The transepithelial voltage of the isolated anterior stomach of mosquito larvae (Aedes aegypti): pharmacological characterization of the serotonin-stimulated cells
School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA
* Author for correspondence (e-mail: onkenh{at}wsu.edu)
Accepted 2 March 2004
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Summary |
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Key words: Aedes aegypti, inhibitor, ion substitution, larva, mosquito, stomach, transepithelial voltage
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Introduction |
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Most information about insect midgut ion transport is based on studies of
the midgut of lepidopteran larvae (for reviews, see
Dow, 1986;
Clements, 1992
;
Klein et al., 1996
), where an
electrogenic V-type H+-pump hyperpolarizes the apical plasma
membranes of the epithelial cells (for a review, see
Wieczorek et al., 1999
). This
electrical potential is thought to energize cation secretion and luminal
alkalization by an electrogenic K+/2H+ antiporter
(Wieczorek et al., 1991
;
Azuma et al., 1995
; for
reviews, see Lepier et al.,
1994
; Wieczorek et al.,
1999
). However, this hypothesis has been questioned on
thermodynamic grounds (Moffett and
Cummings, 1994
; Klein et al.,
1996
; Küppers and Bunse,
1996
; Clark et al.,
1998
).
Like the lepidopteran midgut, the anterior midgut [or `anterior stomach' in
the terminology of Clements
(1992), used in this and
previous publications from this laboratory] of the larval mosquito is known as
an alkalizing organ, and apparently the process is regulated by a neural or
hormonal mechanism (Dadd,
1975
,
1976
). Gill et al.
(1998
) isolated cDNA clones of
two V-ATPase subunits from the stomach of Aedes aegypti larvae, and a
high level of expression of one of these subunits was observed in the anterior
stomach (Filippova et al.,
1998
). Zhuang et al.
(1999
) showed that V-ATPases
are localized on the basal membranes of the anterior stomach, whereas the
ATPase is found in the apical membrane of the caeca and posterior stomach. The
anterior stomach alkalization could be visualized in the living animal by
allowing it to ingest pH indicators added to the rearing medium. When the
rearing medium also contained inhibitors of V-ATPase, carbonic anhydrase or
anion exchangers, alkalization of the anterior stomach was found to be
inhibited (Zhuang et al.,
1999
; Boudko et al.,
2001a
; del Pilar Corena et
al., 2002
). Boudko et al.
(2001b
) used a semi-intact
preparation of mosquito larvae and showed that the basal acid efflux was
inhibited by bafilomycin A, a specific inhibitor of V-ATPases
(Dröse and Altendorf,
1997
). The highest acid and chloride effluxes from the gut of
semi-intact preparations were found in the anterior stomach region and were
also reduced by inhibitors of carbonic anhydrase and anion exchangers when
these drugs were applied to the hemolymph side of the gut
(Boudko et al., 2001a
).
Together, these results indicate a participation of basolateral V-ATPases,
carbonic anhydrase and ion exchangers in the mechanisms of alkalization. Del
Pilar Corena et al. (2002
),
however, studied the localization of carbonic anhydrase in the mosquito
intestinal system and found one isoform of this enzyme only in the caeca and
in the posterior stomach.
Clark et al. (1999) perfused
isolated anterior and posterior stomach segments of Aedes aegypti and
measured the transepithelial voltage (Vte). Interestingly,
the anterior stomach generated a lumen-negative voltage, whereas the posterior
stomach displayed a lumen-positive voltage. The polarity of the voltage
measured in posterior stomach segments is consistent with cation secretion,
driven by an apical V-ATPase, as described for the lepidopteran midgut (see
above). The lumen-negative voltage of the anterior stomach may reflect active
and electrogenic H+ absorption across the basolateral membrane and
HCO3- secretion across the apical membrane. The luminal
accumulation of HCO3- could be one component of luminal
alkalization (up to about pH 8.3), although an additional transepithelial
absorption of H+ would be necessary to reach luminal pH values
above 10 (cf. Boudko et al.,
2001a
). In both segments, the initially high voltages dropped
significantly but could partly be re-established by the addition of
submicromolar doses of serotonin (Clark et
al., 1999
). In a further, more detailed study of the
electrophysiological characteristics of anterior stomach segments, Clark et
al. (2000
) showed the presence
of two different cell types. In the so-called decaying cells, the basolateral
membrane voltage (Vb) almost completely depolarized after
mounting and showed no recovery after application of serotonin. By contrast,
Vb of the so-called stable cells depolarized only slightly
after mounting and hyperpolarized when serotonin was applied. These results
indicated that only the stable cells generated the Vte in
the presence of serotonin.
In the present study, ion substitution experiments and inhibitors of transporters were used in order to obtain more information about the transport mechanisms reflected in the Vte generated by the serotonin-responsive cells. These studies also demonstrated the usefulness of a novel semi-open preparation of the perfused stomach.
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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 l-1): 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. Na+-free saline
was prepared by substituting NaCl by N-methylglucamine. Instead of 5
mmol l-1 NaHCO3, this saline contained 3 mmol
l-1 KHCO3 (no KCl). The pH was adjusted with HCl. In
Cl--free saline, gluconates (Na+, K+,
Ca2+) or sulfate (Mg2+) substituted for the chlorides.
The pH was adjusted with NaOH. The above components were purchased from Sigma
(St Louis, MO, USA), Fisher Scientific (Pittsburgh, PA, USA) or Mallinckrodt
(Hazelwood, MO, USA). Concanamycin and diphenylamine-2-carboxylic acid (DPC or
N-phenylanthranilic acid) were from Fluka (St Louis, MO, USA).
Acetazolamide, amiloride,
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and
furosemide were from Sigma, and BaCl2 was from Mallinckrodt. The
primary solvent for concanamycin, DPC and furosemide was dimethylsulfoxide
(DMSO; Sigma). The final DMSO concentration of 0.1% had no effect on the
Vte (see also Clark et
al., 1999
).
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
Kopf Instruments, Tujunga, CA, USA) was followed by manual elaboration of the
pipette tips (approximately 100 µm in diameter) and by giving the pipette
shaft an L-shaped form. The shaft of the pipette tips was covered with a thin
layer of Sylgard 184 (Dow Corning, Midland, MI, USA).
Preparations and perfusion of anterior stomachs
After the larvae were killed by decapitation, the intestinal system was
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 stomach was left open (semi-open
stomach preparation). The bath (volume 100 µl) was perfused by gravity flow
with oxygenated salines at a rate of 1530 ml h-1. The
perfusion pipette was connected via a set of three-way stopcocks to a
pushpull multi-speed syringe pump (model 120; Stoelting, Wood Dale, IL,
USA), allowing to change between infusion from a syringe with basic mosquito
saline via the pipette and the gut into the bath and withdrawal from
the bath through the open, posterior end of the preparation into a second
syringe. The rate of perfusion was 2060 µl h-1. According
to the physical dimensions of anterior stomach preparations
(Clark et al., 2000), this rate
results in 39 luminal volume exchanges per minute. Semi-open stomach
preparations were only used for further experiments if they showed a marked
increase of Vte with serotonin (see Results; cf. Clark et
al., 1999
,
2000
).
Electrophysiological measurements
The bath and the pipette, reflecting the interior and exterior sides of the
semi-open stomach preparation, were connected via agar bridges (3%
agar in 3 mol l-1 KCl) to calomel electrodes. The
Vte was measured in the lumen with reference to the bath
with the voltmeter of a voltage clamp (VCC 600; Physiological Instruments, San
Diego, CA, USA) and continuously recorded on a chart recorder (model 500;
Linear Instruments, Reno, NV, USA).
Electrophysiology of a semi-open tubular epithelium
The theoretical concept for the voltage generated by active and
electrogenic transport across an epithelium bathed on both sides with
identical salines is based on an equivalent electrical circuit where a
circular current is generated by a transcellular electromotive force
(Ec) and flows via a transcellular conductance
(Gc) and a paracellular conductance
(Gp). The transepithelial electrical potential difference
(PDte) generated by the epithelium is then determined by:
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All epithelial preparations show a certain degree of damage due to
preparation and mounting. In preparations of flat epithelia in Ussing-type
chambers, this is known as edge damage. In the semi-open preparation of a
tubular epithelium, the open end of the tube constitutes a leak conductance
(Gl), parallel to the trans- and paracellular pathways.
Thus, the actual measured Vte is then determined by:
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Statistics
All data are presented as means ± S.E.M. In Figs
2,
3,
4,
5,
6, the Vte
time courses were normalized to percentage of the control. In these cases, the
absolute control values of the shown experiments are indicated in the legends.
Differences between groups were tested with the paired Student's
t-test. In those cases where controls were followed by two
experimental results (e.g. Vte after application of a drug
to the bath and Vte after a subsequent change to
withdrawal mode), one-way analysis of variance (ANOVA) with Tukey's
post-hoc test was performed. Significance was assumed at
P<0.05.
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Results |
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The open end of the stomach preparation permitted changing the flow direction in the perfusion pipette from infusion to withdrawal mode. Changes of the flow direction were often accompanied by fast voltage transients probably due to transient changes of hydrostatic pressure (see Figs 3, 6).Sometimes, Vte was slightly different between infusion and withdrawal modes (see Fig. 6). In these cases, the results were always read after returning to infusion mode. In some pilot experiments, it was recognized that a change to withdrawal mode at high perfusion rate (60 µl h-1) resulted in a dramatic increase of Vte or in its irreversible breakdown. These obvious artifacts are probably due to closure of the pipette or to damage of the tissue and could be avoided by a short perfusion stop before a direction change and by using a lower perfusion rate (20 µl h-1).
When the perfusion pipette was inserted into the anterior end of the
anterior stomach of Aedes aegypti, a high, lumen-negative
Vte of sometimes over 100 mV was detected. This
high initial voltage rapidly decreased, as previously described in Clark et
al. (1999,
2000
). After some minutes and
after fixing the stomach preparation on the pipette, Vte
stabilized at 9±1 mV (range 1 mV to 25 mV;
N=33). Addition of serotonin (0.2 µmol l-1) to the bath
perfusion saline resulted in a rapid increase of the voltage, which stabilized
after 1530 min at a significantly (P<0.05) more negative
value of 27±3 mV (range 9 mV to 65 mV;
N=33; see Fig. 1).
When dinitrophenol (DNP; 2.5 mmol l-1), a well-known uncoupling agent of transmembrane H+ gradients, was added to the bath, Vte was rapidly abolished (from 16±2 mV to 1±0.3 mV; N=6, P<0.05). Interestingly, the effect of DNP on Vte was almost completely reversible if the washout was rapidly initiated (see Fig. 2). However, prolonged bath perfusion with DNP caused irreversible breakdown of Vte.
When Cl--free saline was used on both sides of the epithelium (withdrawal mode) Vte was almost abolished (from 12±2 mV to 0.3±0.4 mV; N=6, P<0.05). In five of these experiments, Cl- was substituted in the bath in a first step and then we changed to withdrawal mode to establish symmetrically Cl--free solutions. In these experiments, Cl- substitution in the bath resulted in a first significant decrease of Vte by 58±9%, from 13±3 mV to 6±2 mV (P<0.05), and the subsequent change to luminally Cl--free solution almost abolished Vte (to 0±0.5 mV; P<0.05). The same experimental protocol as described above was also used with Na+-free saline. A change to Na+-free saline in the bath was followed by a major decrease of the luminally negative Vte to 43±12% of the control, from 27±9 mV to 8±1 mV (N=5, P<0.05). After establishing Na+-free salines on both sides of the epithelium, a mean Vte of 5±1 mV (or 29±6% of the control; N=5) was measured, which is not statistically different from the voltage after Na+ substitution in the bath only (P>0.05). Whereas Cl- substitution consistently abolished Vte (inhibition by 89100% of the control), the effect of Na+ substitution showed a relatively large variation, inhibiting 5092% of the control value. Examples of the effects of ion substitution experiments are demonstrated in time courses of Vte in Fig. 3.
Concanamycin A (10 µmol l-1), a specific inhibitor of
V-ATPases (Dröse and Altendorf,
1997), reduced Vte by 78±6%, from
23±6 mV to 6±3 mV (N=7,
P<0.05), when the drug was applied to the bathing medium. The
effect of the drug, varying between 46% and 94% inhibition, was not reversible
(see Fig. 4). When ouabain (2.5
mmol l-1), a specific inhibitor of the
Na+/K+-ATPase (Skou,
1965
), was added to the bathing medium, a minor but significant
reduction of Vte by 15±2%, from 33±10
mV to 28±8 mV (N=8, P<0.05), was observed.
The inhibition by ouabain varied between 10% and 25% and was reversible (see
Fig. 5). A time course of
Vte showing the subsequent effects of the two ATPase
inhibitors is shown in Fig. 4.
In this experiment, ouabain and concanamycin together almost abolish
Vte.
Acetazolamide, an inhibitor of carbonic anhydrases
(Maren, 1967), caused variable
responses when added to the bathing medium (1 mmol l-1). In five of
the eight experiments, the drug reduced Vte to a variable
extent (between 7% and 91% of the control value). In one case it had no effect
at all, and in two other cases it even increased Vte to
more negative values. On average, Vte before and after
addition of acetazolamide amounted to 28±6 mV and
21±6 mV (N=8), respectively. These values are
statistically not different (P>0.05).
In the next series of experiments, we used the non-specific inhibitors of
anion transporters DIDS and DPC (cf.
Culliford et al., 2003;
Reddy and Quinton, 2002
). When
added to the bathing solution, DIDS (0.1 mmol l-1) effected a
significant (P<0.05) decrease of Vte to
78±3% of the control value (from 29±10 mV to
22±7 mV; N=5). Changing to withdrawal mode, and thus
exposing the luminal side of the epithelium to DIDS, also resulted in a
further significant (P<0.05) decrease of Vte
to 35±4% of the control (10±4 mV; N=5). The
Vte reduction induced by bilateral DIDS was only partially
reversible. A representative time course of the effect of DIDS on
Vte is shown in Fig.
6.
DPC (0.5 mmol l-1) resulted in a decrease of Vte to 49±8% of the control (from 18±3 mV to 8±1 mV; N=11, P<0.05) when the drug was added to the bathing medium. The effects of the drug were, however, very variable from preparation to preparation. DPC initially caused a small, transient hyperpolarization (to 119±5% of the control value; N=8) of Vte in eight of the 11 experiments. The degree of the subsequent inhibition with respect to the control showed a very large scatter (080%). In five experiments, we observed the effects of DPC in the bath and after changing to withdrawal mode. In these cases, a significant (P<0.05) Vte decrease to 52±12% of the control (from 24±4 mV to 11±2 mV) after addition of DPC to the bath was followed by a second Vte reduction (P<0.05) to 23±4% of the control (6±1 mV). The inhibitory effect of DPC was only partially reversible. An example of these experiments is shown in Fig. 6.
Barium (BaCl2; 5 mmol l-1), a well-known blocker of
K+ channels (Van Driessche and
Zeiske, 1985), significantly reduced Vte by
26±5%, from 32±10 mV to 24±7 mV
(N=6, P<0.05), when applied to the bathing medium. The
effect of BaCl2 was reversible. An example of the influence of
Ba2+ ions is shown in a Vte time course
together with an example of the effect of ouabain (see
Fig. 5). A similar degree of
Vte inhibition as with BaCl2 was observed when
furosemide (0.1 mmol l-1), an inhibitor of
Na+/K+/2Cl- and KCl symports as well as
Cl- channels (cf. Culliford et
al., 2003
), was added to the bath perfusion saline. The drug
caused a significant (P<0.05) Vte decrease by
13±3%, from 23±4 mV to 20±5 mV
(N=4).
In 10 experiments, amiloride (0.2 mmol l-1), an inhibitor of
epithelial Na+ channels and various Na+-dependent ion
exchangers (for references, see Garty and
Benos, 1988) as well as of the putative insect
K+/2H+ exchanger
(Wieczorek et al., 1991
), was
applied to the bathing solution of the anterior stomach of Aedes
aegypti larvae. The drug significantly (P<0.05) reduced
Vte by 35±6%, from 27±6 mV to
18±5 mV (N=10). With 1264% of inhibition, the
effect of amiloride showed, however, a large variation. In five of the
experiments, we changed to withdrawal mode in the presence of amiloride. After
a first significant (P<0.05) Vte decrease by
40±10%, from 19±1 mV to 11±1 mV, with
amiloride present in the bath only, the voltage stabilized at
10±2 mV in withdrawal mode with the drug on both sides of the
epithelium. The voltages in the presence of amiloride in the bath and on both
sides of the epithelium are statistically not different (P>0.05).
An example of the effects of amiloride is shown in a Vte
time course in Fig. 5.
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Discussion |
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Changes of the saline's conductance may induce Vte changes of a semi-open preparation without affecting the epithelium but by changing the leak conductance (Gl; cf. Materials and methods). In the present study, the conductance of the saline was certainly decreased when Na+- or Cl--free salines were used. However, in both cases, a decrease of Vte was observed that is opposite to an effect of a reduced Gl alone. Thus, the true effect on the PDte could have been even larger than the Vte change actually observed.
The advantages of the semi-open stomach preparation are evident. The preparation itself is much easier to accomplish. Of particular importance for a very small tissue is that the technique preserves tissue by avoiding the necessity of ligating the stomach segment on a second pipette. Moreover, the open end allows manipulations on the luminal side of the epithelium using withdrawal mode, avoiding the time delay in solution changes imposed by the dead space of the perfusion system in the closed preparation.
The Vte of the anterior stomach of larval Aedes aegypti
The Vte generated by the isolated, anterior stomach of
larval Aedes aegypti was measured with identical salines on both
sides and it was irreversibly inhibited with dinitrophenol, an uncoupling
agent that reduces mitochondrial ATP generation
(Wu and Beyenbach, 2003; see
Results; Fig. 2). Thus,
Vte reflects active transepithelial ion movement.
Interesting, however, is the reversibility of the effect of dinitrophenol when
it was applied only for short times. Because of the capability of
dinitrophenol to decrease transmembrane H+ gradients, this
observation could be interpreted as a short-circuit of a proton gradient
across the basolateral membrane. Thus, this result suggests the presence of a
basolateral proton pump and its importance for the generation of
Vte (see below).
The larval caeca and stomach receive direct serotonergic input from two
large neurons located in the ingluvial ganglion (S.B.M. and D.F.M.,
unpublished observations). All results of the present study were obtained
after stimulation with serotonin (see Results;
Fig. 1). According to Clark et
al. (2000), two populations of
cells were encountered in the anterior stomach: (1) a cell population with a
stable basolateral membrane potential (Vb) that responds
to serotonin with a hyperpolarization of the basolateral and apical membranes
and (2) a population of decaying cells where Vb almost
completely depolarizes with time and does not recover in the presence of
serotonin. Thus, these decaying cells do not contribute to the generation of
Vte of the isolated preparation and it is evident that the
results of the present study are exclusively related to the
serotonin-stimulated cell population.
Which ions are transported?
The Vte polarity indicates transepithelial absorption
of cations and/or secretion of anions. Na+ absorption can be ruled
out because hemolymph-side Na+ substitution affected
Vte whereas luminal substitution had no further effect
(see Results; Fig. 3). Thus,
Vte may reflect Na+-dependent anion secretion.
Substitution of internal Cl- reduced Vte.
However, this decrease could just reflect passive, paracellular diffusion of
Cl- from the lumen to the hemolymph side of the tissue due to the
concentration gradient present under these conditions. Abolition of
Vte was only observed when Cl- was also
substituted on the luminal side (see Results;
Fig. 3). Thus,
Vte seems not to reflect Na+-dependent
Cl- secretion.
The observed results are, however, consistent with the assumption that
Vte reflects HCO3-
secretion/H+ absorption, as has been observed with the anterior
stomach region of intact larvae and semi-intact preparations, respectively
(Zhuang et al., 1999; Boudko
et al.,
2001a
,b
).
Like the alkalization of the anterior stomach lumen and also like the
acidification of the hemolymph-facing surface of the anterior stomach,
Vte of the isolated tissue was inhibited with a V-ATPase
blocker and with non-specific inhibitors of anion transporters (see Results;
Figs 4,
6;
Zhuang et al., 1999
; Boudko et
al.,
2001a
,b
).
Luminal alkalization and contraluminal acidification of living larvae or
semi-intact preparations were observed to depend on a functioning carbonic
anhydrase (Boudko et al.,
2001a,b
;
del Pilar Corena et al.,
2002
), which may serve to rapidly supply H+ and
HCO3- to the respective transporters. Del Pilar Corena
et al. (2002
) localized one
isoform of the carbonic anhydrase in the caeca and in the posterior stomach of
Aedes aegypti but not in the anterior stomach. This result does not
exclude the possibility that other isoforms may be present in the anterior
stomach, and the above-mentioned results obtained under in vivo
conditions clearly indicate the participation of carbonic anhydrase in stomach
alkalization. However, in our experiments, even a high concentration of
acetazolamide had very inconsistent effects on Vte (see
Results). Could it be that the role of the carbonic anhydrase in alkalization
of the anterior stomach is restricted to the very beginning of the anterior
stomach? This could explain our findings, because this part of the stomach was
used for fixing the preparation on the pipette and might have been exposed
only in part of our measurements. Another possibility to explain our results
with respect to acetazolamide could be related to the in vitro
conditions used in the present study. The role of the carbonic anhydrase in
alkalization of the anterior stomach of mosquito larvae and in the generation
of Vte certainly needs to be addressed in further
more-detailed studies.
Altogether, however, it seems obvious to assume that the transepithelial voltage of the isolated anterior stomach reflects HCO3- secretion/H+ absorption involved in the alkalization of the stomach lumen.
Further details of the transport mechanisms
Boudko et al. (2001a)
outlined a model of HCO3- secretion/H+
absorption where a basolateral V-ATPase mediates transbasal H+
absorption and generates a driving force for apical anion exchangers,
resulting in transapical HCO3- secretion. Carbonic
anhydrase rapidly supplies these transporters with their substrates. Since
hemolymph-side DIDS had an effect on Cl- efflux from the cells of
the semi-intact preparation, the authors concluded that Cl-
channels might be present in the basolateral membrane. Our results are
consistent with this basic outline: Vte depends on the
presence of Cl-, it is inhibited by hemolymph-side concanamycin,
DIDS and DPC and by luminal DIDS and DPC (see Results; Figs
3,
4,
6). However, the findings of
the present study add some important details, outlined in
Fig. 7 and discussed below.
|
It is obvious that special care has to be taken when results obtained with
non-specific inhibitors of anion transporters such as DIDS, DPC and furosemide
are interpreted (cf. Culliford et al.,
2003; Reddy and Quinton,
2002
). Apart from the low specificity, these inhibitors show a
significant lipid solubility and it can be argued that they not only act on
the side of application but can also reach the more distant membrane. However,
DIDS, DPC, furosemide and also amiloride always caused an almost immediate
Vte decrease after their application to the bath,
indicating that the site of action was at the basolateral membrane. After
reaching a stabilized, reduced Vte in the presence of
internal DIDS or DPC, these drugs caused additional Vte
decreases when applied also to the luminal side of the epithelium, supporting
the above view.
Despite the above limitations, the results of the present study are
nevertheless useful to elaborate a hypothetical transport model for active and
electrogenic HCO3- secretion/H+ absorption
that needs, of course, further confirmation by future studies. The results
obtained with Na+-free saline (see
Fig. 3) indicate that a part of
the transport processes depends on the presence of hemolymph-side
Na+ whereas another part does not. The Na+-independent
part could be explained by the mechanism proposed by Boudko et al.
(2001a; see above). Instead of
considering a so far unknown electrogenic
Cl-/HCO3--exchanger, we put forward the
alternative idea of a parallel arrangement of an electroneutral anion
exchanger and an anion channel (cf. Fig.
7A). This allows transapical HCO3- secretion
via anion exchange that could be driven by the high cellular
HCO3- concentration (cf.
Boudko et al., 2001a
) and
explains the electrogenicity of the process without assuming a new type of
transporter. At the almost equal concentrations of Cl- and
HCO3- in the tissue of anterior stomachs, even a
considerable amount of HCO3- might be driven through the
anion channels into the lumen (cf.
Cuthbert, 2001
). For the
Na+-dependent part of Vte, we favor the idea of
an Na+-dependent transapical HCO3- secretion
via Na+/23HCO3- symporters
(cf. Fig. 7B). Thus,
hemolymph-side Na+ substitution would affect this part of the
transport by a reduction of cellular Na+ supplied by
Na+-dependent transporters in the basolateral membrane. One of the
latter transporters is most likely an Na+/H+ exchanger,
as the Vte decrease with internal amiloride indicates (see
Results). Apart from supplying cellular Na+, this transporter could
support the V-ATPases to extrude H+ across the basolateral
membrane. The small effects observed with furosemide (see
Fig. 5) may suggest that a
basolateral Na+/K+/2Cl- symporter also
contributes to the Na+ entry via the basolateral membrane.
However, the effect of this drug could just reflect an effect on basolateral
Cl- channels (cf. Culliford et
al., 2003
). It might be argued why the Na+-dependent
part of electrogenic HCO3- secretion is not maintained
in Cl--free salines. However, in the presence of anion channels the
cells would depolarize in Cl--free salines and this would eliminate
the driving force for the apical
Na+/23HCO3- symport.
The small but fast and reversible Vte decreases observed with ouabain and Ba2+ ions (see Results; Figs 4, 5) indicate the presence of basolateral Na+/K+-ATPase and K+ channels and suggest that these transporters support the V-ATPase to generate and maintain cellular negativity. A point to argue may be the relatively small effect of ouabain on a process showing such a marked Na+ dependence. However, as long as the V-ATPase drives just as much Na+ out of the cells (via the apical Na+/23HCO3- symport) as enters (via the basolateral Na+-dependent transporters) the overall process is stable even without a functioning Na+/K+-ATPase.
The effects of certain manipulations showed a particularly high variability: whereas Cl- substitution always almost abolished Vte (inhibition of 89100%), the inhibition by Na+ substitution (5092%), concanamycin (4694%), ouabain (1025%), hemolymph-side DPC (082%) and amiloride (1264%) showed much higher variations. These observations suggest that the relative proportions of Na+-dependent and Na+-independent HCO3- secretion/H+ absorption may markedly vary between individual stomach preparations.
With mosquito saline on both sides of the epithelium, the luminally
negative Vte could supply the driving force for
paracellular movement of Na+ from the hemolymph to the stomach
lumen and/or movement of Cl- in the opposite direction. Altogether,
the serotonin-stimulated cell population could then mediate transapical
HCO3- secretion, transbasal H+ absorption and
transepithelial Na+ secretion and Cl- absorption. These
processes could contribute to the luminal alkalization under in vivo
conditions. However, a 0.1 mol l-1 NaHCO3 solution has
only a pH of about 8.3, thus it is obvious that secretion of NaHCO3
alone cannot explain a luminal pH of 11. It has been repeatedly suggested
(Boudko et al.,
2001a,b
)
that an apical, amiloride-blockable K+/2H+ exchanger
might be involved in stomach alkalization of mosquito larvae. In the present
study, luminal amiloride did not affect Vte (see Results;
Fig. 5). Nevertheless, it could
well be that additional, transepithelial H+ absorption by apical
K+/2H+ antiports and basolateral V-ATPases is a feature
of the decaying cell population. Such a process could then transform the
secretion of HCO3- into a secretion of
CO32, explaining the strong alkalization observed
under in vivo conditions. It is evident that the above hypotheses
need to be investigated in further more-detailed studies in the future.
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References |
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