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Internalization study using EDTA-prepared hepatocytes for receptor-mediated endocytosis of haemoglobin–haptoglobin complex
The International Journal of
PERGAMON
The International Journal of Biochemistry & Cell Biology 30 (1998) 923±931
Biochemistry & Cell Biology
Internalization study using EDTA-prepared hepatocytes for receptor-mediated endocytosis of haemoglobin±haptoglobin complex Jolanta Zuwal/ a-Jagiel/ /lo, Jerzy Osada * Department of Pharmaceutical Biochemistry, Medical Academy, Szewska 38/39, 50-139 Wrocl/ aw, Poland Received 8 August 1997; accepted 4 March 1998
Abstract We have demonstrated the internalization of haemoglobin±haptoglobin (Hb±Hp) complex using rat hepatocytes prepared by EDTA perfusion, followed by Percoll. The isolated hepatocytes exhibited a saturation curve of the binding of ¯uorescein isothiocyanate-labelled haemoglobin±haptoglobin complex (FITC-Hb±Hp). Furthermore, competition between the binding of FITC-Hb±Hp and unlabelled Hb to the hepatocytes, was observed. The cells exhibited 09 104 `high anity sites' (Kd 0 1.2 mM) for the Hb±Hp complex. The data in toto suggest the presence of only one type of receptor i.e. the high anity receptor (in both anity and number of sites per cell). The results were similar to those obtained from rat hepatocytes prepared by collagenase digestion [1]. In order to verify whether EDTA-prepared hepatocytes could be used for the study of receptor-mediated endocytosis, the internalization of pre-bound Hb±Hp in the isolated hepatocytes was assessed by two methods. First, acid-insensitive FITC-Hb±Hp time-dependently increased following incubation at 378C. Secondly, Hb±Hp became inaccessible to the exogenous FITC-anti-haemoglobin antibody. These processes were dependent on ATP, but independent of Ca2+ and stimulated by GTP. The results demonstrate that the receptor-mediated endocytosis of Hb±Hp occurred in the EDTA-prepared hepatocytes. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: EDTA-prepared hepatocytes; Haemoglobin±haptoglobin; Complex-internalization; GTP; ATP
1. Introduction Abbreviations: Ab, antibody, ASOR, asialo-orosomucoid, ATP, adenosine triphosphate, EGTA, ethylene glycol bis(baminoethyl ether)-N, N, N', N'-tetraacetic acid, FITC, ¯uorescein isothiocyanate, Hp, human haptoglobin 1-1, GTP, guanosine triphosphate, Hb, rat haemoglobin, Hb±Hp, haemoglobin±haptoglobin complex, HBS, Hanks' balanced salt solution, HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid, Kd, dissociation constant. * Corresponding author. Fax: +48-71-442277.
Haptoglobin (Hp) is a plasma glycoprotein present in all vertebrates [2]. It can form a very tight complex with haemoglobin (Hb) in vivo and in vitro. The binding of haptoglobin to haemoglobin is one of the strongest known noncovalent interactions in biology. The two proteins form a complex with an association constant that has been too high to measure (greater than 10ÿ15
1357-2725/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 8 ) 0 0 0 3 5 - 1
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M) [3]. Hb±Hp complex disappears rapidly from the circulation being taken up by the liver parenchymal cells through a speci®c receptor for Hb±Hp [1, 4]. Liver metabolism (clearance) of haptoglobin from the Bovidae family and their complexes with haemoglobins was also observed [5±7]. Hb±Hp internalized by receptor-mediated endocytosis is dissociated symmetrically into 82000-Dalton subunits. The organelles with multivesicular body (MVB) or compartment of uncoupling of receptor and ligand (CURL)-like structures are associated with Hb±Hp metabolism [8, 4, 9]. In spite of the information on metabolism of Hb±Hp complexes, the detailed mechanism by which the endocytosis of Hb±Hp is induced is unclear. During receptor-mediated endocytosis, a ligand binds to a speci®c cell surface receptor, and the receptor± ligand complex is internalized in clathrin-coated pits and vesicles. It is dicult to directly approach the mechanism because the components involved in endocytosis are located on the cytoplasmic surface. Therefore, the demonstration of endocytosis in vitro can provide useful information of its mechanism. During the isolation of liver parenchymal cells with collagenase the apparent high anity binding sites for Hb±Hp seemed to be impaired [1]. The presence of proteases in collagenase preparations essential for hepatocyte release may cause surface damage. The liver parenchymal cells in primary culture also lacked the ability to internalize Hb±Hp, although they bound the molecule more extensively than freshly isolated liver cells. An interesting possibility is that collagenase engulfed during perfusion is released in culture, causing protein degradation by the contaminating proteases. It would be reasonable to postulate that impairment of the high anity binding sites for Hb±Hp may be the reason why the isolated hepatocytes lost the ability to internalize the Hb±Hp, although they readily bound the ligand. In this study, we employed the rat hepatocytes prepared by EDTA dissociation, to demonstrate the early stage of endocytosis in the internalization of haemoglobin±haptoglobin complex.
2. Materials and methods 2.1. Animals Male Bualo rats weighing 180±300 g body weight were used. Animals were given food and water ad libitum. 2.2. Materials Materials were obtained as follows: ¯uorescein isothiocyanate (FITC, isomer I), bovine transferrin, human orosomucoid and FITC labelled rabbit anti-human hemoglobin antibody were purchased from Sigma. Asialo-orosomucoid was prepared from human orosomucoid by desialylation with neuraminidase as described elsewhere [10]. All other chemicals were reagent grade. 2.3. FITC-Hb±Hp complex preparation Puri®ed rat haemoglobin (4 mg/ml in 0.1 M NaHCO3, pH 9.5) was incubated with 0.25 mM FITC with stirring at 228C for 1 h. The reaction was stopped by applying the protein-FITC mixture to a GF-5 desalting column (Pierce) equilibrated in 0.1 M NaHCO3, pH 9.5. Hbcontaining fractions were dialyzed against Hanks' balanced salt (HBS) solution and analyzed spectroscopically at 496 nm (FITC) and 280 nm (protein). FITC incorporation into Hb, 1.85 nmol of FITC per nmol Hb, was calculated according to Jobbagy and Kiraly [11]. Human haptoglobin 1-1 was prepared according to the method of Smith et al. [12]. The complex of labelled FITC-Hb with human haptoglobin was obtained in the following way: two fold excess of FITC-Hb was added to 5 mg of haptoglobin dissolved in 2 ml of HBS at room temperature. After 15 min the excess of FITC-Hb was removed by ®ltration through a Sephacryl S-200 column equilibrated with HBS. The fractions containing FITC-Hb± Hp were ®lter-sterilized (0.2 mm) and stored at ÿ208C until use.
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2.4. Preparation of rat hepatocytes Preparation of rat hepatocytes was performed essentially according to Wang et al. [13] as follows. After anesthetizing the rat by i.p. injection of 50 mg/kg Nembutal, the portal vein was cannulated a 19-gauge syringe needle and was secured in place by two ligatures of 4±0 silk. Flow began at 5 ml/min while the chest was opened and an incision made in the heart to allow out¯ow of the perfusate. The ¯ow rate was adjusted to 50±65 ml/min. The duration of perfusion was determined by the appearance of the liver. When the liver appeared softened and buffer was coming through the capsule, perfusion was stopped. Through more than twenty preparations done to date in this laboratory, the perfusion time ranged from 20±40 min, with larger, older rats taking longer. The perfusion buer, wash buer, and Percoll buer were prepared from a 10 stock solution containing 1.4 M NaCl, 50 mM KCl, 8.0 mM MgCl2, 16 mM Na2HPO4, and 4.0 mM KH2PO4. To prepare the perfusate, Buer A, 300 ml of the stock solution was diluted to 3 l with reverse osmosis puri®ed water and NaHCO3 and EDTA were added 25 and 2 mM ®nal concentration, respectively. Carbogen was bubbled through the solution, as the temperature was brought to 378C. Buer B, the wash buer, was Buer A without EDTA and NaHCO3, but containing 1 mM CaCl2. Percoll used to separate intact hepatocytes was made by diluting 87 ml of Percoll (Sigma) to 100 ml with undiluted 10 stock solution. The Percoll and Buer B were stored at 48C until used. The softened liver was removed and washed in Buer B. The liver was placed in a 100 ml beaker and minced with scissors. The liver was washed with Buer B to remove released hepatocytes. By repeated mincing and washing, about 90% of the liver was reduced to material passing a 200 micron ®lter. Centrifugation at 50 g for 2 min was used to collect hepatocytes. After two washes in Buer B, collected hepatocytes were resuspended in Buer B and diluted with 2 volumes of buered Percoll. Hepatocytes were pelleted by centrifugation at 1500 g for 5 min at room
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temperature. The cells were resuspended in hepatocyte modi®ed William's Medium E (Serva Feinbiochemica) containing 10% supplemented calf serum (Serva Feinbiochemica), 100 IU/l potassium penicillin G and 100 mg/l streptomycin sulphate. Cell viability was estimated by assessing the number of cells that excluded trypan blue. Parenchymal cells (hepatocytes) ranging 18±21 mm in diameter and nonparenchymal cells ranging 8±15 mm were readily identi®ed by light microscopy. Parenchymal cell preparations generally contained 5% contamination from nonparenchymal cells. Parenchymal cell preparations were routinely 81% viable. There is no correlation between the percentage of stained cells and the metabolic capability of the isolated hepatocytes [14]. Therefore a number of other viability criteria have been suggested. Among these, the content of ATP is probably the easiest. ATP was assayed using a modi®cation of luciferin/luciferase assay essentially as described by Weigel and Englund [15]. Cellular ATP levels were depleted to 45% normal value (ATP should be >2 mmol/108 cells). 2.5. Hb±Hp binding assay Hb±Hp binding to the receptor of the isolated hepatocytes was measured as follows. The cells were washed twice with a binding buer containing 20 mM HEPES, 150 mM NaCl and 2 mg/ml bovine serum albumin, pH 7.4. The cells were incubated for 2 h at 48C in the binding buer with FITC-Hb±Hp at the indicated concentrations. After the incubation, the cells were washed twice with the binding buer and then released by the addition of 2 M NaOH. The ¯uorescence of the lysate was determined using an LS50 Perkin±Elmer Spectro¯uorimeter (excitation at 485 nm; emission at 520 nm). Relative ¯uorescence units were converted into nmol FITC using ¯uorescein diacetate (Sigma) as standard. Readings were corrected for endogenous ¯uorescence (10±30% of experimental values) measured in freshly isolated liver parenchymal cells.
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2.6. Assay for internalization of Hb±Hp in hepatocytes Internalization of Hb±Hp in the hepatocytes was assessed by two dierent methods as follows: 1. Acid-resistance assay: Isolated rat hepatocytes with pre-bound FITCHb±Hp (prepared as mentioned above) were incubated in the presence of 1 mM ATP for the indicated time periods at 378C. After incubation, the cells were washed twice with the binding buffer and rinsed with 300 ml of 0.5 M NaCl-0.2 M acetic acid for 5 min at 48C. Fluorescence of FITC-Hb±Hp released from or remaining in the cells by this treatment was expressed as cell surface bound or internalized FITC-Hb±Hp, respectively. 2. Antibody inaccessibility assay: This assay was performed by the method of Smythe et al. [16] with some modi®cations. Brie¯y, hepatocytes with pre-bound, unlabelled Hb±Hp were prepared, suspended and incubated as described above. After addition of a stopping buer, 10 ml of FITC-labelled anti-Hb antibody was added to the cells. After incubation for 90 min at 48C unbound antibodies were removed by centrifugation at 10,000 g for 30 s. Fluorescence remaining in the cells by this treatment was expressed as internalized FITC-labelled anti-Hb. Non-speci®c binding was determined in the presence of a 200-fold concentration of unlabelled anti-Hb antibodies, and amounted to less than 20% of the total binding. 2.7. General Protein was determined by the method of Lowry et al. [17] using bovine serum albumin as standard (Sigma). By this assay, isolated hepatocytes contain 1.2 mg of protein/107 cells. 3. Results and discussion In the present study, we demonstrate that haemoglobin (Hb) molecules labelled at their amino termini with ¯uorescent isothiocyanate derivatives serve as excellent reporter groups for the
binding of the Hb±Hp molecule to its cell-surface receptor, and the subsequent internalization of the ligand/receptor complex. An obvious advantage of the isolated intact cells over the isolated plasma membranes for this study is that they better re¯ect the physiological interaction of Hb±Hp with hepatocytes in situ. Hepatic plasma membrane preparations usually include plasma membranes from nonparenchymal cells (endothelial cells, Kuper cells, Ito cells) as well as from hepatocytes. It is possible that nonparenchmal cells may bind Hb±Hp in a manner dierent from hepatocytes. In addition, inside-out membranes present in plasma membrane isolates may increase the fraction of Hb±Hp bound via nonspeci®c ionic bonds obscuring a speci®c Hb±Hp binding `signal'. Kino et al. [1] failed to internalize Hb±Hp into the isolated rat hepatocyte, although asialoglycoprotein was internalized and degraded. The failure of internalization of Hb±Hp might be one of the reasons for the existence of a speci®c mechanism, damaged by the hepatocyte isolation procedure. The presence of protease in collagenase preparations essential for hepatocyte release may cause surface damage. Therefore we chose a method of preparing hepatocytes without collagenase, relying on EDTA to release these cells from the liver. We studied the properties of Hb±Hp receptors of EDTA-prepared hepatocytes by two binding assay systems. First, the equilibrium binding constants for Hb±Hp were determined by incubating FITC-Hb±Hp with cells at 48C with an excess of unlabelled Hb. Speci®c binding in these experiments was >84% of total. In this study FITCHb±Hp binding began saturating at 300 mg Hb± Hp/ml (Fig. 1A). This result was similar to that obtained from the isolated membranes (data not shown). Secondly, in order to determine FITCHb±Hp binding to a speci®c Hb±Hp receptor on the isolated hepatocytes, the competition between the binding of FITC-Hb±Hp and that of unlabelled Hb, transferrin or asialo-orosomucoid (ASOR) was studied. It is interesting to compare the binding of Hb±Hp and transferrin to the same cell type, because haptoglobin alone does not inhibit 125 I-transferrin binding to receptors
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Fig. 1. Equilibrium binding of Hb±Hp complex to hepatocytes. The hepatocytes (107 cells) were incubated with various concentration of FITC-Hb±Hp for 2 h at 48C. Bound FITC-Hb±Hp was measured as described in Section 2. Equilibrium binding constants were determined by the method of Scatchard [38] from the speci®c binding data (Fig. 1A). Isotherms were calculated by linear regression: rhigh anity= ÿ 0.98; rlow anity= ÿ 0.99 (Fig. 1B). Binding constants calculated from these data are presented in the text.
on human placenta [18]. We also tested ASOR's ability to block the binding of Hb±Hp. Limited desialylation of Hp from Hb±Hp complex might account for the increase in FITC-Hb±Hp binding to cells by interaction with the galactosyl receptor [19]. As seen in Fig. 2, the amount of FITC-Hb±Hp bound by hepatocytes decreased 090%, when incubated with <500 molar excess of unlabelled Hb. In contrast, transferrin and ASOR did not compete with FITC-Hb±Hp binding to cells, suggesting that neither transferrin nor galactosyl receptors on these cells were able to bind Hb±Hp. Analyses of the binding data in Fig. 1 indicated that Hb±Hp bound to hepatocytes by a high and low anity component. As calculated from multiple equilibrium binding experiments, hepatocytes expressed 9.0 22.1 104 `high anity sites' (Kd=1.2 2 0.81 mM; n = 3) and 3.9 21.1 105 `apparent low anity sites' (Kd=3.9 2 2.9 mM; n = 3) for FITC-Hb±Hp (Fig. 1B). In the cases with concentrations of FITC-Hb±Hp greater than about 300 mg/ml, Scatchard plots were curvilinear. This was consistent with the inability to saturate completely the binding of FITC-Hb±Hp by hepatocytes
(Fig. 1A). Although this could suggest the presence of a second class of lower anity sites, it is more likely that there is really one type of receptor (designated `high anity sites'), which exhibits negative cooperativity characteristic in its binding at high receptor occupancy [20]. This conclusion is supported by the following observations. (i) The relative dierence in anities (if there were two distinct types of receptors) would be only about 3-fold. In most other cases that have been described [21, 19] the dierence between high and low anity sites is usually several orders of magnitude. (ii) Both the rat Hb±Hp receptor and the complex Hb±Hp are multivalent. The stoichiometry of the in vivo binding reaction between the cell surface receptor and ligand is not known (it was assumed in our experiments to be one for one). Since multivalent interactions should be more likely to occur as the amount of bound ligand increases, this could explain the apparent negative cooperativity of binding when the percentage occupancy of receptors is high (either because of
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Fig. 2. Hb±Hp binding to hepatocytes: eect of transferrin and asialo-orosomucoid. The hepatocytes (107 cells) were incubated for 2 h at 48C with FITC-Hb±Hp with designated amounts of unlabeled haemoglobin, transferrin, or asialo-orosomucoid. Bound FITC-Hb±Hp was measured as described in Section 2. Each symbol represents the mean of duplicate samples that diered by <10%. Estimated molecular weights of ligands: transferrin, 78 kDa; asialo-orosomucoid, 40 kDa.
Fig. 3. Time courses of cell-surface binding or internalization of pre-bound FITC-Hb±Hp in the EDTA-prepared hepatocytes. The hepatocytes (107 cells) were incubated with 500 mg/ ml FITC-Hb±Hp for 2 h at 48C. After unbound ligands were removed, the hepatocytes were incubated in the presence of 1 mM ATP for the indicated time periods at 378C. (R) Cell surface bound or (r) internalized ligands were determined as described in Section 2. Similar results were obtained in two separate experiments.
decreased accessibility or actual modulation of unoccupied receptor sites). (iii) If receptor±receptor interactions occur on the intact cell surface and such interactions are altered when a receptor binds Hb±Hp, then it could also contribute to the appearance of negative cooperative binding. Therefore, although it is possible that there are other types of Hb±Hp receptors, we suggest the presence of only one type of receptor which is represented (in both anity and number of sites per cell) by the high anity receptor. The apparent lower anity receptors are presumed to re¯ect multivalent interactions between receptor (complexes) and ligand. The process of receptor-mediated endocytosis has been studied, using acetic acid treatment. The sensitivity of pre-bound ligand to acetic acid indicates that the receptor-bound ligand is on the cell surface [22]. Using acetic acid treatment, we investigated an internalization of pre-bound Hb± Hp to the receptor in the EDTA-prepared hepatocytes. We found that acid-insensitive Hb±Hp
time-dependently increased during incubation at 378C and was saturated within 30 min (Fig. 3). This result suggests that the internalization of Hb±Hp occurs in the EDTA-prepared hepatocytes at 378C. Furthermore, in order to con®rm this suggestion, we performed an antibody-inaccessible assay by the method of Smythe et al. [16] with some modi®cations. The result of this assay showed that pre-bound Hb±Hp to the isolated hepatocytes became inaccessible to exogenous FITC-anti-Hb antibodies during incubation at 378C (Fig. 4). No antibody bound to the hepatocyte when pre-binding of Hb±Hp to the hepatocytes had not occurred (data not shown). These observations support the ability of the freshly isolated hepatocytes to internalize Hb±Hp. In order to investigate whether this assay system is adequate to approach the mechanism of receptor-mediated endocytosis, we studied the eects of various reagents which aect the endocytosis on the internalization of Hb±Hp. Morgan [23] reported that extracellular Ca2+ is required for receptor-mediated endocytosis of
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growth factor (EGF) to the EGF receptors [24]. In this study, the internalization of Hb±Hp was inhibited by treatment with EDTA (Table 1). No eect on internalization, however, was observed by treatment with EGTA, a speci®c Ca2+-chelating agent (Table 1). These results suggest that the internalization of Hb±Hp in the present assay system requires a metal ion, such as Mg2+, but not Ca2+. Beckers and Balch [25] reported that Ca2+ was required for the transport of intracellular vesicles from endoplasmic reticulum to the Golgi compartment in the secretory pathway in vitro. Furthermore, it was reported that the endocytosed vesicle was transferred from endosomes to a trans-Golgi network in a cell-free assay system [26]. Therefore, the Ca2+-independent stage which occurs prior to transport to the Golgi compartment seems to take place in the present assay system. However, we think that further study needs to be done regarding the role of Ca2+ in the present internalization model because it seems that 5 mM EGTA cannot entirely chelate 5 mM Ca2+. It has been suggested that receptor-mediated endocytosis requires cellular ATP [27, 28]. Recently, Schmid [29, 30] suggested that ATP was required for the formation of coated vesicles, but not required for the formation of invaginated pits in the endocytosis of transferrin in K-562 cells and HeLa cells. In this study, the internalization of Hb±Hp on the EDTA-prepared hepatocytes was inhibited by the removal of ATP (Table 2). These ®ndings suggest that the for-
Fig. 4. Time course of exogenous FITC-anti-Hb antibody-inaccessibility to pre-bound unlabeled Hb±Hp as assessed by the antibody inaccessibility assay in the isolated hepatocytes during incubation at 378C. The hepatocytes (107 cells) were incubated with 500 mg/ml Hb±Hp for 2 h at 48C. After unbound ligands were removed, the hepatocytes were incubated in the presence of 1 mM ATP at 378C. At the indicated time periods, the hepatocytes were incubated with FITC-antiHb antibodies for 90 min at 48C. Binding of FITC-anti-Hb antibodies to the hepatocytes was determined as described in Section 2. Similar results were obtained in three separate experiments.
transferrin in reticulocytes. Stimulation of P2 purinergic receptors on A431 human epidermoid cells with ATP induced a Ca2+-in¯ux into the cells, but inhibited the binding of epidermal
Table 1 Eects of EDTA or EGTA on the internalization of pre-bound FITC-Hb±Hp in the isolated hepatocytes Assay for internalization Incubation time (min)
0 5 10
acid-resistance, FITC-Hb±Hp binding (fmol/107 cells)
antibody-inaccessibility, FITC-anti-Hb Ab binding (fmol/107 cells)
control
EDTA
EGTA
control
EDTA
EGTA
221 211 829 2131 950 2226
244223 689226 5232125a
229 215 777 296 885 2227
15423 13125 12726
1522 4 1432 2b 1412 2b
15123 12722 12423
The isolated hepatocytes (107 cells) were incubated with FITC-Hb±Hp for 2 h at 48C. After unbound ligands were removed, the hepatocytes were incubated in the presence of 1 mM ATP with or without 5 mM EDTA or EGTA for indicated time periods at 378C. Assays for internalization were performed as described in Section 2. Each value is the mean2 S.D. of two samples. Similar results were obtained in two separate experiments. Signi®cantly dierent from control by Students' t-test ap < 0.05, bp < 0.02.
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Table 2 Eects of ATP or GTP on the internalization of pre-bound FITC-Hb±Hp in the isolated hepatocytes Incubation time (min)
0 5 10
Acid-resistance assay FITC Hb±Hp binding (fmol/107 cells) ATP GTP
(+) (ÿ) 2252 8 8982 157 9432 100
(ÿ) (ÿ) 224252 654217 497213b
(+) (+) 21724 1186261 118226a
Antibody-inaccessibility assay FITC-anti-Hb Ab binding (fmol/107 cells) (ÿ) (+) 259211 671226 510226
(+) (ÿ) 15123 127210 125215
(ÿ) (ÿ) 155 21 148 28 154 21b
(+) (+) 149 23 106 23b 92 22b
(ÿ) (+) 154 21 150 23 150 23
The isolated hepatocytes (107 cells) were incubated with FITC-Hb±Hp for 2 h at 48C. After unbound ligands were removed, the hepatocytes were incubated in the presence or absence of 1 mM ATP and/or 500 mM GTP for the indicated time periods at 378C. Internalization of FITC-Hb±Hp was assessed by the acid-resistance assay or the antibody inaccessibility assay. Each assay for internalization was performed as described in Section 2. Each value is the mean2S.D. of two samples. Similar results were obtained in two separate experiments. Signi®cantly dierent from `ATP (+) and GTP (ÿ)' by Students' t-test ap < 0.01, bp < 0.02.
mation of coated vesicles occurs by the isolated hepatocytes in the present assay system. Moreover, it is suggested that early endosome fusion occurs on the parenchymal cell because the fusion is an irreversible process which must require energy (cellular ATP) at some stage [31]. It was reported that rab5 (a small guanosine triphosphate GTP-binding protein) is located at the cytoplasm surface of the plasma membrane and early endosomes [32, 33]. The rab5 would be involved in the process of early endosome fusion [34, 35]. Therefore, in order to investigate whether early endosome fusion occurs, we studied the eect of 500 mM GTP on the internalization of Hb±Hp. GTP stimulated the internalization of Hb±Hp complex on the EDTA-prepared hepatocytes in the presence of ATP, but not in the absence of ATP (Table 2). Colombo et al. [36] reported that the stimulation of early endosome fusion by GTP was observed in vitro. It looks like the early endosome fusion occurs on the hepatocytes in the our assay system. Moreover, Mg2+ may contribute to the activation of GTP-binding protein since Mg2+ is required for the activity of regulatory protein [37]. The biological eects of Hb±Hp complex on hepatic cell function are unknown. This study constitutes the ®rst examination of the early stage of endocytosis in the internalization of Hb± Hp complex. The use of rat hepatocytes prepared by EDTA perfusion followed by Percoll, is available for further studies on the mechanism of
receptor-mediated endocytosis of haemoglobin± haptoglobin complex. References [1] K. Kino, H. Higa, M. Takami, H. Nakajima, Kinetic aspects of hemoglobin±haptoglobin-receptor interaction in rat liver plasma membranes, isolated liver cells, and liver cells in primary culture, J. Biol. Chem. 257 (1982) 4828±4833. [2] B.H. Bowman, A. Kurosky, Haptogobin: The evolutionary product of duplication, unequal crossing over and point mutation, Adv. Hum. Genet. 12 (1982) 189±261. [3] P.K. Hwang, J. Greer, Interaction between hemoglobin subunits in the hemoglobin±haptoglobin complex, J. Biol. Chem. 255 (1980) 3038±3041. [4] S. Oshiro, H. Nakajima, Intrahepatocellular site of the catabolism of hem and globin moiety of hemoglobin± haptoglobin after intravenous administration to rats, J. Biol. Chem. 263 (1988) 16032±16038. [5] J. Osada, Elimination of bovine haptoglobin from rat circulation, Acta Biochim. Pol. 34 (1987) 337±343. [6] J. Osada, Elimination from rat circulation of goat and sheep haptoglobin and their complexes with rat hemoglobin, Acta Biochim. Pol. 35 (1988) 169±175. [7] J. Osada, W. Nowacki, Elimination of goat hemoglobin and its complexes with goat haptoglobin from goat and rat circulation, Acta Biochim. Pol. 36 (1989) 365±369. [8] K. Kino, K. Mizumoto, J. Watanabe, H. Tsunoo, Immunohistochemical studies on hemoglobin±haptoglobin and hemoglobin catabolism sites, J. Histochem. Cytochem. 35 (1987) 381±386. [9] S. Oshiro, Y. Yajima, K. Kawamura, M. Kubota, J. Nishibe, Y. Nishibe, M. Takahama, H. Nakajima, Catabolism of hemoglobin±haptoglobin complex in microsome subfractions, Chem. Pharm. Bull. 40 (1992) 1847±1851.
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cium metabolism in A431 human epidermoid carcinoma cells, Am. J. Physiol. 262 (1992) 635±643. J.M. Beckers, W.E. Balch, Calcium and GTP: Essential components in vesicular tracking between the endoplasmic reticulum and the Golgi apparatus, J. Cell Biol. 108 (1989) 1245±1256. C. Harter, COP-coated vesicles in intracellular protein transport, FEBS Lett. 369 (1995) 89±92. J.M. Lenhard, L. Mayorga, P.D. Stahl, Characterisation of endosome±endosome fusion in a cell-free system using Dictyostelium discoideum, J. Biol. Chem. 267 (1992) 1896±1903. A. Pitt, A.L. Schwartz, Reconstitution of human hepatoma endosome±endosome fusion in vitro: Potential roles for an endoprotease and phosphoprotein phosphatase, Eur. J. Cell. Biol. 55 (1991) 328±335. S.L. Schmid, Stage speci®c assays for coated vesicles budding in vitro, J. Cell Biol. 114 (1991) 869±880. S.L. Schmid, Coated-vesicle formation in vitro: Con¯icting result using dierent assays, Trends Cell Biol. 3 (1993) 145±148. L.S. Mayorga, W. Beron, M.N. Sarrouf, M.I. Colombo, C. Creutz, P.D. Stahl, Calcium-dependent fusion among endosomes, J. Biol. Chem. 269 (1994) 30927±30934. C. Bucci, A. Wandinger-Ness, A. Lutcke, M. Chiariello, C.B. Bruni, M. Zerial, Rab5a is a common component of the apical and basolateral endocytic machinery in polarised epithelial cells, Proc. Natl. Acad. Sci. USA 91 (1994) 5061±5065. P. Chavrier, J.P. Gorvel, E. Stelzer, K. Simons, J. Zerial, M. Zerial, Hypervariable C-terminal domain of rab proteins acts as a targeting signal, Nature 353 (1991) 769±772. M.A. Barbieri, G. Li, M.I. Colombo, P.D. Stahl, Rab5, an early acting endosomal GTPase, supports in vitro endosome fusion without GTP hydrolysis, J. Biol. Chem. 269 (1994) 18720±18722. H. Stenmark, R.G. Parton, O. Steele-Mortimer, A. Gruenberg, J. Gruenberg, M. Zerial, Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis, EMBO J. 13 (1994) 1287±1296. M.I. Colombo, L.S. Mayorga, I. Nashimoto, E.M. Ross, P.D. Stahl, Gs regulation of endosome fusion suggests a role for signal transduction pathways in endocytosis, J. Biol. Chem. 269 (1994) 14919±14923. L. Brinbaumer, J. Abramowitz, A.M. Brown, Receptor± eector coupling by G proteins, Biochem. Biophys. Acta 1031 (1990) 163±224. G. Scatchard, The attractions of protein for small molecules and ions, Ann. NY Acad. Sci. 51 (1949) 660.
PERGAMON
The International Journal of Biochemistry & Cell Biology 30 (1998) 923±931
Biochemistry & Cell Biology
Internalization study using EDTA-prepared hepatocytes for receptor-mediated endocytosis of haemoglobin±haptoglobin complex Jolanta Zuwal/ a-Jagiel/ /lo, Jerzy Osada * Department of Pharmaceutical Biochemistry, Medical Academy, Szewska 38/39, 50-139 Wrocl/ aw, Poland Received 8 August 1997; accepted 4 March 1998
Abstract We have demonstrated the internalization of haemoglobin±haptoglobin (Hb±Hp) complex using rat hepatocytes prepared by EDTA perfusion, followed by Percoll. The isolated hepatocytes exhibited a saturation curve of the binding of ¯uorescein isothiocyanate-labelled haemoglobin±haptoglobin complex (FITC-Hb±Hp). Furthermore, competition between the binding of FITC-Hb±Hp and unlabelled Hb to the hepatocytes, was observed. The cells exhibited 09 104 `high anity sites' (Kd 0 1.2 mM) for the Hb±Hp complex. The data in toto suggest the presence of only one type of receptor i.e. the high anity receptor (in both anity and number of sites per cell). The results were similar to those obtained from rat hepatocytes prepared by collagenase digestion [1]. In order to verify whether EDTA-prepared hepatocytes could be used for the study of receptor-mediated endocytosis, the internalization of pre-bound Hb±Hp in the isolated hepatocytes was assessed by two methods. First, acid-insensitive FITC-Hb±Hp time-dependently increased following incubation at 378C. Secondly, Hb±Hp became inaccessible to the exogenous FITC-anti-haemoglobin antibody. These processes were dependent on ATP, but independent of Ca2+ and stimulated by GTP. The results demonstrate that the receptor-mediated endocytosis of Hb±Hp occurred in the EDTA-prepared hepatocytes. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: EDTA-prepared hepatocytes; Haemoglobin±haptoglobin; Complex-internalization; GTP; ATP
1. Introduction Abbreviations: Ab, antibody, ASOR, asialo-orosomucoid, ATP, adenosine triphosphate, EGTA, ethylene glycol bis(baminoethyl ether)-N, N, N', N'-tetraacetic acid, FITC, ¯uorescein isothiocyanate, Hp, human haptoglobin 1-1, GTP, guanosine triphosphate, Hb, rat haemoglobin, Hb±Hp, haemoglobin±haptoglobin complex, HBS, Hanks' balanced salt solution, HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid, Kd, dissociation constant. * Corresponding author. Fax: +48-71-442277.
Haptoglobin (Hp) is a plasma glycoprotein present in all vertebrates [2]. It can form a very tight complex with haemoglobin (Hb) in vivo and in vitro. The binding of haptoglobin to haemoglobin is one of the strongest known noncovalent interactions in biology. The two proteins form a complex with an association constant that has been too high to measure (greater than 10ÿ15
1357-2725/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 8 ) 0 0 0 3 5 - 1
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M) [3]. Hb±Hp complex disappears rapidly from the circulation being taken up by the liver parenchymal cells through a speci®c receptor for Hb±Hp [1, 4]. Liver metabolism (clearance) of haptoglobin from the Bovidae family and their complexes with haemoglobins was also observed [5±7]. Hb±Hp internalized by receptor-mediated endocytosis is dissociated symmetrically into 82000-Dalton subunits. The organelles with multivesicular body (MVB) or compartment of uncoupling of receptor and ligand (CURL)-like structures are associated with Hb±Hp metabolism [8, 4, 9]. In spite of the information on metabolism of Hb±Hp complexes, the detailed mechanism by which the endocytosis of Hb±Hp is induced is unclear. During receptor-mediated endocytosis, a ligand binds to a speci®c cell surface receptor, and the receptor± ligand complex is internalized in clathrin-coated pits and vesicles. It is dicult to directly approach the mechanism because the components involved in endocytosis are located on the cytoplasmic surface. Therefore, the demonstration of endocytosis in vitro can provide useful information of its mechanism. During the isolation of liver parenchymal cells with collagenase the apparent high anity binding sites for Hb±Hp seemed to be impaired [1]. The presence of proteases in collagenase preparations essential for hepatocyte release may cause surface damage. The liver parenchymal cells in primary culture also lacked the ability to internalize Hb±Hp, although they bound the molecule more extensively than freshly isolated liver cells. An interesting possibility is that collagenase engulfed during perfusion is released in culture, causing protein degradation by the contaminating proteases. It would be reasonable to postulate that impairment of the high anity binding sites for Hb±Hp may be the reason why the isolated hepatocytes lost the ability to internalize the Hb±Hp, although they readily bound the ligand. In this study, we employed the rat hepatocytes prepared by EDTA dissociation, to demonstrate the early stage of endocytosis in the internalization of haemoglobin±haptoglobin complex.
2. Materials and methods 2.1. Animals Male Bualo rats weighing 180±300 g body weight were used. Animals were given food and water ad libitum. 2.2. Materials Materials were obtained as follows: ¯uorescein isothiocyanate (FITC, isomer I), bovine transferrin, human orosomucoid and FITC labelled rabbit anti-human hemoglobin antibody were purchased from Sigma. Asialo-orosomucoid was prepared from human orosomucoid by desialylation with neuraminidase as described elsewhere [10]. All other chemicals were reagent grade. 2.3. FITC-Hb±Hp complex preparation Puri®ed rat haemoglobin (4 mg/ml in 0.1 M NaHCO3, pH 9.5) was incubated with 0.25 mM FITC with stirring at 228C for 1 h. The reaction was stopped by applying the protein-FITC mixture to a GF-5 desalting column (Pierce) equilibrated in 0.1 M NaHCO3, pH 9.5. Hbcontaining fractions were dialyzed against Hanks' balanced salt (HBS) solution and analyzed spectroscopically at 496 nm (FITC) and 280 nm (protein). FITC incorporation into Hb, 1.85 nmol of FITC per nmol Hb, was calculated according to Jobbagy and Kiraly [11]. Human haptoglobin 1-1 was prepared according to the method of Smith et al. [12]. The complex of labelled FITC-Hb with human haptoglobin was obtained in the following way: two fold excess of FITC-Hb was added to 5 mg of haptoglobin dissolved in 2 ml of HBS at room temperature. After 15 min the excess of FITC-Hb was removed by ®ltration through a Sephacryl S-200 column equilibrated with HBS. The fractions containing FITC-Hb± Hp were ®lter-sterilized (0.2 mm) and stored at ÿ208C until use.
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2.4. Preparation of rat hepatocytes Preparation of rat hepatocytes was performed essentially according to Wang et al. [13] as follows. After anesthetizing the rat by i.p. injection of 50 mg/kg Nembutal, the portal vein was cannulated a 19-gauge syringe needle and was secured in place by two ligatures of 4±0 silk. Flow began at 5 ml/min while the chest was opened and an incision made in the heart to allow out¯ow of the perfusate. The ¯ow rate was adjusted to 50±65 ml/min. The duration of perfusion was determined by the appearance of the liver. When the liver appeared softened and buffer was coming through the capsule, perfusion was stopped. Through more than twenty preparations done to date in this laboratory, the perfusion time ranged from 20±40 min, with larger, older rats taking longer. The perfusion buer, wash buer, and Percoll buer were prepared from a 10 stock solution containing 1.4 M NaCl, 50 mM KCl, 8.0 mM MgCl2, 16 mM Na2HPO4, and 4.0 mM KH2PO4. To prepare the perfusate, Buer A, 300 ml of the stock solution was diluted to 3 l with reverse osmosis puri®ed water and NaHCO3 and EDTA were added 25 and 2 mM ®nal concentration, respectively. Carbogen was bubbled through the solution, as the temperature was brought to 378C. Buer B, the wash buer, was Buer A without EDTA and NaHCO3, but containing 1 mM CaCl2. Percoll used to separate intact hepatocytes was made by diluting 87 ml of Percoll (Sigma) to 100 ml with undiluted 10 stock solution. The Percoll and Buer B were stored at 48C until used. The softened liver was removed and washed in Buer B. The liver was placed in a 100 ml beaker and minced with scissors. The liver was washed with Buer B to remove released hepatocytes. By repeated mincing and washing, about 90% of the liver was reduced to material passing a 200 micron ®lter. Centrifugation at 50 g for 2 min was used to collect hepatocytes. After two washes in Buer B, collected hepatocytes were resuspended in Buer B and diluted with 2 volumes of buered Percoll. Hepatocytes were pelleted by centrifugation at 1500 g for 5 min at room
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temperature. The cells were resuspended in hepatocyte modi®ed William's Medium E (Serva Feinbiochemica) containing 10% supplemented calf serum (Serva Feinbiochemica), 100 IU/l potassium penicillin G and 100 mg/l streptomycin sulphate. Cell viability was estimated by assessing the number of cells that excluded trypan blue. Parenchymal cells (hepatocytes) ranging 18±21 mm in diameter and nonparenchymal cells ranging 8±15 mm were readily identi®ed by light microscopy. Parenchymal cell preparations generally contained 5% contamination from nonparenchymal cells. Parenchymal cell preparations were routinely 81% viable. There is no correlation between the percentage of stained cells and the metabolic capability of the isolated hepatocytes [14]. Therefore a number of other viability criteria have been suggested. Among these, the content of ATP is probably the easiest. ATP was assayed using a modi®cation of luciferin/luciferase assay essentially as described by Weigel and Englund [15]. Cellular ATP levels were depleted to 45% normal value (ATP should be >2 mmol/108 cells). 2.5. Hb±Hp binding assay Hb±Hp binding to the receptor of the isolated hepatocytes was measured as follows. The cells were washed twice with a binding buer containing 20 mM HEPES, 150 mM NaCl and 2 mg/ml bovine serum albumin, pH 7.4. The cells were incubated for 2 h at 48C in the binding buer with FITC-Hb±Hp at the indicated concentrations. After the incubation, the cells were washed twice with the binding buer and then released by the addition of 2 M NaOH. The ¯uorescence of the lysate was determined using an LS50 Perkin±Elmer Spectro¯uorimeter (excitation at 485 nm; emission at 520 nm). Relative ¯uorescence units were converted into nmol FITC using ¯uorescein diacetate (Sigma) as standard. Readings were corrected for endogenous ¯uorescence (10±30% of experimental values) measured in freshly isolated liver parenchymal cells.
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2.6. Assay for internalization of Hb±Hp in hepatocytes Internalization of Hb±Hp in the hepatocytes was assessed by two dierent methods as follows: 1. Acid-resistance assay: Isolated rat hepatocytes with pre-bound FITCHb±Hp (prepared as mentioned above) were incubated in the presence of 1 mM ATP for the indicated time periods at 378C. After incubation, the cells were washed twice with the binding buffer and rinsed with 300 ml of 0.5 M NaCl-0.2 M acetic acid for 5 min at 48C. Fluorescence of FITC-Hb±Hp released from or remaining in the cells by this treatment was expressed as cell surface bound or internalized FITC-Hb±Hp, respectively. 2. Antibody inaccessibility assay: This assay was performed by the method of Smythe et al. [16] with some modi®cations. Brie¯y, hepatocytes with pre-bound, unlabelled Hb±Hp were prepared, suspended and incubated as described above. After addition of a stopping buer, 10 ml of FITC-labelled anti-Hb antibody was added to the cells. After incubation for 90 min at 48C unbound antibodies were removed by centrifugation at 10,000 g for 30 s. Fluorescence remaining in the cells by this treatment was expressed as internalized FITC-labelled anti-Hb. Non-speci®c binding was determined in the presence of a 200-fold concentration of unlabelled anti-Hb antibodies, and amounted to less than 20% of the total binding. 2.7. General Protein was determined by the method of Lowry et al. [17] using bovine serum albumin as standard (Sigma). By this assay, isolated hepatocytes contain 1.2 mg of protein/107 cells. 3. Results and discussion In the present study, we demonstrate that haemoglobin (Hb) molecules labelled at their amino termini with ¯uorescent isothiocyanate derivatives serve as excellent reporter groups for the
binding of the Hb±Hp molecule to its cell-surface receptor, and the subsequent internalization of the ligand/receptor complex. An obvious advantage of the isolated intact cells over the isolated plasma membranes for this study is that they better re¯ect the physiological interaction of Hb±Hp with hepatocytes in situ. Hepatic plasma membrane preparations usually include plasma membranes from nonparenchymal cells (endothelial cells, Kuper cells, Ito cells) as well as from hepatocytes. It is possible that nonparenchmal cells may bind Hb±Hp in a manner dierent from hepatocytes. In addition, inside-out membranes present in plasma membrane isolates may increase the fraction of Hb±Hp bound via nonspeci®c ionic bonds obscuring a speci®c Hb±Hp binding `signal'. Kino et al. [1] failed to internalize Hb±Hp into the isolated rat hepatocyte, although asialoglycoprotein was internalized and degraded. The failure of internalization of Hb±Hp might be one of the reasons for the existence of a speci®c mechanism, damaged by the hepatocyte isolation procedure. The presence of protease in collagenase preparations essential for hepatocyte release may cause surface damage. Therefore we chose a method of preparing hepatocytes without collagenase, relying on EDTA to release these cells from the liver. We studied the properties of Hb±Hp receptors of EDTA-prepared hepatocytes by two binding assay systems. First, the equilibrium binding constants for Hb±Hp were determined by incubating FITC-Hb±Hp with cells at 48C with an excess of unlabelled Hb. Speci®c binding in these experiments was >84% of total. In this study FITCHb±Hp binding began saturating at 300 mg Hb± Hp/ml (Fig. 1A). This result was similar to that obtained from the isolated membranes (data not shown). Secondly, in order to determine FITCHb±Hp binding to a speci®c Hb±Hp receptor on the isolated hepatocytes, the competition between the binding of FITC-Hb±Hp and that of unlabelled Hb, transferrin or asialo-orosomucoid (ASOR) was studied. It is interesting to compare the binding of Hb±Hp and transferrin to the same cell type, because haptoglobin alone does not inhibit 125 I-transferrin binding to receptors
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Fig. 1. Equilibrium binding of Hb±Hp complex to hepatocytes. The hepatocytes (107 cells) were incubated with various concentration of FITC-Hb±Hp for 2 h at 48C. Bound FITC-Hb±Hp was measured as described in Section 2. Equilibrium binding constants were determined by the method of Scatchard [38] from the speci®c binding data (Fig. 1A). Isotherms were calculated by linear regression: rhigh anity= ÿ 0.98; rlow anity= ÿ 0.99 (Fig. 1B). Binding constants calculated from these data are presented in the text.
on human placenta [18]. We also tested ASOR's ability to block the binding of Hb±Hp. Limited desialylation of Hp from Hb±Hp complex might account for the increase in FITC-Hb±Hp binding to cells by interaction with the galactosyl receptor [19]. As seen in Fig. 2, the amount of FITC-Hb±Hp bound by hepatocytes decreased 090%, when incubated with <500 molar excess of unlabelled Hb. In contrast, transferrin and ASOR did not compete with FITC-Hb±Hp binding to cells, suggesting that neither transferrin nor galactosyl receptors on these cells were able to bind Hb±Hp. Analyses of the binding data in Fig. 1 indicated that Hb±Hp bound to hepatocytes by a high and low anity component. As calculated from multiple equilibrium binding experiments, hepatocytes expressed 9.0 22.1 104 `high anity sites' (Kd=1.2 2 0.81 mM; n = 3) and 3.9 21.1 105 `apparent low anity sites' (Kd=3.9 2 2.9 mM; n = 3) for FITC-Hb±Hp (Fig. 1B). In the cases with concentrations of FITC-Hb±Hp greater than about 300 mg/ml, Scatchard plots were curvilinear. This was consistent with the inability to saturate completely the binding of FITC-Hb±Hp by hepatocytes
(Fig. 1A). Although this could suggest the presence of a second class of lower anity sites, it is more likely that there is really one type of receptor (designated `high anity sites'), which exhibits negative cooperativity characteristic in its binding at high receptor occupancy [20]. This conclusion is supported by the following observations. (i) The relative dierence in anities (if there were two distinct types of receptors) would be only about 3-fold. In most other cases that have been described [21, 19] the dierence between high and low anity sites is usually several orders of magnitude. (ii) Both the rat Hb±Hp receptor and the complex Hb±Hp are multivalent. The stoichiometry of the in vivo binding reaction between the cell surface receptor and ligand is not known (it was assumed in our experiments to be one for one). Since multivalent interactions should be more likely to occur as the amount of bound ligand increases, this could explain the apparent negative cooperativity of binding when the percentage occupancy of receptors is high (either because of
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Fig. 2. Hb±Hp binding to hepatocytes: eect of transferrin and asialo-orosomucoid. The hepatocytes (107 cells) were incubated for 2 h at 48C with FITC-Hb±Hp with designated amounts of unlabeled haemoglobin, transferrin, or asialo-orosomucoid. Bound FITC-Hb±Hp was measured as described in Section 2. Each symbol represents the mean of duplicate samples that diered by <10%. Estimated molecular weights of ligands: transferrin, 78 kDa; asialo-orosomucoid, 40 kDa.
Fig. 3. Time courses of cell-surface binding or internalization of pre-bound FITC-Hb±Hp in the EDTA-prepared hepatocytes. The hepatocytes (107 cells) were incubated with 500 mg/ ml FITC-Hb±Hp for 2 h at 48C. After unbound ligands were removed, the hepatocytes were incubated in the presence of 1 mM ATP for the indicated time periods at 378C. (R) Cell surface bound or (r) internalized ligands were determined as described in Section 2. Similar results were obtained in two separate experiments.
decreased accessibility or actual modulation of unoccupied receptor sites). (iii) If receptor±receptor interactions occur on the intact cell surface and such interactions are altered when a receptor binds Hb±Hp, then it could also contribute to the appearance of negative cooperative binding. Therefore, although it is possible that there are other types of Hb±Hp receptors, we suggest the presence of only one type of receptor which is represented (in both anity and number of sites per cell) by the high anity receptor. The apparent lower anity receptors are presumed to re¯ect multivalent interactions between receptor (complexes) and ligand. The process of receptor-mediated endocytosis has been studied, using acetic acid treatment. The sensitivity of pre-bound ligand to acetic acid indicates that the receptor-bound ligand is on the cell surface [22]. Using acetic acid treatment, we investigated an internalization of pre-bound Hb± Hp to the receptor in the EDTA-prepared hepatocytes. We found that acid-insensitive Hb±Hp
time-dependently increased during incubation at 378C and was saturated within 30 min (Fig. 3). This result suggests that the internalization of Hb±Hp occurs in the EDTA-prepared hepatocytes at 378C. Furthermore, in order to con®rm this suggestion, we performed an antibody-inaccessible assay by the method of Smythe et al. [16] with some modi®cations. The result of this assay showed that pre-bound Hb±Hp to the isolated hepatocytes became inaccessible to exogenous FITC-anti-Hb antibodies during incubation at 378C (Fig. 4). No antibody bound to the hepatocyte when pre-binding of Hb±Hp to the hepatocytes had not occurred (data not shown). These observations support the ability of the freshly isolated hepatocytes to internalize Hb±Hp. In order to investigate whether this assay system is adequate to approach the mechanism of receptor-mediated endocytosis, we studied the eects of various reagents which aect the endocytosis on the internalization of Hb±Hp. Morgan [23] reported that extracellular Ca2+ is required for receptor-mediated endocytosis of
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growth factor (EGF) to the EGF receptors [24]. In this study, the internalization of Hb±Hp was inhibited by treatment with EDTA (Table 1). No eect on internalization, however, was observed by treatment with EGTA, a speci®c Ca2+-chelating agent (Table 1). These results suggest that the internalization of Hb±Hp in the present assay system requires a metal ion, such as Mg2+, but not Ca2+. Beckers and Balch [25] reported that Ca2+ was required for the transport of intracellular vesicles from endoplasmic reticulum to the Golgi compartment in the secretory pathway in vitro. Furthermore, it was reported that the endocytosed vesicle was transferred from endosomes to a trans-Golgi network in a cell-free assay system [26]. Therefore, the Ca2+-independent stage which occurs prior to transport to the Golgi compartment seems to take place in the present assay system. However, we think that further study needs to be done regarding the role of Ca2+ in the present internalization model because it seems that 5 mM EGTA cannot entirely chelate 5 mM Ca2+. It has been suggested that receptor-mediated endocytosis requires cellular ATP [27, 28]. Recently, Schmid [29, 30] suggested that ATP was required for the formation of coated vesicles, but not required for the formation of invaginated pits in the endocytosis of transferrin in K-562 cells and HeLa cells. In this study, the internalization of Hb±Hp on the EDTA-prepared hepatocytes was inhibited by the removal of ATP (Table 2). These ®ndings suggest that the for-
Fig. 4. Time course of exogenous FITC-anti-Hb antibody-inaccessibility to pre-bound unlabeled Hb±Hp as assessed by the antibody inaccessibility assay in the isolated hepatocytes during incubation at 378C. The hepatocytes (107 cells) were incubated with 500 mg/ml Hb±Hp for 2 h at 48C. After unbound ligands were removed, the hepatocytes were incubated in the presence of 1 mM ATP at 378C. At the indicated time periods, the hepatocytes were incubated with FITC-antiHb antibodies for 90 min at 48C. Binding of FITC-anti-Hb antibodies to the hepatocytes was determined as described in Section 2. Similar results were obtained in three separate experiments.
transferrin in reticulocytes. Stimulation of P2 purinergic receptors on A431 human epidermoid cells with ATP induced a Ca2+-in¯ux into the cells, but inhibited the binding of epidermal
Table 1 Eects of EDTA or EGTA on the internalization of pre-bound FITC-Hb±Hp in the isolated hepatocytes Assay for internalization Incubation time (min)
0 5 10
acid-resistance, FITC-Hb±Hp binding (fmol/107 cells)
antibody-inaccessibility, FITC-anti-Hb Ab binding (fmol/107 cells)
control
EDTA
EGTA
control
EDTA
EGTA
221 211 829 2131 950 2226
244223 689226 5232125a
229 215 777 296 885 2227
15423 13125 12726
1522 4 1432 2b 1412 2b
15123 12722 12423
The isolated hepatocytes (107 cells) were incubated with FITC-Hb±Hp for 2 h at 48C. After unbound ligands were removed, the hepatocytes were incubated in the presence of 1 mM ATP with or without 5 mM EDTA or EGTA for indicated time periods at 378C. Assays for internalization were performed as described in Section 2. Each value is the mean2 S.D. of two samples. Similar results were obtained in two separate experiments. Signi®cantly dierent from control by Students' t-test ap < 0.05, bp < 0.02.
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Table 2 Eects of ATP or GTP on the internalization of pre-bound FITC-Hb±Hp in the isolated hepatocytes Incubation time (min)
0 5 10
Acid-resistance assay FITC Hb±Hp binding (fmol/107 cells) ATP GTP
(+) (ÿ) 2252 8 8982 157 9432 100
(ÿ) (ÿ) 224252 654217 497213b
(+) (+) 21724 1186261 118226a
Antibody-inaccessibility assay FITC-anti-Hb Ab binding (fmol/107 cells) (ÿ) (+) 259211 671226 510226
(+) (ÿ) 15123 127210 125215
(ÿ) (ÿ) 155 21 148 28 154 21b
(+) (+) 149 23 106 23b 92 22b
(ÿ) (+) 154 21 150 23 150 23
The isolated hepatocytes (107 cells) were incubated with FITC-Hb±Hp for 2 h at 48C. After unbound ligands were removed, the hepatocytes were incubated in the presence or absence of 1 mM ATP and/or 500 mM GTP for the indicated time periods at 378C. Internalization of FITC-Hb±Hp was assessed by the acid-resistance assay or the antibody inaccessibility assay. Each assay for internalization was performed as described in Section 2. Each value is the mean2S.D. of two samples. Similar results were obtained in two separate experiments. Signi®cantly dierent from `ATP (+) and GTP (ÿ)' by Students' t-test ap < 0.01, bp < 0.02.
mation of coated vesicles occurs by the isolated hepatocytes in the present assay system. Moreover, it is suggested that early endosome fusion occurs on the parenchymal cell because the fusion is an irreversible process which must require energy (cellular ATP) at some stage [31]. It was reported that rab5 (a small guanosine triphosphate GTP-binding protein) is located at the cytoplasm surface of the plasma membrane and early endosomes [32, 33]. The rab5 would be involved in the process of early endosome fusion [34, 35]. Therefore, in order to investigate whether early endosome fusion occurs, we studied the eect of 500 mM GTP on the internalization of Hb±Hp. GTP stimulated the internalization of Hb±Hp complex on the EDTA-prepared hepatocytes in the presence of ATP, but not in the absence of ATP (Table 2). Colombo et al. [36] reported that the stimulation of early endosome fusion by GTP was observed in vitro. It looks like the early endosome fusion occurs on the hepatocytes in the our assay system. Moreover, Mg2+ may contribute to the activation of GTP-binding protein since Mg2+ is required for the activity of regulatory protein [37]. The biological eects of Hb±Hp complex on hepatic cell function are unknown. This study constitutes the ®rst examination of the early stage of endocytosis in the internalization of Hb± Hp complex. The use of rat hepatocytes prepared by EDTA perfusion followed by Percoll, is available for further studies on the mechanism of
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