Shrinkage-induced activation of Na⫹/H⫹ exchange
in rat renal mesangial cells
MARK O. BEVENSEE,1 ESTHER BASHI,1 WOLF-RÜDIGER SCHLUE,2
GREGORY BOYARSKY,3 AND WALTER F. BORON1
1Department of Cellular and Molecular Physiology, Yale University School of Medicine,
New Haven, Connecticut 06520; 2Institut für Neurobiologie, Heinrich-Heine-Universität
Düsseldorf, 40225 Düsseldorf, Germany; and 3Department of Physiology and Biophysics,
University of Texas Medical Branch, Galveston, Texas 77550
acid-base transport; 28,78-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; chloride; intracellular pH; volume regulation
NORMAL PHYSIOLOGICAL processes challenge the maintenance of constant cell volume (see Ref. 28). For example, the breakdown of proteins, glycogen, and triglycerides into their more osmotically active monomers can
lead to cell swelling. Also, the activation of ion channels
in a cell can elicit volume changes as H2O osmotically
follows the movement of ions. For example, the activation of Na⫹ channels typically leads to the influx of Na⫹
and cell swelling, whereas the activation of K⫹ or Cl⫺
channels generally leads to the efflux of K⫹ or Cl⫺ and
cell shrinkage. Finally, as discussed by Hoffman and
Simonsen (22), excessive oral intake of H2O can create
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.
C674
a hypotonic environment for epithelial cells of the
gastrointestinal tract, and changes in the levels of
antidiuretic hormone lead to changes in the osmolality
of the medullary interstitium of the kidney.
Cells have acquired strategies to combat perturbations in their volume. These strategies can be categorized into chronic responses (hours to days) and acute
responses (seconds to minutes). One important component of the chronic response is typically the synthesis
or degradation of membrane-impermeant solutes (see
Refs. 8 and 28). Also, long-term volume stresses on a
cell may stimulate or inhibit the expression and/or
activation of ion/osmolyte transport processes (see Ref.
20).
Acute responses of a cell to volume disturbances
involve the immediate stimulation of ion transport
across plasma membranes. For example, cell swelling
can stimulate the efflux of ions and thus H2O out of
cells, a process termed volume regulatory decrease (2,
9, 15, 25, 27, 39). Conversely, cell shrinkage stimulates
the influx of ions and thus H2O in cells. The subsequent
increase in cell volume, a volume regulatory increase,
can be caused by stimulation of 1) Na-K-Cl cotransport
and 2) stimulation of Na⫹/H⫹ exchange, which together
with Cl-HCO3 exchange has the net effect of accumulating NaCl (see Refs. 10, 18, 20, 22, 28).
Several groups have studied the shrinkage-induced
activation of Na⫹/H⫹ exchange. Of course, decreased
intracellular pH (pHi ) can also activate Na⫹/H⫹ exchange independently of shrinkage. Both shrinkage
and low pHi activate Na⫹/H⫹ exchange in human
lymphocytes (17), C6 rat glioma cells (24,37), dog erythrocytes (32), barnacle muscle fibers (14), rabbit alveolar
macrophages (21), and rat mandibular salivary glands
(36). In barnacle muscle fibers, a G protein appears to
be an intermediate in the transduction of the shrinkage
signal to the activation of the Na⫹/H⫹ exchanger (13,
23). The presence of intracellular Cl⫺ also appears
necessary for the shrinkage-induced activation of the
transporter in both dog erythrocytes and barnacle
muscle fibers. In the dog erythrocytes, the activation
can be inhibited by removing external Cl⫺, which
presumably depletes intracellular Cl⫺ (31, 33). Indeed,
in the barnacle muscle fiber, shrinkage-induced activation of the Na⫹/H⫹ exchanger requires intracellular Cl⫺
(14, 23) at a step at, or before, activation of a heterotrimeric G protein (23).
In experiments on the pHi dependence of the Na⫹/H⫹
exchanger in barnacle muscle fibers (14), the shrinkageinduced activation of the transporter is approximately
0363-6143/99 $5.00 Copyright r 1999 the American Physiological Society
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
Bevensee, Mark O., Esther Bashi, Wolf-Rüdiger
Schlue, Gregory Boyarsky, and Walter F. Boron. Shrinkage-induced activation of Na⫹/H⫹ exchange in rat renal
mesangial cells. Am. J. Physiol. 276 (Cell Physiol. 45):
C674–C683, 1999.—Using the pH-sensitive dye 28,78-bis(2carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we examined the effect of hyperosmolar solutions, which presumably
caused cell shrinkage, on intracellular pH (pHi ) regulation in
mesangial cells (single cells or populations) cultured from the
rat kidney. The calibration of BCECF is identical in shrunken
and unshrunken mesangial cells if the extracellular K⫹
concentration ([K⫹]) is adjusted to match the predicted intracellular [K⫹]. For pHi values between ⬃6.7 and ⬃7.4, the
intrinsic buffering power in shrunken cells (600 mosmol/
kgH2O) is threefold larger than in unshrunken cells (⬃300
mosmol/kgH2O). In the nominal absence of CO2/HCO3⫺, exposing cell populations to a HEPES-buffered solution supplemented with ⬃300 mM mannitol (600 mosmol/kgH2O) causes
steady-state pHi to increase by ⬃0.4. The pHi increase is due
to activation of Na⫹/H⫹ exchange because, in single cells, it is
blocked in the absence of external Na⫹ or in the presence of 50
µM ethylisopropylamiloride (EIPA). Preincubating cells in a
Cl⫺-free solution for at least 14 min inhibits the shrinkageinduced pHi increase by 80%. We calculated the pHi dependence of the Na⫹/H⫹ exchange rate in cell populations under
normosmolar and hyperosmolar conditions by summing 1)
the pHi dependence of the total acid-extrusion rate and 2) the
pHi dependence of the EIPA-insensitive acid-loading rate.
Shrinkage alkali shifts the pHi dependence of Na⫹/H⫹ exchange by ⬃0.7 pH units.
SHRINKAGE-INDUCED ACTIVATION OF NA⫹/H⫹ EXCHANGE
METHODS
Solutions
All experiments were performed in the nominal absence of
CO2/HCO3⫺. The standard HEPES-buffered solution (⬃305
mosmol/kgH2O) contained (in mM) 125 NaCl, 5 KCl, 1 CaCl2,
1.2 MgSO4, 2 NaH2PO4, 32 HEPES, and 10.5 glucose and was
titrated to 7.4 at 37°C with NaOH. Hyperosmolar solutions
(600 mosmol/kgH2O) were made by adding ⬃300 mM mannitol to a HEPES-buffered solution, thereby increasing the
volume by ⬃3%. In the Na⫹-free solutions, the Na⫹ substitute
was N-methyl-D-glucammonium (NMDG⫹ ). In the Cl⫺-free
solutions, the Cl⫺ substitute was gluconate. In some of the
Cl⫺-free solutions, the Ca2⫹ concentration was increased to 3
mM to compensate for Ca2⫹ chelation by the gluconate. The
inhibitory effect of removing external Cl⫺ on the shrinkageinduced alkalization (see RESULTS) was the same whether or
not we compensated for Ca2⫹ chelation by gluconate. In NH3/
NH4⫹ solutions, NaCl (or NMDG-Cl) was replaced by an equal
concentration of NH4Cl. Both the normosmolar and hyperosmolar dye-calibration solutions, which were based on the
corresponding HEPES-buffered solutions, contained 10 µM
nigericin. In addition, in the normosmolar calibration solution, we replaced Na⫹ with 100 mM K⫹ [final K⫹ concentration ([K⫹]) ⫽ 105 mM] and NMDG⫹. In the hyperosmolar
calibration solution, we replaced the Na⫹ with 205 mM KCl
(final [K⫹] ⫽ 210 mM) and NMDG⫹ and adjusted the osmolality to 600 mosmol/kgH2O by adding ⬃120 mM mannitol. We
measured the osmolality of all solutions with a vaporpressure osmometer (model 5100C; Wescor, Logan, UT).
Ethylisopropylamiloride (EIPA) was diluted 1:1,000 from a 50
mM stock dissolved in dimethyl sulfoxide. The acetoxymethyl
ester of 28,78-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF-AM) was obtained from Molecular Probes (Eugene,
OR). Ethanol was obtained from Pharmco Products (Brookfield, CT). EIPA was obtained from either E. J. Cragoe, Jr.
(Nacogdoches, TX) or Research Biochemicals (Natick, MA).
All other reagents were obtained from Sigma Chemical (St.
Louis, MO).
Cell Preparation
Rat mesangial cells were prepared as previously described
(6, 29). Briefly, cells from passages 3–7 were plated on glass
coverslips and were grown at 37°C in Dulbecco’s modified
Eagle’s medium (GIBCO-BRL, Life Technologies, Gaithersburg, MD) exposed to an atmosphere of 5% CO2-balance air.
The medium was supplemented with 100 U/ml penicillin plus
streptomycin (GIBCO-BRL), 5 µg/ml of insulin plus transferrin plus selenious acid (Becton-Dickinson Labware, Bedford,
MA), and 10% fetal calf serum (Gemini Bioproducts, Calabasas, CA or GIBCO-BRL). To render cells quiescent at the time
of experimentation, the cells were incubated for 16–18 h in a
medium containing only 0.5% fetal calf serum. Experiments
were performed on cells at least 80% confluent. A coverslip
with attached cells was exposed to 10 µM BCECF-AM in the
standard HEPES-buffered solution in an air incubator at
37°C for 20 min. The coverslip was then transferred to a
temperature-regulated, flow-through cuvette and was perfused with the standard HEPES-buffered solution, prewarmed to 37°C, for ⬃5 min before the start of the experiment
to wash nonhydrolyzed BCECF-AM from the cells.
Measurement of pHi
Our technique for measuring pHi in a population of cells
has been previously described (26), and it will only be briefly
summarized here. The flow-through cuvette containing a
coverslip was placed in the excitation light path of a SPEX
Fluorolog-2 spectrofluorometer (model CM1T10E; SPEX Industries, Edison, NJ). Cells loaded with BCECF were alternately excited with light at the pH-sensitive wavelength of
502 nm (I502 ) and the relatively pH-insensitive wavelength of
440 nm (I440 ). The emission wavelength was 526 nm. The
sample integration time was 1 s, and the sample frequency
was once every 3 s. The fluorescence-excitation ratio (I502/I440 )
was normalized and converted to a pHi value using the
high-K⫹/nigericin technique (38), as modified for a singlepoint calibration (6). Background I502 and I440 signals from
cells without dye were subtracted from total I502 and I440
signals. An evaluation of shrinkage on the calibration of
BCECF in mesangial cells is presented in RESULTS.
After each experiment in which a dye calibration was
performed, the perfusion lines were washed with either
ethanol followed by distilled H2O or albumin followed by
ethanol and then H2O. These washing protocols substantially
reduce nigericin contamination from one experiment to the
next (Bevensee, Bashi, and Boron, unpublished observations). Moreover, because we report the Na⫹/H⫹ exchange
rate as the difference between the total and EIPA-insensitive
acid-extrusion rates, our Na⫹/H⫹ exchanger data should be
immune to any residual nigericin contamination (Bevensee,
Bashi, and Boron, unpublished observations).
For some experiments, we measured pHi in single mesangial cells using an inverted Zeiss IM-35 microscope equipped
for epi-illumination. Our technique for measuring pHi in
single cells is described in detail in Ref. 6.
Calculation of Acid-Extrusion and Acid-Loading Rates
Acid-extrusion and acid-loading rates are defined as the
product of the rate of change in pHi (dpHi/dt) and the total
intracellular buffering power (␤Total ). Because it is difficult to
measure the ratio of volume to surface area in many cells, it
has become customary to report acid-extrusion and acidloading rates in units of moles per liter of cell volume per
second (i.e., µM/s), instead of the classical flux units of moles
per unit area per unit time (i.e., µmol · cm⫺2 · s⫺1 ). We use the
symbol ␸ to refer to these acid-extrusion and acid-loading
rates, or ‘‘pseudofluxes.’’
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
threefold greater at relatively acidic values (pHi ⬇ 6.8)
than at relatively alkaline values (pHi ⬇ 7.6). In this
barnacle study, the authors defined the Na⫹/H⫹ exchange rate as the amiloride-sensitive acid-extrusion
rate in experiments in which they measured pHi with
glass microelectrodes. Also, several groups have presented data consistent with the idea that shrinkage
activates the Na⫹/H⫹ exchanger in mammalian cells by
shifting the flux vs. pHi profile of the exchanger in the
alkaline direction (19, 21, 24, 36, 37).
In the present study, we performed experiments
specifically designed to isolate the pHi dependence of
the Na⫹/H⫹ exchange rate in cultured rat mesangial
cells under both normosmolar and hyperosmolar conditions. We found that exposing cells to a hyperosmolar
solution, which presumably causes cell shrinkage, alkali shifts the pHi dependence of the transporter by
⬃0.7 pH units. This shift explains why cell shrinkage
causes steady-state pHi to increase.
Portions of this work have been published in abstract
form (35).
C675
C676
SHRINKAGE-INDUCED ACTIVATION OF NA⫹/H⫹ EXCHANGE
Calculation of Intrinsic Buffering Power
Statistics
Data are reported as means ⫾ SE. Levels of significance
were assessed using the unpaired Student’s t-test. A P value
⬍ 0.05 was considered significant. The pHi dependencies of ␤I
were fitted by straight lines using a least-squares method.
The dpHi/dt values were fitted by a third-order polynomial
using a least-squares method.
RESULTS
Calibration of BCECF in Mesangial Cells Under
Normosmolar and Hyperosmolar Conditions
For maintaining a stable pHi in nigericin calibration
solutions, doubling the osmolality requires doubling the
extracellular [K⫹]. As described in METHODS, we calibrated the pH-sensitive dye BCECF using the high-K⫹/
nigericin technique. In our first series of experiments,
we evaluated the effect of shrinkage on the calibration
of the dye. If the cells were perfect osmometers, then
increasing the extracellular osmolality from ⬃300 to
600 mosmol/kgH2O should decrease cell volume by
Fig. 1. NH3/NH4⫹ solutions were used to compute the intracellular
pH (pHi ) dependence of the intrinsic buffering power (␤I ) in mesangial cells under normosmolar and hyperosmolar (600 mosmol/kgH2O)
conditions. A: representative experiment on mesangial cells exposed
to a hyperosmolar (600 mosmol/kgH2O) solution. In the nominally
CO2/HCO3⫺-free, HEPES-buffered solution, mesangial cells had an
average pHi of ⬃7.1 before point a. Exposing the cells to the
HEPES-buffered solution supplemented with ⬃300 mM mannitol
elicited an initial decrease in pHi (segment ab), followed by a rapid
increase to a value ⬃0.4 pH units higher (segment bc) than the initial
pHi at the onset of the experiment. Cells were exposed to the
hyperosmolar solution for the remainder of the experiment (segment
ck). Removing external Na⫹ elicited a rapid decrease in pHi (segment
cd). When the cells were exposed to 40 mM NH3/NH4⫹ in the
continued absence of external Na⫹, pHi abruptly increased (segment
de). Subsequently, exposing the cells to 20, 10, 5, 2.5, 1.2, and 0 mM
NH3/NH4⫹ solutions caused stepwise decreases in pHi (segments ef, fg,
gh, hi, ij, and jk, respectively). B: pHi dependence of ␤I for mesangial
cells exposed to normosmolar solutions (s) or hyperosmolar solutions
(r). Each symbol represents the mean of at least 3 data points.
Linear least-squares fits to the norm- and hyperosmolar data have
slopes of ⫺14.4 and ⫺40.8 mM/(pHi unit)2, respectively, and
y-intercepts of 113.6 and 323.6 mM/pHi unit, respectively.
one-half and thereby double the concentration of intracellular constituents (e.g., K⫹ ), assuming no change in
membrane permeability. Accordingly, the dye calibration solutions used for such shrunken cells should have
two times the nominal [K⫹] (210 vs. 105 mM) to satisfy
the calibration requirement that extracellular K⫹ concentration ([K⫹]o ) equals the intracellular K⫹ concentration ([K⫹]i ).
To test this hypothesis, we first calibrated BCECF in
mesangial cells under normosmolar conditions using
our standard 105 mM K⫹/nigericin calibration solution
and then monitored pHi as we shrunk the cells in
hyperosmolar (600 mosmol/kgH2O) calibration solu-
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
In the nominal absence of CO2/HCO3⫺, ␤Total is equivalent to
the intrinsic buffering power of the cell (␤I ). Cell shrinkage
will concentrate intracellular H⫹ buffers and likely increase
␤I. Therefore, we used the approach introduced by Boyarsky
et al. (6) to calculate the pHi dependence of ␤I in mesangial
cells exposed to both norm- and hyperosmolar conditions. ␤I
experiments were performed in the absence of external Na⫹
to eliminate pHi changes caused by Na⫹-dependent processes
(e.g., Na⫹/H⫹ exchange). After initially exposing the cells to a
Na⫹-free solution containing 40 mM NH3/NH4⫹, we recorded
step changes in pHi elicited by progressively lowering extracellular [NH3/NH4⫹]. ␤I is defined as the change in intracellular [NH4⫹] per unit change in pHi.
In the experiment shown in Fig. 1A, mesangial cells in the
nominally CO2/HCO3⫺-free, standard HEPES-buffered solution had a pHi of ⬃7.1 before point a. Exposing the cells to the
HEPES-buffered solution supplemented with ⬃300 mM mannitol (total osmolality ⫽ 600 mosmol/kgH2O) elicited an
initial decrease in pHi (segment ab), followed by a larger
increase to ⬃7.5 (segment bc). These osmolality-induced
changes in pHi are described in more detail in RESULTS. For
the remainder of the experiment shown in Fig. 1A (segment
ck), the cells were exposed to hyperosmolar solutions. Removing external Na⫹ caused pHi to decrease to ⬃6.7 (segment cd).
In the continued absence of external Na⫹, switching the cells
to a solution containing 40 mM NH3/NH4⫹ elicited a rapid
increase in pHi (segment de). Subsequently, exposing the cells
to solutions containing progressively lower concentrations of
NH3/NH4⫹ caused stepwise decreases in pHi (segments ef, fg,
gh, hi, ij, and jk). In some experiments (not shown), the pHi
continued to decrease slowly after the initial step changes.
For these experiments, we back-extrapolated the pHi vs. time
record to determine more accurately the initial pHi changes
elicited by the NH3/NH4⫹ solutions (1, 3, 12).
Using data from experiments similar to that shown in Fig.
1A, we plotted the pHi dependence of ␤I for mesangial cells
under hyperosmolar conditions (Fig. 1B). In other experiments, we used the same NH3/NH4⫹ step technique to determine the pHi dependence of ␤I for mesangial cells under
normosmolar conditions (Fig. 1B). As shown by the best-fit
lines to the data, ␤I in shrunken cells is threefold greater than
in unshrunken cells for pHi values between ⬃6.7 and ⬃7.4.
SHRINKAGE-INDUCED ACTIVATION OF NA⫹/H⫹ EXCHANGE
Fig. 2. Calibration of 28,78-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) at pHi 7 is the same in mesangial cells under
normosmolar conditions (presence of 105 mM external K⫹ ) and
hyperosmolar conditions (presence of 210 mM external K⫹ ). Two
populations of cells were exposed to a nigericin-containing calibration
solution throughout the experiment. Exposing one population of cells
to a hyperosmolar calibration solution (600 mosmol/kgH2O) containing 105 mM K⫹ caused a reversible decrease in pHi of ⬃0.25 (segment
abc). In contrast, exposing the other population of cells to a hyperosmolar calibration solution (600 mosmol/kgH2O) containing 210 mM
K⫹ caused only a very small, reversible decrease in pHi of ⬃0.04
(segment ab8c8).
Fig. 3. Calibration of BCECF in mesangial cells exposed to a
normosmolar calibration solution containing 105 mM K⫹ (s) is the
same as the calibration in cells exposed to a hyperosmolar calibration
solution (600 mosmol/kgH2O) containing 210 mM K⫹ (r). Each
symbol represents the mean of 5 or more data points. Combined
norm- and hyperosmolar data were fitted by the equation: pH ⫽ pK ⫹
log[(Rn ⫺ Rmin )/(Rmax ⫺ Rn )], where pK ⫽ 7.098, Rmax ⫽ 1.949, Rmin ⫽
0.243, and Rn ⫽ normalized ratio.
solution (segment b8c8). In 16 experiments similar to
that shown in Fig. 2, shrinking mesangial cells with a
210 mM K⫹/⬃120 mM mannitol calibration solution
caused pHi to decrease from 7.0 to 6.98 ⫾ 0.01 (P ⫽
0.03). Thus, at a pHi of 7.0, the calibration of BCECF in
mesangial cells shrunken in a 600 mosmol/kgH2O
solution containing 210 mM K⫹/nigericin solution is
equivalent to the calibration of the dye in unshrunken
cells incubated in an ⬃300 mosmol/kgH2O solution
containing 105 mM K⫹/nigericin. In similar experiments on BCECF-loaded rat thymic lymphocytes, Grinstein et al. (19) demonstrated that the fluorescence
ratio of the dye remained constant when the cells were
exposed to nigericin-containing solutions (pH 7.0) of
different osmolarities but a constant [K⫹]-to-[Na⫹]
ratio.
BCECF calibration is identical in shrunken and
unshrunken mesangial cells, provided [K⫹]o is adjusted
to match the predicted [K⫹]i. Using an approach similar
to that shown in Fig. 2, we extended our analysis of the
effect of shrinkage and [K⫹]o on the intracellular calibration of BCECF for pHi values between ⬃6 and ⬃8.2. In
Fig. 3, we plot the normalized fluorescence excitation
ratio for the BCECF vs. pHi relationship for cells
calibrated either under normosmolar conditions (⬃300
mosmol/kgH2O) at a [K⫹]o of 105 mM or under hyperosmolar conditions (600 mosmol/kgH2O) with an external
[K⫹] of 210 mM. The best-fit curves are the result of a
nonlinear least-squares curve fit (see legend for Fig. 3).
The two curves are virtually identical to one another. In
other words, a single BCECF calibration can be used in
experiments in which the mesangial cells undergo
volume changes.
Effect of Shrinkage on Steady-State pHi
Shrinkage elicits an increase in steady-state pHi. We
evaluated the effect of shrinkage on the steady-state
pHi of mesangial cells by exposing them to our standard
HEPES-buffered solution supplemented with ⬃300 mM
mannitol (600 mosmol/kgH2O). Before point a in Fig. 4,
the cells had an average pHi of ⬃7.05. Exposing the
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
tions containing either 105 or 210 mM K⫹. Shrinking
cells in the presence of only 105 mM K⫹ would be
expected to lead to a decrease in pHi because [K⫹]o (i.e.,
105 mM) will be less than the predicted [K⫹]i (i.e., 210
mM), thereby promoting the nigericin-mediated exchange of internal K⫹ for external H⫹. On the other
hand, shrinking the cells in a calibration solution
containing 210 mM K⫹ should have no effect on nigericin-mediated pHi changes because [K⫹]o will match
[K⫹]i.
During the initial portions of the recordings shown in
Fig. 2 (prior to point a), both groups of mesangial cells
were exposed to our standard 105 mM K⫹/nigericin
solution (pH 7.0). Thus, by definition, the pHi before
point a is 7.0. Shrinking the cells in the standard
calibration solution supplemented with ⬃300 mM mannitol elicited a pHi decrease of ⬃0.25 (segment ab). This
acidification was likely caused by a nigericin-mediated
exchange of internal K⫹ for external H⫹, due to the
shrinkage-induced increase of [K⫹]i. As indicated by the
broken line, the decrease in pHi was relatively constant
for ⬎40 min. Returning the cells to the normosmolar
calibration solution caused pHi to increase (segment bc)
to near the initial value at the onset of the experiment
(prior to point a). In 10 experiments similar to that
shown in Fig. 2, shrinking mesangial cells with a 105
mM K⫹/⬃300 mM mannitol calibration solution (pH
7.0) caused pHi to decrease from 7.0 to 6.76 ⫾ 0.01 (P ⬍
0.001). Clearly then, BCECF in shrunken cells cannot
be adequately calibrated using the standard calibration
solution containing 105 mM K⫹.
In contrast to what we observed with the 105 mM
K⫹/⬃300 mM mannitol calibration solution (600 mosmol/kgH2O), shrinking cells in a calibration solution
containing 210 mM K⫹/⬃120 mM mannitol (600 mosmol/kgH2O) elicited only a slight decrease in pHi, ⬃0.04
(segment ab8). This small acidification was reversed by
returning the cells to the normosmolar calibration
C677
C678
SHRINKAGE-INDUCED ACTIVATION OF NA⫹/H⫹ EXCHANGE
cells to a hyperosmolar solution containing ⬃300 mM
mannitol consistently elicited a small, transient decrease in pHi (segment ab), presumably due to the effect
of concentrating and then reequilibrating H⫹ and intracellular buffers. This transient pHi decrease was followed by a robust increase in pHi (segment bc) to a
value ⬃0.7 pH units higher than the initial pHi (prior to
point a). The hyperosmolar-induced pHi increase was
at least partially reversed by returning to the normosmolar solution (segment cd). In a total of 54 experiments similar to that shown in Fig. 4, ⬃300 mM
mannitol caused an increase in pHi from 7.17 ⫾ 0.02 to
7.56 ⫾ 0.03 (P ⬍ 0.0001). The average pHi increase of
⬃0.4 was not dependent on the initial steady-state pHi
before exposing the cells to the hyperosmolar solution
(data not shown). Mesangial cells with an initial steadystate pHi of ⬃7.0 were just as likely to alkalinize to the
same extent as cells with an initial steady-state pHi
of ⬃7.4.
Shrinkage-induced increase in pHi is blocked by
removing external Na⫹ or by applying 50 ␮M EIPA.
Using an inverted microscope equipped for epi-illumination, we also performed experiments on single mesangial cells. Exposing single cells (not shown) to a HEPESbuffered solution supplemented with ⬃300 mM
mannitol (600 mosmol/kgH2O) elicited a series of pHi
changes very similar to those in Fig. 4: a transient
decrease in pHi, followed by a rapid and reversible
increase to a value ⬃0.6 pH units greater, on average,
than the pHi at the onset of the experiments. In the
experiment shown in Fig. 5A on a single mesangial cell,
removing external Na⫹ caused pHi to decrease from
⬃7.2 to ⬃6.7 (segment ab), probably due to reversal of
Na⫹/H⫹ exchange and/or unmasking of background
acid loading. In the continued absence of external Na⫹,
exposing the cell to the HEPES-buffered solution supplemented with ⬃300 mM mannitol did not elicit a pHi
increase (segment cd). In other experiments similar to
that shown in Fig. 5B, exposing single cells to 50 µM
EIPA caused pHi to decrease from ⬃7.15 to ⬃6.75
(segment ab), due to unmasking of background acid
loading that is usually balanced by Na⫹/H⫹ exchange at
Fig. 5. In single mesangial cells, the pHi increase elicited by shrinkage can be inhibited by removing external Na⫹ or by applying 50 µM
ethylisopropylamiloride (EIPA). A: shrinkage in the absence of
external Na⫹. At the onset of the experiment, the cell had a
steady-state pHi of ⬃7.2 (point a). Removing external Na⫹ elicited a
decrease in pHi to ⬃6.7 (segment ab). In the absence of external Na⫹,
applying and removing a HEPES-buffered solution supplemented
with ⬃300 mM mannitol (600 mosmol/kgH2O) had no effect on pHi
(segment cde). B: shrinkage in the presence of EIPA. At the onset of
the experiment, the cell had a steady-state pHi of ⬃7.15 at point a.
Applying 50 µM EIPA elicited a decrease in pHi to ⬃6.75 (segment
ab). In the presence of EIPA, applying and removing a HEPESbuffered solution supplemented with ⬃300 mM mannitol (600 mosmol/
kgH2O) had no effect on pHi (segment bcd).
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
Fig. 4. Shrinkage increases steady-state pHi of mesangial cells. At
the onset of the experiment, cells had an average pHi of ⬃7.05 in the
standard HEPES-buffered solution before point a. Exposing the cells
to the HEPES-buffered solution supplemented with ⬃300 mM mannitol (600 mosmol/kgH2O) elicited a small, transient decrease in pHi
(segment ab), followed by a large sustained increase of ⬃0.7 pH units
(segment bc). Returning the cells to the normosmolar solution caused
the pHi to decrease (segment cd) toward the value at the onset of the
experiment.
steady-state pHi. In the continued presence of EIPA,
exposing the cell to a solution supplemented with ⬃300
mM mannitol did not elicit a pHi increase (segment bc).
In summary, the pHi increase elicited by hyperosmolality can be inhibited by either removing external Na⫹ or
by applying 50 µM EIPA. Therefore, shrinkage appears
to increase the steady-state pHi of mesangial cells by
activating the Na⫹/H⫹ exchanger.
Shrinkage-induced increase in pHi is reduced by
incubating the cells for 14 or 90 min in a Cl⫺-free
medium. As noted in the introduction, Cl⫺ is required
for the shrinkage-induced activation of Na⫹/H⫹ exchange in dog erythrocytes and barnacle muscle fibers.
We therefore tested if the shrinkage-induced pHi increase in mesangial cells could be reduced by exposing
the cells to a Cl⫺-free solution. In Fig. 6, we show the
results from two experiments in which cells were
exposed to the HEPES-buffered solution supplemented
with ⬃300 mM mannitol either in the absence or
presence of external Cl⫺. In both experiments, the
SHRINKAGE-INDUCED ACTIVATION OF NA⫹/H⫹ EXCHANGE
mannitol solution elicited a small decrease in pHi
(segment ab), similar to segment ab in Fig. 4. However,
the subsequent pHi increase was 0.36 pH units greater
in the control cells (segment bc) than in those exposed to
the Cl⫺-free medium for ⬃90 min before the experiment
(segment bc8). In five experiments, we found that the
mean pHi increase elicited by hyperosmolality was
0.09 ⫾ 0.05 in the absence of Cl⫺. In seven, weekmatched control experiments conducted in the presence
of Cl⫺, the pHi increase averaged 0.45 ⫾ 0.07. Thus Cl⫺
removal reduced the hyperosmolar-induced pHi increase by 80%.
that is normally balanced by acid extrusion mediated
by Na⫹/H⫹ exchange.
For the normosmolar experiment, we only show the
record for the recovery of pHi from the acid load
(segment f 8g8) and the decrease in pHi from the subsequent application of EIPA (segment g8h8).
It is important to compare the data from these two
experiments at comparable pHi values, because acidbase transport rates can vary markedly with changes
in pHi. In comparing the segment fg and segment f 8g8
pHi recoveries in shrunken and unshrunken cells,
respectively, we see that there is only a narrow pHi
range (near ⬃7.2) in which the two pHi recoveries
overlap. At this single pHi of 7.2, the pHi recovery rate
is substantially greater in shrunken cells (point f) than
in unshrunken cells (point g8).
Shrinkage alkali shifts the pHi dependence of the
Na⫹/H⫹ exchange rate by ⬃0.7 pH units. From segment
fg and segment f 8g8 pHi trajectories similar to those
shown in Fig. 7, we computed the pHi dependence of
total acid extrusion (␸E ) for both shrunken and unshrunken cells (Fig. 8A). We also used the EIPAunmasked acidifications similar to those in segments gh
and g8h8 in Fig. 7 to obtain the pHi dependence of
EIPA-insensitive acid loading (␸EIPA ) for both shrunken
and unshrunken cells (Fig. 8B). Some of the normosmolar ␸EIPA data points at the lower pHi values in Fig. 8B
were obtained by monitoring the recovery of pHi from
an acid load (analogous to segments fg and f 8g8 in Fig. 7)
in the presence of EIPA (data not shown). In principle,
Effect of Shrinkage on pHi Dependence
of Na⫹/H⫹ Exchange
Shrinkage increases the rate of pHi recovery from an
acid load. To determine the effect of shrinkage on the
pHi dependence of Na⫹/H⫹ exchange in mesangial cells,
we used the approach introduced by Boyarsky et al. (5).
For cells subjected to either normosmolar or hyperosmolar conditions, we determined 1) the pHi dependence of
‘‘total acid extrusion’’ (␸E ) during the pHi recovery from
an acid load and 2) the pHi dependence of ‘‘EIPAinsensitive acid loading’’ (␸EIPA ). We obtained the pHi
dependence of the Na⫹/H⫹ exchange rate by adding
␸EIPA to ␸E for identical pHi values.
The results from two experiments, performed on
unshrunken and shrunken mesangial cells, are shown
in Fig. 7. In both cases, we used the NH4⫹-prepulse
technique to acid load the cells (4) and then monitored
the pHi recovery from the acid load. For the hyperosmolar experiment, cells in the standard HEPES-buffered
solution had an average pHi of ⬃7.3. Shrinking the cells
in the solution supplemented with ⬃300 mM mannitol
elicited the usual transient decrease and rapid increase
in pHi (segment abc). Using the NH4⫹-prepulse technique (4), we then acid loaded the cells (segment cdef).
After the acid load, pHi recovered rapidly (segment fg).
After the pHi recovery was complete, applying 50 µM
EIPA elicited a decrease in pHi (segment gh) that
represents an unmasking of background acid loading
Fig. 7. Shrinkage increases the rate of pHi recovery from an acid load
in mesangial cells. For the normosmolar experiment, we only show
the pHi record after the acid load (segment f 8g8h8). In the hyperosmolar experiment, initially exposing the cells to the standard HEPESbuffered solution supplemented with ⬃300 mM mannitol caused the
expected decrease in pHi (segment ab), followed by a robust increase
(segment bc). Exposing the cells to a solution containing 20 mM NH3/
NH4⫹ elicited a rapid increase in pHi (segment cd), due to NH3
entering the cells and consuming H⫹ to form NH4⫹. Subsequently, the
pHi decreased (segment de) due to passive NH4⫹ influx or transport in
the cells. Removing external NH3/NH4⫹ elicited a rapid decrease in
pHi (segment ef), as the NH4⫹ that had accumulated inside the cells
(during segment de) released H⫹ and exited the cells as NH3. In both
the norm- and hyperosmolar experiments, the decrease in pHi was
followed by a recovery (segments fg and f 8g8). After the recovery,
applying 50 µM EIPA caused the pHi to decline slowly in both
experiments (segments gh and g8h8). Broken line at point d represents
the part of the record in which pHi was beyond the pH-sensitive range
of the dye.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
Fig. 6. The pHi increase elicited by shrinkage can be reduced by
incubating cells in a Cl⫺-free medium for 14 or 90 min. In both
experiments, the cells were exposed to a HEPES-buffered solution
supplemented with ⬃300 mM mannitol (600 mosmol/kgH2O). The
pHi increase elicited by the mannitol solution was 0.36 units greater
in the control cells (segment abc) than in the cells preincubated in a
Cl⫺-free solution for 90 min before the experiment (segment abc8). In
a total of 5 experiments, the cells were preincubated in the Cl⫺-free
solution for either 14 min (n ⫽ 3) or 90 min (n ⫽ 2).
C679
C680
SHRINKAGE-INDUCED ACTIVATION OF NA⫹/H⫹ EXCHANGE
we would have obtained normosmolar ␸EIPA data for pHi
values between 6.9 and 6.6 if either EIPA had caused
pHi to fall below pH 6.9 or if pHi had recovered from an
acid load in the presence of EIPA above pH 6.6. In Fig.
8C, we plot the pHi dependence of the Na⫹/H⫹ exchange
rate (␸Na-H ) for both shrunken cells and unshrunken
cells. We obtained the ␸Na-H plots in Fig. 8C by adding
the respective ␸E and ␸EIPA data sets for norm- and
hyperosmolar conditions. For both shrunken and unshrunken cells, ␸Na-H decreases linearly with increasing
values of pHi. However, the plot of ␸Na-H vs. pHi for the
shrunken cells is alkaline shifted by ⬃0.7 pH units
compared with the same plot for the unshrunken cells.
DISCUSSION
Fig. 8. Shrinkage alkali shifts the pHi dependence of the Na⫹/H⫹
exchange rate by ⬃0.7 pH units. A: pHi dependence of the total
acid-extrusion rate (␸E ) for mesangial cells under normosmolar
conditions (s) and hyperosmolar conditions (r). Data were obtained
from segment fg and segment f 8g8 pHi trajectories in Fig. 7. For A–C,
each symbol represents the mean of 3 or more data points. B: pHi
dependence of EIPA-insensitive acid loading (␸EIPA ) for cells under
normosmolar conditions (k) and hyperosmolar conditions (j). Some
of the data were obtained from segment gh and segment g8h8
acidifications elicited by EIPA in Fig. 7. Some of the data at the lower
pHi values were obtained from pHi recoveries from acid loads in the
presence of EIPA. C: pHi dependence of the Na⫹/H⫹ exchange rate
(␸Na-H ) for cells under normosmolar conditions (n) and hyperosmolar
conditions (l). pHi dependence of ␸Na-H was obtained by adding the
pHi dependence of ␸E (A) and the pHi dependence of ␸EIPA (B) for the
respective osmolar condition.
Shrinkage activates the Na⫹/H⫹ exchanger in mesangial cells. In the present study, we demonstrate that
shrinking mesangial cells in a hyperosmolar solution
containing ⬃300 mM mannitol (600 mosmol/kgH2O)
elicits a sustained increase in steady-state pHi of ⬃0.4
pH units. The increase is caused by stimulation of
Na⫹/H⫹ exchange because it can be inhibited by removing external Na⫹ or by applying 50 µM EIPA. The ⬃0.4
pH unit increase is substantially larger than the maximal ⬃0.15 pH unit increase observed when mesangial
cells are stimulated by the growth factor arginine
vasopressin in the nominal absence of CO2/HCO3⫺ (16).
Therefore, shrinkage is a potent stimulator of Na⫹/H⫹
exchange in mesangial cells. In other experiments in
which we monitored the pHi recovery from acid loads in
the presence or absence of EIPA, the pHi dependence of
␸Na-H is alkali shifted by ⬃0.7 pH units in shrunken vs.
unshrunken mesangial cells. As discussed in the APPENDIX, there is no reason to expect that this ⬃0.7 pH unit
alkali shift should be of the same magnitude as the
shrinkage-induced increase in steady-state pHi.
Results from previous studies are consistent with
shrinkage alkali shifting the pHi dependence of ␸Na-H.
As mentioned in the Introduction, several groups have
provided evidence consistent with the idea that cell
shrinkage alkali shifts the pHi dependence of ␸Na-H in
mammalian cells. For example, in acid-loaded human
lymphocytes, shrinkage alkali shifted the Na⫹-dependent acid extrusion vs. pHi relationship by 0.2–0.3 pH
units (19). However, the Na⫹-dependent acid-extrusion
rate includes the contributions of all Na⫹-dependent
acid-base transporters, including the nigericin used to
acid load the cells (Bevensee, Bashi, and Boron, unpublished observations and Ref. 34). In Na⫹-depleted C6 rat
glioma cells, shrinkage alkali shifted the 22Na⫹ flux vs.
pHi relationship by 0.3–0.4 pH units (24). However, the
total, unidirectional 22Na⫹ influx includes the fluxes of
all Na⫹ transport processes and cannot distinguish
Na⫹/Na⫹ exchange from Na⫹/H⫹ exchange. In another
study on acid-loaded rabbit alveolar macrophages,
shrinkage alkali shifted the bafilomycin-resistant acid
extrusion vs. pHi relationship by ⬃0.2 pH units (21).
However, the bafilomycin-resistant acid-extrusion rate
represents the net effect of all acid-loading and acid-
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
Na⫹/H⫹ Exchange in Shrunken Cells
SHRINKAGE-INDUCED ACTIVATION OF NA⫹/H⫹ EXCHANGE
Issues to Consider When Measuring Changes
in Acid-Extrusion and Acid-Loading Rates
Elicited by Changes in Cell Volume
Shrinking mesangial cells does not alter the BCECF
calibration curve. Because shrinkage will increase [K⫹]i,
we calibrated the intracellular dye by exposing the cells
to an extracellular solution with a [K⫹]o chosen to
match the predicted, shrinkage-increased [K⫹]i. We
found that simultaneously doubling both osmolality
(from ⬃300 to 600 mosmol/kgH2O) and [K⫹]o (from 105
to 210 mM) had little effect on the pHi of cells already
clamped with nigericin to 7.0 (segment ab8 in Fig. 2).
Thus the doubling of osmolality must have doubled
[K⫹]i, so that we can conclude that the cells behave as
near-perfect osmometers.
At least under the conditions of our calibration
experiments (i.e., neither Na⫹ nor CO2/HCO3⫺ were
present), shrunken mesangial cells did not exhibit
volume recoveries for at least 40 min after cell shrinkage. The evidence is that pHi failed to increase1 during
segment ab in Fig. 2. On the other hand, cell volume
may have recovered during our other experiments (e.g.,
conducted in the presence of Na⫹ ). If so, then our
postexperiment pHi calibrations would have been erroneously high because [K⫹]i would have been lower than
we thought. However, the size of this error is expected
to be very small because the likely degree of volume
recovery is very low. For example, when we exposed
cells to ⬃300 mM mannitol in Fig. 4, Na⫹/H⫹ exchange
caused pHi to increase by ⬃0.7. This alkalinization
represents the extrusion of ⬃14 mM H⫹ (and thus the
uptake of 14 mM Na⫹ ), assuming a buffering power of
20 mM (Fig. 1). The complete recovery of cell volume
would have required the uptake of ⬃300 mM Na⫹. Thus
the computed uptake of 14 mM represents less
1 If the volume had increased, [K⫹] should have fallen, and
i
nigericin should have mediated an influx of K⫹ and an efflux of H⫹
(i.e., produced a pHi increase).
than a 5% volume recovery. A 5% volume recovery
would lead to a 5% error in the estimated [H⫹]i, which
translates to an overestimation of 0.02 pH units. Thus
it is unlikely that volume recovery seriously affected
either our calibration or its application to physiological
experiments.
In extending our analysis to other pHi values, we
deduced that the calibration of intracellular BCECF is
the same in unshrunken and shrunken cells (600
mosmol/kgH2O) provided that the appropriate [K⫹]o is
used. Therefore, at least in mesangial cells, the calibration of intracellular BCECF is insensitive to cellvolume changes per se. Apparently, the pH sensitivity
of intracellular BCECF in mesangial cells is unaffected
by shrinkage-induced increases in ionic strength and
concentrations of intracellular constituents (excluding
K⫹ ).
Shrinking mesangial cells increases ␤I. We had anticipated that a doubling of osmolality, and a subsequent
halving of cell volume, would have doubled the intracellular concentrations of proton buffers and thus have
increased ␤I by twofold. However, over the pHi range of
6.7–7.4, the apparent ␤I was threefold greater in
shrunken (600 mosmol/kgH2O) vs. unshrunken cells.
One possible explanation for the larger-than-expected
increase in ␤I is cytosolic crowding of proton buffers.
Macromolecular crowding can cause changes in proteinprotein interactions that are disproportionately larger
than the increase in protein concentration (30). If
macromolecular crowding were to cause the pK values
of buffers to shift closer to the physiological pH, the
effect would be an increase in buffering power. Regardless of the mechanism for the increase in ␤I, given
identical pHi recovery rates, the calculated acidextrusion rate will be threefold greater in shrunken vs.
unshrunken mesangial cells. Our ␤I results are at least
qualitatively consistent with those previously reported
in barnacle muscle fibers (14) and rat C6 glioma cells
(37).
Fig. 9. Magnitude of the steady-state pHi increase elicited by an
alkali shift of the pHi dependence of an acid extruder (JE ) will depend
on the pHi dependence of the acid loader (JL ). If the plot of JE vs. pHi
is alkali shifted by 0.7 pH units (JE1 = JE2 ), then steady-state pHi
will 1) increase by 0.7 pH units if the plot of JL vs. pHi is horizontal
(JL2 ), 2) not change if the plot of JL vs. pHi is vertical (JL3 ), or 3)
increase by ⬍0.7 pH units if the plot of JL vs. pHi slopes upward (JL1 ).
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
extruding processes other than the vacuolar H⫹ pump.
Finally, in acid-loaded rat mandibular salivary glands,
shrinkage alkali shifted the total acid-extrusion rate vs.
pHi relationship by 0.15 pH units (36). However, the total
acid-extrusion rate represents the algebraically summed
contributions of all acid-loading and acid-extruding mechanisms. Thus, although the above four studies show that
shrinkage causes an alkali shift in the pHi dependence of
some combination of acid-base parameters, the studies
were not specifically designed to isolate the pHi dependence of ␸Na-H from the pHi dependence of other acidextrusion and acid-loading mechanisms.
Evidence has been presented that cell shrinkage
alkali shifts the pHi dependence of ␸Na-H in rat C6
glioma cells (37). However, the activity of the Na⫹/H⫹
exchanger at alkaline pHi values was underestimated
because there was no compensation for background
acid loading unmasked by amiloride at the high pHi
values reached during cell shrinkage.
C681
C682
SHRINKAGE-INDUCED ACTIVATION OF NA⫹/H⫹ EXCHANGE
2
Imagine that we shrink a cell to one-half its initial volume and
that this shrinkage causes both ␤I and surface-to-volume ratio (S/V)
to double, but has no effect on Na⫹/H⫹ exchange per se. If we now acid
load the cell, the rate of pHi recovery from this acid load will be the
same as that under control conditions because the doubling of ␤I will
tend to cut dpHi/dt in half, whereas the doubling of S/V will tend to
double dpHi/dt. If we report the Na⫹/H⫹ exchange rate in terms of ␸ ⫽
dpHi/dt ⫻ ␤I, we will see that ␸ will double because dpHi/dt is
unchanged and ␤I is doubled. On the other hand, if we report the
Na⫹/H⫹ exchange rate in terms of J ⫽ dpHi/dt ⫻ ␤I/(S/V), we will see
J will be unchanged because the doubling of ␤I is canceled by the
doubling of S/V.
ship, without substantially changing its slope. A ␸-J
effect could affect the magnitude of the transport
parameter at any given pHi but could not produce such
a shift.
In conclusion, shrinkage of mesangial cells leads to a
substantial elevation in steady-state pHi by stimulating the Na⫹/H⫹ exchanger. The pHi increase requires
external Na⫹ and can be inhibited by EIPA. The kinetic
basis of the Na⫹/H⫹ exchange stimulation is a large
alkali shift in the ␸Na-H vs. pHi relationship.
APPENDIX
Relationship Between a Shift in the pHi Dependence of an
Acid-Base Transporter and the Resultant Change in
Steady-State pHi
pHi is controlled by the sum of fluxes or other processes
that extrude acid from the cell (JE ) and those that load acid
into the cell (JL ). As shown in Fig. 9, JE tends to decrease with
increasing pHi, whereas JL tends to increase. Imagine that a
mesangial cell has only one acid loader (e.g., H⫹ influx) and
one acid extruder (e.g., Na⫹/H⫹ exchange). JE1 describes the
kinetics of the Na⫹/H⫹ exchanger under control conditions,
and JL1 describes the kinetics of acid loading. The steadystate pHi of the cell is 7.1, determined by the point where
JE1 ⫽ JL1 ( point a). We now switch to a hyperosmolar
solution, which alkali shifts the pHi dependence of the
Na⫹/H⫹ exchanger by ⬃0.7 pH units (JE1 = JE2 ). Will this 0.7
pH unit alkali shift of JE elicit a 0.7 pH unit increase in
steady-state pHi? In the unlikely event that the plot of JL vs.
pHi is horizontal (JL2 ), then JE1 = JE2 will indeed increase
steady-state pHi by 0.7 pH units, from 7.1 to 7.8 (point a = b).
However, in the equally unlikely event that the plot of JL vs.
pHi is vertical (JL3 ), then JE1 = JE2 will not increase
steady-state pHi at all (point a = c). In all likelihood, the plot
of JL vs. pHi will slope upward, as indicated by JL1 in Fig. 9.
Therefore, the shrinkage stimulus that elicits a 0.7 pH unit
alkali shift of JE will result in a smaller shift of the steadystate pHi (point a = d). Thus the steeper the slope of JL, the
smaller the change in steady-state pHi. Similarly, the steeper
the slope of JE, the smaller the effect on steady-state pHi of
shifting the pHi profile of an acid loader.
If the cell has multiple acid extruders, then, all things
being equal, an alkali shift of the Na⫹/H⫹ exchanger will elicit
an even smaller alkali shift in steady-state pHi, inasmuch as
the Na⫹/H⫹ exchanger makes a smaller contribution to the
overall acid-extrusion rate. One should generally expect the
shift in steady-state pHi to be smaller than the instigating
shift in the pHi profile of either an acid loader or acid
extruder.
We thank Dr. Bruce A. Davis, Emilia M. Hogan, and Dr. Bernhard
M. Schmitt for evaluating the manuscript and providing useful
comments.
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Program Project Grant PO1DK17433.
Address for reprint requests: M. O. Bevensee, Cellular and Molecular Physiology, Yale Univ. School of Medicine, 333 Cedar St., Rm.
B-127 SHM, New Haven, CT 06520 (E-mail: mbevense@biomed.
med.yale.edu).
Received 1 September 1998; accepted in final form 15 December
1998.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
Shrinkage can lead to overestimates in calculating
acid-extrusion and acid-loading rates. One can report
acid-base transport rates either in units of moles per
surface area per time (J) or in units of moles per volume
per time (␸). Shrunken cells have less volume but
presumably the same plasma membrane surface area
as unshrunken cells. Therefore, shrinkage will have
different effects on acid-base transport rates reported
in terms of J vs. ␸.
As discussed in METHODS, it has become customary to
report acid-base fluxes in terms of ␸ for small mammalian cells in which it is difficult to compute the volumeto-surface ratio. In these pseudoflux units, the acidbase transport rate would be higher for the smaller of
two cells, even though the J values for the two cells
would be identical. Ideally, one would correct for cell
volume changes when reporting acid-base fluxes in
terms of ␸. However, it is extremely difficult to measure
volume changes accurately in most small cells. In at
least one example though, Seo et al. (36) used proton
nuclear magnetic resonance spectroscopy to measure
cell-volume changes and then corrected acid-extrusion
rates in perfused rat mandibular salivary glands under
hyperosmolar conditions.
According to the above discussion, acid-extrusion and
acid-loading rates reported in terms of ␸, as in the
present study, should be greater in shrunken (600
mosmol/kgH2O) vs. unshrunken (300 mosmol/kgH2O)
mesangial cells, even if shrinkage had no effect on
acid-base transport per se (i.e., even if shrinkage had no
effect on J). Thus the ␸-J effect would predict that
mesangial cells shrunken to one-half their initial volume would display a doubling of their acid-extrusion
and acid-loading rates when expressed in terms of ␸ but
no change when expressed in terms of J. This doubling
arises because of the effect of shrinkage on surface-tovolume ratio2; any effects on ␤I are automatically
incorporated in the calculated ␸. However, we found
that shrinkage produced a 5- to 12-fold increase in
␸Na-H, 2.5- to 6-fold greater than the doubling expected
because of the ␸-J effect. Thus we conclude that shrinkage increases the Na⫹/H⫹ exchange rate by a factor of
at least 2.5–6.
Two other arguments support our conclusion that
shrinkage does indeed stimulate Na⫹/H⫹ exchange,
independent of a ␸-J effect. First, shrinkage leads to an
increase in steady-state pHi that is blocked by removing Na⫹ or applying EIPA. Second, shrinkage produces
a substantial alkali shift of the ␸Na-H vs. pHi relation-
SHRINKAGE-INDUCED ACTIVATION OF NA⫹/H⫹ EXCHANGE
REFERENCES
21. Heming, T. A., and A. Bidani. Na⫹-H⫹ exchange in resident
alveolar macrophages: activation by osmotic cell shrinkage. J.
Leukoc. Biol. 57: 609–616, 1995.
22. Hoffman, E. K., and L. O. Simonsen. Membrane mechanisms
in volume and pH regulation in vertebrate cells. Physiol. Rev. 69:
315–382, 1989.
23. Hogan, E. M., B. A. Davis, and W. F. Boron. Intracellular Cl⫺
dependence of Na-H exchange in barnacle muscle fibers under
normotonic and hypertonic conditions. J. Gen. Physiol. 110:
629–639, 1997.
24. Jean, T., C. Frelin, P. Vigne, and M. Lazdunski. The Na/H
exchange system in glial cell lines. Eur. J. Biochem. 160: 211–
219, 1986.
25. Jennings, M. L., and R. K. Schulz. Swelling-activated KCl
cotransport in rabbit red cells: flux is determined mainly by cell
volume rather than shape. Am. J. Physiol. 259 (Cell Physiol. 28):
C960–C967, 1990.
26. Kaplan, D., and W. F. Boron. Long-term expression of c-H-ras
stimulates Na-H and Na⫹-dependent Cl-HCO3 exchange in
NIH-3T3 fibroblasts. J. Biol. Chem. 269: 4116–4124, 1994.
27. Knoblauch, C., M. H. Montrose, and H. Murer. Regulatory
volume decrease by cultured renal cells. Am. J. Physiol. 256 (Cell
Physiol. 25): C252–C259, 1989.
28. Lang, F., G. L. Busch, H. Völkl, and D. Häussinger. Cell
volume: a second message in regulation of cell function. News
Physiol. Sci. 10: 18–22, 1995.
29. Lovett, D. H., J. L. Ryan, and R. B. Sterzel. Stimulation of rat
mesangial cell proliferation by macrophage interleukin-1. J.
Immunol. 131: 2830–2836, 1983.
30. Minton, A. P. Influence of macromolecular crowding on intracellular association reactions: possible role in volume regulation. In:
Molecular and Cellular Physiology of Cell Volume Regulation,
edited by K. Strange. Boca Raton, FL: CRC, 1994, p. 181–190.
31. Parker, J. C. Volume-responsive sodium movements in dog red
blood cells. Am. J. Physiol. 244 (Cell Physiol. 13): C324–C330,
1983.
32. Parker, J. C. Interactions of lithium and protons with the
sodium-proton exchanger of dog red blood cells. J. Gen. Physiol.
87: 189–200, 1986.
33. Parker, J. C., and V. Castranova. Volume-responsive sodium
and proton movements in dog red blood cells. J. Gen. Physiol. 84:
379–401, 1984.
34. Richmond, P. H., and R. D. Vaughan-Jones. Assessment of
evidence for K⫹-H⫹ exchange in isolated type-1 cells of neonatal
rat carotid body. Pflügers Arch. 434: 429–437, 1997.
35. Schlue, W.-R., G. Boyarsky, and W. F. Boron. Intracellular pH
regulation in hypertonic solutions by single glomerular mesangial cells. Renal Physiol. Biochem. 13: 175–176, 1990.
36. Seo, J. T., J. B. Larcombe-McDouall, R. M. Case, and M. C.
Steward. Modulation of Na⫹-H⫹ exchange by altered cell volume in perfused rat mandibular salivary gland. J. Physiol.
(Lond.) 487: 185–195, 1995.
37. Shrode, L. D., J. D. Klein, P. B. Douglas, W. C. O’Neill, and
R. W. Putnam. Shrinkage-induced activation of Na⫹/H⫹ exchange: role of cell density and myosin light chain phosphorylation. Am. J. Physiol. 272 (Cell Physiol. 41): C1968–C1979, 1997.
38. Thomas, J. A., R. N. Buchsbaum, A. Zimniak, and E.
Racker. Intracellular pH measurements in Ehrlich ascites
tumor cells utilizing spectroscopic probes generated in situ.
Biochemistry 18: 2210–2218, 1979.
39. Welling, P. A., and R. G. O’Neil. Cell swelling activates
basolateral membrane Cl and K conductances in rabbit proximal
tubule. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27):
F951–F962, 1990.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
1. Aickin, C. C., and R. C. Thomas. Microelectrode measurement
of the intracellular pH and buffering power of mouse soleus
muscle fibres. J. Physiol. (Lond.) 267: 791–810, 1977.
2. Banderali, U., and G. Roy. Activation of K⫹ and Cl⫺ channels
in MDCK cells during volume regulation in hypotonic media. J.
Membr. Biol. 126: 219–234, 1992.
3. Bevensee, M. O., R. A. Weed, and W. F. Boron. Intracellular
pH regulation in cultured astrocytes from rat hippocampus. I.
Role of HCO3⫺. J. Gen. Physiol. 110: 453–465, 1997.
4. Boron, W. F., and P. De Weer. Intracellular pH transients in
squid giant axons caused by CO2, NH3, and metabolic inhibitors.
J. Gen. Physiol. 67: 91–112, 1976.
5. Boyarsky, G., M. B. Ganz, E. J. Cragoe, Jr., and W. F. Boron.
Intracellular pH dependence of Na-H exchange and acid loading
in quiescent and arginine vasopressin-activated mesangial cells.
Proc. Natl. Acad. Sci. USA 87: 5921–5924, 1990.
6. Boyarsky, G., M. B. Ganz, B. Sterzel, and W. F. Boron. pH
regulation in single glomerular mesangial cells. I. Acid extrusion
in absence and presence of HCO3⫺. Am. J. Physiol. 255 (Cell
Physiol. 24): C844–C856, 1988.
8. Burg, M. B. Molecular basis of osmotic regulation. Am. J.
Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F983–F996,
1995.
9. Cala, P. M. Volume regulation by red blood cells. Mechanisms of
ion transport. Mol. Physiol. 4: 33–52, 1983.
10. Cala, P. M. Volume-sensitive alkali metal-H transport in Amphiuma red blood cells. In: Na-H Exchange, Intracellular pH and
Cell Function, edited by P. M. Aronson. and W. F. Boron. Orlando,
FL: Academic, 1986, p. 79–99.
11. Chaillet, J. R., A. G. Lopes, and W. F. Boron. Basolateral
Na-H exchange in the rabbit cortical collecting tubule. J. Gen.
Physiol. 86: 795–812, 1985.
12. Chen, L. K., and W. F. Boron. Acid extrusion in S3 segment of
rabbit proximal tubule. I. Effect of bilateral CO2/HCO3⫺. Am. J.
Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F179–F192,
1995.
13. Davis, B. A., E. M. Hogan, and W. F. Boron. Role of G proteins
in stimulation of Na-H exchange by cell shrinkage. Am. J.
Physiol. 262 (Cell Physiol. 31): C533–C536, 1992.
14. Davis, B. A., E. M. Hogan, and W. F. Boron. Shrinkageinduced activation of Na⫹-H⫹ exchange in barnacle muscle
fibers. Am. J. Physiol. 266 (Cell Physiol. 35): C1744–C1753,
1994.
15. Dunham, P. B., and J. C. Ellory. Passive potassium transport
in low potassium sheep red cells: dependence upon cell volume
and chloride. J. Physiol. (Lond.) 318: 511–530, 1981.
16. Ganz, M. B., G. Boyarsky, W. F. Boron, and R. B. Sterzel.
Effects of angiotensin II and vasopressin on intracellular pH of
glomerular mesangial cells. Am. J. Physiol. 254 (Renal Fluid
Electrolyte Physiol. 23): F787–F794, 1988.
17. Grinstein, S., C. A. Clarke, and A. Rothstein. Activation of
Na⫹/H⫹ exchange in lymphocytes by osmotically induced volume
changes and by cytoplasmic acidification. J. Gen. Physiol. 82:
619–638, 1983.
18. Grinstein, S., and S. J. Dixon. Ion transport, membrane
potential, and cytoplasmic pH in lymphocytes: changes during
activation. Physiol. Rev. 69: 417–481, 1989.
19. Grinstein, S., A. Rothstein, and S. Cohen. Mechanism of
osmotic activation of Na⫹/H⫹ exchange in rat thymic lymphocytes. J. Gen. Physiol. 85: 765–787, 1985.
20. Hallows, K. R., and P. A. Knauf. Principles of cell volume
regulation. In: Cellular and Molecular Physiology of Cell Volume
Regulation, edited by K. Strange. Boca Raton, FL: CRC, 1994, p.
3–29.
C683