1 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520; 2 Institut für Neurobiologie, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany; and 3 Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77550
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ABSTRACT |
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Using the
pH-sensitive dye
2',7'-bis(2-carboxyethyl)-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 shrinkage-induced 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.
acid-base transport; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; chloride; intracellular pH; volume regulation
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INTRODUCTION |
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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 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 shrinkage-induced
activation of the transporter is approximately 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).
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METHODS |
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Solutions
All experiments were performed in the nominal absence of CO2/HCOCell 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 single-point 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 (Calculation of Intrinsic Buffering Power
In the nominal absence of CO2/HCOIn 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/NH+4 elicited a rapid increase in pHi
(segment de). Subsequently, exposing the cells to solutions containing progressively lower concentrations of
NH3/NH+4
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/NH+4
solutions (1, 3, 12).
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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/NH+4
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.
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 ![]() |
RESULTS |
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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 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 solutions 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+.
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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.
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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 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 steady-state 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.
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Shrinkage-induced increase in pHi is
blocked by removing external
Na+ or by
applying 50 µM EIPA.
Using an inverted microscope equipped for epi-illumination, we also
performed experiments on single mesangial cells. Exposing single cells
(not shown) to a HEPES-buffered 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 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.
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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 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
bc'). 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, week-matched
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%.
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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
"EIPA-insensitive acid loading"
(
EIPA). We obtained the
pHi dependence of the
Na+/H+
exchange rate by adding
EIPA to
E for identical
pHi values.
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Shrinkage alkali shifts the pHi
dependence of the
Na+/H+
exchange rate by ~0.7 pH units.
From segment fg and segment
f'g'
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 EIPA-unmasked acidifications similar to those in
segments gh and
g'h'
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'g'
in Fig. 7) in the presence of EIPA (data not shown). In principle, 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.
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DISCUSSION |
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Na+/H+ Exchange in Shrunken Cells
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-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
acid-extrusion and acid-loading mechanisms.
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 ab' 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 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 cell-volume 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
protein-protein 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
acid-extrusion 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).
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APPENDIX |
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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 steady-state 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
|
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.
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ACKNOWLEDGEMENTS |
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We thank Dr. Bruce A. Davis, Emilia M. Hogan, and Dr. Bernhard M. Schmitt for evaluating the manuscript and providing useful comments.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Program Project Grant PO1DK-17433.
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.
1 If the volume had increased, [K+]i should have fallen, and nigericin should have mediated an influx of K+ and an efflux of H+ (i.e., produced a pHi increase).
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.
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{at}biomed.med.yale.edu).
Received 1 September 1998; accepted in final form 15 December 1998.
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