Regulation of the Na+/H+ exchanger in fibroblasts overexpressing the Na+/Ca2+ exchanger

Toshitaro Ikeda, Takahiro Iwamoto, Shigeo Wakabayashi, and Munekazu Shigekawa

Department of Molecular Physiology, National Cardiovascular Research Institute, Osaka 565, Japan

    ABSTRACT
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Abstract
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Procedures
Results
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References

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 alpha -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
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Abstract
Introduction
Procedures
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References

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 alpha -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
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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 (Delta 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
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Abstract
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Procedures
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References

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 (Delta 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 Delta 637-656, which was detected as a protein band of ~110 kDa (Fig. 1B).


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Fig. 1.   Expression of Na+/Ca2+ and Na+/H+ exchangers (NCX1 and NHE1) and a deletion mutant of NHE1 determined by immunoblot analysis. Crude membrane proteins were prepared from CCL-39 or PS120 cells stably transfected with NCX1, NHE1, or a deletion mutant of NHE1 (Delta 637-656). Proteins (50 µg) were subjected to SDS-PAGE followed by immunoblot analysis with anti-NCX1 (A) or anti-NHE1 (B) antibody: control and NCX1-transfected CCL-39 cells (left lanes), PS120 cells transfected with NHE1 and with NHE1 and NCX1 (middle lanes), and PS120 cells transfected with Delta 637-656 and with Delta 637-656 and NCX1 (right lanes). Broad bands showing molecular masses of ~120 and 110 kDa represent mature NCX1 and NHE1 proteins, respectively. Other bands may correspond to immature proteins or proteolytic products.

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).


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Fig. 2.   Thrombin-induced change in intracellular Ca2+ concentration ([Ca2+]i) in control (A) and NCX1-transfected (B) CCL-39 cells. Change in [Ca2+]i was measured using fura 2 fluorescence in medium containing 140 mM NaCl or 140 mM LiCl. Arrows, addition of thrombin (Th, 2 U/ml) to medium.

                              
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Table 1.   Thrombin-induced [Ca2+]i responses in control and NCX1-transfected CCL-39 cells in Na+- or Li+-containing medium

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.


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Fig. 3.   Effect of NCX1 overexpression on time course of cytoplasmic alkalinization induced by thrombin. Thrombin (2 U/ml)-induced change in intracellular pH (pHi) was measured in control (open circle ) and NCX1-transfected CCL-39 cells (bullet ). Resting pHi values before thrombin stimulation were 7.15 ± 0.05 (n = 3) and 7.12 ± 0.03 (n = 3) for control and NCX1-transfected cells, respectively. Values are means ± SD of 3 determinations.


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Fig. 4.   Effect of NCX1 overexpression on cytoplasmic alkalinization induced by various extracellular stimuli. Change in pHi was measured in control and NCX1-transfected CCL-39 cells. A: cells were incubated for 10 min with thrombin (2 U/ml), platelet-derived growth factor-BB (PDGF, 100 ng/ml), sucrose (200 mM), or phorbol 12-myristate 13-acetate (PMA, 1 µM). Resting pHi values before stimulation were 7.19 ± 0.01 (n = 3) and 7.18 ± 0.02 (n = 3) for control and NCX1-transfected CCL-39 cells, respectively. Data for thrombin and PGDF-BB are statistically significant by Student's paired t-test (P < 0.05 vs. control CCL-39 cells). Values are means ± SD of at least 3 determinations. B: thrombin-induced alkalinization was measured at 10 min in medium containing 140 mM NaCl or 140 mM LiCl. Na+o and Li+o, extracellular Na+ and Li+. Values are means ± SD of at least 3 determinations.

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) . 


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Fig. 5.   Effect of prolonged preincubation with PMA on cytoplasmic alkalinization induced by various extracellular stimuli. Cells were incubated for 16 h with or without 0.2 µM PMA in growth medium and for another 5 h with or without 0.2 µM PMA in serum-free medium. Cells were further incubated for 1 h with or without 0.2 µM PMA in serum-free, bicarbonate-free medium buffered with HEPES (pH 7.0). Cells were then subjected to measurement of pHi changes induced by various extracellular stimuli as in Fig. 4A. Values are means ± SD of 3 determinations. A: control cells. Resting pHi values before stimulation were 7.13 ± 0.04 (n = 3) and 7.14 ± 0.03 (n = 3) for control and PMA-pretreated cells, respectively. B: NCX1-transfected CCL-39 cells. Resting pHi values before stimulation were 7.12 ± 0.03 (n = 3) and 7.13 ± 0.03 (n = 3) for control and PMA-pretreated cells, respectively.


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Fig. 6.   Effect of NCX1 overexpression on cytoplasmic alkalinization induced by various extracellular stimuli in PS120 cells expressing NHE1 and its mutants. Change in pHi was measured in PS120 cells cotransfected with NCX1 and NHE1 variants (NHE1, Delta 637-656, or Delta 595). Cells were incubated for 10 min with thrombin (2 U/ml), PDGF-BB (100 ng/ml), sucrose (200 mM), or PMA (1 µM). Resting pHi values before stimulation were 7.27 ± 0.04 (n = 3) and 7.25 ± 0.03 (n = 3) for control and NCX1-transfected cells expressing NHE1, respectively, and 7.30 ± 0.03 (n = 3) and 7.28 ± 0.02 (n = 3) for control and NCX1-transfected cells expressing Delta 637-656, respectively. Values are means ± SD of 3 determinations. Inset: change in pHi measured in Delta 595 transfectant. Resting pHi before stimulation was 7.32 ± 0.02 (n = 3). Cytoplasmic alkalinization caused by thrombin, PGDF-BB, or PMA is statistically significant by Student's paired t-test (P < 0.05, before vs. after stimulation).

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 (Delta 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 Delta 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 Delta 637-656 was inhibited only partially (30-50%) by prolonged PMA pretreatment. This finding was unexpected, because Delta 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 Delta 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 Delta 595 transfectant. However, the response to all these stimuli disappeared in another mutant, Delta 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
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Abstract
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Procedures
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References

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 (Delta 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 Delta 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 Delta 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 Delta 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-gamma , phosphatidylinositol 3'-kinase, and rasGAP (36). Activation of phospholipase C-gamma 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 Delta 637-656 and Delta 595, but not Delta 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 Delta 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 Galpha 12 and Galpha 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 Delta 595 transfectant (Fig. 6), whereas it induced alkalinization normally in the Delta 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, alpha -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
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Abstract
Introduction
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Results
Discussion
References

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Am J Physiol Cell Physiol 274(6):C1537-C1544
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