From the Department of Surgery, The Toronto Hospital
and University of Toronto, Toronto, Ontario M5G 1L7, Canada and the
¶ Department of Physiology and Laboratory of Cellular and
Molecular Physiology, Semmelweis University of Medicine, Budapest 8, P.O. Box 259, H-1444 Budapest, Hungary
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ABSTRACT |
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The signaling pathways by which cell volume
regulates ion transporters, e.g.
Na+/H+ exchangers (NHEs), and affects
cytoskeletal organization are poorly understood. We have previously
shown that shrinkage induces tyrosine phosphorylation in CHO cells,
predominantly in an 85-kDa band. To identify volume-sensitive kinases
and their substrates, we investigated the effect of hypertonicity on
members of the Src kinase family. Hyperosmolarity stimulated Fyn and
inhibited Src. Fyn activation was also observed in
nystatin-permeabilized cells, where shrinkage cannot induce
intracellular alkalinization. In contrast, osmotic inhibition of Src
was prevented by permeabilization or by inhibiting NHE-1. PP1, a
selective Src family inhibitor, strongly reduced the
hypertonicity-induced tyrosine phosphorylation. We identified one of
the major targets of the osmotic stress-elicited phosphorylation as
cortactin, an 85-kDa actin-binding protein and well known Src family
substrate. Cortactin phosphorylation was triggered by shrinkage and not
by changes in osmolarity or pHi and was abrogated by PP1.
Hyperosmotic cortactin phosphorylation was reduced in Fyn-deficient
fibroblasts but remained intact in Src-deficient fibroblasts. To
address the potential role of the Src family in the osmotic regulation
of NHEs, we used PP1. The drug affected neither the hyperosmotic
stimulation of NHE-1 nor the inhibition of NHE-3. Thus, members of the
Src family are volume-sensitive enzymes that may participate in the
shrinkage-related reorganization of the cytoskeleton but are probably
not responsible for the osmotic regulation of NHE.
The maintenance of normal cell volume is an essential homeostatic
function. Most cells are equipped with a variety of volume-sensitive membrane transporters (e.g. isoforms of the
Na+/H+ exchanger,
Na+/K+/Cl Little is known about the volume-dependent signaling
mechanisms and their relationship to the different effector systems
such as the ion carriers or the cytoskeleton. Evidence has been
accumulating that protein-tyrosine kinases may play a pivotal role in
the signaling of both hypo- (5, 10, 11) and hyperosmotic shock
(12-14). Our recent studies using
CHO1 cells (12) as well as
that of Krump et al. on neutrophils (13) have shown that
hypertonicity induces robust tyrosine phosphorylation in various
protein bands. While the phosphorylation pattern differed in the two
cell types studied, the trigger for the phosphorylation of most
proteins was cell shrinkage and not an increase in osmolarity or in
intracellular ion concentrations. In CHO cells, hypertonicity induced
phosphorylation of proteins of ~40, 85, and 110-130 kDa, with the
most prominent response occurring in the ~85-kDa band (p85). While
the ~40-kDa protein proved to be extracellular signal-regulated kinase-2, the identity of p85 and the other higher molecular weight proteins remains to be elucidated. Further, the kinase pathways responsible for these reactions are unknown. Some of the
tonicity-sensitive proteins complexed with Src homology 2 (SH2) and SH3
domains, raising the possibility that the Src family of tyrosine
kinases might be potential mediators of the osmotically induced
phosphorylations. This notion is further strengthened by the finding
that, in neutrophils, hypertonicity altered the activity of Fgr, Hck,
and Lyn (13), members of the Src family expressed specifically in cells
of hematopoietic origin. Furthermore, pharmacological data fostered the
concept that the hypertonicity-stimulated tyrosine phosphorylation
might be causally connected to the osmotic regulation of
Na+/H+ exchange. Specifically, the effect of
hypertonicity on two osmotically sensitive, but oppositely regulated,
isoforms of the Na+/H+ exchanger (15-19) was
abrogated by broad spectrum tyrosine kinase inhibitors; the
hyperosmotic activation of Na+/H+ exchanger-1
(NHE-1) (in neutrophils) was inhibited by genistein (13), whereas the
hyperosmotic inhibition of NHE-3 (in kidney cells) was prevented by
genistein and herbimycin (20). Based on these observations, Krump
et al. (13) suggested that the hypertonic activation of
NHE-1 in neutrophils may be due to the stimulation of certain Src kinases.
To date, no information has been available about the osmotic
responsiveness of the two most widely expressed, ubiquitous Src family
members, p60src and p59fyn. In addition, none of the
major osmosensitive phosphoproteins has been identified. The aim of
this study was to gain further insight into the mechanisms of
volume-dependent signaling by investigating the potential
role of the Src family in the hypertonicity-induced tyrosine
phosphorylation and the subsequent changes in ion transport. Specifically, we intended to investigate whether Src and Fyn can be
regulated by a decrease in cell volume and whether any of the major
phosphoproteins can be identified as a Src family substrate. We also
wished to discern whether the Src family may play an essential role in
the osmotic regulation of NHE-1 or NHE-3. Our results show that members
of the Src family are regulated by cell shrinkage, and one of the major
targets for volume-dependent tyrosine phosphorylation is
the Src family substrate, actin cross-linking protein, cortactin. On
the other hand, the osmotic regulation of NHE-1 and -3 does not appear
to be mediated by Src-like kinases.
Materials--
Me2SO, nystatin (used from a stock
solution of 400,000 units/ml in Me2SO, freshly prepared
before each experiment), nigericin, monensin,
N-methyl-D-glucammonium, and gluconic acid
lactone were purchased from Sigma. Proteinase inhibitor mixture
containing 0.8 mg/ml benzamidine HCl, 0.5 mg/ml aprotinin, 0.5 mg/ml
leupeptin, 0.5 mg/ml pepstatin A, and 50 mM
phenylmethylsulfonyl fluoride in pure ethanol was from PharMingen,
Protein G-Sepharose beads from Amersham Pharmacia Biotech,
2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF)/acetoxymethylester and PP1 were from Calbiochem. Monoclonal
anti-phosphotyrosine (4G10), anti-cortactin, anti-p60src, the
Src assay kit including an Src family-specific substrate peptide, and
the polyclonal anti-Fyn were obtained from Upstate Biotechnology, Inc.
(Lake Placid, NY). Monoclonal anti-Fyn was purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA), and anti-phospho-p38 was from New
England Biolabs. Peroxidase-conjugated anti-mouse and anti-rabbit IgG,
the Enhanced Chemiluminescence kit, and [ Media--
Bicarbonate-free RPMI 1640 was buffered with 25 mM Hepes to pH 7.4 (osmolarity 290 ± 5 mosM). The Iso-Na medium consisted of 140 mM
NaCl, 3 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM glucose, 20 mM Hepes (pH 7.4). When required, Iso-Na was made
hypertonic (Hyper-Na, 600 mosM) by the addition of 300 mM sucrose. The Iso- and Hyper-K media had the same
composition, except NaCl was replaced by KCl. The permeabilization
medium contained 140 mM KCl, 10 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 0.193 mM CaCl2 (100 nM free
Ca2+), 5 mM glucose, 10 mM Hepes
(pH 7.2). To permeabilize the cells, this medium was supplemented with
400 units/ml nystatin and 60 mM sucrose. Sucrose was
included to counterbalance the intracellular colloidosmotic pressure
and thereby prevent swelling of the permeabilized cells, as reported
earlier by us (12). To induce shrinkage, the nystatin-containing
permeabilization buffer was supplemented with 350 mM
sucrose. The Iso-NMG medium was composed of 140 mM NMG, 140 mM gluconic acid lactone, 3 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 0.193 mM CaCl2 (100 nM free
Ca2+), 5 mM glucose, 10 mM Hepes
(pH 7.2). The osmolarity of the isotonic solutions was adjusted to
290 ± 5 mosM with the major salt. Osmolarity was
checked with an Osmette osmometer.
Cell Culture--
For most studies, we used a CHO cell line
(AP-1) devoid of the endogenous NHE and stably transfected with the rat
NHE-1 (referred to as NHE-1 cells) or NHE-3 (referred to as NHE-3
cells), as described previously (15). These cells were grown in
WT, Fyn
Human neutrophils were prepared from healthy volunteers as described
previously (22) except that lysis of red blood cells was carried out
using NH4Cl. Prior to use, cells (106/ml) were
kept in Hepes-buffered RPMI medium at 37 °C.
Preparation of Cell Extracts--
Confluent cultures were
incubated for at least 3 h in serum- and
HCO3 Western Blotting and Immunoprecipitation--
Lysates containing
equivalent amount of protein were either mixed with an equal amount of
2× Laemmli buffer (whole cell lysates) or were clarified by
centrifugation at 12,000 × g for 10 min and further
processed for immunoprecipitation. Extracts were precleared for 1 h using 40 µl of 50% suspension of Protein G-Sepharose beads and
then incubated with the corresponding antibodies (see details in the
figure legends to Figs. 2-6) for 1 h. Immunocomplexes were captured using 40 µl of protein G-Sepharose, and the beads were washed four times with lysis buffer. Immunoprecipitated proteins were
diluted with Laemmli sample buffer, boiled for 5 min, and subjected to
electrophoresis on 10% SDS-polyacrylamide gels. The separated proteins
were transferred to nitrocellulose using a Bio-Rad Mini Protean II
apparatus. To check the effectiveness of transfer and similarity of
protein amount, lanes were visualized by staining with Ponceau S. Blots
were blocked in Tris-buffered saline containing 5% bovine serum
albumin and then incubated with the primary antibody. The binding of
the antibody was visualized by peroxidase-coupled secondary anti-mouse
or rabbit antibody (1:3000 dilution) using the enhanced
chemiluminescence method.
Densitometry--
Quantification of the bands was performed
using a Bio-Rad GS-690 imaging densitometer, and evaluation of data was
carried out with the Molecular Analyst computer program (12).
In Vitro Kinase Assays--
The activity of Src and Fyn was
determined by immunocomplex kinase assays (23, 24). Cell lysates
obtained from iso- or hypertonically treated cells and containing equal
amounts of protein (280-500 µg) were subjected to
immunoprecipitation (see above), and the precipitates were washed with
kinase buffer (20 mM Hepes, 10 mM
MnCl2, 0.25 mM Na3VO4,
pH 7.1). Kinase activity was measured as the phosphorylation of either
the Src family-specific substrate peptide Cdc2-(6-20) or enolase. In
the former case, the Upstate Biotechnology Src kinase assay kit was
used according to the manufacturer's instructions. Briefly, the
immunocomplexes were incubated with 20 µl of reaction buffer (100 mM Tris-HCl, pH 7.2, 125 mM MgCl2 25 mM MnCl2, 2 mM EGTA, 0.25 mM Na3VO4, 2 mM
dithiothreitol), 10 µl of substrate peptide (0.6 mM stock
in H2O), and the reaction was initiated by the addition of
10 µl of manganese/ATP mixture (0.5 mM ATP, 75 mM MnCl2 in reaction buffer) containing 10 µCi [ Measurement and Manipulation of Cytosolic pH--
pHi
was measured fluorometrically using the indicator dye BCECF,
essentially as described (15). Confluent cultures of NHE-1 or NHE-3
cells grown on glass coverslips were loaded with 1 µM
BCECF/acetoxymethylester for 10 min in Iso-Na medium. Ratio fluorometry
was performed on small populations of cells (6-12 cells/measurement)
using an illumination system from Photon Technologies, Inc., in the
dual excitation (495 ± 10 nm/445 ± 10 nm) single emission
(530 ± 30 nm) configuration. Excitation light was reflected to
the cell by a 510-nm dichroic mirror, and emitted light was selected by
a 520-nm longpass filter. Cells were visualized with a Nikon Diaphot
TMD microscope and a Hoffman modulation contrast video system through a
CCD video camera connected to a Panasonic monitor. Dye-loaded NHE-1
cells were preincubated with or without PP1, and then each coverslip
was mounted to form the bottom of a thermostatted, perfusable Leydig
chamber into which 0.5 ml of the same medium was added, and the basal
fluorescence was recorded. The medium was then rapidly exchanged (by
the addition of 10 × 0.5 ml in less than 15 s) to a
hypertonic medium with or without the drug. To follow the osmotic
effects on NHE-3 cells, these cells were first acidified by the
ammonium prepulse technique. After 10 min of dye loading, in the
presence or absence of PP1, cells were washed and then incubated in
Iso- or Hyper-Na medium, supplemented with 20 mM
NH4Cl, and supplemented, where indicated, with PP1 again.
Thereafter, cells were washed with Iso- or Hyper-K medium and placed
under the microscope in the same medium. Recovery of pHi was
initiated with Iso- or Hyper-Na, again in the absence or presence of
the Src inhibitor. After each measurement, fluorescence was calibrated
in terms of pHi by sequential perfusion of the chamber using
nigericin-containing Iso-K medium (or for hypertonic samples Iso-K
supplemented with 150 mM extra KCl), at various pH values
between 6 and 8 with 0.5 pH unit increments. Analysis of the data was
carried out using Felix® software. In neutrophils,
pHi was monitored in cell suspension (0.5 × 106/ml) using a Beckman fluorimeter (22).
Other Methods--
Protein concentration was determined by the
BCA assay (Pierce) using bovine serum albumin as a standard. Data are
presented as representative immunoblots of at least three similar
experiments or as mean ± S.E. of the number of experiments
indicated (n).
The Effect of the Src Kinase Inhibitor, PP1, on
Hypertonicity-induced Tyrosine Phosphorylation--
To assess whether
Src kinases might play a role in the hypertonicity-induced tyrosine
phosphorylation observed in CHO cells, we used PP1, a newly developed,
selective pyrazolo pyrimidine-type inhibitor of this enzyme family (25,
26). Fig. 1A shows that osmotic shock evoked strong tyrosine phosphorylation of several proteins, the predominant response occurring in 80-85- and
110-130-kDa bands, as reported earlier by us (12). PP1 caused a
concentration-dependent inhibition of the
hypertonicity-triggered tyrosine phosphorylation in most bands.
Half-maximal inhibition of p85 phosphorylation was obtained at ~2
µM, whereas 10 µM completely abolished
phosphotyrosine accumulation in this band (Fig. 1B). This
concentration dependence corresponds to the reported in vivo
PP1 sensitivity of Src kinases (25, 26). The drug strongly reduced
tyrosine phosphorylation in the 110-130-kDa region as well, but a part
of the response was persistent even at concentrations as high as
100 µM. By contrast, PP1 (up to 100 µM)
failed to affect the hypertonicity-elicited tyrosine phosphorylation of
p38 stress kinase (Fig. 1C). These findings indicate that
the tonicity-related tyrosine phosphorylations are carried out by at
least two pharmacologically distinguishable signaling pathways, one of
which is PP1-inhibitable, and thus potentially Src
family-dependent, whereas the other is mediated by Src
family-independent mechanisms.
Cell Shrinkage Induces the Tyrosine Phosphorylation of the Src
Family Substrate, Cortactin--
Given the PP1 sensitivity of p85
phosphorylation, we asked whether this band could be identified as a
known Src family substrate. Cortactin, an 80/85-kDa actin cross-linking
protein (27), is a preferred Src substrate, which has been reported to
become intensively tyrosine-phosphorylated in fibroblasts
overexpressing the oncogenic v-Src (28) or the mutationally activated
Fyn (29) as well as in cells lacking Csk, a kinase that inactivates the
Src family (30). Cortactin phosphorylation has also been observed in
normal cells exposed to various stimuli known to activate c-Src or Fyn (31-35).
To address whether hypertonicity induces cortactin phosphorylation,
this protein was immunoprecipitated from lysates of iso- or
hypertonically treated CHO cells, and the immunoprecipitates were
probed with antiphosphotyrosine. Fig.
2A shows that under isotonic
conditions cortactin was not phosphorylated, while marked phosphotyrosine accumulation was detected in cortactin obtained from
hypertonically challenged cells. In CHO cells, both the higher and the
lower molecular weight forms of the protein (i.e. 80 and 85 kDa) became tyrosine phosphorylated. This finding was highly reproducible, occurring in all experiments performed (n = 15). Probing the same blots with an anti-cortactin antibody confirmed that similar amount of cortactin was precipitated from iso- and hypertonic samples. Treatment of the cells with PP1 before and during
the hypertonic stimulation prevented the osmotic shock-induced cortactin phosphorylation, consistent with the involvement of Src-like
kinases.
Hypertonic stimulation of NHE-1-expressing CHO cells causes cytosolic
alkalinization due to osmotic activation of the antiporter (Ref. 15;
see below). Since changes in pHi have been shown to regulate
nonreceptor tyrosine kinases (36), we tested whether cytoplasmic
alkalinization might play a role in hypertonicity-provoked cortactin
phosphorylation. To this end, we used NHE-3-expressing CHO cells in
which hypertonicity does not elicit alkalinization. As shown on Fig.
2B, osmotic challenge triggered an equally strong tyrosine
phosphorylation of cortactin in these cells as well. To further
substantiate that cortactin phosphorylation was caused by cell
shrinkage and not by an increase in osmolarity or a rise in major ionic
constituents of the cytosol, we used an experimental system in which
the decrease in cell volume occurred under iso-osmotic conditions.
Specifically, isotonic shrinkage was achieved by changing the Iso-Na
incubation medium to an isotonic solution composed of impermeant
organic ions, N-methyl-D-glucammonium, and
gluconate (Iso-NMG). As previously reported (12), the cells shrink in this environment due to the concentration gradient-driven efflux of
intracellular ions (mostly K+ and Cl
To assess what portion of the 85-kDa osmosensitive band can be
identified as cortactin, we immunodepleted hypertonic cell lysates with
the anti-cortactin antibody and compared the level of reduction in the
amount of cortactin and in the tyrosine-phosphorylated p85 band. The
addition of 3-µg antibody and protein G-Sepharose beads to the
Triton-soluble cell lysate (400 µg of protein) resulted in a 59 ± 3% decrease in the cortactin content of the sample
(n = 3). Probing of the same blots with
antiphosphotyrosine revealed that the anti-cortactin antibody caused a
34 ± 9% reduction in the p85 band as well, while it did not
affect the intensity of other bands (not shown). Thus, more than half
of the p85 band is composed of phosphocortactin. These studies
therefore show that the Src family substrate cortactin is one of the
major proteins undergoing tyrosine phosphorylation following osmotic
shrinkage of CHO cells.
The Effect of Hypertonicity on the in Vitro Activity of
p60src and p59fyn--
Both our
pharmacological data and the identification of cortactin as a
phosphorylated substrate supported the notion that the Src family is
involved in the mediation of the osmotically induced tyrosine
phosphorylation. In further experiments, we investigated whether
hypertonic treatment could in fact cause detectable changes in the
activity of ubiquitously expressed Src kinases. Covalent modification
(i.e. phosphorylation and dephosphorylation at inhibitory or
activating tyrosine residues) of these kinases represents an important
mechanism by which their activity is regulated (37). Changes in
activity brought about by this type of control can be detected in
vitro. To discern whether hyperosmotic shock may affect members of
the Src family in this manner, we performed in vitro kinase
assays on Src and Fyn immunoprecipitates obtained from iso- and
hypertonically treated cells. Fig. 3
shows that hypertonic exposure of the cells (10 min, 600 mosM) caused a significant decrease (50%) in
p60src activity. This response was not dependent on the Src
substrate applied, since it was readily detectable using either a Src
family-specific peptide (Fig. 3A) or enolase (Fig.
3B). Various stimuli, including thrombin (38), growth
factors (39), and stretch (40) were shown to induce translocation of
Src to the cytoskeleton or complex formation between the kinase and
other molecules. These events may decrease the availability of Src by
the specific antibody. However, we could not detect any significant
difference in the total amount of Src protein immunoprecipitated from
iso- or hypertonic samples (Fig. 3B), and no change was
observed in the amount of Src associated with the Triton
X-100-insoluble (cytoskeletal) extracts obtained after iso- or
hypertonic treatment (Fig. 3D). These findings show that the
decreased activity was due to reduced specific activity of
the kinase. Moreover, the time course of the effect showed that the
inhibition was rapid, clearly detectable after 1 min, arguing against
the possibility that transient activation was followed by a
longer lasting inhibition (Fig. 3C).
In subsequent experiments, we tested whether the hypertonicity-induced
Src inhibition is caused by a decrease in cell volume per se or is due
to other concomitantly changing parameters, such as osmolarity, pH, or
other intracellular ion concentrations. Shrinkage activates NHE-1,
causing a sizable cytosolic alkalinization (
Fyn kinase has also been implicated in the phosphorylation of cortactin
(29, 34). To test whether hypertonicity might differentially regulate
this kinase, its activity was also determined in immunoprecipitates.
Data shown in Fig. 5 summarizes the
effect of hypertonicity on Fyn. 10-min hypertonic treatment caused an approximately 2.9- or 1.7-fold increase in Fyn activity, using the Src
family-specific peptide or enolase as substrate, respectively (Fig. 5,
A and B). PP1 (0.5 µM) added to the
kinase reaction mixture reduced Fyn activity by 90% (not shown).
Hyperosmotic shock elicited an increase in the in vitro
autophosphorylating activity of the kinase as well (Fig.
5C). To test whether the rise in Fyn activity might be
associated with a hypertonicity-induced change in the tyrosine
phosphorylation of this kinase itself, we probed Fyn immunoprecipitates
with anti-phosphotyrosine. The level of tyrosine phosphorylation was
significantly higher in Fyn obtained from the hypertonic than from the
isotonic samples, while no change was detected in the amount of
precipitated protein (Fig. 5D). This phenomenon was present
in NHE-3 cells as well, suggesting that the Fyn tyrosine
phosphorylation was not due to shrinkage-induced alkalinization (not
shown). To verify that Fyn was in fact stimulated by hypertonicity
through a decrease in cell volume, we used nystatin-permeabilized cells. As demonstrated on Fig. 5E, under these conditions
cell shrinkage led to similar stimulation of the kinase as observed in
non-permeabilized cells (Fig. 5, B and E). Taken
together, a decrease in cell volume stimulates the auto- and
heterokinase activity of Fyn, suggesting that this enzyme might be
involved in the signal transduction of osmotic stress.
Comparison of Hypertonicity-induced Cortactin Tyrosine
Phosphorylation in Wild Type, Src
In order to further investigate the potential contribution of Src and
Fyn in the in situ phosphorylation of cortactin, we utilized
mouse fibroblast lines derived from wild type (WT) or Src- or
Fyn-deficient animals (21). To verify the absence of the respective
kinases and to assess whether a compensatory increase occurred in the
expressed Src family member, we immunoprecipitated Src and Fyn from
each cell line (Fig. 6A). As
expected, the WT cells expressed both proteins, whereas the products of
the disrupted genes were entirely missing from the corresponding cell
lines. Interestingly, we found that the Src The Effect of PP1 on the Hyperosmotic Stimulation of NHE-1 and
Inhibition of NHE-3--
Having shown that the Src kinases are
responsive to changes in cell volume, we asked whether shrinkage can
regulate NHE-1 and NHE-3 in CHO cells by activating members of the Src
family. To address this question, we measured the osmotic responses of these antiporters in control and PP1-treated cells loaded with the
fluorescent pH indicator BCECF. In NHE-1 cells (Fig.
7, A and B),
hypertonicity caused a sizable alkalinization ( Cortactin Tyrosine Phosphorylation: Possible Mechanisms and
Significance--
An important finding of the present work is the
identification of the cortical actin-binding protein, cortactin as one
of the major targets of the hyperosmotic shock-induced tyrosine
phosphorylation in CHO cells. We provide evidence that cortactin
phosphorylation is due to a decrease in cell volume and is independent
of the hypertonicity-provoked changes in pHi or in the
concentration of major cytoplasmic ionic constituents. Our observation
that the Src family inhibitor, PP1, abolishes the hypertonicity-induced phosphorylation of many proteins, including cortactin, suggests that
this kinase family plays an important role in the shrinkage-related protein phosphorylation in general and in cortactin phosphorylation in
particular. Specifically, Fyn kinase appears to be one of the enzymes
responsible for the osmotic stress-induced cortactin phosphorylation. This notion is supported by our findings that (a)
hypertonicity stimulates Fyn and (b) the osmotically induced
cortactin phosphorylation is substantially mitigated in Fyn-deficient
cells. The presence of Fyn, however, is not an absolute requirement,
implying the involvement of other kinases. Another tyrosine kinase that
has been suggested to directly phosphorylate cortactin in
erythroleukemia cells is Syk (47). Interestingly, this enzyme can also
be stimulated by hyperosmotic stress (48). However, it is unlikely that
Syk is involved in cortactin phosphorylation in CHO cells because, in
agreement with earlier findings (49), we could not detect any
immunoreactive Syk in CHO cells, and the Syk inhibitor piceatannol was
not effective in preventing the hypertonicity-provoked cortactin phosphorylation (data not shown). On the other hand, we propose that
the hyperosmotic stimulation of Syk in blood cells can be due to Src
family-mediated Syk phosphorylation. In favor of this notion, Src has
been shown to phosphorylate Syk (50), the hyperosmolarity-induced Syk
phosphorylation is not due to autophosphorylation (51), and in
neutrophils the hypertonic Syk phosphorylation can be prevented by
PP1.2 Another candidate is
FER kinase, which was recently suggested to be involved in growth
factor-induced cortactin phosphorylation (52). Future studies should
define whether FER is also a volume-sensitive enzyme that may
participate in the phenomenon. Direct kinase activation, however, may
not be the only mechanism that accounts for the phosphorylation. Cortactin has recently been shown to translocate to the plasma membrane
after growth factor stimulation (53). Our preliminary data suggest that
cell shrinkage may also induce cortactin redistribution to the cell
periphery (not shown). Such relocalization could facilitate phosphorylation by targeting the molecule to the vicinity of
membrane-associated Src kinases.
Cortactin is thought to be involved in the organization of the cortical
actin skeleton, in the regulation of cell-cell contact, cell motility,
and tumor invasiveness (54). While the role of its tyrosine
phosphorylation has not been entirely clarified, certain cytoskeletal
changes have been associated with it. In Src-transformed cells,
cortactin is primarily localized in podosomes, i.e. abnormal
contact sites between the membrane and the substratum (28). In
vitro phosphorylation of cortactin by Src caused a strong
down-regulation of its actin cross-linking activity (55). In keeping
with this, Csk-deficient cells, in which cortactin is
hyperphosphorylated, have a reduced number of stress fibers (30). These
changes are reminiscent of the alterations in the cytoskeleton observed
during hyperosmotic stress. For example, in yeast, osmotic shock
induces the rapid disassembly of the actin cables, followed by a
reassembly in the cortical region (6). This process is similar to the
cycle of stress fiber disassembly/reassembly observed in fibroblasts,
after the stimulation of Src (56). Since actin-cross-linking proteins
of the cortical skeleton were found to be necessary for protection
against osmotic shock in Dictyostelium (57), it is tempting
to speculate that dynamic reorganization of cortactin (perhaps its
redistribution from the stress fibers to the membrane skeleton) may
also serve a similar role. Our future studies are directed to
characterize the shrinkage-induced alteration of the cytoskeleton in
mammalian cells and to assess the role of cortactin in this process.
The Participation of Src Kinases in Stress Signaling and Volume
Regulation--
Members of the Src family have been reported to
participate in the signaling of oxidative (58) and UV-induced cellular
stress (59). This study demonstrates that the two most widely expressed members of the family, Src and Fyn, are responsive to osmotic stress as
well. The involvement of the individual members of the family in the
signaling of oxidative and osmotic stress is selective. For example, in
the same Src-deficient fibroblasts, where the oxidative shock-induced
phosphorylation of big mitogen-activated protein kinase is completely
abolished (58), the osmotic shock-promoted cortactin phosphorylation
remains intact. We found that in CHO cells, hypertonicity decreased Src
and increased Fyn activity. The most plausible explanation for this
differential behavior is that hypertonicity not only alters cell volume
but also activates NHE-1, and the consequent alkalinization may
selectively modify Src activity. This notion is supported by our
findings that (a) the presence of a functional NHE-1 is
required for the hypertonic inhibition of Src and (b)
cytosolic alkalinization inhibits, whereas acidification stimulates,
Src (see also Refs. 23 and 41) even under iso-osmotic conditions. These
observations raise the possibility that NHE-1 may be involved in the
regulation of Src. It must be emphasized, however, that the
NHE-mediated pHi change may not be the only mechanism, and the
contribution of other factors such as a rise in intracellular
Na+ is also possible. When only the volume can change, both
Src and Fyn are stimulated by hypertonicity, although the effect on Src is very modest. In neutrophils, Hck and Fgr were stimulated, whereas Lyn was inhibited, by hypertonicity (13). Further studies should clarify whether the opposite behavior can also be ascribed to the
combined pHi and volume effects.
The emerging picture is that cell shrinkage and swelling affects
specific Src kinases, which can modify various volume-regulating ion
transport processes. Swelling has been recently reported to stimulate
Lck, which in turn phosphorylates and activates chloride channels
leading to regulatory volume decrease (60). In erythrocytes, the
absence of Hck and Fgr augmented the activity of potassium/chloride cotransport, a swelling-stimulatable pathway (61). Rather surprisingly, however, the osmotic regulation of a major volume-regulatory
transporter, NHE-1, does not seem to be mediated by Src kinases. The
hypothesis correlating hypertonicity-induced, presumably Src
family-mediated tyrosine phosphorylation and the activation of NHE-1
was derived from observations made in neutrophils (13). Since these
cells express a number of hematopoietic cell-specific Src members, it was conceivable that in neutrophils certain Src kinases might play an
important role in hypertonic NHE-1 activation. However, we regard this
possibility as unlikely, because PP1, which had a dramatic inhibitory
effect on the hypertonic tyrosine phosphorylation also in neutrophils,
failed to affect the hypertonicity-triggered alkalinization in this
cell type as well (data not shown). These observations suggest that the
vast majority of the hypertonicity-induced tyrosine phosphorylation and
NHE-1 activation are parallel but not causally related processes.
While Src has been shown to control the expression of NHE-3 (41),
apparently it does not participate in the acute osmotic regulation of
this transporter either. The role of two other osmosensitive kinase
pathways (p38 and MEK/extracellular signal-regulated kinase) was also
ruled out on pharmacological grounds (62). Since inhibitor studies
suggest the involvement of tyrosine phosphorylation in the osmotic
control of the antiporters, other kinases must be considered. A
potential candidate is the Jak/Stat pathway which, was recently found
to be stimulated by osmotic stress (63).
The mechanism by which hypertonicity affects Src kinases or the NHE
isoforms is unknown. Shrinkage was shown to induce clustering of cell
surface receptors (64), and this process may turn on many signaling
pathways. It is conceivable that common upstream events lead to the
activation of Src type kinases and other kinases and initiate signaling
toward NHE. Such common factor might be the potential osmotic
activation of small G-proteins. It is noteworthy that Rac and Cdc42
were suggested to play a role in the osmotic shock-induced activation
of stress kinases (65), and their activation by growth factors was
shown to induce membrane translocation of cortactin (53). Furthermore,
Rho has been implicated in NHE regulation (66) and was found to
stimulate myosin light chain phosphorylation (67), a reaction that
seems to be necessary for the osmotic activation of NHE-1 (68). Future
studies should test the intriguing possibility that small G proteins
may be upstream mediators of osmotic shock.
In summary, the present studies show that ubiquitous Src kinases are
regulated by cell volume, that cortactin is a major target of
hypertonicity-induced phosphorylation and that Fyn kinase is involved
in this reaction. The Src family may represent an important link
between cell volume and the organization of the cytoskeleton, whereas
other kinase pathways convey the message to the
Na+/H+ exchanger.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
cotransporter,
K+ and Cl
channels) that can effectively
restore normal cell size after a perturbation caused either by exposure
to an aniso-osmotic environment or by metabolic changes (for reviews,
see Refs. 1 and 2). Alterations in the cell volume are also known to
induce reorganization in the actin skeleton (3-6) and to modulate gene
transcription (7-9). Moreover, the cellular hydration state is not
only a subtly regulated parameter but is also an important regulatory
signal (1). For example, hormones such as glucagon and insulin elicit volume changes which act as "second messengers" necessary for their
cellular effects.
EXPERIMENTAL PROCEDURES
-32P]ATP
(3000 Ci/mmol) were from Amersham Pharmacia Biotech.
-minimal essential medium, containing 25 mM
NaHCO3 and supplemented with 10% fetal calf serum, and 1%
antibiotic suspension (penicillin and streptomycin; Sigma) under a
humidified atmosphere of air/CO2 (19:1) at 37 °C. To
eliminate potential revertants and to maintain the high expression level of the NHE isoforms, cells were selected after every third passage for the Na+/H+
exchange-dependent survival of an acute acid load (15).
/
, Src
/
fibroblasts were isolated from mouse embryos
that were homozygous for disruption in Src or Fyn
gene and were immortalized with large T antigen (21). Cells were kindly provided by Sheila M. Thomas (Fred Hutchinson Cancer Center, Seattle, WA) and were maintained in Dulbecco's modified Eagle's medium. All
other conditions and treatments were similar to those in CHO cells.
-free RPMI 1640 prior to
experiments. Cells were preincubated in Iso-Na medium for 10 min and
then subjected to various treatments as indicated. The medium was then
aspirated, and the cells were vigorously scraped into ice-cold
Triton-containing or modified radioimmune precipitation buffers,
supplemented with 1 mM Na3VO4 and 20 µl/ml
protease inhibitor mixture. The Triton lysis buffer contained 100 mM NaCl, 30 mM Hepes, 20 mM NaF, 1 mM EGTA, 1% Triton X-100, pH 7.5, and the radioimmune
precipitation buffer was composed of 150 mM NaCl, 50 mM Tris-HCl, 1 mM NaF, 2 mM
Tris-EGTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, pH 7.4.
-32P]ATP/sample. After 10 min at 30 °C, 20 µl of trichloroacetic acid was added, and 25 µl of the mixture was
layered on P81 phosphocellulose squares. After extensive washing with
0.85% phosphoric acid, radioactivity bound to the filters was
determined by scintillation counting. Nonspecific binding of
radioactivity to the filters was determined in each experiment by
measuring the activity of samples to which 10 µl of H2O
was added instead of the peptide. The low activity measured in these
samples was subtracted as background. Experiments were repeated at
least three times, and the results were normalized to the amount of
protein content of the initial cell lysate. When enolase was used, the
immunocomplexes were incubated with 8 µl of kinase buffer, 12 µl of
acid-denatured enolase (1.75 µg of enolase/sample), and 10 µl of
ATP mixture (kinase buffer supplemented with 3 µM K-ATP
and 10 µCi of [
-32P]ATP/sample). After 5 or 10 min
at 30 °C for Src and Fyn, respectively, the reaction was terminated
by the addition of 10 µl of 4× Laemmli buffer, and the samples were
boiled and subjected to SDS-polyacrylamide gel electrophoresis. The
gels were dried and used for direct quantification of radioactivity
with a Molecular Dynamics PhosphorImager using ImageQuant software. The
gels were also subjected to radiography with an intensifying screen.
Each experiment was performed at least three times, and each time
duplicates or triplicates were measured. Results are expressed as -fold
increase compared with the controls.
RESULTS
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Fig. 1.
Effect of the Src family inhibitor PP1 on
hypertonicity-induced tyrosine phosphorylation. A, CHO
cells were preincubated for 10 min in Iso-Na medium in the absence or
presence of varying concentrations of PP1, as indicated. The medium was
then replaced either by Iso-Na (I) or by Hyper-Na
(H; Iso-Na plus 300 mM sucrose) supplemented
with the same concentration of PP1 for an additional 10 min. At the end
of the incubation period, cells were lysed with ice-cold Triton lysis
buffer, and the protein content of the samples was determined. After
mixing with an equal amount of 2× Laemmli buffer, the various samples
containing equal amounts of protein were subjected to electrophoresis,
transferred to nitrocellulose, and probed with a monoclonal
anti-phosphotyrosine antibody using enhanced chemiluminescence.
B, concentration dependence of the effect of PP1 on the
hyperosmolarity-induced tyrosine phosphorylation of p85. The intensity
of the 85-kDa band was quantified by densitometry and expressed as
-fold increase compared with the isotonic sample. Data are means ± S.E. of three separate experiments. C,
hyperosmolarity-induced tyrosine phosphorylation of p38 is not
inhibited by PP1. Cells were treated as in A with the
indicated concentrations of PP1. Whole cell lysates were obtained and
probed with a phosphospecific anti-p38 antibody.
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Fig. 2.
Cell shrinkage induces tyrosine
phosphorylation of cortactin. A and B, after
preincubation of cells expressing NHE-1 (A) or NHE-3
(B) for 10 min in Iso-Na, the medium was changed for either
Iso-Na or Hyper-Na (HYP) for 10 min. Where indicated, 10 µM PP1 was present throughout the experiment (HYP + PP1). Cells were then lysed with radioimmune precipitation buffer,
and cortactin was immunoprecipitated from extracts containing equal
amounts of protein (280 µg) as described under "Experimental
Procedures." Immunoprecipitated molecules were separated, blotted
onto nitrocellulose, and probed with anti-phosphotyrosine. To test the
effectiveness of the immunoprecipitation, the same blots were stripped
and reprobed with anti-cortactin. C, NHE-1 cells were
preincubated in Iso-Na, and then the medium was changed to either
Iso-Na or Iso-NMG or Iso-NMG supplemented with 400 units/ml nystatin,
as indicated. After 10 min, cells were lysed, and cortactin was
precipitated and analyzed as above.
),
followed by osmotically obliged water. This process is further facilitated by the addition of nystatin, an ionophore that selectively permeabilizes the membrane for small monovalent ions but does not allow
the permeation of large organic ions. Fig. 2C demonstrates that isotonic shrinkage led to the marked tyrosine phosphorylation of
cortactin. Together, these findings indicate that cell shrinkage is
sufficient to elicit cortactin phosphorylation, independent of
hypertonicity or the type of osmolytes used to achieve it.
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Fig. 3.
The effect of hyperosmolarity on the activity
of p60src kinase. NHE-1 cells were treated with
isotonic solutions or challenged with hypertonicity for 10 min
(A and B) or for the indicated times
(C). Thereafter, cells were lysed with radioimmune
precipitation (A) or Triton buffer (B and
C) and subjected to immunoprecipitation using a monoclonal
anti-Src antibody. Immunocomplex kinase assays were performed using
[ -32P]ATP and either a peptide fragment of Cdc2
(A) or enolase (B and C) as substrate
(see "Experimental Procedures" for details). The peptide was
separated by phosphocellulose filters, and the incorporated activity
was measured by scintillation counting (n = 4, reflecting the Src activity of cell lysates containing 120 µg of
protein). When enolase was used, the samples were subjected to
SDS-polyacrylamide gel electrophoresis followed by radiography, and
phosphorylation was quantified by a PhosphorImager. No radioactive
bands were detected if the primary antibody or enolase was omitted from
the reaction (not shown). Data are expressed as percentage change,
compared with the isotonic activity (100%). Where error
bars are indicated, the results are mean ± S.E. for
3-6 independent determinations. The amount of Src in the
immunoprecipitates (B) and Triton X-100-insoluble
(Tx) fractions (D) obtained from iso- and
hypertonically treated cells were determined by immunoblotting with
anti-Src. In four repeated experiments, no significant difference was
observed between the iso- and hypertonic samples.
0.4 pH unit; see below)
in cells expressing this isoform. Since acidification of the cytoplasm
was reported to stimulate Src in kidney cells (23, 41), it was
conceivable that an NHE-1-mediated cytosolic alkalinization might
contribute to the inhibition of the kinase. To test this assumption,
the effect of hypertonicity was investigated in the presence of HOE
694, a very potent and specific inhibitor of NHE-1. Fig.
4A shows that HOE 694 alone had no significant effect (caused a minimal inhibition) on Src activity. The drug, however, had a strong influence on the
hypertonicity-induced Src response; in HOE-treated cells, the osmotic
challenge not only failed to inhibit Src, but it caused an
approximately 50% increase in its activity. Moreover,
hypertonic exposure of NHE-3 cells did not decrease but rather
moderately increased the activity of precipitated Src (data not shown).
These findings are consistent with a role for NHE-1 in participating in
the hypertonicity-induced inhibition of Src, presumably through the
ensuing cytoplasmic alkalinization. In keeping with this notion, in the
absence of functional NHE-1, shrinkage is known to induce metabolic
acidification (15), which might stimulate Src (23). To address whether
changes in pHi can in fact regulate Src activity in CHO cells, we manipulated pHi under iso-osmotic conditions using ionophores. Intracellular alkalinization was achieved by the
Na+/H+ exchange ionophore monensin, whereas
acidification was induced by the K+/H+
exchanger nigericin. As determined in parallel fluorimetric
experiments, a 2.5-min treatment of the cells with monensin or
nigericin caused an approximately 0.3-unit rise or 0.6-unit drop in the
pHi, respectively (not shown). As shown in Fig. 4B,
monensin caused a significant decrease (~40%), whereas nigericin
induced a substantial increase (~60%) in Src activity. Thus, Src
activity appears to be sensitive to pHi changes in both
directions, and this phenomenon is likely to contribute to the
hypertonic inhibition of the kinase. To test whether Src can be
regulated by the volume change itself, we used nystatin-permeabilized
cells where shrinkage can be induced without any accompanying change in
pHi and in other intracellular monovalent ion concentrations.
For these experiments, cells were kept in a KCl-based
(intracellular-like) medium, in the presence of nystatin. To prevent
swelling of the permeabilized cells (in which the colloidosmotic
pressure of the intracellular proteins is not counterbalanced) 60 mM sucrose was also included in the medium. As shown
earlier, this procedure ensures the maintenance of the resting cell
volume (12). Shrinkage was induced by the addition of an extra 300 mM sucrose. Fig. 4B shows that in the
permeabilized cells a decrease in the volume did not reduce Src
activity, but instead it led to a modest yet reproducible stimulation
of the kinase. Taken together, these findings suggest that
hypertonicity may influence Src activity by a complex mechanism both
through changes in pHi and in the cell volume. However, the
overall effect of hypertonicity on Src under conditions where increased
protein tyrosine phosphorylation and cortactin phosphorylation are
readily detectable is inhibitory.
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Fig. 4.
The involvement of NHE-1-mediated
intracellular alkalinization in the inhibition of Src kinase.
A, NHE-1 cells were pretreated with Iso-Na for 10 min, and
(where indicated) the medium was supplemented with 15 µM
of the NHE inhibitor HOE 694 for the last minute of the preincubation.
Subsequently, the medium was exchanged to Iso- or Hyper-Na, with or
without the drug, and after 2.5 min the cells were lysed with Triton
buffer. In vitro kinase assays and the quantification of
enolase phosphorylation (n = 3) were carried out as in
Fig. 3. B, after a 10-min pretreatment with Iso-Na, the
medium was replaced with Iso-Na without any ionophore
(Control) or with Iso-Na supplemented with either 10 µg/ml
monensin (Alkalinization (mon)) or 10 µg/ml nigericin
(Acidification (nig)). The cells were lysed after 2.5 min,
and the lysates were processed for the Src assay (n = 3). C, after a short pretreatment in Iso-Na, cells were
briefly washed with the permeabilization medium and then were
permeabilized under isovolemic conditions using the same solution
supplemented with 400 units/ml nystatin and 60 mM sucrose.
After 7 min, the medium was aspirated and replaced either by the same
medium (Isovolemic) or by the permeabilization buffer containing an
extra 300 mM sucrose (Shrunken). 10 min later, the cells
were lysed, and the samples were processed for the immunocomplex Src
kinase assays (n = 3).
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Fig. 5.
Hypertonicity activates
p59fyn. Cells were treated with iso- or
hypertonic solutions for 10 min or for the indicated times and lysed.
Fyn was immunoprecipitated from the extracts with a monoclonal
antibody, and its activity was determined essentially as described for
Src under Fig. 4. A, the Cdc2 peptide was used as substrate
(n = 4, reflecting the Fyn activity of cell lysates
containing 270 µg of protein), whereas in B, C,
and E, enolase was applied (for B and
E, n = 5). D, Fyn
immunoprecipitates obtained from iso- and hypertonic samples were
subjected to electrophoresis, blotted onto nitrocellulose, and probed
with anti-phosphotyrosine (top). The same blot was stripped
and reprobed with anti-Fyn (bottom). E, cells
were permeabilized as detailed in the legend to Fig. 4 and were treated
either with the permeabilization buffer ensuring isovolemia
(I) or with this buffer supplemented with 300 mM
extra sucrose, inducing shrinkage (S). After 10 min, cells
were lysed and processed for the Fyn activity determination.
/
, and Fyn
/
Fibroblasts--
Several different mechanisms were reported to
participate in the increased phosphorylation of various Src substrates.
Besides elevated kinase activity, these include the recruitment of the kinase and the substrate in the same compartment (37, 42) and the
noncovalent modification of the substrate, which increases the affinity
of the kinase toward it (43, 44). For example, the Src-mediated
phosphorylation of gelsolin and other actin-binding proteins was
dramatically augmented by phosphatidylinositol 4,5-bisphosphate and
related molecules (43). Interestingly, hypertonicity was shown to
increase the level of inositol phosphates (45), and cortactin contains
a phosphatidylinositol 4,5-bisphosphate binding site (46). Thus, the
decreased in vitro Src activity does not rule out the
potential involvement of this kinase in cortactin phosphorylation.
Moreover, while the hypertonic activation of Fyn is certainly
consistent with a role of this kinase in cortactin phosphorylation, it
does not indicate that Fyn is the only enzyme mediating this reaction.
/
cells overexpressed
Fyn, while Src expression of the Fyn
/
cells was similar to the WT. The different cells were subjected to iso- or hypertonic treatment, and
cortactin was precipitated and probed for tyrosine phosphorylation (Fig. 6B). The expression of cortactin was similar in each
cell line. Hypertonicity caused marked cortactin phosphorylation in the
WT cells, and usually an even stronger response was detected in
Src
/
(but Fyn-overexpressing) cells. This finding clearly shows
that Src kinase is not required for mediating this osmotic response. In
contrast, in Fyn
/
cells, the hypertonicity-elicited cortactin
phosphorylation was substantially weaker than in the WT. These results
suggest that Fyn significantly contributes to osmotic phosphorylation
of cortactin. On the other hand (at least in Src-containing cells), the
presence of Fyn is not an absolute requirement.
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Fig. 6.
Comparison of hypertonicity-induced cortactin
phosphorylation in wild type and Src- and Fyn-deficient
fibroblasts. A, confluent cultures of WT, Src /
, and
Fyn
/
mouse fibroblasts were washed with Iso-Na medium and lysed
with Triton buffer. Src (left) and Fyn (right)
were precipitated out from the lysates using monoclonal anti-Src or
anti-Fyn antibodies. The precipitated proteins were subjected to
electrophoresis followed by Western blotting and probed with the same
monoclonal anti-Src or with a polyclonal anti-Fyn, as indicated.
B, the three groups of cells were serum-deprived and
challenged with Iso-Na (I) or Hyper-Na (H) as
described for the CHO cells. After a 10-min treatment, the cells were
lysed, and cortactin was immunoprecipitated from the lysates. Cortactin
precipitates were probed with anti-phosphotyrosine (PY;
top). The same blot was then stripped and reprobed with
anti-cortactin (bottom).
0.4 pH unit), as
reported earlier (15). Pretreatment of the cells with PP1 had no effect
on their basal pHi and failed to prevent the
hypertonicity-induced response; neither the rate nor the extent of the
alkalinization was different in drug-treated and in control cells.
Thus, the osmotic stimulation of NHE-1 does not seem to depend on the
activation or activity of Src kinases. Next, we performed experiments
to discern whether this enzyme family can be involved in the hypertonic
inhibition of NHE-3 (Fig. 7, C and D). To this
end, control or PP1-treated NHE-3 cells were acidified, and the
sodium-induced recovery of their pHi was monitored under iso-
or hypertonic conditions in the absence or presence of the drug.
Hypertonicity strongly reduced the rate and the amplitude of the
recovery in agreement with earlier reports by us and others (15, 17).
PP1 did not affect the rate of the sodium-induced alkalinization under
isotonic conditions and failed to influence the inhibition of NHE-3 by
hypertonicity. These findings suggest that, at least in CHO cells, the
Src family of tyrosine kinases does not appear to play an essential
role in the hyperosmotic inhibition of NHE-3.
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Fig. 7.
The Src family inhibitor PP1 does not affect
the hyperosmotic stimulation of NHE-1 and hyperosmotic inhibition of
NHE-3 in CHO cells. CHO cells expressing NHE-1 (A and
B) or NHE-3 (C and D) were grown to
confluence on glass coverslips, serum-deprived for 3 h, and loaded
with the fluorescent pH indicator, BCECF, as detailed under
"Experimental Procedures." A, dye-loaded NHE-1 cells
were bathed in Iso-Na medium without or with 10 µM PP1
for 10 min, and their basal fluorescence was recorded. When indicated
by the arrow (HYPER), the medium was rapidly
exchanged to Hyper-Na, in the absence (CONT) or presence of
the drug (PP1). At the end of each run, the fluorescence
response was calibrated in terms of pHi (see "Experimental
Procedures"). B, the initial rate of the
hypertonicity-induced alkalization in the absence (CONT) or
presence of the drug (PP1) is shown (n = 4).
C, NHE-3 cells were loaded with BCECF in Iso-Na medium with
or without PP1. In order to acidify them, the cells were treated with
NH4Cl in Iso-, or Hyper-Na. Where indicated, PP1 was
present during this period as well. The cells were then washed with
Iso- or Hyper-K solutions, their basal pHi was recorded, and
recovery was initiated (at the arrow marked
Na+) by superfusion with Iso- or Hyper-Na, in
the presence or absence of PP1, as shown by the traces. D,
rates of recovery at the various pHi values were determined as
the slope of the linear line fitted to each 0.1 pH segment of the
pHi traces. H+ flux values were calculated by
multiplying the rate of pHi recovery by the buffering capacity
previously determined throughout the pH range studied (15). Data are
expressed as means ± S.E., for 3-6 determinations for each
condition.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. Sergio Grinstein for providing access to the PTI system. We thank Dr. Sheila Thomas, Dr. Bradford Berk, and Dr. Mari Ishida for providing Src- and Fyn-deficient cells. The valuable help of Catherina DiCiano and Samantha Fienberg is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported by a grant from the Banting Foundation, a grant from the Dean's Fund (University of Toronto), Medical Research Council of Canada Grant MT-14789, and National Research Fund of Hungary Grant OTKA F 019715.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.
§ To whom correspondence should be addressed: Toronto Hospital, Dept. of Surgery, Transplantation Research, Rm. CCRW 2-850, 101 College St., Toronto, Ontario M5G 1L7, Canada. Tel.: 416-340-3861; Fax: 416-597-9749; E-mail: akapus{at}transplantunit.org.
This author's research visit to Canada was sponsored by the
Soros Foundation.
2 A. Kapus, K. Szászi, J. Sun, S. Rizoli, and O. D. Rotstein, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: CHO, Chinese hamster ovary; BCECF, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; NHE, Na+/H+ exchanger; NMG, N-methyl-D-glucammonium; pHi, intracellular pH; WT, wild type; WB, Western blot; IP, immunoprecipitation.
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