Intracellular Calcium as a Second Messenger for Human Hepatocyte Growth Factor in Hepatocytes &IRA
KANEK0,l NORIOHAY AS HI,^
HIROHITO TSUBOUCHI,~ YUJI TANAKA,' TOSHIFUMI ITO,l YUTAKA SASAKI,' YASUSHI DAIKUHARA3 AND TAKENOBU KAMADA' HIDEWKIFUSAMOTO,~
'First Department of Medicine, Osaka University Medical School, Osaka 553; 2Second Department of Medicine, Kagoshima University Medical School, Kagoshima 890; and 3Department of Biochemistry, Kagoshima University Dental School, Kagoshima 890, Japan
Human hepatocyte growth factor is a newly discovered substance that stimulates DNA synthesis in vitro. In this study, we examined intracellular Ca2+ movement as one of the second messengers for human hepatocyte growth factor in primary-cultured hepatocytes. The addition of hHGF induced Ca2+oscillation, but the frequency of oscillations varied from cell to cell. We also saw marked intercellular heterogeneity in the initial latent period for the Ca2+ response; the mean latent period was rather longer than those seen with phenylephrine and vasopressin. This difference in the initial latent period may be due to the difference in the pathways of Ca2+elevation. Duration of culture determined the number of human hepatocyte growth factor-responsive cells; their number peaked at 2 to 5 hours of confluent culture, whereas the peak was earlier in a low-density culture. These changes in responsiveness during culture can be explained by the cell cycle-dependent sensitivity to human hepatocyte growth factor of hepatocytes. The Ca2 response to human hepatocyte growth factor was dose dependent; molL hHGF gave the highest Ca2+ response, similar to the doseresponse curve of DNA synthesis. We even observed the Ca2+response in the Ca2+-freebuffer, so the increase in Ca2+ was considered due to release from intracellular Ca2+ stores. These results suggest that human hepatocyte growth factor causes the intracellular Ca2+ elevation in the early stage of the cell cycle and that it plays important roles in the signal transduction systems for human hepatocyte growth factor and the proliferation of 1992;15:1173-1178.) hepatocytes. (HEPATOLOGY +
The liver has strong potential for regeneration; liver regenerates, attaining its original size only 1 wk after two-thirds hepatectomy. Many investigators have studied the mechanism of liver regeneration, but it is
Received April 8, 1991; accepted January 29, 1992. This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. Address reprint requests to: Norio Hayashi, M.D., First Department of Medicine, Osaka University Medical School, Fukushima 1-1-50, Fukushima-ku, Osaka 553, Japan.
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still not well understood. Recently Gohda et al. (1) purified human hepatocyte growth factor (hHGF) from plasma of a patient with fulminant liver failure. Nakamura et al. (2) and Zarnegar and Michalopoulos (3) purified similar substances from rat platelets and rabbit serum and human plasma, respectively, and called them hepatocyte growth factor (HGF) and hepatopoietin A, respectively. Because hHGF stimulates DNA synthesis in primary-cultured hepatocytes more effectively than epidermal growth factor (EGF) (11, it may participate in liver regeneration. Moreover, the blood hHGF level increased markedly in patients with fulminant liver failure (4); thus hHGF was considered to play an important role in liver regeneration in humans. Clarification of the mechanism underlying hepatocyte proliferation due to hHGF will lead to better understanding of proliferation; thus relevant intensive studies are being conducted. External stimuli such as hormones or growth factors are transmitted into cells by signal transduction systems (5, 6). In the liver, phenylephrine, vasopressin and angiotensin I1 activate phospholipase C through guanine nucleotide binding protein (G-protein), and promote the breakdown of phosphatidylinositol 43bisphosphate to yield inositol 1,4,5-triphosphate UP,) and 1,2-diacylglycerol (DAG) (7, 8). IP, releases Ca2+ from intracellular Ca2 stores and DAG activates protein kinase C (PKC), leading to the generation of a physiological response through activation of enzymes or protein phosphorylation. Clarification of the signal transduction pathways is important for elucidating the mechanism underlying cell functional regulation. In spite of intensive studies on hHGF, the signal transduction systems remain unclear. Therefore we studied the movement of intracellular Ca2 as one of the second messengers for hHGF after stimulation by hHGF of primary cultured hepatocytes. +
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MATERIALS AND METHODS Materials. Recombinant hHGF, purified from culture supernatant of Chinese hamster ovary cells that were transfected with cDNA for hHGF, was kindly supplied by Dr. Tadashi Hishida Research Center, Mitsubishi Kasei Corp., Yokohama, Japan. hHGF has almost the same protein structure (with a
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HEPATOLOGY
KANEKO ET AL. 500 1 1 0 InM hHGF
04 0
A
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I5
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0 1nM hHGF
400
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300
G
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Time (min)
Time (min)
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-F 5
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300-
3 0 200-
;
G
100-
0-
0
FIG.1.Ca2+ oscillations, with various frequencies and initial latent periods (A-D),in single hHGF-stimulated hepatocytes. Ca2 was measured every 20 sec for 20 min after addition of hHGF (arrows). +
TABLE1. Mean initial latent period before calcium responses by phenylephrine,vasopressin and hHGF Agonist
Phenylephrine, 2 pmol/L (n = 180) Vasopressin, 1 nmol/L (n = 167) hHGF, 0.1 nmolL (n = 117)
Initial latent period (sec)
7.5 c 4.1“ 10.1 2 3.0b 298 t 218“
All experiments were done in 2 to 5 hr confluent culture. “Mean ? S.D. from more than three experiments. bSignificantly higher than phenylephrine; p < 0.01. “Significantly higher than phenylephrine and vasopressin; p < 0.01.
slight differencein molecular weight of the light chain [9]) and showed the same biological activity to both human and rat hepatocytes in primary culture as that of native hHGF purified from the patients’ plasma (10). Concentrations of recombinant hHGF were determined by an ELISA for hHGF (11). Fura-2 and fura-2 acetoxymethyl ester (fura-21AM) were obtained from Molecular Probes Inc. (Eugene, OR). Collagenase was purchased from Wako Pure Chemical Co. (Osaka, Japan) and collagen (type I) was from Nitta Zeratin Co. (Osaka, Japan). Phenylephrine, vasopressin and all other chemicals were of reagent grade and were obtained from Sigma Chemical Co. (St. Louis, MO). Preparation of Cultured Hepatocytes. Isolated hepatocytes were prepared by the two-step collagenase-perfusionmethod
with male Sprague-Dawleyrats (200 to 300 gm) essentially as described by Seglen (12). Hepatocytes were plated on glass coverslips covered with silicon rings (Flexiperm; Heraeus, Hanau, Germany) and coated with type I collagen (60 pg/cm2) in Williams E medium supplemented with 5% FCS and 1 pmollL dexamethasone, 1 pmol/L insulin, penicillin, streptomycin and amphotericin B. The plating cell density was either lo5 cells/cm2(confluent density) or 3.3 x lo4 cells/cm2 (low density). Cells were then incubated at 37“ C under an atmosphere of 5%CO, and 95%air. Medium was changed after 3 hr. Zntracellular Ca2+ Measurement. Measurement of intracellular Ca2+ was performed essentially as described by Kudo and Ogura (13). Cultured hepatocytes were washed with Hanks’-HEPES buffer (10 mmol/L HEPES, pH 7.4) and then incubated with the same buffer containing 5 pmol/L fura-2/AM at 37” C for 30 to 60 min. After this incubation, the hepatocytes were washed with Hanks’-HEPES buffer to remove the unincorporated fura-2lAM; then the buffer was replaced with fresh Hanks’-HEPES buffer. Hepatocytes were then placed on the stage of an inverted fluorescencemicroscope (TMD;Nikon, Tokyo, Japan). hHGF, phenylephrine or vasopressin was added to the buffer, and fluorescence images were taken every 20 sec for 20 min before and after the addition of hHGF at room temperature. Fluorescence images were obtained with a silicon-intensified-target camera (C2400-08H; Hamamatsu Photonics, Hamamatsu, Japan) with excitation wavelengths of 340 and 380 nm (10 nm band width) and an emission wavelength of 510 nm. The integration time for each image was 0.5 sec. After correction for camera-dark images, ratio images (3401380) were calculated. The ratio images were
Vol. 15, No. 6, 1992
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hHGF-INDUCED Ca MOVEMENT
converted to Ca2+ concentrations according to the Ca2+ calibration curve prepared as described below. Preparation of Ca2+Calibration Curve. A Ca2+ calibration curve was prepared with Ca2 -EGTA buffer essentially as described by Grynkiewicz, Poenie and Tsien (14). Ca2+-EGTA buffer was prepared by mixing 0.5 x n ml CaEGTA solution (100 mmol/L KCl, 10 mmoVL K-MOPS, 10 mmol/L K,CaEGTA and 1 kmol/L fura-2, pH 7.2) and 0.5 x (10 - n) ml EGTA solution (100 mmol/L KC1, 10 mrnol/L K-3-(Nmorpho1ino)propanesulfonic acid (K-MOPS), 10 mrnoVL K,H,EGTA and 1 kmol/L fura-2, pH 7.21, n = 0 to 10. Excitation spectra were checked for each buffer. Fluorescence images, 340 and 380 nm, were obtained for each buffer, and the ratios (340/380) were calculated. The calibration curve of Ca2+ concentration against fluorescence ratio was sigmoid (data not shown).
1001
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Time in culture (hour)
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RESnTS
Figure 1shows the various Ca2 response patterns of primary cultured hepatocytes after addition of hHGF. The final hHGF concentration was determined to be lo-'' moVL because it caused the maximal stimulation of DNA synthesis in primary cultured hepatocytes, as shown by Gohda et al. (1). The Ca2+ response was also maximal at this concentration (describedbelow). We observed repetitive Ca2+ transients, Ca2 oscillations on stimulation by hHGF. The frequency of the oscillations differed between cells, as shown in Figure 1A-C. Some cells showed Ca" oscillations consisting of more than several spikes of Ca2+transients in 20 min (Fig. lA), and others showed only two spikes (Fig. lC> in the same period. Some cells showed no Ca2+ oscillations at all. The initial latent period before Ca2+ elevation after addition of hHGF also varied. Some cells responded within 1min of stimulation (Fig. 1A);others responded after more than 10 min (Fig. lD), even in the same series of measurements. The mean initial latent period before the Ca2+ response was rather longer than those in the cases of phenylephrine and vasopressin, as shown in Table 1. We examined the Ca2 response to hHGF of a primary culture for 24 hr and found that the number of hHGF-responsive cells changed with the progress of the culture. Figure 2 shows the time course of the proportion of hHGF-responsive cells during the culture, which differed with the different cell-plating densities examined. When hepatocytes were plated at the confluent density (lo5 cells/cm2), few responded to hHGF within 1 hr, although we observed an evident Ca2+ response to phenylephrine within that time. The proportion of responsive cells then increased to 80%within 2 to 5 hr, after which it decreased. All disappeared by 24 h r (Fig. 2A). On the other hand, 60% of the cells responded to hHGF within 1hr in a low-density culture (3.3 x lo4 cells/cm2);then the proportion of responsive cells gradually decreased. All disappeared by 24 hr (Fig. 2B). We observed a Ca2 response to phenylephrine in the hHGF-unresponsive cells during the less-responsive period (Fig. 3). The proportion of hHGF-responsive cells was examined with various concentrations of hHGF during +
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Time in culture (hour)
FIG.2. Changes in the proportion of hHGF-responsive hepatocytes during primary culture. (A) Confluent-density culture (lo5 cells/cm2); (B) low-density culture (3.3 x lo4 cells/cm2). Data expressed as means f S.D. More than three experiments were performed for each time point, and we measured the Ca2+ responses of 30 to 60 cells in each experiment.
- "1' ?-
v
5pM Phenylephrine
O.lnM hHGF
4*0{
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FIG. 3. Ca2+ response to phenylephrine of an hHGF-unresponsive hepatocyte in 8 hr of a confluent culture. hHGF and phenylephrine were sequentially added (arrows)for a single hepatocyte.
2 to 5 h r of confluent culture (Fig. 4) because the cells show a good response in this period, as described earlier. Eighty percent of the cells responded with 10-l' m o m hHGF, which causes the maximal stimulation of DNA synthesis in uitro; it also gave the highest Ca2+ response. The proportion of hHGF-responsive cells decreased with lower concentrations, but 20% of the molL hHGF. cells responded to as little as Cytosolic Ca2+ elevation is caused either by release
HEPATOLOGY
KANEKO ET AL.
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mechanisms underlying cellular functional regulation. Ca2+ is one of the second messengers; it plays an important role because it regulates the activities of many enzymes and protein kinases (7, 8). In this study we examined Ca" + movement after stimulation by hHGF of primary cultured hepatocytes. Moreover, recent studies have demonstrated the importance of measurement of Ca2+ at the single-cell level because of the intercellular heterogeneity of Ca"' responses (15, 161, so we used a digital imaging fluorescence microscope to measure Ca2+ in individual cells. Because we observed Ca" elevation after stimulation by hHGF, Ca2+ was considered one of the second messengers for hHGF. The number of hHGF-responsive cells was maximal with mol/L hHGF, which also caused the maximal stimulation of DNA synthesis in primary cultured hepatocytes (l), so Ca2+ movement may be related to the proliferation of hepatocytes in some way. But this Ca2+ movement may also be related to other cell functions, because at less than lo-'' mol/L hHGF, which does not stimulate DNA synthesis, Ca2+ elevation was induced in 20% to 40% of the cells. The addition of hHGF caused Ca2 oscillations,which we have reported before for phenylephrine and vasopressin (17). Ca2+ oscillations are induced by the periodic release of Ca2+from intracellular stores and are considered the means of transmission of external stimuli into cells through a frequency-encoded signaling system (18,191. hHGF induced Ca2+ elevation in the same way. But we do not know why some cells showed no Ca2+ oscillations at all. Kawanishi et al. (16) also observed that some cells did not show Ca2+ oscillations after stimulation by phenylephrine and vasopressin; they suggested an intercellular difference in the rate of Ca" resupply to the intracellular Ca2 stores from extracellular Ca2+. We observed intercellular heterogeneity of the Ca" + response to hHGF (i.e., we saw differences between cells in the initial latent period and the period between oscillations). Some investigators reported similar intercellular heterogeneity of the Ca2+ response to phenylephrine or vasopressin in single hepatocytes (15, 16). They suggested that it was due to the intercellular variation in receptor density, G-protein availability, phospholipase C activity, Ca2+ pool size or pump or channel densities (15).The heterogeneity in the case of hHGF may be due to the same reasons. On the other hand, the initial latent period before the Ca" response induced by hHGF was rather longer than those in the cases of phenylephrine and vasopressin, and this may have been due to the difference in the pathways for Ca2+ elevation. Phenylephrine and vasopressin bind to receptors and activate phospholipase C through G-protein. Phospholipase C promotes the production of IP, and Ca2 release from intracellular stores (5-8).But recently another pathway for Ca release was reported involving EGF (20-23). It was reported that the tyrosine kinase domain of the EGF receptor phosphorylates and activates phospholipase C-y (20,211 and that it induces IPS production and Ca2 release from intracellular stores. +
I ,o-13
10-12
lo-lo
,o-ll
hHGF concentration
l0-9
(M)
FIG. 4. Dose-response curve of hHGF against the proportion of hHGF-responsive hepatocytes. Data expressed as means t S.D. More than three experiments were performed for each point in 2 to 5 hr of a confluent culture.
+
500 O.lnM hHGF
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Time (min)
FIG. 5. Ca2+ response of an hHGF-stimulated hepatocyte in Ca2 -free buffer. Buffer was changed to CaZ -free buffer immediately before the Caz+ measurement. +
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from intracellular Ca2+ stores or by influx of extracellular Ca2 . Different hormones or growth factors induce cytosolic Ca2+ elevation through different mechanisms in the same cells. Thus we measured the Ca"' response in Ca" -free buffer (Ca" -free Hanks'-HEPES buffer containing 5 mmol/L EGTA) to elucidate the mechanism underlying Ca" elevation by hHGF. As shown in Figure 5 , we even observed cytosolic Ca2+ elevation in the Ca2+-free buffer, although most of the cells showed single Ca2+ spikes, not Ca2+ oscillations. +
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DISCUSSION
The production of second messengers is the first step in signal transduction systems, and clarifying the production process is important in elucidating the
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Vol. 15, No. 6, 1992
The phosphorylation of phospholipase C-y by EGF receptor requires 15 min (24), so Ca2' response by EGF would have long latency after the stimulation. There may also be a complicated pathway for hHGF, as in the case of EGF, judging from the long initial latent period. Recently Bottaro et al. (25) reported the possibility that the c-met protooncogene product is the cell-surface receptor for (h)HGF (25). The c-met protooncogene product comprises disulfide-linkedsubunits of 50 kD (a) and 145 kD (p), and the @-subunithas tyrosine kinase domains. Moreover, Rubin et al. (26) showed the tyrosine phosphorylation of a 145-kD protein by a variant of hHGF they isolated from human embryonic lung fibroblasts and suggested the involvement of the tyrosine kinase in its signaling pathways. Considering these facts, it is likely that hHGF has signalingpathways similar to those of EGF. We and others reported previously that hepatocytes in primary culture became less sensitive to phenylephrine and vasopressin after 24-hr culture than they were at an earlier time in the culture (15, 27, 28). This appears to result from partial desensitization of the hormones caused by the decrease in the receptor number because the latter was demonstrated (27), although Rooney, Sass and Thomas (15) proposed the possibility of the presence (or absence) of regulatory factors in the tissue culture medium. A difference was also seen in the sensitivity to hHGF during culture, but the time course of the sensitivity to hHGF was different from time courses in the cases of phenylephrine and vasopressin. Moreover, the time course of Ca2 responsiveness was different for different cell-plating densities. In a confluent culture, few cells responded to hHGF during the first hour of culture, although a remarkable Ca2 response to phenylephrine or vasopressin was seen by that time. Moreover, we observed a Ca2+ response to hHGF in a low-density culture within 1 hr. After the initial lessresponsive period, hepatocytes became sensitive to hHGF; 80% of the cells showed a Ca2+ response on stimulation by hHGF within 2 to 5 hr. Then they became less sensitive again on further culture. Because an evident Ca2 response to phenylephrine or vasopressin was observed during the less hHGF-responsive period, the unresponsiveness to hHGF was concluded to be not due to the changes in the experimental conditions with the progress of the culture (for example, the amount of fura-2 loaded into cells or esterase activity changing fura-21AM to fura-2). On the other hand, when hepatocytes were plated at a low density, we observed no initial less-responsive period; about 60% of the cells showed Ca2+responses to hHGF within 1hr. Then the number of hHGF-responsive cells decreased with the progress of the culture, as in the case of the confluent culture. This change in responsiveness may be due t o a change in the number of receptors for hHGF, as is the case for phenylephrine or vasopressin. This will be clarified by further studies of the hHGF receptor. Thus hHGF induced Ca2+ elevation only within a certain period of a primary culture, a feature characteristic of the Ca2+ Eesponse to hHGF. Nishimoto et al. (29) reported that +
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hHGF-INDUCED Ca MOVEMENT
insulin-like growth factor I1 (IGF-11) induced Ca2 elevation in BALB/c 3T3 cells only in the competent condition primed with platelet-derived growth factor and EGF. They suggested the cell-cycle dependence of the Ca2+ response to IGF-11. Primary cultured hepatocytes traverse from the Goto the GI phase in 40 hr under confluent conditions (30),so hHGF seems t o affect hepatocytes at an early stage of the G, transition. In a lowdensity culture, the G, transition takes only a few hours (30,311. Perhaps we could not observe the less-sensitive period at the early stage of the culture because the transition was so fast in the low-density culture. Two mechanisms exist for Ca2 elevation after stimulation by hormones or growth factors. One involves the influx of extracellular Ca2+ and the other involves release from intracellular Ca2 stores. Because we even observed a Ca2+ response to hHGF in the Ca2+-free buffer, we conclude that increased Ca2* came from intracellular Ca2 stores, not from extracellular Ca2 . Extracellular Ca2 was shown to affect the frequency of Ca2 oscillations, probably because of Ca2+ resupply to the intracellular Ca2+ stores (15, 16). Complete Ca2+ deprivation of the extracellular buffer would decrease the number of cells showing Ca2+ oscillations by the inhibition of the Ca2+ resupply, as in the case of phenylephrine, which has been described by Kawanishi et al. (16). In this study, it was demonstrated that Ca2+ acts as one of the second messengers for hHGF, but other signal transduction pathways for hHGF are not yet clear. One hormone or growth factor may have some signal transduction pathways (as does EGF), and these pathways work together to regulate cell functions. hHGF may also have some signal transduction pathways. Clarification of the other pathways along with that of Ca2 ,is important for elucidation of the cellular mechanisms of hHGF and the proliferation of hepatocytes. +
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REFERENCES 1. Gohda E, Tsubouchi H, Nakayama H, Hirono S, Sakiyama 0, 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. 2. 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. 3. Zarnegar R, Michalopoulos G . Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor for hepatocytes. Cancer Res 1989;49:3314-3320. 4. Tsubouchi H, Hirono S, Gohda E, Nakayama H, Takahashi K, Sakiyama 0, Miyazaki H, et al. Clinical significance of human hepatocyte growth factor in blood from patients with fulminant hepatic failure. HEPATOLOGY 1989;9:875-881. 5. Berridge MJ. Inositol triphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 1987;56:159-193. 6. Berridge MJ, Irvine RF. Inositol phosphates and cell signalling. Nature 1989;341:197-205. 7. Exton JH. Role of phosphoinositides in the regulation of liver function. HEPATOLOGY 1988;8:152-166. 8. Williamson JR, Cooper RH, Joseph SK, Thomas Ap. Inositol triphosphate and diacylglycerolas intracellular second messengers in liver. Am J Physiol 1985;248:C203-C216. 9. Yoshiyama Y, Arakaki N , Naka D,Takahashi K, Hirono S, Kondo J . Nakavama H. et al. Identification of the N-terminal residue of
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the heavy chain of the native and recombinant human hepato- 21. Margolis B, Rhee SG, Felder S, Mervic M, Lydl R, Levitzki A, Ullrich A, et al. EGF induces tyrosine phosphorylation of cyte growth factor. Biochem Biophys Res Commun 1991;175: phospholipase C-11: a potential mechanism for EGF receptor 660-667. signaling. Cell 1989;57:1101-1107. 10. Strain AJ, Ismail T, Tsubouchi H, Arakaki N, Hishida T, Kitamura N, Daikuhara Y, et al. Native and recombinant human 22. Johnson RM, Garrison JC. Epidermal growth factor and angiotensin I1 stimulate formation of inositol 1,4,5- and inositol hepatocyte growth factors are highly potent promoters of DNA 1,3,4-triphosphate in hepatocytes. J Biol Chem 1987;262:17285synthesis in both human and rat hepatocytes. J Clin Invest 17293. 1991;87:1853-1857. 11. Tsubouchi H, Naitani Y, Hirono S, Nakayama H, Gohda E, 23. Bosch F, Bouscarel B, Slaton J , Blackmore PF, Exton JH. Epidermal growth factor mimics insulin effects in rat hepatocytes. Arakaki N, Sakiyama 0, et al. Levels of the human hepatocyte Biochem J 1986239523-530. growth factor in serum of patients with various liver diseases determined by an enzyme-linked immunosorbent assay. HEPA- 24. Goldschmidt-Clermont PJ, Kim JW,Machesky LM, Rhee SG, Pollard TD. Regulation of phospholipase C-y 1 by profilin and TOLOGY 1991;13:1-5. tyrosine phosphorylation. Science 1991;251:1231-1233. 12. Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol 25. Bottaro DP, Rubin JS, Faletto DL, Chan AM-L, Kmiecik TE, 1976;13:29-83. Woude GFV, Aaronson SA. Identification of the hepatocyte growth 13. Kudo Y, Ogura A. Glutamate-induced increase in intracellular factor receDtor as the c-met proto-oncogene product. Science Ca2' concentration in isolated hippocampal neurones. Br J . 1991;251:802-804. Pharmacol 1986;89:191-198. 14. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ 26. Rubin JS, Chan AM-L, Bottaro DP, Burgess WH, Taylor WG, Cech AC. Hirschfield DW, et al. A broad-spectrum human lung indicators with greatly improved fluorescence properties. J Biol fibroblast-derived mitogen is a variant -of hepatocyte growth Chem 1985;260:3440-3450. factor. Proc Natl Acad Sci USA 1991;88:415-419. 15. Rooney TA, Sass EJ, Thomas AP. Characterization of cytosolic calcium oscillations induced by phenylephrine and vasopressin in 27. Bouscarel B, Augert G, Taylor SJ, Exton JH. Alterations in vasopressin and angiotensin I1 receptors and responses during single fura-2-loaded hepatocytes. J Biol Chem 1989;264:17131culture of rat liver cells. Biochim Biophys Acta 1990;1055: 17141. 265-272. 16. Kawanishi T, Blank LM, Harootunian AT, Smith MT, Tsien RY. Ca2 oscillations induced by hormonal stimulation of individual 28. Kaneko A, Hayashi N, TanakaY, Ito T, SasakiY, Fusamoto H, and Kamada T. Modulation of calcium signaling by proliferationfura-2-loaded hepatocytes. J Biol Chem 1989;264:12859-12866. differentiation dynamics in primary cultured hepatocytes [Ab17. Tanaka Y, Hayashi N, Kaneko A, Ito T, Sasaki Y, Fusamoto H, stract]. HEPATOLOGY 1990;12:A913. Sat0 N, et al. Cytosolic calcium oscillations induced by hormonal stimulation of fura-2-loaded hepatocytes [Abstract]. HEPATOLOGY29. Nishimoto I, HataY, Ogata E, Kojima I. Insulin-like growth factor I1 stimulates calcium influx in competent BALB/c 3T3 cells primed 1990;12:4443. with epidermal growth factor. J Biol Chem 1987;262:1212018. Berridge MJ, Galione A. Cytosolic calcium oscillators. FASEB J 12126. 1988;2:3074-3082. 19. Berridge MJ, Cobbold PH, Cuthbertson KSR. Spatial and tem- 30. Nakamura T, Tomita Y, Ichihara A. Density-dependent growth control of adult rat hepatocytes in primary culture. J Biochem poral aspects of cell signalling. Phil Trans R Soc Lond 1988;B320: 1983;94:1029-1035. 325-343. 20. Meisenhelder J, Suh PG, Rhee SG, Hunter T. Phospholipase C-y 31. Ikeda T, Sawada N, Fujinaga K, Minase T, Mori M. c-H-rus gene is expressed at the G1 phase in primary cultures of hepatocytes. is a substrate for the PDGF and EGF receptor protein-tyrosine Exp Cell Res 1989;185:292-296. kinases in uiuo and in uitro. Cell 1989;57:1109-1122. +
KANEK0,l NORIOHAY AS HI,^
HIROHITO TSUBOUCHI,~ YUJI TANAKA,' TOSHIFUMI ITO,l YUTAKA SASAKI,' YASUSHI DAIKUHARA3 AND TAKENOBU KAMADA' HIDEWKIFUSAMOTO,~
'First Department of Medicine, Osaka University Medical School, Osaka 553; 2Second Department of Medicine, Kagoshima University Medical School, Kagoshima 890; and 3Department of Biochemistry, Kagoshima University Dental School, Kagoshima 890, Japan
Human hepatocyte growth factor is a newly discovered substance that stimulates DNA synthesis in vitro. In this study, we examined intracellular Ca2+ movement as one of the second messengers for human hepatocyte growth factor in primary-cultured hepatocytes. The addition of hHGF induced Ca2+oscillation, but the frequency of oscillations varied from cell to cell. We also saw marked intercellular heterogeneity in the initial latent period for the Ca2+ response; the mean latent period was rather longer than those seen with phenylephrine and vasopressin. This difference in the initial latent period may be due to the difference in the pathways of Ca2+elevation. Duration of culture determined the number of human hepatocyte growth factor-responsive cells; their number peaked at 2 to 5 hours of confluent culture, whereas the peak was earlier in a low-density culture. These changes in responsiveness during culture can be explained by the cell cycle-dependent sensitivity to human hepatocyte growth factor of hepatocytes. The Ca2 response to human hepatocyte growth factor was dose dependent; molL hHGF gave the highest Ca2+ response, similar to the doseresponse curve of DNA synthesis. We even observed the Ca2+response in the Ca2+-freebuffer, so the increase in Ca2+ was considered due to release from intracellular Ca2+ stores. These results suggest that human hepatocyte growth factor causes the intracellular Ca2+ elevation in the early stage of the cell cycle and that it plays important roles in the signal transduction systems for human hepatocyte growth factor and the proliferation of 1992;15:1173-1178.) hepatocytes. (HEPATOLOGY +
The liver has strong potential for regeneration; liver regenerates, attaining its original size only 1 wk after two-thirds hepatectomy. Many investigators have studied the mechanism of liver regeneration, but it is
Received April 8, 1991; accepted January 29, 1992. This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. Address reprint requests to: Norio Hayashi, M.D., First Department of Medicine, Osaka University Medical School, Fukushima 1-1-50, Fukushima-ku, Osaka 553, Japan.
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still not well understood. Recently Gohda et al. (1) purified human hepatocyte growth factor (hHGF) from plasma of a patient with fulminant liver failure. Nakamura et al. (2) and Zarnegar and Michalopoulos (3) purified similar substances from rat platelets and rabbit serum and human plasma, respectively, and called them hepatocyte growth factor (HGF) and hepatopoietin A, respectively. Because hHGF stimulates DNA synthesis in primary-cultured hepatocytes more effectively than epidermal growth factor (EGF) (11, it may participate in liver regeneration. Moreover, the blood hHGF level increased markedly in patients with fulminant liver failure (4); thus hHGF was considered to play an important role in liver regeneration in humans. Clarification of the mechanism underlying hepatocyte proliferation due to hHGF will lead to better understanding of proliferation; thus relevant intensive studies are being conducted. External stimuli such as hormones or growth factors are transmitted into cells by signal transduction systems (5, 6). In the liver, phenylephrine, vasopressin and angiotensin I1 activate phospholipase C through guanine nucleotide binding protein (G-protein), and promote the breakdown of phosphatidylinositol 43bisphosphate to yield inositol 1,4,5-triphosphate UP,) and 1,2-diacylglycerol (DAG) (7, 8). IP, releases Ca2+ from intracellular Ca2 stores and DAG activates protein kinase C (PKC), leading to the generation of a physiological response through activation of enzymes or protein phosphorylation. Clarification of the signal transduction pathways is important for elucidating the mechanism underlying cell functional regulation. In spite of intensive studies on hHGF, the signal transduction systems remain unclear. Therefore we studied the movement of intracellular Ca2 as one of the second messengers for hHGF after stimulation by hHGF of primary cultured hepatocytes. +
+
MATERIALS AND METHODS Materials. Recombinant hHGF, purified from culture supernatant of Chinese hamster ovary cells that were transfected with cDNA for hHGF, was kindly supplied by Dr. Tadashi Hishida Research Center, Mitsubishi Kasei Corp., Yokohama, Japan. hHGF has almost the same protein structure (with a
1173
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HEPATOLOGY
KANEKO ET AL. 500 1 1 0 InM hHGF
04 0
A
5
10
0
20
I5
5
0 1nM hHGF
400
G -0
300
G
100
3
200
10
15
20
500 -
500
-? -
5
Time (min)
Time (min)
1
C
T
-F 5
-
400-
300-
3 0 200-
;
G
100-
0-
0
FIG.1.Ca2+ oscillations, with various frequencies and initial latent periods (A-D),in single hHGF-stimulated hepatocytes. Ca2 was measured every 20 sec for 20 min after addition of hHGF (arrows). +
TABLE1. Mean initial latent period before calcium responses by phenylephrine,vasopressin and hHGF Agonist
Phenylephrine, 2 pmol/L (n = 180) Vasopressin, 1 nmol/L (n = 167) hHGF, 0.1 nmolL (n = 117)
Initial latent period (sec)
7.5 c 4.1“ 10.1 2 3.0b 298 t 218“
All experiments were done in 2 to 5 hr confluent culture. “Mean ? S.D. from more than three experiments. bSignificantly higher than phenylephrine; p < 0.01. “Significantly higher than phenylephrine and vasopressin; p < 0.01.
slight differencein molecular weight of the light chain [9]) and showed the same biological activity to both human and rat hepatocytes in primary culture as that of native hHGF purified from the patients’ plasma (10). Concentrations of recombinant hHGF were determined by an ELISA for hHGF (11). Fura-2 and fura-2 acetoxymethyl ester (fura-21AM) were obtained from Molecular Probes Inc. (Eugene, OR). Collagenase was purchased from Wako Pure Chemical Co. (Osaka, Japan) and collagen (type I) was from Nitta Zeratin Co. (Osaka, Japan). Phenylephrine, vasopressin and all other chemicals were of reagent grade and were obtained from Sigma Chemical Co. (St. Louis, MO). Preparation of Cultured Hepatocytes. Isolated hepatocytes were prepared by the two-step collagenase-perfusionmethod
with male Sprague-Dawleyrats (200 to 300 gm) essentially as described by Seglen (12). Hepatocytes were plated on glass coverslips covered with silicon rings (Flexiperm; Heraeus, Hanau, Germany) and coated with type I collagen (60 pg/cm2) in Williams E medium supplemented with 5% FCS and 1 pmollL dexamethasone, 1 pmol/L insulin, penicillin, streptomycin and amphotericin B. The plating cell density was either lo5 cells/cm2(confluent density) or 3.3 x lo4 cells/cm2 (low density). Cells were then incubated at 37“ C under an atmosphere of 5%CO, and 95%air. Medium was changed after 3 hr. Zntracellular Ca2+ Measurement. Measurement of intracellular Ca2+ was performed essentially as described by Kudo and Ogura (13). Cultured hepatocytes were washed with Hanks’-HEPES buffer (10 mmol/L HEPES, pH 7.4) and then incubated with the same buffer containing 5 pmol/L fura-2/AM at 37” C for 30 to 60 min. After this incubation, the hepatocytes were washed with Hanks’-HEPES buffer to remove the unincorporated fura-2lAM; then the buffer was replaced with fresh Hanks’-HEPES buffer. Hepatocytes were then placed on the stage of an inverted fluorescencemicroscope (TMD;Nikon, Tokyo, Japan). hHGF, phenylephrine or vasopressin was added to the buffer, and fluorescence images were taken every 20 sec for 20 min before and after the addition of hHGF at room temperature. Fluorescence images were obtained with a silicon-intensified-target camera (C2400-08H; Hamamatsu Photonics, Hamamatsu, Japan) with excitation wavelengths of 340 and 380 nm (10 nm band width) and an emission wavelength of 510 nm. The integration time for each image was 0.5 sec. After correction for camera-dark images, ratio images (3401380) were calculated. The ratio images were
Vol. 15, No. 6, 1992
1175
hHGF-INDUCED Ca MOVEMENT
converted to Ca2+ concentrations according to the Ca2+ calibration curve prepared as described below. Preparation of Ca2+Calibration Curve. A Ca2+ calibration curve was prepared with Ca2 -EGTA buffer essentially as described by Grynkiewicz, Poenie and Tsien (14). Ca2+-EGTA buffer was prepared by mixing 0.5 x n ml CaEGTA solution (100 mmol/L KCl, 10 mmoVL K-MOPS, 10 mmol/L K,CaEGTA and 1 kmol/L fura-2, pH 7.2) and 0.5 x (10 - n) ml EGTA solution (100 mmol/L KC1, 10 mrnol/L K-3-(Nmorpho1ino)propanesulfonic acid (K-MOPS), 10 mrnoVL K,H,EGTA and 1 kmol/L fura-2, pH 7.21, n = 0 to 10. Excitation spectra were checked for each buffer. Fluorescence images, 340 and 380 nm, were obtained for each buffer, and the ratios (340/380) were calculated. The calibration curve of Ca2+ concentration against fluorescence ratio was sigmoid (data not shown).
1001
A
+
Time in culture (hour)
B
RESnTS
Figure 1shows the various Ca2 response patterns of primary cultured hepatocytes after addition of hHGF. The final hHGF concentration was determined to be lo-'' moVL because it caused the maximal stimulation of DNA synthesis in primary cultured hepatocytes, as shown by Gohda et al. (1). The Ca2+ response was also maximal at this concentration (describedbelow). We observed repetitive Ca2+ transients, Ca2 oscillations on stimulation by hHGF. The frequency of the oscillations differed between cells, as shown in Figure 1A-C. Some cells showed Ca" oscillations consisting of more than several spikes of Ca2+transients in 20 min (Fig. lA), and others showed only two spikes (Fig. lC> in the same period. Some cells showed no Ca2+ oscillations at all. The initial latent period before Ca2+ elevation after addition of hHGF also varied. Some cells responded within 1min of stimulation (Fig. 1A);others responded after more than 10 min (Fig. lD), even in the same series of measurements. The mean initial latent period before the Ca2+ response was rather longer than those in the cases of phenylephrine and vasopressin, as shown in Table 1. We examined the Ca2 response to hHGF of a primary culture for 24 hr and found that the number of hHGF-responsive cells changed with the progress of the culture. Figure 2 shows the time course of the proportion of hHGF-responsive cells during the culture, which differed with the different cell-plating densities examined. When hepatocytes were plated at the confluent density (lo5 cells/cm2), few responded to hHGF within 1 hr, although we observed an evident Ca2+ response to phenylephrine within that time. The proportion of responsive cells then increased to 80%within 2 to 5 hr, after which it decreased. All disappeared by 24 h r (Fig. 2A). On the other hand, 60% of the cells responded to hHGF within 1hr in a low-density culture (3.3 x lo4 cells/cm2);then the proportion of responsive cells gradually decreased. All disappeared by 24 hr (Fig. 2B). We observed a Ca2 response to phenylephrine in the hHGF-unresponsive cells during the less-responsive period (Fig. 3). The proportion of hHGF-responsive cells was examined with various concentrations of hHGF during +
+
Time in culture (hour)
FIG.2. Changes in the proportion of hHGF-responsive hepatocytes during primary culture. (A) Confluent-density culture (lo5 cells/cm2); (B) low-density culture (3.3 x lo4 cells/cm2). Data expressed as means f S.D. More than three experiments were performed for each time point, and we measured the Ca2+ responses of 30 to 60 cells in each experiment.
- "1' ?-
v
5pM Phenylephrine
O.lnM hHGF
4*0{
1
1
1
+
+
0
5
10
20
15
Time
25
30
(rnin)
FIG. 3. Ca2+ response to phenylephrine of an hHGF-unresponsive hepatocyte in 8 hr of a confluent culture. hHGF and phenylephrine were sequentially added (arrows)for a single hepatocyte.
2 to 5 h r of confluent culture (Fig. 4) because the cells show a good response in this period, as described earlier. Eighty percent of the cells responded with 10-l' m o m hHGF, which causes the maximal stimulation of DNA synthesis in uitro; it also gave the highest Ca2+ response. The proportion of hHGF-responsive cells decreased with lower concentrations, but 20% of the molL hHGF. cells responded to as little as Cytosolic Ca2+ elevation is caused either by release
HEPATOLOGY
KANEKO ET AL.
1176
loo
1
mechanisms underlying cellular functional regulation. Ca2+ is one of the second messengers; it plays an important role because it regulates the activities of many enzymes and protein kinases (7, 8). In this study we examined Ca" + movement after stimulation by hHGF of primary cultured hepatocytes. Moreover, recent studies have demonstrated the importance of measurement of Ca2+ at the single-cell level because of the intercellular heterogeneity of Ca"' responses (15, 161, so we used a digital imaging fluorescence microscope to measure Ca2+ in individual cells. Because we observed Ca" elevation after stimulation by hHGF, Ca2+ was considered one of the second messengers for hHGF. The number of hHGF-responsive cells was maximal with mol/L hHGF, which also caused the maximal stimulation of DNA synthesis in primary cultured hepatocytes (l), so Ca2+ movement may be related to the proliferation of hepatocytes in some way. But this Ca2+ movement may also be related to other cell functions, because at less than lo-'' mol/L hHGF, which does not stimulate DNA synthesis, Ca2+ elevation was induced in 20% to 40% of the cells. The addition of hHGF caused Ca2 oscillations,which we have reported before for phenylephrine and vasopressin (17). Ca2+ oscillations are induced by the periodic release of Ca2+from intracellular stores and are considered the means of transmission of external stimuli into cells through a frequency-encoded signaling system (18,191. hHGF induced Ca2+ elevation in the same way. But we do not know why some cells showed no Ca2+ oscillations at all. Kawanishi et al. (16) also observed that some cells did not show Ca2+ oscillations after stimulation by phenylephrine and vasopressin; they suggested an intercellular difference in the rate of Ca" resupply to the intracellular Ca2 stores from extracellular Ca2+. We observed intercellular heterogeneity of the Ca" + response to hHGF (i.e., we saw differences between cells in the initial latent period and the period between oscillations). Some investigators reported similar intercellular heterogeneity of the Ca2+ response to phenylephrine or vasopressin in single hepatocytes (15, 16). They suggested that it was due to the intercellular variation in receptor density, G-protein availability, phospholipase C activity, Ca2+ pool size or pump or channel densities (15).The heterogeneity in the case of hHGF may be due to the same reasons. On the other hand, the initial latent period before the Ca" response induced by hHGF was rather longer than those in the cases of phenylephrine and vasopressin, and this may have been due to the difference in the pathways for Ca2+ elevation. Phenylephrine and vasopressin bind to receptors and activate phospholipase C through G-protein. Phospholipase C promotes the production of IP, and Ca2 release from intracellular stores (5-8).But recently another pathway for Ca release was reported involving EGF (20-23). It was reported that the tyrosine kinase domain of the EGF receptor phosphorylates and activates phospholipase C-y (20,211 and that it induces IPS production and Ca2 release from intracellular stores. +
I ,o-13
10-12
lo-lo
,o-ll
hHGF concentration
l0-9
(M)
FIG. 4. Dose-response curve of hHGF against the proportion of hHGF-responsive hepatocytes. Data expressed as means t S.D. More than three experiments were performed for each point in 2 to 5 hr of a confluent culture.
+
500 O.lnM hHGF
5 5 5
400
1
v
300
.L
0
.-0 200
3
+-'
0"
+
100
+
0
5
15
10
20
Time (min)
FIG. 5. Ca2+ response of an hHGF-stimulated hepatocyte in Ca2 -free buffer. Buffer was changed to CaZ -free buffer immediately before the Caz+ measurement. +
+
from intracellular Ca2+ stores or by influx of extracellular Ca2 . Different hormones or growth factors induce cytosolic Ca2+ elevation through different mechanisms in the same cells. Thus we measured the Ca"' response in Ca" -free buffer (Ca" -free Hanks'-HEPES buffer containing 5 mmol/L EGTA) to elucidate the mechanism underlying Ca" elevation by hHGF. As shown in Figure 5 , we even observed cytosolic Ca2+ elevation in the Ca2+-free buffer, although most of the cells showed single Ca2+ spikes, not Ca2+ oscillations. +
+
+
+
DISCUSSION
The production of second messengers is the first step in signal transduction systems, and clarifying the production process is important in elucidating the
+
+
+
Vol. 15, No. 6, 1992
The phosphorylation of phospholipase C-y by EGF receptor requires 15 min (24), so Ca2' response by EGF would have long latency after the stimulation. There may also be a complicated pathway for hHGF, as in the case of EGF, judging from the long initial latent period. Recently Bottaro et al. (25) reported the possibility that the c-met protooncogene product is the cell-surface receptor for (h)HGF (25). The c-met protooncogene product comprises disulfide-linkedsubunits of 50 kD (a) and 145 kD (p), and the @-subunithas tyrosine kinase domains. Moreover, Rubin et al. (26) showed the tyrosine phosphorylation of a 145-kD protein by a variant of hHGF they isolated from human embryonic lung fibroblasts and suggested the involvement of the tyrosine kinase in its signaling pathways. Considering these facts, it is likely that hHGF has signalingpathways similar to those of EGF. We and others reported previously that hepatocytes in primary culture became less sensitive to phenylephrine and vasopressin after 24-hr culture than they were at an earlier time in the culture (15, 27, 28). This appears to result from partial desensitization of the hormones caused by the decrease in the receptor number because the latter was demonstrated (27), although Rooney, Sass and Thomas (15) proposed the possibility of the presence (or absence) of regulatory factors in the tissue culture medium. A difference was also seen in the sensitivity to hHGF during culture, but the time course of the sensitivity to hHGF was different from time courses in the cases of phenylephrine and vasopressin. Moreover, the time course of Ca2 responsiveness was different for different cell-plating densities. In a confluent culture, few cells responded to hHGF during the first hour of culture, although a remarkable Ca2 response to phenylephrine or vasopressin was seen by that time. Moreover, we observed a Ca2+ response to hHGF in a low-density culture within 1 hr. After the initial lessresponsive period, hepatocytes became sensitive to hHGF; 80% of the cells showed a Ca2+ response on stimulation by hHGF within 2 to 5 hr. Then they became less sensitive again on further culture. Because an evident Ca2 response to phenylephrine or vasopressin was observed during the less hHGF-responsive period, the unresponsiveness to hHGF was concluded to be not due to the changes in the experimental conditions with the progress of the culture (for example, the amount of fura-2 loaded into cells or esterase activity changing fura-21AM to fura-2). On the other hand, when hepatocytes were plated at a low density, we observed no initial less-responsive period; about 60% of the cells showed Ca2+responses to hHGF within 1hr. Then the number of hHGF-responsive cells decreased with the progress of the culture, as in the case of the confluent culture. This change in responsiveness may be due t o a change in the number of receptors for hHGF, as is the case for phenylephrine or vasopressin. This will be clarified by further studies of the hHGF receptor. Thus hHGF induced Ca2+ elevation only within a certain period of a primary culture, a feature characteristic of the Ca2+ Eesponse to hHGF. Nishimoto et al. (29) reported that +
+
+
1177
hHGF-INDUCED Ca MOVEMENT
insulin-like growth factor I1 (IGF-11) induced Ca2 elevation in BALB/c 3T3 cells only in the competent condition primed with platelet-derived growth factor and EGF. They suggested the cell-cycle dependence of the Ca2+ response to IGF-11. Primary cultured hepatocytes traverse from the Goto the GI phase in 40 hr under confluent conditions (30),so hHGF seems t o affect hepatocytes at an early stage of the G, transition. In a lowdensity culture, the G, transition takes only a few hours (30,311. Perhaps we could not observe the less-sensitive period at the early stage of the culture because the transition was so fast in the low-density culture. Two mechanisms exist for Ca2 elevation after stimulation by hormones or growth factors. One involves the influx of extracellular Ca2+ and the other involves release from intracellular Ca2 stores. Because we even observed a Ca2+ response to hHGF in the Ca2+-free buffer, we conclude that increased Ca2* came from intracellular Ca2 stores, not from extracellular Ca2 . Extracellular Ca2 was shown to affect the frequency of Ca2 oscillations, probably because of Ca2+ resupply to the intracellular Ca2+ stores (15, 16). Complete Ca2+ deprivation of the extracellular buffer would decrease the number of cells showing Ca2+ oscillations by the inhibition of the Ca2+ resupply, as in the case of phenylephrine, which has been described by Kawanishi et al. (16). In this study, it was demonstrated that Ca2+ acts as one of the second messengers for hHGF, but other signal transduction pathways for hHGF are not yet clear. One hormone or growth factor may have some signal transduction pathways (as does EGF), and these pathways work together to regulate cell functions. hHGF may also have some signal transduction pathways. Clarification of the other pathways along with that of Ca2 ,is important for elucidation of the cellular mechanisms of hHGF and the proliferation of hepatocytes. +
+
+
+
+
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REFERENCES 1. Gohda E, Tsubouchi H, Nakayama H, Hirono S, Sakiyama 0, 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. 2. 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. 3. Zarnegar R, Michalopoulos G . Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor for hepatocytes. Cancer Res 1989;49:3314-3320. 4. Tsubouchi H, Hirono S, Gohda E, Nakayama H, Takahashi K, Sakiyama 0, Miyazaki H, et al. Clinical significance of human hepatocyte growth factor in blood from patients with fulminant hepatic failure. HEPATOLOGY 1989;9:875-881. 5. Berridge MJ. Inositol triphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 1987;56:159-193. 6. Berridge MJ, Irvine RF. Inositol phosphates and cell signalling. Nature 1989;341:197-205. 7. Exton JH. Role of phosphoinositides in the regulation of liver function. HEPATOLOGY 1988;8:152-166. 8. Williamson JR, Cooper RH, Joseph SK, Thomas Ap. Inositol triphosphate and diacylglycerolas intracellular second messengers in liver. Am J Physiol 1985;248:C203-C216. 9. Yoshiyama Y, Arakaki N , Naka D,Takahashi K, Hirono S, Kondo J . Nakavama H. et al. Identification of the N-terminal residue of
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