Mosquito natriuretic peptide identified as a calcitonin-like diuretic hormone in Anopheles gambiae (Giles)
1 Department of Biology, Birkbeck (University of London), London WC1E 7HX,
UK
2 School of Biological Sciences, University of North Wales, Gwynedd LL57
2UW, UK
3 Biochemistry Department, University of Nevada, Reno, NV 89557,
USA
* Author for correspondence (e-mail: g.coast{at}bbk.ac.uk)
Accepted 22 June 2005
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Summary |
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The cyclic AMP analogue stimulated secretion of Na+-rich urine by An. gambiae Malpighian tubules, reproducing the response to MNP in Ae. aegypti. It also depolarised the principal cell basolateral membrane voltage (Vb) while hyperpolarising the transepithelial voltage (Vt) to a similar extent.
Anoga-DH44 and Anoga-DH31 stimulated production of cyclic AMP, but not cyclic GMP, by Malpighian tubules of An. gambiae. Both peptides had diuretic activity, but only Anoga-DH31 had natriuretic activity and stimulated fluid secretion to the same extent as 8-bromo-cyclic AMP. Likewise, Anoga-DH31 reproduced the effects of cyclic AMP on tubule electrophysiology, whereas Anoga-DH44 initially hyperpolarised Vb and depolarised Vt, which is the opposite of the effect of Anoga-DH31.
Anoga-DH44 and Anoga-DH31 were also tested for effects on fluid secretion and ion transport by Ae. aegypti tubules. As in An. gambiae, the CRF-related peptide Anoga-DH44 had a non-specific effect on the transport of Na+ and K+, whereas the CT-like peptide Anoga-DH31 specifically stimulated transepithelial Na+ transport.
We conclude that the CT-like peptide Anoga-DH31 is the previously uncharacterised mosquito natriuretic peptide.
Key words: excretion, Malpighian tubule, diuretic hormone, natriuresis, mosquito, Anopheles gambiae, Aedes aegypti
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Introduction |
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Endocrine control of this post-prandial diuresis has been extensively
studied in the yellow fever mosquito, Aedes aegypti
(Beyenbach, 2003). Between
blood meals, the female conserves water and voids little or no urine, but,
during the peak phase of diuresis, clear drops of NaCl-rich urine are
eliminated from the anus every 1215 s
(Coast et al., 2002
;
Williams et al., 1983
). The
stimulus for this diuresis and natriuresis is mosquito natriuretic peptide
(MNP), which is released into the haemolymph from structures within the head
when the insect takes a blood meal
(Beyenbach and Petzel, 1987
).
MNP acts via cyclic AMP to stimulate secretion of Na+-rich
primary urine by the five Malpighian (renal) tubules. Its effects are
duplicated by a membrane permeant analogue of cyclic AMP (dibutyryl-cyclic
AMP), which has been shown to depolarise the basolateral membrane voltage
(Vb) of Malpighian tubule principal cells and to
hyperpolarise the transepithelial voltage (Vt) by a
similar amount (Beyenbach,
2003
; Sawyer and Beyenbach,
1985
). These changes are accompanied by reductions in
transepithelial resistance and in the fractional resistance of the basolateral
membrane and have been attributed to an increase in the Na+
conductance of the basolateral membrane
(Beyenbach, 2003
). In addition,
dibutyryl-cyclic AMP activates a bumetanide-sensitive
Na+/K+/2Cl cotransporter
(Hegarty et al., 1991
). The
actions of cyclic AMP make more Na+ available for transport into
the tubule lumen via cation/proton antiports in the principal cell
apical membrane. This additional Na+ (along with
Cl as a counterion) is accompanied by an osmotically
equivalent volume of water, and tubule secretion is elevated >7-fold while
the Na+:K+ concentration
([Na+]:[K+]) ratio of the secreted urine increases from
unity to approximately 10 (Beyenbach,
2003
).
MNP is thought to belong to the corticotropin releasing factor
(CRF)-related family of insect diuretic hormones, which use cyclic AMP as
their second messenger (Beyenbach,
2003). In support of this, a CRF-related diuretic hormone
(Culsa-DH) from the salt-water mosquito Culex salinarius stimulates
cyclic AMP production by Ae. aegypti Malpighian tubules
(Cady and Hagedorn, 1999b
) and
has diuretic and natriuretic activity
(Clark et al., 1998b
), although
the latter effects are small in comparison with those of MNP and exogenous
cyclic AMP. Additionally, Culsa-DH causes a biphasic change in the
transepithelial voltage of isolated perfused Malpighian tubules
(Clark et al., 1998a
), which
resembles the response to MNP, and stimulates urine production in
vivo (Cady and Hagedorn,
1999a
).
Among the regulatory peptide genes identified in the genome of the malaria
mosquito Anopheles gambiae (Holt
et al., 2002; Riehle et al.,
2002
) are those encoding orthologues of peptides known to have
diuretic activity, namely CRF-related DH, myokinins, calcitonin (CT)-like DH,
CAP2b (capa) and tachykinins
(Schooley et al., 2005
). Of
particular interest for the identification of MNP are the CRF-related and
CT-like peptides (Anoga-DH44 and Anoga-DH31,
respectively) since their Drosophila melanogaster homologues act
via cyclic AMP in stimulating Malpighian tubule secretion
(Cabrero et al., 2002
;
Coast et al., 2001
). Here, we
describe the effects of synthetic Anoga-DH44 and
Anoga-DH31 on fluid and electrolyte (Na+ and
K+) transport by Malpighian tubules of female An. gambiae
and Ae. aegypti. We show that the CT-like diuretic hormone is the
mosquito natriuretic factor described by Petzel et al.
(1985
), later designated MNP
(Beyenbach and Petzel,
1987
).
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Materials and methods |
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Fluid secretion assay
Malpighian tubules were removed from adult females at 310 days
post-emergence. Insects were anaesthetised by chilling at 4°C and
decapitated prior to removing the Malpighian tubules under Aedes
saline with the following composition (in mmol l1): NaCl,
150; NaHCO3, 1.8; KCl, 3.4; CaCl2, 1.7;
MgSO4, 4.1; glucose, 5; Hepes
(N-2-hydroxyethylpiperazine-N'2-ethanesulphonic acid),
25; pH adjusted to 7.1 with 1 mol l1 NaOH
(Clark et al., 1998b). Tubule
secretion assays were performed as described by Clark et al.
(1998b
) but using 10 µl
drops of bathing fluid. A number of different salines were tested for their
ability to support fluid secretion (results not shown), and the bathing fluid
selected for An. gambiae Malpighian tubules was one that comprised a
1:1 mixture of K+-free Drosophila saline
(O'Donnell et al., 1996
) and
Schneider's insect medium (GibcoTM; Invitrogen Ltd, Paisley, UK). The
Drosophila saline had the following composition (in mmol
l1): NaCl, 117.5; NaHCO3, 10.2;
NaH2PO4, 4.3; CaCl2, 2.0; MgCl2,
8.5; Hepes, 8.6, glucose, 20.0; pH adjusted to 7.0 with NaOH. Test substances
were dissolved in the bathing fluid at twice their desired final
concentration, and 5 µl were added to the incubation medium, having first
removed a 5 µl sample. Similar procedures were used for Ae.
aegypti Malpighian tubules, but the bathing medium comprised a 1:1
mixture of Aedes saline and Schneider's insect medium.
Urine analysis
Electrolyte concentrations (Na+ and K+) in urine
droplets held beneath water-saturated paraffin oil were measured using
ion-selective microelectrodes (Coast et
al., 2001; Ianowski and
O'Donnell, 2004
). Potassium electrodes were based on potassium
ionophore I, cocktail A (Fluka, Buchs, Switzerland) and were backfilled with
500 mmol l1 KCl. Sodium electrodes were based on sodium
ionophore II, cocktail A (Fluka) and were backfilled with 500 mmol
l1 NaCl. For both measurements, the reference electrode was
filled with 1 mol l1 LiCl. Potassium electrodes (mean slope
56.1±0.4 mV; N=36) were calibrated in mixed solutions of 200
mmol l1 KCl and NaCl, whereas Na+ electrodes
(mean slope 54.0±0.3 mV; N=26) were calibrated in mixed
solutions of 200 mmol l1 NaCl and LiCl. Potassium is known
to interfere with Na+ measurements and this was corrected for as
described by Ianowski and O'Donnell
(2004
).
Electrophysiological measurements
Isolated Malpighian tubules were attached to poly-D-lysine
coated cover slips before being transferred to a small (250 µl) chamber
that was perfused at 1 ml min1 with Aedes saline
containing the following amino acids (in mmol l1): glycine,
1.7; L-proline, 7.0; L-glutamine, 6.16;
L-histidine, 0.95; L-leucine, 0.55; L-lysine,
4.5; L-valine, 1.3. Use of this amino-acid-replete saline, which
contains amino acids at the same concentrations as those in the bathing fluid
for the fluid secretion assay, resulted in more stable voltages and
facilitated the placement of a microelectrode in the tubule lumen. Perfusion
was stopped prior to the addition of test compounds and restarted to wash-off.
Microelectrodes (120140 M resistance when filled with 0.1 mol
l1 KCl) were fabricated from 1 mm o.d. filament glass tubing
(GC100F-75; Clark Electromedical Instruments, Pangbourne, UK) using a
microprocessor-controlled vertical pipette puller (World Precision
Instruments, Sarasota, FL, USA). They were backfilled with 0.1 mol
l1 KCl and connected to a high-impedance electrometer
(M-707A; World Precision Instruments) via an Ag/AgCl half-cell (World
Precision Instruments). An Ag/AgCl reference electrode was placed in a 3 mol
l1 KCl reservoir connected to the perfusion chamber
via a Ringer agar bridge. Basolateral membrane
(Vb) and transepithelial voltages (Vt)
were measured close to the distal (closed) end of tubules, which were observed
using an inverted phase contrast microscope (Nikon TMS, Tokyo, Japan). Large
principal cells were selected for recording Vb, and
microelectrodes were advanced in steps of 37 µm at an oblique angle
using a piezoelectric drive (PM-10; World Precision Instruments) until a
sudden jump in potential indicated that the basolateral membrane had been
impaled. Recordings were deemed successful if the potential remained stable
(±2 mV) for >30 s and returned to 0±2 mV after withdrawal of
the electrode. Similar criteria were adopted for recording
Vt after the microelectrode had been advanced through the
apical membrane into the tubule lumen. Results were recorded digitally using a
data acquisition system (Datacan V; Sable Systems, Henderson, NV, USA).
Cyclic nucleotide assays
Production of cyclic AMP and cyclic GMP by isolated Malpighian tubules was
measured as described by Coast et al.
(2001). Malpighian tubules
were transferred to Eppendorf tubes (5 or 10 per tube) containing
Drosophila saline. After incubating for 10 min at room temperature,
saline containing 5 mmol l1 3-isobutyl-1-methylxanthine
(IBMX; final concentration 0.5 mmol l1) was added to each
tube. This was followed 10 min later by the addition of IBMX saline alone
(controls) or IBMX saline containing either Anoga-DH31 or
Anoga-DH44 (final concentration 1 µmol l1).
The IBMX solution was freshly prepared from a 250 mmol l1
stock solution in DMSO (final DMSO concentration 0.2%). The incubation was
terminated after 010 min by quenching the tubes in liquid nitrogen.
Samples were stored at 80°C until shipped on dry ice to the
University of North Wales for the measurement of cyclic AMP and cyclic GMP by
radioimmunoassay (RIA; Coast et al.,
2001
). The cyclic AMP assay was modified by the inclusion of 10
µl crustacean saline (Webster,
1986
) in the RIA, which improves the slope of the standard curve
(S.G.W., unpublished observation).
Peptide synthesis
The An. gambiae CT-like diuretic hormone was located using the
Ensembl genome browser with Drome-DH31
(Coast et al., 2001) as a
query. The 31-residue peptide Anoga-DH31 has the predicted sequence
TVDFGLSRGYSGAQEAKHRMAMAVANFAGGP-NH2 (84% identical to
Drome-DH31) and is encoded on a 95 amino acid precursor peptide
with accession number XM_321755. The An. gambiae CRF-related diuretic
hormone, a 44-residue peptide with the predicted sequence
TKPSLSIVNPLDVLRQRIILEIARRQMRENTRQVELNKALLREI-NH2 (82% identical
with Drome-DH44) was also found with Ensembl using
Drome-DH44 (Cabrero et al.,
2002
) as a query. There is an intron in the encoding region for
Anoga-DH44, located between Glu33 and Val34;
the gene encoding it is incomplete on both ends, probably due to sequencing
errors. Both peptides were synthesized using
N
-9-fluorenylmethoxycarbonyl (FMOC) chemistry with an Applied
Biosystems 431A synthesiser (Foster City, CA, USA). For each synthesis, Rink
MBHA amide resin (Novabiochem, San Diego, CA, USA) was used on a 0.1 mmol
scale. We activated FMOCamino acids with 1-hydroxybenzotriazole in
1-methyl-2-pyrrolidinone in the presence of dicyclohexylcarbodiimide for amino
acid activation, with a 10-fold molar excess of acylating species. Six 22 min
coupling cycles were employed (King,
1996
;
http://www.abrf.org/ABRFNews/1966/December1996/Long_PepSyn.html);
no double couplings were performed. Protecting groups were
Arg-[2,2,4,6,7-pentamethylbenzo-(2,3-dihydro)-furansulphonyl], Asn(Trityl),
Asp(OtBu), Gln(Trityl), Glu(OtBu), His(Trityl), Lys(Boc), Ser(OtBu), Thr(OtBu)
and Tyr(OtBu). The dry resinpeptide was cleaved using Reagent K
(King et al., 1990
). Crude
peptides were precipitated and then washed with methyl-t-butyl ether.
The dried crude peptides were purified to homogeneity with a ThermoSeparations
P4000 liquid chromatograph (San Jose, CA, USA). Crude peptide was first
purified by cation exchange on a Polysulfoethyl A column (25x2.1 cm;
Poly LC, Columbia, MD, USA) eluted at 10 ml min1 using 20
mmol l1 sodium acetate, pH 4.5, and a gradient from 0 to 1
mol l1 NaCl. This removed Arg deletion peptides, which are
difficult to separate by reversed phase. Purified fractions were re-purified
with a YMC 25x2.0 cm C8 column (Waters Co., Milford, MA, USA)
eluted at 10 ml min1 with an ethanolwater0.1%
trifluoroacetic acid gradient.
Chemicals
Muscakinin (Musdo-K) was a generous gift from Dr R. J. Nachman (USDA,
College Station, TX, USA). Unless otherwise stated, all other chemicals were
obtained from Sigma-Aldrich.
Statistics
Tests for significance were performed using GraphPad Instat v.3.06
(GraphPad Software, San Diego, CA, USA), with P<0.05 being
accepted as significant. Doseresponse curves with variable slope were
fitted using PrismTM v.4.02 (GraphPad Software).
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Results |
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Likewise, the effects of 8-bromo-cyclic AMP on Malpighian tubule electrophysiology in An. gambiae were similar to those reported for dibutyryl-cyclic AMP in Ae. aegypti. Absolute values for Vt (33.7±3.7 mV; N=28) and principal cell Vb (68.5±1.7 mV; N=54) varied considerably between tubules under control conditions. The addition of 100 µmol l1 8-bromo-cyclic AMP resulted in an immediate depolarisation of Vb by 55.8±5.3 mV (N=9) while Vt hyperpolarised by 39.1±10.8 mV (N=5; Fig. 2). These values are not significantly different (P=0.142; unpaired t-test), which suggests that the apical membrane voltage (Va=VtVb) remains relatively constant. However, given the variability of Vt and Vb, which were measured in different Malpighian tubules, a small change in Va might have gone undetected.
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To demonstrate further the different effects of the two diuretic hormones on ion and fluid transport, a group of Malpighian tubules were challenged over 30 min intervals with first Anoga-DH44 alone and then together with Anoga-DH31; the final concentration of both peptides was 1 µmol l1. Under control conditions, tubule secretion was 0.36±0.01 nl min1 (N=5) and the [Na+]:[K+] ratio of the secreted urine was 1.17±0.36. Anoga-DH44 produced a 3-fold increase in fluid secretion but had no effect on the urine [Na+]:[K+] ratio, whereas the ratio increased >7-fold after the addition of Anoga-DH31 to tubules already exposed to Anoga-DH44, and there was a further 2-fold increase in the rate of secretion (Fig. 5). The change in [Na+]:[K+] ratio reflects the markedly different effects of the two peptides on transepithelial Na+ and K+ transport. In the presence of Anoga-DH44, Na+ transport increased from 35±4 pmol min1 to a maximum of 109±17 pmol min1 but reached 399±55.2 pmol min1 after the addition of Anoga-DH31. On the other hand, K+ transport was increased from 36±7 pmol min1 to 105±29 pmol min1 by Anoga-DH44 but then fell to 47±7 pmol min1 following the addition of Anoga-DH31.
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Continuous recording of tubule fluid K+ concentration
Fig. 6 shows real-time
recordings of the K+ concentration in urine secreted by tubules
challenged with either Anoga-DH44 or Anoga-DH31. The
Na+ concentration is calculated on the basis of the sum of
[Na+] and [K+] being virtually constant at 200 mmol
l1. Following a 30 min equilibration period, a drop of
secreted fluid was removed, and a K+ selective microelectrode,
together with a reference electrode, was positioned in the newly secreted
urine. After 23 min, time to establish the K+ concentration
of the urine under control conditions, tubules were challenged with either
Anoga-DH44 or Anoga-DH31 (1 µmol l1
final concentration). The representative recording in
Fig. 6A shows the effect of the
CRF-related peptide Anoga-DH44. The K+ concentration of
the secreted urine was initially 75 mmol l1 but fell to 65
mmol l1 within 10 min of adding the CRF-related diuretic
hormone. This reflects a small increase in Na+ transport relative
to K+, but this was not sustained and the K+
concentration was constant over the next 20 min.
Fig. 6B shows similar data for
a tubule stimulated with Anoga-DH31. The K+
concentration of the secreted urine fell exponentially from about 102 mmol
l1 to 30 mmol l1 over 30 min. The
reciprocal increase in the Na+ concentration of the secreted urine
reflects the natriuretic activity of the calcitonin-like diuretic hormone.
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Effects of Anoga-DH44 and Anoga-DH31 on tubule electrophysiology
Representative recordings of the effects of 100 nmol l1
Anoga-DH44 and 100 nmol l1 Anoga-DH31
on the basolateral membrane voltage of Malpighian tubule principal cells are
shown in Fig. 7. Addition of
Anoga-DH44 resulted in a triphasic change in membrane voltage
(Fig. 7A), which initially
hyperpolarised by 6.1±1.3 mV (N=15; Phase 1) and then
depolarised by 35.4±4.7 mV (Phase 2) before returning to close to its
control value in the continued presence of the peptide (Phase 3). The
transepithelial voltage changed in a similar manner (data not shown), first
depolarising by 13.5±4.1 mV (N=8) and then hyperpolarising by
28.8±6.5 mV before returning to close to the control value. The
magnitude of the changes in transepithelial voltage was not significantly
different from that in the basolateral membrane voltage during both Phase 1
(P=0.124, unpaired t-test with Welch correction) and Phase 2
(P=0.418, unpaired t-test) of the response, which suggests
that the apical membrane voltage is not affected by Anoga-DH44.
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Differences between the actions of Anoga-DH44 and Anoga-DH31 on tubule electrophysiology are highlighted in Fig. 8, which shows the basolateral membrane voltage recorded in a single principal cell challenged sequentially with 100 nmol l1 of each of the peptides and then with 100 µmol l1 8-bromo-cyclic AMP. The triphasic response to Anoga-DH44 is readily distinguishable by the brief hyperpolarising phase that precedes the depolarisation and by the membrane potential repolarising before the peptide is washed off. The initial hyperpolarisation is absent from the responses to Anoga-DH31 and cyclic AMP, and the membrane remains depolarised until the secretagogues are washed off.
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Doseresponse relations for Anoga-DH31
The diuretic activity of Anoga-DH31 was investigated on An.
gambiae tubules at concentrations ranging from 1 nmol
l1 to 1000 nmol l1. Fluid secretion was
measured over 45 minunder control conditions and then for 30 min in the
presence of Anoga-DH31. Finally, 1 mmol l1
8-bromo-cyclic AMP was added to all tubules and fluid secretion measured over
a further 30 min. Diuretic activity was calculated as the difference between
rates of fluid secretion in the control and experimental periods. The length
of tubule within the bathing fluid drop varied considerably and the diuretic
activity of Anoga-DH31 was therefore normalised by expressing it as
a percentage of the diuretic activity of 1 mmol l1
8-bromo-cyclic AMP.
Fluid secretion was significantly stimulated (P<0.05; paired
t-test) by concentrations of Anoga-DH31 of 10 nmol
l1, and the response at 1000 nmol l1 did
not differ from that obtained with 1 mmol l1 8-bromo-cyclic
AMP (P=0.564). The apparent EC50 was 50 nmol
l1 with 95% confidence limits of 4159 nmol
l1.
Cyclic nucleotide production by isolated tubules challenged with Anoga-DH44 and Anoga-DH31
The production of cyclic AMP was measured over 10 min by Malpighian tubules
(five per tube) isolated from An. gambiae and incubated in
Drosophila saline containing 500 µmol l1 IBMX.
Cyclic AMP levels in the saline controls were below the level of detection in
the RIA, which was 15 fmol tube1. However, significant
(P<0.01) amounts of cyclic AMP were detected in the presence of 1
µmol l1 Anoga-DH31, reaching the equivalent of
32.7±7.3 fmol tubule1 (N=8). Cyclic AMP
levels in tubules challenged with 1 µmol l1
Anoga-DH44 were more variable (12.7± 7.5 fmol
tubule1; N=8) and not significantly different from
zero (P=0.134). The experiment was therefore repeated with 10
Malpighian tubules per assay tube and incubating for 0, 2, 5 and 10 min in
saline containing 500 µmol l1 IBMX with or without the
addition of 1 µmol l1 Anoga-DH44. After
incubating for 2 min in the presence of Anoga-DH44, cyclic AMP
levels had increased significantly (P<0.001; MannWhitney
test) from 7.0±1.7 fmol tubule1 (N=10) to
54.2±12.7 fmol tubule1 (N=5). Cyclic AMP
levels were not increased further by incubating for 5 min (32.6± 7.5
fmol tubule1; N=5) and 10 min (40.7±7.8 fmol
tubule1; N=5) but were significantly higher
(P<0.01; MannWhitney test) than the corresponding controls,
which were 2.2±2.2 fmol tubule1 (N=5) and
5.4±1.1 fmol tubule1 (N=5),
respectively.
Following a 10 min incubation in IBMX saline containing either 1 µmol l1 Anoga-DH44 or 1 µmol l1 Anoga-DH31, cyclic GMP levels were equivalent to 0.3±0.1 fmol tubule1 (N=4) and 0.3±0.1 fmol tubule1 (N=5), respectively, which were not significantly different (P=0.965) from the saline controls (0.3±0.1 fmol tubule1; N=6).
Effect of Anoga-DH44 and Anoga-DH31 on ion and fluid transport by Ae. aegypti tubules
Anoga-DH31 has both diuretic and natriuretic activity in An.
gambiae, and this CT-like peptide is the most likely candidate for the
MNP described in Ae. aegypti by Petzel et al.
(1985). We sought to confirm
this by testing both peptides on Malpighian tubules from the yellow fever
mosquito. Secretion was measured over 30 min under control conditions and for
30 min periods in the presence of either Anoga-DH44 or
Anoga-DH31 (final concentration 1 µmol l1)
alone and then in combination with 1 mmol l1 8-bromo-cyclic
AMP. The results were very similar to those obtained with An. gambiae
and are summarised in Table 1. Anoga-DH44 stimulated fluid secretion but had no effect on the
[Na+]:[K+] ratio of the secreted urine. Both parameters
were increased >3-fold after the addition of 8-bromo-cyclic AMP, reflecting
a dramatic rise in transepithelial Na+ transport, whereas
K+ transport was unchanged. In marked contrast, the CT-like peptide
Anoga-DH31 stimulated fluid secretion and transepithelial
Na+ transport to the same extent as exogenous cyclic AMP. To
further illustrate the differing effects of the two peptides, one batch of
tubules was challenged with 1 µmol l1
Anoga-DH44 alone and then in combination with 1 µmol
l1 Anoga-DH31
(Table 1). As shown previously,
Anoga-DH44 increased fluid secretion but had an insignificant
effect on the urine [Na+]:[K+] ratio. Fluid secretion
was further accelerated by the addition of Anoga-DH31 and this was
associated with a marked increase in the [Na+]:[K+]
ratio of the secreted fluid, which reflected the specific stimulation of
transepithelial Na+ transport.
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Effect of IBMX on the response to Anoga-DH44
We have shown that both Anoga-DH44 and Anoga-DH31
stimulate cyclic AMP production by Malpighian tubules isolated from An.
gambiae but that only the CT-like peptide has pronounced natriuretic
activity. Conceivably, this might be because Anoga-DH31 is more
effective than Anoga-DH44 in elevating intracellular levels of
cyclic AMP. To investigate this, the peptides were tested on Malpighian
tubules after cyclic AMP phosphodiesterase activity had been inhibited with
IBMX in order to elevate intracellular levels of the second messenger. The
results are shown in Fig. 9.
Tubule secretion rose during the 50 min incubation with 100 µmol
l1 IBMX, while the [Na+]:[K+] ratio of
the secreted urine was unchanged. The addition of 1 µmol
l1 Anoga-DH44 to the IBMX saline resulted in a
further increase in tubule secretion but had no significant effect
(P>0.05; TukeyKramer Multiple Comparisons Test) on the
[Na+]:[K+] ratio. However, with the inclusion of
Anoga-DH31 (final concentration 1 µmol l1) in
the bathing fluid, the urine [Na+]:[K+] ratio increased
dramatically, which, taken together with the further acceleration of tubule
secretion, represented a 9-fold increase in Na+ transport (from
17.1±2.1 pmol min1 to 180.2± 31.1 pmol
min1) compared with only a 2-fold increase in K+
transport (from 64.5±8.7 pmol min1 to
114.2±15.0 pmol min1).
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Effect of Musdo-K on the activity of Anoga-DH31
The CRF-related peptides Anoga-DH44 and Culsa-DH initially
depolarise the transepithelial voltage of Malpighian tubules from An.
gambiae (present study) and Ae. aegypti
(Clark et al., 1998a),
respectively. Clark et al.
(1998a
) attribute this initial
depolarisation to the Ca2+-dependent opening of a paracellular
Cl conductance, which characterises the response to the
kinin family of insect diuretic peptides and results in a non-selective
increase in Na+ and K+ transport
(Beyenbach, 1995
). Conceivably,
this non-selective response to activation of the Cl
conductance pathway could outweigh a cyclic AMP-dependent stimulation of
Na+ transport and thus account for the different effects of
Anoga-DH31 and Anoga-DH44 on cation transport. To test
this hypothesis, we determined what effect activating the Cl
conductance pathway using a diuretic/myotropic kinin had on the response to
Anoga-DH31. Since none of the An. gambiae kinins were
available to us, we used the housefly (Musca domestica) kinin,
Musdo-K.
Fig. 10 shows a recording
of the basolateral membrane voltage from an An. gambiae principal
cell challenged separately with first 100 nmol l1
Anoga-DH31, then with 100 nmol l1 Musdo-K and
finally with a combination of the two peptides. The basolateral membrane was
depolarised by Anoga-DH31 and hyperpolarised by Musdo-K, which is
consistent with the effect of kinin stimulation in Ae. aegypti
Malpighian tubules (Pannabecker et al.,
1993). A combination of the two peptides produced a triphasic
response similar to that obtained with Anoga-DH44 (cf.
Fig. 7A), although the initial
hyperpolarisation was more pronounced.
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Discussion |
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Effects of Anoga-DH44 and Anoga-DH31 on tubule electrophysiology
Anoga-DH44 and Anoga-DH31 produce characteristic
changes in transepithelial voltage, which are due largely to voltage changes
at the principal cell basolateral membrane. The response to the CRF-related
peptide is very similar to that reported previously for MNP (fraction III in
Petzel et al., 1985) and
Culsa-DH (fig. 2 in Clark et al.,
1998a
), in that the transepithelial voltage briefly depolarises
before hyperpolarising. Interestingly, this transient is not seen with either
Anoga-DH31 or membrane-permeant analogues of cyclic AMP (present
study; Sawyer and Beyenbach,
1985
) and yet they, rather than Anoga-DH44, mimic the
natriuretic activity of MNP. Clark et al.
(1998b
) attribute the
depolarising and hyperpolarising phases of the response to Culsa-DH as being
due to the Ca2+-dependent opening of a paracellular
Cl conductance and the cyclic AMP-mediated stimulation of
active cation transport, respectively. Our findings are consistent with this
interpretation in that a combination of Anoga-DH31 and Musdo-K, a
member of the kinin family of diuretic peptides that use Ca2+ as a
second messenger in activating a Cl conductance pathway
(O'Donnell et al., 1998
;
Yu and Beyenbach, 2002
), mimic
the electrophysiological signature of Anoga-DH44. One way of
accounting for the biphasic effect of MNP on transepithelial voltage is to
suggest that fraction III (which was dubbed MNP by
Petzel et al., 1985
) contained
Anoga-DH31 together with a kinin. In support of this, gel
filtration chromatography of fraction III indicated a molecular mass of 1862
Da (Petzel et al., 1986
),
which is more similar to Aedae-K-I (1593 Da) than to either CT-like or
CRF-related diuretic hormones.
Anoga-DH31 and Anoga-DH44 use cyclic AMP as a second messenger but have different effects on ion transport
A paradox to arise from the present study is that, although
Anoga-DH31 and Anoga-DH44 both stimulate cyclic AMP
production by isolated Malpighian tubules, only the former has natriuretic
activity. As yet, we cannot explain this, but we are able to rule out a number
of possibilities. First, the peptides could be acting on functionally
different populations of principal cells, only one of which responds to a rise
in the level of intracellular cyclic AMP with a selective increase in
transepithelial Na+ transport. However, such functional diversity
among principal cells appears unlikely, because recordings of the basolateral
membrane voltage from a single cell (Fig.
8) show that it responds in characteristic fashion to both
Anoga-DH31 and Anoga-DH44.
Secondly, a quantitative difference in the cyclic AMP signal obtained in
response to Anoga-DH31 and Anoga-DH44 could determine
the extent to which Na+ transport is stimulated. In this context,
it is worth noting that the CRF-related diuretic hormone of D.
melanogaster (Drome-DH44) stimulates cyclic AMP production by
Malpighian tubule principal cells and also activates a cyclic AMP-specific
phosphodiesterase (PDE), which will curtail the second messenger response
(Cabrero et al., 2002). This
was not investigated in the present study, which measured cyclic AMP
production by Malpighian tubules after PDE activity was inhibited by IBMX.
However, a quantitative difference in cyclic AMP signalling is unlikely to
explain why only Anoga-DH31 has natriuretic activity, because
Anoga-DH44 does not selectively stimulate Na+ transport
by Malpighian tubules in which PDE activity was inhibited by IBMX.
The third possibility to be examined was whether the activation of both
paracellular and transcellular pathways by Anoga-DH44 (see above)
militates against a natriuretic response. This also appears unlikely because
Anoga-DH31 selectively stimulates transepithelial Na+
transport by Malpighian tubules in which the paracellular pathway is first
activated by Musdo-K. Similarly, differences in signalling exist for
ß2 and ß1 adrenergic receptors in vertebrate
heart tissue, which are believed to be due to differences in compartmentation
of cyclic AMP in the tissue. Three principles of compartmentation of cyclic
AMP signalling have been postulated: not all cyclic AMP gains access to all
protein kinase A; not all protein kinase A has access to all possible
substrates; and not all cyclic AMP has access to all cellular
phosphodiesterases (Steinberg and Brunton,
2001). There is evidence that most ß2 adrenergic
receptors are localized in caveolae, whereas most ß1
adrenergic receptors are not; this difference in localization in the plasma
membrane may explain differences in the cyclic AMP response observed on
stimulation of these receptors (Steinberg
and Brunton, 2001
). It is likely that the receptors for the
CRF-like DH, vs the CT-like DH, are localized differently in the cell
membrane, giving rise to differences in their signalling. It is also possible
that these receptors couple to different G proteins; Culsa-DH leads to an
apparent activation of both intracellular Ca2+ and cyclic AMP,
depending on the concentration of the ligand
(Clark et al., 1998b
). By
contrast, our data point to Anoga-DH31 leading to only elevation of
cyclic AMP.
In conclusion, the diuretic and natriuretic activities of
Anoga-DH31, a CT-like diuretic hormone common to An.
gambiae and Ae. aegypti, are consistent with it being the MNP
identified by Petzel et al.
(1985). However, we have yet
to show that Anoga-DH31 is released into the circulation when the
insect takes a blood meal.
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References |
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