Mechanisms of K+ transport across basolateral membranes of principal cells in Malpighian tubules of the yellow fever mosquito, Aedes aegypti
Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA
* Author for correspondence (e-mail: kwb1{at}cornell.edu)
Accepted 12 February 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: transepithelial Na+ secretion, transepithelial K+ secretion, transepithelial Cl secretion, barium block, K+ channel, bumetanide, Na+/K+/2Cl cotransport, basolateral membrane voltage, cell input resistance, transepithelial voltage, transepithelial resistance
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study is concerned with identifying the transport pathways that
mediate the entry of K+ from the hemolymph into principal cells.
The K+ conductance of the basolateral membrane, which accounts for
64% of the total membrane conductance, is one pathway for K+ entry
(Beyenbach and Masia, 2002;
Masia et al., 2000
). Although
this K+ conductance can be blocked by barium, the blockade does not
inhibit transepithelial K+ secretion completely
(Masia et al., 2000
). Thus, it
was of interest to identify the K+ entry mechanism remaining in the
presence of Ba2+. Transport via the
Na+/K+/2Cl cotransporter first came to
mind in view of significant effects of bumetanide on transepithelial secretion
of Na+ and K+
(Hegarty et al., 1991
).
In the present study, we have used three experimental methods to probe the
mechanism of K+ entry from hemolymph into principal cells. Using
the fluid secretion assay of Ramsay
(1953), we evaluated the
effects of Ba2+ and bumetanide on the transepithelial secretion of
K+, Na+, Cl and water. In isolated
perfused Malpighian tubules, we studied the effects of Ba2+ and
bumetanide on transepithelial voltage and resistance. Using the methods of
two-electrode voltage clamp (TEVC), we examined the effects of Ba2+
and bumetanide on the basolateral membrane voltage and input resistance of
principal cells (Masia et al.,
2000
). We found two equally important routes for the entry of
K+ into principal cells: (1) an electroconductive route that can be
blocked by Ba2+ and (2) an electroneutral route via
Na+/K+/2Cl cotransport that can be
inhibited by bumetanide. The significant inhibition of transepithelial
Na+ secretion by Ba2+ suggests that basolateral
K+ channels are permeable to Na+. The stimulation of
transepithelial Na+ secretion by bumetanide and reciprocal changes
in the concentrations of Na+ and K+ in secreted fluid
indicate that Na+ can replace K+. However, the tubules
cease epithelial transport and fluid secretion altogether if they are
prevented from secreting K+.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When effects on transepithelial Na+, K+,
Cl and water secretion were of interest, the tubule was
studied by the methods of Ramsay
(1953) and wavelength
dispersive spectroscopy (Williams and
Beyenbach, 1983
). When effects on transepithelial voltage and
resistance were of interest, the tubule was microperfused in vitro
and studied by the method of Helman
(1972
). When effects on the
basolateral membrane voltage and input resistance of principal cells were of
interest, the tubule was studied by the method of TEVC
(Masia et al., 2000
).
Ringer solution and drugs
Ringer solution contained the following: 150 mmol l1
NaCl, 25 mmol l1 Hepes, 3.4 mmol l1 KCl,
1.8 mmol l1 NaHCO3, 1 mmol l1
MgCl2, 1.7 mmol l1 CaCl2 and 5 mmol
l1 glucose. The pH was adjusted to 7.1 with NaOH. The
osmolality was 320 mosmol kg1 H2O.
Ba2+ was used as BaCl2 at a concentration of 5 mmol
l1. In these experiments, the control Ringer solution was
supplemented with 15 mmol l1 mannitol for osmotic balance
with the experimental solution containing 5 mmol l1
BaCl2. In a previous study, we determined that 5 mmol
l1 Ba2+ is a saturating dose for blocking
K+ channels in the basolateral membrane of principal cells
(Masia et al., 2000).
Bumetanide (Sigma, St Louis, MO, USA) was dissolved in Ringer solution and
used at a concentration of 100 µmol l1. Previous attempts
to obtain a doseresponse curve of the effects of bumetanide were
unsuccessful because the effects of bumetanide are cumulative and
irreversible. For this reason, we used the bumetanide concentration (0.1 mmol
l1) employed in a previous study
(Hegarty et al., 1991).
The Ramsay assay
Each Malpighian tubule served as its own control, first under control and
then under experimental conditions. Rates of transepithelial fluid secretion
were measured as described previously
(Hegarty et al., 1991) with
the following modifications. With about 80% of the tubule length remaining in
a 40 µl droplet of Ringer solution, the open end of the tubule was pulled
into the surrounding oil and gently draped around a small steel broach. Thus,
fluid secreted by the tubule exited into oil, forming a droplet. The
dimensions of this droplet were measured over time in order to determine
volume. Cumulative secreted volume was measured every 5 min for an initial
control period (see Fig. 1).
Secreted volume was then removed, and the experimental agent was added to the
peritubular Ringer solution. Thereafter, cumulative volume was measured again
every 5 min for at least 30 min. Secreted volume was collected at the end of
the experimental period. BaCl2 was added to the peritubular Ringer
solution by replacing 10 µl with Ringer solution containing 20 mmol
l1 BaCl2. Due to the limited water solubility of
bumetanide, it was necessary to exchange 20 µl of Ringer solution with an
equal volume containing 200 µmol l1 bumetanide. The
transepithelial fluid secretion rate (Vs) was calculated
as the slope of a least-squares regression line fitted to the plot of time
versus volume secreted by the tubule over at least 30 min control and
experimental periods (Fig. 1A).
In the Ba2+ plus bumetanide study, a quadratic polynomial was
fitted to the data (Fig. 1B).
Here, the rate of fluid secretion was calculated by taking the derivative of
the polynomial and calculating Vs at a specific time.
|
The concentrations of Na+, K+ and
Cl in fluid secreted by Malpighian tubules and in
peritubular Ringer solutions were measured against appropriate standards using
the methods of wavelength dispersive spectroscopy (electron probe), as
described previously (Williams and
Beyenbach, 1983), with the following modifications. Dried sample
spots of 3050 pl volumes were analyzed using a JEOL 8900 electron
microprobe at a beam current of 50 nA. X-rays emitted at the wavelengths of
K+ and Cl were quantified using a pentaerythritol
high-intensity crystal while those emitted at the wavelength of Na+
were measured using a thallium acid phthalate crystal. A set of standard
curves was constructed by plotting known concentrations of Na+,
K+ and Cl, each in a series of six standard
solutions, against the number of X-ray counts per second.
In vitro microperfusion of Malpighian tubules
Malpighian tubules were perfused in vitro for the measurement of
the transepithelial voltage (Vt) and resistance
(Rt), as described previously
(Yu and Beyenbach, 2002). The
peritubular bath (500 µl) was perfused with Ringer solution at a rate of
5.6 ml min1. Vt was measured and
recorded continuously, and Rt was measured periodically
when of interest. Values of Vt and Rt
are steady-state values taken between 10 min and 60 min of control and
experimental periods.
Two-electrode voltage clamp
Measurements of the basolateral membrane voltage (Vbl)
and principal cell input resistance (Rpc) were obtained
using the methods of TEVC, as described by Masia et al.
(2000) and Wu and Beyenbach
(2003
). Again, values of
Vbl and Rpc are steady-state values
taken between 10 min and 60 min of control and experimental periods. The
peritubular bath (500 µl) was perfused with Ringer solution at a rate of
2.6 ml min1. To prevent the movement of the tubule, the
bottom of the lucite bath was coated with poly-L-lysine
(evaporation of 0.125 mg ml1 poly-L-lysine).
Statistical treatment of the data
Data are summarized as means ± S.E.M. (N, number
of observations). Since each Malpighian tubule served as its own control,
experiments were analyzed with the Student's paired t-test.
Significance is defined as P<0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The product of Vs and the ion concentration in secreted fluid yields the rate of transepithelial ion secretion (Fig. 2BD).The mean rate of transepithelial Na+ secretion was 98.1±15.6 pmol min1, the rate of K+ secretion was 99.9±21.9 pmol min1, and the rate of Cl secretion was 166.6±9.5 pmol min1 (N=7 tubules; Fig. 2).
|
The addition of 5 mmol l1 BaCl2 to the peritubular Ringer bath significantly reduced Vs to 44% of control values, from 0.84 nl min1 (control) to 0.37 nl min1, with a range from 0.28 nl min1 to 0.44 nl min1 (Figs 1A, 2A; Table 1). The reduced rate of Vs was constant for as long as it was studied, i.e. 60 min, with negligible fluctuations in Vs in the presence of BaCl2 (Fig. 1A).
Barium affected the ionic composition of secreted fluid (Table 1). The Na+ concentration in secreted fluid significantly increased from 116.8 mmol l1 (control) to 144.6 mmol l1, and the K+ concentration significantly decreased from 119.0 mmol l1 (control) to 54.3 mmol l1. Ba2+ had no significant effect on the Cl concentration in secreted fluid (Table 1).
The product of Vs and ion concentration revealed significant effects of Ba2+ on transepithelial secretion rates of all three ions (Fig. 2BD). The rate of transepithelial Na+ secretion decreased to 55% of control values, from 98.1 pmol min1 to 53.8±4.4 pmol min1. The rate of K+ secretion decreased even more, to 20% of control values, from 99.9 pmol min1 to 19.5±10.6 pmol min1, and the rate of Cl secretion decreased to 43% of control values, from 166.6 pmol min1 to 71.4±6.0 pmol min1 (Fig. 2BD).
Although the Ba2+ block of K+ channels in the basolateral membrane of principal cells substantially reduced transepithelial K+ secretion, it did not completely inhibit K+ secretion (Fig. 2C). For this reason, we searched for the transepithelial K+ transport pathway remaining in the presence of Ba2+. In these experiments, the control rate of fluid secretion was 0.61 nl min1 with a range from 0.56 nl min1 to 0.68 nl min1 (Fig. 1B; Table 1). The addition of 5 mmol l1 Ba2+ plus bumetanide (100 µmol l1) led Vs to decrease progressively, reaching the lowest detectable rate of fluid secretion, 0.06 nl min1, about 25 min after adding these two agents to the peritubular bath (Table 1). After 30 min, Vs had decreased to zero for all six Malpighian tubules tested (Fig. 1B). Since a new steady state was never obtained, we estimated Vs as the derivative of the secreted volume that had accumulated 25 min after the addition of barium and bumetanide to the peritubular bath (Fig. 1B; Table 1).
In the presence of barium plus bumetanide, the Na+ concentration in secreted fluid significantly increased from 93.8 mmol l1 to 120.4 mmol l1, and the K+ concentration significantly decreased from 55.1 mmol l1 to 25.3 mmol l1 (Table 1). The concentration of Cl in secreted fluid did not change significantly: 150.3 mmol l1 versus 140.5 mmol l1 (Table 1). These data are qualitatively similar to those observed in the presence of Ba2+ alone.
The product of Vs and ion concentration in the presence of Ba2+ plus bumetanide showed profound effects on transepithelial ion secretion rates (Fig. 2). After 25 min of the experimental period, transepithelial Na+ secretion had dropped to values 12% of control, from 57.8±5.5 pmol min1 (control) to 7.1±2.9 pmol min1 (N=6; Fig. 2B). At the same time, transepithelial K+ secretion had fallen to 4% of control, from 34.3±5.9 pmol min1 (control) to 1.36±0.5 pmol min1 (N=6; Fig. 2C) and Cl secretion had decreased to 9% of control values, from 92.8±6.3 pmol min1 (control) to 8.1±3.0 pmol min1 (N=6; Fig. 2C). After 30 min in the presence of Ba2+ plus bumetanide, transepithelial transport rates for all three ions were reduced to zero.
Effect of barium and barium plus bumetanide on tubule electrophysiology
In isolated perfused Malpighian tubules, the control transepithelial
voltage (Vt) was 19.4 mV (lumen-positive), and the
transepithelial resistance (Rt) was 6.37 kcm
(Table 1). In principal cells
studied by the methods of TEVC, the control basolateral membrane voltage
(Vbl) was 75.2 mV, and the input resistance of
principal cells (Rpc) was 363.7 k
(Table 1). The difference
between Vt and Vbl is the apical
membrane voltage, 94.6 mV.
Peritubular Ba2+ significantly depolarized
Vt from 19.4 mV to 17.2 mV and significantly increased
Rt from 6.37 kcm to 6.87 k
cm
(Table 1). In addition, 5 mmol
l1 Ba2+ significantly hyperpolarized
Vbl from 75.2 mV to 88.2 mV and
significantly increased Rpc from 363.7 k
to 516.3
k
.
The effects of barium on Vt, Vbl,
Rt and Rpc were immediate, as rapid as
the peritubular batch could be changed to include Ba2+. Moreover,
Ba2+ elicited these effects in a single step, i.e. there were no
secondary time-dependent effects. Likewise, the off-effects upon
Ba2+ washout were immediate and complete, displaying simple
kinetics of an open channel block, as in previous studies
(Beyenbach and Masia, 2002;
Masia et al., 2000
;
Wu and Beyenbach, 2003
).
Peritubular Ba2+ plus bumetanide had the following effects
measured after 25 min of treatment: in isolated perfused Malpighian tubules,
the control Vt was 30.4 mV and the control
Rt was 11.0 kcm
(Table 1). In principal cells
studied by the methods of TEVC, the control Vbl was
78.4 mV while the control Rpc was 314.9 k
(Table 1). The addition of
Ba2+ plus bumetanide to the peritubular bath significantly
hyperpolarized Vt from 30.4 mV to 38.2 mV and
significantly increased Rt from 11.0 k
cm to 13.6
k
cm (Table 1). Vbl significantly depolarized from 78.4 mV to
59.3 mV and significantly increased Rpc from 314.9
k
to 464.9 k
(Table
1). The time course of the electrophysiological effects of
Ba2+ and bumetanide mirrored the time course of the effects on
electrolyte and fluid secretion.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The inhibitory effects of barium on insect Malpighian tubules have been
observed in the locust (Hyde et al.,
2001), ant (Leyssens et al.,
1994
), fruitfly (Wessing et
al., 1993
), beetle (Nicoloson
and Isaacson, 1987
), mealworm
(Wiehart et al., 2003a
),
cricket (Xu and Marshall,
1999a
) and weta (Neufeld and
Leader, 1998
). The present study corroborates the consensus that
Ba2+-sensitive K+ channels mediate the entry of
K+ across the basolateral membrane of principal cells in Malpighian
tubules. The K+ conductance of the basolateral membrane is so large
that intracellular K+ is at or near electrochemical equilibrium
with extracellular K+ in the hemolymph or peritubular Ringer
solution (Ianowski et al.,
2001
; Leyssens et al.,
1993
).
Effects of the loop diuretics bumetanide and furosemide on insect
Malpighian tubules have been reported in the mealworm
(Wiehart et al., 2003b),
blowfly (O'Donnell and Maddrell,
1984
), fruit fly (Linton and
O'Donnell, 1999
), tobacco hornworm
(Audsley et al., 1993
;
Reagan, 1995
), ant
(Leyssens et al., 1994
),
locust (Baldrick et al., 1988
)
and cricket (Xu and Marshall,
1999b
). The consensus of these studies is that bumetanide and
furosemide inhibit Na+/K+/2Cl and/or
K+/Cl cotransport systems
(Baldrick et al., 1988
;
Gillen and Bowles, 2001
;
Leyssens et al., 1994
) in the
basolateral membrane, thereby preventing the entry of NaCl and/or KCl into the
cell. The present study is consistent with this conclusion. However, the
evidence for Na+/K+/2Cl cotransport is
merely pharmacological because this transporter has not been isolated from
and/or cloned in any insect Malpighian tubule. In Malpighian tubules of
Aedes aegypti, bumetanide was found to significantly inhibit
transepithelial K+ secretion without affecting transepithelial
resistance and the fractional resistance of the basolateral membrane, which is
consistent with an effect on a non-conductive transport pathway such as that
of an electroneutral transport system
(Hegarty et al., 1991
).
More importantly, the present study illustrates that inhibitors of K+ transport significantly increase the transepithelial secretion of Na+ and Cl. In what follows, we will seek explanations for the effects of barium and bumetanide on transepithelial Na+, K+ and Cl secretion that are consistent with the present data and present models of transepithelial electrolyte transport in Malpighian tubules of Aedes aegypti.
Inhibition of transepithelial K+ and fluid secretion by barium
The addition of the K+ channel blocker Ba2+ to the
peritubular bath promptly reduced the rate of fluid secretion to 44% of
control values together with significant reductions in the rates of
transepithelial Na+, K+ and Cl
secretion (Fig. 2). The block
of basolateral membrane K+ channels is expected to reduce the entry
of K+ into principal cells, thereby limiting intracellular
K+ for secretion into the tubule lumen
(Fig. 3). Accordingly, the rate
of transcellular K+ transport is inhibited by 80%. Clearly,
K+ channels provide a major route for K+ entry into the
cell. They account for 64% of the total conductance of the basolateral
membrane (Beyenbach and Masia,
2002). When these K+ channels are blocked by
Ba2+ in Malpighian tubules of ants, the intracellular K+
concentration drops from 88 mmol l1 to 73 mmol
l1 (Leyssens et al.,
1993
). A similar drop in the intracellular K+
concentration of Aedes Malpighian tubules reduces the driving force
for K+ extrusion across the apical membrane, explaining in part why
the K+ concentration in secreted fluid drops from 119 mmol
l1 to 54 mmol l1 in the presence of
Ba2+ (Table 1).
Inhibition of transepithelial K+ secretion by bumetanide
In a previous study, we saw no effect of bumetanide (0.1 mmol
l1) on transepithelial fluid secretion
(Hegarty et al., 1991).
However, an examination of secreted fluid revealed significant effects on the
concentrations of Na+ and K+ in secreted fluid
(Table 1). Bumetanide decreased
the K+ concentration by 60 mmol l1 but increased
the Na+ concentration by a similar amount with no change in total
cation concentration and hence no change in the concentration of secreted
Cl. Thus, measures of fluid secretion as the only bioassay
can be misleading. For this reason, the parallel study of ion concentrations
and electrophysiology offers details that otherwise would be missed in the
Ramsay assay.
Since the K+ concentration in secreted fluid decreased and the
Na+ concentration increased, it follows that bumetanide decreased
the rate of transepithelial K+ secretion and increased the rate of
Na+ secretion (Table
1; Fig. 2).
Significantly, bumetanide inhibited K+ secretion by 70%, similar to
the inhibition measured in the presence of Ba2+
(Fig. 2). Bumetanide is known
to inhibit Na+/K+/2Cl cotransport in
Malpighian tubules (Baldrick et al.,
1988; Hegarty et al.,
1991
; Ianowski et al.,
2001
; Leyssens et al.,
1994
; Reagan,
1995
; Wiehart et al.,
2003a
). Hence, the inhibition of K+ secretion to levels
similar to those observed in the presence of Ba2+ reveals that
Na+/K+/2Cl transport is as important a
route for K+ entry as are K+ channels. Moreover, the
complete inhibition of transepithelial K+ secretion by the
co-administration of Ba2+ and bumetanide identifies channel- and
carrier-mediated transport as the two major, if not exclusive, mechanisms for
bringing K+ into the cell across the basolateral membrane.
The inhibition of transepithelial K+ secretion by 80% in the presence of barium and by 70% in the presence of bumetanide suggests that carrier- and channel-mediated K+ entry pathways are functionally coupled, where effects on K+ channels affect Na+/K+/2Cl cotransport and vice versa.
Inhibition of transepithelial Na+ secretion by barium
The significant inhibition of transepithelial Na+ secretion by
barium can be explained by (1) the general reduction in transepithelial
electrogenic ion transport as barium blocks a major conductive pathway and (2)
K+ channels that permit the passage of Na+.
As shown in Fig. 3,
transepithelial secretion of NaCl and KCl can be modeled with an electrical
circuit consisting of a transcellular pathway that mediates active transport
of K+ and Na+ and a paracellular pathway that mediates
passive transport of Cl. Transcellular and paracellular
pathways are electrically coupled, forming an intraepithelial circuit where
cationic current (Na+ and K+) passing through principal
cells is the same as anionic current (Cl) through the
paracellular pathway. The Ba2+ block of K+ channels
significantly increases the input resistance of principal cells from 363.7
k to 516.3 k
, reflecting the substantial increase in the
resistance of the basolateral membrane
(Table 1). The large increase
in basolateral membrane resistance is expected to decrease transcellular
cationic current and, consequently, paracellular current
(Fig. 3). Indeed, estimates of
transcellular and paracellular currents, or the intraepithelial loop current,
show significant reductions in the presence of Ba2+
(Wu and Beyenbach, 2003
).
Thus, as loop current decreases, the rate of K+ and Na+
transport through principal cells decreases and the rate of
Cl transport decreases. Direct measurements of
transepithelial Na+, K+ and Cl
secretion confirm this to be the case (Fig.
2). Moreover, significant reductions in transepithelial NaCl and
KCl secretion have the effect of bringing less water into the tubule lumen,
hence the decrease in fluid secretion in the presence of Ba2+ (Figs
1,
2).
Ba2+ may also affect transepithelial Na+ secretion
directly. Ion channels are never perfectly ion-selective. Some epithelial
K+ channels can be highly selective for K+ with
K+/Na+ permeability ratios as high as 100
(Hurst et al., 1992). Other
K+ channels admit Na+ more readily, with
K+/Na+ permeability ratios as low as 13
(Teulon et al., 1994
), 10
(Suzuki et al., 1994
) or 5
(Labarca et al., 1996
). A
similar low K+/Na+ permeability ratio of K+
channels in the basolateral membrane of Aedes Malpighian tubules
would allow a substantial Na+ influx in view of the high
electrochemical driving force (
100 mV), supporting the entry of
Na+ into the cell. The Ba2+ block of basolateral
membrane K+ channels would then be expected to inhibit not only
K+ entry and transepithelial K+ secretion but also
Na+ entry and transepithelial Na+ secretion.
Accordingly, the significant inhibition of transepithelial Na+
secretion by Ba2+ suggests that K+ channels in the
basolateral membrane of principal cells offer some permeability to
Na+ (Figs 2B,
3).
Stimulation of transepithelial Na+ secretion by bumetanide
The inhibition of Na+/K+/2Cl
cotransport across the basolateral membrane is expected to decrease
Na+ entry into the cell and to decrease transepithelial
Na+ secretion, like K+
(Fig. 3). Paradoxically, the
opposite is observed. Bumetanide significantly stimulates transepithelial
Na+ secretion, from 122 pmol min1 to 169 pmol
min1 (Hegarty et al.,
1991). Hypothetical solutions to this paradox give a glimpse at
the relative roles of several pathways available to Na+ for
entering the cell.
Since Na+/K+/2Cl cotransport is a
major pathway for K+ entry into the cell
(Fig. 3), bumetanide inhibition
is expected to lower the intracellular K+ concentration. Consistent
with this hypothesis is the significant depolarization of the basolateral
membrane voltage from 63 mV to 51 mV, reflecting the drop in the
K+-diffusion potential across the basolateral membrane
(Hegarty et al., 1991). Direct
measurements of intracellular K+ concentrations in Malpighian
tubules of the locust show that furosemide, which blocks
Na+/K+/2Cl cotransport like
bumetanide, causes intracellular K+ concentration to fall and to be
replaced by Na+ (Hopkin et al.,
2001
). Likewise, intracellular Na+ replaces
K+ in Malpighian tubules of Rhodnius that are stimulated
to secrete Na+ in the presence of serotonin
(Ianowski et al., 2001
).
Similar reciprocal changes in intracellular K+ and Na+
concentrations in the presence of bumetanide in Malpighian tubules of
Aedes aegypti would be expected to lead to the observed decrease in
the K+ concentration and the increase in the Na+
concentration in secreted fluid (Fig.
2). Even though bumetanide blocks the entry of both K+
and Na+ into the cell, the intracellular Na+ can still
rise because three other pathways for Na entry remain open: (1)
Na+/H+ exchange transport
(Hegarty et al., 1992
;
Petzel, 2000
), (2) an
Na+ conductance that accounts for 16% of the total conductance of
the basolateral membrane (Beyenbach and
Masia, 2002
) and (3) K+ channels that apparently allow
the passage of some Na+ (Fig.
3). Of these three, Na+ entry via
Na+/H+ exchange is probably the most important pathway
because amiloride, a blocker of Na+/H+ exchange,
inhibits transepithelial Na+ secretion by 70%
(Hegarty et al., 1992
).
Summary and remaining questions
Ba2+ inhibits not only transepithelial K+ secretion
but also transepithelial Na+ and Cl secretion. By
blocking K+ channels that offer some permeability to
Na+, barium reduces the transcellular cationic current from
hemolymph to tubule lumen (Fig.
3, circuit diagram). Since the return current is carried by
Cl, also passing from hemolymph to tubule lumen, it follows
that barium reduces transepithelial Cl secretion.
In as much as basolateral membrane K+ channels are more permeable to K+ than to Na+, barium inhibits transepithelial K+ secretion to a greater degree than Na+ secretion (Fig. 2). Nevertheless, the Na+ concentration in secreted fluid rises because K+ channels provide but one minor pathway for transepithelial Na+ secretion in the presence of multiple other Na+ entry mechanisms across the basolateral membrane (Table 1; Fig. 2). In spite of the increase in the luminal Na+ concentration, transepithelial Na+ secretion decreases because of the overriding reduction in the intraepithelial loop current by barium (Fig. 3, circuit diagram).
By contrast, bumetanide leaves electroconductive pathways intact. As a
result, bumetanide has no effect on transepithelial resistance, no effect on
the fractional resistance of the basolateral membrane
(Hegarty et al., 1991), no
effect on intraepithelial loop current, no effect on transepithelial total
cation secretion and no effect on Cl and fluid secretion.
Total transepithelial cation secretion did not change, but transepithelial
Na+ secretion significantly increased with an equivalent decrease
in K+ secretion. Bumetanide blocks one of two major K+
entry pathways, but only one of four Na+ entry pathways, bringing
about reciprocal changes in intracellular and luminal K+ and
Na+ concentrations and, consequently, the inhibition of
K+ secretion and the stimulation of Na+ secretion.
Finally, the electrophysiological data are consistent with Ba2+ blocking an electroconductive pathway such as that provided by K+ channels in the basolateral membrane of principal cells. Upon the addition of Ba2+ to the peritubular medium, the observed changes in transepithelial voltage and resistance and basolateral membrane voltage and resistance are internally consistent with channel block. By contrast, bumetanide had no effect on epithelial or membrane resistance, and the small changes in transepithelial and membrane voltage can be accounted for by changing intracellular K+ concentrations. Moreover, the kinetics of the inhibitions suggest two distinct mechanisms of action. Rapid kinetics of the effects of Ba2+ evince channel block, and slow kinetics of the effects of bumetanide are consistent with the inhibition of a carrier.
One question that this study leaves open is how bumetanide blocks the cAMP
stimulation of transepithelial Na+ secretion, which we have
observed in a previous study (Hegarty et
al., 1991). In Aedes Malpighian tubules,
corticotropin-releasing factor (CRF)-like diuretic peptides and their second
messenger cAMP selectively increase transepithelial NaCl secretion by
activating an Na+ conductance in the basolateral membrane of
principal cells (Beyenbach,
2001
; Petzel et al.,
1985
,
1987
; Williams and Beyenbach,
1983
,
1984
). How the blockade of the
Na+/K+/2Cl cotransporter brings about
the blockade of Na+ channels is unclear at present. However, it is
known that the Na+/K+/2Cl
cotransporter interacts with channels, such that effects on one will also
affect the other (O'Neill and Steinberg,
1995
; Brzuszczak et al.,
1996
; Marunaka et al.,
1999
; Huang et al.,
2000
; Singh et al.,
2001
; Walter et al.,
2001
; Wang, 2003
).
It is conceivable that a similar coupling of carrier and channels prevents the
activation of Na+ channels when bumetanide has blocked the
Na+/K+/2Cl cotransporter.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Audsley, N., Coast, G. M. and Schooley, D. A.
(1993). The effects of Manduca sexta diuretic hormone on
fluid transport by the Malpighian tubules and cryptonephric complex of
Manduca sexta. J. Exp. Biol.
178,231
-243.
Baldrick, P., Hyde, D. and Anstee, J. H. (1988). Microelectrode studies on Malpighian tubule cells of Locusta migratoria. Effects of external ions and inhibitors. J. Insect Physiol. 34,963 -976.[CrossRef]
Beyenbach, K. W. (1995). Mechanism and regulation of electrolyte transport in Malpighian tubules. J. Insect Physiol. 41,197 -207.[CrossRef]
Beyenbach, K. W. (2001). Energizing epithelial
transport with the vacuolar H+-ATPase. News Physiol.
Sci. 16,145
-151.
Beyenbach, K. W. and Masia, R. (2002). Membrane conductances of principal cells in Malpighian tubules of Aedes aegypti.J. Insect Physiol. 48,375 -386.[CrossRef][Medline]
Beyenbach, K. W. and Petzel, D. H. (1987).
Diuresis in mosquitoes: role of a natriuretic factor. News Physiol.
Sci. 2,171
-175.
Brzuszczak, I. M., Zhao, J., Bell, C., Stiel, D., Fielding, I., Percy, J., Smith, R. and O'Loughlin, E. V. (1996). Cyclic AMP-dependent anion secretion in human small and large intestine. J. Gastroenterol. Hepatol. 11,804 -810.[Medline]
Gillen, C. M. and Bowles, D. W. (2001). Kinetic characterization of a K-Cl cotransporter from the insect cell line Sf9. FASEB J. 15,A412 .
Hegarty, J. L., Zhang, B., Carroll, M. C., Cragoe, E. J. J. and Beyenbach, K. W. (1992). Effects of amiloride on isolated Malpighian tubules of the yellow fever mosquito (Aedes aegypti). J. Insect Physiol. 38,329 -337.[CrossRef]
Hegarty, J. L., Zhang, B., Pannabecker, T. L., Petzel, D. H.,
Baustian, M. D. and Beyenbach, K. W. (1991). Dibutyryl cAMP
activates bumetanide-sensitive electrolyte transport in Malpighian tubules.
Am. J. Physiol. Cell Physiol.
261,C521
-C529.
Helman, S. I. (1972). Determination of electrical resistance of the isolated cortical collecting tubule and its possible anatomical location. Yale J. Biol. Med. 45,339 -345.[Medline]
Hopkin, R., Anstee, J. H. and Bowler, K. (2001). An investigation into the effects of inhibitors of fluid production by Locusta Malpighian tubule type I cells on their secretion and elemental composition. J. Insect Physiol. 47,359 -367.[CrossRef][Medline]
Huang, D. Y., Osswald, H. and Vallon, V.
(2000). Sodium reabsorption in thick ascending limb of Henle's
loop: effect of potassium channel blockade in vivo. Br. J.
Pharmacol. 130,1255
-1262.
Hurst, A. M., Duplain, M. and Lapointe, J. Y. (1992). Basolateral membrane potassium channels in rabbit cortical thick ascending limb. Am. J. Physiol. Cell Physiol. 263,F262 -F267.
Hyde, D., Baldrick, P., Marshall, S. L. and Anstee, J. H. (2001). Rubidium reduces potassium permeability and fluid secretion in Malpighian tubules of Locusta migratoria, L. J. Insect Physiol. 47,629 -637.[CrossRef][Medline]
Ianowski, J. P., Christensen, R. J. and O'Donnell, M. J. (2001). Intracellular ion activities in Malpighian tubule cells of Rhodnius prolixus: evaluation of Na/K/2Cl cotransport across the basolateral membrane. J. Exp. Biol. 205,1645 -1655.
Labarca, P., Santi, C., Zapata, O., Morales, E., Beltr'an, C., Li'evano, A. and Darszon, A. (1996). A cAMP regulated K+-selective channel from the sea urchin sperm plasma membrane. Dev. Biol. 174,271 -280.[CrossRef][Medline]
Leyssens, A., Dijkstra, S., Van Kerkhove, E. and Steels, P.
(1994). Mechanisms of K+ uptake across the basal
membrane of Malpighian tubules of Formica polyctena: the effect of
ions and inhibitors. J. Exp. Biol.
195,123
-145.
Leyssens, A., Zhang, S.-L., Van Kerkhove, E. and Steels, P. (1993). Both dinitrophenol and Ba2+ reduce KCl and fluid secretion in Malpighian tubules of Formica: the role of the apical H+ and K+ concentration gradient. J. Insect Physiol. 39,1061 -1073.[CrossRef]
Linton, S. M. and O'Donnell, M. J. (1999).
Contributions of K+/Cl cotransport and
Na+/K+-ATPase to basolateral ion transport in Malpighian
tubules of Drosophila melanogaster. J. Exp. Biol.
202,1561
-1570.
Marunaka, Y., Niisato, N., O'Brodovich, H., Post, M. and Tanswell, A. K. (1999). Roles of Ca2+ and protein tyrosine kinase in insulin action on cell volume via Na+ and K+ channels and Na+/K+/2Cl cotransporter in fetal rat alveolar type II pneumocyte. J. Membr. Biol. 168,91 -101.[CrossRef][Medline]
Masia, R., Aneshansley, D., Nagel, W., Nachman, R. J. and
Beyenbach, K. W. (2000). Voltage clamping single cells in
intact Malpighian tubules of mosquitoes. Am. J. Physiol. Renal
Physiol. 279,F747
-F754.
Neufeld, D. S. and Leader, J. P. (1998). Electrochemical characteristics of ion secretion in Malpighian tubules of the New Zealand alpine weta (Hemideina maori). J. Insect Physiol. 44,39 -48.
Nicoloson, S. W. and Isaacson, L. C. (1987).
Transepithelial and intracellular potentials in isolated Malpighian tubules of
tenebrionid beetle. Am. J. Physiol. Renal Physiol.
252,F645
-F653.
O'Donnell, M. J. and Maddrell, S. H. P. (1984). Secretion by the Malpighian tubules of Rhodnius prolixus. Electrical events. J. Exp. Biol. 110,275 -290.[Abstract]
O'Neill, W. C. and Steinberg, D. F. (1995).
Functional coupling of Na+-K+-2Cl
cotransport and Ca2+-dependent K+ channels in vascular
endothelial cells. Am. J. Physiol. Cell Physiol.
269,C267
-C274.
Pannabecker, T. L., Hayes, T. K. and Beyenbach, K. W. (1993). Regulation of epithelial shunt conductance by the peptide leucokinin. J. Membr. Biol. 132, 63-76.[Medline]
Petzel, D. H. (2000). Na/H exchange in mosquito
Malpighian tubules. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 279,R1996
-R2003.
Petzel, D. H., Berg, M. M. and Beyenbach, K. W.
(1987). Hormone-controlled cAMP-mediated fluid secretion in
yellow-fever mosquito. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 253,R701
-R711.
Petzel, D. H., Hagedorn, H. H. and Beyenbach, K. W.
(1985). Preliminary isolation of mosquito natriuretic factor.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
249,R379
-R386.
Ramsay, J. A. (1953). Active transport of potassium by the Malpighian tubules of insects. J. Exp. Biol. 93,358 -369.
Reagan, J. D. (1995). Molecular cloning of a putative Na+-K+-2Cl cotransporter from the Malpighian tubules of the tobacco hornworm, Manduca sexta.Insect Biochem. Molec. Biol. 25,875 -880.[CrossRef][Medline]
Singh, S. K., Mennone, A., Gigliozzi, A., Fraioli, F. and Boyer,
J. L. (2001). Cl dependent secretory
mechanisms in isolated rat bile duct epithelial units. Am. J.
Physiol. Gastrointest. Physiol.
281,G438
-G446.
Suzuki, M., Takahashi, K. and Sakai, O. (1994). Regulation by GTP of a Ca2+-activated K+ channel in the apical membrane of rabbit cortical collecting duct cells. J. Membr. Biol. 141,43 -50.[Medline]
Teulon, J., Ronco, P. M. and Vandewalle, A.
(1994). Renal cells transformed with SV40 contain a
high-conductance calcium-insensitive potassium channel. Am. J.
Physiol. Cell Physiol. 267,C940
-C945.
Walter, S. J., Shirley, D. G., Folkerd, E. J. and Unwin, R. J. (2001). Effects of the potassium channel blocker barium on sodium and potassium transport in the rat loop of Henle in vivo. Exp. Physiol. 86,469 -474.[Abstract]
Wang, T. (2003). The effects of the potassium
channel opener minoxidil on renal electrolytes transport in the loop of Henle.
J. Pharm. Exp. Therap.
304,833
-840.
Wessing, A., Bertram, G. and Zierold, K. (1993). Effects of bafilomycin A1 and amiloride on the apical potassium and proton gradients in Drosophila Malpighian tubules studied by X-ray microanalysis and microelectrode measurements. J. Comp. Physiol. B 163,452 -462.[Medline]
Wiehart, U. I., Klein, G., Steels, P., Nicolson, S. W. and Van
Kerkhove, E. (2003a). K+ transport in Malpighian
tubules of Tenebrio molitor L: is a K(ATP) channel involved?
J. Exp. Biol. 206,959
-965.
Wiehart, U. I., Nicolson, S. W. and Van Kerkhove, E.
(2003b). K+ transport in Malpighian tubules of
Tenebrio molitor L: a study of electrochemical gradients and basal
K+ uptake mechanisms. J. Exp. Biol.
206,949
-957.
Williams, J. C. and Beyenbach, K. W. (1983). Differential effects of secretagogues on Na and K secretion in the Malpighian tubules of Aedes aegypti (L.). J. Comp. Physiol. 149,511 -517.
Williams, J. C. and Beyenbach, K. W. (1984). Differential effects of secretagogues on the electrophysiology of the Malpighian tubules of the yellow fever mosquito. J. Comp. Physiol. B 154,301 -309.
Wu, D. S. and Beyenbach, K. W. (2003). The
dependence of electrical transport pathways in Malpighian tubules on ATP.
J. Exp. Biol. 206,233
-243.
Xu, W. and Marshall, A. T. (1999a). Effects of inhibitors and specific ion-free salines on segmental fluid secretion by the Malpighian tubules of the black field cricket Teleogryllus oceanicus.J. Insect Physiol. 45,835 -842.[CrossRef][Medline]
Xu, W. and Marshall, A. T. (1999b). X-ray microanalysis of the Malpighian tubules of the black field cricket Teleogryllus oceanicus: The roles of Na/K ATPase and the Na/K/2Cl cotransporter. J. Insect Physiol. 45,885 -893.[CrossRef][Medline]
Yu, M. J. and Beyenbach, K. W. (2002).
Leucokinin activates Ca2+-dependent signal pathway in principal
cells of Aedes aegypti Malpighian tubules. Am. J. Physiol.
Renal Physiol. 283,F499
-F508.
Yu, M. J., Schooley, D. A. and Beyenbach, K. W. (2003). Cyclic GMP induces a basolateral membrane chloride conductance in principal cells of Aedes aegypti Malpighian tubules. FASEB J. 17,A481 .