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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 |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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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 |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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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 |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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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 · day1
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
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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)
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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.
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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.| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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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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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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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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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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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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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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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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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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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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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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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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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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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 |
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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.
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FOOTNOTES |
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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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Abstract
Introduction
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Discussion
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