Intracellular ion activities in Malpighian tubule cells of Rhodnius prolixus: evaluation of Na+-K+-2Cl- cotransport across the basolateral membrane
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1
* e-mail: ianowsjp{at}mcmaster.ca
Accepted 18 March 2002
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Summary |
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The thermodynamic feasibilities of
Na+-K+-2Cl-, Na+-Cl-
and K+-Cl- cotransporters were evaluated by calculating
the net electrochemical potential (µnet/F) for
each transporter. Our results show that a
Na+-K+-2Cl- or a
Na+-Cl- cotransporter but not a
K+-Cl- cotransporter would permit the movement of ions
into the cell in stimulated tubules. The effects of Ba2+ and
ouabain on Vbl and rates of fluid and ion secretion show
that net entry of K+ through ion channels or the
Na+/K+-ATPase can be ruled out in stimulated tubules.
Maintenance of intracellular Cl- activity was dependent upon the
presence of both Na+ and K+ in the bathing saline.
Bumetanide reduced the fluxes of both Na+ and K+. Taken
together, the results support the involvement of a basolateral
Na+-K+-2Cl- cotransporter in
serotonin-stimulated fluid secretion by Rhodnius prolixus Malpighian
tubules.
Key words: Rhodnius prolixus, Malpighian tubule, ion-selective microelectrode, intracellular ion activity, electrochemical potential, ion transport, Na+-K+-2Cl- cotransporter
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Introduction |
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Entry of Na+, K+ and Cl- through a
basolateral Na+-K+-2Cl- cotransporter has
been proposed for tubules of Rhodnius prolixus on the basis of the
effects of bumetanide, Na+-free saline and Cl--free
saline on fluid secretion and transepithelial potential
(O'Donnell and Maddrell, 1984;
Ianowski and O'Donnell, 2001
).
Na+-K+-2Cl- cotransport has also been
implicated in basolateral entry of ions into Malpighian tubules of other
species, including Aedes aegypti
(Hegarty et al., 1991
),
Formica polyctena (Leyssens et
al., 1994
), Manduca sexta
(Audsley et al., 1993
;
Reagan, 1995
),
Teleogryllus oceanicus (Xu and
Marshall, 1999
) and Locusta migratoria
(Al-Fifi et al., 1998
).
In vertebrates, the cation-Cl- cotransport superfamily includes
two Na+-K+-Cl- (NKCC) isoforms, one
Na+-Cl- (NCC) isoform (bumetanide-insensitive,
K+-independent) and four K+-Cl- (KCC)
isoforms. The Na+-K+-Cl- cotransporter is an
electroneutral transporter with a stoichiometry of
1Na+:1K+:2Cl- in the overwhelming majority of
cases (Haas and Forbush,
2000). Nevertheless, a cotransporter with a stoichiometry of
2Na+:1K+:3Cl- has been described
(Russell, 1983
).
The direction of net ion transport by the
Na+-K+-2Cl- cotransporter may be into or out
of the cell depending on the sum of the chemical potential gradients of the
transported ions, and the transporter can be inhibited by loop diuretics such
as furosemide or bumetanide (Haas and
Forbush, 2000). Several alternative routes for Cl-
and/or K+ entry have been proposed in tubules of other insects,
including a K+-Cl- cotransporter in Drosophila
melanogaster (Linton and O'Donnell,
1999
), Ba2+-sensitive K+ channels in
Formica polyctena
(Leyssens et al., 1994
) and
the Na+/K+-ATPase in Locusta migratoria
(Anstee and Bowler, 1979
;
Anstee et al., 1986
) and
Drosophila melanogaster (Linton
and O'Donnell, 1999
). It is important to point out that our
earlier studies (O'Donnell and Maddrell,
1984
; Ianowski and O'Donnell,
2001
) did not preclude the possible involvement of other
transporters such as K+-Cl- or
Na+-Cl- cotransporters, K+ channels or the
Na+/K+-ATPase.
Critical evaluation of the possible roles of cotransporters, exchangers and
ion channels requires measurement of membrane potential and the intracellular
activities of the ions involved so that electrochemical potentials can be
calculated for each ionic species. The directions of net ion movements for a
particular transporter can then be predicted by summing the electrochemical
potentials of all the participating ions to calculate the net
electrochemical potential. For example, proposals of
Na+-K+-2Cl- cotransport during Malpighian
tubule fluid secretion assume that a favourable electrochemical potential for
Na+ influx will drive the coupled influx of Cl- and
K+ against their electrochemical potentials
(Xu and Marshall, 1999;
Ianowski and O'Donnell, 2001
).
However, in spite of the central role of Na+ in fluid secretion by
Malpighian tubules of blood feeders as well as many other insects, there is to
date a single report of intracellular Na+ activity in tubules of
the weta Hemideina maori (Neufeld
and Leader, 1998
), whereas activities of K+ and
Cl- have been measured in Malpighian tubules of Locusta
migratoria (Morgan and Mordue,
1983
), Formica polyctena
(Leyssens et al., 1993
;
Dijkstra et al., 1995
) and
Hemideina maori (Neufeld and
Leader, 1998
). Measurements of intracellular Na+
activity are also critical to evaluation of the links between the transport of
Na+ and other inorganic ions (H+, Ca2+) or
organic solutes (e.g. sugars, amino acids and organic acids).
This paper describes experiments in which basolateral membrane potential and intracellular activities of Na+, K+ and Cl- were measured simultaneously in Malpighian tubules of Rhodnius prolixus. The results support a role for Na+-K+-2Cl- cotransport and rule out significant contributions of K+-Cl- cotransport, Na+/K+-ATPase or K+ channels to net ion entry during serotonin-stimulated fluid secretion.
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Materials and methods |
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Animals were dissected under the appropriate saline
(Table 1) with aid of a
dissecting microscope. Only the fluid-secreting upper Malpighian tubule, which
comprises the upper two-thirds (approximately 25 mm) of the tubule's length,
was used. In contrast to tubules of dipterans, the upper tubule of
Rhodnius prolixus consists of a single cell type whose secretory
properties are uniform along its length
(Collier and O'Donnell,
1997).
|
Secretion assay
Secretion assays were performed as described previously
(Ianowski and O'Donnell,
2001). Briefly, upper segments of Malpighian tubules were isolated
in 100 µl droplets of bathing saline under paraffin oil. The cut end of the
tubule was pulled out of the saline and wrapped around a fine steel pin pushed
into the Sylgard base of a Petri dish. After stimulation with serotonin
(10-6 moll-1), secreted fluid droplets formed at the cut
end of the tubule and were pulled away from the pin every 5 min for 60-90 min
using a fine glass probe. Secreted droplet volume was calculated from droplet
diameter measured using an ocular micrometer. Secretion rate was calculated by
dividing the volume of the secreted droplet by the time over which it
formed.
Measurement of intracellular ionic activity
An isolated upper Malpighian tubule was attached to the bottom of a
custom-built superfusion chamber pre-coated with poly-L-lysine to facilitate
adherence of the tubules under saline
(Ianowski and O'Donnell,
2001). The fluid in the chamber was exchanged at 6 ml
min-1, sufficient to exchange the chamber's volume every 3 s.
Intracellular ion activity and basolateral membrane potential were measured simultaneously in single cells using ion-selective double-barrelled microelectrodes (ISMEs). The ISMEs were fabricated from borosilicate double-barrelled `Piggy-back' capillary glass (WPI, Sarasota, USA). The capillary glass was washed for 30 min in nitric acid, then rinsed with deionized water and baked on a hot plate at 200°C for 30 min. The capillaries were then removed from the hot plate, and the smaller barrel filamented was filled with a 2-3 cm column of deionized water before pulling on a vertical micropipette puller (PE-2, Narishige, Japan). Retention of the hydrophobic ionophore cocktails requires silanization of the interior of the ion-selective barrel. For this purpose, approximately 300 µl of dimethyldichlorosilane (Fluka) was placed in a glass vial. A 23 gauge syringe needle was passed from inside to outside through the plastic cap of the vial. The syringe needle was placed in the lumen of the larger unfilamented barrel. The glass vial was then placed on a hot plate at 200°C for approximately 8 s to produce a stream of dimethyldichlorosilane vapour through the end of the syringe needle so as to silanize the interior of the larger capillary barrel. The water in the smaller barrel prevented silanization of its interior. The double-barrelled capillary was then removed from the syringe needle and baked for 45 min at 200°C. Finally, a short column of liquid ion exchanger was introduced into the larger barrel and it was backfilled with the appropriate solution. The smaller barrel remained hydrophilic and was filled with the appropriate reference electrode solution (see below).
In some cases, the resistance of the ion-selective electrode was above
1011 , resulting in very slow response times and unstable
voltages. Electrode resistance was therefore reduced by controlled
submicrometre tip breakage. The tip of the electrode was touched to the tubule
surface or to the surface of a piece of tissue paper under saline, as
described by O'Donnell and Machin
(1991
). This process of
controlled tip breakage permitted a two- to fourfold reduction in tip
resistance and consequent improvement in response time without compromising
the quality of subsequent impalements. Electrodes were used for experiments
only when the 90 % response time of the ion-selective barrel to a solution
change was less than 30 s and when the response of the ion-selective barrel to
a 10-fold change in ion activity was more than 49 mV. Approximately 40 % of
K+ electrodes, 30 % of Na+ electrodes and 30 % of
Cl- electrodes met these criteria.
K+-selective microelectrodes were based on potassium ionophore I, cocktail B (Fluka). The K+-selective barrel was backfilled with 500 mmol l-1 KCl. The reference barrel was filled with 1 mol l-1 sodium acetate near the tip and shank and 1 mol l-1 KCl in the rest of the electrode. The K+-selective electrode was calibrated in solutions of (in mmol l-1) 15 KCl:135 NaCl and 150 KCl. The mean slope of the K+ electrodes used in this study was 52±1 mV per decade change in K+ activity (mean ± S.E.M., N=22).
Na+-selective microelectrodes were based on the neutral carrier ETH227 (sodium ionophore I, cocktail A, Fluka). The Na+-selective barrel was backfilled with 500 mmol l-1 NaCl and the reference barrel was filled with 1 mol l-1 KCl. Na+-selective electrodes were calibrated in solutions of (in mmol l-1) 15 NaCl:135 KCl and 150 NaCl. The mean slope of the Na+ electrodes used was 57±1.5 mV per decade change in Na+ activity (mean ± S.E.M., N=21). Since Ca2+ is known to interfere with the Na+ neutral carrier ETH227, the bathing saline in these experiments was initially Ca2+-free saline (Table 1). When the microelectrode electrode had impaled the cell, the bathing saline was replaced with control saline. Preliminary experiments showed that exposure of the tubule to Ca2+-free saline did not affect transepithelial ion transport; secretion rates of serotonin-stimulated upper Malpighian tubules were identical in control saline and in Ca2+-free saline.
Cl--selective microelectrodes were based in ionophore I, cocktail A (Fluka). Both Cl--selective and reference barrels were backfilled with 1 mol l-1 KCl. The electrode was calibrated in 100 mmol l-1 KCl and 10 mmol l-1 KCl. The mean slope of the Cl- electrodes used was 53±1.5 mV per decade change in Cl- activity (mean ± S.E.M., N=22).
Potential differences from the reference (Vref) and
ion-selective (Vi) barrels were measured by a
high-input-impedance differential electrometer (FD 223, WPI).
Vref was measured with respect to a Ag/AgCl electrode
connected to the bath through a 0.5 mol l-1 KCl agar bridge.
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
(approximately 1010) of the ion-selective barrel.
Vref and the difference
Vi-Vref were recorded using an A/D
converter and data-acquisition system (Axotape, Axon Instruments, Burlingame,
CA, USA).
Intracellular recordings were acceptable if the potential was stable to within 1 mV for 30 s or longer. In addition, recordings were acceptable only if the potential of each electrode in the bathing saline after withdrawal differed from the potential before impalement by less than 3 mV. In preliminary experiments using fine-tipped single-barrelled microelectrodes, we established that mean values of basolateral membrane potential before and after serotonin stimulation were -58 mV (95 % confidence interval -61 to -55 mV) and -63 mV (95 % confidence interval -65 to -60 mV) respectively. In experiments using double-barrelled ion-selective electrodes, values of basolateral membrane potential (Vbl) less negative than -55 mV in unstimulated tubules and -60 mV in stimulated tubules were considered indicative of poor-quality impalements, and the data were therefore discarded.
Calibration and calculations
Intracellular ion activity was calculated using the formula:
![]() | (1) |
ab was obtained as:
![]() | (2) |
The ion activity in the calibration solution was calculated as the product
of ion concentration and the ion activity coefficient. The activity
coefficients for the single electrolyte calibration solutions are 0.77 and
0.901 for 100 mmol l-1 KCl and 10 mmol l-1 KCl,
respectively (Hamer and Wu,
1972). For the solutions containing 0.15 mol l-1 KCl or
NaCl and mixed solutions of KCl and NaCl with constant ionic strength (0.15
mol l-1), the activity coefficient is 0.75, calculated using the
Debye-Huckel extended formula and Harned's rule
(Lee, 1981
).
Measurement of K+ and Na+ activities in the
secreted droplet
K+ and Na+ activities of secreted droplets were
measured using single-barrelled ion-selective microelectrodes as described
previously (Maddrell and O'Donnell,
1992; Maddrell et al.,
1993
; O'Donnell and Maddrell,
1995
). The K+- selective and Na+-selective
microelectrodes were silanized using the procedures of Maddrell et al.
(1993
). Filling and
calibration solutions of single-barrelled ion-selective and reference
electrodes were the same as those described above for double-barrelled
microelectrodes.
The activity of an ion in a secreted droplet was calculated using the
formula:
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Ion flux (nmol min-1) was calculated as the product of secretion rate (nl min-1) and ion activity (mmol l-1) in the secreted droplets.
Electrochemical potentials
The electrochemical potential (µ/F, in mV) for an ion
across the basolateral membrane was calculated as:
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Thermodynamic evaluation of ion transporters
Thermodynamic evaluation of a particular ion transporter involves
calculation of the net electrochemical potential
(µnet/F)
(Schmidt and McManus, 1977
;
Haas et al., 1982
;
Loretz, 1995
).
µnet/F is calculated as the sum of the
electrochemical potentials (
µ/F) for all the participating
ions. For the Na+-K+-2Cl- cotransporter, the
net electrochemical potential (
µnet/F) is given
by:
![]() | (5) |
![]() | (6) |
![]() | (7) |
A positive value of µnet/F favours net
movement of ions from cell to bath, whereas a negative value would tend to
promote a net movement from bath to cell. When
µnet/F=0 mV, there is no net force operating on
the cotransporter system (Schmidt and
McManus, 1977
; Haas et al.,
1982
; Loretz,
1995
).
Measurement of basolateral membrane potential
To study the role of K+ channels on K+ transport, the
effect of Ba2+ on Vbl was studied. Electrodes
were pulled from filamented single-barrelled capillary pipettes (WPI,
Sarasota, FL, USA) filled with 3 mol l-1 KCl and connected to an
electrometer (Microprobe system M-707A, WPI, Sarasota, FL, USA).
Microelectrode resistance was typically 20-40 M.
Chemicals
Stock solutions of 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) (Ianowski and
O'Donnell, 2001
). Serotonin and ouabain (Sigma) were dissolved in
the appropriate saline solution (Table
1).
Statistical analyses
Results are expressed as means ± S.E.M. Significant differences were
evaluated using unpaired Student's t-tests (P<0.05).
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Results |
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The negative value of µNa/F indicates that
the electrochemical potential favoured movement of Na+ from the
bathing saline into the cell in both unstimulated and stimulated tubules
(Table 2; Fig. 1A).
|
In unstimulated tubules, the K+ electrochemical potential
favoured K+ movement from the cell to the bath
(Table 2). Stimulation with
serotonin reduced both intracellular K+ activity and
µK/F (Table
2; Fig. 1B). For
stimulated tubules,
µK/F was not significantly
different from 0 mV.
In contrast, serotonin stimulation did not affect intracellular
Cl- activity or µCl/F. The
electrochemical potential favoured Cl- movement from the cell to
the haemolymph in both stimulated and unstimulated tubules
(Table 2;
Fig. 1C).
To determine whether other anions in the cell interfered with the Cl- electrode, the effect of replacing Cl- in the bath with SO42- on intracellular Cl- activity was measured. After 10 min in Cl--free saline, intracellular Cl- activity was reduced to 5±0.8 mmol l-1 (N=3) in stimulated tubules. Thus, the interference of other anions on intracellular Cl- measurements was small.
Evaluation of putative ion transporters:
Na+-K+-2Cl-, Na+-Cl- or
K+-Cl- cotransporters
Net electrochemical potentials were calculated for the three
cation-Cl- cotransporters of interest. The results indicated that
in both unstimulated and serotonin-stimulated tubules the electrochemical
potentials favoured movement of Na+, K+ and
Cl- from the bath into the cell through a cotransporter with a
stoichiometry of Na+-K+-2Cl-
(Fig. 2A). The data were also
consistent with net movement of ions from the bath into the cell through a
Na+-Cl- cotransporter
(Fig. 2B), but not through a
K+-Cl- cotransporter. K+-Cl-
cotransport would have produced net movement of ions in the opposite
direction, from the cell to the bath (Fig.
2C).
|
Effects of bumetanide, Na+-free and K+-free
saline on intracellular Cl- activity
Intracellular Cl- activity in serotonin-stimulated tubules (30
min in 10-6 mol l-1 serotonin) showed a significant
decrease from 30±4 mmol l-1 to a minimum of 8±3 mmol
l-1 (N=4) when the bathing saline was changed from 14.5K
saline (containing 137.1 mmol l-1 Na+) to
Na+-free saline (Table
1) for 1-2 min (Fig.
3A). After the initial rapid decline,
aCli subsequently increased on average by
6±2 mmol l-1 (N=4) during sustained exposure to
Na+-free saline (Fig.
3A). Possible explanations for the decline in
aCli and this subsequent small increase are
considered in the Discussion. Intracellular Cl- activity also
showed a significant decrease from 32±3 mmol l-1 in 14.5K
saline to 10±3 mmol l-1 (N=5) in K+-free
saline (Table 1) after 1-2 min
(Fig. 3B). Intracellular
Cl- activity recovered to 43±2 mmol l-1
(N=4) and 37±6 mmol l-1 (N=5) when
Na+ or K+ concentration, respectively, was restored in
the bathing fluid (Fig. 3A,B).
The salines used in these experiments
(Table 1) were slightly
different from those used in previous experiments to permit depletion of a
single cation (i.e. Na+ or K+) without altering the
concentration of the remaining ions in solution.
|
Intracellular Cl- activity declined from 33±3 mmol l-1 (N=5) in control saline to a minimum of 8±0.6 mmol l-1 (N=5) in saline containing 10-5 mol l-1 bumetanide. The effect of Na+-free or K+-free saline on intracellular Cl- activity was greatly reduced in the presence of 10-5 mol l-1 bumetanide. After addition of bumetanide, Cl- activity was further reduced by only approximately 2 mmol l-1 from approx. 8 to approx. 6 mmol l-1 (N=2) when the tubules were exposed to Na+-free saline (Fig. 4A). Similarly, K+-free saline produced a further decrement in intracellular Cl- activity of only 3 mmol l-1 (from 9±0.8 to 6±0.9 mmol l-1, N=3) in tubules treated with 10-5 mol l-1 bumetanide (Fig. 4B).
|
Effects of bumetanide and ouabain on secretion rate and K+
and Na+ flux
Fluid secretion rates, K+ flux and Na+ flux were all
reduced by the addition of 10-5 mol l-1 bumetanide to
serotonin-stimulated Malpighian tubules. Fluid secretion was reduced by 72%
within 30 min of addition of bumetanide
(Fig. 5A). Over the same
period, K+ flux and Na+ flux were reduced by 69% and
87%, respectively (Fig.
5B,C).
|
In contrast, the addition of ouabain 10-4 moll-1 did not affect either fluid secretion rate (Fig. 6A) or K+ flux (Fig. 6B) in serotonin-stimulated tubules.
|
Effects of Ba2+ on fluid secretion and Vbl
To evaluate the possible role of K+ channels in vectorial
movement of K+ across the basolateral membrane and into the cell,
the effects of the K+ channel blocker Ba2+ on fluid
secretion and basolateral membrane potential (Vbl) were
studied. During this experiment, NaH2PO4 was omitted
from the control saline to prevent the precipitation of barium phosphate.
The addition of 6 mmoll-1 Ba2+ had no effect on the fluid secretion rate of Rhodnius prolixus Malpighian tubules stimulated with 10-6 moll-1 serotonin (Fig. 7). Addition of 6 mmoll-1 Ba2+ caused Vbl to depolarize slightly but significantly by 7±1 mV (N=5) (Fig. 8), which is consistent with the presence of basolateral K+ channels.
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Discussion |
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Intracellular ion activities
Intracellular activities of Na+, K+ and
Cl- in the Malpighian tubule cells of Rhodnius prolixus
fall within the range of activities for these ions seen in other insect
epithelia studied using ISMEs. The intracellular Na+ activity
measured in Rhodnius prolixus tubule cells is very similar to that of
32 mmoll-1 for Hemideina maori, measured in saline of
similar osmolality (Neufeld and Leader,
1998). Values of aNai range from 8
mmoll-1 in rectal cells of Schistocerca gregaria
(Hanrahan and Phillips, 1984
)
to 17 mmoll-1 in unstimulated salivary duct cells of
Periplaneta americana (Lang and
Walz, 2001
).
Intracellular Cl- activities of 32 mmoll-1 measured
in Rhodnius prolixus tubules are very similar to values of 38
mmoll-1 in Locusta migratoria tubules
(Morgan and Mordue, 1983) and
35 mmoll-1 in tubules of Formica polyctena
(Dijkstra et al., 1995
).
Similarly, the intracellular K+ activity in unstimulated
Rhodnius prolixus Malpighian tubules (86 mmoll-1) is
comparable with the values of 71 mmoll-1 measured in Locusta
migratoria tubules (Morgan and
Mordue, 1983) and 61 mmoll-1 in Formica
polyctena tubules (Leyssens et al.,
1993
). For comparisons with studies that reported ion
concentrations, aNai,
aKi and aCli in
other tissues have been calculated assuming an activity coefficient of
0.75.
The values for aioni measured directly with
ISMEs in the present study are also similar to those estimated from total
concentration measurements obtained by X-ray microanalysis
(Gupta et al., 1976).
Estimated activities for Na+, Cl- and K+ in
the main cytoplasm of unstimulated Rhodnius prolixus tubules were 10
mmoll-1, 23 mmoll-1 and 77 mmoll-1,
respectively. After serotonin stimulation, estimated activities for
Na+, Cl- and K+ were 32 mmoll-1,
45 mmoll-1 and 76 mmoll-1, respectively
(Gupta et al., 1976
).
Intracellular levels of Na+ and K+ but not
Cl- are altered by stimulation with serotonin. A dramatic rise in
aNai from 17 to 69 mmoll-1 is also
seen when Periplaneta americana salivary ducts are stimulated with
dopamine (Lang and Walz,
2001). The increase in aNai in the
present study is of similar magnitude to the corresponding decrease in
aKi in response to serotonin stimulation. It is
worth noting that aKi declines from 84 to 30
mmoll-1 when Periplaneta americana salivary ducts are
stimulated with dopamine (Lang and Walz,
2001
). In both Rhodnius prolixus tubules and
Periplaneta americana salivary ducts, therefore, the sum of
aNai and aKi
remains constant when the cells are stimulated, but the
Na+:K+ activity ratio increases.
Basolateral electrochemical potentials
The simultaneous measurement of aNai and
Vbl permits accurate calculation of the electrochemical
potential (µNa/F) for Na+ across the
basolateral membrane. Importantly, these calculations show that passive
Na+ entry into the cell is highly favoured in both unstimulated and
stimulated cells.
The electrochemical potential for Cl-
(µCl/F), in contrast, is outwardly directed.
This indicates that Cl- activity in the cell is higher than
expected on the basis of passive distribution of Cl- across the
basolateral membrane in both unstimulated and serotonin-stimulated cells.
Cl- must therefore be actively transported across the basolateral
membrane into the cell. Outwardly directed electrochemical potentials for
Cl- have also been reported in Locusta migratoria
Malpighian tubules (Morgan and Mordue,
1983
). In contrast,
µCl/F in
Formica polyctena Malpighian tubule cells favours Cl-
movement into the cell across the basolateral membrane
(Dijkstra et al., 1995
).
The electrochemical potential for K+
(µK+/F) across the basolateral
membrane is outwardly directed in unstimulated tubules, whereas values of
µK/F near 0 mV are found after serotonin
stimulation. Similarly, values of
µK/F across
the basolateral membrane not different from zero have been measured for
Malpighian tubule cells of Locusta migratoria
(Morgan and Mordue, 1983
) and
Formica polyctena (Leyssens et
al., 1993
).
Net electrochemical potentials for cation-Cl-
cotransporters
The feasibility of K+-Cl-,
Na+-K+-2Cl- and Na+-Cl-
cotransporters in ion movement across the basolateral membrane was evaluated
by calculating the net electrochemical potential using the electrochemical
potentials for each participating ion
(Schmidt and McManus, 1977;
Haas et al., 1982
;
Loretz, 1995
). The results
show that a K+-Cl- cotransporter would drive net
movement of K+ and Cl- from cell to bath, i.e. in the
opposite direction to that during fluid secretion. It is important to point
out that our data do not preclude the presence of a
K+-Cl- cotransporter and its involvement in other
functions such as cell volume regulation in hypo-osmotic media
(Lauf et al., 1992
). However,
our findings do show that a K+-Cl- cotransporter could
not be involved in vectorial ion transport during fluid secretion by the
Rhodnius prolixus Malpighian tubule.
Both Na+-K+-2Cl- and Na+-Cl- cotransporters favour coupled movement of Na+, K+ and Cl- or of Na+ and Cl-, respectively, from bath to cell in both unstimulated and serotonin-stimulated tubules. Na+-Cl- cotransport, however, is inconsistent with the finding that bumetanide decreases transepithelial fluxes of both Na+ and K+, rather than just that of Na+.
Our results also show that maintenance of
aCli is dependent upon the presence of both
Na+ and K+ and is bumetanide-sensitive. Intracellular
Cl- activity declines in response to bumetanide, K+-free
saline or Na+-free saline. Moreover, our results suggest that
intracellular Cl- activity is near equlibrium across the apical
membrane. For example, in serotonin-stimulated cells with an
aCli of 32 mmol l-1, the measured
apical membrane potential is -31 mV, cell-negative
(Ianowski and O'Donnell,
2001). The latter value is very close to the Nernst equilibrium
potential for Cl- (ECl) across the apical
membrane (-34mV), assuming a luminal Cl- activity of approximately
124 mmol l-1. Moreover, when the apical membrane potential
increases (i.e. as the transepithelial potential becomes more lumen-positive)
in Na+-free saline or K+-free saline or in the presence
of bumetanide (Ianowski and O'Donnell,
2001
), aCli declines, as observed
in the present study (Fig. 3).
We suggest that the subsequent small increase in
aCli after the initial rapid decline in
Na+-free saline (Fig.
3A) reflects changes in apical membrane potential. Earlier studies
(O'Donnell and Maddrell, 1984
)
have shown that transepithelial potential increases to a lumen-positive value
in Na+-free saline, then gradually declines. Taken together, our
results suggest that basolateral Na+-K+-2Cl-
cotransport and apical Cl- channels together play a primary role in
setting the level of intracellular Cl- activity. Future studies
will examine electrochemical gradients across the apical membrane in detail
and will also address possible contributions from other transporters (e.g.
basolateral Cl-/HCO3- exchangers).
There is also molecular biological evidence for cation-Cl-
cotransporters in Malpighian tubules of an insect. A putative
Na+-K+-2Cl- cotransporter cloned from
Manduca sexta tubules (MasBSC)
(Reagan, 1995) shares 40-43%
sequence identity with the shark, rat and mouse bumetanide-sensitive
Na+-K+-2Cl- cotransporter, 40% with human and
mouse thiazide-sensitive Na+-Cl- cotransporters and
25-26% sequence identity with mouse K+-Cl-
cotransporters. MasBSC appears to be one of the oldest members of the family
of Na+-(K+)-Cl- transporters reported to date
(Reagan, 1995
;
Mount et al., 1998
). MasBSC
also shares 52% amino acid sequence identity with the CG2509 gene product of
Drosophila melanogaster, suggesting that this putative
Na+-K+-2Cl- cotransporter may occur in other
insects as well.
K+ channels and the
Na+/K+-ATPase
The involvement of the Na+/K+-ATPase in
serotonin-stimulated fluid secretion can be rejected on the grounds that
ouabain does not reduce fluid secretion rate or K+ flux. We can
also rule the contribution of K+ channels to transport of
K+ from bath to lumen during fluid secretion by Rhodnius
prolixus tubules on the basis of two sets of evidence. First, treatment
with Ba2+ did not affect fluid secretion, suggesting that
K+ channels are not a significant component of transepithelial
K+ transport at physiological concentrations of extracellular
K+. Second, basolateral membrane potential depolarised after
addition of Ba2+. This effect is consistent with blockage of
K+ leakage into the bath, since the basolateral membrane potential
appears to be determined primarily by the K+ conductance (i.e.
Vm is very similar to EK). The
depolarisation of the basolateral membrane potential after addition of
Ba2+ is consistent with a small but positive value for
µK/F that favours movement of K+ from
cell to bath through ion channels. Although the magnitude of
µK/F indicated in
Table 2 (0±2mV) is
indistinguishable from zero, this may reflect the limitations inherent in the
use of double-barrelled ion-selective microelectrodes for measurement of very
small (<1mV) changes in potential. Importantly, since net K+
transport during fluid secretion is in the opposite direction, from bath to
cell, passive K+ movement through channels is not involved in
vectorial ion transport during serotonin-stimulated fluid secretion. It is
also worth noting that, in insect epithelia where basolateral K+
channels play a role in transepithelial K+ secretion driven by
apical H+-ATPases and K+/H+ exchangers,
Ba2+ results not in a depolarisation of Vbl but
in a substantial hyperpolarisation, as discussed in detail by Weltens et al.
(1992
) for Formica
polyctena tubules by Moffett and Koch
(1992
) for the lepidopteran
midgut.
Taken together, our results are consistent with a cardinal role for a
bumetanide-sensitive Na+-K+-2Cl-
cotransporter during serotonin-stimulated fluid secretion by Malpighian
tubules of Rhodnius prolixus. We also found a favourable net
electrochemical potential for this transporter in unstimulated tubules. The
operation of the Na+-K+-2Cl- cotransporter at
a low rate in unstimulated tubules has been suggested previously
(Maddrell and Overton, 1988).
In unstimulated tubules, K+ may enter cells from the bath both
through a ouabain-sensitive Na+/K+-ATPase and a low
level of activity of a Na+-K+-2Cl-
cotransporter (Maddrell and Overton,
1988
). Addition of ouabain reduces one path for K+
entry and blocks the transport of Na+ from cell to bath, with a
resulting increase in Na+ transport from cell to lumen
(Maddrell and Overton,
1988
).
Stimulation with serotonin results in a nearly 1000-fold increase in the
rate of transepithelial ion transport through stimulation of apical ion
transporters as well as the basolateral
Na+-K+-2Cl- cotransporter
(Maddrell and Overton, 1988;
Maddrell, 1991
). Because ion
flux through the basolateral cotransporter is so much greater than that
through the Na+/K+-ATPase, fluid secretion in stimulated
tubules is insensitive to ouabain. Moreover, if the rates of ion transport
through K+ channels and the Na+/K+-ATPase are
negligible relative to the rate of ion influx through the
Na+-K+-2Cl- cotransporter, then levels of
Na+ and K+ in the cell would tend to become equal.
However, it is well known that the apical transporters (i.e. the combined
effects of the H+-ATPase and the alkali cation/H+
exchangers) have a preference for Na+ over K+, resulting
in selective transfer of Na+ into the lumen
(Maddrell, 1978
;
Maddrell and O'Donnell, 1993
).
Under these conditions then, one might expect intracellular Na+
activity to increase, but not to the level of
aKi. This was the pattern of changes in
aNai and aKi
observed in the present study.
The finding of a large inwardly directed electrochemical potential for
Na+ across the basolateral membrane of Rhodnius prolixus
tubules suggests that the Na+ gradient may be utilised for other
Na+-coupled transporter systems in addition to the
Na+-K+-2Cl- cotransporter.
K+-coupled transporters for uptake of organic molecules such as
amino acids have been well described in epithelia from species such as
Manduca sexta with low levels of Na+ in the haemolymph
(Castagna et al., 1997;
Liu and Harvey, 1996
;
Bader et al., 1995
). Future
studies of Rhodnius prolixus Malpighian tubules will address the role
of the basolateral Na+ gradient in processes such as solute uptake
(amino acids, sugars, organic acids) or pH regulation through
Na+/H+ exchange (see
Petzel, 2000
).
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References |
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