Na+ competes with K+ in bumetanide-sensitive transport by Malpighian tubules of Rhodnius prolixus
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada, L8S 4K1
* Author for correspondence (e-mail: odonnell{at}mcmaster.ca)
Accepted 15 July 2004
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Summary |
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Key words: ion transport, Na+/K+/2Cl cotransporter, stoichiometry, K+ homeostasis
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Introduction |
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Rhodnius feeds on blood that is hypo-osmotic to its own haemolymph
and it must therefore produce hypo-osmotic, Na+-rich urine to
maintain homeostasis. This is accomplished by first secreting a near
iso-osmotic fluid containing approximately equimolar NaCl and KCl into the
lumen of the upper Malpighian tubule, then reabsorbing KCl but not water
across the lower Malpighian tubule
(Maddrell and Phillips, 1975).
In the absence of reabsorption, the haemolymph content of K+ would
be exhausted within 1 min (Maddrell et
al., 1993
). The activities of the upper and lower segments of the
tubule are therefore tightly coordinated in order to prevent K+
depletion (Maddrell et al.,
1993
). Two different mechanisms contribute to haemolymph
K+ homeostasis. First, the lower reabsorptive segment is stimulated
more rapidly than the upper segment by diuretic hormones. Second, changes in
the K+ concentration of the haemolymph evoke autonomous regulatory
responses of the tubule itself. A dramatic fall in K+ concentration
in the haemolymph causes a decrease in fluid and K+ secretion rate
by the upper tubule and an increase in K+ reabsorption by the lower
tubule (Maddrell et al.,
1993
).
The current model for ion transport during fluid secretion by the upper
Malpighian tubule proposes that the movement of ions occurs through
transcellular pathways (Fig.
1). Ion transport is driven by an apical vacuolar-type
H+-ATPase that energizes amiloride-sensitive
K+/H+ and/or Na+/H+ exchange. The
movement of Cl into the lumen is proposed to be a passive
consequence of a favourable electrochemical potential across the apical
membrane (Wieczorek et al.,
1989; Maddrell and O'Donnell,
1992
; Ianowski and O'Donnell,
2001
).
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Entry of Na+, K+ and Cl through a
basolateral Na+/K+/2Cl cotransporter
has been proposed on the basis of the electrochemical potentials for
K+, Cl and Na+ and on the effects of
bumetanide on Rhodnius Malpighian tubule cells
(O'Donnell and Maddrell, 1984;
Ianowski and O'Donnell, 2001
).
Secretion of Na+, K+, Cl and fluid is
blocked by bumetanide in the absence of any effects upon basolateral membrane
potential (O'Donnell and Maddrell,
1984
; Ianowski and O'Donnell,
2001
). Recent studies have shown that the intracellular activities
of K+ and Cl are above the values consistent with
electrochemical equilibrium. Thus, both ions must be actively transported into
the cell during fluid secretion (Ianowski
et al., 2002
). On the other hand, the intracellular activity of
Na+ is below electrochemical equilibrium, and the gradient for
passive movement of Na+ from bath to cell is sufficient to drive
the influx of both K+ and Cl through a
Na+/K+/2Cl cotransporter
(Ianowski et al., 2002
). In
addition, Cl transport is linked to transport of both
K+ and Na+ (Ianowski
et al., 2002
). Na+/K+/2Cl
cotransport has also been implicated in basolateral entry of ions into
Malpighian tubules of other species, including the mosquito Aedes
aegypti (Hegarty et al.,
1991
), the ant Formica polyctena
(Leyssens et al., 1994
), the
moth Manduca sexta (Audsley et
al., 1993
; Reagan,
1995
), the cricket Teleogryllus oceanicus
(Xu and Marshall, 1999
) and
the locust Locusta migratoria
(Al-Fifi et al., 1998
).
The results reported in Rhodnius tubules are consistent with a
predominant role for a basolateral bumetanide-sensitive and electroneutral
Na+/K+/2Cl cotransporter during fluid
secretion. The Na+/K+/2Cl
cotransporters studied to date in other systems have consistently demonstrated
sensitivity to bumetanide and its congeners and electroneutrality.
Na+/K+/2Cl cotransporters have also
been shown to require the presence of the three ions on the same side of the
membrane for ion translocation. Furthermore, in the overwhelming majority of
the cases studied, increments in either Na+ or K+
concentration stimulate secretion of each other and do not inhibit (for
reviews, see Russell, 2000;
Haas and Forbush, 2000
;
Mount et al., 1998
).
The pharmacology and electrophysiology of tubule function and the
electrochemical potentials for Na+, K+ and
Cl across the basolateral membrane all suggest the
contribution of an Na+/K+/2Cl
cotransporter to fluid secretion. However, tubules are also capable of
secreting fluid at high rates in the absence of K+ in the bath
(Maddrell, 1969). Moreover,
reduction of bath K+ concentration leads to a decrement in
K+ flux and a corresponding increment of Na+ flux while
total cation flux remains constant. These changes suggest that the tubule is
able to increase Na+ transport at the expense of K+
(Maddrell, 1969
;
Maddrell et al., 1993
), in
conflict with a predominant role for an
Na+/K+/2Cl cotransporter of invariant
stoichiometry.
This paper addresses this conflict through studies of the effects of
changes in the concentration of Na+ and/or K+ in the
bathing saline on upper Malpighian tubules. We have directly tested a previous
proposal that the cation/chloride cotransporter in the Rhodnius
Malpighian tubule may accept stoichiometries other than
1Na+/1K+/2Cl
(O'Donnell and Maddrell,
1984). Furthermore, the possible contribution of a
thiazide-sensitive Na+/Cl cotransport mechanism
has been examined. The results suggest that small changes in bathing saline
K+ or Na+ concentration greatly alter the rate of
Na+ secretion relative to that of K+ through a
bumetanide-sensitive mechanism and that these changes would contribute to
homeostatic regulation of haemolymph K+ concentration during
diuresis.
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Materials and methods |
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Insects were dissected under control saline with the aid of a dissecting
microscope. We used only the fluid-secreting upper tubule, which comprises the
upper two-thirds (25 mm) of the tubule's length. In contrast to tubules
of dipterans, which are comprised of stellate cells and principal cells, the
upper tubule of Rhodnius is comprised of a single cell type whose
secretory properties are uniform along its length
(Collier and O'Donnell, 1997
).
The external diameter of the tubule is
90 µm and the diameter of the
lumen is
70 µm (Maddrell,
1991
).
Physiological salines
The tubules were bathed in one of 15 salines. Control saline consisted of
(in mmol l1): 122.6 NaCl, 14.5 KCl, 8.5 MgCl2, 2
CaCl2, 20 glucose, 10.2 NaHCO3, 4.3
NaH2PO4, 8.6 Hepes. K+-free saline consisted
of (in mmol l1): 122.6 NaCl, 8.5 MgCl2, 2
CaCl2, 20 glucose, 10.2 NaHCO3, 4.3
NaH2PO4, 8.6 Hepes, 14.5
N-methyl-D-glucamine (NMDG). Na+-free saline
consisted of (in mmol l1): 8.5 MgCl2, 2
CaCl2, 20 glucose, 10.2 KHCO3, 4.3
KH2PO4, 8.6 Hepes, 137.1 NMDG. An additional 12 salines
with 6, 8, 10, 12 or 14.5 mmol l1 K+ and 98, 120
or 137.1 mmol l1 Na+ were made by replacing the
control concentrations of Na+ (137.1 mmol l1)
and/or K+ (14.5 mmol l1) with NMDG. It is worth
noting that some previous studies have used K+-free or
Na+-free salines that were made by replacing one cation with the
other. Thus, the Na+-free saline had an excess of K+,
and K+-free saline had an excess of Na+ and, as a
result, the fluid secretion rates reported previously
(Maddrell, 1969) are
significantly different from those described here.
Secretion assay
Malpighian tubule fluid secretion rates were measured using a modified
Ramsay assay (Ramsay, 1954) as
described previously (Ianowski and
O'Donnell, 2001
). Briefly, the 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 (106 mol l1),
secreted fluid droplets formed at the cut end of the tubule and were pulled
away from the pin every 5 min for 4060 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 apical membrane potential
Apical membrane potential (Vap) was measured using
intracellular recording procedures described previously
(Ianowski and O'Donnell,
2001). The reference electrode was placed inside the cell and the
voltage-sensing electrode was positioned in the lumen of the tubule. Since
apical membrane potential is normally defined as the potential of the cell
relative to the lumen, this arrangement of the electrodes measures
Vap. Measurement of Vap
facilitates visual comparison of apical membrane potential with
transepithelial potential (Ianowski and
O'Donnell, 2001
). In recordings of Vap
presented in the Results, upward shifts correspond to more lumen-positive
potentials.
Measurement of luminal ion activity
Luminal K+ activity and transepithelial potential were measured
simultaneously in Malpighian tubules using ion-selective double-barrelled
microelectrodes (ISMEs), which were fabricated as described previously
(Ianowski et al., 2002). The
tip of the K+-selective barrel was filled with potassium ionophore
I, cocktail B (Fluka, Buchs, Switzerland) and the barrel was then backfilled
with 500 mmol l1 KCl. There is negligible interference of
other luminal 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 Na acetate near the tip and shank and 1
mol l1 KCl in the barrel of the electrode. Double-barrelled
ISMEs were used for experiments only when the response of the ion-selective
barrel to a 10-fold 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 an Ag/AgCl electrode connected to the bath through a
0.5 mol l1 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 (>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).
Luminal recordings were acceptable if the potential for 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 differed from the potential before impalement by less than 3 mV and
if transepithelial potential in serotonin-stimulated tubules in control saline
was more negative than 20 mV. Positioning of the microelectrode tip in
the lumen was confirmed by the positive-going change in potential in response
to 105 mol l1 bumetanide
(Ianowski and O'Donnell,
2001).
Measurement of intracellular ion activity
Intracellular K+ activity was measured using double-barrelled
K+-selective microelectrodes as described in Ianowski et al.
(2002). Intracellular
recordings were acceptable if the potential for 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
differed from the potential before impalement by less than 3 mV and if
basolateral membrane potential (Vbl) was more negative
than 60 mV (Ianowski et al.,
2002
). Impalements that produced Vbl values
less negative than 60 mV were considered of poor quality and the data
were discarded.
Calculations
Luminal ion activity was calculated using the formula:
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Intracellular ion activity (ai) was calculated using
the formula:
![]() | (2) |
ab was calculated as:
![]() | (3) |
The ion activity in the calibration solution was calculated as the product
of ion concentration and the corresponding activity coefficient. The activity
coefficient for solutions containing 150 mmol l1 KCl and for
mixed solutions of KCl and NaCl with constant ionic strength (150 mol
l1) is 0.75, calculated using the DebyeHuckel
extended formula and Harned's rule (Lee,
1981).
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
).
K+-selective 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. The tip of each
Na+-selective microelectrode was filled with a neutral carrier
ionophore cocktail (sodium ionophore I, cocktail A; Fluka), and the electrode
was then backfilled with 500 mmol l1 NaCl. Reference
microelectrodes were filled with 1 mol l1 KCl.
Na+-selective electrodes were calibrated in solutions of (in mmol
l1) 15 NaCl:135 KCl and 150 NaCl.
Ion activity in secreted droplets was calculated using the formula:
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Ion flux (nmol min1) was calculated as the product of secretion rate (nl min1) and ion activity (mmol l1) in the secreted droplets.
Drugs
Stock solutions of hydrochlorothiazide and bumetanide (Sigma, St Louis, MO,
USA) were prepared in ethanol so that the maximum final concentration of
ethanol was <1% (v/v). Previous studies have shown that Malpighian tubule
secretion rate is unaffected by ethanol at concentrations of <1% (v/v)
(Ianowski and O'Donnell,
2001). Serotonin (Sigma) was dissolved in the appropriate
saline.
Statistics
Results are expressed as means ±
S.E.M. Significant differences were evaluated
using Student's t-test and one-way or two-way analysis of variance
(ANOVA) as required. Data expressed as percentages were arcsin transformed
prior to statistical analysis. Doseresponse curves were fitted to the
MichaelisMenten equation using SigmaPlot 2000 (SPSS Inc., Chicago, IL,
USA).
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Results |
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Effects of serotonin and bumetanide on Vap of tubules bathed in K+-free or Na+-free saline
Previous studies have shown that stimulation of Malpighian tubules with
serotonin produces a characteristic triphasic change in transepithelial
membrane potential (O'Donnell and
Maddrell, 1984; Ianowski and
O'Donnell, 2001
). This response reflects changes in the apical
membrane potential due to the sequential activation of different ion transport
systems (Fig. 3). Stimulation
of tubules bathed in K+-free saline with 106 mol
l1 serotonin elicited changes in Vap
that consisted of three different phases as reported for tubules in control
saline (Fig. 3). By contrast,
tubules exposed to Na+-free saline showed a biphasic change in
Vap, corresponding to the first two phases of the control
response (Fig. 3). There was no
significant change in Vap in Na+-free saline
for 515 min after the peak value in phase 2 was established
(Fig. 3).
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Vap became more positive by 32±7 mV (N=4) after addition of 105 mol l1 bumetanide to serotonin-stimulated tubules bathed in K+-free saline (Fig. 3). As discussed below, this change in potential is consistent with blockage of basolateral Cl entry. By contrast, there was no change in Vap when 105 mol l1 bumetanide was added to stimulated tubules bathed in Na+-free saline (Fig. 3).
Effects of bumetanide on fluid secretion
Addition of bumetanide reduced fluid secretion rate in a dose-dependent
manner for tubules bathed in control and K+-free salines
(Fig. 4A,B). Fluid secretion by
tubules in Na+-free saline was not affected by addition of
bumetanide (Fig. 4C).
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Bumetanide inhibited tubule fluid secretion to the same extent in control or K+-free saline. At each bumetanide concentration, the percentage inhibition of fluid secretion rate did not differ significantly in the two salines (Student's t-test). Kinetic parameters were obtained by fitting the data to the MichaelisMenten equation (Fig. 5). The values of IC50 for tubules bathed in control and K+-free saline were 2.9x106 mol l1 and 2.6x106 mol l1, respectively. The maximal inhibition was 97% and 96% for tubules in control and K+-free saline, respectively (non-linear regression; SigmaPlot).
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Taken together the results showed that tubules in control or K+-free saline underwent similar changes in Vap in response to stimulation with serotonin, and that transepithelial ion transport mechanisms in the two salines were equally sensitive to bumetanide. By contrast, bumetanide had no effect on fluid secretion rate or Vap of tubules bathed in Na+-free saline.
Hydrochlorothiazide has no effect on fluid secretion rate
Previous reports have shown that the contribution of an
Na+/Cl cotransporter to ion transport in
serotonin-stimulated tubules is thermodynamically feasible
(Ianowski et al., 2002). In
order to test the possible contribution of a thiazide-sensitive
Na+/Cl cotransporter to fluid secretion rate by
tubules bathed in control and K+-free saline, the effect of
hydrochlorothiazide was tested. Addition of 104 mol
l1 hydrochlorothiazide did not alter fluid secretion in
tubules bathed in either control or K+-free saline
(Fig. 6).
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Effect of changes in bathing saline Na+ and K+ concentrations on fluid secretion rate
Fluid secretion rates of Malpighian tubules bathed in saline containing a
constant Na+ concentration (137.1 mmol l1) and
two different concentrations of K+ (10 and 6 mmol
l1) did not differ significantly
(Fig. 7A). Fluid secretion was
equally sensitive to bumetanide in saline containing 6 or 10 mmol
l1 K+ (Fig.
7A). However, K+ and Na+ secretion rates
were significantly different in the two bathing salines. Tubules bathed in
saline containing 10 mmol l1 K+ secreted
Na+ and K+ at almost equal rates
(Fig. 7B), whereas tubules
bathed in saline containing 6 mmol l1 K+ secreted
Na+ at a rate more than 3-fold greater than that of K+
(Fig. 7B). Total cation flux
was equal in both salines (Fig.
7B), indicating that Na+ replaces K+ for
transport when bath K+ concentration is reduced from 10 to 6 mmol
l1.
|
Increasing Na+ concentration in the bathing fluid from 98 mmol l1 to 120 or 137.1 mmol l1 had no effect on fluid secretion rate (Fig. 8). However, statistical analysis using two-way ANOVA and Tukey HSD for unequal sample sizes indicated that increasing Na+ concentration in the bath significantly reduced K+ flux, whereas increasing K+ concentration in the bath significantly increased K+ flux (Fig. 9A). The maximum transport rate (Jmax) and the bathing saline K+ concentration corresponding to 50% of the maximum transport rate (Kt) for K+ transport were derived from double-reciprocal plots of K+ flux against bath K+ concentration in the three different Na+ concentrations (Fig. 9B). Linear regression analysis revealed that the data fit three straight lines with r2 values of >0.98 for tubules bathed in saline containing 98, 120 and 137.1 mmol1 Na+ (Fig. 9B). Maximal transport rates were very similar in salines containing different Na+ concentrations. Values of Jmax were 14.7, 14.1 and 15.1 nmol min1 for tubules in bathing saline containing 98, 120 and 137.1 mmol l1 Na+, respectively. On the other hand, Kt increased with increasing Na+ concentration in the bath. Values of Kt were 5.8, 6.6 and 9.5 mmol l1 K+ for tubules bathed in saline containing 98, 120 and 137.1 mmol l1 Na+, respectively. These results suggest that increases in bathing fluid Na+ concentration reduce the affinity of transepithelial ion transporters for K+ but do not affect the number of transporters in the epithelium, consistent with competitive inhibition of K+ transport by Na+.
|
|
Large changes in bathing saline Na+ and K+ concentrations had little effect on intracellular K+ activity. In tubule cells bathed in saline containing 8 mmol l1 K+ and 137.1 mmol l1 Na+, intracellular K+ activity was 78±6 mmol l1. Intracellular K+ activity increased to 80±6 mmol l1 after 10 min exposure to saline solution containing 14.5 mmol l1 K+ and 98 mmol l1 Na+ (P<0.05, paired t-test, N=5). Simultaneously, luminal K+ activity increased from 55±6 mmol l1 to 65±6 mmol l1 (P<0.05, paired t-test, N=5).
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Discussion |
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Na+ replacement of K+ during fluid secretion by stimulated tubules
Although tubules bathed in saline containing 6 or 10 mmol
l1 K+ and 137.1 mmol l1
Na+ secreted fluid at equal rates, the fluxes of Na+ and
K+ differed greatly. For tubules in saline containing 6 mmol
l1 K+, Na+ flux was >3-fold that of
K+, whereas the fluxes of Na+ and K+ were
equal in saline containing 10 mmol l1 K+. Total
cation (Na++K+) flux was the same in the two salines.
Moreover, addition of bumetanide reduced fluid secretion rate to the same
extent in both salines. These findings are consistent with replacement of
K+ by Na+ in a bumetanide-sensitive cotransport
mechanism during serotonin-stimulated fluid secretion.
In the extreme case of complete removal of K+ from the bathing saline, Malpighian tubules secreted fluid at 45% of the control rate through a mechanism that was insensitive to hydrochlorothiazide but remained sensitive to bumetanide. Moreover, the doseresponse curves relating percentage inhibition of fluid secretion to bumetanide concentration were identical for tubules bathed in control and K+-free saline. These results suggest that fluid secretion involves the same bumetanide-sensitive cotransport system in the presence or absence of K+.
Electrophysiological experiments provide further evidence that the same
cotransporter operates in both control and K+-free salines.
Malpighian tubules bathed in control saline show a characteristic triphasic
change in apical membrane potential in response to stimulation with serotonin
(Ianowski and O'Donnell,
2001). The first phase corresponds to the opening of apical
Cl channels that drives the lumen of the tubules negative.
The second phase corresponds to activation of the apical H+-pump
that drives the lumen positive. The third phase reflects activation of a
basolateral bumetanide-sensitive
Na+/K+/2Cl cotransporter. The
activation of this basolateral Cl entry pathway increases
Cl activity in the cell, thereby increasing the gradient for
Cl to cross the apical membrane through Cl
channels and thus driving the lumen more negative
(O'Donnell and Maddrell, 1984
;
Ianowski and O'Donnell 2001
;
Ianowski et al., 2002
).
Blockage of the Na+/K+/2Cl
cotransporter with bumetanide drives the lumen positive by 65 mV
(O'Donnell and Maddrell, 1984
;
Ianowski and O'Donnell, 2001
)
and reduces intracellular Cl activity from 33 to 8 mmol
l1 (Ianowski et al.,
2002
). These changes are consistent with continued activity of the
H+-ATPase during bumetanide blockade of the basolateral
Cl entry pathway
(Ianowski and O'Donnell, 2001
;
Ianowski et al., 2002
).
Malpighian tubules bathed in K+-free saline also underwent a triphasic change of apical membrane potential in response to stimulation with serotonin. Moreover, addition of bumetanide also resulted in a lumen-positive shift in apical membrane potential. Taken together, our measurements of fluid secretion rates, cation fluxes and apical membrane potentials indicate that Na+ can replace K+ in a single bumetanide-sensitive cotransport mechanism in the basolateral membrane.
By contrast, fluid secretion by tubules bathed in Na+-free saline did not involve a bumetanide-sensitive transport step. The biphasic change in apical membrane potential in response to serotonin in Na+-free saline corresponded to the first two phases of the triphasic response seen in control saline. The absence of the third phase suggests that there is no activation of a bumetanide-sensitive pathway for Cl entry across the basolateral membrane. Moreover, bumetanide had no effect on fluid secretion rate or apical membrane potential in stimulated tubules in the absence of Na+ in the bathing saline.
Na+ competes with K+ for transport
Our kinetic analysis suggests that Na+ competes with
K+ for transport by the basolateral bumetanide-sensitive
cation/Cl cotransporter during fluid secretion.
Increasing Na+ concentration in the bath increases
Kt for K+ transport while the maximum
transepithelial K+ flux (Jmax) remains
constant, consistent with competitive inhibition of K+ transport by
Na+. It is worth emphasizing that this is not a common finding in
studies of bumetanide-sensitive transport mechanisms. There is a single report
of Na+ inhibition of K+ transport in the B variant of
NKCC2 of rabbits expressed in Xenopus oocytes
(Gimé nez et al., 2002).
The most common finding is that increasing bathing saline Na+
concentration produces an increase in K+ flux through
bumetanide-sensitive Na+/K+/2Cl
cotransporters (for reviews, see Russell,
2000
; Haas and Forbush,
2000
; Mount et al.,
1998
). Transport of K+ by the
Na+/K+/2Cl cotransporter is stimulated
by increases in bathing saline Na+ concentration in HeLa cells
(Miyamoto et al., 1986
), duck
erythrocytes (Haas and McManus,
1982
) and renal epithelial cell lines
(Rindler et al., 1982
;
Brown and Murer, 1985
).
Affinity of the Na+/K+/2Cl
cotransporter for the K+ surrogate Rb+ increases in HeLa
cells when bathing saline Na+ concentration increases
(Miyamoto et al., 1986
).
Double-reciprocal plots of Rb+ uptake vs Rb+
concentration in the bath show that increases in Na+ concentration
in the bath reduce Kt for K+ transport without
affecting Jmax
(Miyamoto et al., 1986
).
The kinetic parameters Kt and Jmax
reported here were based on measurements of transepithelial K+ flux
and therefore include the contributions of both apical and basolateral
transporters. The competition between K+ and Na+ for
entry into the cell is best explained on the basis of competition for binding
to a bumetanide-sensitive transporter. In particular, an increase in bathing
saline K+ concentration from 8 to 14.5 mmol l1
represents an 81% increase in the availability of this ion for a basolateral
transporter. However, our measurements of changes in intracellular
K+ activity in response to changes in bathing saline Na+
and K+ concentration indicate that intracellular K+
levels are tightly regulated and that an 81% increase in extracellular
K+ concentration is associated with a change of only 2% in
intracellular K+ activity. The secreted fluid K+
concentration increases by 20% in response to an increase in bathing saline
K+ concentration from 8 to 14.5 mmol l1. It seems
unlikely that a 2% change in intracellular K+ activity could have a
very large effect on the activity of the apical K+ and
Na+ transporters. This is particularly true for Rhodnius
tubules because the apical transporters show a preference for Na+
over K+ (Maddrell and
O'Donnell, 1993). Instead, our data are most consistent with
regulation of the rate of K+ transport by the apical transporters
by mechanisms that do not involve sustained changes in intracellular
K+ activity. In the early distal tubule of the frog kidney, an NaCl
reabsorbing epithelium, coordination of Na+ uptake across the
apical membrane and Na+ transport across the apical membrane is
achieved not by Na+ availability in the intracellular compartment
but by the intracellular second messenger Ca2+
(Cooper et al., 2001
;
Fowler et al., 2004
). Changes
in intracellular chloride have been proposed to function as a second
messenger, mediating the release of calcium from intracellular stores
(Cooper et al., 2001
;
Fowler et al., 2004
). We have
shown large changes in intracellular Cl activity in
Rhodnius Malpighian tubules in response to bumetanide,
K+-free-media and Na+-free media
(Ianowski et al., 2002
). Thus,
a mechanism analogous to that in the frog kidney may mediate basolateral to
apical cross-talk in Rhodnius tubules. Changes in bathing saline
K+ concentration may thus control not only transport across the
basolateral membrane (i.e. NKCC) but may also regulate the apical transporters
through intracellular signalling mechanisms.
The role of the bumetanide-sensitive cation/Cl cotransporter in K+ homeostasis
Our evidence for competition between K+ and Na+ for
basolateral transport reveals a new aspect of the homeostatic mechanisms for
autonomous regulation of haemolymph K+ concentration by
Rhodnius Malpighian tubules
(Maddrell et al., 1993).
Previous studies have shown that the upper tubule responds to reductions in
haemolymph K+ concentration by reducing the K+
concentration in the secreted fluid. This reduction enhances reabsorption of
K+ by the lower tubule, thereby contributing to homeostatic
regulation of haemolymph K+.
Homeostatic mechanisms for autonomous regulation of haemolymph
K+ concentration have also been described in Malpighian tubules of
the ant F. polyctena (Leyssens et al.,
1992,
1994
). Malpighian tubules of
F. polyctena respond to increased K+ levels in the
haemolymph by increasing the rate of fluid and K+ secretion by the
tubules, thereby lowering haemolymph K+ concentration
(Van Kerkhove et al., 1989
).
The underlying mechanisms for the changes in fluid secretion rate and
K+ transport involve the activation of different ion transport
systems according to the haemolymph K+ concentration (Leyssens et
al., 1992
,
1994
). At low K+
concentration (
5 mmol l1), fluid secretion involves an
Na+/K+/2Cl cotransporter. Fluid
secretion involves a K+/Cl cotransporter when
K+ concentration is
50 mmol l1. At very high
K+ concentrations (113 mmol l1), fluid secretion
involves basolateral K+ channels (Leyssens et al.,
1992
,
1994
).
Our study shows that in Rhodnius tubules, competition between Na+ and K+ for transport by a single bumetanide-sensitive cotransporter in the upper tubule provides a mechanism for reducing secreted fluid K+ concentration. Importantly, the increase in Na+ flux when saline K+ concentration is reduced not only minimizes the loss of K+ but at the same time permits the rate of urine production to remain very high.
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