(Received for publication, April 16, 1997)
From the Division of Cell Biology, Research Institute, the Hospital for Sick Children, Toronto M5G 1X8, Canada
The ubiquitous isoform of the Na+/H+ exchanger (NHE1) is essential for the regulation of cellular volume. The underlying molecular mechanism, which is poorly understood, was studied in human polymorphonuclear leukocytes (PMN). Suspension of PMN in hypertonic media induced rapid cellular shrinkage and activation of NHE1, which is measurable as a cytosolic alkalinization. Concomitantly, hypertonic stress also induced extensive tyrosine phosphorylation of several proteins. Pretreatment of PMN with genistein, a tyrosine kinase inhibitor, prevented not only the tyrosine phosphorylation in response to a hypertonic shock but also the activation of NHE1. The signal elicited by hyperosmolarity that induces activation of tyrosine kinases and NHE1 was investigated. Methods were devised to change medium osmolarity without altering cell volume and vice versa. Increasing medium and intracellular osmolarity in normovolemic cells failed to activate tyrosine kinases or NHE1. However, shrinkage of cells under iso-osmotic conditions stimulated both tyrosine phosphorylation and NHE1 activity. These findings imply that cells detect alterations in cell size but not changes in osmolarity or ionic strength. The identity of the proteins that were tyrosine-phosphorylated in response to cell shrinkage was also investigated. Unexpectedly, the mitogen-activated protein kinases SAPK, p38, erk1, and erk2 were not detectably phosphorylated or activated. In contrast, the tyrosine kinases p59fgr and p56/59hck were phosphorylated and activated upon hypertonic challenge. We propose that cells respond to alterations in cell size, but not to changes in osmolarity, with increased tyrosine phosphorylation, which in turn leads to the activation of NHE1. The resulting changes in ion content and cytosolic pH contribute to the restoration of cell volume in shrunken cells.
The Na+/H+ exchanger isoform 1 (NHE1)1 is a ubiquitously expressed cation antiporter that is involved in the regulation of cell volume and intracellular pH (pHi). NHE1 is nearly quiescent in resting cells but becomes activated upon cytosolic acidification or by treatment of the cells with a variety of hormones and growth factors (see Ref. 1 for review). Phosphorylation of the exchanger was suggested to induce its activation, since treatment with growth promoters was found to increase the phosphoserine content of NHE1 (2, 3). Moreover, increased phosphorylation and functional activation were also induced by inhibitors of Ser/Thr phosphatases, such as okadaic acid (3).
NHE1 is also rapidly stimulated when cells are made to shrink in
hypertonic solutions (4). It is unclear whether increased osmolarity or
reduced cell volume are the signals that trigger activation of the
exchanger. The osmotic stimulation of Na+/H+
exchange requires intracellular ATP and is not additive with that
induced by growth factors (5). These observations suggested that
phosphorylation was also involved in the osmotic activation of NHE1.
However, the phosphorylation state of the exchanger was found to be
unaffected during osmotic challenge (4). Moreover, osmotic stimulation
could still be observed following truncation of all the putative
phosphorylation sites of NHE1 (6). Thus, the mechanism responsible for
osmotically induced stimulation of the exchanger remains unclear. It is
possible that phosphorylation of ancillary regulatory proteins is
involved. In this context, calcineurin B
homolog protein (CHP), a substrate of Ser/Thr
kinases, was reported to bind to the cytosolic tail of the antiporter
(7). Also, a polypeptide of 24 kDa, the approximate size of CHP, is constitutively associated with NHE1 in several cell types (8).
Osmotic shrinkage of mammalian cells is a powerful stimulant of MAPK including the stress kinases p38 and SAPK (JNK) (9, 10) and in some instances Erk (11). MAPK have recently been invoked as possible regulators of the activity of NHE1 in platelets (12) and fibroblasts (13) treated with various agonists. The precise mechanism whereby shrinkage stimulates the kinases is unknown, as is their relationship to the osmotic stimulation of NHE1.
In this report, we investigated the relationship between the stimulation of protein kinases and the activation of NHE1, and we attempted to determine whether reduced cell volume or increased cytosolic osmolarity were the signals leading to the activation of these effectors. To this end we used human blood neutrophils, which express NHE1 (14) and are known to respond vigorously to changes in medium osmolarity (15).
Dextran T-500 and Ficoll-Paque were from Pharmacia Biotech Inc. Genistein and erbstatin analog were from Calbiochem. BCECF was from Molecular Probes Inc. Nystatin was from Sigma and was freshly dissolved in dimethyl sulfoxide before each experiment. All other chemicals used were of the highest purity available. The enhanced chemiluminescence detection system and horseradish peroxidase-coupled anti-rabbit and anti-mouse antibodies were from Amersham Corp. Phosphotyrosine monoclonal antibody (4G10) was from Upstate Biotechnology Inc. Polyclonal anti-paxillin antibody was from Zymed Inc. and anti-c-cbl was from Transduction Laboratories Inc. Polyclonal antibody against p38 was the generous gift of Dr. Brent Zanke (Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Canada). MAPKAPK-2 polyclonal antibody was the kind gift of Dr. Steven L. Pelech (Kinetek Pharmaceuticals Inc., Vancouver, British Columbia, Canada). A GST-c-Jun construct was provided by Dr. James Woodgett (Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Canada). Phospho-specific Erk polyclonal antibody was from New England Biolab. lyn, fgr, and hck polyclonal antibodies were generously provided by Dr. Joseph B. Bolen (DNAX Research Institute, Palo Alto, CA).
SolutionsBicarbonate-free RPMI 1640 was buffered to pH 7.4 with 10 mM Hepes. Isotonic NaCl buffer contained (in
mM) 5 KCl, 10 glucose, 140 NaCl, 1 CaCl2, 1 MgCl2, 10 Hepes, pH 7.4. Isotonic KCl buffer contained 10 glucose, 145 KCl, 1 CaCl2, 1 MgCl2, and 10 Hepes, pH 7.4. Hypertonic NaCl buffer contained 5 KCl, 10 glucose, 240 NaCl, 1 CaCl2, 1 MgCl2, and 10 Hepes, pH 7.4. Hypertonic KCl buffer was similar to hypertonic NaCl buffer, except
that NaCl was replaced with KCl. Hypotonic NaCl buffer contained 5 KCl,
10 glucose, 50 NaCl 1 CaCl2, 1 MgCl2, and 10 Hepes, pH 7.4. Iso-osmotic sucrose buffer contained 5 KCl, 10 glucose,
280 sucrose, 1 CaCl2, 1 MgCl2, and 10 Hepes, pH
7.4. Ca2+ and Mg2+ were omitted from all
buffers that were used during permeabilization with nystatin. The
iso-osmolar buffers were adjusted to 290 ± 5 mOsm with either
water or the major salt. All buffers used for cell incubations were
nominally HCO3-free. Laemmli sample buffer (LSB)
contained 10% glycerol, 5% 2-mercaptoethanol, 2% SDS, 0.025%
bromphenol blue, 62.5 mM Tris, pH 6.8. Nonidet P-40 buffer
contained 1% Nonidet P-40, 1 mM EGTA, 150 mM
NaCl, and 50 mM Tris, pH 8.0.
Human PMN were isolated from fresh blood drawn by venipuncture into heparinized tubes. Isolation of cells was performed using dextran sedimentation and centrifugation on Ficoll-Paque cushions as described previously (16). Cells were resuspended in Hepes-buffered RPMI 1640 and kept on a rotary shaker at room temperature until use. When immunoprecipitation was performed, PMN were pretreated with 1 mM diisopropylfluorophosphate for 30 min to minimize proteolysis. Cell volume and counts were assessed with a Coulter Counter (model ZM) equipped with a Channelyzer.
Immunoprecipitation and ImmunoblottingTreatments were stopped by the addition of 2 volumes of ice-cold buffer of the corresponding osmolarity, and the PMN were rapidly sedimented in a microcentrifuge. For experiments where whole cell anti-phosphotyrosine blotting was performed, the cell pellet was resuspended in hot LSB and boiled for 10 min. For immunoprecipitation, the cell pellet was dissolved in ice-cold Nonidet P-40 buffer containing protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µM sodium vanadate, and 1 mM NaF) and kept on ice for at least 10 min. Immunoprecipitation and immunoblotting were performed as described previously (17). Samples were subjected to 10% SDS-PAGE, transferred to poly(vinylidene difluoride) membranes, and blotted with the appropriate antibody.
Kinase AssaysTyrosine kinase activity was assayed in vitro using enolase as the substrate, as described previously (18). SAPK assays using GST-c-jun as a substrate were performed essentially as described (19). Reaction products were separated by 10% SDS-PAGE, and incorporated 32P was quantified with a PhosphorImager equipped with ImageQuant software (Molecular Dynamics Inc.).
Intracellular pH MeasurementsPMN (107/ml) were incubated with 2 µM of the acetoxymethyl form of BCECF for 15 min at 37 °C, sedimented, and resuspended (at 2 × 107/ml) in the appropriate buffer. Where indicated, the cells were pretreated with nystatin (50 µg/ml) to increase the permeability of the plasmalemma to small monovalent ions (see "Results"). An aliquot of the cell suspension (106 cells) was added to 1 ml of prewarmed (37 °C) buffer of the required osmolarity in the cuvette compartment of a spectrofluorimeter (Perkin-Elmer model 650-40). Measurements of BCECF emission and the calibration of fluorescence versus pHi were performed as described previously (20).
Statistical AnalysisAll experiments were performed at least in triplicate. Data are presented as means ± S.E. or illustrated as representative traces or blots. Significance was assessed using Student's paired t test. A score of p < 0.05 was considered significant.
Correlation between Tyrosine Phosphorylation and NHE1 Activation
We tested the effect of hypertonic solutions on PMN volume,
measured electronically, and pHi, estimated from the
fluorescence of BCECF. Increasing the osmolarity of the medium from 290 to 475 mOsm by addition of 100 mM NaCl caused a rapid
reduction of median cell volume from 327 ± 3 fl to 273 ± 2 fl (means ± S.E., n = 5, p < 0.01).2 As shown in Fig.
1A, hypertonic stress also induced an
alkalinization of the cytosol ranging from 0.2 to 0.3 pH units, which
was evident at 30 s and stabilized within 5 min. As in other
cells, this alkalinization was mediated by the NHE, since it was
abolished by omission of external Na+ (not shown) or by
addition of the specific inhibitor compound HOE694 (see below).
Phosphorylation of tyrosine residues is one of the earliest events in a
variety of signaling cascades. We questioned whether tyrosine
phosphorylation was also involved in signaling the osmotic activation
of NHE1. To address this possibility, the content of tyrosine-phosphorylated proteins was analyzed by immunoblotting in PMN
subjected to hypertonic stress. Fig. 1B shows that osmotic shrinkage was associated with a remarkable increase in the
phosphotyrosine content of several proteins, which was clearly apparent
at 30 s, attained maximum levels by 2 min, and persisted for up to
30 min. Polypeptides of 210, 125, 74, 60, 42, and 40 kDa were
consistently tyrosine-phosphorylated in all of our experiments.
We next investigated whether tyrosine phosphorylation was a consequence
or the cause of NHE1 activation. To determine if activation of
Na+/H+ exchange was required for induction of
tyrosine phosphorylation, PMN were pretreated with 2 µM
HOE694, a concentration predicted to produce almost complete inhibition
of NHE1 (21) and subjected to a hypertonic shock. As shown in Fig.
2, while the inhibitor largely eliminated the cytosolic
alkalinization, the accumulation of phosphotyrosine induced by
hyperosmolarity was unaffected. A comparable degree of tyrosine
phosphorylation was also obtained in cells suspended in a hypertonic
KCl (Na+-free) medium. The absence of Na+, the
external substrate for NHE, precluded cytosolic alkalinization (results
not shown). These experiments imply that stimulation of tyrosine
phosphorylation by hyperosmolar solutions is not a consequence of
activation of NHE1.
We therefore considered whether tyrosine phosphorylation was instead
the cause of NHE1 activation. Cells were pretreated with 100 µM genistein, a potent tyrosine kinase inhibitor, and
subjected to hypertonicity. Under these conditions, both the cytosolic
alkalinization (Fig. 3A) and tyrosine
phosphorylation were inhibited (Fig. 3B). Similar results
were obtained by pretreating PMN with 10 µg/ml erbstatin analog, a
structurally unrelated tyrosine kinase inhibitor (not shown). These
findings suggest that phosphotyrosine accumulation is required for the
hypertonic activation of NHE1.
Role of Osmolarity in the Induction of Tyrosine Phosphorylation
We next investigated the signal that triggers phosphotyrosine accumulation in cells exposed to hypertonic media. In principle, the response could be initiated by osmosensors that detect the change in medium or intracellular tonicity. Alternatively, the signal for phosphorylation could be the cellular shrinkage that results from the net loss of cytosolic water. The experiments described below were designed to discern between these alternative models.
Fig. 4A illustrates the protocol used to
increase the intracellular osmolarity while keeping the cellular volume
constant. PMN were suspended in isotonic KCl medium and treated with 50 µg/ml nystatin, a pore-forming molecule that allows the passage of
small monovalent ions across the plasma membrane (22). Sucrose (50 mM), which cannot permeate through nystatin, was added to the medium to prevent swelling due to the presence of impermeant osmolytes within the cells (20). After 9 min, the time required for
adequate permeabilization, an additional 125 mM KCl was
introduced to render both the extracellular and intracellular solutions
hyperosmotic. Because both K+ and Cl permeate
readily through nystatin, cell shrinkage is minimal (step
III in Fig. 4A). Cells were then washed at 37 °C to
remove extracellular as well as membrane-associated nystatin, resulting in rapid and effective resealing of the membrane, and the hypertonic KCl was replaced with hypertonic NaCl (step IV). Sizing with
the Coulter-Channelyzer confirmed that, following nystatin treatment in
the hypertonic buffer, the volume of the cells was similar to that of
untreated PMN in isotonic solution (cf. columns I and IV in Fig. 4B). This contrasts with the shrinkage
noted when cells were suspended in hypertonic NaCl or KCl in the
absence of nystatin2 (II in Fig.
4, A and B). Fig. 4C confirms that
cell shrinkage induced by the hypertonic media (in the absence of
nystatin) stimulated tyrosine phosphorylation of multiple proteins,
regardless of the solute used (lanes labeled II in Fig.
4C). By contrast, exposure to hyperosmotic solutions under
conditions where shrinkage was prevented (i.e. in
nystatin-treated cells) resulted in a substantially lower level of
tyrosine phosphorylation (cf. lane IV). It is noteworthy that the residual increase in phosphorylation may have been caused by a
transient shrinkage of the cells that likely occurred when the
osmolarity of the medium was raised. Despite the presence of nystatin,
some efflux of water from the cells may have preceded entry and
equilibration of hyperosmotic KCl into the cells.
We also used a second approach to assess the effect of increasing the
osmolarity on protein tyrosine phosphorylation in the absence of
significant cell volume changes. For these experiments osmolarity was
increased adding 200 mM urea, a rapidly permeating solute,
to cells suspended in isotonic NaCl buffer. Fig.
5A illustrates the protocol used. The
addition of urea did not alter the steady state volume of the cells
(measured after 5 min; II in Fig. 5), which contrasts with
the sustained shrinkage induced by an equimolar concentration of
sucrose (IV in Fig. 5, A and B) or 100 mM NaCl (e.g. Fig. 4). In parallel experiments,
tyrosine phosphorylation was assessed in cells exposed to hyperosmotic
urea and was found to be similar to that of cells maintained in
isotonic NaCl medium throughout (Fig. 5C). That urea did not
exert an inhibitory effect on tyrosine phosphorylation was tested by
treating cells with either 200 mM sucrose or 100 mM NaCl in the presence (III in Fig. 5A) or absence (IV in Fig. 5A) of
urea. As shown in Fig. 5B, the impermeant osmolytes induced
shrinkage both in the presence and absence of urea. More importantly,
both sucrose (cf. lanes 3 and 4 in Fig.
5C) and NaCl (cf. lanes 5 and 6)
activated tyrosine phosphorylation to comparable degrees whether
urea was present or not. These results suggest that urea does not
per se prevent phosphotyrosine accumulation and that an
increase in the osmolarity of the medium and/or the intracellular space
is not sufficient to induce tyrosine phosphorylation of PMN
proteins.
Role of Cell Shrinkage in the Induction of Tyrosine Phosphorylation
In the next series of experiments, we analyzed the contribution of
cell volume changes to the induction of tyrosine phosphorylation. To
this end, we attempted to induce cell shrinkage while maintaining iso-osmolar conditions. Fig. 6A illustrates
the first method used; PMN were resuspended in an ice-cold iso-osmotic
sucrose medium and permeabilized with nystatin for 10 min. While the
extracellular sucrose is unable to diffuse through the nystatin pores,
intracellular KCl readily diffuses out of the cells (II in
Fig. 6A). The net efflux of KCl is accompanied by
osmotically obliged water, thus causing a reduction in cell volume
(cf. I and III in Fig. 6, A and
B). Interestingly, the resulting shrinkage of
nystatin-permeabilized PMN in the isotonic sucrose buffer caused a
marked increase in tyrosine phosphorylation (Fig. 6C, lane
4), which was in fact greater than that caused by hypertonic NaCl
buffer (cf. lane 5). The effect of sucrose was not due to
the reduction in ionic strength, since in the absence of nystatin
tyrosine phosphorylation was not stimulated. As expected, cell volume
was unaffected under these conditions (Fig. 6B). Moreover,
the stimulation of phosphorylation was not due to nystatin itself,
because cells treated with the pore former under conditions intended to
keep cell volume constant (125 mM NaCl plus 50 mM sucrose; see Fig. 6B) did not show increased phosphorylation. It is also noteworthy that treatment with nystatin in
isotonic NaCl buffer, which induced cell swelling (Fig. 6B), decreased tyrosine phosphorylation below the level noted in untreated (isotonic) cells (Fig. 6C, cf. lanes 1 and
2).
A second method used to dissociate the effects of cell shrinkage and
hypertonicity is illustrated in Fig. 7A. PMN
were suspended in hypotonic NaCl buffer (50% of the normal
osmolarity), thereby causing the cells to swell (II in Fig.
7, A and B). This initial passive swelling was
followed by a gradual loss of volume, reaching near normal size after
approximately 30 min (III in Fig. 7, A and
B). This secondary volume loss, known as regulatory volume decrease, is thought to be mediated by increased permeability to
K+ and anions (23). Subsequent addition of 90 mM NaCl to the medium, which restored the osmolarity to the
initial (iso-osmotic, 290 mOsm) level, caused the cells to shrink
(IV in Fig. 7). Such shrinkage under iso-osmotic conditions
was accompanied by a marked phosphotyrosine accumulation, usually
exceeding that induced by comparable hypertonic shrinkage (Fig.
7C). The combined results of Figs. 6 and 7 demonstrate that
tyrosine phosphorylation can be promoted in PMN by reducing the volume
of the cells, regardless of the osmolarity of the medium or
cytosol.
Role of Cell Volume and Hypertonicity in the Activation of NHE1
The preceding data indicate that tyrosine phosphorylation was
triggered by a reduction of the cell volume and not by hypertonicity per se. It was therefore of interest to define whether cell
volume, as opposed to medium osmolarity, is responsible for activation of NHE1. Protocols like those employed above were used to
differentially alter cell volume and osmolarity while measuring
pHi to evaluate the state of activation of NHE1. Fig.
8 shows that a significant cytosolic alkalinization,
comparable to that observed during hypertonic stress, was caused by
reducing cell volume isotonically using nystatin/sucrose, or by
restoring iso-osmolarity after regulatory volume decrease. Conversely,
increasing osmolarity while keeping the volume constant, using either
nystatin/KCl or urea, failed to activate the antiporter. This pattern
correlates closely with that of tyrosine phosphorylation and is
consistent with the notion that NHE1 activation lies downstream of
phosphotyrosine accumulation.
Identity of Tyrosine-phosphorylated Proteins in Shrunken PMN
MAPKBecause the activation of NHE1 appears to be dependent
on phosphotyrosine accumulation, we tried to identify some of the
proteins that become tyrosine-phosphorylated when PMN shrink. The
stimulation of NHE1 by growth factors has recently been reported to be
partially dependent on the erk1 and erk2 MAPK
(p42/44MAPK) pathway (13). Moreover, it is well established
that kinases of the MAPK family require phosphorylation on tyrosine
residues to become active (24). Since hypertonic stress has been shown to induce the activation of erk1 and erk2 in
other cell types (11, 13), we investigated whether these MAPK are the
40-42-kDa tyrosine-phosphorylated proteins observed in shrunken
PMN. Cells were subjected to hypertonic stress for up to 30 min, and
whole cell lysates were immunoblotted with an antibody that
specifically recognizes the phosphorylated form of erk1 and
erk2. Fig. 9A shows that neither
erk1 nor erk2 were tyrosine-phosphorylated in PMN in response to hypertonic stress. The sensitivity of the
phospho-specific antibody and the responsiveness of the cells were
assessed by stimulation with 100 nM fMLP, a well documented
activator of erk1 and erk2 in PMN (25, 26). As
shown in Fig. 9, comparable amounts of cell lysate revealed sizable
amounts of phosphorylated erk1 and erk2 after
treatment with the chemoattractant. We conclude that erk1
and erk2 are not phosphorylated during hypertonic challenge and are therefore unlikely to mediate the activation of NHE1.
Another member of the MAPK family, p38, has been shown to be activated
by hypertonic stress in other cells (10) and was recently detected in
fMLP-stimulated human PMN (17, 27). To test whether this kinase is
phosphorylated and activated by shrinkage also in PMN we
immunoprecipitated p38 and blotted the precipitates with
anti-phosphotyrosine antibodies (Fig. 10A).
Unlike other cells, PMN did not show evidence of p38 phosphorylation
upon shrinking. As before, the effectiveness and sensitivity of the
procedure were confirmed in parallel samples stimulated with fMLP
(rightmost lane in Fig. 10A). That p38 was
activated by chemoattractant but not by osmotic challenge was also
confirmed in experiments where whole cell lysates were blotted with an
anti-MAPKAPK-2 antibody (Fig. 10C). This kinase, a substrate
of p38, undergoes an upward shift in electrophoretic mobility when
phosphorylated (17). A distinct shift was noted for fMLP-stimulated
samples but not in osmotically shrunken cells. We conclude that p38 is
not phosphorylated or activated by hypertonic challenge in PMN.
Hypertonic stress activates SAPK in a number of cells (e.g. Ref. 9). To investigate if SAPK was similarly stimulated in PMN, this kinase was precipitated from cell lysates using GST-c-jun-coupled to Sepharose beads and its activity tested in vitro. SAPK failed to phosphorylate GST-c-jun following hypertonic stress in PMN (results not shown). It is unclear whether SAPK is not activated or not expressed by human PMN, since we were also unable to demonstrate activation upon treatment of these cells with anisomycin, a well known activator of SAPK.
Src Family KinasesKinases of the src family are
themselves regulated by phosphorylation on tyrosine residues and may
account for the phosphotyrosine accumulation in the 60-kDa range in
shrunken PMN. We therefore investigated the ability of cell shrinkage
to induce the phosphorylation and activation of three src
family kinases that are comparatively abundant in PMN, namely
fgr (59 kDa), hck (56/59 kDa), and lyn (59 kDa). PMN were osmotically stimulated for 1 min and lysed, and the
three tyrosine kinases were individually immunoprecipitated. The
immunoprecipitates were subsequently separated by SDS-PAGE and blotted
with a phosphotyrosine-specific antibody. As shown in Fig.
11A, all three kinases were significantly
phosphorylated in untreated cells, and cell shrinkage promoted
increased tyrosine phosphorylation of fgr and
hck, whereas a slight decrease was noted for lyn.
The effect of volume changes on the activity of these kinase assays was
also tested, performing in vitro assays with
immunoprecipitates from control and shrunken cells. We assessed the
ability of the kinases to autophosphorylate as well as to phosphorylate
the exogenous substrate enolase. Consistent with the phosphotyrosine
immunoblots of Fig. 11A, both auto-phosphorylation and
enolase kinase activity increased for fgr and hck
but decreased slightly for lyn (Fig. 11B).
The identity of other tyrosine-phosphorylated proteins was also probed using sequential immunoprecipitation and blotting as in Fig. 11A. We failed to detect tyrosine phosphorylation of paxillin (67 kDa) or c-cbl (120 kDa) in PMN stimulated hypertonically (results not shown).
PMN are exposed to a wide range of dynamic physical forces during
their active life span, particularly during passage through narrow
capillaries and across vascular walls and during chemotaxis. Such
mechanical stress causes shape and volume alterations that need to be
compensated in order for the cells to function optimally (28). Such
regulation of shape and volume can occur in part via the movement of
ions and osmotically obliged water across the cell membrane. The
current study investigated the mechanism that regulates the activation
of a major, volume-sensitive ion transporter in human PMN, namely NHE1.
The salient observations were (i) that a moderate reduction of the cell
volume (16%) induced the tyrosine phosphorylation of several
proteins and (ii) that such tyrosine phosphorylation is seemingly
required for the activation of NHE1.
Several hypotheses exist regarding the mechanism(s) whereby cells detect osmotic stress (reviewed in Refs. 29-31). First, cells may sense the ionic strength or total osmolarity of the medium or of the intracellular milieu. This explanation cannot account for the observed phosphotyrosine accumulation in PMN for several reasons. Tyrosine phosphorylation could be induced by shrinkage at constant osmolarity and ionic strength (Figs. 6 and 7). Moreover, increasing the osmolarity and ionic strength at constant volume had minimal effect on phosphotyrosine formation (Figs. 4 and 5). It has also been suggested that changes in cytoskeletal architecture upon shrinking may mediate activation of the cells. While we cannot dismiss this possibility, our data suggest that assembly of microtubules and de novo F-actin polymerization are not essential, since neither colchicine nor cytochalasin B prevented the volume-induced tyrosine phosphorylation (results not shown).
An interesting hypothesis stipulates that cells perceive their volume by sensing macromolecular crowding (29); small changes in cell volume can lead to large increases in the thermodynamic activity of macromolecules (32). One form of crowding, leading to such disproportionate increases in activity, may be the aggregation of surface receptors recently reported by Rosette and Karin (33). These authors found that osmotic shrinkage of HeLa cells induced clustering of interleukin-1, epidermal growth factor, and tumor necrosis factor receptors despite the absence of their ligands. Clustering of receptors is known to be crucial to their activation (34), and accordingly, receptor stimulation was found in the shrunken HeLa cells (33). In PMN, engagement and cross-linking of Fc receptors or of integrins lead to the activation of the tyrosine kinases fgr and hck (35-38), which were also found to be stimulated osmotically in this study. It is tempting to speculate that shrinkage of PMN induces the activation of fgr and hck through clustering of Fc receptors, integrins, and/or other tyrosine kinase (associated) receptors.
The similarity in the pattern of osmotic activation of tyrosine phosphorylation and of NHE1, together with the inhibitory effects of genistein and erbstatin, suggests that stimulation of tyrosine kinases precedes and is necessary for activation of ion exchange. A causal relationship between these events has in fact been postulated for several cell types (e.g. Ref. 39) including PMN where phagocytic stimuli (14) and chemotactic peptides (40) regulate pHi in a tyrosine kinase-dependent manner. In the context of macromolecular crowding, it is noteworthy that cross-linking of Fc receptors and integrins can in fact activate NHE1 in PMN and in other cells (14, 41, 42). It is, however, unlikely that NHE1 itself is the target of the tyrosine kinases for the following reasons. First, only serine residues have been found to be phosphorylated in this isoform (2, 3). Second, in Chinese hamster ovary cells no increase in the phosphorylation of NHE1 was detected following activation by osmotic stress (6). Therefore, other intervening steps are likely situated between the tyrosine kinases and NHE1. Potential regulators of NHE1 include Ca2+/calmodulin, protein kinase C, phosphatidylinositol 3-kinase, and heterotrimeric G proteins (43-46). We found, however, that depletion of Ca2+ had no effect on either tyrosine phosphorylation or NHE1 activation in response to hypertonic stimulation. Moreover, pretreatment of PMN with bis-indolylmaleimide (a protein kinase C inhibitor), wortmannin (a phosphatidylinositol 3-kinase inhibitor), or pertussis toxin (a heterotrimeric G protein inhibitor) all failed to inhibit NHE1 or the tyrosine phosphorylation stimulated by hypertonic stress.3
Hooley et al. (44) demonstrated that RhoA was involved in the activation of NHE1 in fibroblasts. Interestingly, a connection between tyrosine kinases and RhoA had been previously established (47). It is therefore conceivable that the pathway leading to osmotic activation of NHE1 involves stimulation of RhoA through src-related tyrosine kinases. The mechanism by which RhoA activates NHE1 is currently unknown, but some information can be gleaned from the recent identification of Rho-binding proteins. Of relevance, the phosphorylation of myosin light chain was found to be regulated by a RhoA-dependent kinase (48). This observation is important in that Shrode et al. (49) demonstrated that inhibitors of myosin light chain kinase were potent blockers of the osmotic activation of NHE1. One can therefore envisage the following sequence: cell shrinkage may lead to receptor clustering and activation of tyrosine phosphorylation. This would in turn activate Rho leading to stimulation of NHE1, possibly via phosphorylation of the light chain of myosin. It is noteworthy that MAPKs are seemingly not components of this signaling cascade.
In conclusion, the current study showed that the shrinkage of PMN induced the tyrosine phosphorylation of several proteins, two of which were identified as fgr and hck. Given the ability of tyrosine kinase inhibitors to block the stimulation of NHE1, we propose that tyrosine kinases, including fgr and hck, are involved in the osmotic activation of the antiporter, through some as yet unidentified intermediate(s).
We thank John H. Brumell for help with tyrosine kinase assays and Lamara D. Shrode for helpful comments on the manuscript.