- Email: [email protected]
Differential Ca2+ signaling in neonatal and adult rat hepatocyte doublets
Journal of Hepatology 1998; 28: 221–230 Printed in Denmark ¡ All rights reserved Munksgaard ¡ Copenhagen
Copyright C European Association for the Study of the Liver 1998
Journal of Hepatology ISSN 0168-8278
Differential Ca2π signaling in neonatal and adult rat hepatocyte doublets Nobuyuki Enomoto, Tsuneo Kitamura, Miyoko Hirose, Kenichi Ikejima, Sumio Watanabe and Nobuhiro Sato Department of Gastroenterology, Juntendo University School of Medicine, Tokyo, Japan
Background/Aims: Intracellular Ca2π ([Ca2π]i) is important in various cellular functions, including cellular proliferation and differentiation. To elucidate the relationship between [Ca2π]i oscillations and physiological hepatocyte proliferation, phenylephrine-evoked [Ca2π]i responses were sequentially investigated using short-term cultured hepatocyte doublets obtained from 1-, 3-, 6- and 8-week-old rats. Methods/Results: DNA synthesis in hepatocytes, determined by BrdU incorporation, was ∂20% in 1week-old rats, and decreased to ∞1% as the rats aged. Correspondingly, [Ca2π]i responses evoked by 10 mmol/l phenylephrine in hepatocyte doublets shifted from transient to sinusoidal-type [Ca2π]i oscillations and then to a sustained increase in [Ca2π]i, followed by a gradual return to baseline. The incidence of [Ca2π]i oscillations was 100∫0.0%, 83.3∫16.7%, 38.7∫0.6% and 5.5∫5.0% in 1-, 3-, 6- and 8-weekold rats, respectively. Removal of extracellular Ca2π
did not abolish [Ca2π]i oscillations, indicating that [Ca2π]i oscillations were caused primarily by Ca2π mobilization from internal sites of the cells. The [Ca2π]i level in each of the adjacent cells was synchronous in sustained increase in [Ca2π]i, but asynchronous in [Ca2π]i oscillations. In proliferating doublets obtained from 1-week-old rats, the frequency of oscillations increased in a dose-dependent manner for phenylephrine concentrations of 1 to 100 mmol/l. Conclusions: Phenylephrine-evoked [Ca2π]i oscillations were directly related to hepatocyte proliferation and were mediated by frequency modulation. These results suggest that phenylephrine-evoked [Ca2π]i oscillations may contribute to cell-cycle progression of hepatocytes in physiological liver growth.
evidence indicates that intracellular Ca2π ([Ca2π]i) is a key player in regulation of cell motility (1), secretion (2), contraction (3) and possibly gene expression (4). Recently, much attention has focused on these findings in relation to temporal and spatial propagation of [Ca2π]i that are called [Ca2π]i oscillations and waves, respectively. The importance of periodic changes in [Ca2π]i has been emphasized in many aspects of cell growth as well. For instance, [Ca2π]i oscillations have been reported in mouse oocytes that are activated by sperm (5) and in fertilized hamster eggs (6). Oscillations of [Ca2π]i could be also detected in lymphocytes during the action of growth factors (7) and in fibroblasts
transformed with the ras oncogene when stimulated with bradykinin (8). Possible mechanisms for the oscillations in ras-transformed cells include an enhancement of inositol 1,4,5-triphosphate (InsP3) formation or an increased calcium influx (9). Recently, the role of [Ca2π]i in cell proliferation has been highlighted by anti-cancer therapies directed towards this Ca2π signaling pathway (10). These results suggest that intracellular Ca2π signal transduction, particularly in relation to [Ca2π]i oscillations or waves, may extensively involve cell-cycle control that includes developmental, physiological and transformed cell growth in various types of cells. The liver has strong potential for proliferation in regeneration, and it may therefore be assumed that Ca2π signal transduction plays an important role in hepatocyte proliferation. We recently reported characteristic [Ca2π]i oscillations in proliferating rat hepatocytes, after two-thirds hepatectomy (11), suggesting the importance of calcium signal transduction in hepatocyte
I
Received 6 August; revised 19 September; accepted 23 September 1997
Correspondence: Nobuhiro Sato, Department of Gastroenterology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Tel: 81 (03) 5802-1058. Fax: 81 (03) 3813-8862.
Key words: Cell-cycle; Intracellular calcium; Physiological liver growth.
221
N. Enomoto et al.
proliferation during liver regeneration. However, it is not known how [Ca2π]i modulates physiological hepatocyte growth in the developing liver. In the present study, to elucidate the relationship between [Ca2π]i oscillations and physiological hepatocyte proliferation, [Ca2π]i responses evoked by phenylephrine, a known co-mitogen, were sequentially investigated using short-term cultured hepatocyte doublets obtained from 1-, 3-, 6- and 8-week-old rats. Most studies that have evaluated the [Ca2π]i signaling have used conventional monolayered hepatocytes in culture. However, it is generally recognized that differentiated cell function is rapidly decreased under these conditions, suggesting that these cells may not accurately reflect the true nature of cell function in vivo. For this reason, short-term cultured hepatocyte doublets were used in the present study, because they retain various in vivo functions including cell-to-cell communication (12,13), bile canalicular contraction (14,15) and organic anion transport system (16,17).
Materials and Methods Materials Phenylephrine, ionomycin, 5-bromo-2ø-deoxy-uridine (BrdU), monoclonal anti-BrdU antibody and type I collagenase were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fura-2 acetomethoxy ester (fura-2/AM) and pluronic F127 were purchased from Molecular Probes (Junction City, OR, USA). All other chemicals were of analytical grade.
dishes (Becton Dickinson Labware, Lincoln Park, NJ, USA) at 37æC. Viability was assessed by the trypan blue exclusion test. All experiments were performed using cells showing viability of ±90%. Cells were maintained in L-15 containing 10% fetal cow serum (FCS), penicillin (100 IU/ml) and streptomycin (100 mg/ml). Hepatocyte doublets were identified by phase microscopy after cells had been cultured at 37æC for 4 h. Approximately 10% of hepatocytes appeared in pairs that enclosed circular spaces easily identified as bile canaliculi (16). BrdU staining of hepatocytes in developing rat livers After incubation of hepatocytes from 1-, 3-, 6- and 8week-old rats for 4 h, the medium was replaced with fresh medium containing BrdU, 10 mg/ml, for 1 h, and then cells were stained by indirect immunofluorescence methods using monoclonal anti-BrdU antibody (19). Serial quantitative analysis of [Ca2π]i in doublets using a fluorescence microscope imaging system All experiments were performed after cells had been incubated at 37æC for 4 h. Fura-2/AM with pluronic F127 was dissolved in phosphate saline solution (PSS; 135 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l CaCl2, 1 mmol/l MgCl2, 5.5 mmol/l glucose, 10 mmol/l HEPES) containing 1.0 mmol/l Ca2π, in which final concentrations of fura-2/AM and pluronic F127 were 4 mmol/l and 0.05%, respectively. After loading with fura-2/AM
Animals Female Wistar rats, 1, 3, 6 and 8 weeks old, were housed in a temperature-controlled room with lightdark periods of 12 h each and maintained on Purina rat chow and water ad libitum. The study protocol was approved by the Animal Care Committee of Juntendo University, which conforms to the National Institutes of Health guidelines. Preparation of primary cultured hepatocytes Monolayer cultures of rat hepatocytes were prepared from 1-, 3-, 6- and 8-week-old rats, using a modification of the method of Seglen (18). The portal vein was perfused with L-15 medium (Gibco/BRL, Life Technologies, Inc., New York, NY, USA) for 10 min at 4–40 ml/min, followed by the perfusion of a 0.05% solution of type I collagenase for 10 min. The liver was excised, and hepatocytes were detached. Isolated cells were centrifuged at 50 g for 2 min and resuspended in the fresh cold L-15 medium. Cells were filtered through nylon gauze, recentrifuged and incubated at 3¿105 cells per dish on a coverslip in 35-mm Falcon culture
222
Fig. 1. DNA synthesis determined by BrdU incorporation in hepatocytes from 1-, 3-, 6- and 8-week-old rats. Values are expressed as mean∫S.E.M. from three separate preparations (1 vs. 3 weeks, p∞0.005; 3 vs. 6 weeks, p∞0.005; 6 vs. 8 weeks, pΩN.S.).
Ca2π signaling in hepatocyte doublets
Fig. 2. Phenylephrine-evoked [Ca2π]i responses in hepatocyte doublets from 1- (A), 3- (B), 6- (C) and 8- (D) -week-old rats. After loading with 4 mmol/l fura-2/AM, hepatocyte doublets were perfused with PSS containing 10 mmol/l phenylephrine in the presence of extracellular Ca2π. Thereafter, [Ca2π]i was measured every 10 s for 5 min. Representative data are shown from at least 20 experiments in each age group.
solution at 37æC for 30 min, hepatocyte doublets on coverslips were placed in a Dvorak-Stotler Culture Chamber (Nicholson Precision Instruments, Gaithersburg, MD, USA) at 37æC and perfused with PSS in the presence or absence of Ca2π (1.0 mmol/l) at 0.2 ml/min using a Harvard pump (Harvard Apparatus 22, South Natik, MA, USA). The chamber was installed in a fluorescence microscope (Diaphot, Nikon, Tokyo, Japan) with a 100-W xenon arc lamp as a light source. The objective lens was a Nikon Fluor ¿100. A siliconintensified target camera (C-2400, Hamamatsu Photonics, Hamamatsu, Japan) was linked to a computer (MAXY DT2, Mitsubishi, Tokyo, Japan), printer (BJ10V, Cannon, Tokyo, Japan) and video recorder (UP5100, Sony, Tokyo, Japan). Fluorescence intensity of fura-2/AM was quantified every 10 s for 6 min in userselected regions of uniform pixel value with a 256-level gray scale corresponding to a uniform site in cytoplasm. Wavelengths of 340 nm and 380 nm for excitation and 520 nm for emission were used, and frame
averaging for noise reduction was performed. Sixteen frames were averaged for each measurement. The interval between excitation filters was 2 s. To reduce bleaching caused by the high intensity of the xenon lamp, a neutral-density filter (1/16) was used. Background fluorescence and cellular autofluorescence were not detectable at the setting used for gain and sensitivity. [Ca2π]i was determined by the following equation (20): [Ca2π]iΩKd([Ro–Rmin]/[Rmax –Ro]) B Kd, the Ca2π dissociation constant for fura-2, was confirmed as 224 nmol/l. R represents fluorescence intensity at 340 nm excitation divided by that at 380 nm excitation (Ro, experimental data; Rmin, R in 2 mmol/l ethylene glycol-bis [b aminoethyl ether] N,N,Nø,Nøtetra acetic acid (EGTA) and 1 mmol/l ionomycin; Rmax is R in 10 mmol/l Ca and 1 mmol/l ionomycin). B is the ratio of fluorescence intensity at 380 nm in the absence of Ca2π versus a saturating concentration of Ca2π. Although reduction of fluorescence intensity
223
N. Enomoto et al.
The incidence of [Ca2π]i oscillations was assessed as the ratio of cells showing [Ca2π]i oscillations to those presenting [Ca2π]i responses in each experiment. Statistical methods Statistical analysis was performed using Student’s ttest. All results are expressed as mean∫S.E.M.
Results BrdU staining of rat hepatocytes DNA synthesis was determined by BrdU incorporation in hepatocytes from 1-, 3-, 6- and 8-week-old rats (nΩ3). The BrdU labeling index was 18.9∫2.4% in 1week-old rats, and then decreased to 0.8∫0.02% in 8week-old rats (Fig. 1). Fig. 3. Incidence of [Ca2π]i oscillations in phenylephrineevoked [Ca2π]i responses in hepatocyte doublets from 1-, 3-, 6- and 8-week-old rats. Values are expressed as mean∫S.E.M. from three to six separate preparations, each comprising at least 5 doublets (1 vs. 3 weeks, p∞0.005; 3 vs. 6 weeks, p∞0.005; 6 vs. 8 weeks, pΩN.S.).
Fig. 4. The correlation between BrdU labeling index and incidence of oscillations. Mean value shown in Fig. 1 and 3 was plotted by linear regression (rΩ0.92).
caused by bleaching or dye leakage was maximally 20% at 340 nm and 380 nm, the ratio R was highly stable over 6 min in each experiment. Because intracellular Ca2π was calculated from the ratio R, the fading of fluorescence did not interfere with the results. The effects of phenylephrine (1 to 100 mmol/l) on [Ca2π]i were investigated using hepatocyte doublets from 1-, 3-, 6and 8-week-old rats.
224
Fig. 5. Effect of extracellular Ca2π on phenylephrineevoked [Ca2π]i oscillations in hepatocyte doublets. After loading with 4 mmol/l fura-2/AM, hepatocyte doublets from 1-week-old rats were perfused with PSS containing 10 mmol/l phenylephrine in the presence (A) and absence (B) of extracellular Ca2π. Thereafter, [Ca2π]i was measured every 10 s for 5 min. Representative data are shown from five experiments with and without extracellular Ca2π.
Ca2π signaling in hepatocyte doublets
Fig. 6. Phenylephrine-evoked [Ca2π]i responses in hepatocyte doublets from 1-week-old rats. Doublets were stimulated with different concentrations of phenylephrine (A;1 mmol/l, B;10 mmol/l, C;100 mmol/l). Representative data are shown from at least 20 experiments at each phenylephrine concentration.
Effect of rat aging on phenylephrine-evoked [Ca2π]i response pattern in hepatocyte doublets After loading cells with fura-2/AM for 30 min, [Ca2π]i was serially measured in the cytoplasmic region of each cell in doublets. [Ca2π]i response was observed in all examined doublets, following stimulation of cells with phenylephrine. In the presence of extracellular Ca2π, addition of 10 mmol/l phenylephrine to 1-week-old hepatocyte doublets resulted in transient-type [Ca2π]i oscillations, which were defined by the repetitive increase in [Ca2π]i that always returned to the basal [Ca2π]i levels (Fig. 2A). Transient-type [Ca2π]i oscillations, observed in 1-week-old rats, were replaced by sinusoidaltype changes in 3- and 6-week-old rats (Fig. 2B and 2C), and a sustained elevation in [Ca2π]i followed by a gradual return to baseline in 8-week-old rats (Fig. 2D). Similar results were obtained when cells were stimulated with 1 or 100 mmol/l phenylephrine. During these experiments, there was no significant difference in the basal level of [Ca2π]i between 1-, 3-, 6-, and 8-week-old rats (1 w, 108.3∫11.4 nmol/l, nΩ10; 3 w, 113.5∫12.8 nmol/l, nΩ10; 6 w, 107.2∫10.9 nmol/ l, nΩ10; 8 w, 118.3∫12.5 nmol/l, nΩ10).
Fig. 7. Frequency of phenylephrine-evoked [Ca2π]i oscillations in hepatocyte doublets from 1-week-old rats. Doublets were stimulated with different concentrations of phenylephrine. Values are expressed as mean∫S.E.M. from three to six separate preparations, each comprising at least 5 doublets.
225
N. Enomoto et al.
226
Ca2π signaling in hepatocyte doublets
Effect of rat aging on incidence of phenylephrineevoked [Ca2π]i oscillations In 1-week-old rat hepatocyte doublets, the addition of 10 mmol/l phenylephrine resulted in [Ca2π]i oscillations in all cells examined in the presence of extracellular Ca2π. The incidence of [Ca2π]i oscillations decreased proportionally as the rats aged (Fig. 3), and was linearly correlated with BrdU labeling index as shown in Fig. 4 (rΩ0.92). All the cells examined in 1-week-old hepatocytes displayed [Ca2π]i oscillations, but no sustained increase in [Ca2π]i, when stimulated with maximal concentration of phenylephrine (100 mmol/l). Effect of extracellular Ca2π on phenylephrine-evoked [Ca2π]i oscillations Phenylephrine-evoked [Ca2π]i oscillations were examined in the presence and absence of extracellular Ca2π in 1-week-old rat hepatocyte doublets. Removal of extracellular Ca2π did not abolish [Ca2π]i oscillations in 1-week-old rats, although the amplitude of [Ca2π]i oscillations was attenuated, presumably due to decreased recruitment of Ca2π from outside of cells (Fig. 5). This indicated that [Ca2π]i oscillations were caused primarily by Ca2π mobilization from internal sites of the cells. Effect of phenylephrine concentrations on the oscillation frequency Phenylephrine-evoked [Ca2π]i responses were investigated in 1-week-old hepatocyte doublets in the presence of extracellular Ca2π and 1 to 100 mmol/l phenylephrine. The increasing concentrations of phenylephrine enhanced the oscillation frequency with no significant change in oscillation amplitudes (Fig. 6 and 7). The frequency modulation was observed only in 1week-old hepatocytes, but not in more mature hepatocytes, suggesting that different Ca2π signaling between neonatal and adult rat hepatocytes is not due to the sensitivity of the cells to phenylephrine. Image analyses of phenylephrine-evoked [Ca2π]i responses in hepatocyte doublets Image analyses in phenylephrine-evoked [Ca2π]i responses are shown in Fig. 8. When stimulated with 10 mmol/l phenylephrine, 1-week-old rat hepatocyte doublets presented the asynchronous change in [Ca2π]i in each adjacent cell during the time course of experiment
(Fig. 8A). In contrast, [Ca2π]i responses were synchronous in each cell of 8-week-old rat hepatocyte doublets (Fig. 8B). Although we do not have any evidence which could explain the cell size difference between neonatal and adult rats, it may be partly due to the role played by cell volume in the regulation of osmotic balance controlled by plasma membrane Naπ-Kπ ATPase.
Discussion The orderly sequence of events that constitutes the cell cycle is carefully regulated by a number of factors, including the ubiquitous calcium signaling system in various types of cells (21). In hepatocytes, we recently reported that phenylephrine-evoked [Ca2π]i oscillations, which are temporal fluctuations in [Ca2π]i, might regulate cellular proliferation in regenerating rat liver in response to partial hepatectomy (11). It has not yet been determined, however, whether phenylephrine-evoked [Ca2π]i oscillations are specifically related to hepatocyte proliferation in liver regeneration after partial hepatectomy or are involved in hepatocyte proliferation in different states as well. In the present report we have shown that phenylephrineevoked [Ca2π]i oscillations are directly related to hepatocyte proliferation in neonatal but not adult rats, suggesting that [Ca2π]i oscillations play a crucial role in hepatocyte proliferation in developing liver. Fig. 1 shows that DNA synthesis occurred in ∂20% of 1-week-old rat hepatocytes, providing direct evidence of proliferation of neonatal rat hepatocytes. DNA synthesis in hepatocytes decreased as rats grew older, indicating that cellular proliferation and differentiation in vitro virtually parallel those in vivo. Based on these results, we examined [Ca2π]i responses in growing rat hepatocyte doublets following stimulation of cells with phenylephrine that is known to be a co-mitogen for epidermal growth factor (EGF) in hepatocytes (22,23). Growth stimulation in neonatal hepatocytes is more dependent upon exogenous growth factors than in adult hepatocytes (24–26). EGF can still cause quiescent (G0) hepatocytes kept in calcium-free medium to enter and traverse G1 phase. Ca2π-deprived cells, however, stop just before entering DNA synthesis phase (S), and only the replacement of calcium can induce them to cross the G1/S boundary (27). The observations described above strongly suggest
Fig. 8. Image analysis of phenylephrine-evoked [Ca2π]i responses in hepatocyte doublets. Following stimulation with 10 mmol/l phenylephrine, distribution of [Ca2π]i is shown as sequential images during the time course in hepatocyte doublets from 1- (A) and 8- (B) week-old rats. Time intervals between the images are 20 to 40 s. Images are serially shown from the left top to the right bottom. Representative data are shown from at least 20 experiments at each age.
227
N. Enomoto et al.
that Ca2π functions as a key factor in regulating G1/ S transition in physiological hepatocyte proliferation. Since phenylephrine is a co-mitogen of EGF (22,23), it may contribute to hepatocyte proliferation in neonatal rats by modulation of [Ca2π]i signal transduction. As shown in Fig. 2, in the presence of extracellular Ca2π, the addition of 10 mmol/l phenylephrine to hepatocyte doublets from 1–8-week-old rats resulted in [Ca2π]i oscillations or a non-oscillatory sustained increase in [Ca2π]i, followed by a gradual return to baseline. As the rats aged, however, phenylephrine-evoked [Ca2π]i responses shifted from transient-type [Ca2π]i oscillations to sinusoidal-type, and then to a sustained increase in [Ca2π]i, followed by a gradual return to baseline (Fig. 2 and 3). Although we cannot exclude the possibility that the sensitivity of the cells to phenylephrine shifts as the liver matures, the data in Fig. 1–4 strongly suggest that phenylephrine-evoked [Ca2π]i oscillations are directly related to hepatocyte proliferation in neonatal rats as well as in those that underwent partial hepatectomy (11). Most proliferative growth factors are capable of generating a number of signaling pathways, one of which is the hydrolysis of inositol lipids (28,29). The hydrolysis of phosphatidyl inositol 4,5-bis phosphate produces both diacylglycerol (DAG) and inositol 1,4,5-triphosphate (InsP3). The InsP3 that is released into the cytosol is responsible for setting up the [Ca2π]i oscillations (30). As shown in the present study, extracellular Ca2π was not required for [Ca2π]i oscillations (Fig. 5), indicating that the oscillations were caused primarily by Ca2π mobilization from the internal sites of the cells. This is consistent with most previous findings showing that internal Ca2π stores play an important role in maintaining the calcium signaling system, although the process has been suggested to depend upon the influx of external calcium (31,32). Indeed, extracellular calcium providing these stores with Ca2π is required for hepatocytes to proliferate both in vivo and in vitro (33). Furthermore, thapsigargin, a Ca2π-ATPase inhibitor of endoplasmic reticulum (ER), has been shown to inhibit proliferation perhaps by disrupting the role of the ER in protein synthesis (34). Interestingly, the oscillatory phase of [Ca2π]i was independent in each cell of doublets from neonatal rats, whereas the [Ca2π]i level was synchronous in doublet cells from adult rats (Fig. 8). Although the responsible mechanisms are unclear, this may be partly explained by the finding that gap junction protein, regulating cell-cell communication, is decreased in proliferating hepatocytes (35). Because gap junction conductance has also been suggested to be important for coordinat-
228
ing [Ca2π]i responses between adjacent cells (12,13), impaired cell-cell communication may participate in asynchronous [Ca2π]i responses in proliferating doublets from neonatal rats. There are a number of studies dealing with the role of Ca2π in mediating cell cycle progression (36). One of most important Ca2π regulatory roles is the activation of the immediate early genes responsible at both the G0/G1 transition and late G1 (37). However, there is much less information on the targets of Ca2π signaling, which is thought to occur in late G1. If Ca2π plays a role at this late stage, it is likely to be interacting with some component of the cell cycle proteins that control the onset of DNA synthesis. Indeed, calmodulin, the primary intracellular receptor for Ca2π, may be involved in this process, because its concentration is elevated as quiescent cells are stimulated to reenter the proliferative cycle and increases twofold at the G1/S boundary (38,39). Recently, Takuwa et al. reported that Ca2π signal in late G1 might act through cdc2 kinase activation to induce the phosphorylation of the product of the retinoblastoma (Rb) gene (40). When dephosphorylated, Rb functions as a tumor suppressor gene to prevent entry into S phase. As cells approach the G1/S interface, Rb becomes heavily phosphorylated through a mechanism that might be regulated by calcium (41). There is no phosphorylation of Rb and little DNA synthesis when cells are stimulated with serum with low amounts of calcium. Despite these findings supporting the importance of Ca2π in cell growth, physiological roles of [Ca2π]i oscillations still remain unclear. As shown in Fig. 6 and 7, [Ca2π]i oscillations observed in neonatal, but not adult, rat hepatocyte doublets displayed the frequency modulation that is dependent upon agonist concentrations. This phenomenon has been believed to be advantageous in regulation of various cellular functions in many cell types (42,43). Furthermore, Dolmestsch et al. recently reported that the spike and and plateau phases of [Ca2π]i response selectively controlled activation of the transcriptional regulators such as NF-kB, suggesting frequency-specific effects of [Ca2π]i oscillations on gene transcription (44). Because phenylephrine, a co-mitogen for hepatocytes, does not promote cell-cycle progression in hepatocytes by itself, it was difficult to demonstrate a direct cause-effect relationship between [Ca2π]i oscillations and cell proliferation in the present study. However, our recent data indicated that phenylephrineevoked [Ca2π]i signaling was modulated by preincubation of hepatocytes with EGF (45), thus suggesting that the differential [Ca2π]i signaling in neonatal and
Ca2π signaling in hepatocyte doublets
adult hepatocytes may play a role in cellular regulation during physiological liver growth. The exact mechanisms of [Ca2π]i oscillations are complicated and controversial. The Ca2π-sensitive InsP3 receptor in ER, but not the ryanodine receptor, which participates in calcium-induced calcium release, has been implicated in the production of [Ca2π]i oscillations (46). The role of the InsP3 receptor in the production of [Ca2π]i oscillations is not universally accepted and may depend upon the cell type (47). Molecular mechanisms of [Ca2π]i oscillations related to hepatocyte proliferation require further study.
13.
14.
15.
16.
Acknowledgements This work was supported by Grant-in-Aid (08670622) from the Ministry of Education, Science, and Culture of Japan.
17.
References
18.
1. Gilbert SH, Perry K, Fay FS. Mediation of chemoattractantinduced changes in [Ca2π]i and cell shape, polarity and locomotion by InsP3, DAG and protein kinase C. J Cell Biol 1994; 127: 489–503. 2. Petersen OH. Stimulus-secretion coupling: cytoplasmic calcium signals and the control of ion channels in exocrine acinar cells. J Physiol 1992; 448: 1–51. 3. Cheng H, Lederer WJ, Cannell MB. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 1993; 262: 740–4. 4. Peunova N, Enikolopov G. Amplification of calcium-induced gene transcription by nitric oxide in neuronal cells. Nature 1993; 364: 450–453. 5. Cuthbertson KSR, Cobbold PH. Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2π. Nature 1985; 316: 541–2. 6. Igusa Y, Miyazaki S-I. Periodic increase of cytoplasmic free calcium in fertilized hamster eggs measured with calciumsensitive electrodes. J Physiol (Lond) 1986; 377: 193–205. 7. Hess SD, Oortgiesen M, Cahalan MD. Calcium oscillations in human T and natural killer cells depend upon membrane potential and calcium influx. J Immunol 1993; 150: 2620–33. 8. Hashii M, Nozawa Y, Higashida H. Bradykinin-induced cytosolic Ca2π oscillations and inositol tetrakisphosphate-induced Ca2π influx in voltage-clamped ras-transformed NIH/ 3T3 fibroblasts. J Biol Chem 1993; 268: 19403–410. 9. Tao FU, Okano Y, Nozawa Y. Differential pathways (Phospholipase C and Phospholipase D) of bradykinin-induced biphasic 1,2-diacylglycerol formation in non-transformed and K-ras-transformed NIH-3T3 fibroblasts. Biochem J 1992; 283: 347–54. 10. Benzaquen LR, Brugnara C, Byers HR, Gattoni-Celli S, Halperin JA. Clotrimazole inhibits cell proliferation in vitro and in vivo. Nature Med 1995; 1: 534–40. 11. Kitamura T, Watanabe S, Ikejima K, Hirose M, Miyazaki A, Yumoto A, et al. Different features of Ca2π oscillations in differentiated and undifferentiated hepatocyte doublets. Hepatology 1995; 21: 1395–404. 12. Watanabe S, Phillips MJ. Ca2π causes active contraction of
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
bile canaliculi: direct evidence from microinjection studies. Proc Natl Acad Sci USA 1984; 81: 6164–8. Nathanson MH, Burgstahler AD. Coordination of hormone induced calcium signals in isolated rat hepatocyte couplets: demonstration with confocal microscopy. Mol Biol Cell 1992; 3: 113–21. Kitamura T, Brauneis U, Gatmaitan Z, Arias IM. Extracellular ATP, intracellular calcium and canalicular contraction in rat hepatocyte doublets. Hepatology 1991; 14: 640–7. Wilton JC, Chipman JK, Lawson CJ, Strain AJ, Coleman R. Periportal- and perivenous-enriched hepatocyte couplets: differences in canalicular activity and in response to oxidative stress. Biochem J 1993; 292: 773–9. Kitamura T, Jansen P, Hardenbrook C, Kamimoto Y, Gatmaitan Z, Arias IM. Defective ATP-dependent bile canalicular transport of organic anions in mutant (TR-) rats with conjugated hyperbilirubinemia. Proc Natl Acad Sci USA 1990; 87: 3557–61. Kitamura T, Gatmaitan Z, Arias IM. Serial quantitative image analysis and confocal microscopy of hepatic uptake, intracellular distribution and biliary secretion of a fluorescent bile acid analogue in rat hepatocyte doublets. Hepatology 1990; 10: 1358–64. Seglen PO. Preparation of isolated rat liver cells. In: Prescott DM, editor. Methods in Cell Biology, Vol. XIII. New York: Academic Press; 1976. p. 29–83. Gratzner HG. Monoclonal antibody to 5-bromo- and 5iododexyuridine: a new reagent for detection of DNA replication. Science 1982; 218: 474–5. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2π indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440–50. Whitfield JF, MacManus JP, Rixon RH, Boynton AL, Youdale T, Swierenga S. The positive control of cell proliferation by the interplay of calcium ions and cyclic nucleotides. In Vitro 1976; 12: 1–18. Cruise JL, Houck KA, Michalopoulos GK. Induction of DNA synthesis in cultured rat hepatocytes through stimulation of a1 adrenoreceptor by norepinephrine. Science 1985; 227: 749–51. Cruise JL, Michalopoulos GK. Norepinephrine and epidermal growth factor: dynamics of their interaction in the stimulation of hepatocyte DNA synthesis. J Cell Physiol 1985; 125: 45–50. Draghi E, Armato U, Andreis PG, Mengato L. The stimulation by epidermal growth factor (urogastrone) of the growth of neonatal rat hepatocytes in primary tissue culture and its modulation by serum and associated pancreatic hormones. J Cell Physiol 1980; 103: 129–47. Tomomura A, Sawada N, Sattler GL, Kleinman HK, Pitot HC. The control of DNA synthesis in primary cultures of hepatocytes from adult and young rats: interactions of extracellular matrix components, epidermal growth factor, and the cell cycle. J Cell Physiol 1987; 130: 221–7. Leffert HL. Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. VII. Hormonal control of DNA synthesis and its possible significance to the problem of liver regeneration. J Cell Biol 1974; 62: 767–79. Armato U, Andreis PG, Whitfield JF. The calcium-dependence of the stimulation of neonatal rat hepatocyte DNA synthesis and division by epidermal growth factor, glucagon and insulin. Chem Biol Interact 1983; 45: 203–22. Osada S, Saji S, Nakamura T, Nozawa Y. Cytosolic calcium
229
N. Enomoto et al.
29.
30.
31.
32.
33.
34.
35.
36. 37.
38.
oscillasions induced by hepatocyte growth factor (HGF) in single fura-2-loaded cultured hepatocytes: effects of extracellular calcium and protein kinase C. Biochim Biophys Acta 1992; 1135: 229–32. Kaneko A, Hayashi N, Tsubouchi H, Tanaka Y, Ito T, Sasaki Y, et al. Intracellular calcium as a second messenger for human hepatocyte growth factor in hepatocytes. Hepatology 1992; 15: 1173–8. Hansen CA, Yang L, Williamson JR. Mechanisms of receptor-mediated Ca2π signaling in rat hepatocytes. J Biol Chem 1991; 266: 18573–9. Woods NM, Cuthbertson KSR, Cobbold PH. Repetitive transient rises in cytoplasmic free calcium in hormone stimulated hepatocytes. Nature 1986; 319: 600–2. Rooney TA, Sass EJ, Thomas AP. Characterization of cytosolic oscillations induced by phenylephrine and vasopressin in single fura-2-loaded hepatocytes. J Biol Chem 1989; 264: 17131–41. Walker PR, Whitfield JF. Regulation of the prereplicative changes in the synthesis and transport of messenger and ribosomal RNA in regenerating livers of normal and hypocalcemic rats. J Cell Physiol 1981; 108: 427–37. Short AD, Bian J, Ghosh TK, Waldron RT, Rybak SL, Gill DL. Intracellular Ca2π pool content is linked to control of cell growth. Proc Natl Acad Sci USA 1993; 90: 4986–90. Dermietzel R, Yancey SB, Traub O, Willecke K, Revel J-P. Major loss of the 28-kD protein of gap junction in proliferating hepatocytes. J Cell Biol 1987; 105: 1925–34. Lu KP, Means AR. Regulation of the cell cycle by calcium and calmodulin. Endocrine Rev 1993; 14: 40–58. Hazelton B, Mitchell B, Tupper J. Calcium, magnesium and growth control in the WI-38 fibroblast cell. J Cell Biol 1979; 83: 487–98. Chafouleas JG, Bolton WE, Boyd III AE, Means AR. Cal-
230
39.
40.
41.
42.
43.
44.
45.
46.
47.
modulin and the cell cycle: involvement in regulation of cell cycle progression. Cell 1982; 28: 41–50. Chafouleas JG, Bolton WE, Boyd III AE, Means AR. Effects of anti-calmodulin drugs on the re-entry of plateau phase (G0) cells into the cell cycle. Cell 1984; 36: 73–81. Takuwa N, Zhou W, Kumada M, Takuwa Y. Ca2π-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts. J Biol Chem 1993; 268: 138–45. Chen P-L, Scully P, Shew J-Y, Wang JYJ, Lee W-H. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell 1989; 22: 1193–8. Berridge MJ. Inositol triphosphate-induced membrane potential oscillations in Xenopus oocytes. J Physiol 1988; 403: 589–99. Goldbeter A, Dupont G, Berridge MJ. Minimal model for signal-induced Ca2π oscillations and for their frequency encoding through protein phosphorylation. Proc Natl Acad Sci USA 1990; 87: 1461–5. Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI. Differential activation of transcription factors induced by Ca2π response amplitude and duration. Nature 1997; 386: 855–8. Kitamura T, Watanabe S, Hirose M, Enomoto N, Yamada T, Oide H, et al. [Ca2π]i oscillations mediate comitogenic effect of phenylephrine for epidermal growth factor (EGF) in rat hepatocyte doublets [abstract]. Hepatology 1996; 24: 360. Iino M, Endo M. Calcium-dependent immediate feedback control of inositol 1,4,5-triphosphate-induced Ca2π release. Nature 1992; 360: 76–8. Hua SY, Tokimasa T, Takasawa S, Furuya Y, Nohmi M, Okamoto H, et al. Cyclic ADP-ribose modulates Ca2π release channels for activation by physiological Ca2π entry in bullfrog sympathetic neurons. Neuron 1994; 12: 1073–9.
Copyright C European Association for the Study of the Liver 1998
Journal of Hepatology ISSN 0168-8278
Differential Ca2π signaling in neonatal and adult rat hepatocyte doublets Nobuyuki Enomoto, Tsuneo Kitamura, Miyoko Hirose, Kenichi Ikejima, Sumio Watanabe and Nobuhiro Sato Department of Gastroenterology, Juntendo University School of Medicine, Tokyo, Japan
Background/Aims: Intracellular Ca2π ([Ca2π]i) is important in various cellular functions, including cellular proliferation and differentiation. To elucidate the relationship between [Ca2π]i oscillations and physiological hepatocyte proliferation, phenylephrine-evoked [Ca2π]i responses were sequentially investigated using short-term cultured hepatocyte doublets obtained from 1-, 3-, 6- and 8-week-old rats. Methods/Results: DNA synthesis in hepatocytes, determined by BrdU incorporation, was ∂20% in 1week-old rats, and decreased to ∞1% as the rats aged. Correspondingly, [Ca2π]i responses evoked by 10 mmol/l phenylephrine in hepatocyte doublets shifted from transient to sinusoidal-type [Ca2π]i oscillations and then to a sustained increase in [Ca2π]i, followed by a gradual return to baseline. The incidence of [Ca2π]i oscillations was 100∫0.0%, 83.3∫16.7%, 38.7∫0.6% and 5.5∫5.0% in 1-, 3-, 6- and 8-weekold rats, respectively. Removal of extracellular Ca2π
did not abolish [Ca2π]i oscillations, indicating that [Ca2π]i oscillations were caused primarily by Ca2π mobilization from internal sites of the cells. The [Ca2π]i level in each of the adjacent cells was synchronous in sustained increase in [Ca2π]i, but asynchronous in [Ca2π]i oscillations. In proliferating doublets obtained from 1-week-old rats, the frequency of oscillations increased in a dose-dependent manner for phenylephrine concentrations of 1 to 100 mmol/l. Conclusions: Phenylephrine-evoked [Ca2π]i oscillations were directly related to hepatocyte proliferation and were mediated by frequency modulation. These results suggest that phenylephrine-evoked [Ca2π]i oscillations may contribute to cell-cycle progression of hepatocytes in physiological liver growth.
evidence indicates that intracellular Ca2π ([Ca2π]i) is a key player in regulation of cell motility (1), secretion (2), contraction (3) and possibly gene expression (4). Recently, much attention has focused on these findings in relation to temporal and spatial propagation of [Ca2π]i that are called [Ca2π]i oscillations and waves, respectively. The importance of periodic changes in [Ca2π]i has been emphasized in many aspects of cell growth as well. For instance, [Ca2π]i oscillations have been reported in mouse oocytes that are activated by sperm (5) and in fertilized hamster eggs (6). Oscillations of [Ca2π]i could be also detected in lymphocytes during the action of growth factors (7) and in fibroblasts
transformed with the ras oncogene when stimulated with bradykinin (8). Possible mechanisms for the oscillations in ras-transformed cells include an enhancement of inositol 1,4,5-triphosphate (InsP3) formation or an increased calcium influx (9). Recently, the role of [Ca2π]i in cell proliferation has been highlighted by anti-cancer therapies directed towards this Ca2π signaling pathway (10). These results suggest that intracellular Ca2π signal transduction, particularly in relation to [Ca2π]i oscillations or waves, may extensively involve cell-cycle control that includes developmental, physiological and transformed cell growth in various types of cells. The liver has strong potential for proliferation in regeneration, and it may therefore be assumed that Ca2π signal transduction plays an important role in hepatocyte proliferation. We recently reported characteristic [Ca2π]i oscillations in proliferating rat hepatocytes, after two-thirds hepatectomy (11), suggesting the importance of calcium signal transduction in hepatocyte
I
Received 6 August; revised 19 September; accepted 23 September 1997
Correspondence: Nobuhiro Sato, Department of Gastroenterology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Tel: 81 (03) 5802-1058. Fax: 81 (03) 3813-8862.
Key words: Cell-cycle; Intracellular calcium; Physiological liver growth.
221
N. Enomoto et al.
proliferation during liver regeneration. However, it is not known how [Ca2π]i modulates physiological hepatocyte growth in the developing liver. In the present study, to elucidate the relationship between [Ca2π]i oscillations and physiological hepatocyte proliferation, [Ca2π]i responses evoked by phenylephrine, a known co-mitogen, were sequentially investigated using short-term cultured hepatocyte doublets obtained from 1-, 3-, 6- and 8-week-old rats. Most studies that have evaluated the [Ca2π]i signaling have used conventional monolayered hepatocytes in culture. However, it is generally recognized that differentiated cell function is rapidly decreased under these conditions, suggesting that these cells may not accurately reflect the true nature of cell function in vivo. For this reason, short-term cultured hepatocyte doublets were used in the present study, because they retain various in vivo functions including cell-to-cell communication (12,13), bile canalicular contraction (14,15) and organic anion transport system (16,17).
Materials and Methods Materials Phenylephrine, ionomycin, 5-bromo-2ø-deoxy-uridine (BrdU), monoclonal anti-BrdU antibody and type I collagenase were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fura-2 acetomethoxy ester (fura-2/AM) and pluronic F127 were purchased from Molecular Probes (Junction City, OR, USA). All other chemicals were of analytical grade.
dishes (Becton Dickinson Labware, Lincoln Park, NJ, USA) at 37æC. Viability was assessed by the trypan blue exclusion test. All experiments were performed using cells showing viability of ±90%. Cells were maintained in L-15 containing 10% fetal cow serum (FCS), penicillin (100 IU/ml) and streptomycin (100 mg/ml). Hepatocyte doublets were identified by phase microscopy after cells had been cultured at 37æC for 4 h. Approximately 10% of hepatocytes appeared in pairs that enclosed circular spaces easily identified as bile canaliculi (16). BrdU staining of hepatocytes in developing rat livers After incubation of hepatocytes from 1-, 3-, 6- and 8week-old rats for 4 h, the medium was replaced with fresh medium containing BrdU, 10 mg/ml, for 1 h, and then cells were stained by indirect immunofluorescence methods using monoclonal anti-BrdU antibody (19). Serial quantitative analysis of [Ca2π]i in doublets using a fluorescence microscope imaging system All experiments were performed after cells had been incubated at 37æC for 4 h. Fura-2/AM with pluronic F127 was dissolved in phosphate saline solution (PSS; 135 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l CaCl2, 1 mmol/l MgCl2, 5.5 mmol/l glucose, 10 mmol/l HEPES) containing 1.0 mmol/l Ca2π, in which final concentrations of fura-2/AM and pluronic F127 were 4 mmol/l and 0.05%, respectively. After loading with fura-2/AM
Animals Female Wistar rats, 1, 3, 6 and 8 weeks old, were housed in a temperature-controlled room with lightdark periods of 12 h each and maintained on Purina rat chow and water ad libitum. The study protocol was approved by the Animal Care Committee of Juntendo University, which conforms to the National Institutes of Health guidelines. Preparation of primary cultured hepatocytes Monolayer cultures of rat hepatocytes were prepared from 1-, 3-, 6- and 8-week-old rats, using a modification of the method of Seglen (18). The portal vein was perfused with L-15 medium (Gibco/BRL, Life Technologies, Inc., New York, NY, USA) for 10 min at 4–40 ml/min, followed by the perfusion of a 0.05% solution of type I collagenase for 10 min. The liver was excised, and hepatocytes were detached. Isolated cells were centrifuged at 50 g for 2 min and resuspended in the fresh cold L-15 medium. Cells were filtered through nylon gauze, recentrifuged and incubated at 3¿105 cells per dish on a coverslip in 35-mm Falcon culture
222
Fig. 1. DNA synthesis determined by BrdU incorporation in hepatocytes from 1-, 3-, 6- and 8-week-old rats. Values are expressed as mean∫S.E.M. from three separate preparations (1 vs. 3 weeks, p∞0.005; 3 vs. 6 weeks, p∞0.005; 6 vs. 8 weeks, pΩN.S.).
Ca2π signaling in hepatocyte doublets
Fig. 2. Phenylephrine-evoked [Ca2π]i responses in hepatocyte doublets from 1- (A), 3- (B), 6- (C) and 8- (D) -week-old rats. After loading with 4 mmol/l fura-2/AM, hepatocyte doublets were perfused with PSS containing 10 mmol/l phenylephrine in the presence of extracellular Ca2π. Thereafter, [Ca2π]i was measured every 10 s for 5 min. Representative data are shown from at least 20 experiments in each age group.
solution at 37æC for 30 min, hepatocyte doublets on coverslips were placed in a Dvorak-Stotler Culture Chamber (Nicholson Precision Instruments, Gaithersburg, MD, USA) at 37æC and perfused with PSS in the presence or absence of Ca2π (1.0 mmol/l) at 0.2 ml/min using a Harvard pump (Harvard Apparatus 22, South Natik, MA, USA). The chamber was installed in a fluorescence microscope (Diaphot, Nikon, Tokyo, Japan) with a 100-W xenon arc lamp as a light source. The objective lens was a Nikon Fluor ¿100. A siliconintensified target camera (C-2400, Hamamatsu Photonics, Hamamatsu, Japan) was linked to a computer (MAXY DT2, Mitsubishi, Tokyo, Japan), printer (BJ10V, Cannon, Tokyo, Japan) and video recorder (UP5100, Sony, Tokyo, Japan). Fluorescence intensity of fura-2/AM was quantified every 10 s for 6 min in userselected regions of uniform pixel value with a 256-level gray scale corresponding to a uniform site in cytoplasm. Wavelengths of 340 nm and 380 nm for excitation and 520 nm for emission were used, and frame
averaging for noise reduction was performed. Sixteen frames were averaged for each measurement. The interval between excitation filters was 2 s. To reduce bleaching caused by the high intensity of the xenon lamp, a neutral-density filter (1/16) was used. Background fluorescence and cellular autofluorescence were not detectable at the setting used for gain and sensitivity. [Ca2π]i was determined by the following equation (20): [Ca2π]iΩKd([Ro–Rmin]/[Rmax –Ro]) B Kd, the Ca2π dissociation constant for fura-2, was confirmed as 224 nmol/l. R represents fluorescence intensity at 340 nm excitation divided by that at 380 nm excitation (Ro, experimental data; Rmin, R in 2 mmol/l ethylene glycol-bis [b aminoethyl ether] N,N,Nø,Nøtetra acetic acid (EGTA) and 1 mmol/l ionomycin; Rmax is R in 10 mmol/l Ca and 1 mmol/l ionomycin). B is the ratio of fluorescence intensity at 380 nm in the absence of Ca2π versus a saturating concentration of Ca2π. Although reduction of fluorescence intensity
223
N. Enomoto et al.
The incidence of [Ca2π]i oscillations was assessed as the ratio of cells showing [Ca2π]i oscillations to those presenting [Ca2π]i responses in each experiment. Statistical methods Statistical analysis was performed using Student’s ttest. All results are expressed as mean∫S.E.M.
Results BrdU staining of rat hepatocytes DNA synthesis was determined by BrdU incorporation in hepatocytes from 1-, 3-, 6- and 8-week-old rats (nΩ3). The BrdU labeling index was 18.9∫2.4% in 1week-old rats, and then decreased to 0.8∫0.02% in 8week-old rats (Fig. 1). Fig. 3. Incidence of [Ca2π]i oscillations in phenylephrineevoked [Ca2π]i responses in hepatocyte doublets from 1-, 3-, 6- and 8-week-old rats. Values are expressed as mean∫S.E.M. from three to six separate preparations, each comprising at least 5 doublets (1 vs. 3 weeks, p∞0.005; 3 vs. 6 weeks, p∞0.005; 6 vs. 8 weeks, pΩN.S.).
Fig. 4. The correlation between BrdU labeling index and incidence of oscillations. Mean value shown in Fig. 1 and 3 was plotted by linear regression (rΩ0.92).
caused by bleaching or dye leakage was maximally 20% at 340 nm and 380 nm, the ratio R was highly stable over 6 min in each experiment. Because intracellular Ca2π was calculated from the ratio R, the fading of fluorescence did not interfere with the results. The effects of phenylephrine (1 to 100 mmol/l) on [Ca2π]i were investigated using hepatocyte doublets from 1-, 3-, 6and 8-week-old rats.
224
Fig. 5. Effect of extracellular Ca2π on phenylephrineevoked [Ca2π]i oscillations in hepatocyte doublets. After loading with 4 mmol/l fura-2/AM, hepatocyte doublets from 1-week-old rats were perfused with PSS containing 10 mmol/l phenylephrine in the presence (A) and absence (B) of extracellular Ca2π. Thereafter, [Ca2π]i was measured every 10 s for 5 min. Representative data are shown from five experiments with and without extracellular Ca2π.
Ca2π signaling in hepatocyte doublets
Fig. 6. Phenylephrine-evoked [Ca2π]i responses in hepatocyte doublets from 1-week-old rats. Doublets were stimulated with different concentrations of phenylephrine (A;1 mmol/l, B;10 mmol/l, C;100 mmol/l). Representative data are shown from at least 20 experiments at each phenylephrine concentration.
Effect of rat aging on phenylephrine-evoked [Ca2π]i response pattern in hepatocyte doublets After loading cells with fura-2/AM for 30 min, [Ca2π]i was serially measured in the cytoplasmic region of each cell in doublets. [Ca2π]i response was observed in all examined doublets, following stimulation of cells with phenylephrine. In the presence of extracellular Ca2π, addition of 10 mmol/l phenylephrine to 1-week-old hepatocyte doublets resulted in transient-type [Ca2π]i oscillations, which were defined by the repetitive increase in [Ca2π]i that always returned to the basal [Ca2π]i levels (Fig. 2A). Transient-type [Ca2π]i oscillations, observed in 1-week-old rats, were replaced by sinusoidaltype changes in 3- and 6-week-old rats (Fig. 2B and 2C), and a sustained elevation in [Ca2π]i followed by a gradual return to baseline in 8-week-old rats (Fig. 2D). Similar results were obtained when cells were stimulated with 1 or 100 mmol/l phenylephrine. During these experiments, there was no significant difference in the basal level of [Ca2π]i between 1-, 3-, 6-, and 8-week-old rats (1 w, 108.3∫11.4 nmol/l, nΩ10; 3 w, 113.5∫12.8 nmol/l, nΩ10; 6 w, 107.2∫10.9 nmol/ l, nΩ10; 8 w, 118.3∫12.5 nmol/l, nΩ10).
Fig. 7. Frequency of phenylephrine-evoked [Ca2π]i oscillations in hepatocyte doublets from 1-week-old rats. Doublets were stimulated with different concentrations of phenylephrine. Values are expressed as mean∫S.E.M. from three to six separate preparations, each comprising at least 5 doublets.
225
N. Enomoto et al.
226
Ca2π signaling in hepatocyte doublets
Effect of rat aging on incidence of phenylephrineevoked [Ca2π]i oscillations In 1-week-old rat hepatocyte doublets, the addition of 10 mmol/l phenylephrine resulted in [Ca2π]i oscillations in all cells examined in the presence of extracellular Ca2π. The incidence of [Ca2π]i oscillations decreased proportionally as the rats aged (Fig. 3), and was linearly correlated with BrdU labeling index as shown in Fig. 4 (rΩ0.92). All the cells examined in 1-week-old hepatocytes displayed [Ca2π]i oscillations, but no sustained increase in [Ca2π]i, when stimulated with maximal concentration of phenylephrine (100 mmol/l). Effect of extracellular Ca2π on phenylephrine-evoked [Ca2π]i oscillations Phenylephrine-evoked [Ca2π]i oscillations were examined in the presence and absence of extracellular Ca2π in 1-week-old rat hepatocyte doublets. Removal of extracellular Ca2π did not abolish [Ca2π]i oscillations in 1-week-old rats, although the amplitude of [Ca2π]i oscillations was attenuated, presumably due to decreased recruitment of Ca2π from outside of cells (Fig. 5). This indicated that [Ca2π]i oscillations were caused primarily by Ca2π mobilization from internal sites of the cells. Effect of phenylephrine concentrations on the oscillation frequency Phenylephrine-evoked [Ca2π]i responses were investigated in 1-week-old hepatocyte doublets in the presence of extracellular Ca2π and 1 to 100 mmol/l phenylephrine. The increasing concentrations of phenylephrine enhanced the oscillation frequency with no significant change in oscillation amplitudes (Fig. 6 and 7). The frequency modulation was observed only in 1week-old hepatocytes, but not in more mature hepatocytes, suggesting that different Ca2π signaling between neonatal and adult rat hepatocytes is not due to the sensitivity of the cells to phenylephrine. Image analyses of phenylephrine-evoked [Ca2π]i responses in hepatocyte doublets Image analyses in phenylephrine-evoked [Ca2π]i responses are shown in Fig. 8. When stimulated with 10 mmol/l phenylephrine, 1-week-old rat hepatocyte doublets presented the asynchronous change in [Ca2π]i in each adjacent cell during the time course of experiment
(Fig. 8A). In contrast, [Ca2π]i responses were synchronous in each cell of 8-week-old rat hepatocyte doublets (Fig. 8B). Although we do not have any evidence which could explain the cell size difference between neonatal and adult rats, it may be partly due to the role played by cell volume in the regulation of osmotic balance controlled by plasma membrane Naπ-Kπ ATPase.
Discussion The orderly sequence of events that constitutes the cell cycle is carefully regulated by a number of factors, including the ubiquitous calcium signaling system in various types of cells (21). In hepatocytes, we recently reported that phenylephrine-evoked [Ca2π]i oscillations, which are temporal fluctuations in [Ca2π]i, might regulate cellular proliferation in regenerating rat liver in response to partial hepatectomy (11). It has not yet been determined, however, whether phenylephrine-evoked [Ca2π]i oscillations are specifically related to hepatocyte proliferation in liver regeneration after partial hepatectomy or are involved in hepatocyte proliferation in different states as well. In the present report we have shown that phenylephrineevoked [Ca2π]i oscillations are directly related to hepatocyte proliferation in neonatal but not adult rats, suggesting that [Ca2π]i oscillations play a crucial role in hepatocyte proliferation in developing liver. Fig. 1 shows that DNA synthesis occurred in ∂20% of 1-week-old rat hepatocytes, providing direct evidence of proliferation of neonatal rat hepatocytes. DNA synthesis in hepatocytes decreased as rats grew older, indicating that cellular proliferation and differentiation in vitro virtually parallel those in vivo. Based on these results, we examined [Ca2π]i responses in growing rat hepatocyte doublets following stimulation of cells with phenylephrine that is known to be a co-mitogen for epidermal growth factor (EGF) in hepatocytes (22,23). Growth stimulation in neonatal hepatocytes is more dependent upon exogenous growth factors than in adult hepatocytes (24–26). EGF can still cause quiescent (G0) hepatocytes kept in calcium-free medium to enter and traverse G1 phase. Ca2π-deprived cells, however, stop just before entering DNA synthesis phase (S), and only the replacement of calcium can induce them to cross the G1/S boundary (27). The observations described above strongly suggest
Fig. 8. Image analysis of phenylephrine-evoked [Ca2π]i responses in hepatocyte doublets. Following stimulation with 10 mmol/l phenylephrine, distribution of [Ca2π]i is shown as sequential images during the time course in hepatocyte doublets from 1- (A) and 8- (B) week-old rats. Time intervals between the images are 20 to 40 s. Images are serially shown from the left top to the right bottom. Representative data are shown from at least 20 experiments at each age.
227
N. Enomoto et al.
that Ca2π functions as a key factor in regulating G1/ S transition in physiological hepatocyte proliferation. Since phenylephrine is a co-mitogen of EGF (22,23), it may contribute to hepatocyte proliferation in neonatal rats by modulation of [Ca2π]i signal transduction. As shown in Fig. 2, in the presence of extracellular Ca2π, the addition of 10 mmol/l phenylephrine to hepatocyte doublets from 1–8-week-old rats resulted in [Ca2π]i oscillations or a non-oscillatory sustained increase in [Ca2π]i, followed by a gradual return to baseline. As the rats aged, however, phenylephrine-evoked [Ca2π]i responses shifted from transient-type [Ca2π]i oscillations to sinusoidal-type, and then to a sustained increase in [Ca2π]i, followed by a gradual return to baseline (Fig. 2 and 3). Although we cannot exclude the possibility that the sensitivity of the cells to phenylephrine shifts as the liver matures, the data in Fig. 1–4 strongly suggest that phenylephrine-evoked [Ca2π]i oscillations are directly related to hepatocyte proliferation in neonatal rats as well as in those that underwent partial hepatectomy (11). Most proliferative growth factors are capable of generating a number of signaling pathways, one of which is the hydrolysis of inositol lipids (28,29). The hydrolysis of phosphatidyl inositol 4,5-bis phosphate produces both diacylglycerol (DAG) and inositol 1,4,5-triphosphate (InsP3). The InsP3 that is released into the cytosol is responsible for setting up the [Ca2π]i oscillations (30). As shown in the present study, extracellular Ca2π was not required for [Ca2π]i oscillations (Fig. 5), indicating that the oscillations were caused primarily by Ca2π mobilization from the internal sites of the cells. This is consistent with most previous findings showing that internal Ca2π stores play an important role in maintaining the calcium signaling system, although the process has been suggested to depend upon the influx of external calcium (31,32). Indeed, extracellular calcium providing these stores with Ca2π is required for hepatocytes to proliferate both in vivo and in vitro (33). Furthermore, thapsigargin, a Ca2π-ATPase inhibitor of endoplasmic reticulum (ER), has been shown to inhibit proliferation perhaps by disrupting the role of the ER in protein synthesis (34). Interestingly, the oscillatory phase of [Ca2π]i was independent in each cell of doublets from neonatal rats, whereas the [Ca2π]i level was synchronous in doublet cells from adult rats (Fig. 8). Although the responsible mechanisms are unclear, this may be partly explained by the finding that gap junction protein, regulating cell-cell communication, is decreased in proliferating hepatocytes (35). Because gap junction conductance has also been suggested to be important for coordinat-
228
ing [Ca2π]i responses between adjacent cells (12,13), impaired cell-cell communication may participate in asynchronous [Ca2π]i responses in proliferating doublets from neonatal rats. There are a number of studies dealing with the role of Ca2π in mediating cell cycle progression (36). One of most important Ca2π regulatory roles is the activation of the immediate early genes responsible at both the G0/G1 transition and late G1 (37). However, there is much less information on the targets of Ca2π signaling, which is thought to occur in late G1. If Ca2π plays a role at this late stage, it is likely to be interacting with some component of the cell cycle proteins that control the onset of DNA synthesis. Indeed, calmodulin, the primary intracellular receptor for Ca2π, may be involved in this process, because its concentration is elevated as quiescent cells are stimulated to reenter the proliferative cycle and increases twofold at the G1/S boundary (38,39). Recently, Takuwa et al. reported that Ca2π signal in late G1 might act through cdc2 kinase activation to induce the phosphorylation of the product of the retinoblastoma (Rb) gene (40). When dephosphorylated, Rb functions as a tumor suppressor gene to prevent entry into S phase. As cells approach the G1/S interface, Rb becomes heavily phosphorylated through a mechanism that might be regulated by calcium (41). There is no phosphorylation of Rb and little DNA synthesis when cells are stimulated with serum with low amounts of calcium. Despite these findings supporting the importance of Ca2π in cell growth, physiological roles of [Ca2π]i oscillations still remain unclear. As shown in Fig. 6 and 7, [Ca2π]i oscillations observed in neonatal, but not adult, rat hepatocyte doublets displayed the frequency modulation that is dependent upon agonist concentrations. This phenomenon has been believed to be advantageous in regulation of various cellular functions in many cell types (42,43). Furthermore, Dolmestsch et al. recently reported that the spike and and plateau phases of [Ca2π]i response selectively controlled activation of the transcriptional regulators such as NF-kB, suggesting frequency-specific effects of [Ca2π]i oscillations on gene transcription (44). Because phenylephrine, a co-mitogen for hepatocytes, does not promote cell-cycle progression in hepatocytes by itself, it was difficult to demonstrate a direct cause-effect relationship between [Ca2π]i oscillations and cell proliferation in the present study. However, our recent data indicated that phenylephrineevoked [Ca2π]i signaling was modulated by preincubation of hepatocytes with EGF (45), thus suggesting that the differential [Ca2π]i signaling in neonatal and
Ca2π signaling in hepatocyte doublets
adult hepatocytes may play a role in cellular regulation during physiological liver growth. The exact mechanisms of [Ca2π]i oscillations are complicated and controversial. The Ca2π-sensitive InsP3 receptor in ER, but not the ryanodine receptor, which participates in calcium-induced calcium release, has been implicated in the production of [Ca2π]i oscillations (46). The role of the InsP3 receptor in the production of [Ca2π]i oscillations is not universally accepted and may depend upon the cell type (47). Molecular mechanisms of [Ca2π]i oscillations related to hepatocyte proliferation require further study.
13.
14.
15.
16.
Acknowledgements This work was supported by Grant-in-Aid (08670622) from the Ministry of Education, Science, and Culture of Japan.
17.
References
18.
1. Gilbert SH, Perry K, Fay FS. Mediation of chemoattractantinduced changes in [Ca2π]i and cell shape, polarity and locomotion by InsP3, DAG and protein kinase C. J Cell Biol 1994; 127: 489–503. 2. Petersen OH. Stimulus-secretion coupling: cytoplasmic calcium signals and the control of ion channels in exocrine acinar cells. J Physiol 1992; 448: 1–51. 3. Cheng H, Lederer WJ, Cannell MB. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 1993; 262: 740–4. 4. Peunova N, Enikolopov G. Amplification of calcium-induced gene transcription by nitric oxide in neuronal cells. Nature 1993; 364: 450–453. 5. Cuthbertson KSR, Cobbold PH. Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2π. Nature 1985; 316: 541–2. 6. Igusa Y, Miyazaki S-I. Periodic increase of cytoplasmic free calcium in fertilized hamster eggs measured with calciumsensitive electrodes. J Physiol (Lond) 1986; 377: 193–205. 7. Hess SD, Oortgiesen M, Cahalan MD. Calcium oscillations in human T and natural killer cells depend upon membrane potential and calcium influx. J Immunol 1993; 150: 2620–33. 8. Hashii M, Nozawa Y, Higashida H. Bradykinin-induced cytosolic Ca2π oscillations and inositol tetrakisphosphate-induced Ca2π influx in voltage-clamped ras-transformed NIH/ 3T3 fibroblasts. J Biol Chem 1993; 268: 19403–410. 9. Tao FU, Okano Y, Nozawa Y. Differential pathways (Phospholipase C and Phospholipase D) of bradykinin-induced biphasic 1,2-diacylglycerol formation in non-transformed and K-ras-transformed NIH-3T3 fibroblasts. Biochem J 1992; 283: 347–54. 10. Benzaquen LR, Brugnara C, Byers HR, Gattoni-Celli S, Halperin JA. Clotrimazole inhibits cell proliferation in vitro and in vivo. Nature Med 1995; 1: 534–40. 11. Kitamura T, Watanabe S, Ikejima K, Hirose M, Miyazaki A, Yumoto A, et al. Different features of Ca2π oscillations in differentiated and undifferentiated hepatocyte doublets. Hepatology 1995; 21: 1395–404. 12. Watanabe S, Phillips MJ. Ca2π causes active contraction of
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
bile canaliculi: direct evidence from microinjection studies. Proc Natl Acad Sci USA 1984; 81: 6164–8. Nathanson MH, Burgstahler AD. Coordination of hormone induced calcium signals in isolated rat hepatocyte couplets: demonstration with confocal microscopy. Mol Biol Cell 1992; 3: 113–21. Kitamura T, Brauneis U, Gatmaitan Z, Arias IM. Extracellular ATP, intracellular calcium and canalicular contraction in rat hepatocyte doublets. Hepatology 1991; 14: 640–7. Wilton JC, Chipman JK, Lawson CJ, Strain AJ, Coleman R. Periportal- and perivenous-enriched hepatocyte couplets: differences in canalicular activity and in response to oxidative stress. Biochem J 1993; 292: 773–9. Kitamura T, Jansen P, Hardenbrook C, Kamimoto Y, Gatmaitan Z, Arias IM. Defective ATP-dependent bile canalicular transport of organic anions in mutant (TR-) rats with conjugated hyperbilirubinemia. Proc Natl Acad Sci USA 1990; 87: 3557–61. Kitamura T, Gatmaitan Z, Arias IM. Serial quantitative image analysis and confocal microscopy of hepatic uptake, intracellular distribution and biliary secretion of a fluorescent bile acid analogue in rat hepatocyte doublets. Hepatology 1990; 10: 1358–64. Seglen PO. Preparation of isolated rat liver cells. In: Prescott DM, editor. Methods in Cell Biology, Vol. XIII. New York: Academic Press; 1976. p. 29–83. Gratzner HG. Monoclonal antibody to 5-bromo- and 5iododexyuridine: a new reagent for detection of DNA replication. Science 1982; 218: 474–5. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2π indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440–50. Whitfield JF, MacManus JP, Rixon RH, Boynton AL, Youdale T, Swierenga S. The positive control of cell proliferation by the interplay of calcium ions and cyclic nucleotides. In Vitro 1976; 12: 1–18. Cruise JL, Houck KA, Michalopoulos GK. Induction of DNA synthesis in cultured rat hepatocytes through stimulation of a1 adrenoreceptor by norepinephrine. Science 1985; 227: 749–51. Cruise JL, Michalopoulos GK. Norepinephrine and epidermal growth factor: dynamics of their interaction in the stimulation of hepatocyte DNA synthesis. J Cell Physiol 1985; 125: 45–50. Draghi E, Armato U, Andreis PG, Mengato L. The stimulation by epidermal growth factor (urogastrone) of the growth of neonatal rat hepatocytes in primary tissue culture and its modulation by serum and associated pancreatic hormones. J Cell Physiol 1980; 103: 129–47. Tomomura A, Sawada N, Sattler GL, Kleinman HK, Pitot HC. The control of DNA synthesis in primary cultures of hepatocytes from adult and young rats: interactions of extracellular matrix components, epidermal growth factor, and the cell cycle. J Cell Physiol 1987; 130: 221–7. Leffert HL. Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. VII. Hormonal control of DNA synthesis and its possible significance to the problem of liver regeneration. J Cell Biol 1974; 62: 767–79. Armato U, Andreis PG, Whitfield JF. The calcium-dependence of the stimulation of neonatal rat hepatocyte DNA synthesis and division by epidermal growth factor, glucagon and insulin. Chem Biol Interact 1983; 45: 203–22. Osada S, Saji S, Nakamura T, Nozawa Y. Cytosolic calcium
229
N. Enomoto et al.
29.
30.
31.
32.
33.
34.
35.
36. 37.
38.
oscillasions induced by hepatocyte growth factor (HGF) in single fura-2-loaded cultured hepatocytes: effects of extracellular calcium and protein kinase C. Biochim Biophys Acta 1992; 1135: 229–32. Kaneko A, Hayashi N, Tsubouchi H, Tanaka Y, Ito T, Sasaki Y, et al. Intracellular calcium as a second messenger for human hepatocyte growth factor in hepatocytes. Hepatology 1992; 15: 1173–8. Hansen CA, Yang L, Williamson JR. Mechanisms of receptor-mediated Ca2π signaling in rat hepatocytes. J Biol Chem 1991; 266: 18573–9. Woods NM, Cuthbertson KSR, Cobbold PH. Repetitive transient rises in cytoplasmic free calcium in hormone stimulated hepatocytes. Nature 1986; 319: 600–2. Rooney TA, Sass EJ, Thomas AP. Characterization of cytosolic oscillations induced by phenylephrine and vasopressin in single fura-2-loaded hepatocytes. J Biol Chem 1989; 264: 17131–41. Walker PR, Whitfield JF. Regulation of the prereplicative changes in the synthesis and transport of messenger and ribosomal RNA in regenerating livers of normal and hypocalcemic rats. J Cell Physiol 1981; 108: 427–37. Short AD, Bian J, Ghosh TK, Waldron RT, Rybak SL, Gill DL. Intracellular Ca2π pool content is linked to control of cell growth. Proc Natl Acad Sci USA 1993; 90: 4986–90. Dermietzel R, Yancey SB, Traub O, Willecke K, Revel J-P. Major loss of the 28-kD protein of gap junction in proliferating hepatocytes. J Cell Biol 1987; 105: 1925–34. Lu KP, Means AR. Regulation of the cell cycle by calcium and calmodulin. Endocrine Rev 1993; 14: 40–58. Hazelton B, Mitchell B, Tupper J. Calcium, magnesium and growth control in the WI-38 fibroblast cell. J Cell Biol 1979; 83: 487–98. Chafouleas JG, Bolton WE, Boyd III AE, Means AR. Cal-
230
39.
40.
41.
42.
43.
44.
45.
46.
47.
modulin and the cell cycle: involvement in regulation of cell cycle progression. Cell 1982; 28: 41–50. Chafouleas JG, Bolton WE, Boyd III AE, Means AR. Effects of anti-calmodulin drugs on the re-entry of plateau phase (G0) cells into the cell cycle. Cell 1984; 36: 73–81. Takuwa N, Zhou W, Kumada M, Takuwa Y. Ca2π-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts. J Biol Chem 1993; 268: 138–45. Chen P-L, Scully P, Shew J-Y, Wang JYJ, Lee W-H. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell 1989; 22: 1193–8. Berridge MJ. Inositol triphosphate-induced membrane potential oscillations in Xenopus oocytes. J Physiol 1988; 403: 589–99. Goldbeter A, Dupont G, Berridge MJ. Minimal model for signal-induced Ca2π oscillations and for their frequency encoding through protein phosphorylation. Proc Natl Acad Sci USA 1990; 87: 1461–5. Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI. Differential activation of transcription factors induced by Ca2π response amplitude and duration. Nature 1997; 386: 855–8. Kitamura T, Watanabe S, Hirose M, Enomoto N, Yamada T, Oide H, et al. [Ca2π]i oscillations mediate comitogenic effect of phenylephrine for epidermal growth factor (EGF) in rat hepatocyte doublets [abstract]. Hepatology 1996; 24: 360. Iino M, Endo M. Calcium-dependent immediate feedback control of inositol 1,4,5-triphosphate-induced Ca2π release. Nature 1992; 360: 76–8. Hua SY, Tokimasa T, Takasawa S, Furuya Y, Nohmi M, Okamoto H, et al. Cyclic ADP-ribose modulates Ca2π release channels for activation by physiological Ca2π entry in bullfrog sympathetic neurons. Neuron 1994; 12: 1073–9.