Department of Molecular Physiology, National Cardiovascular Research Institute, Osaka 565, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To assess the role of Ca2+
in regulation of the
Na+/H+
exchanger (NHE1), we used CCL-39 fibroblasts overexpressing the
Na+/Ca2+
exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca2+ rise and
long-lasting cytoplasmic alkalinization (60-80% inhibition) induced by -thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant with
the calmodulin (CaM)-binding domain deleted (amino acids 637-656),
suggesting that the effect of NCX1 transfection involves Ca2+-CaM binding. Expression of
NCX1 only slightly inhibited platelet-derived growth factor BB-induced
alkalinization and did not affect hyperosmolarity- or phorbol
12-myristate 13-acetate-induced alkalinization. Downregulation of
protein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely in
NCX1-transfected cells, suggesting that the thrombin effect is mediated
exclusively via Ca2+ and PKC. On
the other hand, deletion mutant study revealed that PKC-dependent
regulation occurs through a small cytoplasmic segment (amino aids
566-595). These data suggest that a mechanism involving direct
Ca2+-CaM binding lasts for a
relatively long period after agonist stimulation, despite apparent
short-lived Ca2+ mobilization, and
further support our previous conclusion that Ca2+- and PKC-dependent mechanisms
are mediated through distinct segments of the NHE1 cytoplasmic domain.
sodium/hydrogen exchange; sodium/calcium exchange; growth factors; calmodulin; protein kinase C
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE UBIQUITOUS Na+/H+ exchanger isoform 1 (NHE1) is one of the important plasma membrane transporters regulating intracellular pH (pHi) and cell volume (11, 32, 41). An interesting feature is that NHE1 is rapidly activated in response to a wide variety of extracellular stimuli ranging from growth factors to mechanical stress such as cell shrinkage. Activation of NHE1 results in sustained cytoplasmic alkalinization (10, 22, 26, 29), most prominent in the absence of bicarbonate (9). Since the first molecular identification of NHE1 (34), the molecular aspect of NHE1 regulation has extensively been investigated (see Ref. 41 for review). An important finding is that NHE1 is regulated via multiple intracellular signaling mechanisms, although these mechanisms remain to be precisely characterized.
Growth factors such as -thrombin activate their specific receptors
in the cell membrane, triggering hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol trisphosphate and diacylglycerol (2).
These agents in turn induce intracellular
Ca2+ mobilization and activation
of protein kinase C (PKC), both of which are thought to be involved in
growth factor-induced activation of NHE1 (11, 41). We have provided
evidence suggesting that direct binding of calmodulin (CaM) to NHE1 is
a crucial event in the
Ca2+-induced activation of NHE1
(3, 38). We further showed that deletion of the CaM-binding domain of
NHE1 markedly decreases thrombin-induced cytoplasmic alkalinization
(3). However, we are not certain about the extent to which
Ca2+ contributes to the
long-lasting alkalinization induced by thrombin, because the
thrombin-induced rise in the intracellular
Ca2+ concentration
([Ca2+]i)
is usually transient. We also observed that deletion of the CaM-binding
domain abolished most of the cytoplasmic alkalinization induced by
hyperosmotic stress (3). It is also not clear whether this reduction in
alkalinization is due to the lack of the CaM-binding domain, which has
an autoinhibitory function (38), or whether Ca2+ (and CaM) is required for
hyperosmolarity-induced activation of NHE1.
Using fibroblastic (CCL-39 and PS120) cells overexpressing the cardiac Na+/Ca2+ exchanger NCX1 (18), we have analyzed the role of intracellular Ca2+ and PKC in NHE1 activation caused by growth factors and hyperosmolarity. In these cells, Ca2+ signaling in the subplasmalemmal region seems to be intensely inhibited, as evidenced by our failure to detect a rise in thrombin-induced Ca2+ concentration in the peripheral cytoplasm of these cells by use of the confocal fluorescence Ca2+ imaging technique as well as by our finding that activation of plasma membrane Ca2+-dependent K+ channels in response to thrombin or ionomycin was markedly suppressed in these cells (18a). Thus these cells seem to provide a unique experimental system in which suppression of Ca2+ signaling is achieved by increasing the expression of a normal cell Ca2+ extrusion system (Na+/Ca2+ exchanger), the activity of which can be controlled easily by changing the extracellular Na+ concentration. The use of these NCX1-overexpressing cells to examine the role of [Ca2+]i would have an advantage over the use of intracellular Ca2+ chelators such as 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, because in our experience the latter agents sometimes cause artifacts such as cytoplasmic acidification.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. [7-14C]benzoic acid and fura 2-AM were purchased from DuPont NEN and Dojindo Chemical, respectively. Polyclonal rabbit antibodies against NCX1 and NHE1 proteins were prepared using maltose-binding protein fusion proteins containing portions of the cytoplasmic domains of NCX1 and NHE1, respectively, as described previously (3, 19). The NHE1-specific antibody was affinity purified as described previously (3), whereas the IgG fraction containing the NCX1-specific antibody was purified from immunized serum by use of a protein A-Sepharose column (Bio-Rad). All other chemicals were of the highest purity available.
Cells and culture condition. Chinese hamster lung fibroblast (CCL-39) cells, its Na+/H+ exchanger-deficient derivative PS120 (33) cells, and their transfectants were maintained in DMEM (Life Technologies) containing 25 mM NaHCO3 and supplemented with 7.5% (vol/vol) FCS, penicillin (50 U/ml), and streptomycin (50 mg/ml). Cells were maintained at 37°C in the presence of 5% CO2.
Stable expression of NCX1, NHE1, and NHE1 mutants.
The canine cardiac NCX1 cDNA cloned into the mammalian expression
vector pKCRH (designated pKCRH-NCX1) was transfected into CCL-39 cells
by the calcium-phosphate coprecipitation method, and single cell clones
expressing the highest levels of NCX1 were chosen as described
previously (18). PS120 cells stably expressing human NHE1 and its
deletion mutant (637-656) missing amino acids 637-656 were described previously (3, 40). These stable transfectants were further transfected with pKCRH-NCX1 by the calcium-phosphate coprecipitation method. Two days after transfection, cells were exposed
to 10 µM ionomycin for 30 min at 37°C in the growth medium. This
procedure ("Ca2+ killing")
eliminates most of the nontransfected cells (18a). Cells were subjected
to successive
Ca2+-killing
selection three to four times over a period of 3 wk. Resulting cell
populations highly express NCX1 and NHE1 (or its variants).
Measurement of [Ca2+]i change. [Ca2+]i was monitored with use of fura 2 as a fluorescent Ca2+ indicator, essentially as described previously (19). Cells attached to glass coverslips were loaded for 15 min at 37°C with 4 µM fura 2-AM in the culture medium. Loaded cells were washed twice with a medium composed of (in mM) 140 NaCl, 1 MgCl2, 2 CaCl2, 5 KCl, 1 NaHPO4, 5 glucose, and 20 HEPES-Tris (pH 7.4). In some experiments, 140 mM NaCl was replaced by 140 mM LiCl. The glass coverslip was fixed to a mount that was inserted diagonally into a cuvette filled with 2.2 ml of the above medium. The fluorescence signal was monitored at 510 nm with excitation wavelengths alternating between 345 and 375 nm with use of a SPEX Fluorolog-2 spectrofluorometer. [Ca2+]i was calculated as described previously (19) after correction for fluorescence from extracellular fura 2 and autofluorescence.
Measurement of pHi change. A change in pHi was estimated from the distribution of [7-14C]benzoic acid, as described previously (22). The stable transfectants grown to confluence in 24-well dishes were incubated for 5 h at 37°C in a serum-free, bicarbonate-free DMEM to maintain the Na+/H+ exchanger in the resting state. Cells were further incubated for 1 h in DMEM buffered with 20 mM HEPES-Tris (pH 7.0) and then placed in the same medium containing [14C]benzoic acid at 7 kBq/ml with or without various stimuli for the indicated period of time. For some experiments (see Fig. 4B), pHi was measured using an NaCl- or LiCl-containing solution composed of (in mM) 140 NaCl or 140 LiCl, 1 MgCl2, 2 CaCl2, 5 KCl, 5 glucose, and 20 HEPES-Tris (pH 7.0). Cells were then rapidly washed four times with ice-cold PBS and solubilized in 0.1 N NaOH, and 14C radioactivity was counted.
Crude membrane preparation and immunoblot analysis. As described previously (3), confluent cells in 150-mm dishes were washed once with ice-cold PBS and harvested in 10 ml of a hypotonic solution containing 20 mM HEPES (pH 7.4), 5 mM EDTA, and protease inhibitors (1 mM benzamidine and 1 mM phenylmethylsulfonyl fluoride). Cells were then homogenized (Physcotron, Nition) and centrifuged for 10 min at 700 g. The supernatant was centrifuged again for 30 min at 70,000 g. The pellet was washed once with a solution containing 20 mM HEPES-Tris (pH 7.4) and 150 mM NaCl by centrifugation for 30 min at 70,000 g. Isolated crude membranes were subjected to SDS-PAGE on a 7.5% acrylamide gel and then elecrophoretically transferred to an Immobilon membrane (Millipore) in 25 mM Tris, 0.19 M glycine, and 10% methanol. The blots were blocked in PBS containing 5% nonfat dry milk and incubated overnight at 4°C with anti-NCX1 or anti-NHE1 antibody (1:100 dilution) in PBS-milk. After they were washed several times, the blots were incubated for 1 h with PBS-milk containing horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1,000 dilution). After the blots were washed, immunoreaction was visualized using the enhanced chemiluminescence detection system (Amersham).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we stably transfected a plasmid carrying the dog cardiac
NCX1 cDNA into CCL-39 cells expressing a low level of endogenous NHE1
or PS120 cells lacking an endogenous NHE1 but transfected with NHE1
cDNA or its mutant with the CaM-binding domain deleted
(637-656). Figure 1
shows results of immunoblot analysis. In the NCX1-transfected cells, a
high level of NCX1 protein was detected as a broad band of ~120 kDa,
although parental CCL-39 and PS120 cells expressed almost undetectable
levels of endogenous NCX1 (Fig.
1A). High expression of NCX1 did
not significantly influence expression of NHE1 or
637-656,
which was detected as a protein band of ~110 kDa (Fig.
1B).
|
Figure 2 shows intracellular Ca2+ mobilization induced by thrombin at 2 U/ml in NCX1-transfected or -nontransfected CCL-39 cells. In control CCL-39 cells the thrombin-induced rise in [Ca2+]i was not different in the presence or absence of extracellular Na+ (Fig. 2A, Table 1). However, this [Ca2+]i rise was markedly inhibited in NCX1-transfected cells in Na+-containing medium, whereas such inhibition did not occur in cells placed in Li+-containing medium (Fig. 2B). Because Li+ does not serve as a transport substrate for Na+/Ca2+ exchange, it is most likely that overexpressed NCX1 rapidly extruded the Ca2+ released from intracellular stores from the cytoplasm. The resting [Ca2+]i was also found to be significantly lower in NCX1-transfected cells than in nontransfected cells (Table 1).
|
|
Figure 3 shows time courses of thrombin-induced cytoplasmic alkalinization in NCX1-transfected and control CCL-39 cells in Na+-containing medium. Thrombin induced a large cytoplasmic alkalinization for up to 20 min, which was abolished completely in the presence of 0.1 mM ethylisopropylamiloride, an inhibitor of the Na+/H+ exchanger (data not shown). With 22Na+ uptake measurement, we previously showed that NHE1 is very rapidly (<1 min) activated in response to the increase in [Ca2+]i (38). However, a rapid (<1 min) increase in pHi is not detected because of a lag time required for H+ extrusion. Interestingly, cytoplasmic alkalinization was strongly inhibited by expression of NCX1 during the whole period tested (Fig. 3), although the resting pHi before thrombin stimulation was similar for control and NCX1-transfected cells. The thrombin-induced alkalinization at 10 min was suppressed by 60-80% in NCX-transfected cells on the basis of the results from five independent experiments (Fig. 4A). To examine whether the inhibition of thrombin-induced alkalinization is due to reduced Ca2+ mobilization caused by expression of NCX1, the same experiment was carried out in Li+-containing medium, since Li+ is known to be a substrate for NHE1. When the extracellular Na+ was replaced by Li+, thrombin induced a large cytoplasmic alkalinization even in NCX1-transfected cells (Fig. 4B), consistent with the finding that a significant thrombin-induced [Ca2+]i rise was observed in Li+-containing medium (Fig. 2B). Thus the data suggest that intracellular Ca2+ plays a major role in thrombin-induced activation of NHE1.
|
|
We also examined the effect of NCX1 expression on cytoplasmic alkalinization induced by a 10-min stimulation with other stimuli (Fig. 4A). We found that cytoplasmic alkalinization caused by platelet-derived growth factor (PDGF)-BB at 100 ng/ml was slightly inhibited by expression of NCX1, suggesting a small contribution of Ca2+ to the PDGF response. Consistent with this observation, we did not detect a significant Ca2+ mobilization in CCL-39 cells in response to the same concentration of PDGF-BB. On the other hand, expression of NCX1 did not affect cytoplasmic alkalinization induced by 200 mM sucrose (hyperosmotic stress) or 1 µM phorbol 12-myristate 13-acetate (PMA; Fig. 4A), indicating that the intracellular Ca2+ plays no apparent role in the alkalinization induced by these stimuli.
PKC is known to be involved in growth factor-induced activation of NHE1. To assess the role of PKC in the alkalinization induced by various stimuli, cells were pretreated with PMA for 22 h. As shown in Fig. 5A, PMA-induced cytoplasmic alkalinization in CCL-39 cells was abolished by prior PMA treatment, indicating that phorbol ester-sensitive PKC was completely downregulated. The alkalinization by thrombin or PDGF-BB was inhibited by 30-50% in PMA-pretreated compared with control cells (Fig. 5A). Importantly, downregulation of PKC in NCX1-transfected cells resulted in complete inhibition of thrombin-induced cytoplasmic alkalinization (Fig. 5B), suggesting that the thrombin-induced activation of NHE1 occurs exclusively via parallel Ca2+- and PKC-dependent signaling pathways in CCL-39 cells. In contrast, ~40% of the PDGF-BB-induced alkalinization was still resistant to the PMA pretreatment in NCX1-transfected cells (Fig. 5B). Furthermore, hyperosmolarity-induced alkalinization was not inhibited by downregulation of PKC in NCX1-transfected and control CCL-39 cells (Fig. 5). Essentially the same results were obtained using PS120 cells transfected with NHE1 or NHE1 and NCX1 (data not shown; Fig. 6) .
|
|
Finally, we compared the effect of NCX1 expression on cytoplasmic
alkalinization induced by various stimuli in PS120 cells that had been
transfected with NHE1 or its mutants, including one with the
CaM-binding domain deleted (637-656). As shown in Fig. 6,
coexpression of NCX1 markedly reduced thrombin-induced cytoplasmic
alkalinization in NHE1-transfected PS120 cells. Deletion of the
CaM-binding domain from NHE1 also reduced thrombin-induced cytoplasmic
alkalinization markedly (Fig. 6). This is due to activation of NHE1 by
removal of the CaM-binding domain that has an autoinhibitory function
(3, 38). In this
637-656 mutant, coexpression of NCX1 did not
significantly change the extent of cytoplasmic alkalinization induced
by thrombin or PDGF-BB, as well as by sucrose or PMA (Fig. 6).
Thrombin-induced alkalinization in cells transfected with
637-656 was inhibited only partially (30-50%) by
prolonged PMA pretreatment. This finding was unexpected, because
637-656 was activated by PMA to the same level as that for
intact NHE1 (Fig. 6; data not shown). We also examined cytoplasmic
alkalinization induced by various stimuli in PS120 cells transfected
with
595, a mutant with two-thirds (amino acids 596-815) of the
cytoplasmic tail of NHE1 truncated (Fig. 6,
inset). Thrombin, PDGF-BB, and PMA,
but not sucrose, induced cytoplasmic alkalinization in the
595
transfectant. However, the response to all these stimuli disappeared in
another mutant,
566, in which amino acids 567-815 were deleted
(data not shown) (39, 40). Finally, the expression levels of these
deletion mutants are enough to mediate a change in
pHi, because the
22Na+
uptake activities of cells expressing these mutants at an acidic pHi are higher than the activity
of CCL-39 cells.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we analyzed the roles of intracellular Ca2+ and PKC in the activation of NHE1 caused by growth factors and hyperosmolarity. We found that overexpression of the cardiac Na+/Ca2+ exchanger NCX1 strongly inhibited a relatively long-lasting cytoplasmic alkalinization induced by thrombin in CCL-39 cells expressing an endogenous NHE1 and PS120 cells expressing an exogenous NHE1 (Figs. 3, 4, and 6). This inhibition is due to high Ca2+ extrusion activity of NCX1 expressed in the plasma membrane, because replacement of external Na+ with Li+ could restore a large amount of cytoplasmic alkalinization (Fig. 4B). These results are consistent with our finding obtained from a recent confocal Ca2+ imaging study that a thrombin-induced rise in Ca2+ concentration in the cell periphery was markedly inhibited in the NCX1-overexpressing cells (18a). All these findings strongly suggest that the intracellular Ca2+ is a major second messenger for thrombin-induced activation of NHE1 not only in its early phase but also in its late phase, although the thrombin-induced rise in mean cytoplasmic [Ca2+]i monitored by fura 2 fluorescence is short lived. This is consistent with recent reports that intracellular or extracellular Ca2+ depletion with Ca2+ chelators inhibits growth factor-induced activation of NHE1 (13, 15, 16, 25, 28, 31).
The Ca2+-dependent activation of
NHE1 can be explained by several mechanisms:
1) direct
Ca2+-CaM binding to NHE1 (3),
2)
Ca2+-CaM-dependent protein kinase,
3)
Ca2+-induced cell shrinkage (42),
4)
Ca2+-dependent activation of
tyrosine kinase (8), and 5)
Ca2+-dependent activation of
conventional PKCs. We previously showed that the observed
Ca2+ effect is not due to
Ca2+-induced cell shrinkage in
PS120 cells (38). Among the above mechanisms, direct
Ca2+-CaM binding to NHE1 seems to
be a major mechanism for the
Ca2+-dependent activation of NHE1,
because the effect of expression of NCX1 on thrombin-induced
alkalinization disappeared in cells expressing the NHE1 mutant
(637-656) depleted of the CaM-binding domain (Fig. 6). This
conclusion is consistent with our previous finding that deletion of the
CaM-binding domain completely abolished thrombin-induced activation of
NHE1 in the early phase (<1 min) of stimulation (38).
Expression of NCX1 did not totally abolish cytoplasmic alkalinization
induced by a long stimulation with thrombin (Figs. 3 and 4). In
addition, thrombin was still able to induce alkalinization in cells
expressing 637-656 (Fig. 6). This residual activation of NHE1
is due to phorbol ester-sensitive PKCs, because downregulation of PKC
almost completely abolished thrombin-induced alkalinization in CCL-39
cells expressing NCX1 or in PS120 cells coexpressing NHE1 and NCX1
(Fig. 4) (see RESULTS). Involvement
of PKC in NHE1 activation was suggested previously using various cell
types (1, 12, 20, 26, 28, 44). Several isoforms of PKC require Ca2+ for its full activation (27).
Therefore, it is possible that Ca2+ is involved in PKC-dependent
activation of NHE1. However, the contribution of PKC to the
Ca2+ requirement of NHE1
activation appears to be small, because expression of NCX1 did not
significantly change the thrombin response of the
637-656
transfectant. This conclusion is also supported by the present finding
that PKC downregulation greatly suppressed PDGF-BB-induced
alkalinization in CCL-39 cells, although PDGF-BB did not induce a
detectable Ca2+ mobilization under
the equivalent conditions (see
RESULTS). We noted that PKC
downregulation only partially inhibited thrombin-induced alkalinization
in PS120 cells cotransfected with
637-656 and NCX1 (see
RESULTS). We do not know why this
inhibition was incomplete in these cells, unlike CCL-39 cells
transfected with NCX1.
Unlike the thrombin response, PDGF-BB-induced cytoplasmic
alkalinization was only slightly inhibited by expression of NCX1. PDGF-BB-induced autophosphorylation at tyrosine residues of the PDGF
receptor presumably activates downstream signaling molecules such as
phospholipase C-, phosphatidylinositol 3'-kinase, and rasGAP
(36). Activation of phospholipase C-
leads to polyphosphoinositide breakdown, which in turn activates PKC and intracellular
Ca2+ mobilization. However, a
PDGF-BB-induced rise in
[Ca2+]i
was not detected in CCL-39 cells, consistent with the minimum observed
effect of NCX1 expression on the response induced by this agent (Fig.
4A). Because downregulation of PKC
could not completely abolish PDGF-BB-induced alkalinization in
NCX1-transfected CCL-39 cells (Fig. 5), another mechanism (or
mechanisms) is also involved in the NHE1 activation. Ma et al. (24),
using mutant PDGF receptors, reported that phosphatidylinositol
3'-kinase- and PKC-dependent pathways are required for
PDGF-induced activation of NHE1.
It is important to note that deletion mutants 637-656 and
595, but not
566, were activated by PMA (Fig. 6) (17, 40). Thus
the amino acid 566-595 domain appears to be a PKC-responsible region that is part of the "pH-maintenance" domain identified previously (17). Agents such as thrombin, PDGF-BB, and okadaic acid
were also able to induce cytoplasmic alkalinization in cells expressing
595 (Fig. 6) (see RESULTS).
Therefore, phosphorylation-dependent signaling pathways converge to the
specific segment (amino acids 566-595) of the pH-maintenance
domain. Thus separate cytoplasmic subdomains, amino acids 566-595
and 637-656 (CaM-binding domain), mediate
phosphorylation-dependent and
Ca2+-dependent signals,
respectively. Although intracellular signaling pathways leading to NHE1
activation are still not fully understood, recent reports suggest that
the mitogen-activating protein kinase cascade is one of the
intermediary steps linking receptor activation by agonists to NHE1
activation (5, 14). PKC has been reported to activate the
mitogen-activating protein kinase cascade through phosphorylation of
Raf-1 kinase (21). In addition, it has been shown that NHE1 can be
activated by expression of constitutively active heterotrimeric G
proteins G
12 and
G
13 or small G proteins rho,
rac, and cdc42 (7, 14, 37). These signaling molecules may transmit
signals to a regulatory factor (or factors) that directly binds to
specific segments in the cytoplasmic domain of NHE1. The calcineurin
homologue protein, one of such regulatory factors, has recently been
identified (23).
Response of cells to hyperosmotic stress is completely different from
response to growth factors. Expression of NCX1 or PKC downregulation
did not inhibit osmotic stress-induced cytoplasmic alkalinization.
Therefore, cell shrinkage-induced activation of NHE1 does not require
Ca2+ or PKC. This is consistent
with previous data obtained with other cell types, including bone cells
(6), astrocytes (35), or Ehrlich ascites tumor cells (30). Deletion of
the CaM-binding domain markedly (80%) reduced the extent of osmotic
stress-induced alkalinization (3) (cf. Figs. 4 and 6). In addition,
osmotic stress failed to induce cytoplasmic alkalinization in the
595 transfectant (Fig. 6), whereas it induced alkalinization
normally in the
698 transfectant (data not shown) (4). Therefore,
the CaM-binding domain may be essential for cell shrinkage-induced activation of NHE1, although a region within or near the membrane domain may also be important (4). The present finding that Ca2+ is not involved in osmotic
activation suggests that CaM-binding itself is not involved in the
osmotic activation. It is possible, however, that another molecule (or
molecules) binds to the CaM-binding domain of NHE1 in response to
osmotic stress and thus activates NHE1 via removal of its
autoinhibition. Recently,
-actinin has been shown to bind to the
N-methyl-D-glucamine
receptor in competition with CaM (43). Such a dual regulation by CaM
and a cytoskeletal protein may also occur in NHE1.
In summary, we conclude that direct Ca2+-CaM binding to NHE1 is a major intracellular signaling pathway for Ca2+-dependent activation of NHE1 in response to growth factors such as thrombin. Growth factors also activate PKC-dependent and PKC-, Ca2+-independent signaling pathways. Growth factor-induced cytoplasmic alkalinization lasts for a relatively long period of time, despite the rapid rise and decline of average [Ca2+]i. One possible interpretation is that subplasmalemmal Ca2+ concentration is maintained at a relatively high level for a long period after agonist stimulation. Alternatively, it is also possible that such an apparent discrepancy between [Ca2+]i and pHi might be explained by a "memory" effect, i.e., maintenance of the activated state of NHE1 long after CaM is dissociated from the autoinhibitory domain. Further studies are needed to clarify this point. Using cells coexpressing NHE1 and NCX1, we thus showed that [Ca2+]i is an important regulator of pHi. This relationship between pHi and [Ca2+]i should be important for control of cell function under physiological and pathophysiological conditions, particularly in cardiac or smooth muscle cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. J. Pouysségur (Nice, France) for the gift of PS120 cells.
![]() |
FOOTNOTES |
---|
This work was supported by Grant-in-Aid on Priority Area 321 and Grant-in-Aid for Scientific Research C from the Ministry of Education, Science, and Culture, and Special Coodination Funds Promoting Science and Technology (Encouragement System of Center of Excellence) from the Ministry of Science and Technology.
Address for reprint requests: S. Wakabayashi, Dept. of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishirodai 5-7-1, Suita, Osaka 565, Japan (phone: 81-6-833-5012; FAX: 81-6-872-7485; E-mail: wak{at}ri.ncvc.go.jp).
Received 22 October 1997; accepted in final form 18 February 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Berk, B. C.,
M. B. Taubman,
E. J. Cragoe, Jr.,
J. W. Fenton II,
and
K. K. Griendling.
Thrombin signal transaction mechanisms in rat vascular smooth muscle cells: calcium and protein kinase C-dependent and -independent pathways.
J. Biol. Chem.
265:
17334-17340,
1990
2.
Berridge, M. J.,
and
R. I. Irvine.
Inositol phosphates and cell signaling.
Nature
341:
197-205,
1989[Medline].
3.
Bertrand, B.,
S. Wakabayashi,
T. Ikeda,
J. Pouysségur,
and
M. Shigekawa.
The Na+/H+ exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Identification and characterization of calmodulin-binding sites.
J. Biol. Chem.
269:
13703-13709,
1994
4.
Bianchini, L.,
A. Kapus,
G. Lukacs,
S. Wasan,
S. Wakabayashi,
J. Pouysségur,
F. H. Yu,
J. Orlowski,
and
S. Grinstein.
Responsiveness of mutants of NHE1 isoform of Na+/H+ antiport to osmotic stress.
Am. J. Physiol.
269 (Cell Physiol. 38):
C998-C1007,
1995
5.
Bianchini, L.,
G. L'Allemain,
and
J. Pouysségur.
The p42/p44 mitogen-activated protein kinase cascade is determinant in mediating activation of the Na+/H+ exchanger (NHE1 isoform) in response to growth factors.
J. Biol. Chem.
272:
271-279,
1997
6.
Dascalu, A.,
A. Nevo,
and
R. Korenstein.
Hyperosmotic activation of the Na+/H+ exchanger in a rat bone cell line: temperature dependence and activation pathways.
J. Physiol. (Lond.)
456:
503-518,
1992[Abstract].
7.
Dhanasekaran, N.,
M. B. V. S. Vara Prasad,
S. J. Wadsworth,
J. M. Dermott,
and
G. Van Rossum.
Protein kinase C-dependent and -independent activation of Na+/H+ exchanger by G12 class of G proteins.
J. Biol. Chem.
269:
11802-11806,
1994
8.
Fukushima, T.,
T. K. Waddell,
S. Grinstein,
G. G. Goss,
J. Orlowski,
and
G. P. Downey.
Na+/H+ exchange activity during phagocytosis in human neutrophils: role of Fcr receptors and tyrosine kinases.
J. Cell Biol.
132:
1037-1052,
1996[Abstract].
9.
Ganz, M. B.,
G. Boyarsky,
R. B. Sterzel,
and
W. F. Boron.
Arginine vasopressin enhances pHi regulation in the presence of HCO3 by stimulating three acid-base transport systems.
Nature
337:
648-651,
1989[Medline].
10.
Grinstein, S.,
A. Rothstein,
and
S. Cohen.
Mechanism of osmotic activation of Na+/H+ exchange in rat thymic lymphocytes.
J. Gen. Physiol.
85:
765-787,
1985[Abstract].
11.
Grinstein, S.,
D. Rotin,
and
M. J. Mason.
Na+/H+ exchange and growth factor-induced cytosolic pH changes. Role in cellular proliferation.
Biochim. Biophys. Acta
988:
73-97,
1989[Medline].
12.
Gupta, A.,
C. J. Schwiening,
and
W. F. Boron.
Effects of CGRP, forskolin, PMA, and ionomycin on pHi dependence of Na-H exchange in UMR-106 cells.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1088-C1092,
1994[Abstract].
13.
Hendey, B.,
M. D. Mamrack,
and
R. W. Putnam.
Thrombin induces a calcium transient that mediates an activation of the Na+/H+ exchanger in human fibroblasts.
J. Biol. Chem.
264:
19540-19547,
1989
14.
Hooley, R.,
C. Y. Yu,
M. Symons,
and
D. L. Barber.
G13 stimulates Na+-H+ exchange through distinct Cdc42-dependent and RhoA-dependent pathways.
J. Biol. Chem.
271:
6152-6158,
1996
15.
Huang, C.-L.,
M. G. Cogan,
E. J. Cragoe, Jr.,
and
H. E. Ives.
Thrombin activation of the Na+/H+ exchanger in vascular smooth muscle cells. Evidence for a kinase C-independent pathway which is Ca2+-dependent and pertussis toxin-sensitive.
J. Biol. Chem.
262:
14134-14140,
1987
16.
Hubel, C. A.,
and
R. F. Highsmith.
Endothelin-induced changes in intracellular pH and Ca2+ in coronary smooth muscle: role of Na+/H+ exchange.
Biochem. J.
310:
1013-1020,
1995[Medline].
17.
Ikeda, T.,
B. Schmitt,
J. Pouysségur,
S. Wakabayashi,
and
M. Shigekawa.
Identification of cytoplasmic subdomains that control pH-sensing of the Na+/H+ exchanger (NHE1): pH-maintenance, ATP-sensitive, and flexible loop domains.
J. Biochem.
121:
295-303,
1997[Abstract].
18.
Iwamoto, T.,
Y. Pan,
S. Wakabayashi,
T. Imagawa,
H. L. Yamanaka,
and
M. Shigekawa.
Phosphorylation-dependent regulation of cardiac Na+/Ca2+ exchanger via protein kinase C.
J. Biol. Chem.
271:
13609-13615,
1996
18a.
Iwamoto, T., S. Wakabayashi, T. Imagawa, and M. Shigekawa. Na+/Ca2+ exchanger
overexpression impairs calcium signaling in fibroblasts: inhibition of
the [Ca2+] increase at the cell periphery and retardation
of cell adhesion. Eur. J. Cell Biol. In press.
19.
Iwamoto, T.,
S. Wakabayashi,
and
M. Shigekawa.
Growth factor-induced phosphorylation and activation of aortic smooth muscle Na+/Ca2+ exchanger.
J. Biol. Chem.
270:
8996-9001,
1995
20.
Kimura, M.,
J. P. Gardner,
and
A. Aviv.
Agonist-evoked alkaline shift in the cytosolic pH set point for activation of Na+/H+ antiport in human platelets.
J. Biol. Chem.
265:
21068-21074,
1990
21.
Kolch, W.,
G. Hidecker,
P. Lioyd,
and
U. R. Rapp.
Raf-1 protein kinase is required for growth of induced NIH/3T3 cells.
Nature
349:
426-428,
1991[Medline].
22.
L'Allemain, G.,
S. Paris,
and
J. Pouysségur.
Growth factor action and intracellular pH regulation in fibroblasts. Evidence for a major role of the Na+/H+ antiporter.
J. Biol. Chem.
259:
5809-5815,
1984
23.
Lin, X.,
and
D. L. Barber.
A calcineurin homologous protein inhibits GTPase-stimulated Na-H exchange.
Proc. Natl. Acad. Sci. USA
93:
12631-12636,
1996
24.
Ma, Y.-H.,
H. P. Reusch,
E. Wilson,
J. A. Escobedo,
W. J. Fantl,
L. T. Williams,
and
H. E. Ives.
Activation of Na+/H+ exchange by platelet-derived growth factor involves phosphatidylinositol 3'-kinase and phospholipase C.
J. Biol. Chem.
269:
30734-30739,
1994
25.
McSwine, R. L.,
J. Li,
and
M. L. Villereal.
Examination of the role for Ca2+ in regulation and phosphorylation of the Na+/H+: antiporter NHE1 via mitogen and hypertonic stimulation.
J. Cell. Physiol.
168:
8-17,
1996[Medline].
26.
Moolenaar, W. H.,
R. Y. Tsien,
P. T. van der Saag,
and
S. W. de Laar.
Na+/H+ exchange and cytoplasmic pH in the action of growth factors in human fibroblasts.
Nature
304:
645-648,
1983[Medline].
27.
Newton, A. C.
Regulation of protein kinase C.
Curr. Opin. Cell Biol.
9:
161-167,
1997[Medline].
28.
Nieuwland, R.,
G. V. Willigen,
and
J.-W. N. Akkerman.
Different pathways for control of Na+/H+ exchange via activation of the thrombin receptor.
Biochem. J.
297:
47-52,
1994[Medline].
29.
Paris, S.,
and
J. Pouysségur.
Growth factors activate the Na+/H+ antiporter in quiescent fibroblasts by increasing its affinity for intracellular H+.
J. Biol. Chem.
259:
10989-10994,
1984
30.
Pedersen, S. F.,
B. Kramhøft,
N. K. Jørgensen,
and
E. K. Hoffmann.
Shrinkage-induced activation of the Na+/H+ exchanger in Ehrlich ascites tumor cells: mechanisms involved in the activation and a role of the exchanger in cell volume regulation.
J. Membr. Biol.
149:
141-159,
1996[Medline].
31.
Poch, E.,
A. Botey,
J. Gaya,
A. Cases,
F. Rivera,
and
L. Revert.
Intracellular calcium mobilization and activation of the Na+/H+ exchanger in platelets.
Biochem. J.
290:
617-622,
1993[Medline].
32.
Pouysségur, J.
The growth factor-activatable Na+/H+ exchange system: a genetic approach.
Trends Biochem. Sci.
10:
453-455,
1985.
33.
Pouysségur, J.,
C. Sardet,
A. Franchi,
G. L'Allemain,
and
S. Paris.
A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH.
Proc. Natl. Acad. Sci. USA
81:
4833-4837,
1984[Abstract].
34.
Sardet, C.,
A. Franchi,
and
J. Pouysségur.
Molecular cloning, primary structure and expression of the human growth factor-activatable Na+/H+ antiporter.
Cell
56:
271-280,
1989[Medline].
35.
Shrode, L. D.,
J. D. Klein,
W. C. O'Neill,
and
R. W. Putnam.
Shrinkage-induced activation of Na+/H+ exchange in primary rat astrocytes: role of myosin light-chain kinase.
Am. J. Physiol.
269 (Cell Physiol. 38):
C257-C266,
1995
36.
Van der Geer, P.,
T. Hunter,
and
R. A. Lindberg.
Receptor protein-tyrosine kinases and their signal transduction pathways.
Annu. Rev. Cell Biol.
10:
251-338,
1994.
37.
Voyno-Yasenetskaya, T.,
B. R. Conklin,
R. L. Gilbert,
R. Hooley,
H. R. Bourne,
and
D. L. Barber.
G13 stimulates Na-H exchange.
J. Biol. Chem.
269:
4721-4724,
1994
38.
Wakabayashi, S.,
B. Bertrand,
T. Ikeda,
J. Pouysségur,
and
M. Shigekawa.
Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H+-sensitive and Ca2+ regulation-defective.
J. Biol. Chem.
269:
13710-13715,
1994
39.
Wakabayashi, S.,
B. Bertrand,
M. Shigekawa,
P. Fafournoux,
and
J. Pouysségur.
Growth factor activation and "H+-sensing" of the Na+/H+ exchanger isoform 1 (NHE1). Evidence for an additional mechanism not requiring direct phosphorylation.
J. Biol. Chem.
269:
5583-5588,
1994
40.
Wakabayashi, S.,
P. Fafournoux,
C. Sardet,
and
J. Pouysségur.
The Na+/H+ antiporter cytoplasmic domain mediates growth factor signals and controls "H+-sensing."
Proc. Natl. Acad. Sci. USA
81:
4833-4837,
1984[Abstract].
41.
Wakabayashi, S.,
M. Shigekawa,
and
J. Pouysségur.
Molecular physiology of vertebrate Na+/H+ exchangers.
Physiol. Rev.
77:
51-74,
1997
42.
Wöll, E.,
M. Ritter,
W. Scholz,
D. Heussinger,
and
F. Lang.
The role of calcium in cell shrinkage and intracellular alkalinization by bradykinin in Ha-ras oncogene expressing cells.
FEBS Lett.
322:
261-265,
1993[Medline].
43.
Wyszynski, M.,
J. Lin,
A. Rao,
E. Nigh,
A. H. Beggs,
A. M. Craig,
and
M. Sheng.
Competitive binding of -actinin and calmodulin to the NMDA receptor.
Nature
385:
439-442,
1996.
44.
Yasutake, M.,
R. S. Haworth,
A. King,
and
M. Avkiran.
Thrombin activates the sarcolemmal Na+/H+ exchanger. Evidence for a receptor-mediated mechanism involving protein kinase C.
Circ. Res.
79:
705-715,
1996