Basolateral Na+ pump
modulates apical Na+ and
K+ conductances in rabbit
cortical collecting ducts
Shigeaki
Muto1,
Yasushi
Asano1,
Donald
Seldin2, and
Gerhard
Giebisch3
1 Department of Nephrology,
Jichi Medical School, Tochigi 329-0498, Japan;
2 Department of Internal
Medicine, University of Texas Southwestern Medical Center at
Dallas, Dallas, Texas 75235; and
3 Department of Cellular and
Molecular Physiology, Yale University School of Medicine, New Haven,
Connecticut 06520
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ABSTRACT |
Previous studies indicated that an acute elevation of
peritubular K+ enhances
K+ secretion and
Na+ reabsorption in the isolated
perfused cortical collecting duct (CCD) from rabbit kidneys [S.
Muto, G. Giebisch, and S. Sansom. Am. J. Physiol. 255 (Renal Fluid Electrolyte
Physiol. 24): F108-F114, 1988]. To determine
the underlying cellular mechanisms, we used microelectrode techniques
to assess the membrane properties of collecting duct cells in isolated
perfused CCDs of control and desoxycorticosterone acetate
(DOCA)-treated rabbits following acute stimulation of the basolateral
Na+-K+
pump by rapidly increasing the bath solution from 2.5 to 8.5 mM
K+. This induced in both groups of
tubules, first, a short-lasting hyperpolarization and, second, a
sustained phase of depolarization of transepithelial, basolateral, and
apical membrane voltages. Whereas the transepithelial conductance
(GT) and
fractional apical membrane resistance
(fRA) remained
unchanged during the initial phase of hyperpolarization, during the
depolarization,
GT increased and
fRA decreased.
Perfusion of the lumen with solutions containing either amiloride or
Ba2+ attenuated the high
K+-induced apical electrical
changes, and basolateral strophanthidin abolished both apical and
basolateral electrical responses during elevation of
K+ in the bath. From these results
we conclude the following: 1) acute
elevation of basolateral K+
activates the basolateral
Na+-K+
pump, which secondarily elevates the apical
Na+ and
K+ conductances;
2) DOCA pretreatment increases the
basolateral K+ conductance and
augments the response to the rise of
K+ of both basolateral
Na+-K+
pump activity and apical cation conductances.
sodium conductance; potassium conductance; sodium-potassium pump; acute potassium adaptation; membrane crosstalk
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INTRODUCTION |
THE MAMMALIAN CORTICAL collecting duct (CCD) plays a
dominant role in regulating K+
excretion by the nephron (45). K+
secretion in collecting duct (CD) cells of the CCD is an active process
directly linked to active Na+
reabsorption via the basolateral membrane
Na+-K+
pump, followed by passive diffusion of
K+ from the cell into the lumen
down a favorable electrochemical gradient via an apical membrane
K+ conductance (13, 19, 20,
22-24, 27, 28, 30-32, 45). In addition,
K+ recycles across the basolateral
membrane by passive diffusion along a favorable electrochemical
gradient via a basolateral membrane K+ conductance (19, 23, 30, 31).
On the other hand, Na+ is
reabsorbed passively from lumen to cell along its electrochemical gradient via an apical membrane
Na+ conductance and is transported
actively from cell to blood by the basolateral membrane
Na+-K+
pump. Both K+ secretion and
Na+ reabsorption in the CCD are
dependent on the mineralocorticoid state of the animal (4, 18, 22, 31,
32, 45).
The kidney adapts to high concentrations of peritubular
K+ by increasing urinary
K+ excretion along the late distal
tubule (initial CD) and the CCD (11, 22, 30-32, 45). This segment
is composed of at least two cell types (11, 12, 17, 21, 25, 26, 29, 37, 38), i.e., CD and intercalated cells. In vivo micropuncture studies
have shown that an elevation in plasma
K+ concentration induces a
saturable increase in K+ secretion
in the perfused rat distal tubule (38). Studies in isolated perfused
CCDs from both control and deoxycorticosterone acetate (DOCA)-treated
rabbits show that acute elevation of peritubular K+ from 2.5 to 8.5 mM greatly
enhances transcellular K+
secretion and Na+ reabsorption
(21). Such an increase in K+ and
Na+ transport following elevation
of bath K+ is sharply enhanced in
the CCDs of DOCA-treated rabbits (21).
The cell mechanisms responsible for the increase in
K+ transport remain incompletely
understood, especially with respect to the coordinated changes in
basolateral pump stimulation and apical conductance changes. To resolve
the problem, the present study addressed the following issues.
1) Are there changes of
Na+ and
K+ conductances in the apical
membrane after elevation of bath
K+, and are
Na+-K+
pump activity and K+ conductance
correlated in the basolateral membrane?
2) How are high
K+-induced electrical changes
modulated by chronic DOCA treatment?
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METHODS |
Isolation and Perfusion of Tubules
Female Japanese white rabbits (1.5-2.5 kg) were maintained on a
standard rabbit diet (Clea Japan, Tokyo, Japan) and tap
water ad libitum, and after a period of acclimation, they were divided into control and DOCA-treated groups. DOCA-treated rabbits were given
DOCA (Sigma Chemical, St. Louis, MO) intramuscularly at a dosage of 2 mg · kg
1 · day
1
for 7-10 days before the experiment.
The animals of both groups were anesthetized with intravenous
pentobarbital sodium (35 mg/kg), and both kidneys were removed. Slices
of 1-2 mm were taken from the coronal section of each kidney and
transferred to a dish containing a cold intracellular-fluid-like solution having the following composition (in mM): 14 KCl, 44 K2HPO4,
14 KH2PO4,
9 NaHCO3, and 160 sucrose. This
intracellular-fluid-like dissection medium was selected because it had
been shown to improve the kidney tissue function (19, 20, 23, 25).
Segments of CCDs were dissected from the cortex and transferred to a
bath chamber mounted on an inverted microscope (Diaphot; Nikon, Tokyo, Japan). Each tubule was perfused in vitro according to the techniques of Burg et al. (2), as modified in this laboratory for the use of
intracellular microelectrodes (19, 20, 23, 25). The details of the
technique have been published previously (19, 20, 23, 25); accordingly,
these will be presented here only briefly. After suspending the tubules
between two pipettes, we perfused the lumen at a rate exceeding 20 nl/min in all tubules. The distal end of each tubule was held in a
collecting pipette treated with unpolymerized Sylgard 184 (Dow Corning,
Midland, MI). Each tubule was perfused in a bath chamber of ~100 µl
to permit rapid exchange of the bath solution within 5 s. The bath solution flowed by gravity at a rate of 5-15 ml/min from the
reservoirs through a water jacket to stabilize the bath temperature at
37°C.
Electrical Measurements
The transepithelial and cellular electrical properties of the tubule
were measured using techniques described previously by Muto et al. (19,
20, 23, 25). The transepithelial voltage (VT) was
measured via the perfusion pipette, connected to one channel of a
dual-channel electrometer (Duo 773; World Precision Instruments,
Sarasota, FL) with a 3 M KCl-3% agar bridge and a calomel half-cell
electrode. The basolateral membrane voltage (VB) was
measured with microelectrodes filled with 0.5 M KCl. These were
fabricated from borosilicate glass capillaries (GD-1.5; 1.5 mm OD, 1.0 mm ID; Narishige Scientific Laboratory, Tokyo, Japan) by using a
vertical puller (PE-2, Narishige Scientific Laboratory). Both voltages
were referenced to the bath and recorded on a four-pen chart recorder
(model R64; Rikadenki, Tokyo, Japan). The electrical potential
difference across the apical membrane (VA) was
calculated according to the following formula
Cable analysis was used to calculate the transepithelial
conductance
(GT) and the
fractional apical membrane resistance
(fRA), as
described in detail previously (19, 20, 22-25, 27, 28, 31, 32).
Constant-current pulses of 50 nA (300-ms duration, 10-s intervals) were
injected into the tubule lumen via the perfusion pipette. The
fRA value was
estimated from the ratio of the voltage deflection across the apical
membrane and the entire epithelium at the point of impalement.
The conductances of the apical and basolateral membranes
(GA and
GB, respectively)
and the tight junction conductance
(GTj) were
estimated by the following equation described previously (19,
22-24, 31)
which
is the equation of a straight line with a slope of
GB and intercept
of GTj. Because
Ba2+ has been shown to cause a
selective decrease in the apical membrane conductance only (22, 28,
32), the effects of Ba2+ on
fRA and
GT can be used to
obtain an estimate of
GB from the slope
of the relation between the parameters.
The partial apical membrane Na+
conductance (GNaA) and
K+ conductance
(GKA) were estimated as the
amiloride- and Ba2+-sensitive
apical membrane conductances, respectively (22, 31, 32).
Identification of CD Cells
Cell impalements in this study were limited to CD cells, which were
electrophysiologically distinguished from intercalated cells according
to the criteria described previously by Muto et al. (19, 20,
22-25); i.e., CD cells have a lower
fRA and higher VB, apical
Na+ and
K+ conductances, and basolateral
K+ and
Cl
conductances, whereas intercalated cells have a higher
fRA, lower VB, a dominant
basolateral Cl
conductance,
and no detectable apical Na+ or
K+ conductances. The CD cells were
electrophysiologically identified by the depolarization of
VA and the
decrease in fRA
upon raising the luminal perfusate
K+ concentration. In addition, the
CD cells showed a depolarization of
VA and an
increase in fRA
upon addition of luminal Ba2+,
which is a K+-channel inhibitor.
In sharp contrast, the intercalated cells did not show any significant
changes in VA or
fRA upon the
raising of the luminal perfusate
K+ concentration and the addition
of luminal Ba2+.
Solutions and Materials
Table 1 shows the composition of the
solutions used. Each tubule was initially perfused with a solution
containing 5.0 mM K+ and bathed
with a solution containing 2.5 mM
K+. After a 60-min equilibration
period, CD cells were impaled with microelectrodes; the bath solution
was then rapidly changed to one containing 8.5 mM
K+. After several minutes, the
initial solution containing 2.5 mM K+ was restored. All solutions had
an osmolality between 285 and 295 mosmol/kgH2O and were equilibrated
with 95% O2-5%
CO2 adjusted to pH 7.4 at
37°C.
Amiloride (Sigma) was added to the luminal perfusate to achieve a final
concentration of 50 µM. BaCl2
was used in either the lumen or the bath at a final concentration of 2 mM. Strophanthidin (Sigma) was used in the bath at a concentration of
200 µM.
Statistics
The data are expressed as means ± SE. Comparisons were performed
either by the paired or nonpaired Student's
t-test, as needed. Only
P < 0.05 was considered
statistically significant.
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RESULTS |
Electrophysiological Data in Control Conditions
(2.5 mM K+
in Bath)
The CCDs from control and DOCA-treated rabbits had an average length of
997 ± 78 (n = 25) and 1,025 ± 50 µm (n = 25), and their inner and
outer diameters were 28.6 ± 1.3 (n = 25) and 29.8 ± 1.1 µm (n = 25), respectively. As shown in Table 2, the
lumen-negative VT
and VB values of
the CCDs from DOCA-treated rabbits were significantly (P < 0.001) greater than those of
the CCDs from control rabbits. However, the calculated
VA of the tubules
from DOCA-treated rabbits was not significantly different from that
observed in tubules from untreated control rabbits. The
GT in the tubules
from DOCA-treated rabbits was significantly
(P < 0.001) greater than that in the tubules from control rabbits, but there was no significant difference in fRA values
between the two groups. These electrical properties in the CCDs from
both control and DOCA-treated rabbits are similar to values previously
reported (30-32, 45).
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Table 2.
Effects of raising bath K+ concentration from
2.5 to 8.5 mM on barrier voltages and conductances in control and
DOCA-treated rabbits
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Effects of Acute Increase in Bath
K+ on
Barrier Voltages and Conductances of CD Cells from Control and
DOCA-Treated Rabbits
We first examined the effects of raising bath
K+ concentration on barrier
voltages (VT and
VB) and
conductances in the tubules from control and DOCA-treated animals.
Typical tracings of
VT and
VB upon raising
bath K+ concentration are shown in
Fig. 1, and summaries are presented in
Table 2. In the tubules from both control and DOCA-treated animals, an
increase in the bath K+
concentration from 2.5 to 8.5 mM induced a biphasic response of
VT and
VB, consisting of
an initial hyperpolarization followed by a late depolarization. The
initial hyperpolarization of
VT and
VB peaked within
10 s after a solution change from low to high
K+ in the bath. Subsequently,
VT and
VB slowly
depolarized to approach a new steady state over the next minute.
Changes in VA
(see Table 2) paralleled those of
VT and
VB; when the bath
K+ concentration was returned to
2.5 mM, VT and
VB returned to
control levels, and these high
K+-induced potential and
conductance changes could be repeated.

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Fig. 1.
Typical tracings showing effects of raising bath
K+ on transepithelial voltage
(VT) and
basolateral membrane voltage
(VB) of
collecting duct (CD) cells in cortical collecting ducts (CCD) of
control (A) and deoxycorticosterone
acetate (DOCA)-treated (B)
rabbits.
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Our observation that both
GT and
fRA did not
change during the "initial" hyperpolarization (Table
2) suggests that the observed increase in the cell-negative potential
was generated exclusively by activation of electrogenic
Na+-K+
pump (see also Effects of bath
strophanthidin, below).
In sharp contrast, during the "late" depolarization phase,
GT significantly
increased, whereas
fRA significantly
decreased (Table 2). These findings are consistent with the
interpretation that raising bath
K+ affects the conductive pathway
of the apical membrane more than that of the basolateral membrane. This
notion was further supported by the observation that in tubules of both
groups, GA
significantly increased, whereas neither
GB nor
GTj was changed
(see Fig. 2).

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Fig. 2.
Effects of raising bath K+ on
conductances of apical and basolateral membranes
(GA and
GB, respectively)
and the tight junction
(GTj) in the
late depolarization phase (DEPO).
* P < 0.01 compared with 2.5 mM bath K+ values.
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Changes in barrier voltages
(VT,
VB, and
VA) during the
initial hyperpolarization phase were significantly smaller in
DOCA-treated animals compared with those in control animals (Fig.
3). However, changes in barrier voltages as
well as conductances
(GT and
fRA) in the
"late" depolarization phase were significantly greater in the
DOCA group than those in the control group (Fig. 3). These findings can
be explained by the known stimulation of both
Na+-K+
pump and basolateral K+
conductance in CD cells by mineralocorticoids (14, 18, 30-32). The
observation that the initial hyperpolarization is smaller in the DOCA
group is consistent with an increase in basolateral K+ conductance and the fact that
the membrane potential exceeds the
K+ equilibrium potential (30). The
resulting positive current from bath to cell opposes the pump-generated
current and reduces the magnitude of the hyperpolarization. Since the
late depolarization is strongly affected by the transmembrane
concentration difference of K+,
the increase in the conductance of
K+ and decline in that of
Cl
after chronic DOCA
treatment would amplify the depolarizing effect of increasing the
concentration of K+ in the bath.

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Fig. 3.
Comparison of changes ( ) in electrical parameters of both phases
upon raising bath K+ in the two
groups. HYPER, initial hyperpolarization phase; DEPO, late
depolarization phase.
VA, electrical
potential difference across the apical membrane;
GT,
transepithelial conductance; and
fRA, fractional
apical membrane resistance. * P < 0.005 and ** P < 0.001, compared with control.
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It should be noted that small voltage changes might occur across the
basolateral and apical membranes due to circular current flow produced
by raising bath K+ and
establishing a concentration difference for
K+ between bath and lumen. Since
the paracellular shunt permeability to
K+ in the rabbit CCD is low (27),
the voltage drop would result in a small hyperpolarization
of the basolateral
(VB more
negative) and apical membranes
(VA more
positive). However, these changes must be quite small because no change
in either VB or
VA was observed in control or DOCA-treated tubules after raising bath
K+ in the presence of amiloride in
the lumen, a condition expected to impede current flow in the opposite
direction and thus interfere with closing of the transcellular current
loop (see Table 3).
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Table 3.
Effects of raising bath K+ in absence or
presence of luminal amiloride on barrier voltages and conductances
in control and DOCA-treated rabbits
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Effects of Acute Increase in Bath
K+
Concentration on
Na+ and
K+
Transport Properties of the Apical Membrane of CD Cells From Control
and DOCA-Treated Rabbits
The GA of the CD
cell is composed of a small Na+
conductance and a dominant K+
conductance (13, 19, 20, 22, 23, 27, 28, 30-32). We examined
whether the Na+ conductance
and/or K+ conductance in
the apical membrane changed upon raising bath K+.
Effects of luminal amiloride. In these
experiments, the Na+ channel
inhibitor amiloride was added to the luminal perfusate, and the bath
K+ concentration was raised.
Typical tracings of
VT and
VB in tubules of
both control and DOCA-treated rabbits and the effects of the bath
K+ concentration, in the absence
or presence of luminal amiloride, are illustrated in Fig.
4. Summaries of barrier voltages as well as
conductances are given in Table 3. Upon addition of luminal amiloride
(50 µM), VT and
VB in the tubules
of both groups rapidly depolarized, resulting in a significant
hyperpolarization of
VA. These results
confirm previous observations (13, 19, 20, 22, 23, 32).

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Fig. 4.
Typical tracings showing effects of raising bath
K+ in absence or presence of
luminal amiloride on
VT and
VB of the CD cell
in CCDs of control (A) and
DOCA-treated (B) rabbits.
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Amiloride also significantly decreased
GT and increased
fRA. Importantly,
the amiloride-inhibitable changes in
VA (12.0 ± 1.0 mV, n = 11, P < 0.001),
GT (3.9 ± 0.5 mS/cm2,
n = 7, P < 0.001), and
fRA (0.18 ± 0.02, n = 7, P < 0.001) in the DOCA group were
significantly greater than those in the control group
(
VA = 5.1 ± 1.0 mV, n = 8;
GT = 1.6 ± 0.3 mS/cm2,
n = 7; and
fRA = 0.08 ± 0.01, n = 7). These results
confirm previous observations that the apical amiloride-sensitive
Na+ conductance of CD cells under
basal conditions was stimulated by chronic DOCA treatment (13, 31, 32).
The initial hyperpolarization of
VT,
VB, and
VA was not
observed in the tubules of both control and DOCA-treated animals, when
bath K+ was increased in the
presence of luminal amiloride (Fig. 4; Table 3). Thereafter,
VT increased and
VB depolarized in
CD cells of both groups, resulting in a significant depolarization of
VA (Table 3). At
this time, GT
significantly increased and
fRA significantly decreased (Table 3). It should be noted that the high
K+-induced changes in
VT,
VB, and
VA, as well as
GT and
fRA in tubules of
both groups, were significantly smaller in the presence of luminal
amiloride compared with those observed in its absence (see Fig.
5). Therefore, luminal amiloride attenuated
the high K+-induced electrical
changes in both phases. As shown in Fig. 6, the estimated apical membrane Na+
conductance (GNaA) in both
groups of tubules was significantly increased during the late
depolarization phase. Moreover, during the late depolarization, the
amiloride-inhibitable changes in
VA
(
VA = 4.3 ± 0.7 mV, n = 11, P < 0.01) and
GT
(
GT = 1.6 ± 0.3 mS/cm2,
n = 7, P < 0.05) in tubules from
DOCA-treated animals were significantly greater than those in tubules
of control animals
(
VA = 1.5 ± 0.3 mV, n = 8;
GT = 0.5 ± 0.1 mS/cm2,
n = 7). These data indicate that the
increase in apical membrane Na+
conductance upon raising the concentration of
K+ in the bath was greater in the
DOCA group. When the bath K+
concentration was increased from 2.5 to 8.5 mM (late phase), the
increase of GNaA in the DOCA
group (4.8 ± 0.7 mS/cm2,
n = 7, P < 0.05) was
significantly greater than that in the control group (2.4 ± 0.6 mS/cm2,
n = 7).

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Fig. 5.
Comparison of changes in electrical parameters during both phases upon
raising bath K+ in absence or
presence of luminal amiloride. HYPER, initial hyperpolarization phase;
DEPO, late depolarization phase.
* P < 0.05, ** P < 0.01, P < 0.005, and
 P < 0.001, compared
with absence of amiloride ( amiloride). The number of
measurements of
VT,
VB, and
VA in control
and DOCA groups is 8 and 11, respectively. The number of measurements
of GT and
fRA in control
and DOCA groups is 7.
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Fig. 6.
Effects of raising bath K+ on
partial apical membrane Na+
conductance (GNaA) and
K+ conductance
(GKA) in late depolarization
phase (DEPO). * P < 0.01, ** P < 0.005, and
P < 0.001, compared with
2.5 mM bath K+ values.
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Effects of luminal
Ba2+.
To test whether the apical membrane
K+ conductance changes with
basolateral activation of the
Na+-K+
pump, we added the K+ channel
inhibitor Ba2+ to the luminal
perfusate. Typical tracings of
VT and
VB upon raising
bath K+ concentration in the
absence or presence of luminal
Ba2+ are illustrated in Fig.
7, and summaries of barrier voltages and
conductances are shown in Table 4. It can
be seen that addition of 2 mM Ba2+
to the luminal perfusate led to rapid hyperpolarization of the lumen-negative
VT, which
subsequently slowly depolarized to reach a new steady state.
VB, in contrast,
first rapidly depolarized before slowly depolarizing further toward a
new steady state. Thus
VA also rapidly
depolarized during this fast phase, then slowly depolarized to a new
steady state. The biphasic effects of
Ba2+ on
VT,
VB, and
VA are similar to
those reported previously (19, 22, 32). The transient current induced
by luminal addition of Ba2+ is due
to blocking of the K+ current
directed from cell to lumen. The
VT becomes more
negative and the
VA becomes
depolarized at first, because after blocking the opposing
K+ current, the net current is now
only composed of the Na+
reabsorptive current. The circular
Na+ current flow produced at the
apical membrane causes the
VB to depolarize.
In the second phase, the Na+
current relaxed probably due to several factors; i.e., first, a
decrease in the driving force for
Na+ entry, since
VA is depolarized
by ~40 mV; second, changes in intracellular ion content; and,
finally, cell volume changes, since
K+ exit is eliminated
by Ba2+ (22, 32). Thus both
VT and
VB slowly
depolarize to a new steady state.

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Fig. 7.
Typical tracings showing effects of raising bath
K+ in absence or presence of
luminal Ba2+ on
VT and
VB of the CD cell
in CCDs of control (A) and
DOCA-treated (B) rabbits.
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Table 4.
Effects of raising bath K+ in absence or
presence of luminal Ba2+ on barrier voltages
and conductances in control and DOCA-treated rabbits
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Following addition of Ba2+ to the
lumen, GT
significantly decreased and
fRA significantly
increased. Similar to the apical conductance of
Na+, in the DOCA group the
Ba2+-inhibitable changes in
VA (36.5 ± 1.8 mV, n = 21, P < 0.005) and
GT (6.2 ± 0.8 mS/cm2,
n = 10, P < 0.005) significantly
exceeded those in the control group
(
VA = 28.9 ± 1.1 mV, n = 20;
GT = 3.2 ± 0.3 mS/cm2,
n = 11). These findings indicate that
under basal conditions, DOCA increases the apical
Ba2+-sensitive
K+ conductance (13, 31, 32).
When the bath K+ concentration was
raised in the presence of luminal
Ba2+,
VT and
VB initially
hyperpolarized in the tubules of both groups without any changes in
VA,
GT, or
fRA (Table 4). As
shown in Fig. 7, the high
K+-induced changes in
VT and
VB were
significantly smaller in the presence of luminal
Ba2+ than those in its absence.
VT,
VB, and
VA then
depolarized significantly in parallel with an increase in
GT and a decrease
in fRA (Table 4).
The high K+-induced changes in
barrier voltages and conductances were also significantly smaller in
the presence of luminal Ba2+ than
those in its absence (Fig. 8), showing that
luminal Ba2+ partially inhibited
the high K+-induced electrical
changes in both phases of the experiments. These findings indicate that
the apical Ba2+-sensitive
K+ conductance in the tubules from
both groups of animals was stimulated upon raising bath
K+ concentration. In fact, as
shown in Fig. 6, the estimated apical membrane
K+ conductance
(GKA) in both groups of
tubules was significantly increased during the late depolarization
phase. Moreover, in the late depolarization phase, the
Ba2+-inhibitable changes in
VA
(
VA = 5.5 ± 0.6 mV, n = 21, P < 0.001) and
GT
(
GT = 2.4 ± 0.4 mS/cm2,
n = 10, P < 0.005) in the tubules from
DOCA-treated animals were significantly greater than those in the
control animals
(
VA = 1.3 ± 0.4 mV, n = 20;
GT = 0.7 ± 0.3 mS/cm2,
n = 11). Thus the increase in
apical membrane K+ conductance
upon raising bath K+ concentration
is significantly greater in the DOCA group.

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Fig. 8.
Comparison of changes in electrical parameters during both phases upon
raising bath K+ in absence or
presence of luminal Ba2+. HYPER,
initial hyperpolarization phase; DEPO, late depolarization phase.
* P < 0.05 and
** P < 0.001, compared with
absence of Ba2+
( Ba2+). The number of
measurements of
VT,
VB, and
VA in control
and DOCA groups is 20 and 21, respectively. The number of measurements
of GT and
fRA in control
and DOCA groups is 11 and 10, respectively.
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Effects of Acute Increase in Bath
K+
Concentration on
Na+ and
K+
Transport Properties of the Basolateral Membrane of CD Cells From
Control and DOCA-Treated Rabbits
Effects of bath strophanthidin. Next,
we examined whether the basolateral
Na+-K+
pump was responsible for the high
K+-induced electrical changes. To
this end, we added a
Na+-K+
pump inhibitor, strophanthidin, to the bath and then raised the bath
K+ concentration from 2.5 to 8.5 mM. Typical tracings of
VT and VB are
illustrated in Fig. 9, and summaries of
barrier voltages are shown in Table 5. In
both groups of tubules, addition of 200 µM strophanthidin resulted in
a two-phase depolarization of VB, with an
initial rapid depolarization followed by a slow and more prolonged
depolarization. We note that the initial peak changes in
VB were
significantly greater in the DOCA group (DOCA, 19.3 ± 0.8 mV,
n = 9, P < 0.001; control, 8.5 ± 1.4 mV, n = 6). These results
are consistent with the notion that under basal conditions, the
Na+-K+
pump activity in the tubules from DOCA-treated rabbits was stimulated, since the initial fast-depolarization phase has been attributed to
direct inhibition of electrogenic
Na+-K+
pump activity (13, 31). This interpretation is also supported by the
fact that
VB in the
DOCA group was significantly greater by ~25 mV (see Table 2).

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Fig. 9.
Typical tracings showing effects of raising bath
K+ in absence or presence of bath
strophanthidin on
VT and
VB of the CD cell
in CCDs of control (A) and
DOCA-treated (B) rabbits.
|
|
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Table 5.
Effects of raising bath K+ in absence or
presence of bath strophanthidin on barrier voltages in control and
DOCA-treated rabbits
|
|
It is of interest that in both groups of tubules, the addition of bath
strophanthidin completely inhibited the high
K+-induced voltage changes in both
phases of the experiments (Table 5). These observations indicate that
the basolateral
Na+-K+
pump is tightly coupled to the high
K+-induced electrical changes
during both phases.
Effects of bath
Ba2+.
Finally, we investigated whether the basolateral
K+ conductance may contribute to
the high K+-induced electrical
changes. For this purpose, we added 2 mM
Ba2+ to the bath and subsequently
raised the bath K+ concentration.
Typical tracings of
VT and
VB upon raising
bath K+ concentration in the
absence or presence of bath Ba2+
are illustrated in Fig. 10, and summaries
of barrier voltages and conductances are shown in Table
6. In control tubules, addition of
Ba2+ to the bath had no
significant effect on
VT or
VB, although it caused both GT
and fRA to
decrease significantly. These findings indicate that
K+ is close to equilibrium across
the basolateral membrane. In sharp contrast, when 2 mM
Ba2+ was added to the bath of the
DOCA-treated tubules,
VT and
VB rapidly
hyperpolarized within several seconds by ~4 mV, in parallel with
decreases in GT
and fRA. These
observations indicate that Ba2+
blocks a K+ current from the bath
into the cell in the tubules of DOCA-treated animals. These results are
in agreement with reports of observations in the CCDs from control and
DOCA-treated rabbits (30, 31). Furthermore, the
Ba2+-inhibitable changes in
VB (3.5 ± 0.5 mV, n = 8, P < 0.05) and GT (2.3 ± 0.6 mS/cm2,
n = 6, P < 0.05) in the tubules of
DOCA-treated rabbits were significantly greater than those in the
tubules of control rabbits (
VB = 0.8 ± 1.4 mV, n = 7;
GT = 0.5 ± 0.6 mS/cm2,
n = 7), indicating that under basal
conditions, basolateral K+
conductance is stimulated by long-term DOCA treatment.

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Fig. 10.
Typical tracings showing effects of raising bath
K+ in absence or presence of bath
Ba2+ on
VT and
VB of the CD cell
in CCDs of control (A) and
DOCA-treated (B) rabbits.
|
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Table 6.
Effects of raising bath K+ in absence or
presence of bath Ba2+ on barrier voltages and
conductances in control and DOCA-treated rabbits
|
|
Addition of bath Ba2+ to the
tubules of control rabbits had no significant effects on the high
K+-induced electrical changes
during either phase (Fig. 10; Table 6). This finding indicates that
changes of the basolateral K+
conductance are not responsible for the high
K+-induced electrical changes. In
the tubules of DOCA-treated rabbits, by contrast, raising the bath
K+ concentration in the presence
of Ba2+ initially hyperpolarized
VT,
VB, and
VA without any
changes in GT or
fRA (Table 6).
However, as shown in Figs. 10 and 11, DOCA treatment increased the
magnitude of the initial hyperpolarization of
VT,
VB, and
VA in the
presence of bath Ba2+ to values
significantly greater than that seen in its absence. During the second
phase, values of
VT,
VB, and
VA significantly depolarized, in parallel with an increase in
GT and a decrease in fRA (Table 6).
These high K+-induced changes in
barrier voltages and conductances were significantly smaller in the
presence of bath Ba2+ than those
seen in its absence (Fig. 11). Thus, in
tubules from DOCA-treated animals,
Ba2+ in the bath magnified the
high K+-induced voltage changes
during the initial hyperpolarization phase and partially inhibited
these electrical changes in the late depolarization phase. This
demonstrates that an increase in basolateral
K+ conductance after raising the
concentration of K+ in the bath
occurs only in tubules from DOCA-treated rabbits.

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Fig. 11.
Comparison of changes in electrical parameters during both phases upon
raising bath K+ in absence or
presence of bath Ba2+. HYPER,
initial hyperpolarization phase; DEPO, late depolarization phase.
* P < 0.05, ** P < 0.01, P < 0.005, and
 P < 0.001, compared
with Ba2+. The number of
measurements of
VT,
VB, and
VA in control
and DOCA groups is 7 and 8, respectively. The number of measurements of
GT and
fRA in control
and DOCA groups is 7 and 6, respectively.
|
|
 |
DISCUSSION |
A model of CD cells explains K+
secretion as a process involving two distinct transport steps. First is
the net movement of K+ across the
basolateral membrane, which is accounted for by the balance between
active uptake of K+ via
Na+-K+
pump and passive backleak through
K+ channels, and second is passive
K+ diffusion from cell to lumen
via K+ channels along a favorable
electrochemical gradient. The latter is strongly dependent on the
apical membrane potential, which varies with the
Na+ concentration difference
between lumen and cell (45). We have previously shown in isolated
perfused rabbit CCD that an acute increase in the concentration of
K+ in the bath stimulates both
transcellular K+ secretion and
Na+ absorption; both processes are
significantly enhanced in tubules from DOCA-treated animals (21). In
the present study, we explore and identify the individual transport
mechanisms mediating acute transport stimulation by
K+. We conclude that raising
peritubular K+ initiates a
sequence of events that involves, initially, rapid activation of the
basolateral
Na+-K+
pump followed by a delayed response that involves an increase of both
apical and basolateral K+
conductances; moreover, the Na+
conductance of the apical membrane of CD cells is also enhanced.
Immediate Response to Elevation of
K+
The view that increasing basolateral
K+ acts directly on the
Na+-K+
pump in CD cells is consistent with our observation of a rapid initial
hyperpolarization of the basolateral membrane potential that reflects
stimulation of electrogenic
Na+-K+-ATPase
activity (21). The interpretation that basolateral
Na+-K+
pump activity responds to changes in
K+ concentration is also supported
by the ability of strophanthidin to completely inhibit the
K+-induced initial voltage
changes. The findings in our study that the application of luminal
amiloride suppressed the initial hyperpolarization after raising bath
K+ concentration is further
evidence of involvement of peritubular Na+-K+
pump stimulation, because luminal amiloride caused a decrease in the
electrical driving force for Na+
entry, and the subsequent inhibition of apical
Na+ entry would be expected to
prevent a turnover to basolateral Na+-K+
pump activity after raising basolateral
K+. The finding that luminal
Ba2+ attenuates the high
K+-induced voltage changes in the
initial phase after increasing bath
K+ is best explained by the
significant decline in apical Na+
entry caused by the sharp depolarization of the apical membrane. Such
voltage changes would be expected to lower the favorable electrochemical gradient for Na+
entry and thus compromise the optimal response of electrogenic Na+-K+-ATPase
activity to respond to an increase of
K+ in the bath.
Flux studies of ouabain-sensitive rubidium uptake across isolated rat
CCD have also shown that increasing external
K+ over a range from 2-7 mM
led to a rapid increase of
Na+-K+-ATPase
activity (6). Such transport stimulation was independent of changes in
hormone secretion and cell Na+,
did not involve the recruitment of new pump units, and occurred over a
much greater concentration range of external
K+ than that observed in purified
enzyme preparations of
Na+-K+-ATPase
in which half-saturation of maximal transport stimulation occurred at
concentrations as low as 0.5 mM (10). Previous studies had already
shown that raising peritubular K+
stimulated net secretion of K+ in
isolated perfused rat distal tubule in vivo progressively, until
concentrations as high as 7 mM were reached (38). Thus the kinetics of
basolateral
Na+-K+-ATPase
activity in intact CCDs differ significantly from isolated enzyme or
membrane preparations, because half-saturation of
Na+-K+
pump activity takes place at a significantly higher concentration of
external K+ (6). An increased
K+ concentration might have a
greater effect on
Na+-K+-ATPase
activity in the intact tubule because neither external K+ nor internal
Na+ are presumably under near saturation.
Both GT and
fRA remained
unchanged in the initial phase of membrane hyperpolarization. These
findings further support the conclusion that hyperpolarization of the
basolateral membrane following the acute elevation of
K+ results from stimulation of
electrogenic
Na+-K+
pump activity and not from changes in membrane conductances.
Delayed Response to Elevation of
K+
The initial hyperpolarization after raising
K+ was followed by a protracted
period of depolarization, best explained by the changes in
transmembrane K+ concentration
gradient following the acute elevation of peritubular K+. Importantly, additional
effects were observed. These included an increase in
GT and
GA and a decline
of the fRA.
Whereas the application of either luminal amiloride or
Ba2+ only partly reduced the
membrane response, the suppression of basolateral
Na+-K+-ATPase
by strophanthidin completely inhibited the high
K+-induced electrical changes.
Estimates of apical Na+ and
K+ conductances demonstrate that
both increase significantly (see Fig. 6). This tightly coordinated
K+-induced stimulation of
basolateral
Na+-K+-ATPase
turnover with apical Na+ and
K+ conductances provides a
satisfactory explanation for the enhancement of both
Na+ absorption and
K+ secretion in a previous study
in which net transport rates of Na+ and
K+ were measured under similar
experimental conditions (21).
Estimates of Net Driving Force for
Na+ and
K+ Across
the Apical Membrane
Estimates of net driving force for
Na+ and
K+ across the apical membrane,
upon raising bath K+, were derived
by using equivalent circuit analysis as reported (22, 31, 32). The net
driving force for Na+ entry across
the apical membrane,
VA
ENaA (ENaA is the Nernst
equilibrium potential for Na+
across the apical membrane), was not significantly changed after raising bath K+ from 2.5 to 8.5 mM
(late phase) (control, 129.1 ± 11.7 to 134.7 ± 11.7 mV,
n = 7; DOCA, 122.7 ± 10.6 to 134.8 ± 13.2 mV, n = 7). Therefore, the
increased Na+ reabsorption in both
groups of tubules, upon raising bath
K+, is primarily due to an
increase in apical membrane Na+
conductance. Similarly, the net driving force for
K+ secretion across the apical
membrane, VA
EKA (EKA is the Nernst equilibrium
potential for K+ across the apical
membrane), was also not influenced by raising bath
K+ from 2.5 to 8.5 mM (late phase)
(control, 15.4 ± 3.0 to 18.8 ± 3.1 mV,
n = 7; DOCA, 21.0 ± 3.8 to 24.2 ± 3.6 mV, n = 7).
Therefore, the increased K+
secretion in both groups of tubules can be explained by an increase in
apical membrane K+ conductance.
The effects of acute stimulation of basolateral
Na+-K+
pump activity on the apical Na+
and K+ conductances is
qualitatively quite similar to that observed in a variety of conditions
in which either systemic K+ and
Na+ balance was changed. An
increase in apical Na+ and
K+ conductances was observed in
animals chronically treated with mineralocorticoids (14, 31, 32) or
receiving exogenous loads of K+
when changes in circulating aldosterone levels were prevented (24). The
effects of vasopressin in CCD include an increase in apical
Na+ permeability (33, 35), and
this hormone has also been reported to enhance
K+ channel activity in the apical
membrane of CD cells (3). Common to all of these conditions is the
simultaneous activation of basolateral Na+-K+
pump and apical Na+ and
K+ conductances. It should be
noted that the simultaneous increase of
K+ and
Na+ conductances minimizes the
effects of an increase in K+
conductance on the apical membrane potential (39). Were it not for the
depolarizing effect of the increase in
Na+ conductance, stimulation of
the apical K+ conductance alone
would tend to hyperpolarize the apical membrane potential and diminish
the driving force of passive K+
movement from cell to lumen. These two opposing effects on
K+ secretion are minimized when
both cation conductances increase simultaneously.
Effects of DOCA
Mineralocorticoid treatment led not only to significant changes in the
control levels of apical ion conductances but also to significant
modifications of the response to the acute elevation of
K+. In addition to augmenting the
basal levels of apical cation conductances, an effect that had been
previously observed in tubules from mineralocorticoid-pretreated
animals (14, 27), we now observed that the
K+-induced increments in apical
Na+ and
K+ conductances following
basolateral
Na+-K+
pump stimulation were greater than those in control tubules (see Fig.
6). This observation is also consistent with our results obtained in
another study in which the rates of
K+ secretion and
Na+ absorption following elevation
of peritubular K+ were observed to
be much greater in tubules from rabbits that had received DOCA (21).
However, in contrast to tubules obtained from untreated animals, our
present studies also show that the initial phase of hyperpolarization
is modified by DOCA treatment. The fact that addition of
Ba2+ to the bath enhanced the
K+-induced hyperpolarization is
consistent with an increase of both electrogenic
Na+-K+-ATPase
activity and augmentation of basolateral
K+ conductance. It is safe to
conclude that, in the absence of
Ba2+, the full display of
hyperpolarization induced by stimulation of the basolateral
Na+-K+
pump is partially masked by the inwardly directed
K+ flux that reflects an increased
K+ conductance. It should be noted
that the basolateral membrane potential of DOCA-treated tubules exceeds
that measured in control conditions by ~25 mV. Such strong
hyperpolarization has been shown to exceed the
K+ equilibrium potential in CD
cells (30, 31) so that the net driving force for
K+ reverses and favors
K+ uptake into the cell via a
mineralocorticoid-dependent K+
conductance. That DOCA significantly increased the basolateral K+ conductance is further
supported by the greater
K+-induced depolarization of the
basolateral membrane compared with that in control tubules. The
interpretation that DOCA increased the basolateral
K+ conductance and that
K+ uptake across the basolateral
membrane by a favorable electrochemical gradient contributes to
K+ secretion is also consistent
with our observation that bath
Ba2+ inhibited
K+ secretion in DOCA-treated
tubules after an acute increase in bath
K+, whereas the same maneuver was
ineffective in control tubules (21). Thus the response to a
K+ challenge is maximized by
mineralocorticoids through effects on both active and passive
components of the K+ secretory
system. Two parallel transport pathways across the basolateral
membrane, stimulation of active K+
uptake by
Na+-K+-ATPase
and diffusion into the cell along a favorable electrochemical gradient
and increased K+ conductance lead
to enhanced cell uptake. Simultaneously, an increase in
K+ conductance of the apical
membrane accelerates K+ diffusion
into the tubule lumen while the rise in
Na+ conductance effectively
stabilizes membrane potential. The fact that the apical
Na+ conductance increases with the
elevation of basolateral K+ is
also consistent with previous studies in which we showed that both
Na+ and
K+ net transport increase with the
rise in peritubular K+ (21).
Possible Mechanisms for Coupling Between
Basolateral
Na+-K+
Pump and Ion Conductances
The present study expands the number of examples demonstrating that
ion conductances are functionally linked to active
exchange of Na+ for
K+ in epithelia. Tight coupling
between basolateral
Na+-K+
pump turnover and apical K+
conductance has been shown in CD cells of the rabbit and rat (41) CCD
and in other epithelia (36). A similar relationship also exists between
Na+-K+
pump activity and basolateral K+
conductance. These synchronized mechanisms, also referred to as
"crosstalk," between active pump rate and passive ion
conductances prevent rapid and possibly deleterious disturbances of
cell volume, ion concentrations, and cell potential with changes of
vectorial Na+ or
K+ transport or in
pathophysiological conditions in which ion transport and
metabolism are seriously compromised (15, 36). Possible mechanisms that could account for coupling between basolateral and
apical transport include modulation of cell pH (5, 43), cell
Ca2+ (36, 43), cell ATP (9, 40,
43, 44), nitric oxide (43), and membrane polarization (1, 7, 8, 16, 27, 31, 32, 34, 40, 44). Further studies are required to determine the
contribution of individual coupling mechanisms that coordinate
basolateral with apical transport functions.
Basolateral Regulation of
K+
Excretion
The present study underscores the importance of changes in basolateral
K+ concentration for the
regulation of K+ secretion in CD
cells. Since lumen Na+ is constant
in the present experimental setting, our findings point to regulation
of K+ secretion independent of
Na+ reabsorption. Under normal
circumstances, the steady-state basolateral regulation by plasma
K+ is highly sensitive so as not
to be associated with dramatic elevations in cell
K+ or plasma
K+. By contrast, the inappropriate
coupling of high distal Na+
delivery and high aldosterone accelerates
K+ excretion in a manner so as to
deplete cell K+, lower plasma
K+, and generate metabolic
alkalosis. Under normal circumstances, then, it would appear that the
principal regulator of K+
excretion is the dietary load of
K+.
Conclusion
The response of CD cells of isolated CCD to an acute elevation of
basolateral K+ involves several
mechanisms including an initial brief phase of
Na+-K+
pump-dependent membrane hyperpolarization followed by prolonged augmentation of both apical and basolateral
K+ conductances. In addition, the
apical Na+ conductance is also
increased. Such synchronized coupling of apical and basolateral
conductance changes in response to altered activity mimics that
initiated by mineralocorticoids but can, as shown in the present study,
also be elicited by changes in basolateral
K+ concentrations alone. Possible
mechanisms underlying such complex membrane crosstalk may involve
interactions between active
Na+-K+-ATPase
turnover, cell pH, ATP, Ca2+,
nitric oxide, and membrane polarization.
 |
ACKNOWLEDGEMENTS |
This work was supported by a grant from the Japanese Kidney
Foundation (Jinkenkyukai), by the Salt Science Foundation, by the
Science Research Promotion Fund of the Japan Private School Promotion
Foundation, by Grants-in-Aid for Scientific Research from the Ministry
of Education, Science and Culture, Japan, and by National Institute of
Diabetes and Digestive and Kidney Diseases Grant DK-17433.
 |
FOOTNOTES |
A portion of this work was presented at the Annual Meeting of the
American Society of Nephrology in New Orleans, LA, in 1996 and has been
published in abstract form (J. Am. Soc.
Nephrol. 7: 1286, 1996).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: S. Muto, Dept. of Nephrology, Jichi
Medical School, Minamikawachi, Kawachi, Tochigi 329-0498, Japan.
Received 8 July 1998; accepted in final form 24 September 1998.
 |
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