut responds to kinins in a similar fashion as other insect hindguts [5,14,15,16,30,33,37]. Extraction and separation of the 5th instar Rhodnius CNS indicates the presence of serotonin and kinin-like peptides in the 30% eluent. Previously, we have shown that over 90% of the secretion caused by the 30% eluate is blocked by the serotonin antagonist, ketanserin [35]. The RP-HPLC separation of the 30% C18 Sep-Pak eluate indicated several fractions that were positive for kinin-like material tested using RIA. However, those fractions which contained kininlike material did not increase secretion rate of Rhodnius Malpighian tubules. Several RP-HPLC fractions collected at the beginning of the RP-HPLC run (fractions 6,7) increased secretion and cAMP content of the Malpighian tubules and likely reflect the activity of serotonin, which elutes from the C18 column at a time consistent with these active fractions (Fig. 3A). The nature and identity of the activity seen in fraction 12, which increased both secretion and cAMP content of Malpighian tubules, is not known. The 60% eluent from C18 Sep-Pak was run on RP- HPLC and three fractions, 43, 44 and 45, increased secretion and cAMP content of Rhodnius 5th instar Malpighian tubules. Fraction 44 gave a dose-dependent increase in the rate of secretion of Malpighian tubules. The active fractions elute at concentrations of acetonitrile consistent with that found in other studies looking at CRF-like peptides [29] and CRFlike standards (Fig. 4). The CRF-like peptides, Locusta-DH ([7], Te Brugge unpublished), Dippu-DH46 and Zoone-DH each increased secretion and cAMP content of Malpighian tubules. Taken together, the evidence suggests that there is a CRF-like peptide present in Rhodnius with diuretic activity, which may be similar to other members of the CRF-like family of peptides. The CRF-like and kinin-like peptides which are co-localized in the neurohemal terminals of the abdominal nerves have the potential to be released together and independent of serotonin. Serotonin concentrations reach a peak of 10⫺7 M at 5 min after the commencement of feeding. The serotonin concentration decreases to a level close to 10⫺8 M over the next 15 min, remaining at low levels for the next few hours [20]. Preliminary results suggest the presence of a CRF-like peptide in the hemolymph at 5 min and 2 h after feeding (unpublished data). It is possible then, that several potential diuretic factors could be present in the hemolymph at the same time. Coast [6] has demonstrated that the interaction of Locusta CRF-like and kinin-like peptides on Locusta tubules results in a synergistic response in tubule secretion. These factors together caused the dose-response curve to be steeper and shifted to the left. Maddrell et al. [24] found that serotonin and either MTGM extracts or
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forskolin also had a synergistic interaction on the rate of Malpighian tubule secretion. In the present study we found some synergism between MTGM homogenates or forskolin and serotonin on the rate of secretion. This was strongest using the pharmacological agent forskolin in combination with serotonin (3.9 fold greater rate of secretion than the sum of the rates of secretion of the two factors). This confirms the results of Maddrell et al. [24] and verifies our technique for looking at synergism on Malpighian tubule secretion. Forskolin was used since it increases cAMP content of the Malpighian tubules, as do both serotonin and the ‘diuretic peptide’ known to be involved in diuresis [24]. The combination of serotonin and forskolin was used to test whether two agents that increase cAMP could have synergistic effects on secretion. However, this synergism between serotonin and forskolin on Malpighian tubule secretion was not evident in experiments looking at the cAMP content of Malpighian tubules. CRF-like analogues and RP- HPLC fraction 44 tested in the presence of serotonin did not elicit a diuretic response significantly greater than an additive response, when tested for rate of secretion or cAMP content of Malpighian tubules. Why there is such strong synergism between serotonin and forskolin on Malpighian tubule secretion but not cAMP content is unclear. It may indicate that the synergism is caused by other cAMP- independent pharmacological effects of forskolin [22], the interaction of different second messenger systems or pathways downstream of cAMP formation. Tissue extracts tested in the presence of serotonin demonstrated synergism at 10⫺8 M serotonin (2 fold greater response than the sum of two diuretic factors) and at 2 ⫻ 10⫺8 M serotonin (1.7 fold greater response than the sum of the two diuretic factors), and the dose-response curve for serotonin was shifted to the left. However, the results obtained are not as dramatic as those seen with serotonin and forskolin. The other “diuretic” factor tested, kinin-like peptides, did not cause any change in the rate of secretion when tested in combination with either serotonin and/or CRF-like peptides at low concentrations. As there was some evidence of synergism between the tissue homogenates and serotonin, there may be other diuretic factors that play a role in the rapid post-feeding diuresis. The interactions of various diuretic factors on Malpighian tubules may also affect the various characteristics of diuresis, such as ion composition, speed of response and duration of the response, in addition to the rate of secretion. Serotonin, CRF-like and kinin-like peptides have the potential to be released from neurohemal sites into the hemolymph. Thus, these factors are possibly exposed to many tissues such as salivary gland, cuticle, crop and hindgut, as well as Malpighian tubules. The present work indicates that there are multiple diuretic factors present in Rhodnius CNS, but the kinins, which have diuretic activity in other insects, do not appear to be active on Rhodnius Malpighian tubule secretion. The kinins do, however, have an action on hind-
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gut muscle contraction; the hindgut being involved with the voiding of fluid following diuresis.
Acknowledgments This work was funded through an NIH grant (GM 48172) to D.A.S. and I.O. and through an NSERC grant to I.O.
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