Basolateral ion transport mechanisms during fluid secretion by Drosophila Malpighian tubules: Na+ recycling, Na+:K+:2Cl cotransport and Cl conductance
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada, L8S 4K1
* Author for correspondence (e-mail: odonnell{at}mcmaster.ca)
Accepted 28 April 2004
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Summary |
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Key words: Drosophila melanogaster, Malpighian tubule, ion-selective microelectrode, K+ conductance, Cl conductance, electrochemical potential, cation-coupled chloride cotransporter, intracellular K+ activity, intracellular Cl activity
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Introduction |
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The physiology of ion transport by the Malpighian tubules has been
extensively investigated in a number of insect species. Current models propose
that cations are transported through the transcellular pathway, but several
different transport pathways for anions have been described in tubules from
different species. Anion transport may involve paracellular pathways or
transcellular pathways, either through the same cell type as for cations or
through a different cell type (Beyenbach,
2003; Ianowski et al.,
2002
; Linton and O'Donnell,
1999
).
Ion transport in tubules is driven primarily by a vacuolar-type
H+-ATPase, which generates a proton gradient across the apical
membrane. This gradient, in turn, energizes apical amiloride-sensitive
K+/H+ and/or Na+/H+ exchange,
driving net movement of K+ and Na+ from cell to lumen
and, in some species, generating a large positive transepithelial potential
that drives passive transepithelial Cl transport
(Maddrell and O'Donnell,
1992).
The basolateral membrane transport systems involved in fluid secretion by
Malpighian tubules differ among species. Transport of both K+ and
Cl across the basolateral membrane during secretion of
Na+-rich fluid by blood-feeding insects is driven by the
Na+ electrochemical potential. K+, Cl
and Na+ ions are transported across the basolateral membrane
through a Na+-driven
Na+:K+:2Cl cotransporter in
Rhodnius prolixus (Ianowski and
O'Donnell, 2001; Ianowski et
al., 2002
). KCl is subsequently reabsorbed in the lower tubule
(Haley and O'Donnell, 1997
).
In the mosquito, Aedes aegypti, both Na+ channels and
Na+:K+:2Cl cotransport have been
implicated in fluid secretion (Hegarty et
al., 1991
; Williams and
Beyenbach, 1984
). In contrast, Malpighian tubules in species that
are not blood feeders, secrete K+-rich fluids. In the ant
Formica polyctena, several basolateral ion transporters have been
proposed, including K+ channels, a
K+:Cl cotransporter and a
Na+:K+:2Cl cotransporter (Leyssens et
al., 1993b
,
1994
). K+ channels,
a Na+:K+:2Cl cotransporter and the
Na+/K+-ATPase have been implicated in fluid secretion by
Tenebrio molitor (Wiehart et al.,
2003a
,b
).
K+ channels and Na+ channels are the main routes for
cation entry across the basolateral membrane in tubules of the New Zealand
alpine weta Hemideina maori
(Neufeld and Leader,
1998
).
In Malpighian tubules of dipterans both K+ and Na+
are transported against their transepithelial electrochemical gradients across
the principal cells, whereas transepithelial transport of Cl
involves passive movement (Pannabecker et
al., 1993; O'Donnell et al.,
1996
,
1998
). Two models for ion
transport across the basolateral membrane of the principal cells have been
proposed in unstimulated tubules (i.e. in the absence of hormonal or second
messenger stimulation of fluid secretion) of Drosophila melanogaster.
One model suggests that K+ transport across the basolateral
membrane of the principal cells occurs through K+ channels, on the
grounds that the K+ channel blocker Ba2+ blocks fluid
secretion and causes hyperpolarization of the basolateral membrane potential
consistent with blockage of K+ entry. Cl
transport is proposed to occur solely across the stellate cells (Dow et al.,
1994a
,b
;
O'Donnell et al., 1996
).
A more recent model suggests that Cl moves through both
principal and stellate cells. K+ crosses the basolateral membrane
during fluid secretion through the Na+/K+-ATPase and
through a Na+-independent K+:Cl
cotransporter sensitive to [(dihydroindenyl)oxy] alkanoic acid (DIOA) and the
loop diuretic bumetanide. Addition of either DIOA or bumetanide reduces fluid
secretion rate. Furthermore, exposure to high concentrations of bumetanide
alone also reduces K+ secretion, but has no effect on
Na+ secretion (Linton and
O'Donnell, 1999).
A direct test of the thermodynamic feasibility of transepithelial
K+ secretion through basolateral K+ channels or
K+:Cl cotransporters requires measurement of the
electrochemical potentials for both ions across the basolateral membrane.
Intracellular K+ activity must be below equilibrium if
transepithelial K+ secretion involves K+ channels or
K+-driven Cl uptake. These mechanisms require
reduction of K+ activity through the actions of apical ion
transporters because the basolateral ouabain-sensitive
Na+/K+-ATPase will tend to increase intracellular
K+ activity (Linton and
O'Donnell, 1999). Alternatively, K+ might enter through
K+:Cl cotransport driven by a favourable gradient
for Cl entry (Linton and
O'Donnell, 1999
). This gradient could be produced by the large
lumen-positive apical membrane potential favouring cell to lumen movement of
Cl through channels. A sufficiently large apical membrane
potential and a significant apical Cl permeability could
thereby reduce intracellular Cl levels to the point where
there is a favourable electrochemical potential for Cl entry
across the basolateral membrane (Linton
and O'Donnell, 1999
).
This study examines the possible contributions of K+ channels and cation:Cl cotransporters to ion transport during fluid secretion. Intracellular K+ and Cl activity and basolateral membrane potential were measured simultaneously using double-barrelled ion-selective microelectrodes. These data permit calculation of the corresponding electrochemical potentials across the basolateral membrane of the principal cells. We also studied the effects of ion substitution and ion transport inhibitors on fluid secretion rates, net transepithelial ion flux, basolateral membrane potential and intracellular ion activity. The results have been incorporated in a revised model of the mechanisms of basolateral ion transport during fluid secretion by the principal cells of Malpighian tubules of D. melanogaster.
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Materials and methods |
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Measurement of intracellular ion activity
Intracellular ion activity and basolateral membrane potential were measured
simultaneously in principal cells using ion-selective double-barrelled
microelectrodes (ISMEs), which were fabricated as described previously
(Ianowski et al., 2002).
Double-barrelled K+-selective microelectrodes were based on potassium ionophore I, cocktail B (Fluka, CH-9471 Buchs, Switzerland) and were backfilled with 500 mmol l1 KCl. There is negligible interference of other intracellular cations on measurements made with these electrodes, which are 8000 times more selective to K+ relative to Na+ and 40 000 times more selective to K+ relative to Mg2+. The K+-selective electrode was calibrated in solutions of (in mmol l1) 15 KCl:135 NaCl and 150 KCl. The reference barrel was filled with 1 mol l1 sodium acetate near the tip and shank and 1 mol l1 KCl in the barrel of the electrode.
Cl-selective microelectrodes were based on ionophore I, cocktail A (Fluka). The electrodes are 30 times more selective to Cl relative to HCO3 and 20 times more selective to Cl relative to acetate. Both Cl-selective and reference barrels were backfilled with 1 mol l1 KCl. The electrode was calibrated in 100 mmol l1 KCl and 10 mmol l1 KCl.
Double-barrelled ISMEs were used for experiments only when the response of the ion-selective barrel to a tenfold change in ion activity was >49 mV and the 90% response time to a solution change was <30 s.
Potential differences from the reference (Vref) and
ion-selective barrel (Vi) were measured by a high input impedance
differential electrometer (FD 223, World Precision Instruments, Sarasota, FL,
USA). Vi and Vref were measured with respect to a
Ag/AgCl electrode connected to the bath through a 0.5 mol l1
KCl agar bridge. Preliminary experiments showed that using free flowing
electrodes (Neher, 1992) or
0.5 mol l1 KClagar bridges produce identical results.
Vi was filtered through a low-pass RC filter with a time constant
of 1 s to eliminate noise resulting from the high input impedance
(>1010
) of the ion-selective barrel. Vref and
the difference (ViVref) were recorded using an AD
converter and data-acquisition system (Axotape, Axon Instruments, Burlingame,
CA, USA).
Intracellular recordings were acceptable if the potential of each barrel
was stable to within ±2 mV for 30 s. In addition, recordings were
acceptable only if the potential of each barrel in the bathing saline after
withdrawal from the cell differed from the potential before impalement by less
than 3 mV, and if Vbl was more negative than 40 mV. The
latter value was selected since the published mean value for basolateral
membrane potential recorded with fine-tipped voltage-sensitive microelectrodes
in principal cells of D. melanogaster tubules is 44±0.5
mV (N=122; O'Donnell et al.,
1996
). Impalements that produced Vbl values less
negative than 40 mV were considered of poor quality and the data were
discarded.
Calculations
Intracellular ion activity was calculated using the formula:
![]() | (1) |
ab was calculated as:
![]() | (2) |
The ion activity in the calibration solution was calculated as the product
of ion concentration and the ion activity coefficient. Activity coefficients
for single electrolyte calibration solutions of 100 mmol l1
KCl and 10 mmol l1 KCl are 0.77 and 0.901, respectively
(Hamer and Wu, 1972). For
solutions containing 150 mmol l1 KCl and mixed solutions of
KCl and NaCl with constant ionic strength (150 mmol l1), the
activity coefficient is 0.75, calculated using the DebyeHuckel extended
formula and Harned's rule (Lee,
1981
).
Electrochemical potentials
The electrochemical potential (µ/F, in mV) for an ion
across the basolateral membrane was calculated as:
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Measurement of K+ and Na+ activities in secreted droplets
K+ and Na+ activities in secreted droplets collected
from isolated tubules set up in the Ramsay assay were measured using
single-barrelled ion-selective microelectrodes as described previously
(Maddrell et al., 1993;
O'Donnell and Maddrell, 1995
).
The K+ and Na+-selective microelectrodes were silanized
using the procedures of Maddrell et al.
(1993
). Filling and
calibration solutions of single-barrelled K+-selective and
reference electrodes were the same as those described above for
double-barrelled K+-selective microelectrodes.
K+ activity in secreted droplets was calculated using the
formula:
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Na+-selective microelectrodes were based on the neutral carrier
ETH157 (sodium ionophore II, cocktail A, Fluka). The Na+-selective
barrel was backfilled with 500 mmol l1 NaCl and the
reference barrel was filled with 1 mol l1 LiCl.
Na+-selective electrodes were calibrated in solutions of (in mmol
l1) 15 NaCl:135 LiCl and 150 NaCl. K+ is known to
interfere with the Na+ neutral carrier ETH157. The interference for
each secreted fluid droplet was corrected for using the
NicolskyEisenman equation (Ammann,
1986) and the measured value of secreted fluid K+
activity for the same droplet:
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Ion flux (pmol min1) was calculated as the product of secretion rate (nl min1) and ion activity (mmol l1) in the secreted droplets.
Chemicals
Stock solutions of ouabain and bumetanide (Sigma) were prepared in ethanol
so that the maximum final concentration of ethanol was 0.1% (v/v).
Previous studies have shown that Malpighian tubule secretion rate is
unaffected by ethanol at concentrations
1% (v/v)
(Linton and O'Donnell,
1999
).
Statistics
Values are expressed as mean ±
S.E.M. for the indicated number (N)
of measurements. Data were compared using paired and unpaired Student's
t-tests and differences were considered significant when
P<0.05.
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Results |
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Intracellular K+ and Cl activities and electrochemical potentials
Intracellular K+ activity was 121±7 mmol
l1 (N=10 of 35 impalements) and bath K+
activity was 15±0.6 mmol l1 (N=10). The
corresponding Vbl was 43±0.9 mV (N=10)
(Fig. 1A). The calculated
K+ electrochemical potential (µK/F)
across the basolateral membrane of principal cells was positive in all 10
experiments and the mean value was 9±1 mV. This value indicates that
K+ movement from cell to bath was favoured, and that K+
was actively transported into the cell.
|
Intracellular Cl activity was 30±2 mmol
l1 (N=9 of 80 impalements) and bath
Cl activity was 104±4 (N=9). The
corresponding Vbl was 42±1 mV (N=9)
(Fig. 1B). The calculated
Cl electrochemical potential
(µCl/F) across the basolateral membrane of
principal cells was positive in all 9 experiments and the mean value was
10±1 mV. This indicates that Cl movement from cell to
bath was favoured. To determine if other intracellular anions interfered with
the Cl electrode, the effect of replacing
Cl in the bath with SO42
(Table 1) on intracellular
Cl activity was measured. After 10 min in
Cl-free saline, intracellular Cl activity
was reduced to 4±1 mmol l1 (N=3 of 32
impalements). The electrochemical potential for Cl was then
corrected by subtraction of the measured level of interference. The corrected
value of
µCl/F was positive in all nine
experiments and the mean value was 7±1 mV. Thus, correction for
Cl interference did not alter the finding that
Cl movement from cell to bath was favoured, and that
Cl was actively transported into the cell.
Effects of bumetanide on K+ flux, Na+ flux and fluid secretion rate.
Previous studies have shown that exposing unstimulated tubules to ouabain
increases net transepithelial Na+ flux because inhibition of the
basolateral Na+/K+-ATPase permits Na+ that
enters the tubules to be transported out across the apical membrane
(Linton and O'Donnell, 1999).
Since a proportionately larger Na+ flux would make the effect of
bumetanide on Na+ flux more visible, Malpighian tubules were first
exposed to 104 mol l1 ouabain for 30 min
and 104 mol l1 bumetanide was then added
for a further 30 min. Fluid secretion rate was reduced by 36% when bumetanide
was added to tubules that had undergone prior exposure to saline containing
ouabain (Fig. 2A). Bumetanide
reduced K+ flux by 46% (Fig.
2B) and also reduced Na+ flux by 29%
(Fig. 2C). The results suggest
that ion transport across the basolateral membrane of principal cells involves
a Na+-driven Na+:K+:2Cl
cotransporter.
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Effects of ion substitution and Ba2+ on Vbl
Basolateral membrane potential hyperpolarized by 27±6 mV
(N=4, paired t-test, P<0.05) when K+
concentration in the bathing saline was reduced tenfold from 20 to 2 mmol
l1 (Fig. 3A).
A purely K+-selective membrane would hyperpolarize by 59 mV in
response to a tenfold reduction in bathing saline K+ concentration,
provided that the intracellular K+ level remained constant.
However, a gradual reduction in intracellular K+ level in response
to a reduction in bath K+ concentration would result in a
corresponding gradual reduction in the magnitude of the hyperpolarization of
Vbl, as seen previously
(O'Donnell et al., 1996). The
pattern of changes in Vbl over time after a change to low
K+ saline or back to control saline were consistent with changes in
intracellular K+ activity when saline K+ levels were
altered. These intracellular changes would result in a less than tenfold
change in K+ activity across the basolateral memebrane, and the
contribution of K+ to setting Vbl
(
27/59x100=46%) would therefore be underestimated.
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A tenfold reduction in bath Cl concentration produced a hyperpolarization of 5±1 mV (N=4, paired t-test, P<0.05) in basolateral membrane potential (Fig. 3B). The membrane potential did not change significantly when bath Na+ concentration was reduced tenfold (N=5, Fig. 3C). Taken together, the data show that the basolateral membrane of the principal cells has a high conductance to K+, a low conductance to Cl and a negligible conductance to Na+.
Addition of 6 mmol l1 Ba2+ to the bathing saline hyperpolarized the basolateral membrane potential by 10±2 mV (N=13, paired t-test, P<0.05, Fig. 3). NaH2PO4 was omitted from the salines containing Ba2+ to prevent precipitation of barium phosphate. A tenfold reduction in bath K+ concentration depolarized Vbl by 6±1 mV (N=4, paired t-test, P<0.05) in the presence of Ba2+, consistent with a reduction of K+ conductance (Fig. 3A). By contrast, a tenfold reduction of bath Cl concentration produced a much larger effect on the membrane potential of 31±7 mV (N=4, paired t-test, P<0.05, Fig. 3B) after addition of Ba2+. Changes in bath Na+ activity did not produce a significant change in membrane potential in the presence of Ba2+ (3±2 mV, N=5, Fig. 3C).
These results revealed that in control conditions the basolateral membrane potential of the principal cells showed a dominant K+ conductance, but that blockade of K+ channels with Ba2+ unmasked a smaller Cl conductance. The Na+ conductance of the basolateral membrane was negligible in the presence or absence of Ba2+.
Effects of Ba2+ on intracellular K+ and Cl activity
K+ channels have been proposed as a pathway for K+
movement across the basolateral membrane of D. melanogaster tubules
(Dow et al.,
1994a,b
).
Blocking of K+ channels might then lead to a decrement in
intracellular K+ activity if K+ continues to be
transported into the lumen across the apical membrane. To test this
hypothesis, the effect of 6 mmol l1 Ba2+ on
intracellular K+ was determined. The results showed that addition
of Ba2+ had no effect on intracellular K+ (N=4
of 12 impalements; Fig. 4). We
also used double-barrelled Clselective microelectrodes to
determine if the change in Vbl in response to 6 mmol
l1 Ba2+ altered
aCli. Although Vbl hyperpolarized by
15 mV, there was no change in aCli in response
to Ba2+ (N=2 of 20 impalements).
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Effects of Ba2+ on Vbl in tubules before and after exposure to ouabain, K+-free saline or Cl-free saline
As discussed below, the hyperpolarization of the basolateral membrane
potential after addition of Ba2+ to tubules of A. aegypti
and F. polyctena has been explained on the basis of an increased
resistance to the entrance of positive charges (i.e. K+ ions) into
the cell (Weltens et al.,
1992; Masia et al.,
2000
). In D. melanogaster tubules the electrochemical
gradient for K+ favours transport from cell to bath, in the
opposite direction to that proposed to explain the effect of Ba2+
on Vbl in tubules of A. aegypti and F. polyctena.
Therefore, current must flow either through another conductance or through an
electrogenic transporter to account for the hyperpolarization of the
basolateral membrane potential of D. melanogaster tubules in response
to the addition of Ba2+. Possible contributions of currents
generated by the Na+/K+-ATPase, K+ channels
and Cl channels to the hyperpolarization produced by
Ba2+ were therefore examined.
The effect of Ba2+ (6 mmol l1) on Vbl was measured before and after exposure to ouabain, K+-free saline or Cl-free saline (Table 1). The hyperpolarization caused by Ba2+ increased from 20±2 mV (N=5) in control saline to 31±2 mV (N=5) during exposure to 104 mol l1 ouabain (paired t-test, P<0.05, Fig. 5A).
|
The basolateral membrane potential showed a large transient hyperpolarization in K+-free saline, then recovered within 5 min (Fig. 5B). The hyperpolarization of Vbl produced by 6 mmol l1 Ba2+ increased from 14±1 mV in control saline to 23±3 mV during exposure to K+-free saline (N=5, paired t-test, P<0.05, Fig. 5B). By contrast, exposure to Cl-free saline blocked the hyperpolarization produced by Ba2+ almost completely, reducing the change in Vbl from 18±5 mV in control saline to 0.1±0.7 mV in Clfree saline (N=5, paired t-test, P<0.05, Fig. 5C).
The effect of Ba2+ on Vbl has been shown to be
dependant upon the activity of the apical H+-ATPase. Addition of
the H+-ATPase inhibitor bafilomycin blocks the effect of
Ba2+ on Vbl in F. polyctena tubules
(Weltens et al., 1992). To
test if exposure to Cl-free saline blocks the apical
H+-ATPase, the effect of Clfree saline on fluid
secretion was measured. Exposure to Clfree saline reduced
fluid secretion rate from 0.50±0.06 nl min1
(N=6) to 0.20±0.04 nl min1 (N=8)
after 90 min. These results indicate that in Cl-free saline
the apical H+-ATPase is still functional and drives transepithelial
fluid secretion for sustained periods, albeit at a reduced rate.
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Discussion |
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Calculations of electrochemical potentials for K+ and Cl rule out a role for K+ channels or K+:Cl cotransport in fluid secretion
Intracellular K+ activity in the principal cells of D.
melanogaster tubules (121 mmol l1) is very similar to
that reported in tubule cells of H. maori (110 mmol
l1; Neufeld and Leader,
1998) but is higher than that measured in tubule cells of R.
prolixus, (86 mmol l1;
Ianowski et al., 2002
), L.
migratoria (71 mmol l1;
Morgan and Mordue, 1983
) and
F. polyctena (61 mmol l1; Leyssens et al.,
1993a
,b
).
Calculation of the K+ electrochemical potential across the
basolateral membrane revealed that passive movement of K+ from cell
to bath is favoured, indicating that K+ is actively transported
into the cell. Given that passive net K+ flux from bath to cell is
not feasible, a direct contribution of K+ channels to net
transepithelial fluid secretion can be ruled out. Similar results were
reported in unstimulated Malpighian tubules of R. prolixus
(Ianowski et al., 2002). In
Malpighian tubules of other species K+ channels may play a role in
transepithelial ion transport if intracellular K+ activity is below
electrochemical equilibrium across the basolateral membrane, as proposed for
Malpighian tubule cells of F. polyctena
(Leyssens et al., 1993a
) and
H. maori (Neufeld and Leader,
1998
).
Intracellular Cl activity in the principal cells of
D. melanogaster tubules (30 mmol l1) is very
similar to that reported in tubule cells of R. prolixus (32 mmol
l1; Ianowski et al.,
2002), L. migratoria (38 mmol l1;
Morgan and Mordue, 1983
) and
F. polyctena (35 mmol l1;
Dijkstra et al., 1995
) but is
higher than that measured in tubule cells of H. maori (21 mmol
l1; Neufeld and Leader,
1998
).
The Cl electrochemical potential indicates that passive
Cl movement from cell to bath is favoured and that
Cl is actively accumulated in the cell. Outwardly directed
electrochemical potentials for Cl have also been reported in
Malpighian tubule cells of L. migratoria
(Morgan and Mordue, 1983) and
R. prolixus (Ianowski et al.,
2002
). On the other hand, an inwardly directed electrochemical
potential for Cl has been reported in tubules of F.
polyctena (Dijkstra et al.,
1995
). In H. maori tubules intracellular
Cl activity is very low and Cl is at
equilibrium across the basolateral membrane
(Neufeld and Leader,
1998
).
Given that the electrochemical potentials for both Cl and K+ favour movement of these ions from cell to bath, the contribution of a K+:Cl cotransporter to net transepithelial fluid secretion can be ruled out. Entry of these ions into the cell therefore requires an ATP-dependent pump (e.g. Na+/K+-ATPase) or secondary active transport driven by a favourable electrochemical potential for the entry of another ion (e.g. Na+:K+:2Cl).
Bumetanide inhibits K+ flux, Na+ flux and fluid secretion
In this paper we have exploited an earlier finding that Malpighian tubules
secrete fluid with nearly equimolar concentrations of Na+ and
K+ when treated with the Na+/K+-ATPase
inhibitor ouabain (Linton and O'Donnell,
1999). Our results show that when secreted fluid Na+
concentration is elevated from
23 mmol l1 in control
saline to
45 mmol l1 by pre-exposure of tubules to
saline containing 104 mol l1 ouabain,
bumetanide reduces fluid secretion rate and the transepithelial fluxes of both
K+ and Na+. These results are consistent with inhibition
of a Na+-driven Na+:K+:2Cl
cotransporter.
The earlier hypothesis for a role for K+:Cl
cotransport in fluid secretion was based on the finding that in the absence of
ouabain, the effect of bumetanide is to reduce fluid secretion rate and
K+ flux but not Na+ flux
(Linton and O'Donnell, 1999).
In the absence of ouabain most of the Na+ transported by the
Na+:K+:2Cl cotransporter is recycled
through the Na+/K+-ATPase to the bath. The
Na+ activity in the secreted fluid is therefore low (
23 mmol
l1) and it is difficult to observe reduction in secreted
fluid Na+ activity in response to bumetanide
(Linton and O'Donnell, 1999
).
Pre-exposure to ouabain prevents Na+ recycling to the bath, thereby
increasing transepithelial Na+ flux and making the effect of
bumetanide on Na+ flux more evident. The hypothesis of a
K+:Cl cotransporter can be ruled out on the basis
of the electrochemical potentials reported above, and also on the basis of the
effects of bumetanide in the presence of ouabain. It is worth noting that
recent analysis of the D. melanogaster genome has revealed five genes
encoding for a cation:Cl cotransporter. However, none of
these putative transporters has been characterized and their function remains
unknown (for review, see Pullikuth et al.,
2003
).
Conductive pathways of the basolateral membrane
Ion substitution experiments revealed that in control saline the
basolateral membrane of the principal cells of D. melanogaster
tubules has a large K+ conductance that can be blocked with
Ba2+. Dominant Ba2+-sensitive K+ conductances
have been described in basolateral membranes of Malpighian tubules cells of
most insects studied to date (Morgan and
Mordue, 1983; O'Donnell and
Maddrell, 1984
; Baldrick et
al., 1988
; Leyssens et al.,
1992
; Neufeld and Leader,
1998
).
On the other hand, the results show that the basolateral membrane of D.
melanogaster tubules does not have a significant Na+
conductance. In contrast, a large Na+ conductance, which
contributes to the increase in Na+ excretion rate after a blood
meal, has been described in tubules of the mosquito A. aegypti
(Hegarty et al., 1991;
Williams and Beyenbach,
1984
).
A key finding of the present work is the evidence for a
Cl conductance in the basolateral membrane of Malpighian
tubule principal cells. The results indicate that the cells have a
Cl conductance smaller than that for K+, but that
blockage of K+ channels with Ba2+ increases the relative
contribution of the Cl conductance to Vbl.
Cl conductances may also exist in Malpighian tubules of
other insects. Principal cells of A. aegypti tubules have basolateral
conductances for both K+ and Na+, and for a third
unidentified ion that could be Cl
(Beyenbach and Masia, 2002).
Furthermore, Yu et al. (2003
)
have reported preliminary results consistent with the existence of a
Cl conductance on the basolateral membrane of the principal
cells of A. aegypti Malpighian tubules. Tubules of F.
polyctena also show a dominant K+ conductance in the
basolateral membrane but a Cl conductance has not been
excluded (Weltens et al.,
1992
).
The effect of Ba2+
Ba2+ has been shown to block fluid secretion and to
hyperpolarize the basolateral membrane potential in Malpighian tubules of
several species. These results lead to proposals of vectorial K+
transport through K+ channels in Malpighian tubules of T.
molitor (Wiehart et al.,
2003b), A. aegypti
(Masia et al., 2000
), L.
migratoria (Hyde et al.,
2001
), H. maori
(Neufeld and Leader, 1998
) and
D. melanogaster (Dow et al.,
1994a
,b
).
Our results show that in D. melanogaster Malpighian tubules the K+ electrochemical potential across the basolateral membrane is outwardly directed, from cell to bath. Thus, transepithelial K+ secretion cannot involve basolateral K+ channels. Furthermore, addition of Ba2+ does not affect intracellular K+ activity, suggesting that K+ entry is not dependent upon basolateral K+ channels. Inhibition of fluid secretion and hyperpolarization of the basolateral membrane potential by Ba2+ must therefore reflect a process other than blockade of electrodiffusive K+ entry.
The effect of Ba2+ on Vbl: a role for basolateral Cl channels
Electrophysiological studies of tubules of F. polyctena
(Weltens et al., 1992;
Leyssens et al., 1992
) and
A. aegypti (Pannabecker et al.,
1992
; Masia et al.,
2000
) propose that Vbl is determined not only by
diffusion potentials and electrogenic pumps across the basolateral membrane,
but also by a loop current flowing through the basolateral membrane resistance
(Leyssens et al., 1992
;
Pannabecker et al., 1992
).
Equivalent circuit analysis shows that increasing basolateral membrane
resistance with Ba2+ will cause the loop current to drive the
basolateral membrane potential more negative
(Leyssens et al., 1992
;
Pannabecker et al., 1992
;
Weltens et al., 1992
;
Masia et al., 2000
;
Wiehart et al., 2003b
). In
F. polyctena and A. aegypti tubules it has been proposed
that the loop current is carried by inward K+ flow through
basolateral K+ channels
(Leyssens et al., 1992
;
Weltens et al., 1992
;
Masia et al., 2000
).
Our measurements of electrochemical potentials show that inward flow of K+ through basolateral channels is not feasible in D. melanogaster tubules. An alternative explanation is thus required to explain the effect of Ba2+ on Vbl. In order to test possible mechanisms of action of Ba2+ we investigated the contributions of K+ conductance, Cl conductance and the electrogenic Na+/K+-ATPase to the Ba2+-induced hyperpolarization of Vbl.
Blocking the Na+/K+-ATPase by pre-exposure to ouabain
did not block the hyperpolarization of Vbl in response to
Ba2+. The effect of Ba2+ is therefore independent of the
current produced by the electrogenic activity (3Na+/2K+)
of this basolateral pump. Similarly, blockage of K+ currents by
prior exposure K+-free saline did not block the effect of
Ba2+ on the basolateral membrane potential. This result indicates
that the current responsible for the hyperpolarization is not carried by
K+, in contrast to proposals for tubules of F. polyctena
and A. aegypti (Leyssens et al.,
1992; Pannabecker et al.,
1992
; Weltens et al.,
1992
; Masia et al.,
2000
). On the other hand, prior exposure to
Cl-free saline containing 20 mmol l1
K+ blocked the hyperpolarization of Vbl in response to
Ba2+. This finding suggests that Cl carries the
loop current responsible for the hyperpolarization produced by Ba2+
in D. melanogaster tubules. It is important to point out that
Cl flow from cell to bath would produce a current of the
same sign as K+ flow from bath to cell, proposed as the basis for
the Ba2+-induced hyperpolarization of Vbl in tubules of
F. polyctena and A. aegypti
(Leyssens et al., 1992
;
Pannabecker et al., 1992
;
Weltens et al., 1992
;
Masia et al., 2000
). Taken
together, our results suggest that the effect of Ba2+ on the
basolateral membrane potential in D. melanogaster tubules is the
result of an increased influence of the loop current on Vbl caused
by the increased membrane resistance. The current responsible for the
hyperpolarization is carried not by K+, as proposed for tubules of
A. aegypti and F. polyctena, but by
Cl.
The effect of Ba2+ on fluid secretion
Previous studies have shown that fluid secretion by D.
melanogaster tubules is inhibited by Ba2+ (Dow et al.,
1994a,b
).
Our results indicate that this inhibition cannot be explained as a result of
blockage of K+ influx through K+ channels. However, it
is important to note that Ba2+ has been shown to affect several
cellular functions other than basolateral K+ channels.
Ba2+ inhibits the Na+/K+-ATPase in proximal
tubules of the mammalian kidney (Kone et
al., 1989
) and alters mitochondrial function by blocking
mitochondrial megachannels (Szabo et al.,
1992
). Ba2+ is also known to activate acid phosphatases
in Culex tarsalis (Houk and
Hardy, 1987
), to enhance phospholipase A2 activity
(Blache and Ciavatti, 1987
) and
to interfere with Ca2+/calmodulin control of exocytosis
(Verhage et al., 1995
).
A revised model for ion transport across of principal cells
Our results can be summarized in the revised model shown in
Fig. 6. K+ and
Cl are actively transported into the cell through a
Na+-driven Na+:K+:2Cl
cotransporter. A role for K+ channels or
K+:Cl cotransport can be ruled out. Most of the
K+ that enters the cell is transported into the lumen through a
K+/H+ exchanger. Most of the Na+ that enters
through the Na+:K+:2Cl cotransporter
is recycled back to the bath through a Na+/K+-ATPase,
while a smaller portion is transported into the lumen through an apical
Na+/H+ exchanger. Blockage of the basolateral
Na+/K+-ATPase with ouabain prevents Na+
transport back to the bath and increases the availability of Na+
ions for transport into the lumen, thereby increasing net transepithelial
Na+ secretion. These results suggest that in unstimulated tubules
the basolateral Na+/K+-ATPase and
Na+:K+:2Cl cotransporter may act in
concert to set the ratio of Na+ to K+ in the secreted
fluid. Downregulation of Na+/K+-ATPase activity, for
example, will enhance elimination of Na+. It is worth noting that
regulation of the activity of the Na+/K+-ATPase by
protein kinase C has been reported in Malpighian tubule cells of R.
prolixus (Caruso-Neves et al.,
1998).
|
Due to the small basolateral Cl conductance, only a small portion of the intracellular Cl can be recycled back to the bath through Cl channels (Fig. 6). The model shown in Fig. 6 does not preclude other basolateral transport systems for Cl (e.g. Cl/HCO3 exchange). After Cl crosses the basolateral membrane it may also cross into the lumen through apical Cl channels (not shown), driven by the large lumen-positive apical membrane potential generated by the H+-ATPase (Fig. 6).
It is important to point out the D. melanogaster tubule secretes
even when bathed in K+-free or Na+-free saline (Linton
and O'Donnell, 1999,
2000
). Moreover, fluid
secretion in the absence of either cation is insensitive to high
concentrations of bumetanide (104 mol l1).
Thus, it is clear that in K+-free or Na+-free saline a
different set of basolateral membrane transport systems mediate ion influx
across the basolateral membrane. In K+-free saline the entry of
Na+ could involve a Na+:organic anion cotransporter
(Linton and O'Donnell, 2000
)
and/or a Na+-dependent
Cl/HCO3 exchanger
(Sciortino et al., 2001
). In
Na+-free saline the K+ gradient may change and
K+ influx may involve K+ channels or
K+:Cl cotransporters.
Tubules secreted fluid, albeit at lower rates, when bathed in Cl-free saline. The composition of the secreted fluid has not been analyzed but Cl must be replaced by another anion such as HCO3 or H2PO4. Na+ and K+ could enter the cell in Cl-free saline through one of the transport systems proposed above.
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References |
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