Hyperosmotic mannitol activates basolateral NHE in proximal
tubule from P-glycoprotein null mice
Yukio
Miyata,
Yasushi
Asano, and
Shigeaki
Muto
Department of Nephrology, Jichi Medical School, Tochigi
329-0498, Japan
 |
ABSTRACT |
Using the pH-sensitive fluorescent dye
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein
acetoxymethyl ester, we examined the effects of hyperosmotic mannitol
on basolateral Na+/H+ exchange (NHE) activity
in isolated nonperfused proximal tubule S2 segments from mice lacking
both the mdr1a and mdr1b genes (KO) and wild-type
mice (WT). All experiments were performed in
CO2/HCO
-free HEPES solutions. Osmolality
of the peritubular solution was raised from 300 to 500 mosmol/kgH2O by the addition of mannitol. NHE activity was
assessed by Na+-dependent acid extrusion rates
(JH) after an acid load with NH4Cl prepulse. Under isosmotic conditions, JH values
at a wide intracellular pH (pHi) range of 6.20-6.90
were not different between the two groups. In WT mice, hyperosmotic
mannitol had no effect on JH at the wide
pHi range. In contrast, in KO mice, hyperosmotic mannitol increased JH at a pHi range of
6.20-6.45 and shifted the
JH-pHi relationship by 0.15 pH units
in the alkaline direction. In KO mice, hyperosmotic mannitol caused an
increase in maximal velocity without changing the Michaelis-Menten
constant for peritubular Na+. Exposure of cells from WT
mice to the hyperosmotic mannitol solution including the P-gp inhibitor
cyclosporin A increased JH (at pHi
6.30) to an extent similar to that in cells from KO mice exposed to
hyperosmotic mannitol alone. In KO mice, staurosporine and calphostin C
inhibited the hyperosmotic mannitol-induced increase in
JH. The stimulatory effect of hyperosmotic
mannitol on JH was mimicked by addition to the
isosmotic control solution, including phorbol 12-myristate 13-acetate
(PMA; the PKC activator). In WT mice, hyperosmotic mannitol with PMA
increased JH. We conclude that, in the absence
of P-gp activity, hyperosmotic mannitol activates basolateral NHE via
protein kinase C, whereas in the presence of P-gp activity, it does not.
isolated nonperfused tubule; regulatory volume increase; intracellular pH measurement; sodium-hydrogen exchange
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INTRODUCTION |
P-GLYCOPROTEIN (P-GP)
WAS initially identified through its ability to confer multidrug
resistance (MDR) in mammalian tumor cells (reviewed in Ref.
11). P-gp is a member of the ATP-binding cassette
superfamily of transporters (11) and utilizes ATP to pump
hydrophobic drugs out of the cells, decreasing their intracellular concentrations and hence their toxicity. Humans have one
drug-transporting P-gp (MDR1), whereas mice have two genes
encoding drug-transporting P-gps, mdr1a (also called
mdr3) and mdr1b (also called mdr1)
(11, 14, 27). In the mouse, mdr1a and
mdr1b together fulfill the same function as MDR1
in humans, and similar levels of mdr1a and mdr1b
expression are observed in the kidney (5, 28). In the kidney, the apical membrane of the proximal tubule epithelium is
particularly rich in P-gp (7, 30), placing this pump in the correct location to mediate the active excretion of xenobiotics. Consistent with a role for P-gp as an excretory transporter, our laboratory (31) recently reported that in the isolated
perfused mouse proximal tubule, P-gp-mediated drug efflux capacity
indeed exists in the apical membrane of the proximal tubule S2 segment from wild-type (WT) mice but is lacking in that of mice in which both
the mdr1a and mdr1b genes were disrupted (KO mice).
In a variety of cell types, Na+/H+ exchange
(NHE) is activated by shrinkage of cells in a hyperosmotic solution,
resulting in Na+ entry into the cell. Osmotic uptake of
water due to this Na+ entry leads to cell swelling, a
process termed regulatory volume increase (RVI) (reviewed in Refs.
17 and 20). In sharp contrast, when rabbit proximal tubule
cells are suddenly exposed to hyperosmotic mannitol, NaCl, or raffinose
solutions, they rapidly shrink but remain reduced in size (9,
19). However, the underlying mechanisms responsible for the lack
of RVI have not been fully demonstrated. Recently, our laboratory
(21) used isolated nonperfused proximal tubule S2 segments
from WT and KO mice to demonstrate that exposure of tubules from WT
mice to hyperosmotic mannitol did not result in RVI, whereas exposure
of tubules from KO mice to hyperosmotic mannitol elicited RVI. We
observed that in tubules from WT mice, the peritubular addition of P-gp
inhibitors (verapamil and cyclosporin A) resulted in RVI, whereas in
the tubules of the KO mice it had no effect on RVI (21).
Therefore, in the mouse proximal tubule, P-gp modulates RVI during the
exposure to hyperosmotic mannitol. We also observed that the
P-gp-induced modulation of RVI was abolished by both removing
peritubular Na+ and adding peritubular
ethylisopropylamiloride (EIPA; the specific NHE inhibitor)
(21). These findings indicate that basolateral NHE
contributes to the P-gp-induced modulation of RVI. However, it is not
known whether basolateral NHE is actually activated in P-gp-induced
cell volume regulation during exposure to hyperosmotic mannitol. NHE in
the basolateral membrane of the mouse proximal tubule under isosmotic
conditions also has not yet been characterized.
Therefore, we used isolated nonperfused proximal tubule S2 segments
from KO and WT mice to examine 1) whether the basolateral membrane of mouse proximal tubule cells possesses an NHE under isosmotic conditions and, if so, 2) whether and how
basolateral NHE is modulated by hyperosmotic mannitol.
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METHODS |
Solutions.
The compositions of solutions are given in Table
1. All solutions were nominally
CO2/HCO
free. Solutions
1-3 were adjusted to pH 7.4 at 37°C. The osmolality of all
solutions was measured before the experiment and was verified to be
within a range of 300 ± 5 mosmol/kgH2O. A
hyperosmotic mannitol solution (500 mosmol/kgH2O) was made
by adding mannitol to solutions 1 or 2. The
nigericin-calibrating solutions were titrated to different pH values at
37°C with either HCl or N-methyl-D-glucamine
(NMDG). For Na+-free solutions (solutions
2-4, Table 1), Na+ was replaced with NMDG
titrated with the appropriate acid.
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was prepared as a 10 mM
stock solution and diluted 1:1,000 to a final concentration of 10 µM.
EIPA was prepared as a 100 mM stock solution in methanol and diluted
1:1,000 to a final concentration of 100 µM. Nigericin was prepared as a 10 mM stock solution in ethanol and diluted 1:1,000 into
solution 4 (Table 1) to a final concentration of 10 µM.
Cyclosporin A was dissolved in ethanol at 0.1% final concentration.
Phorbol 12-myristate 13-acetate (PMA), staurosporine, and calphostin C were each dissolved in DMSO at 0.1% final concentrations. Equivalent concentrations of vehicle were added as a control for individual protocols.
Animals.
KO mice used in this study were originally described by Schinkel et al.
(27). Male KO and FVB (WT) mice, serving as controls (body
wt 25-40 g), were purchased from Taconic Engineering (Germantown, NY). The animals were maintained under a controlled environment and had
free access to standard rodent chow and tap water ad libitum until the
experiments began. Ages of KO animals were matched with their WT controls.
In vitro microperfusion.
Both groups of mice were anesthetized with an intraperitoneal injection
of pentobarbital sodium (4 mg/100 g body wt), 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 KH2PO4, 44 K2HPO4, 15 KCl, 9 NaHCO3, and 160 sucrose. Proximal tubule S2 segments
were then dissected with fine forceps under a stereomicroscope and
transferred to a bath chamber mounted on an inverted microscope (IMT-2,
Olympus, Tokyo, Japan).
Both proximal and distal ends of the tubules were drawn into and
crimped by glass micropipettes as described by Dellasega and Grantham
(6) and Miyata et al. (21). In this
preparation, both ends of the tubules are occluded so that the lumen
becomes and remains collapsed. A flow-through bath system was utilized to permit rapid exchange of bath fluid. The bathing solution flowed by
gravity at 4.5-5.5 ml/min from the reservoirs through a water jacket to stabilize the bath temperature at 37°C. After tubules were
tightly crimped between the two glass micropipettes, they were bathed
in the isosmotic control solution (solution 1, Table 1) at
37°C for 10-15 min to allow them to equilibrate. After the
equilibration period, intracellular pH (pHi) was measured as described below.
Measurement of pHi.
Both groups of tubules were exposed from the bath to the isosmotic
control HEPES-buffered solution (solution 1, Table 1) containing the fluorescent pH-sensitive dye BCECF-AM (10 µM). After a
15-min dye-loading period at 37°C, the dye was washed out.
Single-cell measurements of pHi were performed using a
microscopic fluorometer (OSP3, Olympus) as described previously
(3, 22, 25). Measurements were made at ×100
magnification, and the diameter of the beam of light focused on the
cells was ~7.5 µm. The light source was a 75-W xenon lamp. The
fluorescent dye was excited alternatively at 440 and 490 nm by spinning
the sector mirror at 300 rpm and measured at a wavelength of 530 nm.
Because it takes 10 ms to obtain one fluorescence-excitation ratio
(I490/I440) with this
apparatus, each cell was exposed to light for 1 s to obtain one
mean I490/I440. We used
only cells that had at least a 20-fold greater fluorescence intensity
than that of the background. To minimize dye bleaching and cell damage,
protocols were made as short as possible.
Calibration of pHi.
pHi was calculated using the nigericin calibration
technique as described previously (3, 22, 25). At the end
of each experiment, cells were exposed to a Na+-free
solution (solution 4, Table 1) containing 10 µM of the H+/K+ exchanger (nigericin) and 105 mM
K+. If internal and external K+ concentration
are equal, then nigericin should equalize pHi and extracellular pH. Figure 1A is
a plot of I490/I440 vs.
time for a single cell exposed to a series of nigericin solutions at
different pH values.
I490/I440 and pH data
from this experiment are summarized in Fig. 1B. The data
were normalized to make the ratio at pH 7.0 equal to unity. The ratio
data can be described by a pH titration curve of the form
|
(1)
|
Inasmuch as the curve was constrained to pass through the point
having the coordinates
I490/I440 = 1.0 and pH = 7.0, we fitted the data to a variant of the pH titration equation that
forces the curve through this standard point
|
(2)
|
where a and b are the lower asymptote of
the curve and the distance between the upper and lower asymptotes of
the curve, respectively, and pK is the dissociation
constant.

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Fig. 1.
pH calibration of intracellular dye. A: time
course of fluorescence-excitation ratio
(I490/I440) during
exposure to high-extracellular K+ concentration-nigericin
solutions at different pH values. At the beginning of recording,
proximal tubule cells from wild-type (WT) and mdr1a and
mdr1b null (KO) mice were exposed to a pH 7.0 nigericin-containing solution (solution 4, Table 1, with 10 µM nigericin added). At indicated times, bathing solution was
switched to nigericin-containing solutions at different pH values.
pHo, extracellular pH. B: dependence of
normalized fluorescence-excitation ratio
(I490/I440) on
intracellular pH (pHi). , Data from 5 experiments in WT mice; , data from 7 experiments in KO
mice. Curves are results of a nonlinear least-squares fit of data to pH
titration curve that forces curve to pass through
(I490/I440) = 1.0 at
pHi of 7.0.
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The curve drawn through the points in Fig. 1B is the result
of a nonlinear least-squares fit of the normalized data to Eq. 2. The fitted values in the WT mice were 7.15 ± 0.01 (n = 10) and 1.56 ± 0.01 (n = 10)
for pK and b, respectively. The corresponding values in KO mice for pK and b were 7.15 ± 0.04 (n = 10) and 1.55 ± 0.03 (n = 10). The advantage of this normalization procedure is that it allows
us to obtain a one-point nigericin calibration for a cell. At the end
of each experiment, the cell was exposed to a nigericin solution at pH
7.0, and the I490/I440
data from the entire experiment were divided by the value at pH 7.0. We used Eq. 2 to calculate pHi from these
normalized I490/I440
values and the fitted values for pK and
b.
Determination of intracellular buffering power.
Intrinsic buffering power (
I) of proximal tubule cells
from WT and KO mice was determined by the method described previously (3, 22, 25). As shown in Fig.
2A, acid-loaded cells were exposed to a series of nominally Na+-free solutions
(solution 2, Table 1) that contained 20, 10, 5, 2.5, 1.0, 0.5, and 0 mM total ammonium
(NH3/NH
). Total
NH3/NH
-containing solutions were prepared
by adding NH4Cl, with replacement of NMDG in
Na+-free solution. With each stepwise decrease in
extracellular NH3/NH
concentration
([NH3/NH
]o), the amount of
protons delivered to the cytoplasm (
[acid]i) was
considered equal to the resultant change in intracellular
NH
concentration
([NH
]i). If it is assumed that
[NH3]i equals
[NH3]o, and that the acidic pK
governing NH3/NH
equilibrium (8.9 at 37°C) is the same in the cytoplasm as in the extracellular fluid,
[NH
]i can be calculated from the
observed pHi.
pHi was taken as the change in
pHi produced by the stepwise decrease in
[NH3/NH
]o.
I
was then calculated as 
[acid]i/
pHi
(3, 22, 25).
I was assigned to the mean of
the two pHi values used for its calculation.
I vs. pHi data were fitted by a straight
line for both groups of cells.

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Fig. 2.
pHi dependence of intrinsic intracellular
buffering power ( I). A: a typical experiment
in which we determined the buffering power of proximal tubule cells
from WT mice in HCO /CO2-free,
HEPES-buffered solution. Na+ was removed, and NMDGCl was
then replaced with 20 mM NH4Cl. NH4Cl
concentration was decreased step by step (horizontal bar). Restoration
of extracellular Na+ concentration to 142 mM caused
pHi to increase, indicating Na+/H+
exchange (NHE) activity. From these data, I was
calculated as described in METHODS. B:
pHi dependence of I for proximal tubule
cells from WT ( ) and KO ( ) mice.
Straight lines fit data from 8 proximal tubule cells from 3 WT mice and
6 proximal tubule cells from 3 KO mice. The equations of the best fit
line in the proximal tubule cells from WT and KO mice were
I = 441.0 61.5 × pHi
(r = 0.985) and I = 429.6 60.0 × pHi (r = 0.992) at a range of
physiological pHi, respectively.
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Computation of Na+-dependent acid
extrusion rates.
Na+-dependent acid extrusion rates
(JH) in proximal tubule cells from WT and KO
mice were calculated from rates of pHi increase (dpHi/dt) and
I in the following
experiment: proximal tubule cells from WT and KO mice were incubated in
the Na+-containing isosmotic solution (solution
1, Table 1) and then exposed to the Na+-free solution
containing 20 mM NH4Cl for 2 min (solution 3,
Table 1). During the pulse, pHi increased due to the entry
of NH3 into the cell and then tended to decrease toward
baseline due to slower influx of NH
. In the continued
presence of the Na+-free solution, NH4Cl was
abruptly removed from the peritubular solution, causing pHi
to decrease. After that, readdition of Na+ to the
peritubular solution gave rise to a rapid increase in pHi.
For the experiment, the pHi increase was divided into two or three sections, each of which was fitted to a third- or fourth-order polynomial. The derivatives of these equations (i.e.,
dpHi/dt values) were calculated at
pHi intervals of 0.05. At each pHi, data from
four or more experiments were averaged to produce a plot of mean
JH-pHi data (3, 22,
25). The H+ flux due to NHE (i.e.,
JH) was then determined from the equation JH = dpHi/dt
×
I.
Drugs and chemicals.
All chemicals were obtained from Wako (Osaka, Japan) except HEPES and
BCECF-AM, which were from Dojindo (Kumamoto, Japan), and EIPA, NMDG,
PMA, staurosporine, nigericin, and cyclosporin A, which were from Sigma
(St. Louis, MO).
Data analysis and statistics.
The data are expressed as means ± SE. Comparisons were performed
by Student's t-test or one-way ANOVA in combination with Fisher's protected least significant difference test where
appropriate. P values <0.05 were considered significant.
 |
RESULTS |
Steady-state pHi in proximal tubule cells from WT and
KO mice.
The study of pHi was carried out in
HCO
-free HEPES-buffered solutions to minimize the
contribution of HCO
-dependent transport mechanisms. When collapsed proximal tubule cells from WT and KO mice were peritubularly perfused with the Na+-containing
HEPES-buffered solution (solution 1, Table 1) at a rate of
4.5-5.5 ml/min at 37°C, the steady-state pHi values were 7.00 ± 0.01 (n = 52) and 6.98 ± 0.01 (n = 54), respectively, and were not statistically
significant between the two groups.
I in proximal tubule cells from WT and KO mice.
The pHi dependence of
I was determined from
experiments such as those illustrated in Fig. 2A. The
pHi dependence of such
I data is summarized
in Fig. 2B. The equations of the best fit line of data for
proximal tubule cells from WT and KO mice were
I = 441.0
61.5 × pHi (r = 0.985)
and
I = 429.6
60.0 × pHi (r = 0.992) at a range of physiological
pHi, respectively. These data indicate that in both groups
of proximal tubule cells,
I decreases with increasing
pHi. Furthermore,
I at comparable
pHi values was not significantly different between the two
groups of proximal tubule cells.
Na+-dependent pHi
recovery from an acid load in proximal tubule cells from WT and KO mice
under isosmotic conditions.
Next, we observed Na+-dependent pHi recovery
from an acid load with NH4Cl in proximal tubule cells from
WT (Fig. 3A) and KO (Fig.
3B) mice. Proximal tubule cells from WT and KO mice were first bathed in the solution 1 (Table 1) and were then
incubated in the Na+-free HEPES-buffered solution
containing 20 mM NH4Cl (solution 3, Table 1). At
this time, the pHi increased rapidly due to the rapid
diffusion of NH3 into the cell. During the exposure to
NH4Cl for 2 min, pHi tended to decrease toward
baseline due to slow inward diffusion of NH
. After
that, removing NH4Cl from the Na+-free
HEPES-buffered solution rapidly decreased the pHi, because accumulated internal NH
dissociates into
H+ and NH3 (which exits from the cell). At this
time, pHi values of proximal tubule cells from WT and KO
mice were 6.16 ± 0.05 (n = 8) and 6.15 ± 0.03 (n = 10), respectively, and were not significantly different between the two groups. Subsequent addition of
Na+ to the peritubular solution caused pHi to
rapidly increase to values similar to those of the initial steady-state
pHi. In fact, the pHi values of the final
steady state in cells from WT and KO mice were 6.95 ± 0.03 (n = 8) and 6.97 ± 0.03 (n = 10),
respectively, and were not significantly different from those of the
initial steady-state (WT mice: 7.03 ± 0.02, n = 8; KO mice: 6.97 ± 0.02, n = 10). Figure
4, A and B, shows
the effects of peritubular addition of EIPA (100 µM) to proximal
tubule cells from WT and KO mice on Na+-dependent
pHi recovery from an acid load, respectively. When proximal
tubule cells from WT and KO mice were exposed to EIPA, steady-state
pHi values significantly decreased from 6.97 ± 0.04 (n = 4) and 7.02 ± 0.05 (n = 6)
to 6.95 ± 0.04 (n = 4, P < 0.05) and 6.99 ± 0.05 (n = 6, P < 0.05), respectively. In the continued presence of EIPA, readdition of
Na+ produced a much smaller pHi increase (WT
mice: 0.27 ± 0.02, n = 4, P < 0.001; KO mice: 0.31 ± 0.07, n = 6, P < 0.001) than in its absence (WT mice: 0.79 ± 0.03, n = 8; KO mice: 0.82 ± 0.04, n = 10). These findings indicate that basolateral NHE
indeed exists in proximal tubule cells from both WT and KO mice and
contributes to Na+-dependent cell alkalinization from an
acid load.

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Fig. 3.
Na+-dependent pHi recovery from
an acid load under is- and hyperosmotic conditions in proximal tubule
cells from WT (A and C) and KO (B and
D) mice. Both groups of cells were first bathed in
Na+-containing HEPES-buffered solution (solution
1, Table 1) and were then incubated in Na+-free
HEPES-buffered solution containing 20 mM NH4Cl
(solution 3, Table 1) for 2 min. During the pulse,
pHi increased (due to entry of NH3 into the
cell) and then tended to decrease toward baseline due to slower influx
of NH . Thereafter, the abrupt removal of
NH4Cl from the Na+-free solution caused a
substantial and sustained decrease in pHi. In the absence
and presence of the hyperosmotic mannitol solution, readdition of
peritubular Na+ to both groups of cells caused
pHi to rapidly increase to values similar to initial
steady-state pHi values. In both groups of cells in the
absence of peritubular Na+, hyperosmotic mannitol had no
effect on pHi. However, when cells from KO mice were
exposed to the hyperosmotic mannitol solution, the
Na+-dependent pHi recovery was faster than when
cells from WT mice were exposed to hyperosmotic mannitol or when cells
from KO mice were exposed to the isosmotic solution.
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Fig. 4.
Na+-dependent pHi recovery from
an acid load in the presence of peritubular
ethylisopropylamiloride (EIPA) under is- and hyperosmotic conditions in
proximal tubule cells from WT (A and C) and KO
(B and D) mice. It should be noted that in both
groups of cells under isosmotic conditions, EIPA (100 µM) alone
decreased pHi. In both groups of cells under is- and
hyperosmotic conditions, Na+-dependent pHi
recovery in the presence of EIPA was substantially slower, and final
steady-state pHi values after the addition of EIPA were
much smaller than those of the initial steady state.
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Na+-dependent pHi
recovery from an acid load in proximal tubule cells from WT and KO mice
under hyperosmotic conditions.
Next, we examined whether the hyperosmotic mannitol solution affected
Na+-dependent pHi recovery from an acid load in
the proximal tubule cells from WT (Fig. 3C) and KO (Fig.
3D) mice. Both groups of proximal tubule cells were first
incubated in solution 1 and were then bathed in
solution 3 for 2 min. After that, NH4Cl was
rapidly removed from solution 3, leading to a rapid decrease
in pHi. Thereafter, both groups of cells were exposed to
the Na+-free HEPES-buffered solution (solution
2, Table 1) treated with 200 mM mannitol (500 mosmol/kgH2O). At this time, the pHi values in
either group of cells were unchanged (WT mice: from 6.11 ± 0.03 to 6.13 ± 0.03, n = 18; KO mice: from 6.13 ± 0.04 to 6.16 ± 0.03, n = 15) (Fig. 3,
C and D). After that, when external
Na+ was readded in the presence of hyperosmotic mannitol,
pHi recovery in proximal tubule cells from KO mice was
substantially faster than in its absence, although final steady-state
pHi values (7.01 ± 0.03, n = 15) were
not significantly different from those in the initial steady state
(7.01 ± 0.02, n = 15) (see Fig. 3, B and D). At this time, the Na+-dependent
pHi recovery rate in the presence of hyperosmotic mannitol (124.4 ± 10.7 pH/s × 104, P < 0.001, n = 15) was significantly greater than that in
its absence (76.8 ± 4.6 pH/s × 104,
n = 10). Furthermore, under hyperosmotic conditions,
the Na+-dependent pHi recovery in proximal
tubule cells from KO mice was faster than that in cells from WT mice
(see Fig. 3, C and D). At this time, the
Na+-dependent pHi recovery rate in KO mice
(124.4 ± 10.7 pH/s × 104, P < 0.001, n = 15) was also significantly greater than that in cells from WT mice (88.8 ± 7.2 pH/s × 104,
n = 18). In sharp contrast to proximal tubule cells of
KO mice, in proximal tubule cells from WT mice,
Na+-dependent pHi recovery under hyperosmotic
conditions was similar to that under isosmotic conditions, and final
steady-state pHi values (6.94 ± 0.04, n = 18) were not significantly different from those of
the initial steady state (6.99 ± 0.03, n = 18)
(see Fig. 3, A and C).
Effects of peritubular addition of EIPA to proximal tubule cells of WT
and KO mice on Na+-dependent pHi recovery under
hyperosmotic conditions are shown in Fig. 4, C and
D, respectively. In both groups of proximal tubule cells
treated with hyperosmotic mannitol, Na+-dependent
pHi recovery from an acid load in the presence of EIPA (100 µM) was substantially slower than in its absence (see Figs. 3,
C and D, and 4, C and D).
In cells from WT and KO mice under hyperosmotic conditions, final
steady-state pHi values after readdition of Na+
in the presence of EIPA were 6.51 ± 0.03 (n = 6)
and 6.45 ± 0.06 (n = 7), respectively, and were
not significantly different between the two groups. Also, in both
groups of cells, the Na+-dependent pHi increase
in the presence of EIPA (WT mice: 0.31 ± 0.04, n = 6, P < 0.001; KO mice: 0.25 ± 0.05, n = 7, P < 0.001) was much smaller
than in its absence (WT mice: 0.81 ± 0.05, n = 18; KO mice: 0.84 ± 0.03, n = 15). At this time,
in both groups of cells, the Na+-dependent pHi
recovery rate in the presence of EIPA (WT mice: 29.1 ± 2.9 pH/s × 104, n = 6, P < 0.001; KO mice: 28.8 ± 6.2 pH/s × 104,
n = 7, P < 0.001) was also
significantly smaller than in its absence (WT mice: 88.8 ± 7.2 pH/s × 104, n = 18; KO mice:
124.4 ± 10.7 pH/s × 104, n = 15).
From dpHi/dt and
I, we calculated
the relationship between JH and pHi
under is- and hyperosmotic conditions in both groups of proximal tubule
cells, as shown in Fig. 5. In both groups
of cells under is- and hyperosmotic conditions,
JH decreased as pHi increased. In
cells from WT mice, JH values under hyperosmotic conditions were not significantly different from those under isosmotic conditions over the entire range of pHi studied
(6.20-6.90). In sharp contrast, in cells from KO mice,
JH values under hyperosmotic conditions were
significantly (P < 0.05) greater than those under isosmotic conditions at a pHi range of 6.20-6.45. In
cells from KO mice, maximal JH values under
hyperosmotic conditions were also significantly greater than under
isosmotic conditions. Furthermore, under hyperosmotic conditions, cells
from KO mice had significantly (P < 0.05) greater
JH values than those of cells from WT mice at
pHi values of 6.20-6.45, although, under isosmotic
conditions, JH values in cells from KO mice were
not significantly different from those in cells of WT mice over the
entire range of pHi studied (6.20-6.90). Therefore, we
conclude that in proximal tubule cells from KO mice, the hyperosmotic
mannitol solution enhanced JH after an
intracellular acid load through basolateral NHE at low pHi (6.20-6.45), whereas in proximal tubule cells from WT mice, it had
no effect on basolateral NHE activity over the entire wide pHi range examined (6.20-6.90). Under isosmotic
conditions, the Na+-dependent acid extrusion rates through
basolateral NHE were not different between the two groups of cells.

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Fig. 5.
pHi dependence of the
Na+-dependent acid extrusion rate
(JH) under is- and hyperosmotic conditions in
proximal tubule cells from WT and KO mice. Plots were computed from
experiments such as those illustrated in Fig. 3. Values are means ± SE; n = no. of tubules examined. Isosm.,
isosmolality; Hyperosm., hyperosmolality. *P < 0.05 compared with cells from KO mice that were exposed to the isosmotic
control solution, at comparable pHi values.
P < 0.05 compared with cells from WT mice that were
exposed to the hyperosmotic mannitol solution, at comparable
pHi values.
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Next, we examined whether the changes in Michaelis-Menten constant
(Km) values for peritubular Na+
concentrations and/or maximal velocity (Vmax)
are involved in basolateral NHE activation induced by hyperosmotic
mannitol. For this purpose, the kinetics of basolateral NHE were
determined by measuring JH at pHi of
6.30 on readdition of varying concentrations of Na+ (0, 7, 14, 43, 100, and 142 mM) to the peritubular side of tubules from KO
mice after peritubular Na+ removal and an acid load with
NH4Cl prepulse under is- and hyperosmotic conditions.
Results are shown in Fig. 6A.
As shown in Fig. 6B, a Lineweaver-Burk plot of the data
obtained showed that, under isosmotic conditions, the apparent
Km for peritubular Na+
concentrations was 21.1 mM with a Vmax of 370.6 µM/s. Under hyperosmotic conditions, the apparent
Km for peritubular Na+ was similar
(22.7 mM), but Vmax increased to 528.4 µM/s.

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Fig. 6.
Kinetics of basolateral NHE under is- and hyperosmotic
conditions. A: after the removal of peritubular
Na+ and an acid load with NH4Cl prepulse,
varying concentrations of Na+ ([Na+]; 0, 7, 14, 43, 100, and 142 mM) were added to the peritubular side of tubules
from KO mice, and JH at pHi of 6.30 was estimated. Each point represents the mean ± SE of at least 4 determinations. B: Lineweaver-Burk analysis of data
indicates that exposure of cells from KO mice to the hyperosmotic
mannitol solution had no effect on the apparent Michaelis-Menten
constant values for peritubular Na+ (from 21.1 to 22.7 mM),
but increased maximal velocity from 370.6 to 528.4 µM/s.
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Na+-dependent pHi
recovery from an acid load in proximal tubule cells from WT and KO mice
under is- and hyperosmotic conditions in the presence of cyclosporin A.
Next, we examined the effects of cyclosporin A (a P-gp inhibitor) on
Na+-dependent pHi recovery from an acid load in
proximal tubule cells from WT and KO mice under is- and hyperosmotic
conditions. Representative pHi recordings are shown in Fig.
7. When cyclosporin A (5 µM) alone, in
the presence of Na+, was added to cells from WT or KO mice,
pHi values were not influenced at all (WT mice: from
6.96 ± 0.05 to 6.96 ± 0.05, n = 7; KO mice: 7.00 ± 0.05 to 6.98 ± 0.04, n = 6) (Fig. 7,
A and B). When cyclosporin A (5 µM) plus
hyperosmotic mannitol, in the absence of Na+, were added to
cells from WT or KO mice, pHi values were also not affected
at all (WT mice: from 6.17 ± 0.04 to 6.20 ± 0.05, n = 11; KO mice: from 6.17 ± 0.04 to 6.17 ± 0.04, n = 8; see Fig. 7, C and
D). When external Na+ was readded to cells from
WT mice in the continued presence of hyperosmotic mannitol and
cyclosporin A, pHi recovery from an acid load was
substantially faster than that in the continued presence of cyclosporin
A alone, although final steady-state pHi values (7.00 ± 0.04, n = 11) were not significantly different from
those in the initial steady state (6.96 ± 0.03, n = 11) (see Fig. 7, A and C). In cells from WT
mice, Na+-dependent pHi recovery from an acid
load in the continued presence of hyperosmotic mannitol and cyclosporin
A was also faster than that in the continued presence of hyperosmotic
mannitol alone (see Figs. 3C and 7C). When
Na+ was readded to tubules from KO mice in the continued
presence of hyperosmotic mannitol and cyclosporin A, pHi
recovery from an acid load was also faster than that in the continued
presence of cyclosporin A alone, although final steady-state
pHi values (7.04 ± 0.04, n = 8) were
not significantly different from those of the initial steady state
(7.05 ± 0.03, n = 8) (Fig. 7, B and D). In the presence of cyclosporin A under isosmotic
conditions, in cells from KO mice, Na+-dependent
pHi recovery from an acid load was similar to that in the
cells of the WT mice (see Fig. 7, A and B). From
dpHi/dt and
I, we computed
JH at pHi of 6.30 and compared them
in the absence and presence and cyclosporin A under is- and
hyperosmotic conditions in cells from WT and KO mice, as shown in Fig.
8. In the absence of cyclosporin A, in
the cells from WT mice, JH values at
pHi of 6.30 under hyperosmotic conditions (379.1 ± 32.8 µM/s, n = 17) were not significantly different
from those under isosmotic conditions (378.0 ± 57.0 µM/s,
n = 7), whereas in cells from KO mice,
JH values at pHi of 6.30 under
hyperosmotic conditions (565.0 ± 59.5 µM/s, P < 0.05, n = 12) were significantly greater than those
under isosmotic conditions (360.0 ± 28.6 µM/s,
n = 10). In the presence of cyclosporin A, in cells
from WT mice, hyperosmotic mannitol significantly (P < 0.05) increased JH (at pHi of 6.30) to 579.8 ± 45.6 µM/s (n = 5), values that were
not different from those in KO mice in the presence of hyperosmotic
mannitol alone. Furthermore, in cells from WT mice under hyperosmotic
conditions, JH values at pHi of 6.30 in the presence of cyclosporin A were significantly greater than those
in its absence, although in cells from WT mice under isosmotic
conditions, JH values at pHi of 6.30 in the presence of cyclosporin A (398.3 ± 68.6 µM/s,
n = 6) were not different from those in its absence. In
cells from KO mice in the presence of cyclosporin A, hyperosmotic
mannitol significantly increased JH (at
pHi of 6.30) to 570.2 ± 15.9 µM/s
(n = 5, P < 0.05), values that were
not different from those in KO mice in the presence of hyperosmotic
mannitol alone. In cells from KO mice under isosmotic conditions,
JH values at pHi of 6.30 in the presence of cyclosporin A were not different from those in its absence.

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Fig. 7.
Na+-dependent pHi recovery from
an acid load in the presence of peritubular cyclosporin A under is- and
hyperosmotic conditions in proximal tubule cells from WT (A
and C) and KO (B and D) mice. In the
presence of peritubular cyclosporin A (5 µM) under is- and
hyperosmotic conditions, readdition of peritubular Na+ to
both groups of cells caused pHi to rapidly increase to
pHi values similar to those of the initial steady state. In
both groups of cells in the absence of peritubular Na+, the
hyperosmotic mannitol solution involving cyclosporin A had no effect on
pHi. It should be noted that when cells from WT mice were
exposed to the hyperosmotic mannitol solution including cyclosporin A,
Na+-dependent pHi recovery was faster than when
cells from WT mice were exposed to the isosmotic solution including
cyclosporin A. Also, when cells from KO mice were exposed to the
hyperosmotic mannitol solution including cyclosporin A,
Na+-dependent pHi recovery was faster than when
cells from KO mice were exposed to the isosmotic solution including
cyclosporin A.
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Fig. 8.
JH at pHi of 6.30 under is- and hyperosmotic conditions in the absence and presence of
cyclosporin A in proximal tubule cells from WT and KO mice. Values are
means ± SE taken from experiments such as those illustrated in
Figs. 3 and 7; no. of tubules examined are in parentheses.
Isosmotic, isosmolality; Hyperosmotic, hyperosmolality; CyA,
cyclosporin A; NS, not significant.
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Role of protein kinase C in hyperosmotic mannitol-induced NHE
activation.
Very recently, Miyata et al. (23) have reported that
exposure of tubules from WT mice to a hyperosmotic mannitol solution including PMA elicited RVI, whereas exposure to the hyperosmotic mannitol solution alone did not. Also, we found that the addition of
tubules from KO mice to the hyperosmotic mannitol solution including
either of two protein kinse C (PKC) inhibitors (staurosporine and
calphostin C) abolished RVI (23). Furthermore, we have
demonstrated that exposure to the hyperosmotic mannitol solution
activated PKC in tubules from KO mice but not in those from WT mice,
whereas exposure of tubules from WT mice to the hyperosmotic mannitol solution containing PMA activated PKC (23). These findings
suggest the possibility that PKC may be involved in hyperosmotic
mannitol-induced NHE activation. To explore this possibility, we added
the hyperosmotic mannitol solution in the presence of either
staurosporine (100 nM) or calphostin C (500 nM) to cells from KO mice
and then observed the Na+-dependent pHi
recovery. Representative pHi recordings are shown in Fig.
9, A (for the
staurosporine-treated cell) and B (for the calphostin
C-treated cell). When staurosporine or calphostin C alone in the
presence of Na+ was added to cells from KO mice,
pHi values were not influenced at all (staurosporine: from
6.92 ± 0.02 to 6.94 ± 0.02, n = 9; calphostin C: 6.95 ± 0.06 to 6.94 ± 0.05, n = 5). When external Na+ was readded to cells from KO mice
in the presence of the hyperosmotic mannitol solution plus either
staurosporine or calphostin C, pHi recovery from an acid
load was substantially slower than that in the presence of the
hyperosmotic mannitol solution alone (see Figs. 3D and 9,
A and B). From dpHi/dt and
I, we computed JH values at
pHi of 6.30 and compared them in the absence and presence of the two PKC inhibitors under hyperosmotic conditions in cells from
KO mice, as shown in Fig. 10. When
cells from KO mice were treated with the hyperosmotic mannitol solution
plus either staurosporine or calphostin C, JH
values at pHi of 6.30 were 355.1 ± 34.6 (n = 9) or 310.3 ± 29.3 (n = 5)
µM/s, respectively. These values were significantly smaller than
those in cells treated with the hyperosmotic mannitol solution alone
(536.7 ± 58.6 µM/s, n = 13) but were not
significantly different from those in cells treated with the isosmotic
control solution (332.7 ± 27.9 µM/s, n = 8). Next, we examined whether the exposure of cells from KO mice to PMA
(the specific activator of PKC) under isosmotic conditions increases
basolateral NHE activity. Representative pHi recordings are
shown in Fig. 9C. When PMA (100 nM) in the absence of
Na+ was added to cells from KO mice, pHi values
were not influenced at all (from 6.19 ± 0.03 to 6.15 ± 0.04, n = 4). When external Na+ was readded
to cells from KO mice in the continued presence of PMA, pHi
recovery from an acid load was substantially faster than in its absence
(see Fig. 3B), although final steady-state pHi values (6.98 ± 0.02, n = 4) were not
significantly different from those in the initial steady state
(6.96 ± 0.05, n = 4). At this time,
JH at pHi of 6.30 was 516.9 ± 29.6 µM/s (n = 4). These values were significantly
greater than those in cells treated with the isosmotic control solution
alone but were not significantly different from those in cells treated
with the hyperosmotic mannitol solution alone (Fig. 10). Finally, we
examined whether exposure of cells from WT mice to the hyperosmotic
mannitol solution plus PMA increases basolateral NHE activity.
Representative pHi recordings are shown in Fig.
9D. When external Na+ was readded to cells from
WT mice in the presence of the hyperosmotic mannitol solution plus PMA,
pHi recovery from an acid load was substantially faster
than that in the presence of the hyperosmotic mannitol solution
alone. At this time, JH values at
pHi of 6.30 were 524.1 ± 51.2 µM/s
(n = 5), values that were significantly greater than
those in cells exposed to the hyperosmotic mannitol solution alone
(365.9 ± 32.0 µM/s, n = 16) and were
significantly greater than those in cells exposed to the isosmotic
control solution (330.9 ± 38.0 µM/s, n = 6) (
Fig. 10). These values were not significantly different from those in
cells from KO mice in the presence of the hyperosmotic mannitol alone.

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Fig. 9.
Role of protein kinase C (PKC) in the
Na+-dependent pHi recovery from an acid load
under is- and hyperosmotic conditions in proximal tubule cells from KO
(A, B, and C) and WT (D)
mice. When cells from KO mice were exposed to the hyperosmotic mannitol
in the presence of either of 2 PKC inhibitors, staurosporine
(A) or calphostin C (B), the
Na+-dependent pHi recovery was slower than in
its absence (see Fig. 3D). C: when cells from KO
mice were exposed to the isosmotic control solution in the presence of
phorbol 12-myristate 13-acetate (PMA), Na+-dependent
pHi recovery was faster than in its absence (see Fig.
3B) D: when cells from WT mice were exposed to
the hyperosmotic mannitol solution in the presence of PMA, the
Na+-dependent pHi recovery was faster than in
its absence (see Fig. 3C).
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Fig. 10.
JH at pHi of 6.30 under is- and hyperosmotic conditions in the absence and presence of
PMA in proximal tubule cells from WT and KO mice. Values are means ± SE taken from experiments such as those illustrated in Figs. 3 and
9; no. of tubules examined are in parentheses. Stauro, staurosporine;
Cal, calphostin C.
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 |
DISCUSSION |
Our laboratory has recently reported that the exposure of isolated
nonperfused proximal tubules from WT mice to a hyperosmotic mannitol
solution did not elicit RVI after the initial cell shrinkage (21). On the other hand, RVI was observed in tubules in
the hyperosmotic mannitol solution when P-gp activity was acutely suppressed by P-gp inhibitors (verapamil and cyclosporin A) or when
both the mdr1a and mdr1b genes were genetically
knocked out (21). We also reported that when tubules from
WT mice were exposed to the hyperosmotic mannitol solution including
either of the two P-gp inhibitors, in the absence of peritubular
Na+ or in the presence of peritubular EIPA, they did not
exhibit RVI (21). Furthermore, in proximal tubules from KO
mice, both removing peritubular Na+ and adding peritubular
EIPA inhibited RVI induced by the hyperosmotic mannitol solution. These
findings indicate that basolateral NHE partly contributes to the
P-gp-induced modulation of RVI under hyperosmotic stress. In the
present study, we extend our previous study to determine whether and
how basolateral NHE is activated in the P-gp-induced modulation of RVI.
Evidence for basolateral NHE in proximal tubule cells from WT and
KO mice.
In contrast to apical NHE, which has been widely documented in the
proximal tubule, basolateral NHE is more controversial. Studies in
basolateral membrane vesicles prepared from the renal cortex of rabbits
(15) and rats (26) have provided no evidence for basolateral NHE. On the other hand, in intact proximal tubules, previous evidence for basolateral NHE has been described in the salamander (2), rabbit proximal tubule S3 segment
(16), and rabbit juxtamedullary S1 and S2 segments
(10). However, it has not yet been demonstrated whether
the basolateral membrane of the mouse proximal tubule possesses NHE. In
the present study, we observed that in proximal tubule cells from both
WT and KO mice, readdition of Na+ to the bath after an acid
load caused a rapid pHi increase that was significantly
inhibited by pretreatment with EIPA, as shown in Figs. 3, A
and B, and 4, A and B. Therefore, in
proximal tubule cells from both WT and KO mice, basolateral
Na+-dependent pHi recovery is mediated by NHE.
When proximal tubule cells from both WT and KO mice were treated with
EIPA, steady-state pHi values significantly decreased, as
shown in Fig. 4, A and B. These findings indicate
that in the nominal absence of
CO2/HCO
, basolateral NHE in
proximal tubule cells from both WT and KO mice must be active in the
normal steady-state pHi to balance a substantial rate of
intracellular acid loading.
Basolateral NHE activity under is- and hyperosmotic conditions.
As shown in Fig. 5, in cells from WT and KO mice under both is- and
hyperosmotic conditions, JH, through an NHE
process, decreased as pHi increased from the acidic to the
normal range. Under isosmotic conditions, JH
values in cells from KO mice were not significantly different from
those from WT mice over the entire range of pHi studied
(6.20-6.90), which is consistent with the notion that under these
conditions, NHE activity in the basolateral membrane of proximal tubule
cells from KO mice is similar to that in cells from WT mice. However,
in cells from KO mice, JH values under hyperosmotic conditions were significantly greater than those under
isosmotic conditions at a pHi range of 6.20-6.45,
although in cells from WT mice, JH values under
hyperosmotic conditions were not significantly different from those
under isosmotic conditions over the entire range of pHi
examined (6.20-6.90). In sharp contrast, under hyperosmotic
conditions, JH values in cells from KO mice were
significantly greater than those in cells from WT mice at pHi values of 6.20-6.45. In cells from KO mice,
maximal JH values under hyperosmotic conditions
were also significantly greater than under isosmotic conditions.
Furthermore, in proximal tubule cells from KO mice, the hyperosmotic
mannitol solution shifted the JH-pHi
relationship by ~0.15 pH units in the alkaline direction at low
pHi values. Therefore, in proximal tubule cells from KO mice, hyperosmolality greatly enhances the pHi sensitivity
of the NHE at low pHi, with an increase in maximal
JH. Similarly, Parker (24) has
demonstrated that NHE in dog erythrocytes is activated by low
pHi, although they did not demonstrate that
shrinkage-induced activation of NHE is mediated via a shift in
pHi sensitivity. In contrast, Miyata et al.
(22) reported that in cultured mesangial cells from rat
kidneys, a hyperosmotic mannitol solution shifted the
JH-pHi relationship by 0.15-0.3
pH units in the alkaline direction at a wide pHi range of
6.40-6.95, with increasing maximal JH. Similarly, Grinstein et al. (13) studied the
Na+-dependent component of pHi recovery from an
acid load in thymic lymphocytes, finding that shrinkage shifts the
JH-pHi relationship by 0.2-0.3
pH units in the alkaline direction over the entire range of
pHi between ~6.2 and ~7.2.
In the present study, we observed that the addition of the hyperosmotic
mannitol solution including cyclosporin A (the P-gp inhibitor) to cells
from WT mice activated basolateral NHE at magnitude similar to that for
the addition of hyperosmotic mannitol alone to cells from KO mice.
Cyclosporin A alone had no effect on NHE activity in cells from WT mice
exposed to the isosmotic solution or in the cells from KO mice exposed
to the hyperosmotic mannitol solution. Taken together, when P-gp
activity is acutely inhibited by the P-gp inhibitor (cyclosporin A) or
when both the mdr1a and mdr1b genes are
genetically disrupted, hyperosmotic mannitol activates basolateral NHE
in mouse proximal tubule cells and consequently elicits RVI. However,
the mechanisms responsible for the activation of basolateral NHE under
the above conditions are presently unclear. Because the pHi
sensitivity of the exchange system appears to be largely determined by
an allosteric modifier site (1, 12) located on the
cytoplasmic surface of the membrane, one of the mechanisms for
shrinkage-induced NHE activation in proximal tubule cells from KO mice
is a shift in pHi dependence of the antiport.
In Madin-Darby canine kidney cells, hyperosmolality induced by NaCl or
raffinose has been shown to enhance inositol 1,4,5-triphosphate levels
and thereby activate PMA-sensitive PKC (29). In Ehrlich mouse ascites tumor cells, PKC is involved in activation of the Na+-K+-2Cl
cotransporter induced
by hyperosmolality (18). Treatment of NIH/3T3 cells with
hyperosmotic NaCl solution has also been reported to trigger
phospholipase C activation and then induce an increase in
diacylglycerol levels, and as a consequence, PKC activation (32). Very recently, Miyata et al. (23)
demonstrated that exposure of proximal tubules from KO mice to
staurosporine and calphostin C abolished RVI. We also demonstrated that
exposure of proximal tubules from KO mice to a hyperosmotic mannitol
solution activated PKC (23). The present studies have
shown that exposure of proximal tubule cells from KO mice to
staurosporine and calphostin C inhibited basolateral NHE activation
induced by the hyperosmotic mannitol solution (see Figs. 9,
A and B, and 10). We have also shown that the
addition of PMA to cells from KO mice under isosmotic conditions
mimicked the stimulatory effects of the hyperosmotic mannitol solution
on basolateral NHE activity (see Figs. 9C and 10). In sharp
contrast to that in cells from KO mice, exposure of cells from WT mice
to hyperosmotic mannitol did not induce PKC activation
(23), had no stimulatory effect on NHE activity (see Fig.
5), and did not result in RVI (23). On the other hand, exposure of cells from WT mice to the hyperosmotic mannitol solution including PMA activated PKC (23), increased basolateral
NHE activity (see Figs. 9D and 10), and elicited RVI
(23). Taking these observations together, we conclude that
the effects of a hyperosmotic mannitol solution on basolateral NHE
activity and RVI indeed occur via PKC.
The apparent Km (21.1 mM) for peritubular
Na+ of NHE in the basolateral membrane of proximal tubule
cells from KO mice under isosmotic conditions was lower than that found
for the basolateral membrane of the rabbit proximal tubule S3 segment
(16) and rat thymic lymphocytes (12) but was
similar to that reported for the basolateral membrane of the rabbit
cortical collecting duct (4) and human endothelial cells
(8). In the present study, we found that exposure of cells
from KO mice to the hyperosmotic mannitol solution increased
Vmax from 370.6 to 528.4 µM/s without changing
the Km for peritubular Na+,
indicating that under hyperosmotic conditions, the affinity for
peritubular Na+ remained the same, but the number of the
NHEs increased. These findings also contribute to the hyperosmotic
mannitol-induced increase in basolateral NHE activity. Very recently,
we reported that the addition of the microtubule disruptor (colchicine)
and the microfilament disruptor (cytochalasin B) to tubules from WT mice inhibited the PMA-induced RVI after the initial cell shrinkage (23). We have also shown that the addition of colchicine
and cytochalsin B to tubules from KO mice abolished RVI
(23). From our previous and present findings, we propose
that under hyperosmotic conditions, PKC and/or NHE itself may be
shuttled to the basolateral membrane via colchicine- and cytochalasin
B-sensitive processes to activate NHE. Also, it is possible that PKC
activation may promote the exocytic insertion into the basolateral
membrane of NHE previously stored in the cytoplasm. These possibilities
will have to await further investigation.
From the above data, we conclude that under isosmotic conditions, the
basolateral membrane of proximal tubule cells from both WT and KO mice
possesses an NHE. We also clearly demonstrated that in the absence of
P-gp activity, hyperosmotic mannitol activates basolateral NHE via PKC,
whereas in the presence of P-gp activity, it does not. Therefore,
basolateral NHE indeed contributes to the P-gp-induced modulation of
RVI after initial cell shrinkage.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by a grant from the Japanese
Kidney Foundation (Jinkenkyukai), by a grant from the Salt Science Foundation, and Grants-in-Aid for Scientific Research from the Ministry
of Education, Science, and Culture (Japan).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: S. Muto,
Dept. of Nephrology, Jichi Medical School, Minamikawachi, Tochigi
329-0498 Japan (E-mail: smuto{at}jichi.ac.jp).
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. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00183.2001
Received 21 March 2001; accepted in final form 1 November 2001.
 |
REFERENCES |
1.
Aronson, PS,
Nee J,
and
Suhm MA.
Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles.
Nature
299:
161-163,
1982[ISI][Medline].
2.
Boron, WF,
and
Boulpaep EL.
Intracellular pH regulation in the renal proximal tubule of the salamander. Na-H exchange.
J Gen Physiol
81:
29-52,
1983[Abstract].
3.
Boyarsky, G,
Ganz MB,
Sterzel RB,
and
Boron WF.
pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO
.
Am J Physiol Cell Physiol
255:
C844-C856,
1988[Abstract/Free Full Text].
4.
Chaillet, JR,
Lopes AG,
and
Boron WF.
Basolateral Na-H exchange in the rabbit cortical collecting tubule.
J Gen Physiol
86:
795-812,
1985[Abstract].
5.
Croop, JM,
Raymond M,
Harber D,
Devault A,
Arceci RJ,
Gros P,
and
Houseman DE.
The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissue.
Mol Cell Biol
9:
1346-1350,
1989[ISI][Medline].
6.
Dellasega, M,
and
Grantham JJ.
Regulation of renal tubule cell volume in hypotonic media.
Am J Physiol
224:
1288-1294,
1973[ISI][Medline].
7.
Ernest, S,
Rajaraman S,
Megyesi J,
and
Bello-Reuss E.
Expression of MDR1 (multidrug resistance) gene and its protein in normal human kidney.
Nephron
77:
284-289,
1997[ISI][Medline].
8.
Escobales, N,
Longo E,
Cragoe EJ,
Danthuluri NR,
and
Brock TA.
Osmotic activation of Na+-H+ exchange in human endothelial cells.
Am J Physiol Cell Physiol
259:
C640-C646,
1990[Abstract/Free Full Text].
9.
Gagnon, J,
Ouimet D,
Nguyen H,
Laprade R,
Le Grimellec C,
Carriere S,
and
Cardinal J.
Cell volume regulation in the proximal convoluted tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
243:
F408-F415,
1982[Abstract/Free Full Text].
10.
Geibel, J,
Giebisch G,
and
Boron WF.
Basolateral sodium-coupled acid-base transport mechanisms of the rabbit proximal tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
257:
F790-F797,
1989[Abstract/Free Full Text].
11.
Gottesman, M,
and
Pastan I.
Biochemistry of multidrug resistance mediated by multidrug transporter.
Annu Rev Biochem
62:
385-427,
1993[ISI][Medline].
12.
Grinstein, S,
Goetz JD,
and
Rothstein A.
22Na+ fluxes in thymic lymphocytes. II. Amiloride-sensitive Na+/H+ exchange pathway; reversibility of transport and asymmetry of the modifier site.
J Gen Physiol
84:
585-600,
1984[Abstract].
13.
Grinstein, S,
Rothstein A,
and
Cohen S.
Mechanism of osmotic activation of Na+/H+ exchange in rat thymic lymphocytes.
J Gen Physiol
85:
765-787,
1985[Abstract].
14.
Hsu, SI,
Lothstein L,
and
Horwitz SB.
Differential overexpression of three mdr gene family members in multidrug-resistant J774.2 mouse cells.
J Biol Chem
264:
12053-12062,
1989[Abstract/Free Full Text].
15.
Ives, HE,
Yee JV,
and
Warnock DG.
Asymmetric distinction of the Na+/H+ antiport in the renal proximal tubule cell.
J Biol Chem
258:
13513-13516,
1983[Abstract/Free Full Text].
16.
Kurtz, I.
Basolateral membrane Na+/H+ antiport, Na+/base cotransport, and Na+-independent Cl
/base exchange in the rabbit S3 proximal tubule.
J Clin Invest
83:
616-622,
1989[ISI][Medline].
17.
Lang, F,
Busch GL,
Ritter M,
Volkl H,
Waldegger S,
Gulbins E,
and
Haussinger D.
Functional significance of cell volume regulatory mechanisms.
Physiol Rev
78:
247-306,
1998[Abstract/Free Full Text].
18.
Larsen, AK,
Skaaning B,
and
Hoffmann EK.
Activation of protein kinase C during cell volume regulation in Ehrlich mouse ascites tumor cells.
Biochim Biophys Acta
1222:
477-482,
1994[ISI][Medline].
19.
Lohr, JW,
and
Grantham JJ.
Isovolumetric regulation of isolated S2 proximal tubules in anisosmotic media.
J Clin Invest
78:
1165-1172,
1986[ISI][Medline].
20.
McCarty, NA,
and
O'Neil RG.
Calcium signaling in cell volume regulation.
Physiol Rev
72:
1037-1061,
1992[Abstract/Free Full Text].
21.
Miyata, Y,
Asano Y,
and
Muto S.
Effects of P-glycoprotein on cell volume regulation in mouse proximal tubule.
Am J Physiol Renal Physiol
280:
F829-F837,
2001[Abstract/Free Full Text].
22.
Miyata, Y,
Muto S,
Yanagiba S,
and
Asano Y.
Extracellular Cl
modulates shrinkage-induced activation of Na+/H+ exchanger in rat mesangial cells.
Am J Physiol Cell Physiol
278:
C1218-C1229,
2000[Abstract/Free Full Text].
23.
Miyata, Y,
Okada K,
Ishibashi S,
Asano Y,
and
Muto S.
P-gp-induced modulation of regulatory volume increase occurs via PKC in mouse proximal tubule.
Am J Physiol Renal Physiol
282:
F65-F76,
2002[Abstract/Free Full Text].
24.
Parker, JC.
Interaction of lithium and protons with the sodium-proton exchanger of dog red blood cells.
J Gen Physiol
84:
379-401,
1984[Abstract].
25.
Roos, A,
and
Boron WF.
Intracellular pH.
Physiol Rev
61:
296-434,
1981[Free Full Text].
26.
Sabolic, I,
and
Burckhardt J.
Proton pathways in rat renal brush-border and basolateral membranes.
Biochim Biophys Acta
734:
210-220,
1983[ISI][Medline].
27.
Schinkel, AH,
Mayer U,
Wagenaar E,
Mol CAAM,
van Deemter L,
Smit JJM,
van der Valk M,
Voordouw AC,
Spit H,
van Tellingen O,
Zijlamans JMJM,
Fibbe WE,
and
Borst P.
Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins.
Proc Natl Acad Sci USA
15:
4028-4033,
1997.
28.
Schinkel, AH,
Smit JJM,
van Tellingen O,
Beijinen JH,
Wagenaar E,
van Deemter L,
Mol CAAM,
van der Valk MA,
Robanus-Maandag EC,
te Riele HPT,
Berns AJ,
and
Borst P.
Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs.
Cell
77:
491-502,
1994[ISI][Medline].
29.
Terada, Y,
Tomita K,
Homma MK,
Nonoguchi H,
Yang T,
Yamada T,
Yuasa Y,
Krebs EG,
Sasaki S,
and
Marumo F.
Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells.
J Biol Chem
269:
31296-31301,
1994[Abstract/Free Full Text].
30.
Thiebaut, F,
Tsuruo T,
Hamada H,
Gottesman MM,
Pastan I,
and
Willingham MC.
Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues.
Proc Natl Acad Sci USA
84:
7735-7738,
1987[Abstract].
31.
Tsuruoka, S,
Sugimoto K,
Imai M,
Fujimura A,
Asano Y,
and
Muto S.
P-glycoprotein-mediated drug secretion in mouse proximal tubule perfused in vitro.
J Am Soc Nephrol
12:
177-181,
2001[Abstract/Free Full Text].
32.
Zhuang, S,
Hirai S,
and
Ohno S.
Hyperosmolality induced activation of cPKC and nPKC, a requirement for ERK1/2 activation in NIH/3T3 cells.
Am J Physiol Cell Physiol
278:
C102-C109,
2000[Abstract/Free Full Text].
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