Ouabain reduces net acid secretion and increases
pHi by inhibiting
NH+4 uptake on rat tIMCD
Na+-K+-ATPase
Susan M.
Wall
Division of Renal Diseases and Hypertension, University of Texas
Medical School at Houston, Houston, Texas 77030
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ABSTRACT |
In the rat terminal inner medullary collecting duct (tIMCD),
Na+ pump inhibition reduces
transepithelial net acid secretion (JtAMM) [JH = total
CO2 absorption
(JtCO2) + total
ammonia secretion] and increases resting intracellular pH
(pHi). The increase in pHi and reduction in
JH that follow
ouabain addition do not occur in the absence of
NH+4 nor when NH+4 is substituted with another weak base. The purpose of this study was to
explore the mechanism of the NH+4-dependent reduction in
JtCO2 and
increase in pHi that follow
ouabain addition. We hypothesized that NH+4
enters the tIMCD cell through the
Na+-K+-ATPase
with proton release in the cytosol. To test this hypothesis, tIMCDs
were dissected from deoxycorticosterone-treated rats and perfused in
vitro with symmetrical physiological saline solutions containing 6 mM
NH4Cl. Since
K+ and
NH+4 compete for a common binding site on the
Na+ pump, increasing extracellular
K+ should limit
NH+4 (and hence net
H+) uptake by the
Na+ pump. Upon increasing
extracellular K+ concentration
from 3 to 12 mM, the NH+4-dependent, ouabain-induced increase in pHi
and reduction in
JtCO2 were
attenuated. In the presence but not in the absence of
NH+4, reducing
Na+ pump activity by limiting
Na+ entry reduced
JtCO2 and
attenuated ouabain-induced alkalinization. Ouabain-induced
alkalinization was not dependent on the presence of
/CO2
and was not reproduced with BaCl2 or bumetanide addition. Therefore, ouabain-induced alkalinization is
not mediated by the
Na+-K+-2Cl
cotransporter or a
transporter
and is not mediated by changes in membrane potential. In conclusion, on
the basolateral membrane of the tIMCD cell,
NH+4 uptake is mediated by the
Na+-K+-ATPase.
These data provide an explanation for the reduction in net acid
secretion in the tIMCD observed following administration of amiloride
or with dietary K+ loading.
ammonia; sodium-potassium-chloride cotransport; potassium channels; acidification
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INTRODUCTION |
THE TERMINAL inner medullary collecting duct (tIMCD) is
the final nephron site of urinary acidification within the mammalian kidney. Ammonium (NH+4) secretion in the
tIMCD occurs, in part, through active proton transport in parallel with the passive diffusion of NH3 (15).
However, our laboratory has demonstrated an important role of direct
NH+4 transport in this segment. In cultured
IMCD cells, NH+4 and
K+ compete for a common binding
site on the
Na+-K+-adenosinetriphosphatase
(Na+-K+-ATPase)
(34, 37). In native IMCD cells in suspension, both ions support
ouabain-sensitive ATP hydrolysis (34). Thus both NH+4 and
K+ are transported directly by the
Na+ pump.
To test the significance of Na+
pump-mediated NH+4 uptake on proton
secretion, the effect of ouabain on transepithelial net acid secretion
was examined in rat tIMCD tubules perfused in vitro. Since
deoxycorticosterone pivalate (DOCP) increases Na+ pump activity in the rat tIMCD
(30), DOCP-treated rats were studied. In the absence of
NH4Cl, total
CO2 absorption
(JtCO2) was low and not affected by ouabain addition to the bath (30). Baseline
JtCO2 was higher
in the presence of NH+4 than in its absence
(30). Moreover, in the presence of
NH4Cl, JtCO2 was
significantly inhibited upon ouabain addition to the bath (30). It was
reasoned that NH+4 enters the tIMCD cell on
the basolateral membrane through an
Na+-K+-ATPase-dependent
pathway with release of protons in the cytosol. NH+4 serves as a source of
NH3 and
H+, which are secreted across the
apical membrane. If such a model were true, then blockade of
NH+4 uptake through Na+ pump inhibition should
decrease net proton entry and alkalinize the cell. To test this
hypothesis, the effect of ouabain on intracellular pH
(pHi) was examined. Resting
pHi was lower in the presence than in the absence of NH4Cl (30).
Moreover, in the presence of
NH4Cl, addition of ouabain to the
bath alkalinized the cell (30). Ouabain-induced alkalinization was not
observed in the absence of NH4Cl
or when NH+4 was substituted with another
weak base (30). Thus NH+4 and the
Na+-K+-ATPase
are important determinants of both net acid secretion and resting
pHi.
However, the mechanism for the increase in
pHi and the reduction in
JtCO2 observed
following ouabain addition could be due to blockade of
Na+-K+-ATPase-mediated
NH+4 uptake or through changes in ion
gradients generated by the Na+
pump. The Na+ pump could generate
ion gradients that affect other
NH+4/OH
/H+/
transporters independent of direct
Na+-K+-ATPase-mediated
NH+4 uptake. The purpose of this study was to
further characterize the NH+4-dependent increase in pHi and reduction in
JtCO2 that follow
ouabain addition and to determine whether these observations can be
explained by a transport mechanism other than
Na+-K+-ATPase-mediated
NH+4 uptake.
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METHODS |
Tissue preparation. tIMCD tubules were
dissected from pathogen-free male Sprague-Dawley rats weighing
65-120 g (Rm. 205G; Harlan, Indianapolis, IN). All animals were
housed in microisolator cages and fed a
low-Na+, 0.8%
K+ diet (Ziegler Brothers,
Gardners, PA) (36). Rats were injected with 5 mg DOCP (CIBA-Geigy
Animal Health, Greensboro, NC) by intramuscular injection 5-7 days
prior to death. DOCP was employed to increase Na+-K+-ATPase
activity, as described previously (30). Animals were injected with
furosemide (5 mg/100 g body wt ip) 45 min before death by decapitation
to induce a rapid diuresis. This furosemide-induced diuresis reduces
the inner medullary axial solute concentration gradient (36) and
attenuates changes in the extracellular osmolality of the tubule.
Unless otherwise stated, all experiments were performed in
bicarbonate-buffered solutions (Table 1),
gassed with 95% air-5% CO2
before use. The measured osmolalities of all solutions are listed
(Table 1).
Coronal slices were cut from the kidneys and placed into a dissection
dish containing the chilled experimental solution (11°C). IMCDs
were dissected from the middle third of the inner medulla as described
previously (36). Tubules were mounted on concentric glass pipettes and
perfused in vitro at 37°C. Experiments were performed with
identical solutions in the perfusate and bath. In some experiments,
ouabain (2.5 or 5 mM) or bumetanide (100 µM) was added to the bath
fluid only. To maintain the desired CO2 concentration, the perfusate
was passed through jacketed concentric tubing through which 95%
air-5% CO2 was blown in a
countercurrent direction around the perfusate line. To maintain pH in
-containing solutions, the bath
fluid was constantly bubbled with 95% air-5% CO2. In
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES)-buffered solutions, bath fluid was bubbled with 100%
O2. Bath pH was measured continuously during all experiments as described previously (36). Bumetanide was prepared as a 100 mM stock in 500 mM
tris(hydroxymethyl)aminomethane (Tris). Ouabain was
dissolved directly into the bath solution.
Measurement of bicarbonate flux.
Tubule fluid samples were collected under oil in calibrated
constriction pipettes. Flow rate was determined as described previously
(36). Total CO2
(tCO2) concentration
was measured in the collected fluid
(CL) and perfusate
(Co) using a continuous flow
fluorometer (30). The CO2 reagent
was purchased as a kit (no. 132-A; Sigma, St. Louis, MO) and diluted to
50% strength with water. Using this method, bicarbonate (total
CO2, tCO2)
concentration differences of less than 1 mM can be
detected using a pipette of 8 nl (30). Bicarbonate flux,
JtCO2, was
calculated according to the equation
where
Co and
CL are the perfusate and
collected fluid tCO2
concentration. VL is
the flow rate (in nl/min), and L is
the tubule length (in mm). This equation assumes zero net
fluid transport in the absence of an imposed osmolality gradient (36).
To eliminate detection of tCO2
flux which represents artifact generated by loss of
CO2 in the collection pipette,
Co was estimated by measuring
tCO2 concentration in the
collected fluid at very fast flow rates (>50
nl · mm
1 · min
1).
Thus CO2 loss was matched in
Co and
CL measurements, allowing the
loss terms to cancel (36). Amiloride at a 1 µM concentration did not
affect the fluorescence signal of the
CO2 reagent
(n = 3, data not shown).
Measurement of pHi.
pHi was measured using the
esterified form of
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein
(BCECF) (30). The detailed methodology for measurement of
pHi in tubules perfused in vitro has been described previously (30).
Tubules were cannulated on concentric glass pipettes and then perfused
for 20 min at 37°C. The bath solution was then changed to include 5 µM of the acetoxymethyl ester of BCECF (BCECF-AM). Tubules were
perfused with BCECF present in the bath solution for 20 min. BCECF was
then removed from the bath. Measurements of
pHi were performed at least 10 min
following removal of BCECF from the bath solution.
The excitation light source was a 75-W xenon short-arc lamp (Photoscan
II; Nikon, Melville, NY) (30). The excitation light hit a rotating
chopper disc (30), allowing light to pass alternately through 440- and
495-nm band-pass filters (Omega Optical, Brattleboro, VT) at a
frequency of 60 Hz. The excitation light was reflected by a dichroic
mirror with 50% reflectance at 515 nm (Omega Optical) and passed
through the ×40 objective to strike the tubule (30). The emitted
light was collected by the objective and passed through the dichroic
mirror and long-pass filter with transmission >535 nm (Omega Optical)
(30). The transmitted light was detected with a photomultiplier tube
(Photoscan II, Nikon) (30). This detected signal was sampled at 20 points/s (30).
In most experiments, pHi was
measured in three periods. In period 1 no inhibitor was present. In period 2,
ouabain was present in the bath. In period
3 ouabain was removed from the bath fluid. Each period
was begun with a rapid bath exchange, performed by introduction of new
solution (preheated and pregassed) from a separate closed reservoir at
a rate of >30 ml/min. At the same time, the new solution was
introduced to the bath exchange reservoir. Bath fluid was exchanged
continuously at 0.5 ml/min. Thus bath fluid could be exchanged
completely in less than 10 s (30). Unless otherwise stated,
fluorescence was measured for 90 s, beginning 4 min after the bath was
exchanged. The fluorescence recorded over each time period was fit to a
line digitally by the method of least squares. The fluorescence for
that period was taken to be the fluorescence value given by that line
at the midpoint of the recording.
A two-point standard curve was constructed for each tubule by perfusing
the lumen and peritubular space at 37°C, with a
high-K+ containing solution, pH
6.9-7.4, buffered with HEPES-Tris (30). The full composition of
this calibration solution is given in Table 1
(solution 8). In addition, the bath
contained 14 µM nigericin. After 10 min of exposure to nigericin,
fluorescence was recorded. Dark current values were obtained by taking
readings in the absence of transmitted light. Dark current values were
subtracted from the unknown and the standard curve values.
Transepithelial potential difference.
To measure transepithelial potential difference
(VT), the
solution in the perfusion pipette was connected to an electrometer
(model KS-700; World Precision Instruments, New Haven, CT) through an
agar bridge saturated with 0.16 M NaCl and a calomel cell as described
previously (30). The reference was an agar bridge from the bath to a
calomel cell. VT
was recorded 1 h after warming the tubule and then 20-30 min after
the addition of amiloride to the perfusate.
Statistical analysis. For each tubule
wherein JtCO2 was
measured, two to four measurements were averaged to obtain a single value for each experimental condition. For
pHi measurements, a single
measurement was made for each condition. Mean values were used in the
statistical analysis. Statistical significance was determined by a
paired or unpaired two-tailed Student's
t-test, as appropriate, with
P < 0.05 indicating statistical
significance. Data are displayed as means ± SE.
 |
RESULTS |
Effect of limiting
Na+ entry
on JtCO2 and resting
pHi.
It was hypothesized that across the basolateral membrane of the rat
tIMCD, NH+4 uptake is mediated by the Na+-K+-ATPase.
NH+4 thus provides a source of
H+ and
NH3 for luminal secretion and the
titration of other luminal buffers (30). In the presence of
NH4Cl, the reduction in
JtCO2 and the
increase in pHi observed following
ouabain addition (30) can be explained by inhibition of
Na+ pump-mediated
NH+4 uptake with reduced net
H+ entry and increased
pHi. However,
Na+ pump inhibition affects other
H+/OH
/
/NH+4
pathways. Ouabain-induced changes in activity of these other
transporters could also explain the above observation. The purpose of
this study was to explore further the mechanism responsible for the
NH+4-dependent, ouabain-induced reduction in
JtCO2 and
increase in pHi observed previously (30).
On the apical membrane of the tIMCD,
Na+ enters the cell through
amiloride-sensitive Na+ channels
(17, 28) and exits across the basolateral membrane through the
Na+ pump (17, 19). Increased
intracellular Na+
(Na+i) facilitates
Na+ pump-mediated
NH+4 or
K+ uptake. If
NH+4-dependent, ouabain-induced changes in
pHi and
JtCO2 reflect
Na+ pump-mediated
NH+4 transport, then reducing
Na+i by limiting
Na+ entry should reduce
JtCO2 and
increase pHi.
To determine the effect of limiting Na+i
entry on Na+ pump-mediated
NH+4 uptake,
JtCO2 was
measured in the presence and the absence of the
Na+ channel inhibitor, amiloride.
In the presence of 3 mM KCl and 6 mM
NH4Cl in the bath and perfusate
(solution 1), baseline
JtCO2 was 3.7 ± 0.7 and then fell to 2.0 ± 0.9 pmol · mm
1 · min
1
upon the addition of 1 µM amiloride to the perfusate
(n = 5, P < 0.05; Fig.
1 and Table 2). This
reduction in
JtCO2 was not observed in time controls [Fig. 1, Table 2;
n = 3, P = not significant (NS)] and
was not observed in the absence of
NH4Cl (solution
3, Table 2; n = 3, P = NS). To test whether the
amiloride-induced reduction in
JtCO2 results
from a membrane potential-induced change in paracellular transport,
VT was measured
in the presence and absence of 1 µM amiloride
(solution 1). Baseline
VT was 0.0 ± 0.2 and +0.1 ± 0.1 mV (n = 3) upon
the addition of 1 µM amiloride to the perfusate
(P = NS). Thus the reduction in
JtCO2 observed with amiloride addition is NH+4 dependent and is not mediated by a nonspecific effect of amiloride on
VT that drives
changes in paracellular transport.

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Fig. 1.
Effect of luminal amiloride on total
CO2 absorption
(JtCO2) with
NH4Cl present in bath and
perfusate. A: effect of 1 µM
amiloride on
JtCO2. During
control period, mean flux was 3.7 ± 0.7 pmol · mm 1 · min 1.
After addition of 1 µM amiloride to the luminal perfusate, flux
declined to 2.0 ± 0.9 (n = 5, P < 0.05).
B: experiment in
A was repeated, but with a mock
perfusate exchange, i.e., amiloride was not introduced into luminal
perfusate. Baseline
JtCO2 was 3.4 ± 1.1 and 3.6 ± 0.8 pmol · mm 1 · min 1
after perfusate exchange (n = 3, P = NS).
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The effect of luminal amiloride on resting
pHi was tested. It was reasoned
that if limiting apical Na+ entry
limits Na+ pump-mediated
NH+4 uptake, then in the presence of
NH4Cl luminal amiloride should
decrease net H+ uptake and
alkalinize the cell. To test this hypothesis, the effect of amiloride
on pHi was tested (Table
3). In the presence of
NH4Cl (solution
1), following the addition of amiloride to the
perfusate, a small increase in pHi
was observed. This amiloride-induced alkalinization was not observed in
the absence of NH4Cl
(solution 3). However, these
amiloride-induced changes in pHi
were very small. Therefore, the effect of limiting
Na+ availability on
pHi was explored further.
Effect of limiting
Na+ entry
on
NH+4-dependent,
ouabain-induced changes in pHi.
If Na+ pump-mediated
NH+4 entry supplies the cell cytosol with
H+ and
NH+4, then
Na+ pump blockade should limit
NH+4 uptake, attenuating net
H+ entry and alkalinizing the
cell. The effect of ouabain on resting pHi was therefore explored.
Results were compared when the experiment was repeated with
substitution of ouabain for its vehicle (solution 1). In the presence of 3 mM KCl, 6 mM
NH4Cl, and 25 mM
NaHCO3/5% CO2 in the bath and
perfusate (solution 1), resting
pHi averaged 7.18 ± 0.02 (n = 16). As shown in
Fig. 2 (solution 1), with ouabain addition to the bath, a prompt and sustained increase in
pHi was observed. Since
pHi remained elevated, compared
with control (vehicle), for at least 5.5 min following the addition of
ouabain to the bath, pHi was
measured 4 min after the addition of ouabain to the peritubular bath.
These results confirm our previous observations that ouabain addition
to the bath results in increased
pHi when perfused in the presence
of NH4Cl (30). The effect of ion
substitution on ouabain-induced alkalinization was studied when each
tubule was used as its own control.

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Fig. 2.
Effect of ouabain on resting intracellular pH
(pHi) with
NH4Cl present in bath and
perfusate. Tubules were perfused with symmetrical solutions containing
3 mM KCl + 6 mM NH4Cl
(solution 1). Bath was exchanged
with the introduction of 2.5 mM ouabain and
pHi measured every 45 s. Results
are the mean of 3 tubules studied. In separate tubules, the experiment
was repeated, but with a mock bath exchange and without introduction of
ouabain. Ouabain vehicle was solution
1. Results are the mean of 3 experiments.
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The mechanism responsible for the
NH+4-dependent, amiloride-induced reduction
ion in JtCO2 was
studied further. In principal cells, amiloride attenuates but does not
fully inhibit the increase in Na+i that
follows Na+ pump inhibition (25).
These results imply that inhibiting
Na+ uptake through the apical
Na+ channel attenuates but does
not fully inhibit activity of the Na+ pump. These results might
explain the greater increase in
pHi and the greater decline in
JtCO2 observed
following the addition of ouabain to the bath than that observed
following the addition of amiloride to the perfusate.
In principal cells, limiting Na+
entry from both the bath and perfusate by removal of extracellular
Na+ fully inhibits the
Na+ pump (25). Therefore removal
of Na+ from the bath and perfusate
should fully inhibit ouabain-induced alkalinization in the tIMCD, if
mediated by NH+4 uptake on the
Na+-K+-ATPase.
The effect of removing Na+ from
the bath and perfusate on NH+4-dependent, ouabain-induced alkalinization was therefore studied. With
Na+ present in the bath and
perfusate (solution 1; Fig.
3 and Table 4),
pHi increased 0.07 ± 0.01 pH
units (P < 0.05) following ouabain addition and returned to baseline upon ouabain withdrawal, as reported
previously (30). In the absence of extracellular
Na+ (solution
2), pHi did not
increase following ouabain addition to the bath (Fig. 3).

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Fig. 3.
Effect of limiting Na+ entry on
NH+4-dependent, ouabain-induced
alkalinization. Tubules were perfused and bathed with
NH4Cl present in bath and
perfusate (solution 1). Baseline
pHi was measured. Bath solution
was exchanged to include ouabain (2.5 mM), and
pHi was measured 4 min after the
exchange. Bath was again exchanged, removing ouabain, and
pHi was measured 4 min later
(recovery). In the same tubules, perfusate and bath was exchanged,
removing extracellular Na+
(solution 2). Ouabain-induced
alkalinization was measured as above. Dotted line indicates that the
experiment was performed in the reversed order, i.e., ouabain-induced
alkalinization was measured first in absence and then in presence of
extracellular Na+.
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In the tIMCD, upon the removal of extracellular
Na+, a prompt and sustained
reduction in Na+i is observed (35). In
the present experiment, when Na+
was removed from the bath and perfusate (solution
2; Fig. 3 and Table 4),
pHi decreased
(n = 3).
1 A reduction in
Na+ pump activity under
Na+-free conditions could be due
to reduced Na+i or the reduced
pHi (37). The
pHi dependency of the
Na+ pump in mouse tIMCD has been
reported by our laboratory (37). With decreasing
pHi, a reduction in
ouabain-sensitive Rb+ uptake was
observed. However, in the present study, the decline in
pHi upon
Na+ removal was modest (7.22 ± 0.02 to 6.98 ± 0.03, n = 3). Over this pHi range,
ouabain-sensitive Rb+ uptake is
reduced by only ~15% (37). Therefore, the absence of ouabain-induced
alkalinization that follows extracellular
Na+ removal cannot be explained
fully by a pHi-induced change in Na+ pump activity. Thus limiting
Na+ uptake by the tIMCD attenuates
NH+4-dependent, ouabain-induced
alkalinization.
Effect of extracellular
K+ on
ouabain-induced alkalinization.
Our laboratory has demonstrated that NH+4 and
K+ are competitive inhibitors for
a common extracellular binding site of the
Na+-K+-ATPase
(34, 37). Thus increased extracellular
K+ concentration attenuates
NH+4 uptake by the tIMCD cell (34, 37). Using
the model of Kurtz and Balaban (18) and employing kinetic values
measured in our laboratory (34), we were able to estimate
NH+4 uptake through the Na+-K+-ATPase.
The model predicts NH+4 uptake through the
Na+ pump to be increased two- to
threefold when the extracellular K+ concentration is reduced from
12 to 3 mM. We reasoned that if the model were true, then in the
presence of NH4Cl, increasing extracellular K+ concentration
should attenuate ouabain-induced alkalinization as well as total and
ouabain-sensitive
JtCO2. To test
this hypothesis, NH+4-dependent,
ouabain-induced alkalinization was measured at two extracellular
K+ concentrations (3 and 12 mM).
In the presence of NH4Cl,
pHi was 7.10 ± 0.06 at a
K+ concentration of 3 mM
(solution 1, Table 4) and 7.16 ± 0.05 (n = 4) at a
K+ concentration of 12 mM
(solution 4, Table 4). These results compare with
JtCO2 flux rates
of 3.4 ± 0.4 (n = 11) and 2.5 ± 0.3 pmol · mm
1 · min
1
(n = 7) when the extracellular
K+ concentrations were 3 and 12 mM, respectively (Table 2). Thus, on average,
JtCO2 was
increased, and resting pHi was
reduced, at lower extracellular K+
concentrations. These differences, however, did not reach statistical significance.
To evaluate the mechanism of NH+4 uptake in
the tIMCD further, NH+4-dependent,
ouabain-induced changes in
JtCO2 and resting
pHi were measured when the
extracellular K+ concentration was
either 3 or 12 mM. In the presence of 3 mM KCl + 6 mM
NH4Cl
(solution 1), resting
pHi rose 0.07 ± 0.01 pH units
(n = 4, P < 0.05) with ouabain addition and
fell to baseline with ouabain withdrawal (Fig.
4; Table 4). However, at a
K+ concentration of 12 mM no
change in pHi was detected
following the addition or withdrawal of ouabain
(n = 4, P = NS; Fig. 4 and Table 4).

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Fig. 4.
Effect of increased extracellular
K+ concentration on
ouabain-induced alkalinization. Ouabain-induced alkalinization was
measured in presence of 3 mM KCl + 6 mM
NH4Cl (solution
1), as outlined in Fig. 3. Ouabain concentration was
5 mM. Perfusate and bath were then exchanged to solution containing 12 mM KCl + 6 mM NH4Cl
(solution 4), and ouabain-induced
alkalinization was again measured. In 2 tubules, the experiment was
performed in the reverse order, i.e., ouabain-induced alkalinization
was first measured with 12 mM KCl + 6 mM
NH4Cl present. Results are means ± SE of 4 experiments.
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We have demonstrated previously that in DOCP-treated rats, in the
presence of 3 mM NH4Cl + 6 mM
NH4Cl in the bath and perfusate (solution 1), baseline
JtCO2 was 3.8 ± 0.5 and then 1.6 ± 0.3 pmol · mm
1 · min
1
(n = 7) following the addition of
ouabain to the bath (30). These previously published data are given in
Fig. 5A.
In the present study, we asked whether ouabain-sensitive
JtCO2 could be
detected when NH+4 concentration was held
constant but extracellular K+ was
increased to 12 mM (solution 4). As
shown in Table 2 and Fig. 5B, at a
K+ concentration of 12 mM a
reduction in
JtCO2 could not
be detected with the addition of ouabain to the bath. These results
support the hypothesis that K+ and
NH+4 compete for a common binding site on the
Na+-K+-ATPase.
Upon increasing extracellular K+
concentration, Na+ pump-mediated
NH+4 uptake is attenuated, which results in a
decrease in the ouabain-sensitive component of
JtCO2 and resting
pHi.

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Fig. 5.
Effect of ouabain on
JtCO2 when the
extracellular K+ concentration is
increased. A: effect of ouabain on
JtCO2 in presence
of 3 mM KCl + 6 mM NH4Cl. During
control period, mean flux was 3.8 ± 0.5 pmol · mm 1 · min 1.
After addition of 2.5 mM ouabain to the bath, flux declined to 1.6 ± 0.3 pmol · mm 1 · min 1
(n = 7, P < 0.05). These data were taken
from Ref. 30. B: experiment in
A was repeated, but in presence of 12 mM KCl + 6 mM NH4Cl
(solution 4). Baseline
JtCO2 was 2.9 ± 0.4 and 2.9 ± 0.3 pmol · mm 1 · min 1
following the addition of 5 mM ouabain to bath
(n = 4, P = NS).
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Role of
Ba2+-sensitive
K+
channels.
Increasing extracellular K+
concentration could increase pHi
by limiting Na+ pump-mediated
NH+4 entry. An alternative hypothesis, however, is that increasing extracellular
K+ depolarizes the cell, which
alters the activity of other
H+/OH
transporters. If this hypothesis were true, then
NH+4-dependent, ouabain-induced
alkalinization results from membrane depolarization, which in turn
alters electrogenic
H+/OH
transport rather than
Na+-K+-ATPase-mediated
NH+4 uptake. At a concentration of 1 mM,
BaCl2 induces a rapid and
sustained cellular depolarization through
K+ channel blockade in rat IMCD
tubules perfused in vitro (26). Thus the effect of
BaCl2 on
pHi was explored to test the
effect of changes in membrane potential on
pHi, independent of direct Na+ pump blockade or changes in
extracellular K+ concentration.
Resting pHi was examined in the
presence and the absence of Ba2+
(solution 5). Following the addition
of 1 mM Ba2+ to the peritubular
bath (n = 5),
pHi was similar to that observed in time controls (n = 3, solution 5,
Ba2+ absent; Fig.
6 a nd Table 5). Thus
Ba2+ does not induce cellular
alkalinization commensurate with that observed upon ouabain addition
(solution 5; Fig.
6B and Table 5).2
Moreover, NH+4-dependent, ouabain-induced
alkalinization was not abolished when 1 mM
BaCl2 was present in the bath
solution (Table 5).

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Fig. 6.
Effect of Ba2+ on resting
pHi.
A: tubules were perfused in presence
of 3 mM KCl + 6 mM NH4Cl but in
absence of and
(solution
5) (see footnote 2.). Then, baseline
pHi was measured
(left). Bath solution was exchanged
with the introduction of 1 mM
BaCl2.
pHi was measured 4 min later. In a
time control series (right), the
experiment above was repeated, but NaCl was substituted for
BaCl2 isosmotically
(n = 3).
B: ouabain-induced alkalinization was
measured under the same conditions as above (solution
5), but in absence of
Ba2+. Baseline
pHi was measured.
pHi was again measured 4 min after
ouabain addition (2.5 mM) and then 4 min following ouabain
withdrawal.
|
|
The K+ channel present on the
basolateral membrane of the IMCD recycles
K+ (26), taken up by the
Na+-K+-ATPase
(26). It is possible that this K+
channel mediates NH+4 efflux as well (39). Thus ouabain addition could alter
K+ or
NH+4 transport through a
Ba2+-sensitive pathway such as
K+ channels. However,
ouabain-induced alkalinization was observed in the presence of
Ba2+ (Table 5). Therefore,
NH+4 transport through Ba2+-sensitive pathways cannot
fully explain NH+4-dependent, ouabain-induced
alkalinization.
Role of
transport.
Another explanation for the observed
NH+4-dependent reduction in
JtCO2 and
increase in pHi observed following ouabain addition is through changes in
transport. The
Cl
/
exchanger and
Na+-
symporter are located on the basolateral membrane of the rat tIMCD (12,
33). In other cell types such as the proximal tubule,
NH3 uptake "back-titrates"
intracellular protons and thus increases
pHi (29). The
NH3-induced increase in
pHi could stimulate
exit through either of the above
pathways. Moreover, in other cells the Na+ pump modulates both
Cl
/
exchange and
Na+-
symport activity (27, 31). Thus the increase in
pHi and the reduction in
JtCO2, which
follow ouabain addition, could be explained by changes in either
Na+-
or
Cl
/
-mediated
efflux.
To resolve this question, NH+4-dependent,
ouabain-induced alkalinization was tested in the absence of
/CO2. To do so, bicarbonate-free, HEPES-buffered solutions
(solutions 6 and
7) were employed, which were bubbled
with ultrapure O2 (<3 ppm
CO2). In the presence of 3 mM
KCl + 6 mM NH4Cl in the bath and
perfusate (solution 6),
pHi rose 0.08 ± 0.01 pH units
(n = 5) with ouabain
addition and fell to baseline with ouabain withdrawal (Fig.
7; Table 4). This ouabain-induced
alkalinization, however, was not observed in the absence of
NH4Cl (solution
7; Fig. 8 and Table 4). Thus
ouabain-induced alkalinization was independent of
/CO2
in the extracellular media but was completely dependent on the presence
of
NH4Cl.

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Fig. 7.
Effect of ouabain on resting pHi
in absence of
/CO2.
Tubules were perfused and bathed in presence of 3 mM KCl + 6 mM
NH4Cl but in absence of
/CO2
(solution 6). Baseline
pHi was measured. Resting
pHi was then measured following
addition and then withdrawal of ouabain (2.5 mM) to peritubular bath.
Bath was then exchanged to include 0.1 mM ethoxzolamide, a carbonic
anhydrase inhibitor. Resting pHi
was again measured in presence and absence of ouabain (2.5 mM) in
peritubular bath. Results from 5 separate tubules are displayed.
|
|

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Fig. 8.
Effect of ouabain on resting pHi
in absence of both
/CO2
and NH4Cl. Tubules were perfused
and bathed in presence of 3 mM KCl, but without
NH4Cl (solution
7). Baseline pHi
was measured. pHi was again
measured following addition and then withdrawal of ouabain (2.5 mM) in
peritubular bath. Results obtained from 3 separate tubules are
displayed.
|
|
Considerable CO2 can be generated
through cellular metabolism, even in the nominal absence of
/CO2
(3). In other cell types, the hydration of
CO2, catalyzed by carbonic anhydrase, can produce cellular
and hence substantial
Na+-
or
Cl
/
-mediated
transport (3). Therefore the role
of
transport in ouabain-induced
alkalinization was further explored. To do so,
NH+4-dependent, ouabain-induced
alkalinization was measured when 0.1 mM ethoxzolamide, a membrane
permeant inhibitor of carbonic anhydrase, was added to the bath
(solution 6). As shown (Fig. 7;
Table 4), carbonic anhydrase inhibition did not abolish ouabain-induced
alkalinization. Thus changes in
transport cannot explain NH+4dependent,
ouabain-induced alkalinization.
Effect of
Na+-K+-2Cl
inhibition on JtCO2 and resting
pHi.
Our laboratory has shown that NH+4 and
K+ compete for a common binding
site on the
Na+-K+-2Cl
cotransporter in mouse tIMCD cells (37). Furthermore, both ions are
transported through this carrier (37). These results are in keeping
with a recent report of high levels of
Na+-K+-2Cl
cotransport expression in the mouse tIMCD (14). In many cell types,
increased Na+i reduces
K+ (or
NH+4) uptake through the
Na+-K+-2Cl
cotransporter (27, 40). Thus the reduction in
JtCO2 and the increase in resting pHi observed
with ouabain addition could occur from reduced
Na+-K+-2Cl
cotransport-mediated NH+4 uptake. If this
hypothesis were true, then addition of an
Na+-K+-2Cl
cotransport inhibitor, such as bumetanide, should inhibit
JtCO2 the same or
more than that observed with ouabain addition. Bumetanide at a
concentration of 100 µM fully inhibits the
Na+-K+-2Cl
cotransporter in the rat tIMCD (11). Thus bumetanide in a 100 µM
concentration was employed, and its effects on
JtCO2 and resting pHi were tested (Table 2; Fig.
9). Our laboratory reported previously that
in tIMCD tubules from DOCP-treated rats perfused in vitro in the
presence of 3 mM KCl + 6 mM NH4Cl
(solution 1),
JtCO2 fell from
3.8 ± 0.5 to 1.6 ± 0.3 pmol · mm
1 · min
1
upon the addition of ouabain to the bath
(n = 7, P < 0.05) (30). We asked ether the
reduction in
JtCO2 observed
following ouabain addition could be reproduced with bumetanide. Under
these conditions (solution 1),
baseline JtCO2
was 3.1 ± 0.4 and 2.8 ± 0.5 pmol · mm
1 · min
1
with the application of 100 µM bumetanide to the bath
(n = 3, P = NS). Thus, although
ouabain addition to the bath reduced
JtCO2, the
addition of bumetanide did not.

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Fig. 9.
Effect of bumetanide on
JtCO2. Tubules
were perfused and bathed in presence of 3 mM KCl + 6 mM
NH4Cl (solution
1). During control period, mean flux was 3.1 ± 0.4 pmol · mm 1 · min 1.
After addition of 100 µM bumetanide to peritubular bath, flux was 2.8 ± 0.5 pmol · mm 1 · min 1
(n = 3, P = NS).
|
|
As an independent assessment of the effect of bumetanide on
NH+4 transport,
pHi was measured following the addition of bumetanide to the bath. Under conditions
identical to those above (solution
1), pHi did not
increase following bumetanide addition to the bath (Table
6). We conclude that the effect of ouabain on
JtCO2 and
pHi cannot be explained by
NH+4 transport on the
Na+-K+-2Cl
cotransporter. These results are also consistent with
immunolocalization studies that indicate low levels of expression of
Na+-K+-2Cl
cotransport protein (BSC-2) in the rat tIMCD (9), which contrasts with
the high levels of expression reported in the tIMCD of the mouse (14).
 |
DISCUSSION |
Our laboratory has demonstrated previously that net acid secretion in
the tIMCD is greater in the presence of
NH4Cl than in its absence (30).
Furthermore, in the presence but not in the absence of
NH4Cl, ouabain addition reduces
net acid secretion and increases
pHi. These observations cannot
be explained by differences in buffering of the luminal
fluid or the cytosol which might occur in the presence and absence of
NH4Cl3
(30). The present study demonstrates that the
NH+4- dependent, ouabain-induced
alkalinization and reduction in
JtCO2 occur
through inhibition of NH+4 uptake by the Na+-K+-ATPase.
We conclude that
Na+-K+-ATPase-mediated
NH+4 uptake is an important determinant of
pHi and net acid secretion in the
rat tIMCD.
In mouse tIMCD cells in culture, a
K+/NH+4
exchanger has been reported (1). Since the driving force for K+
exit/NH+4 uptake, or
K+/NH+4
exchange (Fig. 10) (1), is decreased with Na+ pump inhibition,
NH+4- dependent, ouabain-induced alkalinization might be explained by changes in
K+/NH+4
exchange. This question cannot be tested directly, since no specific
inhibitors of
K+/NH+4
exchange are available (1, 38). However, it is unlikely that
ouabain-induced changes in pHi and
JtCO2, as
observed in this study, are mediated by changes in
K+/NH+4
exchange activity. Inhibition of the
Na+ pump decreases the driving
force for
K+/NH+4
exchange by reducing intracellular
K+
(K+i) concentration. In many cells,
including the proximal tubule (22, 41), following the addition of
ouabain, Na+i increases, and hence
K+i decreases, over a time period of at
least 20 min. However, as shown in Fig. 2, the alkalinization observed
following ouabain addition is complete within 2 min. Thus
pHi changes that follow ouabain
addition do not parallel expected changes in
K+i. Moreover, we have shown that both
NH+4 and
K+ support equivalent rates of
ouabain-sensitive ATP hydrolysis in permeabilized, native rat IMCD
cells (34). Thus both cations are transported directly by the
Na+-K+-ATPase.
Since these experiments were performed in permeabilized cells,
inhibition of NH+4 transport following ouabain addition occurred independent of changes in intracellular ion
composition.
In the rat tIMCD, two K+-ATPases
have been identified (16). One of these
K+-ATPases, like the gastric
H+-K+-ATPase,
is sensitive to low concentrations of Sch-28080 but insensitive to
ouabain. The other K+-ATPase is
insensitive to Sch-28080 but sensitive to ouabain and thus resembles
the "colonic"
H+-K+-ATPase
isoform. NH+4 transport by the
H+-K+-ATPase
has been described (8). Therefore, it is possible that the
ouabain-sensitive "colonic"
H+-K+-ATPase
transports NH+4 and therefore mediates the
NH+4-dependent, ouabain-induced increase in pHi and decrease in
JtCO2 observed in
the present study. However, activity of the colonic
H+-K+-ATPase
is insensitive to changes in Na+
availability (4) and therefore cannot explain the observations of the
current study. We have reported Sch-28080-sensitive bicarbonate absorption in the rat tIMCD (38). The transporter mediating Sch-28080-sensitive bicarbonate absorption, however, is distinct from
the transport mechanism that mediates the
NH+4-dependent, ouabain-sensitive changes in
pHi and
JtCO2 described
herein (38).
Our results do not support significant
K+ channel-mediated
NH+4 efflux on the basolateral membrane of
the rat tIMCD. However, they cannot exclude channel-mediated
NH+4 efflux on the apical membrane.
ATP-sensitive K+ channels
sensitive to intracellular but not extracellular
Ba2+ have been localized to the
apical membrane of the mouse IMCD (24). Similarly, a nonspecific,
amiloride-sensitive cation channel on the apical membrane has been
reported (20). Both of these channels transport
NH+4. Thus depolarization of the cell with
ouabain addition could facilitate NH+4 efflux
across the apical membrane mediated by these channels. The result would
be increased NH+4 secretion into the luminal
fluid upon ouabain addition. However, in a previous study (30), when
ouabain was applied to the peritubular bath, total ammonium secretion
fell from
0.3 ± 0.1 to
0.1 ± 0.1 pmol · mm
1 · min
1.
Thus ouabain does not increase channel-mediated
NH+4 efflux across the apical membrane. Thus
changes in K+ channel-mediated
NH+4 transport cannot explain the
observations of the present study.
In epithelia that utilize carbonic acid as the main proton source, net
luminal proton secretion occurs when proton secretion across the apical
membrane is accompanied by bicarbonate (base) secretion across the
basolateral membrane (31). However, cytosolic carbonic anhydrase
activity is less abundant in the tIMCD than in other nephron segments
(32, 33). Therefore, the regulation of basolateral
exit and apical proton secretion in parallel may be less important in the tIMCD than in other nephron segments. Our results show that NH+4, rather
than carbonic acid, provides the primary source of protons in the
tIMCD.

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Fig. 10.
Proposed NH+4 pathway in rat terminal inner
medullary collecting duct (tIMCD). NH+4 is
taken up on basolateral membrane by the
Na+-K+-ATPase.
Intracellular NH+4 provides a source of
H+ and
NH3 for the cell.
H+ is secreted across the apical
membrane through the
H+-K+-ATPase.
NH3 leaves the cell by passive
diffusion across the apical membrane, or it recycles across the
basolateral membrane. Other
NH+4/OH /H+/
transport pathways reported in tIMCD are shown (1, 11-13, 20, 24,
26, 28, 30, 35, 38).
|
|
In the tIMCD, NH+4 is an important
H+ source. Our data show that
NH+4 is taken up across the basolateral
membrane by the Na+ pump,
providing a source of H+ and
NH3. Protons are then secreted
across the apical membrane. However, since the
pKa for
NH3/NH+4
is 9.03 (15), in the range of physiological
pHi, only ~1% of
NH+4 releases a proton (39). Thus
NH+4 uptake alone should not result in
measurable changes in pHi.
However, rapid entry of NH+4 coupled with
NH3 exit across the basolateral
membrane should provide significant net proton uptake with a
substantial fall in pHi (30, 39).
Across the basolateral membrane, NH+4 uptake
with NH3 efflux would provide an
NH3 shuttle, with net proton
uptake. Across the apical membrane, NH3 secretion in parallel with
H+ secretion by the
H+-K+-ATPase
(or H+-ATPase) (38) would trap
NH+4 and facilitate net acid secretion (Fig.
10).
Amiloride administration decreases
K+ excretion and impairs urinary
acidification (5, 7). These defects in
H+ and
K+ secretion generate a
hyperkalemic, hyperchloremic metabolic acidosis (7). In the cortical
collecting duct (21) and turtle bladder (2), amiloride administration
reduces the lumen-negative potential difference and decreases proton
secretion. The acidification defect that follows amiloride
administration is felt to occur from elimination of this lumen-negative
potential difference, which decreases the driving force for
H+ secretion. In the turtle
bladder, if the lumen-negative potential difference is restored, then
the defect in proton secretion that follows amiloride administration is
reversed, despite the ongoing presence of amiloride (2). Thus the
amiloride-induced reduction in proton secretion has been attributed to
a "voltage defect." In the rat tIMCD, micropuncture studies have
demonstrated a reduction in net acid secretion with luminal amiloride
(5). In bicarbonate-loaded rats, DuBose and Caflisch (5) observed that
the papillary urine-to-blood PCO2
gradient, an index of proton secretion, was reduced with amiloride
administration. The present study demonstrates a reduction in
JtCO2 upon
addition of amiloride to the perfusate that cannot be explained by
changes in VT.
The present study, therefore, provides an explanation for this impaired
proton secretion in the tIMCD. Amiloride reduces
Na+i availability, which attenuates Na+ pump-mediated
NH+4 uptake and therefore reduces net proton
secretion.4
Luminal acidification in the tIMCD is thus dependent on apical Na+ entry.
Hyperkalemia is frequently associated with metabolic acidosis (6).
Hyperkalemia reduces ammonium production by the proximal tubule and
reduces ammonium absorption by the thick ascending limb, which together
attenuate net acid excretion (6). In rats receiving dietary
K+ loading, DuBose and Good (6)
have observed a decrease in the interstitial
NH3 concentration and therefore a
reduction in the NH3 gradient from
the interstitium to the collecting duct lumen. However, in rats which
received a high-K+ diet, no net
transfer of ammonium from the interstitium to the lumen was observed,
despite the presence of an NH3
gradient that should favor NH3
diffusion into the collecting duct lumen. This observation suggested a
defect in NH+4 transfer from the interstitium
and the collecting duct lumen. The present study shows that this
transfer defect can be explained, at least in part, by competition
between NH+4 and
K+ for the extracellular binding
site on the Na+ pump. Increasing
extracellular K+ reduces
NH+4 uptake by the tIMCD cell and hence reduces acid secretion.
In conclusion, NH+4 uptake across the
basolateral membrane of the tIMCD is mediated by the
Na+ pump.
NH+4 uptake provides a source of
H+ for apical
H+ secretion and the titration of
luminal buffers. The reduction in acid secretion in the rat tIMCD
observed following the administration of amiloride or upon dietary
K+ loading can be explained, at
least in part, by reduced NH+4 uptake by the
Na+-K+-ATPase.
 |
ACKNOWLEDGEMENTS |
I thank Drs. Steve Sansom, Andrew Kahn, and Roger O'Neil for
helpful suggestions. I am again grateful to Dr. Thomas D. DuBose, Jr.,
for suggestions and continued support.
 |
FOOTNOTES |
This work was supported by a National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-46493.
1
Our laboratory has demonstrated that removal of
extracellular Na+ results in a
prompt reduction in pHi and
Na+i, mediated by
Na+/H+
exchange (35).
2
In experiments that explored the effect of
Ba2+ on
pHi,
and
were removed from the
perfusate and bath. These anions were removed, since both BaSO4 and
BaPO4 are poorly soluble in
aqueous solution.
3
Changes in
pHi are dependent on cellular
buffering capacity. An increase in buffering capacity should attenuate
pHi changes measured following
changes in net proton transport. However, buffering capacity is greater
in the presence of NH+4 than in its absence
(22). Thus the NH+4-dependent, ouabain-induced increase in pHi
observed in the present study differs directionally from expected
pHi changes that represent an
NH+4-induced change in buffering capacity. The observation that ouabain-induced alkalinization occurs only in the
presence of NH4Cl therefore cannot
be explained by differences in cellular
H+ buffering capacities in cells
exposed to NH4Cl.
4
In the presence of
NH4Cl, luminal amiloride reduces
JtCO2 and
increases pHi through inhibition
of Na+ pump-mediated
NH+4 uptake. However, we cannot exclude an
additional mechanism responsible for the amiloride-induced reduction in
JtCO2.
Address for reprint requests: S. M. Wall, Division of Renal Diseases
and Hypertension, Univ. of Texas Medical School at Houston, 6431 Fannin, MSB 4.148, Houston, TX 77030.
Received 3 January 1997; accepted in final form 24 July 1997.
 |
REFERENCES |
1.
Amlal, H.,
and
M. Soleimani.
K+/NH+4 antiporter: a unique ammonium carrying transporter in the kidney inner medulla.
Biochim. Biophys. Acta
1323:
319-333,
1997[Medline].
2.
Arruda, J. A. L.,
K. Subbarayudu,
G. Dytko,
R. Mola,
and
N. A. Kurtzman.
Voltage-dependent distal acidification defect induced by amiloride.
J. Lab. Clin. Med.
95:
407-416,
1980[Medline].
3.
Brookes, N.,
and
R. J. Turner.
K+-induced alkalinization in mouse cerebral astrocytes mediated by reversal of electrogenic Na+-
cotransport.
Am. J. Physiol.
267 (Cell Physiol. 36):
C1633-C1640,
1994[Abstract/Free Full Text].
4.
Codina, J.,
B. C. Kone,
J. T. Delmas-Mata,
and
T. D. DuBose, Jr.
Functional expression of the colonic H+,K+-ATPase
-subunit.
J. Biol. Chem.
271:
29759-29763,
1996[Abstract/Free Full Text].
5.
DuBose, T. D., Jr.,
and
C. R. Caflisch.
Validation of the difference in urine and blood carbon dioxide tension during bicarbonate loading as an index of distal nephron acidification in experimental models of distal renal tubular acidosis.
J. Clin. Invest.
75:
1116-1123,
1985[Medline].
6.
DuBose, T. D., Jr.,
and
D. W. Good.
Chronic hyperkalemia impairs ammonium transport and accumulation in the inner medulla of the rat.
J. Clin. Invest.
90:
1443-1449,
1992[Medline].
7.
DuBose, T. D., Jr.
Experimental models of distal renal tubular acidosis.
Semin. Nephrol.
10:
174-180,
1990[Medline].
8.
Fryklund, J.,
K. Gedda,
D. Scott,
G. Sachs,
and
B. Wallmark.
Coupling of H+-K+-ATPase activity and glucose oxidation in gastric glands.
Am. J. Physiol.
258 (Gastrointest. Liver Physiol. 21):
G719-G727,
1990[Abstract/Free Full Text].
9.
Ginns, S. M.,
M. A. Knepper,
C. A. Ecelbarger,
J. Terris,
X. He,
R. A. Coleman,
and
J. B. Wade.
Immunolocalization of the secretory isoform of Na-K-2Cl cotransporter in rat renal intercalated cells.
J. Am. Soc. Nephrol.
7:
2533-2542,
1996[Abstract].
10.
Good, D. W.,
C. R. Caflisch,
and
T. D. DuBose, Jr.
Transepithelial ammonia concentration gradients in inner medulla of the rat.
Am. J. Physiol.
252 (Renal Fluid Electrolyte Physiol. 21):
F491-F500,
1987[Abstract/Free Full Text].
11.
Grupp, C.,
I. Pavendstadt-Grupp,
R. W. Grunewald,
C. Bevan,
J. B. Stokes III,
and
R. K. H. Kinne.
A Na-K-Cl cotransporter in isolated rat papillary collecting duct cells.
Kidney Int.
36:
201-209,
1989[Medline].
12.
Hart, D.,
and
E. P. Nord.
Polarized distribution of Na+/H+ antiport and Na+/
cotransport in primary cultures of renal inner medullary collecting duct cells.
J. Biol. Chem.
266:
2374-2382,
1991[Abstract/Free Full Text].
13.
Husted, R. F.,
K. A. Volk,
R. D. Sigmund,
and
J. B. Stokes.
Anion secretion by the inner medullary collecting duct.
J. Clin. Invest.
95:
644-650,
1995[Medline].
14.
Kaplan, M. R.,
M. D. Plotkin,
D. Brown,
S. C. Hebert,
and
E. Delpire.
Expression of the mouse Na-K-2Cl cotransporter, mBSC2, in the terminal inner medullary collecting duct, the glomerular and extraglomerular mesangium and the glomerular afferent arteriole.
J. Clin. Invest.
98:
723-730,
1996[Abstract/Free Full Text].
15.
Knepper, M. A.,
R. Packer,
and
D. W. Good.
Ammonium transport in the kidney.
Physiol. Rev.
69:
179-249,
1989[Free Full Text].
16.
Kone, B. C.
Renal H,K-ATPase: structure, function, and regulation.
Miner. Electrolyte Metab.
22:
349-365,
1996[Medline].
17.
Kudo, L. H.,
A. A. Van Baak,
and
A. S. Rocha.
Effect of vasopressin on sodium transport across inner medullary collecting duct.
Am. J. Physiol.
258 (Renal Fluid Electrolyte Physiol. 27):
F1438-F1447,
1990[Abstract/Free Full Text].
18.
Kurtz, I.,
and
R. S. Balaban.
Ammonium as a substrate for Na+-K+-ATPase in rabbit proximal tubules.
Am. J. Physiol.
250 (Renal Fluid Electrolyte Physiol. 19):
F497-F502,
1986[Abstract/Free Full Text].
19.
Laplace, J. R.,
R. F. Husted,
and
J. B. Stokes.
Cellular responses to steroids in the enhancement of Na+ transport by rat collecting duct cells in culture.
J. Clin. Invest.
90:
1370-1378,
1992[Medline].
20.
Light, D. B.,
F. V. McCann,
T. M. Keller,
and
B. A. Stanton.
Amiloride-sensitive cation channel in apical membrane of inner medullary collecting duct.
Am. J. Physiol.
255 (Renal Fluid Electrolyte Physiol. 24):
F278-F286,
1988[Abstract/Free Full Text].
21.
McKinney, T. D.,
and
M. B. Burg.
Bicarbonate absorption by rabbit cortical collecting tubules in vitro.
Am. J. Physiol.
234 (Renal Fluid Electrolyte Physiol. 3):
F141-F145,
1978[Medline].
22.
Negulescu, P. A.,
A. Harootunian,
R. Y. Tsien,
and
T. E. Machen.
Fluorescence measurements of cytosolic free Na concentration, influx and efflux in gastric cells.
Cell Regul.
1:
259-268,
1990[Medline].
23.
Roos, A.,
and
W. F. Boron.
Intracellular pH.
Physiol. Rev.
61:
296-434,
1981[Free Full Text].
24.
Sansom, S. C.,
T. Mougouris,
S. Ono,
and
T. D. DuBose, Jr.
ATP-sensitive K+-selective channels of inner medullary collecting duct cells.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F489-F496,
1994[Abstract/Free Full Text].
25.
Sauer, M.,
A. Flemmer,
K. Thurau,
and
F.-X. Beck.
Sodium entry routes in principal and intercalated cells of the isolated perfused cortical collecting duct.
Pflügers Arch.
416:
88-93,
1990[Medline].
26.
Stanton, B. A.
Characterization of apical and basolateral membrane conductances of rat inner medullary collecting duct.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F862-F868,
1989[Abstract/Free Full Text].
27.
Turner, R. J.,
and
J. N. George.
Cl
-
exchange is present with Na+-K+-Cl
cotransport in rabbit parotid acinar basolateral membranes.
Am. J. Physiol.
254 (Cell Physiol. 23):
C391-C396,
1988[Abstract/Free Full Text].
28.
Volk, K. A.,
R. D. Sigmund,
P. M. Snyder,
F. J. McDonald,
M. J. Welsh,
and
J. B. Stokes.
rENaC is the predominant Na+ channel in the apical membrane of the rat renal inner medullary collecting duct.
J. Clin. Invest.
96:
2748-2757,
1995[Medline].
29.
Volkl, H.,
and
F. Lang.
Electrophysiology of ammonia transport in renal straight proximal tubules.
Kidney Int.
40:
1082-1089,
1991[Medline].
30.
Wall, S. M.
NH+4 augments net acid secretion by a ouabain-sensitive mechanism in isolated perfused inner medullary collecting ducts.
Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol. 39):
F432-F439,
1996[Abstract/Free Full Text].
31.
Wall, S. M.
Ammonium transport and the role of the Na,K-ATPase.
Miner. Electrolyte Metab.
22:
311-317,
1996[Medline].
32.
Wall, S. M.,
M. F. Flessner,
and
M. A. Knepper.
Distribution of luminal carbonic anhydrase activity along rat inner medullary collecting duct.
Am. J. Physiol.
260 (Renal Fluid Electrolyte Physiol. 29):
F738-F748,
1991[Abstract/Free Full Text].
33.
Wall, S. M.,
and
M. A. Knepper.
Acid-Base transport in the inner medullary collecting duct.
Semin. Nephrol.
10:
148-158,
1990[Medline].
34.
Wall, S. M.,
and
L. M. Koger.
NH+4 transport mediated by Na+-K+-ATPase in rat inner medullary collecting duct.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F660-F670,
1994[Abstract/Free Full Text].
35.
Wall, S. M.,
J. A. Kraut,
and
S. Muallem.
Modulation of Na+-H+ exchange activity by intracellular Na+,H+ and Li+ in IMCD cells.
Am. J. Physiol.
255 (Renal Fluid Electrolyte Physiol. 24):
F331-F339,
1988[Abstract/Free Full Text].
36.
Wall, S. M.,
J. M. Sands,
M. F. Flessner,
H. Nonoguchi,
K. R. Spring,
and
M. A. Knepper.
Net acid transport by isolated perfused inner medullary collecting ducts.
Am. J. Physiol.
258 (Renal Fluid Electrolyte Physiol. 27):
F75-F84,
1990[Abstract/Free Full Text].
37.
Wall, S. M.,
H. N. Trinh,
and
K. E. Woodward.
Heterogeneity of NH+4 transport in mouse terminal inner medullary collecting duct.
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
F536-F544,
1995[Abstract/Free Full Text].
38.
Wall, S. M.,
A. V. Truong,
and
T. D. DuBose, Jr.
H+,K+-ATPase mediates net acid secretion in the rat terminal inner medullary collecting duct.
Am. J. Physiol.
271 (Renal Fluid Electrolyte Physiol. 40):
F1037-F1044,
1996[Abstract/Free Full Text].
39.
Watts, B. A., III,
and
D. W. Good.
Effects of ammonium on intracellular pH in rat medullary thick ascending limb: mechanisms of apical membrane NH+4 transport.
J. Gen. Physiol.
103:
917-936,
1994[Abstract].
40.
Whisenant, N.,
M. Khademazad,
and
S. Muallem.
Regulatory interaction of ATP Na+ and Cl
in the turnover cycle of the NaK2Cl cotransporter.
J. Gen. Physiol.
101:
889-908,
1993[Abstract].
41.
Wong, P. S. K.,
P. L. Barclay,
M. J. Newman,
and
E. J. Johns.
The influence of acetazolamide and amlodipine on the intracellular sodium content of rat proximal tubular cells.
Br. J. Pharmacol.
112:
881-886,
1994[Abstract].
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