The anterior stomach of larval mosquitoes (Aedes aegypti): effects of neuropeptides on transepithelial ion transport and muscular motility
School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA
* Author for correspondence (e-mail: onkenh{at}wsu.edu)
Accepted 26 July 2004
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Summary |
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Key words: Aedes aegypti, allatostatin, allatotropin, anterior stomach, head peptide, larva, midgut, mosquito, neuropeptide F, peristalsis, proctolin, transepithelial voltage
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Introduction |
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The rates and mechanisms of acidbase relevant transport processes in
the stomach of mosquito larvae are poorly understood. Alkalization in the
anterior stomach seems to be reflected in a luminally negative transepithelial
voltage (Clark et al., 1999,
2000
) and is partly based on
transapical HCO3 secretion and transbasolateral
H+ absorption energized by V-type H+ pumps in the
basolateral membrane (Zhuang et al.,
1999
; Boudko et al.,
2001a
,b
;
Onken et al., 2004
). Since
active accumulation of luminal HCO3 only explains
alkalization up to about pH 8.5, additional transepithelial absorption of acid
equivalents has to be evoked to account for alkalization to values above pH
10.
Regulatory systems involved in controlling the activities of the intestinal
tract in response to feeding, and in integrating its function and whole-animal
homeostasis with behavioral responses such as eating, drinking and breathing,
are known only in a general way (cf.
Sehnal and Zitnan, 1996).
Possible regulatory inputs may come via innervation from the central
nervous system, via neuroendocrine factors emanating from the central
nervous system or from the numerous enteroendocrine cells, each expressing one
or more peptide hormones (Brown et al.,
1985
). Previous studies showed that the transepithelial voltage of
the isolated anterior stomach of the larval yellow fever mosquito Aedes
aegypti declined precipitously within minutes after mounting, but could
be partly restored by submicromolar concentrations of serotonin
(Clark et al., 1999
). In a
later, more detailed study (Clark et al.,
2000
) two cell types were discovered. In one cell type the
basolateral membrane voltage was stable after mounting of the isolated tissue,
whereas it dramatically depolarized in the other cell type. It could be that
the two cell types may be related to the two processes that seem to be
involved in anterior stomach alkalization (see above). With respect to
regulation, the above-described results suggest that endogenous serotonin and
some additional chemical messengers were critical for authentic stomach
function and were lost when the stomach was isolated and perfused with
artificial saline.
The present study evaluated several candidate neuropeptides for their potential roles in controlling stomach function. Initially, these studies focused on ion transport of the anterior stomach, using changes in the transepithelial voltage as an indicator of effects on ion transport. However, the muscular motility of the preparations produced a dynamic component of the transepithelial voltage, which also allowed us to evaluate myotropic effects of the peptides.
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Materials and methods |
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Solutions and chemicals
The basic saline used was based on larval Aedes hemolymph
composition (Edwards,
1982a,b
)
and consisted of (in mmol l1): NaCl, 42.5; KCl, 3.0;
MgCl2, 0.6; CaCl2, 5.0; NaHCO3, 5.0; succinic
acid, 5.0, malic acid, 5.0; L-proline, 5.0; L-glutamine,
9.1; L-histidine, 8.7; L-arginine, 3.3; dextrose, 10.0;
Hepes, 25. The pH was adjusted to 7.0 with NaOH. The above components were
purchased from Sigma (St Louis, MO, USA), Fisher Scientific (Pittsburgh, PA,
USA) or Mallinckrodt (Hazelwood, MO, USA). Serotonin and proctolin were
purchased from Sigma. A. aegypti head peptide I (Aedae-HP-I), A.
aegypti head peptide III (Aedae-HP-III), A. aegypti short
neuropeptide F (Aedae-sNPF also known as Aedae-LRLFa or new head peptide) and
A. aegypti neuropeptide F (Aedae-NPF) were provided by Dr M. R. Brown
(University of Georgia, Athens, USA). A. aegypti allatotropin
(Aedae-AT) was provided by Dr J. A. Veenstra (Université de Bordeaux,
Talence, France) and five different A. aegypti allatostatins type A
(Aedae-AST-A 15) were provided by Dr F. G. Noriega (University of
Arizona, Tucson, USA). The amino acid sequences of the peptides used are shown
in Table 1. The peptides and
concentrated stock solutions in water or saline were stored at 80°C
before their use in the experiments.
|
Perfusion pipettes
Perfusion pipettes were made from glass capillary pipettes (100 µl, VWR,
West Chester, PA, USA). A pull on a vertical pipette puller (model 700B, David
Knopf Instruments, Tujunga, CA, USA) was followed by manual elaboration of the
pipette tips (approximately 100 µm in diameter) to give the pipette shaft
an L-shaped form. The shaft of the pipette tips was covered with a thin layer
of cured Sylgard 184 (Dow Corning, Midland, MI, USA) to improve the electrical
seal between the pipette and the tissue.
Preparations and perfusion of anterior stomachs
The larvae were decapitated and the intestinal system then isolated and
transferred to the bath of a perfusion chamber. The caeca and the posterior
stomach were cut off and the anterior stomach was mounted with its anterior
end on the tip of the perfusion pipette, held by a micromanipulator
(Brinkmann, Westbury, NY, USA). The preparations were tied in place with a
fine human hair and the posterior end of the anterior stomach was left open
(semi-open preparation; see Fig.
1). The bath (volume 100 µl) was perfused by gravity flow with
oxygenated salines at a rate of 1530 ml h1. The
perfusion pipette was connected via a set of 3-way stopcocks to a
pushpull multi-speed syringe pump (model 120, Stoelting, Wood Dale, IL,
USA). The rate of perfusion was 2060 µl min1.
According to the physical dimensions of anterior stomach preparations
(Clark et al., 2000), this rate
results in 39 luminal volume exchanges per minute.
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Electrophysiological measurements
The bath and the pipette, reflecting the hemolymph side and the lumen of
the semi-open stomach preparation, were connected via agar bridges
(3% agar in 3 mol l1 KCl) to calomel electrodes (see
Fig. 1). The transepithelial
voltage (Vte) was measured in the lumen with reference to
the bath (hemolymph side of the tissue) with the voltmeter of a voltage clamp
(VCC 600, Physiologic Instruments, San Diego, CA, USA) and continuously
recorded on a chart recorder (model 500, Linear Instruments, Reno, NV, USA).
In a prior study (Onken et al.,
2004), the validity of the semi-open preparation for voltage
measurements was verified and discussed in detail.
Statistics
All data are presented as means ± standard error of the mean
(S.E.M.). Differences between groups were
tested, using one-way analysis of variance (ANOVA) with Tukey's post-test.
Significance was assumed at P<0.05.
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Results |
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Effects of neuropeptides on the transepithelial voltage in presence of serotonin
In a first series of experiments (N=5), A. aegypti
allatostatins A1 to A5 (Aedae-AST-A, see
Table 1;
Veenstra et al., 1997) were
applied at a concentration of 107 mol l1
to the bath. All five different Aedae-AST-A affected Vte
in a very similar way, inducing a small but significant (P<0.05)
depolarization of the luminally negative voltage by 1015%. The voltage
decreases were partly reversible. A time-course of one representative
experiment is shown in Fig.
2.
In the next series of experiments, individual anterior stomach preparations
were exposed to A. aegypti allatotropin (Aedae-AT, see
Table 1;
Veenstra and Costes, 1999), to
two different A. aegypti head peptides (Aedae-HP-I, Aedae-HP-III, see
Table 1; cf.
Matsumoto et al., 1989
;
Veenstra, 1999
) or to short
neuropeptide F (Aedae-sNPF, see Table
1; cf. Riehle et al.,
2002
) at stepwise increasing concentrations between
1016 and 106 mol l1. The
time of exposure to each concentration of a peptide was between 5 and 10 min.
Neither Aedae-AT (N=5) nor either one of the head peptides
(N=8 and 6, respectively) nor Aedae-sNPF (N=5) caused a
significant change of Vte at any concentration
(P>0.05). The results are summarized in
Fig. 3.
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In another series of experiments the individual tissues were exposed to
increasing concentrations (1016 to 106 mol
l1) of A. aegypti neuropeptide F (Aedae-NPF; see
Table 1; Stanek et al., 2002). The
results are summarized in Fig.
4. At 1010, 108 and
106 mol l1, the peptide significantly
reduced Vte by 22±5, 26±6 and 31±7%
of the control value, respectively (P<0.05). The effect of
Aedae-NPF showed a large scatter among individual stomach preparations. At
106 mol l1, voltage reductions between 9%
and 68% of the control value were measured. The effectiveness of the peptide
showed no relation to the magnitude of Vte under control
conditions. The effect of the peptide was only partly reversible. However, it
must be taken into consideration that in this kind of experiment the tissue is
exposed to increasing concentrations of the modulator for 3060 min,
which certainly does not favor a successful washout.
|
In the next series of experiments (N=6) we tested whether
proctolin (for a review, see Konopinska
and Rosinski, 1999) affects Vte of the
anterior stomach of A. aegypti larvae. In all six experiments the
individual tissues were subsequently exposed to hemolymph-side proctolin at
increasing concentrations between 1016 and
106 mol l1. The results are summarized in
Fig. 4. Proctolin effected an
almost continuous decrease of Vte with increasing
concentration. However, only at concentrations between 1010
and 106 mol l1 did the results satisfy the
criteria for statistically significant difference from the initial control
value (P<0.05). As with Aedae-NPF, the voltage reduction induced
by proctolin showed a large scatter (978% of the control at
106 mol l1), which was independent of the
magnitude of Vte before addition of the peptide. The
effects of proctolin were only partly reversible.
Effects of neuropeptides on stomach motility
Within 2 h after addition of serotonin (0.2 µmol l1),
many preparations generated fluctuations of the transepithelial voltage
(Vte) that were independent of the magnitude of
Vte. At the same time when
Vte
appeared, muscular motility of the mounted tissue could be observed through
the preparation microscope. The motility included regular peristaltic waves.
However, often it was observed to contain less regular contractions and seemed
to reflect pump-like mixing of stomach contents instead of directional
transport by peristaltic activity. With different preparations different
patterns of
Vte were observed (see
Fig. 5A). In some preparations,
the muscular motility was reflected in a relatively regular sinusoidal wave.
In others, their appearance resembled a double saw, where two waves of
different frequency and amplitude appeared to overlap. In some cases bursts of
high wave activity were regularly interrupted by phases of much lower
activity. Both
Vte and muscular motility were
simultaneously and reversibly affected by washout of serotonin or by addition
of peptides, indicating a direct relationship between
Vte and motility. In order to quantitatively assess
Vte the voltage signal was recorded at increased
gain (0.51.0 mV cm1) and paper velocity (0.2 cm
s1). The amplitude of the voltage oscillation was determined
in 15 time intervals of 1 s and afterwards averaged. The frequencies were
determined by counting oscillation peaks during a time period of 15 s. In nine
experiments the preparations generated fluctuations of Vte
with an amplitude of 0.65±0.20 mV (mean ±
S.E.M.) and frequency of 1.42±0.05
s1 (mean ±
S.E.M.).
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The Vte fluctuations and the stomach motility are not
instantaneously induced after the first addition of serotonin to the bathing
medium. However, once initiated (60120 minafter addition of the drug)
they were strictly dependent on the presence of serotonin
(Fig. 5B). Washout of serotonin
rapidly abolished Vte and stomach motility, and
re-addition of the drug re-established them (N=7).
Like Aedae-HP-I (see Fig.
5C), Aedae-HP-III, Aedae-sNPF and Aedae-NPF also reversibly
inhibited Vte and the stomach motility in a
dose-dependent way. To quantify the influence of the neuropeptides,
experiments were performed in which a peptide was applied at increasing
concentrations (108 and 106 mol
l1) before and after recording
Vte under control conditions. Analyzing the
amplitudes of
Vte under control conditions and
comparing them with those measured in the presence of different concentrations
of the neuropeptides clearly demonstrated the dose-dependent inhibitory effect
of Aedae-HP-I, Aedae-sNPF and Aedae-NPF (see
Fig. 6). For these peptides,
the amplitudes of
Vte at 108 mol
l1 were significantly lower than those under control
conditions (P<0.05). At 106 mol
l1 these peptides caused a further decrease of the
amplitudes of
Vte, which were found to be
significantly lower than the amplitudes under control conditions and in
presence of the lower neuropeptide concentration (P<0.05).
Aedae-HP-III also caused a reduction of the amplitudes of
Vte. However, in this case the two different
peptide concentrations did not result in significantly different effects on
the amplitude of
Vte (see
Fig. 6), suggesting that
Aedae-HP-III already exerts its maximal influence on stomach motility at
108 mol l1.
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Analysing the influence of neuropeptides on the frequencies of
Vte (N=57) showed that this
parameter is affected to a minor degree by the peptides. When compared with
the frequencies under control conditions, the lower peptide concentrations
(108 mol l1) did not result in significant
changes (P>0.05) of the frequencies. Only at 106
mol l1 did Aedae-HP-I (0.52±0.14
s1), Aedae-sNPF (0.33±0.13 s1) and
Aedae-NPF (0.53±0.31 s1) significantly reduce the
frequencies of
Vte (P<0.05) when
compared with the control (1.47±0.04 s1).
The influences of Aedae-AST-A, Aedae-AT and proctolin on
Vte were not studied in detail. During the
experiments with Aedae-AT voltage fluctuations were never observed. In one of
the five experiments with the different Aedae-AST-A, Vte
fluctuations were observed and not inhibited by 107 mol
l1 of the peptides. In one experiment with proctolin,
Vte fluctuations and motility were observed directly after
administration of 1016 mol l1 of the
peptide; increasing the proctolin concentration did not further modify
Vte. In the other five experiments with proctolin,
no
Vte or motility were observed.
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Discussion |
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Methodological aspects
The validity and advantages of the semi-open preparation of the isolated
and perfused anterior stomach of larval mosquitoes have been demonstrated and
discussed in detail in a previous study
(Onken et al., 2004). With
respect to the regulation of transport rates the qualitative aspect of the
transepithelial voltage (Vte) is not ideal and the
development of a technique to quantitatively monitor ion transport in the
mosquito stomach remains a major task. Nevertheless, Vte
measurements are certainly useful for detecting changes induced by transport
modulators, although their interpretation can be more complicated and insight
into the mechanism of action of a modulator is difficult without simultaneous,
area-specific conductance measurements. Vte is determined
by paracellular and transcellular parameters and a hormone-induced change of
Vte could reflect a change of the transport rates
via an effect on the paracellular conductance, as has been proposed
for the leucokinin-induced stimulation of fluid secretion by Malpighian
tubules of A. aegypti (cf.
Beyenbach, 2003
;
Yu and Beyenbach, 2004
). When
a modulator affects only the paracellular resistance the changes of
Vte and transport rates are opposite. Reduction of the
paracellular resistance results in an increased transport rate at decreased
Vte, whereas an increased paracellular resistance causes
reduction of transport rates at increased Vte. However,
the vast majority of hormonal modulators seems to act on electromotive force
and/or conductance of the transcellular pathway, and in this case an
increasing Vte reflects transport stimulation and a
decreasing Vte reduction of the transport rate.
As in a previous study with the semi-open preparation of the larval
anterior stomach of A. aegypti
(Onken et al., 2004),
Vte under control conditions and after stimulation with
serotonin showed considerable variability. Of course, we cannot rule out that
this variation is partly based on different voltage decrements via
the open ends of the preparations, reflecting a different longitudinal
resistance of preparations of different length. Interestingly, however, the
effects of neuropeptide F and proctolin on Vte also showed
a large scatter from preparation to preparation, and the effectiveness showed
no relation to the magnitude of Vte under control
conditions before addition of the peptides (see Results). This observation
suggests a significant variability between individual preparations that seems
not to be related to the method. Instead, the differences may reflect a
variability of the characteristics of the tissue from individual to
individual. Such differences could be related, for example, to age and/or
feeding behavior of fourth instar larvae and need further, more detailed
studies.
During the present study, we noticed an additional feature of the
preparation that is of particular importance for the evaluation of possible
modulators of larval mosquito stomach functions. The stomach motility appears
to be reflected in small, but properly recordable Vte
deflections with different patterns (see
Fig. 5A). The dependence of
Vte fluctuations and of the observable stomach motility on
the presence of serotonin and their inhibition by peptides (see below) is an
indication that the dynamic Vte component reflects
muscular motility. In fact, the Vte fluctuations cannot be
related to the force of muscular activity, as is possible with other bioassays
that use microforce transducers. We do not know how the motility generates
fluctuations of Vte. It is certainly possible that the
stomach motility could generate waves of small hydrostatic pressure changes in
the lumen that could in turn influence ion movement across ion-selective
paracellular pathways. However, we cannot exclude direct effects of pressure
gradients on the transcellular ion movement
(Wang et al., 2003) or other
explanations. More detailed experiments are needed to uncover the link between
motility and Vte. Nevertheless, as the results of the
present study indicate, the Vte fluctuations seem to
directly reflect muscular motility. Thus, the semi-open preparation of stomach
segments could be an especially powerful tool for studying the effects of
possible modulators on midgut function, because it seems to allow simultaneous
monitoring of transepithelial ion transport (reflected in
Vte) and stomach motility (reflected in its
fluctuations).
Effects of peptides
The number of gastrointestinal endocrine cells of insects, their degree of
FMRFamide-immunoreactivity and the hemolymph concentration of the
immunoreactive agent were observed to change as a response to feeding
(Brown et al., 1986;
Jenkins et al., 1989
). For
gastrointestinal functions like muscular motility
(Schoofs et al., 1993
),
salivary gland fluid secretion (Duve et
al., 1992
), midgut secretion of digestive enzymes
(Fusé et al., 1999
;
Nachman et al., 1997
), fluid
secretion by Malpighian tubules (Beyenbach,
2003
; Coast et al.,
2002
) and reabsorption by the hindgut
(Coast et al., 2002
),
regulation by peptides is well documented. In contrast, to our knowledge only
a single study was performed that is related to the regulation of midgut ion
transport by neuropeptides (Lee et al.,
1998
).
All effects of peptides on Vte and its fluctuations observed in the present study appeared quickly after addition of the modulators to the bathing medium (see Fig. 1). In the experiments where effects of Aedae-NPF and proctolin on Vte were observed the reversibility was limited, probably due to the long exposure of the tissue to the respective peptide. Interestingly, stomach motility and Vte fluctuations appeared only 60120 min after addition of serotonin. This observation could be explained if liberated endogenous peptides inhibit peristaltic activity until they are washed out.
Allatotropin and allatostatin
Peptide factors of the nervous system are known to stimulate (allatotropin)
or inhibit (allatostatin) the synthesis of juvenile hormone in the corpora
allata. Both peptide types were also shown to have myotropic effects,
including on gut peristalsis (Lange et
al., 1993; Duve and Thorpe,
1994
; Veenstra et al.,
1994
). Moreover, allatostatin was shown to stimulate midgut
carbohydrate enzyme activity (Fusé
et al., 1999
) and allatotropin was demonstrated to inhibit ion
transport across Manduca sexta posterior midgut
(Lee et al., 1998
). An
Aedae-AT (Veenstra and Costes,
1999
) and different Aedae-AST-A
(Veenstra et al., 1997
) have
been isolated from adult A. aegypti. The identification of a partial
prepro-allatostatin cDNA from a midgut cDNA library indicated that this gene
is also expressed in the mosquito midgut
(Veenstra et al., 1997
). As
shown in the present study (see Fig.
2), the five allatostatins acted in an almost identical way on
Vte of the anterior stomach, causing a slight but
significant reduction. In contrast, Aedae-AT was without significant effect on
Vte in the whole concentration range studied (see
Fig. 3).
Although the effects of Aedae-AT and Aedae-AST-A on stomach motility were not studied in detail, voltage fluctuations were never observed after addition of Aedae-AT. Thus, this peptide apparently does not induce muscular motility in the anterior stomach of A. aegypti. In one of the five experiments with the Aedae-AST-A, muscular contractions and Vte fluctuations were observed and not inhibited by the peptides, suggesting that allatostatins do not inhibit motility in this tissue. Nevertheless, additional studies are needed to verify that Aedae-AT and Aedae-AST-A do not affect stomach motility in larval A. aegypti.
Head peptides and short neuropeptide F
Different head peptides have been isolated from A. aegypti
(Matsumoto et al., 1989;
Veenstra, 1999
). Antiserum
against Aedae-HP-I reacted with gastrointestinal endocrine cells of adult
mosquitoes (Brown et al., 1994
)
and the gene is expressed in larvae, adult males and adult females in response
to a blood meal (Stracker et al.,
2002
). Aedae-HP-I was demonstrated to induce supression of
host-seeking behaviour in adult females
(Brown et al., 1994
). A gene
encoding short neuropeptide F (sNPF) has been found in Drosophila
melanogaster and Anopheles gambiae
(Riehle et al., 2002
), and the
Aedae-sNPF was isolated from an adult abdomen extract based on its
immunoreactivity in a NPF radioimmunoassay. In the present study, Aedae-HP-I,
Aedae-HP-III and Aedae-sNPF did not induce significant changes of
Vte across the anterior stomach of larval A.
aegypti (see Fig. 3),
indicating that these peptides do not interfere with ion transport
via the serotonin-stimulated cells. However, all three peptides
clearly inhibited stomach motility (see Figs
5C,
6). Aedae-HP-I and Aedae-sNPF
supressed the amplitude of the Vte fluctuations apparently
more effectively than Aedae-HP-III. The finding of a myotropic effect of head
peptide is contradictory to the results of a study on hindguts of adult A.
aegypti where Aedae-HP-I was without effect on peristalsis. It could be,
however, that muscular motility in different regions of the gut is modulated
by different peptides.
Neuropeptide F
Neuropeptide F (NPF), a member of the neuropeptide F/Y superfamily, was
isolated from adult A. aegypti and NPF-like immunostaining was
observed in brain and midgut of adults and larvae
(Stanek et al., 2002). In
adult females the NPF hemolymph concentration dropped immediately after a
blood meal but showed a peak 24 h after the blood meal, suggesting a relation
to digestive processes and the reproductive cycle
(Stanek et al., 2002
). In
larvae, submicromolar concentrations of the peptide induced a decrease of
Vte across the anterior stomach (see
Fig. 4). In addition, a strong
inhibitory effect on stomach motility was observed (see
Fig. 6). Thus, Aedae-NPF is
evidently involved in the regulation and coordination of two major functions
of the anterior stomach of mosquito larvae: alkalization and motility.
Proctolin
Proctolin is the best known and most investigated myotropic neuropeptide in
insects, and it is regarded as the main neuromuscular transmitter/modulator in
the gut of insects (Konopinska and
Rosinski, 1999). In a variety of insects, proctolin has been shown
to induce or stimulate muscle contractions, including gut peristalsis
(Orchard et al., 1989
). In the
present study, proctolin induced a dose-dependent reduction of
Vte (see Fig.
4), indicating that it affects transepithelial ion transport
via the serotonin-stimulated cells at submicromolar doses. Thus,
besides the promotion of vitellogenesis in the cockroach oocyte
(Goudey-Perriere et al.,
1994
), the present finding is one of the rare examples of
proctolin affecting a non-excitable cell type.
In the present investigation, possible effects of proctolin on
Vte fluctuations, and thus stomach motility, were not
studied in detail. In one experiment proctolin was observed to apparently
induce Vte fluctuations at 1016 mol
l1 and the Vte fluctuations were not
further modified when the concentration of the peptide was increased. However,
this effect was not reproduced in the other five experiments. Thus, it appears
that proctolin does not influence muscular motility in the anterior stomach of
A. aegypti, but this observation needs verification in future.
Nevertheless, it is noteworthy that in a comparative study
(Messer and Brown, 1995)
cricket hindguts responded in the expected way with a stimulation of
peristalsis to proctolin, whereas with the hindguts of adult mosquitoes,
proctolin did not affect peristalsis.
Effects of peptides related to their amino acid sequences
The amino acid sequences of the peptides used in the present study (see
Table 1) show clear
similarities between Aedae-HP-I, Aedae-HP-II and Aedae-sNPF that cannot be
neglected. In fact, there has been a proposal to name all peptides with the
R(K)-X1-R-X2amide C-terminal motif as short NPFs (cf.
Mertens et al., 2002). It
might be that they all bind with different affinities to the same receptor to
exercise their inhibitory effect on stomach motility. In D.
melanogaster, different G-protein coupled receptors have been identified
for short and long NPFs (Garczynski et
al., 2002
; Mertens et al.,
2002
; Feng et al.,
2003
). The same can be anticipated for A. aegypti, based
on the observation that long NPF inhibited ion transport
(Fig. 4), whereas head
peptides/short NPF did not (Fig.
3). That the five Aedae-AST-A produced almost the same inhibitory
effect on Vte may also be attributable to their action on
the same receptor. Among the three types of peptides that reduced
Vte, similarities in their sequence can hardly be seen and
it seems reasonable to assume that proctolin, Aedae-NPF and Aedae-AST-A use
different receptors to unfold their action on ion transport. Since the effects
of peptides on ion transport and motility were antagonistic to the effects of
serotonin, it seems likely that the peptides interfere with the signal
transduction pathways of the biogenic amine.
Effects on transport and/or motility
The effects of serotonin and of neuropeptides observed in the present study
are summarized in Fig. 7. Four
groups of modulators can be distinguished: (1) stimulants of transepithelial
transport and stomach motility (serotonin), (2) inhibitors of transepithelial
transport and stomach motility (neuropeptide F), (3) inhibitors of
transepithelial transport (proctolin and allatostatin) and (4) inhibitors of
stomach motility (head peptides and short neuropeptide F). The identified
modulators offer the possibility of an effective regulation and coordination
of ion transport (alkalization) and muscular motility. However, it cannot of
course be excluded that direct neuronal influence and further
hormones/neuropeptides are also involved in the regulation and coordination of
the anterior stomach functions.
|
Although the present study provides new insights into the regulation of the
activity of the anterior stomach of larval A. aegypti, it has
revealed even more gaps in our knowledge. We still have not identified the
modulator (hormonal or non-hormonal) that re-establishes proper function of
the cells with depolarizing basolateral membrane voltage (cf.
Clark et al., 2000), which seem
to be crucial for alkalization to pH 1012 (see Introduction). Moreover,
future studies need to determine the mode of action of the identified
regulators, how their effects are relayed to the intracellular level and which
transporters are affected. Another question to be addressed is where the
peptides are liberated and which factors govern their liberation.
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Acknowledgments |
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References |
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