K+ transport in Malpighian tubules of Tenebrio molitor L.: a study of electrochemical gradients and basal K+ uptake mechanisms
1 Department of Zoology and Entomology, University of Pretoria, Pretoria
0002, South Africa
2 Laboratory of Physiology, Biomed CMK, Limburgs Universitair Centrum, B3590
Diepenbeek, Belgium
* Author for correspondence (e-mail: emmy.vankerkhove{at}luc.ac.be)
Accepted 10 December 2002
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Summary |
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Key words: K+ transport, K+ uptake, Malpighian tubules, Tenebrio molitor, K+ channel, Na+/K+/2Cl- cotransporter, Na+/K+-ATPase, basolateral membrane, apical membrane, fluid secretion rate, membrane potential, transepithelial potential
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Introduction |
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Basolateral K+ transport systems must permit adequate
K+ uptake to maintain fluid secretion. The existence of
Ba2+-sensitive K+ channels has been confirmed in tubules
of Onymacris (Nicolson and
Isaacson, 1987), Formica
(Weltens et al., 1992
),
Aedes (Masia et al.,
2000
) and Locusta
(Hyde et al., 2001
). Uptake
mechanisms for K+ and/or Na+ at the haemolymph side may
differ according to the species. In tubules of the forest ant Formica
polyctena, alternative routes for basal K+ entry appear to be
implicated over different ranges of bathing saline [K+]. In the
presence of high [K+], entry occurs via high-conductance,
Ba2+-sensitive channels. At lower [K+],
K+/Cl- and/or
Na+/K+/2Cl- cotransporters become functional
(Leyssens et al., 1994
;
Van Kerkhove, 1994
). Although
there has been some controversy about the presence of a basolateral
Na+/K+-ATPase in insect epithelia, this
ouabain-sensitive electrogenic pump has been implicated in facilitating
K+ transport in Locusta
(Anstee et al., 1986
) and
Na+ transport in Rhodnius
(Maddrell and Overton, 1988
)
tubule cells. However, fluid secretion rates of Drosophila
melanogaster (Dow et al.,
1994
) and Formica
(Leyssens et al., 1994
)
tubules remain unaffected by ouabain and thus, if present, the
Na+/K+-ATPase does not appear to play a role in fluid
secretion.
The present study focuses on possible K+ uptake pathways across the basolateral membrane of Tenebrio tubule cells and demonstrates the impact of the apical membrane on passive K+ uptake via the Ba2+-sensitive K+ channels. Fluid secretion rates and potentials across basolateral and apical membranes have been measured at different bath [K+] and in the presence and absence of various blockers. The results obtained indicate the direction of the electrochemical gradient for K+, the influence of the apical membrane potential on this gradient and the relative importance of the alternative K+ uptake routes. This study provides a basis for future studies in which (1) the nature of the K+ channels involved in basolateral K+ uptake is further investigated and (2) the various K+ uptake mechanisms are implicated as sites for regulation by endogenous factors.
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Materials and methods |
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Artificial salines
The composition of the bathing solutions is summarised in
Table 1. Solutions were freshly
prepared each week, filtered through 0.22 µm Millipore filters and kept at
2°C until use. The pH was measured daily before use. In experiments
containing Ba2+, NaH2PO4 was omitted from all
salines to maintain constant osmolality and prevent precipitation. Control
experiments in which NaH2PO4 was omitted showed no
change in secretion rate or electrical profile. In low Na+
experiments, salines contained a maximum of 6 mmol l-1
Na+.
|
The following pharmacological substances were tested on Malpighian tubule preparations: barium chloride (Sigma, Bornem, Belgium), ouabain (Fluka Buchs, Switzerland) and bumetanide (Sigma).
Fluid secretion experiments
Malpighian tubules from larval Tenebrio molitor were isolated as
described by Wiehart et al.
(2002). These were the free
segments of the tubules, severed near the midgut and the rectal complex.
Droplets of physiological saline (50 µl) were placed in a Petri dish coated
with Sylgard (10:1 base to curing agent) and covered with water-saturated
liquid paraffin. Two tubules were placed into each saline drop. The two ends
of each tubule were pulled out of the bathing fluid and wrapped around Minuten
pins, where they continued to secrete, and the urine was collected as discrete
droplets in the liquid paraffin. Secreted drops were removed with a fine glass
pipette and their diameters measured with a calibrated eyepiece graticule. The
volume, and therefore the rate of secretion, was determined assuming that the
droplets were spherical. The tubules were allowed to equilibrate for 20 min
before three control readings were made at 15 min intervals. The bathing
solution was then replaced with the experimental solution containing a high or
low K+ concentration and/or the test substances. For the purpose of
this study, the effect of the various K+ concentrations and/or the
test substances was tested on unstimulated tubules. Measurements were then
taken over a 45-60 min period. Rates of secretion were expressed as a
percentage of the third control rate reading.
Measurement of basolateral (Vbl) and
transepithelial (Vte) potentials
Immediately after dissection, a portion of a Malpighian tubule (3-5 mm) was
suspended in a Ringer bath between two holding pipettes. The peritubular bath
(volume 300 µl) was perfused with Ringer solution at a rate of 1 ml
min-1. Intracellular (Vbl) and transepithelial
(Vte) potentials were measured with 3 mol l-1
KCl-filled microelectrodes (borosilicate filament glass, Harvard; o.d. 1.2 mm,
i.d. 0.69 mm; tip diameter <0.5 µm; resistance 20-40 M) connected
to a Micro Probe System M-707 electrometer (World Precision Instruments, New
Haven, USA) via Ag/AgCl wire. The reference electrode was a coarse,
low-resistance glass electrode (1 M
) filled with 3 mol l-1
KCl/agar (2%) connected to earth via Ag/AgCl wire. Cell impalement
was accepted if a sudden drop in potential occurred, if the potential was
stable for at least a few minutes and if the electrode potential differed by
not more than 3 mV from the baseline after withdrawal. Transepithelial
potential was measured by advancing the microelectrode through the cell layer
into the lumen of the tubule. The apical membrane potential
(Vap) was calculated as the difference between the
measured transepithelial and basolateral potentials.
Statistics
Results are presented as means ± S.E.M., with the number of tubules
(N) or number of measurements (n) in parentheses. The
statistical significance of differences in fluid secretion or electrode
potentials was evaluated by paired or unpaired Student's t-tests
(two-tailed). A value of P<0.05 was accepted as statistically
significant.
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Results |
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The effect of changing external [K+] on fluid
secretion
Fig. 1 summarises the change
in fluid secretion rates observed after changing [K+] and
[Na+] in the bath. An increase in [K+] from 50 mmol
l-1 (control) to 140 mmol l-1 (Na+ replaced
by K+) increased the fluid secretion rate from 5.38±0.58 nl
min-1 to 7.64±0.60 nl min-1 (P<0.02,
N=8). Replacing the high [K+] Ringer with control Ringer
reversed this effect. With a 10-fold drop in K+ concentration
(K+ replaced by Na+), secretion rates dropped from
6.38±0.95 nl min-1 to 1.48±0.52 nl min-1
(P=0.01, N=8). Fluid secretion rates did not recover after
the low K+ medium was replaced with control Ringer, and some
tubules stopped secreting altogether, illustrating the importance of
K+ to the normal secretion of Tenebrio Malpighian
tubules.
|
The effect of [K+] on membrane potentials
Fig. 2A shows the response
of Vbl to varying K+ concentrations. The
microelectrode was then advanced into the lumen and the effect of the same
series of K+ concentrations was measured on
Vte. The basolateral membrane was clearly sensitive to the
bath [K+]. It depolarized from -24 mV in control Ringer (50 mmol
l-1 K+) to -13 mV in the presence of a high
K+ concentration (140 mmol l-1) and hyperpolarized to
approximately -50 mV in 5 mmol l-1 K+. The response of
Vbl to a change in bath [K+] was immediate and
is consistent with the presence of a significant K+ conductance in
the basolateral membrane of the tubule cells, with the change in
Vbl being 28 mV decade-1
(Fig. 2B).
|
Transepithelially, there was an increase in potential as the K+ concentration increased from 5 mmol l-1 to 140 mmol l-1 K+ (Fig. 2A). Fig. 3 summarises the results. From the electrical potential profile presented in this figure, it is clear that there is a correlation between Vap and Vbl. The overall result of increasing bath [K+] from 5 mmol l-1 to 140 mmol l-1 was depolarisation of both Vbl and Vap, the change in Vap being 60-77% of the change observed in Vbl.
|
The effect of barium
The presence of K+ channels in the basolateral membrane of
Tenebrio tubules and their relative importance in fluid secretion
were investigated by the addition of 6 mmol l-1 Ba2+, a
known K+ channel blocker, to the control bathing solution (50 mmol
l-1 K+). Fluid secretion rates decreased significantly
from 4.17± 0.88 nl min-1 to 0.76±0.24 nl
min-1 within 45 min in the presence of Ba2+
(Fig. 4A; N=8,
P<0.004). This drop in secretion rate was immediate, but the
return of secretion rates to control levels after washout of Ba2+
took approximately 45 min.
|
Fig. 4B shows a typical response of Vbl to Ba2+. Adding 6 mmol l-1 Ba2+ to control bath Ringer caused a hyperpolarization of the basolateral membrane from -24±0.4 mV to -52±1.9 mV and a slight decrease of Vte (not shown) from 24±1.4 mV to 21±1.9 mV (n=47 and 34, respectively). The hyperpolarization of Vbl was sudden in some cells and more sluggish in others, the mean time being 4.6±0.4 min (n=47). The effect of Ba2+ on the potential profile was completely reversible after washout for 5-8 min, with Vbl and Vte not significantly different from the initial control potentials. The addition of 6 mmol l-1 Ba2+ to 140 mmol l-1 K+ Ringer hyperpolarized Vbl significantly from -9±1.2 mV to -40±3.6 mV over a period of 11.6±2.2 min and caused a small but non-significant decrease in Vte from 30±7 mV to 27±7.8 mV (n=7 and 5, respectively; results not shown).
Two different responses were seen when 6 mmol l-1 Ba2+ was added to a low bath [K+] (5 mmol l-1). In eight of the 21 impalements, the Vbl hyperpolarized significantly from a mean value of -52±2 mV to -62±3 mV (P<0.01), while in the remaining 13 impalements a significant depolarisation from -60±2 mV to -46±3 mV was seen (P<0.001). Fig. 5 illustrates both these responses. Possible reasons for this will be discussed later. The changes in the electrical profiles of Malpighian tubule cells bathed in the different K+ concentrations in the presence of Ba2+ are summarised in Fig. 6.
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The effect of rubidium on the basolateral membrane potential
To further investigate the nature of the potassium conductance of the
basolateral membrane, K+ ions were replaced by rubidium, a commonly
used `permeant' substitute for potassium. However, replacing 40 mmol
l-1 K+ of the 50 mmol l-1 K+
control Ringer solution by 40 mmol l-1 rubidium caused a
hyperpolarization of the Vbl of 35 mV (results not shown).
The addition of 6 mmol l-1 rubidium to control Ringer had no effect
on Vbl (n=4). Clearly, rubidium is not able to
substitute for potassium in the Malpighian tubules of Tenebrio.
The effect of bumetanide on Vbl
and Vte
The possibility of K+ entering the cell through the
Na+/K+/2Cl- cotransporter was investigated by
means of the loop diuretic bumetanide. Usually, a concentration of 10 µmol
l-1 bumetanide is sufficient to block the
Na+/K+/2Cl- cotransporter, and higher
concentrations are needed to block the K+/Cl-
cotransporter (Palfrey and O'Donnell,
1992). Bumetanide (10 µmol l-1) significantly
decreased fluid secretion rates of unstimulated tubules from 2.08±0.22
nl min-1 to 0.55±0.09 nl min-1
(P<0.01, n=14). This inhibitory effect was reversible and
after a washout period of 45 min, fluid secretion rates had recovered to
1.56±0.16 nl min-1 (Fig.
7). The effect of bumetanide on Vbl and
Vte was investigated in the presence and absence of
Ba2+. None of the bumetanide treatments showed any significant
effect on Vbl or Vte (n=5 and
7, respectively).
|
The effect of ouabain on Vbl and
fluid secretion
The contribution of the basolateral Na+/K+-ATPase to
K+ uptake was investigated by blocking this ATP-dependent pump with
1 mmol l-1 ouabain. K+ ions and ouabain compete for the
same binding sites (Baker and Willis,
1970); therefore, a high bath [K+] would decrease the
effect of ouabain. Because, as previously shown, a low [K+] (5 mmol
l-1) irreversibly inhibits fluid secretion rates, the secretion
assay was carried out in control Ringer (50 mmol l-1
K+). The secretion rates of unstimulated tubules decreased from
5.6±0.93 nl min-1 to 3.0±0.6 nl min-1
after 15 min in the presence of 1 mmol l-1 ouabain
(Fig. 8). A further decrease to
2.26±0.36 nl min-1 was observed after an additional 45 min
(n=8). The inhibitory effect of ouabain on tubule secretion rates was
irreversible. Basolateral and transepitelial membrane potentials showed no
visible changes 10 min after the addition of 1 mmol l-1 ouabain
(n=5).
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Discussion |
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Sodium-dependent fluid secretion is primarily found in blood-sucking
insects (e.g. Rhodnius and Aedes). These insects take huge,
but infrequent, blood meals and need to rid themselves as soon as possible of
the high NaCl and water load, which render them sluggish and easy prey.
However, when not stimulated, tubules of Rhodnius secrete a
K+-rich fluid containing only low levels of sodium ions
(Maddrell and O'Donnell,
1992).
K+ dependence of
Vbl
The potential profile in Malpighian tubules of Tenebrio under
control conditions is Vbl -24 mV, with reference to the
bath, and Vte 24 mV, lumen positive. The result is a mean
Vap of 48 mV, cell interior negative. This potential
profile closely resembles that described for tubules of Onymacris
plana, another tenebrionid beetle
(Nicolson and Isaacson,
1987).
Vbl was very responsive to the K+ concentration in the bath. Decreasing bath [K+] caused an immediate hyperpolarization, which was followed by an instant depolarization when the K+ concentration was again increased. Although Vbl recovered, fluid secretion of tubules previously treated with a low [K+] did not (Fig. 1). A possible reason for this could be cell shrinking, but there is no microscopical evidence for this. It must be remembered that Vbl only measures the electrical effects of a low [K+] at the level of the basolateral membrane, but for fluid secretion in general a low [K+] could affect numerous transport mechanisms. Lack of availability of K+ ions could slow down the putative apical V-ATPase, which would in turn affect the performance of the putative apical cation/nH+ antiporter. During fluid secretion, fluid is secreted over the entire tubule length and it would therefore take a longer period of time for the mechanisms to recover and produce enough fluid within the tubule lumen to actually be measurable.
Basal K+ permeability is high in Malpighian tubule cells of most
insect species studied so far (for a review, see
Nicolson, 1993) and only the
tubule cells of the yellow fever mosquito Aedes aegypti show a
detectable Na+ permeability, which is further increased in the
presence of cAMP (Sawyer and Beyenbach,
1985
).
When Vbl was plotted as a function of log
[K+], a linear function was found with a slope of 28 mV
decade-1 (Fig. 2B)
and not 58 mV decade-1, as expected if K+ alone
determined the membrane potential. Either other ions play a role and/or the
intracellular K+ concentration drops in low [K+]. If
other ions (e.g. Na+) had some importance, the dependence of
Vbl to log [K+] would be expected to level off
at lower [K+]. This is not the case. Thus, a drop in intracellular
[K+] seems more likely. Leyssens et al.
(1993) have shown that the
intracellular [K+] in tubule cells of Formica can drop to
75% of the initial value when the [K+] in the bath is decreased
10-fold. In Tenebrio cells, a changing intracellular [K+]
dependent on the bath K+ concentration thus seems feasible, but
this will have to be confirmed by ion-selective microelectrode measurements.
On the other hand, the intracellular [K+] in Malpighian tubule
cells of Locusta seemed to remain fairly constant and was not
affected by the bath [K+]
(Baldrick et al., 1988
).
Furthermore, changing the bath K+ concentration indicated that
Vap closely followed Vbl (60-77% of
the change observed in Vbl). This has also been reported
for tubules of Formica (Leyssens
et al., 1993). Weltens et al.
(1992
) have measured the
resistance of the apical and basolateral membranes in Formica tubule
cells and found that the resistance ratio of the apical membrane over the
basolateral membrane is high. From the electrochemical model of an epithelium,
it can be understood that the circular current, caused by the different
electromotive forces across the basolateral and apical membranes and across
the paracellular shunt, will contribute to the actual measured basolateral and
apical voltage differences (Weltens et
al., 1992
). The effect will be small in the membrane with the
lowest resistance and high in the membrane with the relative higher
resistance. The basolateral membrane is dominated by a high K+
conductance (low resistance) so that the circular current will have little
impact on the measured potential and Vbl approaches the
equilibrium potential for K+. The apical membrane, on the other
hand, will be influenced appreciably by a change in electromotive force (and
thus of circular current) and will `follow' this change. This mechanism may
also be operative in the tubules of Tenebrio.
The effect of barium on secretion rate and
Vbl
Barium is a frequently used K+ channel blocker and has been
shown to reduce K+ movement across the basolateral membrane in
Malpighian tubule cells of Formica
(Weltens et al., 1992),
Rhodnius (lower tubule; Haley and
O'Donnell, 1997
), Drosophila
(O'Donnell et al., 1996
),
Aedes (Masia et al.,
2000
) and Locusta
(Hyde et al., 2001
). Applying
barium will increase the basolateral resistance and therefore make the effect
of the epithelial circular current more `visible' on this membrane, as
explained in the previous section. The electromotive force of the apical
V-ATPase sends a positive current into the lumen, making the cell interior
more negative. In control [K+], the electrochemical gradient seems
to be cell inward and, in the presence of barium, it becomes more difficult
for K+ to cross the basolateral membrane and to compensate for this
loss of positive ions from the cell. Also, the inward current across this
larger resistance will cause a bigger potential drop across the membrane,
hyperpolarizing it. This was substantiated by the study of Weltens et al.
(1992
) where the authors have
shown that the hyperpolarization of the basolateral membrane in the presence
of Ba2+ was abolished by bafilomycin A1, an inhibitor of
the V-ATPase.
In the present study, Ba2+ reversibly reduced fluid secretion by 83%. In control saline, basolateral and apical membranes responded by a marked hyperpolarization and Vte decreased slightly. This is most probably due to (1) blocking of the K+ channels in the basolateral membrane, (2) the increase in electrical potential difference created across the apical membrane, possibly by a putative proton pump (V-ATPase), and (3) increases in the basolateral resistance and the hyperpolarizing effect of the circular current as explained above.
The hyperpolarization of the basolateral membrane in some cells of Tenebrio tubules was notably slower than in others. This could indicate impeded access of Ba2+ to its site of interaction, possibly along the lateral spaces.
Different K+ channel types are mostly classified according to
their single channel conductance or ligands rather than by their gating
kinetics. The basolateral barium-sensitive K+ channels found in
Tenebrio tubule cells appear to be specific for K+ ions
and are impermeable to rubidium. This became evident when the equimolar
substitution of K+ by rubidium caused a hyperpolarization of the
basolateral membrane potential, similar to the hyperpolarization found in the
presence of a low [K+]. If rubidium were able to substitute for
K+, a slight depolarization of the basolateral membrane potential
would have been expected after the addition of 6 mmol l-1 rubidium
to control Ringer, as this would have been sensed as an increase in total
K+ concentration, but no effect was seen. In comparable studies,
substitution of K+ ions with rubidium caused a 50% decrease in
fluid secretion of Locusta tubules and a hyperpolarization of
Vbl (Hyde et al.,
2001; Pivovarova et al.,
1994
). This is in contrast to K+ channels found in the
midgut of the tobacco hornworm Manduca sexta, where rubidium
substituted for K+ ions to a greater extent
(Schirmanns and Zeiske, 1994
),
and in the Malpighian tubules of the black field cricket Teleogyllus
oceanicus, where a concentration of 8.6 mmol l-1 rubidium
caused a 10% increase in fluid secretion rates with rubidium almost completely
replacing K+ in the secreted fluid
(Marshall and Xu, 1999
). In
Locusta tubule cells, an increase in intracellular rubidium was seen
when K+ was replaced by rubidium, but the rubidium was not
transferred to the lumen via the apical K+/H+
exchanger, indicating that the selectivity to K+ ions does not lie
within the K+ channel but rather the putative apical
K+/H+ exchanger responsible for transporting
K+ to the lumen (Pivovarova et
al., 1994
). In contrast to the K+/H+
exchanger of Teleogyllus tubule cells, this exchanger in
Locusta cells appears to have a much higher affinity for
K+ ions than for rubidium. Whether this is the same for tubule
cells of Tenebrio has yet to be determined.
The effect of barium in low K+ concentrations
In the presence of a low bath [K+] (5 mmol l-1),
Ba2+ either hyperpolarized or depolarized Vbl.
Providing that Vbl follows the Nernst potential for
K+, for a Vbl of -52 mV, K+ would be
at equilibrium if the intracellular [K+] were 40 mmol
l-1. This value is close to that found by Leyssens et al.
(1993) in tubule cells of
Formica. Any intracellular concentration below 40 mmol l-1
K+ would cause K+ to move into the cell. Blocking this
inward movement with barium would result in a hyperpolarization (see
Weltens et al., 1992
). Cells
that show a depolarization of the basolateral membrane in the presence of
barium most probably have an outward electrochemical gradient for
K+, the favourable electrochemical gradient created by the apical
proton pump being too low to draw K+ ions into the cell. The
addition of barium blocks the K+ ions from leaving the cell
via the K+ channels and results in a depolarization of
Vbl. This has also been found in the lower tubules of
Rhodnius (Haley and O'Donnell,
1997
), which are responsible for ion reabsorption, and in the
upper secreting tubule of the same insect
(Ianowski et al., 2002
).
Secretion by the upper Malpighian tubules of Rhodnius is not
inhibited by Ba2+ because it is driven by Na+ rather
than K+ (Ianowski et al.,
2002
). Clearly, at physiological (50 mmol l-1) and
higher bath K+ concentrations, which we have shown to increase
fluid secretion, the hyperpolarization of the basolateral membrane indicates
that the net movement of K+ ions is from the bath into the
cell.
The effect of bumetanide
Basolateral entry of ions via the
Na+/K+/2Cl- cotransporter has been implicated
in Malpighian tubules of other insect species. In the present study, we tested
the effect of bumetanide, which blocks this cotransporter, on fluid secretion
rates of Tenebrio Malpighian tubules and found strong inhibition in
non-stimulated tubules. Bumetanide also inhibits fluid secretion in
cAMP-stimulated tubules of Rhodnius
(O'Donnell and Maddrell, 1984;
Ianowski et al., 2002
) and
Aedes (Hegarty et al.,
1991
), in stimulated and unstimulated tubules of
Drosophila (Linton and O'Donnell,
1999
) and in unstimulated tubules of Locusta
(Baldrick et al., 1988
) and
Formica (Leyssens et al.,
1994
). However, due to the relatively high concentration of
bumetanide required to partially inhibit fluid secretion in
Drosophila, Linton and O'Donnell
(1999
) suggested that
bumetanide inhibited a K+/Cl- cotransporter rather than
an Na+/K+/2Cl- cotransporter. According to
the results of the fluid secretion assay, it seems highly likely that
K+ ions cross the basolateral membrane of Tenebrio tubules
via the Na+/K+/2Cl- cotransporter,
in addition to passage through channels.
Bumetanide affects electroneutral transport mechanisms, and the lack of a
visible effect on membrane potentials supports this characteristic. This
result is in accordance with findings of previous studies on Locusta
(Baldrick et al., 1988) and
Formica (Leyssens et al.,
1994
).
The effect of ouabain
The role of an Na+/K+-ATPase in the Malpighian
tubules of Tenebrio has been substantiated, given that ouabain, a
specific inhibitor of the Na+/K+-ATPase, decreased fluid
secretion irreversibly by 52%. Fluid secretion by unstimulated tubules of
Aedes (Hegarty et al.,
1991) and Locusta
(Anstee and Bowler, 1979
) is
similarly affected in the presence of 1 mmol l-1 ouabain. By
contrast, ouabain stimulates fluid secretion in tubules of Rhodnius
(Maddrell and Overton, 1988
)
and Drosophila (Linton and
O'Donnell, 1999
). Inhibiting the
Na+/K+-ATPase disrupts its active role of transporting
three Na+ ions from the cell interior to the outside and two
K+ ions from the outside into the cell
(De Weer, 1992
), resulting in
an increase in intracellular Na+ concentration
(Maddrell and O'Donnell,
1992
). In stimulated tubules of Rhodnius, when fluid
secretion is predominantly driven by the presence of a high [Na+],
the presence of ouabain increases the secretion rate as well as the
Na+ concentration in the secreted fluid
(Maddrell and Overton, 1988
).
In both Rhodnius and Drosophila, Vbl depolarized
in the presence of ouabain, indicative of an increase in intracellular
Na+ concentration and, possibly, a decrease in intracellular
K+ levels. Considering that the Na+/K+-ATPase
provides a route of K+ entry into the tubule cells, blocking this
pump decreases fluid secretion in insects, where K+ is the main
player in driving fluid secretion.
In this study we have shown that basolateral K+ uptake is an important factor determining fluid secretion rates of Tenebrio Malpighian tubules. K+ ions are transported across the basolateral membrane via barium-sensitive K+ channels and via the electroneutral Na+/K+/2Cl- cotransporter and the Na+/K+-ATPase. The nature of the basolateral K+ channels and the possible regulation of the various K+ uptake mechanisms by endogenous factors are subjects for further investigation.
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Acknowledgments |
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References |
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