Activation of Na+H+ exchanger by hepatocyte growth factor in hepatocytes

Activation of Na+/H + Exchanger by Hepatocyte Growth Factor in Hepatocytes AI~RA KANEKO, NORIO HAYASHI, YUJI TANAKA, MASAYOSHI HORIMOTO, TOSHIFUM! ITO, YUTAKA SASAKI, HIDEYUKI FUSAMOTO, AND TAKENOBU KAMADA

The effect o f t h e h e p a t o c y t e g r o w t h factor (HGF) o n t h e Na+/H ÷ e x c h a n g e r w a s s t u d i e d u s i n g p r i m a r y cult u r e d h e p a t o c y t e s . HGF i n d u c e d intracellular pH (pHi) e l e v a t i o n of 0.10 pH u n i t s in h e p a t o c y t e s c u l t u r e d for 4 to 7 hours; t h e r e s p o n s e w a s l o w e r after o t h e r culture periods. E v e n w i t h t h e s a m e culture period, intercellular h e t e r o g e n e i t y w a s f o u n d in the r e s p o n s i v e n e s s to HGF. This h e t e r o g e n e i t y m a y be partially a c c o u n t e d for by the w e a k but significant c o r r e l a t i o n o b s e r v e d b e t w e e n the basal p h i level a n d the d e g r e e of pHi e l e v a t i o n c a u s e d by HGF in h e p a t o c y t e s . The pHi e l e v a t i o n c a u s e d by HGF w a s b l o c k e d o n p r e t r e a t m e n t o f t h e h e p a t o c y t e s w i t h amiloride, s u g g e s t i n g that HGF activates t h e Na*/ H ÷ e x c h a n g e r . This h y p o t h e s i s w a s c o n f i r m e d by t h e fact that HGF i n c r e a s e d t h e initial rapid rate o f cell alkalization of acid-loaded h e p a t o c y t e s . The t y r o s i n e k i n a s e inhibitor, genistein, also b l o c k e d t h e elevation, c o n s i s t e n t w i t h the fact that HGF receptor/c-met h a s a t y r o s i n e kin a s e domain. To clarify t h e signal t r a n s d u c t i o n p a t h w a y from t y r o s i n e k i n a s e to the Na÷/H ÷ e x c h a n g e r , w e examined the effects o f inhibitors o f o t h e r k i n a s e s (H-7, H-8, a n d W-7) o n the HGF-induced p h i e l e v a t i o n a n d f o u n d that o n l y W-7 b l o c k e d it. This p h i e l e v a t i o n w a s also prevented on preincubation of the hepatocytes with thapsigargin, w h i c h b l o c k s t h e c a l c i u m r e s p o n s e c a u s e d by HGF. T h e s e results s u g g e s t that HGF activates t h e Na÷/H ÷ e x c h a n g e r in h e p a t o c y t e s t h r o u g h a t y r o s i n e kin a s e - e a l c i u m / c a l m o d u l i n - d e p e n d e n t p a t h w a y . (HEPATOLOGY 1995;22:629-636.)

Hepatocyte growth factor (HGF) is one of the most potent mitogens for hepatocytes, ~3 and the mechanism underlying hepatocyte proliferation caused by HGF is being studied. We previously showed that calcium acts as a second messenger for HGF, leading to HGF-in-

Abbreviations: HGF, hepatocyte growth factor; pHi, intracellular pH. From the First Department of Medicine, Osaka University Medical School, Osaka, Japan. Received March 22, 1994; accepted March 30, 1995. Supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. Address reprint requests to: Norio Hayashi, MD, First Department of Medicine, Osaka University Medical School, Yamadaoka 2-2, Suita, Osaka 565, Japan. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2202-003653.00/0

duced calcium oscillations. 4 This calcium response caused by HGF is considered to be caused by the activation of phospholipase C-T by tyrosine kinase, which is within the HGF receptor~c-met. In fact, tyrosine phosphorylation of phospholipase C-T in HGF-stimulated hepatocytes has recently been reported. 5 The receptors of most growth factors have tyrosine kinase domains, and the binding of a growth factor to its receptor activates a number of signal pathways through tyrosine kinase as well as the phospholipase C-T-calcium pathway. 6 Activation of the Na+/H ÷ exchanger occurs in a variety of cells after growth factor stimulation, 7 and is considered to be one of the important pathways in cell proliferation. Recent studies have shown the tissue distribution of Na÷/H ÷ exchanger isoforms, and NHE-1, which was cloned by Sardet et al s as a growth factoractivated Na+/H ÷ exchanger, was also shown to be expressed in liver. 9 Another isoform, NHE-2, is also expressed in liver, 1° whereas NHE-3 and NHE-4 are not. 9'11 Whether or not HGF induces activation of the Na÷/H ÷ exchanger in hepatocytes is not known, which led us to examine the changes in the intracellular pH (pHi) level in hepatocytes after H G F stimulation. Many attempts have been made to clarify the signal transduction pathways for Na+/H ÷ exchanger activation, but the results are controversial. In some cells, activation of protein kinase C leads to Na÷/H ÷ exchanger activation, '2'13 but in others, elevation of the intracellular calcium concentration causes the activation. 14-17 In addition, in some cells, different reagents use different signal pathways to activate the Na+/H ÷ exchanger even in the same cell. ls-2° It is possible that different cells activate the Na+/H ÷ exchanger through different signal pathways and that different pathways for the activation exist even in the same cell. In hepatocytes, little is known about the signal transduction pathways for the activation of the Na÷/H ÷ exchanger. In this study, we examined the pathway for HGF-induced Na÷/H ÷ exchanger activation in hepatocytes using various kinase inhibitors and some other reagents. MATERIALS A N D M E T H O D S Animals. Male Sprague-Dawley rats (Japan SLC Inc., Shizuoka, Japan) were housed under controlled temperature, humidity, and light conditions, and received food and water ad libitum.

629

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ET AL

Materials. Recombinant human HGF, purified from the culture supernatant of Chinese hamster ovary cells transfected with complementary DNA for human HGF, was kindly supplied by Dr Tadashi Hishida, Research Center, Mitsubishi Kasei Corporation, Yokohama, Japan, and Dr Hirohito Tsubouchi, Second Department of Medicine, Miyazaki Medical University, Miyazaki, Japan. 21 2',7'-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 acetoxymethyl ester (fura-2/AM), and fura-2 were obtained from Molecular Probes Inc. (Eugene, OR). Collagenase was purchased from Wako Pure Chemical Co. (Osaka, Japan), and collagen (type I) from Nitta Zeratin Co. (Osaka, Japan). H-7, H-8, and W-7 were from Seikagaku Kogyo (Tokyo, Japan), and genistein from Gibco BRL (Grand Island, NY). Thapsigargin, amiloride, and all other chemicals were of reagent grade and obtained from Sigma Chemical Co. (St. Louis, MO). Preparation o f Cultured Hepatoeytes. Isolated hepatocytes were prepared by the two-step collagenase perfusion method from male Sprague-Dawley rats (weighing 200 to 300 g), as described previously.4'22'23 The hepatocytes were plated on glass coverslips, which had been covered with silicon rings (Flexiperm; Heraeus, Hanau, Germany) and coated with type I collagen (60 #g/cm2), at a cell density of 10~ cells/cm2 in Williams E medium supplemented with 5% fetal bovine serum, 1 #mol/L of dexamethasone, and 1 #mol/L of insulin. The cells were then incubated at 37°C under an atmosphere of 5% CO2 and 95% air. The medium was changed after 3 hours. Dye Loading. Cultured hepatocytes were washed with Hanks-HEPES buffer (10 mmol/L of HEPES, without bicarbonate, pH 7.4) and then incubated with the same buffer containing 5 #mol/L of BCECF-AM or 5 #mol/L of fura-2/AM at 37°C for 80 minutes. After this incubation, the hepatocytes were washed with Hank's-HEPES buffer to remove the unincorporated fluoroprobes and then the buffer was replaced with fresh Hank's-HEPES buffer. Next, the hepatocytes were placed on the stage of an inverted fluorescence microscope (TMD; Nikon, Tokyo, Japan) for 20 minutes before the fluorescence measurement. A small change in the basal hepatocyte pHi was observed when the cells were moved from the 5% C Q and 95% air atmosphere to 100% air, but the basal pHi became equilibrated within 20 minutes on the stage. In some experiments, the hepatocytes were preincubated with amiloride for 20 minutes, with genistein, H-7, H-8, or W-7 for 60 minutes, or with thapsigargin for 100 minutes before the measurement. Even after their preincubation with these inhibitors, they remained normal in appearance, and were considered to be intact because they took up sufficient amounts of fluorescent dyes without subsequent efflux of the dyes. These hepatocytes also maintained a normal intracellular calcium concentration. Intracellular pH Measurement. HGF was added to BCECF-loaded hepatocytes, and then fluorescence images were taken every 40 seconds for 20 minutes at room temperature. Fluorescence images were obtained with a silicon-intensified target camera (C2400-08H; H a m a m a t s u Photonics, Hamamatsu, Japan), with excitation wavelengths of 490 and 450 nm (10 nm bandwidth), and an emission wavelength of 540 nm. The integration time for each image was 0.5 seconds. After correction for camera dark images, ratio images (490/ 450) were calculated. At the end of each experiment, in situ calibration curves between pH 7.0 and 7.8 were generated for the same cells by the addition of 12.5 #mol/L of nigericin,

HEPATOLOGY

August 1995

TABLE 1. E f f e c t o f C u l t u r e P e r i o d o n H G F - I n d u c e d pHi Elevation Culture Period

n

Basal pHi

ApHi

2-4 hr 4-7 hr 7-10 hr

43 42 36

7.29 ± 0.07a 7.33 +_ 0.08b 7.44 _+ 0.07c

0.03 ÷ 0.04d 0.10 ± 0.05e 0.05 ± 0.04f

NOTE. HGF (0.1 nmol/L) was added to BCECF-loaded hepatocytes after various culture periods, and then the changes in pHi (ApHi) were measured, n denotes the number of cells from more than 3 hepatocyte preparations. Values are means ± SD. Significance was determined by Student's unpaired t-test. a VS. c, b VS. c, d VS. e, e VS. f; P < .0001. a vs. b, d vs. fi P < .05.

a K÷/H ÷ ionophore, in 140 mmolfL of K ÷ buffer to equalize the pHi and extracellular pH. 24 The ratio images (490/450) were converted to pHi according to the individual in situ calibration curves, and we could determine the movement in pHi of individual hepatocytes. We could examine 15 to 20 hepatocytes on each coverslip and perform three measurements for each hepatocyte preparation. During the 20-minute measurement at room temperature, no leakage of the fluorescent dye was observed, judging from the changes in the two fluorescence strengths of the cells and that of the cellfree field. To minimize photobleaching, the strength of the excitation light was lowered using neutral-density filters. In the experiments on the initial rapid rate of cell alkalization in acid-loaded hepatocytes, hepatocytes were acid-loaded by means of pulse exposure to 30 mmol/L of NH4C1. For these experiments, 30 mmol/L of NH4C1 replaced equimolar amounts of NaC1 in the Hank's-HEPES buffer. BCECFloaded hepatocytes (HGF-treated and untreated) were exposed to this NH4C1 buffer for 4 minutes and then the buffer was replaced with Hank's-HEPES buffer. The rate of change of pHi (ApHYmin) during the initial 40 seconds was calculated as the initial rapid rate of pHi recovery. Intraeellular Ca Measurement. Fluorescence images of fura-2-1oaded hepatocytes were obtained every 20 seconds for 20 minutes at room temperature with excitation wavelengths of 340 and 380 nm (10 nm bandwidth), and an emission wavelength of 510 nm. After correction for camera dark images, ratio images (340/380) were calculated. The ratio images were converted to Ca 2÷ concentrations according to the Ca 2÷ calibration curve, which was previously prepared with Ca2÷-EGTA buffer as described previously.4'23'25 Statistical Analysis. Data are expressed as means _+ SD. Significance was determined by means of Student's unpaired t-test. Analysis of the linear correlation between variables was performed by the least squares method. RESULTS

T h e a d d i t i o n of 0.1 n m o l / L of H G F i n d u c e d p H i e l e v a t i o n in p r i m a r y c u l t u r e d h e p a t o c y t e s , b u t t h e d e g r e e of p H i e l e v a t i o n i n d u c e d b y H G F w a s d e p e n d e n t on t h e t i m e in t h e c u l t u r e , w i t h h e p a t o c y t e s c u l t u r e d for 4 to 7 h o u r s b e i n g t h e m o s t r e s p o n s i v e to H G F ( T a b l e 1). B e c a u s e h e p a t o c y t e s c u l t u r e d for o t h e r p e r i o d s w e r e l e s s r e s p o n s i v e to H G F , we p e r f o r m e d t h e f o l l o w i n g e x p e r i m e n t s u s i n g h e p a t o c y t e s f r o m 4- to 7 - h o u r cul-

HEPATOLOGY Vol. 22, No. 2, 1995

KANEKO ET AL

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was between 7.19 to 7.47, although that after 7 to 10 hours of culture was a little higher (7.30 to 7.60). The pHi in most of the hepatocytes began to increase within a few minutes after stimulation and reached a plateau within 20 minutes. The degree of pHi elevation caused by HGF differed from cell to cell even among cells from the same culture. Some cells showed a pHi increase of 0.20 pH units (Fig. 1A), some showed none (Fig. 1C), and others showed an intermediate increase (Fig. 1B). The average increase was 0.10 pH units (Table 1). We found a weak but significant correlation between the basal pHi and the degree of pHi elevation caused by HGF (Fig. 2). We also found that 1 nmol/L of epidermal growth factor induced a similar degree of pHi elevation, 2~ whereas the vehicle (Hank's-HEPES buffer) did not induce any (Table 2). To clarify the mechanism underlying pHi elevation induced by HGF, we examined the effect of amiloride,

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FIG. 1. Various pHi response patterns after the addition of 0.1 nmol/L of HGF in hepatocytes after 4 to 7 hours of culture. The buffer of BCECF-loaded hepatocytes was replaced with bicarbonate-free Hank's-HEPES buffer and then the hepatocytes were placed on the stage of an inverted fluorescence microscope for 20 minutes. Next, 0.1 nmol/L of HGF was added to the buffer and fluorescence images were obtained for 20 minutes. At the end of each measurement, perfusion with calibration buffers of between pH 7.0 and pH 7.8 was performed to obtain in situ calibration curves. Some cells showed a pHi increase of 0.20 pH units (A), some showed none (C), and others showed an intermediate increase (B).

tures. Figure 1 shows three representative pHi response patterns of these hepatocytes after the addition of 0.1 nmol/L of HGF. The basal p h i of the hepatocytes

TABLE 2. E f f e c t s o f H G F a n d E p i d e r m a l G r o w t h Factor on pHi of Hepatocytes Agonist

n

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ApHi

Control 0.1 nmol/L HGF 1 nmol/L epidermal growth factor

40 42

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32

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0.09 _+ 0.05I"

NOTE. HGF (0.1 nmol/L), epidermal growth factor (1 nmol/L), or vehicle (control) was added to BCECF-loaded hepatocytes, and then the changes in pHi (April) were measured. All experiments were performed with hepatocytes after 4 to 7 hours of culture, n denotes the number of cells from more t h a n 3 hepatocyte preparations. Values are means _+ SD. Significance was determined by Student's unpaired t-test. * Not significant vs. control. ¢ P < .0001 vs. control.

632 KANEKO ET AL

HEPATOLOGY August 1995

TABLE 3. E f f e c t s o f V a r i o u s A g e n t s o n I t G F - I n d u c e d pHi Elevation pHi

Agent

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Basal

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41

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0.4

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0.10 0.00 0.02 -0.01 0.11 0.09

± 0.05 ± 0.055 ± 0.035 _+ 0.075 ± 0.05* ± 0.06*

an inhibitor of the Na÷/H ÷ exchanger, on the HGFinduced pHi elevation. The pHi elevation induced by H G F was blocked on pretreatment of the hepatocytes with amiloride, as shown in Table 3, suggesting that HGF induced pHi elevation through activation of the Na÷/H ÷ exchanger. To confirm this hypothesis, we examined the effect of H G F on the initial rapid rate of cell alkalization in acid-loaded hepatocytes, which was inhibited by amiloride (mean ApH/minute: untreated cells, 0.12; amiloride-treated cells, 0.056). Figure 3 3 0 m M NH4CI l

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shows representative examples of pHi recovery from an acid load in HGF-treated and untreated hepatocytes. The initial rate of pHi recovery from an acid load in the HGF-treated cells was higher than that in the untreated cells. Figure 4 shows a comparison of the initial rates of pHi recovery from an acid load in HGFtreated and untreated cells. The former was significantly higher than the latter, suggesting Na÷/H ÷ exchanger activation by HGF. We next examined the effects of various kinase inhibitors on the HGF-induced pHi elevation to clarify the signal transduction pathways for Na÷/H ÷ exchanger activation. HGF receptor~c-met has a tyrosine kinase domain, and its activation is considered to be the first step in signal transduction. First, we examined the effect of a tyrosine kinase inhibitor, genistein, which inhibited the HGF-induced calcium response (HGF-responsive cells: untreated cells, 83.0%; genistein-treated cells, 6.8%), and found that it blocked the pHi elevation induced by H G F (Table 3). Tyrosine kinase seems to participate in the activation of the Na÷/H ÷ exchanger, but because this activation was reported to be induced on the phosphorylation of its serine r e s i d u e s y it is not likely that tyrosine kinase directly phosphorylates and activates the Na÷/H ÷ exchanger. This led us to examine the effects of other kinase inhibitors on the HGF-induced pHi elevation to clarify the signal pathway between tyrosine kinase and the Na÷/H ÷ exchanger. Preincubation of hepatocytes with H-7 (a protein kinase C

HEPATOLOGYVol. 22, No. 2, 1995

inhibitor) or H-8 (a protein kinase A inhibitor) did not affect the pHi elevation caused by HGF, but that with W-7 (a calmodulin inhibitor) completely blocked it (Table 3). These results indicated the existence of a calcium/ calmodulin-dependent pathway for Na÷/H + exchanger activation by HGF. To confirm this hypothesis, we examined the effects of thapsigargin, which inhibits calcium-ATPase in the endoplasmic reticulum and depletes the intracellular calcium pool (i.e., endoplasmic reticulum), on HGF-induced calcium and pHi movement. The basal intracellular calcium concentration in hepatocytes pretreated with thapsigargin for 100 minutes was similar to that in untreated hepatocytes. The addition of 0.1 nmol/L of HGF induced calcium oscillations in untreated hepatocytes, as shown in Fig. 5A, but this response was blocked on pretreatment of the hepatocytes with thapsigargin (Fig. 5B) because the intracellular calcium pool had been depleted on the thapsigargin pretreatment. Under such conditions, the hepatocytes did not show HGF-induced pHi elevation (Fig. 5C and Table 3), which supported the hypothesis of calcium/calmodulin-dependent activation of the Na÷/ H ÷ exchanger. DISCUSSION

The Na÷/H ÷ exchanger is considered to play an important role in cell proliferation from the fact that most growth factors induce its activation in many types of cells, v In the case of hepatocytes, there has only been one report that a growth factor (epidermal growth factor) stimulates the Na÷/H ÷ exchanger to induce pHi elevation, 2s but the concentration of epidermal growth factor used was extremely high (30 nmol/L). Although HGF is one of the most potent mitogens for hepatocytes, whether or not HGF induces activation of the Na÷/H ÷ exchanger and pHi elevation of hepatocytes is not known. Therefore, we first examined the pHi change in hepatocytes after the addition of HGF and found that hepatocyte pHi elevation was induced by 0.1 nmol/L of HGF, which caused maximal stimulation of DNA synthesis in primary cultured hepatocytes. 13 The degree of pHi elevation induced by 0.1 nmol/L of HGF was similar to that by 1 nmol/L of epidermal growth factor. In this study, we measured the pHi changes in hepatocytes in bicarbonate-free buffer because bicarbonatecontaining buffer would not allow the detection of Na*/ H ÷ exchanger activation-induced pHi changes because of the buffering action of bicarbonate-dependent pHi regulating systems. 7'2~-31 In fact, some laboratories have reported that growth factor-induced pHi elevation is not observed in bicarbonate-containing buffer although the Na÷/H ÷ exchanger is activated. 7'32'33 This suggests that what is important in the activation of the Na÷/H ÷ exchanger is not pHi elevation caused by H ÷ efflux but Na ÷ influx coupled with H ÷ efflux. The pHi elevation caused by HGF depended on the time in culture, as we previously reported for the HGF-

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FIG. 5. Effects ofthapsigargin on HGF-induced calcium and pHi changes. Hepatocytes were pretreated with thapsigargin for 100 minutes (B and C) before HGF (0.1 nmol/L) stimulation. (A) Calcium; no pretreatment. (B) Calcium; pretreatment with 3 #mol/L ofthapsigargin. (C) pHi; pretreatment with 3 #mol/L of thapsigargin.

induced calcium response, 4 and the period for a good response was also similar. There are some possible reasons for these changes in responsiveness during culture. First, they may be related to the cell cycle, with early events of proliferation, such as calcium and pHi

634 KANEKO ET AL changes, occurring in the early stage. Nishimoto et a134 have reported similar events in cell cycle-dependent cell signaling. Another possible reason is de-differentiation of the hepatocytes during culture. The damage to the receptor function caused by collagenase used to isolate hepatocytes and recovery of the function during culture may be partially responsible for the lower responsiveness to HGF in a 2- to 4-hour culture, although we shortened the period of collagenase perfusion to avoid damage to the receptor function. The lower responsiveness in a 7- to 10-hour culture may be related to the elevated basal pHi. It is also unclear why the basal pHi in hepatocytes after this culture period was higher than that in earlier periods. The Na÷/H ÷ exchanger or other pH regulating systems may be activated spontaneously with progression of a culture to induce the basal pHi elevation. We further observed intercellular heterogeneity of the pHi elevation caused by HGF (i.e., the degree of pHi elevation caused by HGF differed between cells even in the same culture), as in the case of the calcium response to HGF (i.e., the frequency of calcium oscillations differed individually and some cells showed no calcium response). 4 The difference in the basal pHi in hepatocytes may be partially responsible for the intercellular heterogeneity of the responsiveness to HGF because there was a weak but significant correlation between the basal pHi and the degree of pHi elevation caused by HGF. Other possible reasons are intercellular variation in receptor density, the degree of intracellular signal pathway activation including calcium, and the Na÷/H ÷ exchanger density. The similarity in the culture period for a good response for calcium and pHi supports the first two explanations. Further experiments are needed on the relationship between the calcium response and pHi elevation caused by HGF in the same cell. However, this would require four excitation and two emission filters as well as a controller to measure calcium and pHi simultaneously, which is not possible with our present measuring system. We next tried to clarify the mechanism underlying the pHi elevation caused by HGF, and concluded that the HGF-induced pHi elevation resulted from activation of the Na÷/H ÷ exchanger because it was inhibited on pretreatment of the hepatocytes with amiloride, an inhibitor of the Na+/H ÷ exchanger. This hypothesis was confirmed by the fact that HGF increased the initial rapid rate of cell alkalization in acid-loaded hepatocytes. We further tried to clarify the signal transduction pathways for the Na÷/H ÷ exchanger. HGF receptor/cm e t has a tyrosine kinase domain, 35 and activation of the tyrosine kinase is considered to induce many intracellular events. We first examined the effect of a tyrosine kinase inhibitor, genistein, on the HGF-induced pHi elevation and found that this inhibitor blocked it. Thus, tyrosine kinase seems to participate in the activation of the Na+/H ÷ exchanger. But it is unlikely that tyrosine kinase directly phosphorylates and acti-

HEPATOLOGYAugust 1995 vates the Na+/H ÷ exchanger because Sardet et a127 reported that growth factor-stimulated Na+/H ÷ exchanger phosphorylation occurs at its serine residues. Thus, some signal pathways involving serine kinase(s) are likely to operate between tyrosine kinase and the Na÷/H ÷ exchanger. There have been many studies on the signal pathways for Na÷/H ÷ exchanger activation, but the results are controversial. In many cell types, activation of protein kinase C induces activation of the Na+/H ÷ exchanger. 12'13 But with some cell types, the Na+/H ÷ exchanger has been shown to be activated via a calcium/calmodulin-dependent pathway. 14-17 Fliegel et al 3~ reported phosphorylation of the C-terminal domain of the Na÷/H ÷ exchanger by calcium/calmodulindependent protein kinase II in vitro. Moreover, some laboratories have shown that different reagents activate the Na+/H ÷ exchanger via a protein kinase C dependent pathway and a calcium/calmodulin-dependent pathway in the same cell. ls2° These findings suggest that the intracellular signal pathways for Na+/H ÷ exchanger activation may differ in different cell types, and even in the same cell different pathways may be used for different types of stimulation. In hepatocytes, Na*/H ÷ exchanger activation by HGF is considered to be dependent on the calcium/calmodulin-dependent pathway because the HGF-induced pHi elevation was blocked on pretreatment of the hepatocytes with a calmodulin inhibitor, W-7, but not with H-7 (protein kinase C inhibitor) or H-8 (protein kinase A inhibitor). This hypothesis was confirmed by the finding that the HGF-induced pHi elevation was also blocked on depletion of the intracellular calcium pool and on the blockage of the HGF-induced calcium response on pretreatment of hepatocytes with thapsigargin. This hypothesis favors the previously stated similarity between HGF-induced calcium and pHi changes (i.e., the culture period for a good response was similar and both showed intercellular heterogeneity). Anwer and Atkinson 37 also showed calcium/calmodulin-dependent Na÷/ H ÷ exchanger activation in phenylephrine and arginine vasopressin-stimulated hepatocytes. Figure 6 shows the proposed mechanism for the Na+/H ÷ exchanger activation by H G F in hepatocytes. Calmodulin is known to activate some protein kinases and one of them may be involved in activation of the Na+/H ÷ exchanger. Calcium/calmodulin-dependent protein kinase II is one such candidate, taking into consideration the report by Fliegel et al. 36 It is also possible that more than one protein kinase is involved in the pathway between calmodulin and the Na÷/H ÷ exchanger. Recently, Anwer 3s showed ionomycin-induced pHi elevation using a BCECF-loaded hepatocyte suspension. We also attempted to determine the effect of ionomycin on the pHi in hepatocytes, but all the cultured cells became detached from the coverslips during perfusion with the pH-calibration buffer because of the cell damage caused by ionomycin and nigericin. Thus, we were not able to duplicate the results. Anwer reported that ionomycin-induced pHi elevation was not affected by

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KANEKO ET AL

HGF

DG

IP3

1

1

Ca2+

> calmodulin

/

protein kinase(s)

FIG. 6. Proposed mechanism for Na+/H + exchanger activation by HGF in hepatocytes. PLC-T, phospholipase C-y; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-triphosphate; DG, 1,2diacylglycerol; PKC, protein kinase C; ER, endoplasmic reticulum; R, receptor; P, phosphate.

amiloride, indicating the existence of a pathway other than that of the Na+/H + exchanger, and proposed ionomycin-mediated electroneutral Ca2+/2H ÷ exchange across the hepatocyte plasma membrane. This Ca2+/ 2H ÷ exchange may affect the HGF-induced pHi increase because calcium influx through the plasma membrane is thought to be concerned with calcium oscillations. Further experiments are needed to address these issues. REFERENCES

1. Zarnegar R, Michalopoulos G. Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor for hepatocytes. Cancer Res 1989;49:3314-3320. 2. Gohda E, Tsubouchi H, Nakayama H, Hirono S, Sakiyama O, Takahashi K, Miyazaki H, et al. Purification and partial characterization of hepatocyte growth factor from plasma of a patient with fulminant hepatic failure. J Clin Invest 1988;81:414-419. 3. Nakamura T, Nawa K, Ichihara A, Kaise N, Nishino T. Purification and subunit structure of hepatocyte growth factor from rat platelets. FEBS Lett 1987;224:311-316. 4. Kaneko A, Hayashi N, Tsubouchi H, Tanaka Y, Ito T, Sasaki Y, Fusamoto H, et al. Intracellular calcium as a second messenger for human hepatocyte growth factor in hepatocytes. HEPATOLOGY 1992; 15:1173-1178. 5. Okano Y, Mizuno K, Osada S, Nakamura T, Nozawa Y. Tyrosine phosphorylation of phospholipase CT in c-meffHGF receptorstimulated hepatocytes: comparison with HepG2 hepatocarcinoma cells. Biochem Biophys Res Commun 1993; 190:842-848. 6. Cantley LC, Auger KR, Carpenter C, Duckworth B, Graziani A, Kapeller R, Soltoff S. Oncogenes and signal transduction. Cell 1991;64:281-302. 7. Grinstein S, Rotin D, Mason MJ. Na÷/H + exchange and growth factor-induced cytosolic pH changes. Role in cellular proliferation. Biochim Biophys Acta 1989;988:73-97. 8. Sardet C, Franchi A, Pouyssegur J. Molecular cloning, primary structure, and expression of the human growth factor-activatable Na÷/H ÷ antiporter. Cell 1989;56:271-280. 9. Orlowski J, Kandasamy RA, Shull GE. Molecular cloning of putative members of the Na/H exchanger gene family. J Biol Chem 1992;267:9331-9339.

635

10. Collins JF, Honda T, Knobel S, Bulus NM, Conary J, DuBois R, Ghishan FK. Molecular cloning, sequencing, tissue distribution, and functional expression of a Na÷/H * exchanger (NHE-2). Proc Natl Acad Sci U S A 1993;90:3938-3942. 11. Tse CM, Brant SR, Walker MS, Pouyssegur J, Donowitz M. Cloning and sequencing of a rabbit cDNA encoding an intestinal and kidney-specific Na÷/H ÷ exchanger isoform (NHE-3). J Biol Chem 1992; 267:9340-9346. 12. Grinstein S, Rothstein A. Mechanisms of regulation of the Na÷/ H ÷ exchanger. J Membr Biol 1986;90:1-12. 13. Moolenaar WH. Effects of growth factors on intracellular pH regulation. Ann Rev Physiol 1986;48:363-376. 14. Hendey B, Mamrack MD, Putnam RW. Thrombin induces a calcium transient that mediates an activation of the Na+/H + exchanger in human fibroblasts. J Biol Chem 1989;264:1954019547. 15. Manganel M, Turner RJ. Agonist-induced activation of Na+/H ~ exchange in rat parotid acinar cells is dependent on calcium but not on protein kinase C. J Biol Chem 1990;265:4284-4289. 16. Okada M, Saito Y, Sawada E, Nishiyama A. Microfluorimetric imaging study of the mechanism of activation of the Nat/H + antiport by muscarinic agonist in rat mandibular acinar cells. Pflugers Arch 1991;419:338-348. 17. Owen NE, Villereal ML. Evidence for a role of cahnodulin in serum stimulation of Na ~ influx in human fibroblasts. Proc Natl Acad Sci U S A 1982;79:3537-3541. 18. Bierman AJ, Koenderman L, Tool AJ, DeLaat SW. Epidermal growth factor and bombesin differ strikingly in the induction of early responses in Swiss 3T3 cells. J Cell Physiol 1990; 142:441448. 19. Green J, Muallem S. A common mechanism for activation of the Na+/H + exchanger by different types of stimuli. FASEB J 1989;3:2408-2414. 20. Ober SS, Pardee AB. Both protein kinase C and calcium mediate activation of the Na ~/H+ antiporter in chinese hamster embryo fibroblasts. J Cell Physiol 1987;132:311-317. 21. Yoshiyama Y, Arakaki N, Naka D, Takahashi K, Hirono S, Kondo J, Nakayama H, et al. Identification of the N-terminal residue of the heavy chain of the native and recombinant human hepatocyte growth factor. Biochem Biophys Res Commun 1991; 175:660-667. 22. Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol 1976; 13:29-83. 23. Tanaka Y, Hayashi N, Kaneko A, Ito T, Miyoshi E, Sasaki Y, Fusamoto H, et al. Epidermal growth factor induces dose-dependent calcium oscillations in single fura-2-1oaded hepatocytes. HEPATOLOGY 1992; 16:479-486. 24. Paradiso AM, Negulescu PA, Machen TE. N a - H + and C1 -OH (HCO.,~) exchange in gastric glands. Am J Physiol 1986; 250: G524-534. 25. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca 2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985;260:3440-3450. 26. Tanaka Y, Hayashi N, Kaneko A, Ito T, Horimoto M, Sasaki Y, Kasahara A, et al. Characterization of signaling pathways to Na-/H- exchanger activation with epidermal growth factor in hepatocytes. HEPATOLOGY1994;20:966-974. 27. Sardet C, Counillon L, Franchi A, Pouyssegur J. Growth factors induce phosphorylation of the Na+/H + antiporter, a glycoprotein of 110 kD. Science 1990;247:723-726. 28. Moule SK, McGivan jD. Epidermal growth factor and cyclic AMP stimulate Na+/H + exchange in isolated rat hepatocytes. Eur J Biochem 1990; 187:677-682. 29. Boyer JL, Graf J, Meier PJ. Hepatic transport systems regulating pHi, cell volume, and bile secretion. Ann Rev Physiol 1992;54:415-438. 30. Gleeson D, Smith ND, Boyer JL. Bicarbonate-dependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes. Evidence for Na+-HCO:~ cotransport. J Clin Invest 1989;84:312-321. 31. Fitz JG, Lidofsky SD, Xie M, Cochran M, Scharschmidt BF. Plasma membrane H~-HCO:~ transport in rat hepatocytes: a

636

KANEKO ET AL

principal role for Na÷-coupled HCO3 transport. Am J Physiol 1991;261:G803-G809. 32. Cassel D, Whiteley B, Zhuang YX, Glaser L, Mitogen-independent activation of Na+/H + exchange in human epidermoid carcinoma A431 cells: regulation by medium osmolarity. J Cell Physiol 1985; 122:178-186. 33. Kakinuma Y, Sakamaki Y, Ito K, Cragoe EJ, Igarashi K. Relationship among activation of the Na+/H * antiporter, ornithine decarboxylase induction, and DNA synthesis. Arch Biochem Biophys 1987;259:171-178. 34. Nishimoto I, Hata Y, Ogata E, Kojima I. Insulin-like growth factor II stimulates calcium influx in competent BALB/c 3T3 cells primed with epidermal growth factor. J Biol Chem 1987; 262:12120-12126.

HEPATOLOGYAugust 1995 35. Bottaro DP, Rubin JS, Faletto DL, Chan AM-L, Kmiecik TE, Woude GFV, Aaronson SA. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 1991;251:802-804. 36. Fliegel L, Walsh MP, Singh D, Wong C, Barr A. Phosphorylation of the C-terminal domain of the Na+/H ÷ exchanger by Ca2÷/calmodulin-dependent protein kinase II. Biochem J 1992;282:139145. 37. Anwer MS, Atkinson JM. Intracellular calcium-mediated activation of hepatic Na÷/H ÷ exchange by arginine vasopressin and phenylephrine. HEPATOLOGY1992;15:134-143. 38. Anwer MS. Mechanism of ionomycin-induced intracellular alkalinization of rat hepatocytes. HEPATOLOGY 1993;18:433439.