Extracellular Cl
modulates
shrinkage-induced activation of Na+/H+
exchanger in rat mesangial cells
Yukio
Miyata,
Shigeaki
Muto,
Satoru
Yanagiba, and
Yasushi
Asano
Department of Nephrology, Jichi Medical School,
Minamikawachi, Kawachi, Tochigi 329-0498, Japan
 |
ABSTRACT |
To examine the
effect of hyperosmolality on Na+/H+ exchanger
(NHE) activity in mesangial cells (MCs), we used a
pH-sensitive dye,
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, to measure intracellular pH (pHi) in a single MC from rat
glomeruli. All the experiments were performed in
CO2/HCO
3-free HEPES
solutions. Exposure of MCs to hyperosmotic HEPES solutions (500 mosmol/kgH2O) treated with mannitol caused cell
alkalinization. The hyperosmolality-induced cell alkalinization was
inhibited by 100 µM ethylisopropylamiloride, a specific NHE
inhibitor, and was dependent on extracellular Na+. The
hyperosmolality shifted the Na+-dependent acid extrusion
rate vs. pHi by 0.15-0.3 pH units in the
alkaline direction. Removal of extracellular Cl
by
replacement with gluconate completely abolished the rate of cell
alkalinization induced by hyperosmolality and inhibited the Na+-dependent acid extrusion rate, whereas, under isosmotic
conditions, it caused no effect on Na+-dependent
pHi recovery rate or Na+-dependent acid
extrusion rate. The Cl
-dependent cell alkalinization
rate under hyperosmotic conditions was partially inhibited by
pretreatment with 5-nitro-2-(3-phenylpropylamino)benzoic acid, DIDS,
and colchicine. We conclude: 1) in MCs, hyperosmolality activates NHE to cause cell alkalinization, 2) the acid
extrusion rate via NHE is greater under hyperosmotic conditions than
under isosmotic conditions at a wide range of pHi,
3) the NHE activation under hyperosmotic conditions, but not
under isosmotic conditions, requires extracellular
Cl
, and 4) the
Cl
-dependent NHE activation under hyperosmotic
conditions partly occurs via Cl
channel and
microtubule-dependent processes.
intracellular pH; chloride ion; chloride channel; microtubule
 |
INTRODUCTION |
THE SODIUM HYDROGEN EXCHANGER (NHE) is a plasma
membrane transport protein found in a broad range of biological
systems, including glomerular mesangial cells (MCs) (4, 42). In MCs,
entry of Na+ into cells in exchange for an intracellular
H+ is the main effect of NHE, which therefore is involved
in the regulation of intracellular pH (pHi) and initiation
of cell growth and proliferation (12, 13).
Cell volume regulation generally involves the movement of ions and
organic osmolytes across the surface membrane and is often mediated by
pHi-regulating transporters (reviewed in Refs. 6 and 19).
In a variety of cells (8, 10, 15, 16, 20, 30-32, 37, 38), one such
transporter, the NHE, is activated by shrinkage 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. Activation of
NHE also results in H+ extrusion, which causes cell
alkalinization. However, it is not yet known whether hyperosmolality
activates NHE in MCs.
MCs are rich in contractile fibers and smooth muscle-like cells (1,
21). They are located in the intercapillary space of the glomerulus,
and may regulate, through its contraction, the intraglomerular
hemodynamics and thus the glomerular filtration rate (GFR) (reviewed in
Ref. 23). Some vasoactive peptides, angiotensin II (ANG II) and
arginine vasopressin (AVP), cause MC contraction through an increase in
intracellular Ca2+ and consequently reduce glomerular
ultrafiltration (23). Because the sensitivity of contractile proteins
to Ca2+ is reduced at low pHi in skeletal and
cardiac muscles (11), pHi change might influence MC
contractility, and subsequently GFR. Cultured MCs have also been shown
to change morphologically, altering their growth characteristics and
metabolic activity during periods of cyclic stretching-relaxation
(18). Therefore, the ability to regulate cell volume is
especially important for MCs, in which cell volume changes can alter
glomerular hemodynamics.
In dog erythrocytes (31, 32) and in barnacle muscle fibers (10), it has
been demonstrated that hyperosmolality-induced activation of NHE is
inhibited by removing Cl
from the extracellular
fluid. In the apical membrane vesicle of rat colonic crypt cells, NHE
activity under isosmotic conditions has been reported to be dependent
on extracellular Cl
(33). In addition, both increase
in intracellular Ca2+ concentration and MC contraction
induced by ANG II and AVP are attenuated when the extracellular
Cl
concentration is reduced (28). It is also well
known that Cl
is an important mediator of
tubuloglomerular feedback (TGF) regulation of glomerular filtration by
the distal tubular flow (35). These reports suggest the possibility
that MC NHE activity may be modified by extracellular
Cl
under iso- and/or hyperosmotic conditions.
Therefore, we isolated and cultured MCs from rat glomeruli to address
the following issues: 1) whether hyperosmolality activates NHE
activity, and 2) whether and how NHE activity under iso- or hyperosmotic conditions is modulated by extracellular
Cl
.
 |
METHODS |
Solutions.
The composition of the solutions is given in Table
1. All solutions were nominally
CO2/HCO
3 free and 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. The nigericin calibrating
solutions were titrated to different pH values at 37°C with either
HCl or N-methyl-D-glucamine (NMDG). For the
Na+-free solutions (solutions 2 and 3,
Table 1), Na+ was replaced with NMDG titrated
with the appropriate acid. In Cl
-free (solutions
3 and 4, Table 1) and low-Cl
(solutions 5-8, Table 1) solutions, Cl
was replaced with gluconate. Because of the chelation of
Ca2+ in gluconate-containing solutions, we compensated by
increasing total extracellular Ca2+ concentrations, as
shown in Table 1.
2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM (BCECF-AM) was prepared as a 10 mM stock solution and diluted 1:1,000
to a final concentration of 10 µM. Ethylisopropylamiloride (EIPA) was
prepared as a 100 mM stock solution in methanol and was diluted 1:1,000
to a final concentration of 100 µM. Nigericin was prepared as a 10 mM
stock solution in ethanol and was diluted 1:1,000 into solution
13 of Table 1 to a final concentration of 10 µM.
MC culture.
Glomerular MCs were obtained by the methods previously described in our
laboratory (27). In brief, kidneys were removed from 150- to 200-g male
Sprague-Dawley rats. All subsequent steps were performed with sterile
conditions under a laminar flow hood. Renal cortices from two to three
rats were pooled and minced with a sterile razor blade. The tissue was
collected and suspended in PBS. The sediments were gently poured onto a
series of stainless steel sieves of decreasing pore sizes (250, 150, and 75 µm; NBC Kogyo, Tokyo, Japan). The filtrates were
collected and resuspended in PBS. The sediments comprised a large
number of glomeruli that were generally free of capsular and tubular
debris. The glomerular suspension was passed through successive sieves
and was resuspended in PBS in a culture tube. After centrifugation at
1,000 rpm for 5 min, the supernatant was discarded and the glomeruli
were then plated onto 100-mm culture dishes (Becton Dickinson, Rathway, NJ). The cells were cultured in RPMI 1640, buffered with 10 mM HEPES at
pH 7.4, and supplemented with 20% fetal bovine serum (FBS), 2 mg/ml
NaHCO3, 100 U/ml penicillin, 100 µg/ml streptomycin, and
15.5 µg/ml insulin-transferrin-selenium in a 5% CO2
incubator at 37°C. Glomerular attachment was 20-30% after 48 h. Under these conditions, epithelial cells started to grow from the
glomeruli within 2-3 days. MCs started to grow rapidly after
7-10 days, but the number of epithelial cells continuously
decreased. MCs grew to confluence after 21-28 days. By this time,
the cultures were virtually free of epithelial cells and showed
positive staining for
-actin, myosin, and desmin, and showed
negative staining for an endothelial cell marker factor VIII. ANG
II-induced contraction of MCs was also detected by phase-contrast microscopy.
Confluent cultures were passaged through trypsin-EDTA and seeded at a
1:3 ratio in 100-mm culture dishes (Becton Dickinson). The passages
were performed at 5- to 7-day intervals after confluence of the cells.
Because major phenotypic changes (e.g., failure to
contract in response to AVP) occur in MCs after multiple
passages (e.g., Ref. 21), we only studied cells passages
3-5.
Measurement of pHi.
For pHi measurements, MCs were plated on 35-mm petri dishes
containing a glass coverslip bottom (MatTec, Ashland, MA) and were used
5-7 days later. Cells were incubated in 0.5% FBS-containing RPMI
1640 for 24 h before use. Before each experiment, the cells grown to
subconfluence were incubated for 20 min in 0.5% FBS-containing RPMI
1640 treated with 10 µM BCECF-AM at 37°C. The petri dish was then
placed on the stage of an inverted epifluorescence microscope (IMT-2;
Olympus, Tokyo, Japan) and was then continuously perfused with
HEPES-buffered solutions by gravity. A water jacket was used to
maintain temperature in the dish at 37°C.
Single-cell measurement of pHi was performed using a
microscopic fluorometer (OSP3; Olympus) as described previously (39). Measurements were made ×100 magnification, and the diameter of the beam of light focused on the MC 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 in this apparatus it takes 10 ms to
obtain one fluorescence ratio (I490/I440), the MC was exposed to light for 1 s to obtain one mean
I490/I440. We used only MCs that had at least
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.
Conversion of the I490/I440 ratios to
pHi values was performed according to the
high-K+/nigericin technique as described previously (4,
34). At the end of each experiment, the cell was exposed to a
high-K+ solution (solution 13, Table 1), titrated
to pH 7.0, to which 10 µM nigericin was added. The
I490/I440 ratios of the entire experiment were
normalized by dividing them by the I490/I440
ratio corresponding to pH 7.0. The pHi was then calculated
using the following equation (4)
where
b is the distance between the upper and lower asymptote of the
curve. The values of pK and b were determined from
experiments in which MCs were exposed to 10 µM nigericin-containing
solutions at different pH, always including pH 7.0. We fitted
calibration data to the above equation, using a nonlinear least-squares
method, and obtained best-fit values for b and pK of
1.54 ± 0.02 (SE) and 7.12 ± 0.02 (SE), respectively.
Determination of intracellular buffering power.
Intrinsic buffering power (
I) of MCs was determined
using the method of Boyarsky et al. (4). As shown in Fig.
1A, acid-loaded MCs were exposed to
a series of nominally Na+-free solutions (solution
2) that contained 20, 10, 5, 2.5, 1.0, 0.5, and 0 mM total ammonium
(NH+4/NH3). Total NH+4/NH3-containing solutions
were prepared by adding NH4Cl with replacement of NMDG in
Na+-free solution. With each stepwise decrease in
[NH+4/NH3]o, the amount of protons delivered to the cytoplasm
(
[acid]i) was considered equal to the
resultant change in
[NH+4]i. If it is
assumed that [NH3]i equals
[NH3]o, and that the
pKa governing the
NH3/NH+4 equilibrium (8.9 at
37°C) is the same in the cytoplasm as in the extracellular fluid,
[NH+4]i can be
calculated from the observed pHi.
pHi was
taken as the change in pHi produced by the stepwise
decrease in
[NH3/NH+4]o.
I was then calculated as

[acid]i/
pHi (34).
I was assigned to the mean of the two pHi
values used for its calculation. Figure 1B shows the
pHi dependence of
I. The buffering power
data were grouped into pH intervals of 0.2. Each closed circle
represents the mean buffering power of 0.2 pH interval, and the line
represents the least-squares fit to all 56 individual data points. We
used this straight line to determine
I as a function of
pHi in buffering power calculations in the present study.
The equation of the best-fit line is
I = 147.2
19.7 × pHi (r = 0.997) at a range of physiological pHi, indicating that
I decreases with
increasing pHi.

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Fig. 1.
Intracellular pH (pHi) dependence of intrinsic
intracellular buffering power ( I). A: typical
experiment. Cell was bathed in HEPES solution (solution 1,
Table 1). Na+ was removed, and then NMDG Cl was replaced
with 20 mM NH4Cl. NH4Cl concentration was then
decreased step by step, as indicated concentrations. Restoration of
[Na+]o to 142 mM caused
pHi to increase, indicating the activity of
Na+/H+ exchanger (NHE). From these data,
I is calculated as described in METHODS.
B: I vs. pHi. Data are summarized
from 11 experiments similar to that of A. , mean of
buffering power values calculated for data within pHi
intervals of 0.2. Bars, SE for mean pHi values and for mean
buffering power in each pH interval. Solid line, results of linear
least-squares fit to all 56 individual buffering power values;
y-intercept of 147.2 mM/pH and a slope of 19.7
(mM/pH)/pH, r = 0.997.
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Computation of net acid extrusion rates.
Net acid extrusion rates (JH) in MCs were calculated from
rates of pHi increase (dpHi/dt) and
I in two kinds of experiments. First, Na+
was removed from the extracellular solution, causing pHi to
decrease. We analyzed the pHi increase elicited by
readdition of Na+. Second, we analyzed pHi
increase when, at a steady-state pHi, MCs were exposed to
hyperosmotic solutions containing mannitol, sucrose, or urea. For both
experiments, we fitted a third or a fourth-degree polynomial to the
pHi increase using a least-squares method, as described
previously (7). At each pHi, data from four or more
experiments were averaged to produce a plot of mean net acid extrusion
rate vs. pHi (4, 34).
Drugs and chemicals.
All chemicals were obtained from Wako (Osaka, Japan) unless noted as
follows: RPMI 1640, penicillin, streptomycin,
insulin-transferrin-selenium, and FBS were obtained from GIBCO BRL
(Rockville, MD); HEPES and BCECF-AM were from Dojindo (Kumamoto,
Japan); EIPA and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)
were from Research Biochemicals International (Natick, MA); and NMDG,
nigericin, DIDS, colchicine, and cytochalasin B were from
Sigma (St. Louis, MO).
Statistical analysis.
The data are expressed as means ± SE. Comparisons were performed by
paired or nonpaired Student's t-test or one-way ANOVA where
appropriate. P values <0.05 were considered significant.
 |
RESULTS |
Steady-state pHi.
When MCs were perfused with the HEPES-buffered solution (solution
1, Table 1) at a rate of 4.5-5.5 ml/min at 37°C,
their pHi values were gradually decreased and sustained at
a steady-state level. In MCs, mean steady-state pHi values
were 6.96 ± 0.01 (n = 227).
pHi regulation of MCs under isosmotic conditions.
At first we observed pHi recovery after removal of
extracellular Na+ (Fig.
2A). Cells were first bathed in the
Na+-containing HEPES-buffered solution (solution 1,
Table 1) and were then incubated in the Na+-free solution
(solution 2, Table 1). The removal of extracellular Na+ caused a substantial and sustained decrease in
pHi from 7.01 ± 0.07 to 6.48 ± 0.02 (P < 0.001, n = 5). Subsequent addition of Na+ to the
extracellular solution caused pHi to rapidly increase to
similar values of the initial steady-state pHi (7.00 ± 0.06, n = 5). When a specific inhibitor of NHE, EIPA (100 µM), was added in the continued absence of external Na+,
pHi was not changed at all (Fig. 2B). Readdition of
Na+ to the extracellular solution in the continued presence
of EIPA also caused no effect on pHi (Fig. 2B).
When cells were treated with EIPA after readdition of Na+
to the extracellular solution, no further increase in pHi
was observed (Fig. 2C). These results indicate that MCs possess
NHE and that these cells exclusively regulate their pHi
through a NHE mechanism in the nominal absence of
CO2/HCO
3.

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Fig. 2.
Evidence for NHE in mesangial cells (MCs). A: MCs were first
bathed in the Na+-containing HEPES-buffered solution (300 mosmol/kgH2O) and were then incubated in the
Na+-free solution. The removal of external Na+
caused a substantial and sustained decrease in pHi.
Readdition of external Na+ caused pHi to
rapidly increase to similar values of the initial steady-state
pHi. B: readdition of external Na+ in
the presence of ethylisopropylamiloride (EIPA) (100 µM) completely
inhibited the Na+-dependent pHi increase.
C: when MCs were treated with EIPA (100 µM) after readdition
of external Na+, no further increase in pHi was
observed. D: removing extracellular Cl by
replacement with gluconate caused no effect on
Na+-dependent pHi increase after acid load.
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pHi regulation of MCs under hyperosmotic conditions.
We next examined whether hyperosmolality affects pHi in
MCs. MCs were incubated in HEPES-buffered Na+-containing
solution (solution 1, 300 mosmol/kgH2O) and were
then exposed to the HEPES-buffered solution treated with 200 mM
mannitol (500 mosmol/kgH2O). As shown in Fig.
3A, pHi significantly
increased from 6.86 ± 0.03 to 7.26 ± 0.02 (P < 0.001, n = 11). When MCs were treated with EIPA (100 µM)
in the continued presence of hyperosmotic mannitol solution, no further
increase in pHi was observed (Fig. 3B). When MCs
were treated with EIPA (100 µM) in the absence of hyperosmotic
mannitol solution, pHi significantly decreased from 6.99 ± 0.06 to 6.91 ± 0.06 (P < 0.001, n = 5; Fig.
3C), indicating that in the nominal absence of
CO2/HCO
3, the NHE must be
active in the normal steady-state pHi to balance a substantial rate of intracellular acid loading. In the continued presence of EIPA, exposure of MCs to the hyperosmotic mannitol solution
did not elicit a pHi increase at all (Fig. 3C). We
next examined whether cell alkalinization induced by hyperosmotic
mannitol is dependent on external Na+. Exposure of the
cells to hyperosmotic mannitol solution in the continued absence of
external Na+ caused no increase in pHi (Fig.
4A). However, when external
Na+ was readded in the presence of hyperosmotic mannitol,
pHi recovery was substantially faster, and the steady-state
pHi values (7.35 ± 0.06, P < 0.01, n = 7) were greater than in its absence (6.97 ± 0.07, n = 7; see
Figs. 2A and 4A). These findings are consistent with
the notion that hyperosmotic mannitol activates NHE to cause cell
alkalinization in MCs. From the Na+-dependent
pHi recovery rate and
I, we calculated the
relationship between Na+-dependent acid extrusion rate
(JH) and pHi under iso- and hyperosmotic conditions (Fig. 5). Under the two
conditions, Na+-dependent JH decreased as
pHi increased. However, the cells exposed to the
hyperosmotic mannitol solution had a significantly greater JH than those exposed to the isosmotic solution at
comparable pHi values. Furthermore, hyperosmolality shifted
the JH vs. pHi by 0.15-0.3
pH units in the alkaline direction.

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Fig. 3.
Cell shrinkage causes an EIPA-sensitive cell alkalinization in MCs.
A: exposure of MCs to hyperosmotic HEPES solution (500 mosmol/kgH2O) containing 200 mM mannitol caused cell
alkalinization. B: when MCs were treated with EIPA (100 µM)
in the continued presence of hyperosmotic mannitol solution, no further
increase in pHi was observed. C: addition of EIPA
(100 µM) to the MCs caused pHi to decrease. In the
continued presence of EIPA, exposure of MCs to hyperosmotic mannitol
solution did not elicit a pHi increase at all.
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Fig. 4.
Effect of cell shrinkage on Na+-dependent pHi
increase after acid load with or without extracellular
Cl . A: in the presence of extracellular
Cl , shrinkage-induced cell alkalinization was
inhibited by Na+-free solution. When external
Na+ was readded, pHi increased markedly.
B: removal of extracellular Cl by
replacement with gluconate caused no effect on pHi, but
reduced the Na+-dependent pHi increase induced
by cell shrinkage.
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Fig. 5.
pHi dependence of the Na+-dependent acid
extrusion rates (JH) under iso- and hyperosmotic conditions
in the presence and absence of extracellular Cl . The
plots were computed from experiments such as illustrated in Figs. 2,
A and D, and 4, A and B. Data represent
means ± SE. Isosmo, isosmolality; hyperosmo, hyperosmolality.
* P < 0.05, ** P < 0.005 compared with
isosmolality in the presence of Cl at comparable
pHi values. P < 0.05,  P < 0.005 compared with
hyperosmolality in the presence of Cl at comparable
pHi values. Note that under hyperosmotic conditions, the
JH is not only pHi dependent but also
Cl dependent.
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To determine the effects of different osmolytes on pHi, we
used other osmolytes, sucrose and urea. For this purpose, MCs were incubated in HEPES-buffered solution (solution 1, 300 mosmol/kgH2O, Table 1), and were then exposed to the
HEPES-buffered solution treated with 200 mM sucrose or urea (500 mosmol/kgH2O). As shown in Fig.
6A, hyperosmotic sucrose solution
caused steady-state pHi to increase from 6.93 ± 0.03 to
7.29 ± 0.04 (P < 0.001, n = 6). EIPA (100 µM)
caused pHi to decrease from 7.11 ± 0.12 to 7.05 ± 0.13 (P < 0.001, n = 4), but completely inhibited the
hyperosmotic sucrose-induced cell alkalinization (Fig. 6B). By
sharp contrast, hyperosmotic urea solution had no effect on
pHi (6.86 ± 0.06 to 6.86 ± 0.06, n = 8; Fig.
6C). Hyperosmotic mannitol and sucrose solutions showed
significant initial rates of cell alkalinization (initial
dpHi/dt), but hyperosmotic urea solution failed to
detect the initial dpHi/dt (Fig. 6D).

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Fig. 6.
Effects of hyperosmotic mannitol, sucrose, or urea on NHE activity in
MCs. A: exposure of MCs to hyperosmotic HEPES solution (500 mosmol/kgH2O) containing 200 mM sucrose caused cell
alkalinization. B: EIPA (100 µM) caused steady-state
pHi to decrease and completely inhibited the
sucrose-induced cell alkalinization. C: exposure of MCs
to hyperosmotic HEPES solution (500 mosmol/kgH2O)
containing 200 mM urea caused no effect on pHi. D:
initial rate of cell alkalinization (initial
dpHi/dt) induced by hyperosmotic mannitol, sucrose,
and urea. Data represent means ± SE; number of experiments in
parentheses. Initial pHi values at which we estimated
initial dpHi/dt induced by hyperosmotic mannitol,
sucrose, and urea, were 6.86 ± 0.03 (n = 11), 6.93 ± 0.03 (n = 6), and 6.86 ± 0.06 (n = 8),
respectively, and were not significantly different among them. Initial
dpHi/dt in the hyperosmotic sucrose solution was
significantly (P < 0.05) smaller than that of the
hyperosmotic mannitol solution.
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Glomerular MCs require Cl
for the development of a
variety of metabolic and functional properties (22, 28, 29). Also, in
canine erythrocytes (31, 32) and barnacle muscle fibers (10),
hyperosmolality-induced NHE activation has been reported to require
extracellular Cl
. Therefore, we next examined
whether NHE activity in MCs under iso- or hyperosmotic conditions is
dependent on extracellular Cl
. For this purpose,
extracellular Cl
was replaced with a nonpermeant
anion, gluconate (solutions 3 and 4, Table 1). Removal
of extracellular Cl
by replacement with gluconate
did not influence pHi (Fig.
7A). When hyperosmotic mannitol
solution was added in the continued absence of extracellular
Cl
, pHi was not changed at all (Fig.
7A). However, when extracellular Cl
was
readded in the continued presence of hyperosmotic mannitol, pHi significantly increased from 6.92 ± 0.03 to 7.30 ± 0.02 (P < 0.001, n = 12; Fig. 7A
and Table 2). EIPA (100 µM) alone caused the steady-state pHi to decrease by 0.08. When
extracellular Cl
was readded to the MCs treated with
hyperosmotic mannitol in the continued presence of EIPA, the
Cl
-dependent cell alkalinization was completely
inhibited (Fig. 7B). We next examined whether removal of
extracellular Cl
affects the
Na+-dependent pHi recovery in the presence of
hyperosmotic mannitol. As shown in Fig. 4B, the
Na+-dependent pHi recovery rate in the absence
of extracellular Cl
(56.1 ± 6.4 pH units/s × 104, P < 0.005, n = 9) was
significantly smaller than that in its presence (92.7 ± 9.1 pH
units/s × 104, n = 7). The relationship
between Na+-dependent JH and pHi
under hyperosmotic conditions in the absence of extracellular
Cl
is shown in Fig. 5. Removing extracellular
Cl
under hyperosmotic conditions caused a
significant decrease in JH at comparable pHi
values. By sharp contrast, when extracellular Cl
was
removed under isosmotic conditions, either pHi,
Na+-dependent pHi recovery rate (Fig.
2D), or Na+-dependent JH at different
pHi values (Fig. 5) were not affected.

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Fig. 7.
Cl -dependent cell alkalinization induced by cell
shrinkage. A: removal of extracellular Cl by
replacement with gluconate completely abolished the cell alkalinization
induced by hyperosmotic mannitol (500 mosmol/kgH2O). When
external Cl was readded, cell alkalinization
occurred. B: EIPA (100 µM) caused steady-state
pHi to decrease and completely inhibited the
Cl -dependent cell alkalinization induced by cell
shrinkage. C: 5-nitro-2-(3-phenylpropylamino)benzoic acid
(NPPB; 100 µM) caused no effect on steady-state pHi, but
partially inhibited the Cl -dependent cell
alkalinization induced by cell shrinkage. D: DIDS (100 µM)
caused no effect on steady-state pHi, but partially
inhibited the Cl -dependent cell alkalinization
induced by cell shrinkage. E: colchicine (100 µM) caused no
effect on steady-state pHi, but partially inhibited the
Cl -dependent cell alkalinization induced by cell
shrinkage. F: cytochalasin B (100 µM) caused no effect on
steady-state pHi or Cl -dependent cell
alkalinization induced by cell shrinkage.
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Table 2.
Effect of various inhibitors on Cl -dependent
initial rate of pHi increase after hyperosmotic
mannitol solution
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To examine the dose-dependent effect of Cl
on the
shrinkage-induced NHE activation, MCs were exposed to hyperosmotic
mannitol solution (500 mosmol/kgH2O) with different
extracellular Cl
concentrations (0, 13, 36, 64, 93, and 132 mM) by replacement with gluconate (solutions 1,
4, and 5-8, Table 1). Figure
8 shows the relationship between
extracellular Cl
concentrations and the rate of cell
alkalinization (dpHi/dt) induced by the
hyperosmotic mannitol at pHi of 6.85. The
dpHi/dt decreased in a sigmoidal fashion as
extracellular Cl
concentrations decreased. The
apparent 50% inhibitory concentration of extracellular
Cl
for the dpHi/dt induced by
hyperosmotic mannitol was 69.2 mM.

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Fig. 8.
Relationship between extracellular Cl
concentrations and the rate of cell alkalinization
(dpHi/dt) induced by the hyperosmotic mannitol at
pHi of 6.85. The dpHi/dt decreased in a
sigmoidal fashion, as extracellular Cl concentrations
decreased. Data represent means ± SE; number of experiments
in parentheses.
|
|
We next examined whether the Cl
-dependent NHE
activation induced by hyperosmotic mannitol occurs through
Cl
-dependent transport processes, including
Cl
channel and Cl
/base exchange.
For this purpose, we added NPPB (100 µM), a Cl
channel inhibitor, or DIDS (100 µM), an inhibitor of
Cl
/base exchange, to the MCs, and then observed the
Cl
-dependent cell alkalinization induced by
hyperosmotic mannitol. NPPB or DIDS alone caused no effects on
pHi values (Fig. 7, C and D). When MCs were
treated with hyperosmotic mannitol in the presence of NPPB,
pHi significantly increased from 6.94 ± 0.03 to 7.25 ± 0.07 (P < 0.001, n = 8). However, the final
pHi values (7.25 ± 0.07, P < 0.05, n = 8) in the presence of NPPB were significantly smaller than those in its
absence (7.30 ± 0.02, n = 12; Fig. 7, A and
C, Table 2). Furthermore, in the presence of NPPB, the Cl
-dependent initial alkalinization rate induced by
hyperosmotic mannitol (initial dpHi/dt; 41.4 ± 6.8 pH units/s × 104, P < 0.005, n = 8) was significantly smaller than that in its absence (73.3 ± 8.0 pH units/s × 104, n = 12; Table 2).
When MCs were treated with hyperosmotic mannitol in the presence of
DIDS, pHi significantly increased from 6.95 ± 0.02 to
7.36 ± 0.05 (P < 0.001, n = 8; Fig. 7D and
Table 2). However, in the presence of DIDS, the initial
dpHi/dt under hyperosmotic conditions (51.8 ± 7.1 pH units/s × 104, P < 0.05, n = 8) was significantly smaller than that in its absence, although the
final pHi values (7.36 ± 0.05, n = 8) in the
presence of DIDS were not significantly different from those in its
absence (Table 2). It has been reported that the interactions between
the cytoskeleton and the plasma membrane regulate the activity of many
ion channels and transport proteins, including NHE (8, 42, 43). Thus we
examined whether the cytoskeletal elements are involved in the
Cl
-dependent NHE activation induced by hyperosmotic
mannitol. For this purpose, we added colchicine (100 µM), a disruptor
of microtubules, or cytochalasin B (100 µM), a disruptor of
filamentous actin, to the MCs, and then observed the
Cl
-dependent cell alkalinization induced by
hyperosmotic mannitol. Colchicine or cytochalasin B alone had no
effects on pHi values (Fig. 7, E and F,
Table 2). When MCs were treated with hyperosmotic mannitol in the
presence of colchicine, pHi significantly increased from
6.95 ± 0.02 to 7.12 ± 0.02 (P < 0.001, n = 7).
However, the final pHi values (7.12 ± 0.02, P < 0.001, n = 7) in the presence of colchicine were significantly
smaller than those in its absence (Fig. 7, A and E,
Table 2). Furthermore, in the presence of colchicine, the initial
dpHi/dt under hyperosmotic conditions (32.7 ± 9.5 pH units/s × 104, P < 0.001, n = 7)
was significantly smaller than that in its absence (Table 2). In sharp
contrast, pretreatment with cytochalasin B caused no effect on
Cl
-dependent pHi increase induced by
hyperosmotic mannitol (Fig. 7F and Table 2).
To further examine the specificity of extracellular
Cl
for the shrinkage-induced NHE activation, 125 mM
Cl
in the solution was substituted with equimolar
gluconate, I
, Br
,
SCN
, or F
(solutions 5 and 9-12, Table 1). Effects of substitution of extracellular Cl
with I
,
Br
, SCN
, or F
on pHi under iso- and hyperosmotic conditions are
illustrated in Fig. 9, A-D,
respectively. Substitution of Cl
with
I
, Br
, or SCN
under isosmotic conditions had no effect on pHi. However,
when the cells were exposed to hyperosmotic mannitol (500 mosmol/kgH2O) in the presence of I
,
Br
, and SCN
, the pHi
significantly increased from 6.82 ± 0.01 to 7.35 ± 0.03 (P < 0.001, n = 9), from 6.82 ± 0.04 to 7.26 ± 0.02 (P < 0.001, n = 6), and from 6.79 ± 0.02 to 6.95 ± 0.05 (P < 0.001, n = 6), respectively. Pretreatment with EIPA (100 µM) completely inhibited the pHi increase induced by hyperosmotic mannitol in the
presence of I
, Br
, and
SCN
(Fig. 9, E-G, respectively). By sharp
contrast, substitution of Cl
with
F
under isosmotic conditions caused a significant
decrease in pHi from 6.83 ± 0.02 to 6.61 ± 0.03 (P < 0.001, n = 9). When hyperosmotic mannitol was
added in the presence of F
, pHi was
increased to 6.81 ± 0.02 (P < 0.001, n = 9).
However, these pHi values (6.81 ± 0.02) were not
significantly different from basal pHi values (6.83 ± 0.02). When MCs were treated with EIPA under isosmotic conditions,
pHi significantly decreased from 6.89 ± 0.03 to 6.75 ± 0.02 (P < 0.001, n = 5; Fig. 9H). When MCs were exposed to the solution containing F
in the
continued presence of EIPA, pHi further decreased to 6.62 ± 0.02 (P < 0.001, n = 5). However, the
EIPA-sensitive pHi decrease in the presence of
F
(
pHi: 0.13 ± 0.02, P < 0.05, n = 5) was significantly smaller than that in its absence
(
pHi: 0.22 ± 0.03, n = 9). Pretreatment with
EIPA completely inhibited the pHi increase induced by
hyperosmotic mannitol in the presence of F
(Fig.
9H). We calculated acid extrusion rates (initial
JH) at the initial point of cell alkalinization induced by
hyperosmotic mannitol solution with different anions. As shown in Fig.
10, these studies yielded an apparent
specificity of Cl
Br
I
> SCN
> F
= gluconate for the initial JH induced
by hyperosmolality. When 125 mM Cl
in the solution
was substituted with equimolar cyclamate, the initial JH
induced by hyperosmolality were similar to those observed in the
gluconate solution (data not shown).

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Fig. 9.
Effects of substitution of extracellular Cl with
different anions on pHi under iso- and hyperosmotic
conditions. 125 mM Cl was replaced by equimolar
I , Br , SCN , or
F . Substitution of extracellular
Cl with I (A),
Br (B), or SCN
(C) in the isosmotic solution had no effect on pHi,
whereas it caused cell alkalinization in the hyperosmotic mannitol
solution (500 mosmol/kgH2O). Substitution of
extracellular Cl with F
(D) under isosmotic conditions caused cell acidification. When
hyperosmotic mannitol (500 mosmol/kgH2O) was added in the
presence of F , pHi returned to basal
pHi level. EIPA (100 µM) caused steady-state
pHi to decrease, but completely inhibited the
pHi increase induced by hyperosmotic mannitol in
the presence of I (E),
Br (F), SCN
(G), or F (H).
|
|

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Fig. 10.
Initial JH at the initial point of cell alkalinization
induced by hyperosmotic mannitol solution with different anions. Data
represent means ± SE; number of experiments in parentheses.
|
|
Figure 9, D and H, showed that, under isosmotic
conditions, F
caused pHi to decrease and
that the decreased pHi induced by F
was
partially inhibited by EIPA. These data suggest the possibility that,
under isosmotic conditions, F
may partially inhibit
NHE activity. To demonstrate this possibility, we examined
Na+-dependent pHi increase after acid load in
the absence and presence of F
. When MCs were first
bathed in the Na+-containing HEPES solution (control,
solution 1, Table 1) and were then incubated in the
Na+-free solution (solution 2, Table 1),
pHi was decreased to ~6.50. Thereafter, MCs were exposed
to the Na+-containing control solution (solution 1,
Table 1) or the Na+- and F
-containing
solution (solution 12, Table 1). As shown in Fig. 11A, subsequent addition of
Na+ to the extracellular solution caused a rapid increase
in pHi in MCs exposed to both control and
F
-containing solutions, but the rise in
pHi in the presence of F
(19.2 ± 4.7 pH units/s × 104, P < 0.005, n = 9) was significantly smaller than that in its absence (56.2 ± 11.1 pH units/s × 104, n = 11).
Furthermore, the Na+-dependent JH at
pHi of 6.60 after acid load in the presence of F
(36.6 ± 9.7 µM/s, P < 0.05, n = 9) was significantly smaller than that in its absence
(110.6 ± 22.6 µM/s, n = 11; Fig. 11B). In MCs
exposed to both control and F
-containing solutions,
the Na+-dependent pHi increase after acid load
was completely inhibited by EIPA (100 µM; Figs. 2B and
11A, respectively). The control solution contained 132 mM
Cl
, whereas the F
-containing
solution contained 13 mM Cl
(Table 1). Because
removing extracellular Cl
had no effect on NHE
activity under isosmotic conditions (Fig. 5), it is unlikely that the
decreased JH in the presence of F
is due
to decreased Cl
concentration of the solution.
Rather, the above data indicate that F
decreased the
Na+-dependent JH via an NHE process.

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Fig. 11.
Effect of F on NHE activity under isosomotic conditions.
A: Na+-dependent pHi increase after
acid load in the absence and presence of F . MCs were
first bathed in the Na+-containing HEPES-buffered solution,
and were then incubated in the Na+-free solution. The
removal of external Na+ caused a substantial and sustained
decrease in pHi. Readdition of external Na+ in
the absence and presence of F (125 mM) caused
pHi to rapidly increase, but the pHi increase
in the presence of F was slower and smaller than that in
its absence. The pHi increase in the presence of
F was completely inhibited by addition of EIPA (100 µM). B: The Na+-dependent JH in the
absence and presence of F at the initial point of
cell alkalinization after acid load. JH was obtained from
the product of dpHi/dt and I at
pHi of 6.60. Data represent means ± SE; number of
experiments in parentheses. * P < 0.05 compared with
control.
|
|
 |
DISCUSSION |
The present study was designed to determine whether hyperosmolality
activates NHE in MCs and, if so, to determine the underlying mechanisms, especially the role of extracellular Cl
.
We demonstrate that hyperosmotic mannitol and sucrose activate NHE to
cause cell alkalinization, but hyperosmotic urea causes no effect. The
acid extrusion rate via a NHE process is greater under hyperosmotic
conditions than under isosmotic conditions. Furthermore, NHE activation
under hyperosmotic conditions requires the presence of extracellular
Cl
, whereas NHE activity under isosmotic conditions
is independent of extracellular Cl
. The
Cl
-dependent NHE activation under
hyperosmotic conditions occurs, at least in part, via
Cl
channel- and microtubule-dependent processes.
Substitution of extracellular Cl
with different
anions modulates NHE activation induced by hyperosmolality.
Evidence for NHE in MCs
The present study demonstrates that, in the nominal absence of
CO2/HCO
3, pHi
recovery rate from an acid load was dependent on external
Na+ and was completely inhibited by a specific NHE
inhibitor (EIPA; see Fig. 2, A-C). Thus MCs exclusively
regulate their pHi through a NHE mechanism in the nominal
absence of CO2/HCO
3. These
findings confirm previous studies (4, 13).
pHi Regulation of MCs Under Iso- and
Hyperosmotic Conditions
pHi dependence of NHE. Our results show that
cell alkalinization induced by hyperosmotic mannitol is due to
activation of NHE because it is dependent on external Na+
and is inhibited by EIPA (see Figs. 3 and 4A). As shown in Fig. 5, under both iso- and hyperosmotic conditions,
Na+-dependent JH through a NHE process
decreased as pHi increased from the acidic to the normal
range. However, the cells exposed to the hyperosmotic mannitol solution
had a significantly greater JH than those exposed to the
isosmotic solution at comparable pHi values. In other
words, hyperosmolality shifted the JH vs. pHi
by 0.15-0.3 pH units in the alkaline direction.
Similarly, in Na+-depleted glial cells, Jean et al. (20)
reported that shrinkage shifted the flux vs. pHi profile by
0.3-0.4 pH units in the alkaline direction. In addition, Grinstein
et al. (15) studied the Na+-dependent component of a
pHi recovery from an acid load in lymphocytes, finding that
shrinkage shifts the flux vs. pHi relationship by 0.2-0.3 pH units in the alkaline direction. Similar findings have been reported in barnacle muscle fibers exposed to hyperosmolality (10)
and in C6 glioma cells treated with hyperosmotic mannitol solution (37). Because the pHi sensitivity of the exchange
system appears to be largely determined by an allosteric modifier site (2) located on the cytoplasmic face of the membrane, the mechanisms for
the shrinkage-induced NHE activation seem to be a shift in the
pHi dependence of the antiport.
Effects of different osmolytes.
In the present study, hyperosmotic mannitol and sucrose solutions
showed significant initial rate of cell alkalinization, whereas
hyperosmotic urea solution failed to detect it (see Figs. 3 and 6). In
addition, the cell alkalinization induced by hyperosmotic mannitol and
sucrose was completely inhibited by pretreatment with EIPA (see Figs.
3C and 6B). Therefore, these findings indicate that, at
steady-state pHi, both hyperosmotic mannitol and sucrose activate NHE, but hyperosmotic urea does not. Because mannitol and
sucrose are relatively impermeant, it is most likely that they make the
solution hypertonic. In contrast, urea is freely permeant and raises
the osmolality but does not affect the solution tonicity (40).
Accordingly, solution tonicity rather than absolute osmolality is the
important factor for the NHE activation at steady-state pHi. Similarly, Muto et al. (24) reported that, in vascular smooth muscle cells, hyperosmotic glucose or mannitol media stimulated Na+-K+-ATPase
1- and
1-mRNA accumulation,
1- and
1-subunit protein accumulation, and
Na+-K+-ATPase activity, whereas hyperosmotic
urea medium caused no effect. Similar actions of hyperosmotic NaCl,
raffinose, or urea on accumulation of organic osmolytes have been
reported in Madin-Darby canine kidney cells (25, 26): NaCl- or
raffinose-induced hyperosmotic stress resulted in the accumulation of
betaine, glycerophosphorylcholine, and myo-inositol, but
urea-induced hyperosmotic stress failed to induce osmolyte accumulation.
Effects of removing extracellular Cl
.
NHE activity in the apical membrane of the colonic crypt cell under
isosmotic conditions is dependent on extracellular
Cl
(33). In dog erythrocytes (31, 32) and in
barnacle muscle fibers (10), NHE activation under hyperosmotic
conditions is inhibited by removing extracellular
Cl
. Therefore, the present study examined whether
the MC NHE activity under iso- or hyperosmotic conditions is dependent
on extracellular Cl
. The MCs exposed to the
hyperosmotic mannitol solution in the absence of extracellular
Cl
had a significantly smaller JH than
that in its presence at comparable pHi values (see Fig. 5).
Furthermore, extracellular Cl
modulates the
shrinkage-induced NHE activation in a dose-dependent manner (Figs. 2
and 9), whereas, under isosmotic conditions, it causes no effect on NHE
activity (Fig. 2, A and D, and Fig. 5). Therefore, the
NHE activation under hyperosmotic conditions is not only
pHi dependent but also Cl
dependent.
Also, NHE in MCs is distinct from that of the apical membrane in the
colonic crypt cell.
The mechanisms by which extracellular Cl
affects the
shrinkage-induced NHE activation are not clear presently. The effects of extracellular Cl
may be through changes in
intracellular Cl
, as reported in barnacle muscle
fibers treated with hyperosmotic mannitol solution (10). Several
mechanisms would be proposed for the Cl
-dependent
NHE activation induced by hyperosmolality. Removal of extracellular
Cl
by replacement with gluconate did not influence
NHE activity under isosmotic conditions (see Fig. 2, A and
D, and Fig. 5), whereas it inhibited NHE activity under
hyperosmotic conditions (Figs. 4 and 5). On the contrary, when external
Cl
was readded in the continued presence of
hyperosmotic mannitol, NHE activation occurred (Fig. 7A).
Therefore, it is unlikely that this inhibition was due to the
introduction of gluconate as the extracellular Cl
replacement. Rather, Cl
might be required for some
aspects of the shrinkage-induced activation process, the sensor or
signal transduction system, as Davis et al. (10) suggested. Regarding
the latter, the activation of G proteins has been
reported to be involved in the shrinkage-induced activation of NHE in
barnacle muscle fibers (9). Regarding the former, we found that the
Cl
-dependent NHE activation induced by
hyperosmolality was partially inhibited by pretreatment with NPPB (the
Cl
channel inhibitor; see Fig. 7, A and
C, Table 2). These results indicate that the shrinkage-induced
NHE activation partly occurs through Cl
channel
activation. Pretreatment with DIDS (the inhibitor of Cl
/base exchange) also partially inhibited the
Cl
-dependent NHE activation induced by
hyperosmolality (see Fig. 7, A and D, Table 2). In the
nominal absence of
CO2/HCO
3, removing
extracellular Cl
caused no increase in
pHi, as shown in Fig. 7A. Thus, under these conditions, the Cl
/base exchange process is indeed
inoperative, although, in the presence of
CO2/HCO
3, the MC anion
exchanger has been reported to transport base (5). Rather, in the
nominal absence of
CO2/HCO
3, DIDS may act on
MCs as a Cl
channel inhibitor. In other cell
systems, DIDS has been reported to be one of the Cl
channel inhibitors (17, 36). As shown in Fig. 7, E and
F, and Table 2, colchicine, but not cytochalasin B, partially
inhibited the Cl
-dependent NHE activation induced by
hyperosmolality. These findings indicate that microtubule, but not
filamentous actin, is involved in the Cl
-dependent
NHE activation induced by hyperosmolality, and that under these
conditions, a colchicine-sensitive process may be activated, and
microtubule may regulate the exocytic insertion of the NHE
protein-containing vesicles into the plasma membrane. Similarly, Wagner
et al. (41) reported in rat proximal tubules that ANG II stimulated
H+-ATPase activity and that this stimulation was inhibited
by removing external Cl
, NPPB, and colchicine. Their
report suggests that ANG II stimulates proton extrusion via
H+-ATPase by a Cl
-dependent process
involving brush-border insertion of vesicles. The mechanisms by which
the Cl
-dependent NHE activation induced by
hyperosmolality occurs via a colchicine-sensitive process are still unclear.
Effects of substitution of Cl
with different
anions.
The present study further examined the Cl
specificity for the shrinkage-induced NHE activation. When
extracellular Cl
under isosmotic conditions was
replaced with Br
, I
, or
SCN
, basal pHi values were not affected
(see Fig. 9, A-C). Substitution of extracellular
Cl
with different anions yielded an apparent
specificity of Cl
Br
I
> SCN
> gluconate for the
shrinkage-induced acid extrusion rates through a NHE process (Fig. 10).
The hyperosmolality-induced cell alkalinization in the presence of
Cl
, Br
, I
,
SCN
, and gluconate was completely inhibited by
pretreatment with EIPA (Figs. 3C and 9, E-G). These
results indicate that both Br
and
I
, like Cl
, modulate the
shrinkage-induced NHE activation. Therefore, these three anions may
share common metabolic and/or transport pathway(s), as discussed above.
On the other hand, when Cl
was replaced with
SCN
, the acid extrusion rate induced by
hyperosmolality was 38.9% of that of Cl
. These
findings indicate that SCN
partially inhibits the
Cl
-dependent NHE activation induced by
hyperosmolality. When external Cl
under isosmotic
conditions was substituted with F
, basal
pHi values were significantly decreased (Fig. 9D).
Pretreatment with EIPA partially inhibits the
F
-induced cell acidification (Fig. 9H).
Furthermore, as shown in Fig. 11, the Na+-dependent
JH at pHi of 6.60 after acid load in the
presence of F
was significantly smaller than that in
its absence, and the Na+-dependent pHi recovery
was completely inhibited by EIPA. Thus, at steady-state
pHi, F
inhibits NHE activity, and
consequently causes cell acidification, although the possibility that
the F
-induced cell acidification may occur via
mechanisms other than inhibition of the NHE cannot be excluded. NaF
(more specifically AlF4
) at a
concentration of 10 mM is a well-known pharmacological probe for
establishing the significance of G protein activation in cellular
systems (14, 30). The concentration of NaF used in the present study
was 125 mM. It is not known whether this concentration of NaF actually
acts on MC NHE as a G protein activator. We found that the
hyperosmolality-induced cell alkalinization in the presence of
F
was completely inhibited by pretreatment with EIPA
(Fig. 9H) and that the initial JH in the
hyperosmotic F
solution was similar to that in the
hyperosmotic gluconate solution (Fig. 10). These findings indicate that
the hyperosmolality-induced cell alkalinization in the presence of
F
is due to NHE activation and that
F
does not influence the
Cl
-dependent NHE activation induced by
hyperosmolality. In contrast to MCs, in vascular smooth muscle cells,
NaF has been shown to stimulate NHE activity under both iso- and
hyperosmotic conditions (30). From our present findings, different
anions modulate Cl
-dependent NHE activation induced
by hyperosmolality. However, the underlying mechanisms will be required
to be elucidated.
In conclusion, the present study clearly demonstrates that, at
steady-state pHi, hyperosmolality by the poorly permeating solutes (mannitol and sucrose) activates NHE to cause cell
alkalinization, whereas the rapidly permeating solute (urea) has no
effect, and shrinkage-induced NHE activation requires extracellular
Cl
and is modulated by substitution of
Cl
with different anions. The
Cl
-dependent NHE activation induced by
hyperosmolality partly occurs via Cl
channel- and
microtubule-dependent processes. The Cl
-dependent
NHE activation under hyperosmotic conditions may be important for cell
volume regulation, intraglumerular hemodynamics, and/or TGF mechanisms.
 |
ACKNOWLEDGEMENTS |
We thank H. Kasakura for expert secretarial assistance in preparing
the manuscript.
 |
FOOTNOTES |
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.
A portion of this work was presented at the 1998 Annual Meeting of the
American Society of Nephrology in Philadelphia, PA, and has been
published in abstract form (23a).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Muto, Dept.
of Nephrology, Jichi Medical School, 3311-1 Yakushiji,
Minamikawachi, Tochigi 329-0498, Japan (E-mail:
smuto{at}jichi.ac.jp).
Received 25 February 1999; accepted in final form 7 January 2000.
 |
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