Regulation of cytosolic free calcium concentration by extracellular nucleotides in human hepatocytes

Christof Schöfl1, Martin Ponczek1, Thilo Mader1, Mark Waring1, Heike Benecke1, Alexander von zur Mühlen1, Heiko Mix2, Markus Cornberg2, Klaus H. W. Böker2, Michael P. Manns2, and Siegfried Wagner2

1 Departments of Clinical Endocrinology and 2 Gastroenterology and Hepatology, Medizinische Hochschule Hannover, 30623 Hannover, Germany


    ABSTRACT
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Abstract
Introduction
Materials and methods
Results
Discussion
References

The effects of extracellular ATP and other nucleotides on the cytosolic free Ca2+ concentration ([Ca2+]i) have been studied in single primary human hepatocytes and in human Hep G2 and HuH-7 hepatoma cells. ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S), and UTP caused a concentration-dependent biphasic increase in [Ca2+]i with an initial peak followed by a small sustained plateau in most cells. In some cells, however, repetitive Ca2+ transients were observed. The rank order of potency was ATP >=  UTP > ATPgamma S, and complete cross-desensitization of the Ca2+ responses occurred between ATP and UTP. The initial transient peak in [Ca2+]i was resistant to extracellular Ca2+ depletion, which demonstrates mobilization of internal Ca2+ by inositol 1,4,5-trisphosphate whose formation was enhanced by ATP and UTP. In contrast, the sustained plateau phase required influx of external Ca2+. Ca2+ influx occurs most likely through a capacitative Ca2+ entry mechanism, which was shown to exist in these cells by experiments performed with thapsigargin. On the molecular level, specific mRNA coding for the human P2Y1, P2Y2, P2Y4, and P2Y6 receptors could be detected by RT-PCR in Hep G2 and HuH-7 cells. However, ADP and UDP, which are agonists for P2Y1 and P2Y6 receptors, respectively, caused no changes in [Ca2+]i, demonstrating that these receptors are not expressed at a functional level. Likewise, alpha ,beta -methylene-ATP, beta ,gamma -methylene-ATP, AMP, and adenosine were inactive in elevating [Ca2+]i, suggesting that the ATP-induced increase in [Ca2+]i was not caused by activation of P2X or P1 receptors. Thus, on the basis of the pharmacological profile of the nucleotide-induced Ca2+-responses, extracellular ATP and UTP increase [Ca2+]i by activating P2Y2 and possibly P2Y4 receptors coupled to the Ca2+-phosphatidylinositol signaling cascade in human hepatocytes. This suggests that extracellular nucleotides from various sources may contribute to the regulation of human liver cell functions.

adenosine triphosphate; uridine triphosphate; nucleotide receptor; intracellular calcium; human liver


    INTRODUCTION
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Abstract
Introduction
Materials and methods
Results
Discussion
References

EXTRACELLULAR ATP AND related compounds have significant biological actions on many tissues and cell types, including hepatocytes (3-5, 11, 13, 18). ATP and other nucleotides have been shown to act on specific P2 receptors coupled to the phosphatidylinositol (PI)-Ca2+ signaling pathway and to increase cytosolic free Ca2+ concentration ([Ca2+]i) in hepatocytes from several species (7, 9, 10, 12, 19, 25, 31, 41). An increase in [Ca2+]i is one of the key signals for the regulation of hepatic enzymes, such as glycogen phosphorylase, the rate-limiting enzyme for glycogenolysis, and extracellular nucleotides have been reported (6, 20-23) to activate glycogen phosphorylase and to stimulate glycogenolysis, most likely by binding to specific P2 receptors in both rat and human hepatocytes. Furthermore, extracellular ATP has been suggested to be involved in canalicular contraction of hepatocyte doublets (25) and in intercellular communication between hepatocytes and between hepatocytes and bile duct cells in the rat liver (37). This clearly suggests a potential role of extracellular nucleotides in the control of human hepatocyte functions. Several P2 receptors exist that are classified as ligand-gated cationic channels or P2X receptors and G protein-coupled P2Y receptors (3, 5, 11, 13, 14). On the basis of the relative potencies of various ATP analogs and nucleotides, P2Y and P2X receptors can be further subclassified and up to seven P2X and P2Y receptor subtypes have been cloned so far (3, 14, 24). P2Y receptors coupled to the PI-Ca2+ signaling pathway have been described on hepatocytes from various species (7, 9, 10, 12, 19, 25, 31, 41), and P2X receptors have been shown on guinea pig hepatocytes (7). Because differences between species in the regulation of hepatic functions exist (8, 23, 38), we characterized the P2 receptors expressed on human hepatocytes and investigated the signal transduction mechanisms involved. Because P2 receptors are coupled to an increase in [Ca2+]i, we measured [Ca2+]i in single fura 2-loaded primary human hepatocytes and in the differentiated human hepatoma cell lines Hep G2 (19, 20) and HuH-7 (30). A single cell approach was chosen because this circumvents several problems of population measurements in primary cells, including contamination with other cell types that are known to express P2 receptors (2, 17, 35, 43).


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Materials. Fura 2-AM and Pluronic F-127 were purchased from Molecular Probes (Eugene, OR). Nifedipine was generously provided by Bayer (Leverkusen, Germany). Williams' medium E, DMEM, FCS, and antibiotics were from Life Technologies (Berlin, Germany). Thapsigargin was from Calbiochem (Bad Soden, Germany), and collagenase type H and hexokinase were from Boehringer (Mannheim, Germany). All other substances were from Sigma Chemical (Munich, Germany). Stock solutions were prepared in water or as follows. Thapsigargin (5 mM) was prepared in DMSO and nifedipine (10 mM) in ethanol.

Because ADP and UDP preparations from commercial sources might be contaminated with the respective nucleotide triphosphates, we preincubated stock solutions of ADP and UDP (1 mM) with or without hexokinase (10 U/ml) and 10 mM glucose for 1 h at 37°C as described previously (28, 32), and hexokinase (1 U/ml) was added to the medium in experiments with nucleotide diphosphates. This protocol has been shown to effectively convert any contaminating nucleotide triphosphate to the respective nucleotide diphosphate and to prevent possible conversion of nucleotide diphosphates to nucleotide triphosphates by membranous nucleoside diphosphokinase (27, 32).

Isolation of primary human hepatocytes. Pieces of liver tissue were obtained from macroscopically normal regions of five lobectomies performed for unilocular metastases at the Department of Abdominal and Transplantation Surgery, Medical School Hannover. Cut vessels on the surface of the liver tissue were cannulated, and hepatocytes were prepared by collagenase perfusion, using a protocol described previously for the isolation of rat hepatocytes by Seglen (40). The viability of human hepatocytes obtained was 70-85% as indicated by trypan blue exclusion. Isolated hepatocytes were suspended in Williams' medium E supplemented with 10% FCS (vol/vol), plated on glass coverslips coated with poly-L-lysine, and incubated for 4-24 h at 37°C in 5% CO2 (vol/vol) and 95% air (vol/vol) to allow attachment of the cells. [Ca2+]i measurements were done within 4-24 h after cell isolation.

Cell culture. HuH-7 and Hep G2 cells were grown in DMEM medium supplemented with 10% FCS (vol/vol), 100 units of penicillin/ml, and 100 µg streptomycin/ml at 37°C in 5% CO2 (vol/vol) and 95% air (vol/vol). For Ca2+ measurements, cells were seeded on glass coverslips and used after 2-3 days. Human osteoblastic MG-63 cells were cultured in RPMI 1640 medium supplemented with 10% FCS (vol/vol), 100 units of penicillin/ml, and 100 µg streptomycin/ml at 37°C in 5% CO2 (vol/vol) and 95% air (vol/vol).

Measurement of [Ca2+]i. Primary hepatocytes and Hep G2 or HuH-7 cells attached on glass coverslips were loaded with 5 µM fura 2-AM for 30 min at 37°C in medium containing 130 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.5 mM CaCl2, 10 mM glucose, 20 mM HEPES, 2% BSA (wt/vol), and 0.1% Pluronic F-127 (wt/vol), gassed with 100% O2 (vol/vol), pH 7.4. After loading, the coverslips were mounted in a temperature-controlled superfusion chamber (37°C; Intracel, Royston Herts, United Kingdom) and placed on the stage of a Zeiss Axiovert IM 135 microscope equipped with a ×40 Achrostigmat oil immersion objective (Zeiss, Jena, Germany). The chamber was superfused at a flow rate of 0.8 ml/min using a similar medium as described above but with 0.1% BSA (wt/vol) and without Pluronic F-127. Ca2+ measurements were done on cells that were morphologically clearly identified as hepatocytes and of healthy appearance (round in shape, no membrane blebs). Fura 2 fluorescence from a single cell was recorded with a dual wavelength excitation spectrofluorometer system (Deltascan 4000, Photon Technology Instruments, Wedel, Germany), and [Ca2+]i was calculated as previously described (16, 39).

Measurement of inositol 1,4,5-trisphosphate. Hep G2 and HuH-7 cells grown to confluency in petri dishes (100 × 20 mm) were preincubated for 30 min in medium containing 130 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.5 mM CaCl2, 10 mM glucose, 20 mM HEPES, 0.1% BSA (wt/vol), and 10 mM LiCl, gassed with 100% O2 (vol/vol), pH 7.4, at 37°C. Cells were washed and incubated for 20 s with or without the respective test agents in 1 ml of medium. The incubation period was stopped by adding 1 ml of 10% ice-cold HClO4, and the cells were put on ice for 20 min. The cells were detached and centrifuged, and 800 µl of the supernatant were added with 200 µl of 10 mM EGTA (pH 7.0). The samples were neutralized by adding 600 µl of a 1:1 (vol/vol) mixture of 1,1,2-trichlorotrifluoroethane and tri-n-octylamine, followed by vigorous mixing and centrifugation. A 800-µl portion of the upper phase was removed, and intracellular inositol 1,4,5-trisphosphate (IP3) was evaluated using a commercial radioreceptor assay (Amersham, Braunschweig, Germany).

RNA isolation. RNA was extracted from confluent cultures of Hep G2, HuH-7, and MG-63 cells using a commercial RNA isolation kit (QIAGEN RNeasy, Hilden, Germany). Isolation was performed according to the manufacturer's guidelines.

RT-PCR for evaluation of P2Y subtype mRNA expression. Total RNA (2.5 µg) was used as a template for first-strand cDNA synthesis in a 20-µl reaction volume containing the following reagents: 5 nmol dNTP (dATP, dTTP, dCTP, and dGTP), 10 pmol oligo(dT)12-18 (Pharmacia, Freiburg, Germany), 20 units rRNasin (Promega, Heidelberg, Germany), 200 units Superscript RTase (Life Technologies, Berlin, Germany), 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 10 mM dithiothreitol in diethyl pyrocarbonate-treated distilled and deionized water. The reaction was incubated at 42°C for 30 min and terminated by heating at 90°C for 5 min. PCR reactions were carried out in a 50-µl reaction volume containing the following reagents: 4 µl of cDNA preparation, 20 mM Tris · HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of dATP, dGTP, dCTP, and dTTP, 2.5 U of Taq DNA polymerase (Life Technologies), and 0.5 µM of sense and antisense primer (Pharmacia). PCR was performed on a RoboCycler Gradient 96 (Stratagene, Heidelberg, Germany), using the following conditions for denaturation, annealing, and extension (35 cycles): 94°C for 1 min, 62°C for 1 min, and 72°C for 2 min, followed by 72°C for 10 min. PCR products were separated by electrophoresis on a 1.8% agarose gel stained with ethidium bromide. Specificity of the amplified fragments was confirmed by sequencing the PCR products with the T7 sequencing kit (Pharmacia), according to the manufacturer's suggested protocol. To verify that the amplified products were from mRNA and not genomic DNA contamination, we performed negative controls by omitting the RT. In the absence of RT, no products were observed. The primer oligonucleotides of the different P2Y genes were selected from published cDNA sequences (Ref. 29; Table 1). Glyceraldehyde-3-phosphate dehydrogenase was used as a housekeeping gene (sense, 5'-GGT-CGG-AGT-CAA-CGG-ATT-TGG-TCG-3'; antisense, 5'-CCT-CCG-ACG-CCT-GCT-TCA-CCA-C-3'), yielding a 782-bp product.

                              
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Table 1.   Primers used to identify human P2Y receptor subtypes

Statistics. Unless representative tracings are shown, values are means ± SE. Statistical analysis was performed using Student's t-test for unpaired or paired data. EC50 values were calculated using GraphPad Prism software (San Diego, CA).


    RESULTS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Effects of extracellular ATP and UTP on [Ca2+]i in primary human hepatocytes. In single primary human hepatocytes, resting [Ca2+]i was 225 ± 17 nM (n = 40). Stimulation with ATP at low micromolar concentrations caused a rapid increase in [Ca2+]i with a large initial peak followed by a sustained plateau in most cells (Fig. 1A). The peak increase in [Ca2+]i was dependent on the concentration of extracellular ATP (see Fig. 3A). The estimated EC50 for the ATP-induced increase in [Ca2+]i was ~1.8 µM with a threshold concentration <1 µM and a maximal effective concentration of 10-30 µM ATP. In some cells, however, repetitive Ca2+ transients were observed in response to ATP (Fig. 1C). The pyrimidine nucleotide UTP elicited a biphasic increase in [Ca2+]i or repetitive Ca2+ transients that was indistinguishable from that seen after ATP stimulation (Fig. 1, B and D). UTP (30 µM) increased [Ca2+]i by 223 ± 12 nM (n = 3), which was similar to the effect of ATP (30 µM) in the same preparation (189 ± 15 nM; n = 3). Pretreatment of cells with maximal concentrations of ATP or UTP (30 µM) abolished the Ca2+ response to supramaximal concentrations of UTP or ATP (100 µM), as shown in Fig. 1, A and B, suggesting a common P2 receptor present on primary human hepatocytes. The nucleotide-induced Ca2+ responses were observed as early as 4 h after the preparation of primary hepatocytes, and no increase in the magnitude of the [Ca2+]i changes occurred during 24 h of short-term culture (not shown). Furthermore, in primary hepatocytes the ATP- or UTP-induced increases in [Ca2+]i were of a magnitude similar to those in the hepatoma cell lines (see below). Together, these data indicate that the nucleotide-induced changes in [Ca2+]i in the primary cells reflect P2 receptor expression in vivo rather than P2 receptor upregulation during short-term culture as has been reported in rat salivary gland cells (44).


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Fig. 1.   Effect of extracellular ATP and UTP on intracellular Ca2+ concentration ([Ca2+]i) in single primary human hepatocytes. In most cells ATP and UTP caused a biphasic increase in [Ca2+]i and complete cross-desensitization occurred between ATP and UTP (A and B). In 20-30% of cells, repetitive Ca2+ transients were observed in response to ATP or UTP (C and D). Bars indicate the presence of the respective agents in the superfusion medium. Representative tracings are shown from 4 to 13 cells.

Effects of extracellular nucleotides on [Ca2+]i in single Hep G2 and HuH-7 cells. Basal [Ca2+]i was 226 ± 10 nM (n = 50) and 139 ± 4 nM (n = 50) in the differentiated human hepatoma cell lines Hep G2 and HuH-7, respectively. As occurred in primary human hepatocytes, extracellular ATP caused a concentration-dependent increase in [Ca2+]i with a large initial peak followed by a sustained plateau in the vast majority of Hep G2 cells (Fig. 2A), while in most HuH-7 cells only a single transient increase in [Ca2+]i was observed (Fig. 2B). In some cells of both cell lines, however, ATP-induced repetitive Ca2+ transients were seen (not shown). The P2 receptor was further characterized pharmacologically by investigating the effects on [Ca2+]i of UTP, adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S), ADP, UDP, AMP, adenosine, alpha ,beta -methylene-ATP, and beta ,gamma -methylene-ATP. As in primary hepatocytes, UTP elicited an increase in [Ca2+]i that was indistinguishable from that seen after ATP stimulation and complete cross-desensitization occurred between both nucleotides in Hep G2 and HuH-7 cells, respectively (Fig. 2, C and D). Likewise, ATPgamma S, which is resistant to hydrolysis, caused an increase in [Ca2+]i, although this compound was less potent than ATP or UTP (Fig. 3, B and C). The order of potency of the nucleotides for their effects on [Ca2+]i was ATP >=  UTP > ATPgamma S (Fig. 3, B and C, Table 2). ADP and UDP at higher concentrations (>10 µM) also increased [Ca2+]i in a concentration-dependent fashion (Fig. 3, B and C). Because ADP and UDP preparations from commercial sources might be contaminated with the respective nucleotide triphosphates, stock solutions of ADP and UDP were preincubated with or without (controls) 10 U/ml hexokinase and 10 mM glucose for 1 h at 37°C to convert any contaminating nucleotide triphosphate to the respective nucleotide diphosphate (28, 32). Furthermore, hexokinase (1 U/ml) was added to the medium in experiments with nucleotide diphosphates to prevent possible conversion of nucleotide diphosphates to nucleotide triphosphates by membranous nucleoside diphosphokinase (32). As depicted in Fig. 2, E and F, pretreatment of nucleotide diphosphates with hexokinase abolished the increase in [Ca2+]i, whereas UDP or ADP treated in the same way but without hexokinase increased [Ca2+]i in both cell lines (n = 7 each). This demonstrates that the nucleotide diphosphate-induced increase in [Ca2+]i was caused by contaminating ATP or UTP rather than by ADP or UDP itself. AMP, adenosine, alpha ,beta -methylene-ATP, and beta ,gamma -methylene-ATP at concentrations as high as 500 µM caused no changes in [Ca2+]i in either Hep G2 or HuH-7 cells (n = 4-8; not shown).


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Fig. 2.   Effect of extracellular nucleotides on [Ca2+]i in single Hep G2 and HuH-7 cells. Effect of ATP on [Ca2+]i in Hep G2 (A) and HuH-7 cells (B) is shown. Complete cross-desensitization occurred between ATP and UTP in Hep G2 (C) and HuH-7 cells (D). Pretreatment of commercial preparations of ADP (E) or UDP (F) with hexokinase (ADP-HK or UDP-HK, respectively) as described in MATERIALS AND METHODS abolished the increase in [Ca2+]i caused by stimulating Hep G2 cells with untreated nucleotide diphosphate. Identical results were obtained in HuH-7 cells (not shown). Bars indicate the presence of the respective agents in the superfusion medium. Representative tracings are shown from 6 to 21 cells.


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Fig. 3.   Dose-response curves for extracellular nucleotides on [Ca2+]i in primary hepatocytes and Hep G2 and HuH-7 cells. A: dose-response curve of ATP-induced increase in [Ca2+]i in primary hepatocytes. B and C: dose-response curves for various nucleotides on [Ca2+]i in Hep G2 (B) and HuH-7 cells (C). [Ca2+]i denotes peak increase above basal. ADP and UDP, untreated commercial preparation; ADP + HK and UDP + HK, ADP and UDP pretreated with hexokinase and glucose as described in MATERIALS AND METHODS. ATPgamma S, adenosine 5'-O-(3-thiotriphosphate). Data points represent means ± SE from 3 to 21 cells of at least 3 different preparations.

                              
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Table 2.   EC50 values for effects of various extracellular nucleotides on [Ca2+]i in Hep G2 and HuH-7 cells

Mechanisms of nucleotide-induced Ca2+-signals in human hepatocytes. In Ca2+-free medium (2.5 mM EGTA), the initial peak in [Ca2+]i in response to extracellular ATP or UTP (100 µM) was mainly preserved and amounted to ~60% of the peak increase in the presence of extracellular Ca2+ in primary hepatocytes and Hep G2 and HuH-7 cells (Fig. 4, A and C). This indicates that the initial increase in [Ca2+]i is caused by mobilization of Ca2+ from intracellular pools most likely by IP3 whose formation was determined in Hep G2 and HuH-7 cells to exclude errors arising from contamination of primary human hepatocytes with other cell types. Stimulation of Hep G2 and HuH-7 cells with ATP (100 µM) or UTP (100 µM) for 20 s increased cellular IP3 levels by 25 ± 5 and 30 ± 8%, respectively, above that of control levels (n = 6 each, P < 0.05). In contrast, ATP or UTP caused no plateau increase in [Ca2+]i in the absence of extracellular Ca2+, and withdrawal of extracellular Ca2+ by adding EGTA (2.5 mM) rapidly abolished the sustained plateau in primary hepatocytes and Hep G2 and HuH-7 cells (Fig. 4, A and C). This indicates that the sustained plateau increase in [Ca2+]i requires Ca2+ influx from the extracellular space. Ca2+ influx could occur through voltage-sensitive (VSCC) or voltage-insensitive Ca2+ channels (VICC). To test whether VSCC are present on human hepatocytes and whether they participate in the cytosolic Ca2+ response induced by the nucleotides, we depolarized cells with high K+ (45 mM) and investigated the effects of the VSCC blockers nifedipine or verapamil on the sustained plateau. High K+ (45 mM) caused no changes in [Ca2+]i, and nifedipine (10 µM) or verapamil (50 µM) had no effects on the ATP (30 or 100 µM)-induced sustained plateau increase in [Ca2+]i in primary hepatocytes and Hep G2 and HuH-7 cells (n = 4 each; not shown). This clearly demonstrates that human hepatocytes are nonexcitable cells and do not express VSCC. In nonexcitable cells, depletion of the IP3-sensitive Ca2+ store causes an influx of Ca2+ across the plasma membrane to the cytosol, termed capacitative Ca2+ entry, that is thought to be the basis for sustained Ca2+ responses in these cells (34). A major tool used to study capacitative Ca2+ entry is the microsomal Ca2+-ATPase inhibitor thapsigargin, which releases intracellular Ca2+ by preventing reuptake into the IP3-sensitive pool (42). In the presence of extracellular Ca2+, thapsigargin (2 µM) caused a sustained increase in [Ca2+]i, whereas in Ca2+-free medium the increase in [Ca2+]i was transient in primary hepatocytes and Hep G2 and HuH-7 cells, respectively, (Fig. 4, B and D, Table 3). Thus thapsigargin depletes intracellular Ca2+ stores and stimulates influx of extracellular Ca2+, which is consistent with capacitative Ca2+ entry being operational in human hepatocytes. After intracellular Ca2+ stores were discharged by successive stimulation with increasing concentrations of ATP in Ca2+-free medium, thapsigargin was still capable of evoking further release of intracellular Ca2+ (Fig. 4C). The thapsigargin-induced increase in [Ca2+]i, however, was greatly diminished and amounted to 30-50% of the Ca2+ response seen in Hep G2 or HuH-7 cells that had not been pretreated with ATP in Ca2+-free medium (n = 7 each). In contrast, depletion of intracellular Ca2+ stores by thapsigargin abolished the increase in [Ca2+]i in response to high concentrations of ATP (Fig. 4D). This demonstrates that there is a significant overlap between the ATP- and thapsigargin-releasable intracellular Ca2+ stores with the latter being larger than the ATP-sensitive store.


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Fig. 4.   A: effect of extracellular ATP on [Ca2+]i in the absence of extracellular Ca2+ in single primary hepatocytes. B: in the presence of extracellular Ca2+ the endoplasmic Ca2+-ATPase inhibitor thapsigargin caused a sustained increase in [Ca2+]i, which was abolished by withdrawal of extracellular Ca2+ in primary hepatocytes. C: effect of depletion of ATP-sensitive Ca2+ stores on the thapsigargin-induced increase in [Ca2+]i in Ca2+-free medium in Hep G2 cells. D: effect of thapsigargin on [Ca2+]i in Ca2+-free medium and on ATP-induced mobilization of internal Ca2+ in Hep G2 cells. Bars indicate the presence of the respective agents in the superfusion medium. Representative tracings are shown from 3 to 9 cells.

                              
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Table 3.   Effect of thapsigargin on [Ca2+]i in primary hepatocytes and Hep G2 and HuH-7 cells in the presence or absence of external Ca2+

Expression of P2Y receptor subtypes in Hep G2 and HuH-7 cells. The expression of specific P2Y1, P2Y2, P2Y4, and P2Y6 receptor mRNA was further investigated in Hep G2 and HuH-7 cells to avoid problems arising from contamination of primary hepatocytes with other cell types. Human P2Y receptor subtype-specific primer pairs were used as described in MATERIALS AND METHODS, and mRNA from human MG-63 cells, which have been previously shown to express P2Y1, P2Y2, P2Y4, and P2Y6 receptors (29), served as a positive control. PCR amplification of cDNA derived from RNA extracts of Hep G2 and HuH-7 cells and sequencing of the PCR products showed that all P2Y receptor subtypes were expressed in both cell lines (Fig. 5).


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Fig. 5.   RT-PCR analysis of P2Y receptor subtype expression in the human hepatoma cell lines Hep G2 and HuH-7. Primer pairs specific for the amplification of P2Y1, P2Y2, P2Y4, and P2Y6 receptor subtypes were used as previously described (28). Control amplifications were done using glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primers as a housekeeping gene. In Hep G2 (A) and HuH-7 cells (B), P2Y1, P2Y2, P2Y4, and P2Y6 receptor-specific transcripts were detected. C: in osteoblastic MG-63 cells, which served as positive controls, all P2Y receptor subtypes are expressed as described previously (28).


    DISCUSSION
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Abstract
Introduction
Materials and methods
Results
Discussion
References

In this study, we report for the first time that extracellular ATP at low micromolar concentrations elevates [Ca2+]i in primary human hepatocytes and in two differentiated human hepatoma cell lines, Hep G2 and HuH-7. The concentration range over which ATP increased [Ca2+]i is similar to the dissociation constant value for binding of ATP to human liver plasma membranes (21), indicating the expression of specific P2 receptors on human hepatocytes. P2 receptors are subdivided into two main groups based on their mode of signal transduction. The P2X receptors are members of the transmitter ion channel receptors, whereas the P2Y receptor subtypes are members of the G protein-coupled receptor superfamily, which are further subclassified pharmacologically by their differing responses to nucleotide analogs in various tissues (3, 5, 11, 13, 14). We characterized the P2 receptor expressed on human hepatocytes by investigating the effects on [Ca2+]i of UTP, ATPgamma S, ADP, UDP, alpha ,beta -methylene-ATP, beta ,gamma -methylene-ATP, AMP, and adenosine. UTP and ATPgamma S elicited an increase in [Ca2+]i that was indistinguishable from that seen after ATP stimulation, and complete cross-desensitization occurred between UTP and ATP in primary hepatocytes and in Hep G2 and HuH-7 cells. The order of potency of the nucleotides for their effects on [Ca2+]i was ATP >=  UTP > ATPgamma S. ADP and UDP at higher concentrations (>10 µM) also increased [Ca2+]i in a concentration-dependent fashion. Because commercial ADP and UDP preparations might be contaminated with the respective nucleotide triphosphates, stock solutions of ADP and UDP were preincubated with hexokinase and glucose to convert any contaminating nucleotide triphosphate to the respective nucleotide diphosphate and hexokinase was added to the medium in experiments with nucleotide diphosphates to prevent possible conversion of nucleotide diphosphates to nucleotide triphosphates by membranous nucleoside diphosphokinase (28, 32). The latter point, however, appears to be of minor importance in the present study as the cells were continuously perfused with fresh medium. ADP or UDP treated in such a way, however, caused no changes in [Ca2+]i, indicating that the observed increases in [Ca2+]i after stimulation with commercial ADP or UDP were produced by contaminating ATP or UTP rather than by ADP or UDP itself. The P2X receptor agonists alpha ,beta -methylene-ATP and beta ,gamma -methylene-ATP at concentrations as high as 500 µM caused no changes in [Ca2+]i in either Hep G2 or HuH-7 cells. This contrasts with a recent report (7) demonstrating the expression of P2X receptors in guinea pig hepatocytes, which could be activated by ATP and alpha ,beta -methylene-ATP. Because not all the P2X receptor subtypes are equally sensitive to alpha ,beta -methylene-ATP or beta ,gamma -methylene-ATP (3, 5, 14), we cannot rule out with certainty that a specific P2X receptor subtype insensitive to alpha ,beta -methylene-ATP or beta ,gamma -methylene-ATP is expressed. No evidence, however, could be found for an ATP-gated cationic channel or P2X receptor on human hepatocytes, since the shape and time course of the nucleotide-induced changes in [Ca2+]i were identical and complete cross-desensitization of the Ca2+ changes occurred between ATP and UTP with no further increase in [Ca2+]i in response to high concentrations of ATP after maximal UTP stimulation. Likewise, AMP and adenosine had no effects on [Ca2+]i in Hep G2 and HuH-7 cells, which indicates that the ATP-induced increase in [Ca2+]i was not caused by P1 receptors.

According to a recent classification of P2 receptors, this pharmacological profile is characteristic of P2Y2 receptors present on human hepatocytes (3, 5, 11, 13, 14). This is consistent with the expression of mRNA coding for the cloned P2Y2 receptor in human liver tissue (33). However, liver tissue such as isolated primary hepatocytes could be contaminated with cell types other than hepatocytes. As the nucleotide-induced Ca2+ responses were virtually identical in primary hepatocytes and in the hepatoma cell lines, the expression of P2Y receptor subtype-specific mRNA was investigated in Hep G2 and HuH-7 cells. Because recent evidence suggests (24) that the P2Y receptors designated as P2Y5 and P2Y7 do not belong to the family of nucleotide receptors, we used primer pairs for the human P2Y1, P2Y2, P2Y4, and P2Y6 receptors. In accordance with the pharmacological characterization at the functional level, P2Y2 receptor-specific mRNA could be detected by RT-PCR in both Hep G2 and HuH-7 cells. However, specific mRNA for the other three P2Y receptor subtypes could also be found in both cell lines. Because ADP and UDP, which are the most potent agonists for P2Y1 and P2Y6 receptors, respectively, caused no changes in [Ca2+]i, this strongly suggests that these receptor subtypes are not expressed at a functional level. This points to posttranscriptional processes that appear to be of prime importance for the regulation of functional P2Y receptor protein expression. Thus caution is needed when specific P2Y mRNA expression is provided to suggest functional receptor expression. Because P2Y2 and P2Y4 receptors are only sensitive to ATP and UTP, albeit with different potencies, demonstration of functional coexpression of both receptors in the same cell is hard to achieve. Therefore, the possibility exists that functional P2Y4 receptors are coexpressed with P2Y2 receptors and contribute to the nucleotide-induced changes in [Ca2+]i in the human hepatoma cell lines.

P2Y receptor subtypes belong to the G protein-coupled receptor superfamily (3, 5, 11, 13, 14), and the mechanisms involved in the generation of the nucleotide-induced Ca2+-signals in human hepatocytes were investigated in the present study. ATP and the other nucleotides caused a biphasic increase in [Ca2+]i with an initial peak followed by a sustained plateau in the presence of extracellular Ca2+ in the majority of cells. In some cells, however, repetitive Ca2+ transients were observed as has been reported from single rat hepatocytes at lower nucleotide concentrations (12). In the absence of extracellular Ca2+ only a transient increase in [Ca2+]i was seen. This indicates mobilization of Ca2+ from intracellular stores during the initial peak, most likely mediated by IP3 whose formation was enhanced after stimulation of Hep G2 and HuH-7 cells with ATP or UTP. The nucleotide-stimulated increase in cellular IP3 levels in the hepatoma cells was similar to that previously reported from rat hepatocytes (9, 22). In addition to mobilization of intracellular Ca2+, influx of Ca2+ from the extracellular space is required as demonstrated by the absence of the sustained plateau increase in [Ca2+]i in Ca2+-free medium. Because the plateau increase in [Ca2+]i was insensitive to blockers of VSCC of the L-type, Ca2+ influx does not occur through L-type Ca2+ channels. The lack of effect on [Ca2+]i of membrane depolarization by high extracellular K+ further indicates that VSCC channels are not expressed on human hepatocytes. Other routes of Ca2+ influx involve VICC, and Ca2+ influx across the plasma membrane to the cytosol is stimulated by depletion of the IP3-sensitive Ca2+ store. This mechanism has been termed capacitative Ca2+ entry and is thought to be the basis for sustained Ca2+ responses in nonexcitable cells (34). Consistent with capacitative Ca2+ entry being operational in human hepatocytes, thapsigargin, which depletes intracellular Ca2+ stores without formation of IP3, caused a sustained increase in [Ca2+]i in the presence of extracellular Ca2+, whereas in Ca2+-free medium the increase in [Ca2+]i was transient. Because the thapsigargin- and the ATP-releasable Ca2+ stores overlap, ATP-induced mobilization of internal Ca2+ might also activate capacitative Ca2+ entry. Thus activation of phospholipase C and concomitant formation of IP3 could explain the mobilization of Ca2+ from intracellular stores by ATP and UTP, which is in agreement with a P2Y2 and possibly P2Y4 receptor coupled to the PI-Ca2+ signaling cascade as has been demonstrated in other tissues (3, 5, 11, 13, 14). Depletion of the IP3-sensitive Ca2+ store might then activate influx of extracellular Ca2+ across the plasma membrane via a capacitative Ca2+ entry mechanism shown to exist in human hepatocytes. This might be the basis for the sustained increase in [Ca2+]i observed in response to ATP or UTP as has been shown for other Ca2+-mobilizing agonists in a variety of nonexcitable cells (34).

The range of concentrations over which ATP increased [Ca2+]i is similar to the concentrations required for ATP to stimulate glycogen phosphorylase activity in human hepatocytes, which is the rate-limiting step in liver glycogenolysis (20). This clearly offers a functional role for extracellular ATP in human liver metabolism. In addition, activation of the PI-Ca2+ signaling cascade and an increase in [Ca2+]i, which is a major intracellular signal in many cells, may be involved in the regulation of other hepatic functions such as protein synthesis or gene expression (15). Furthermore, nucleotides such as ATP have been recently shown (37) to be involved in intercellular signal propagation between hepatocytes and between hepatocytes and bile duct cells in the rat liver. Given the pronounced effects of ATP or UTP on [Ca2+]i, the regulated release of both nucleotides, e.g., from sympathetic nerve endings, where ATP is coreleased with norepinephrine, from purinergic nerves in the autonomic nervous system, from hepatocytes by cell volume-controlled autocrine or paracrine secretion, or from platelets and damaged cells during inflammation, could prove to be physiologically important in the control of human hepatic functions (5, 11, 18, 36).


    ACKNOWLEDGEMENTS

We thank Dr. N. Schütz, University of Würzburg, for the supply of MG-63 cells.


    FOOTNOTES

This work was supported by Deutsche Forschungsgemeinschaft Grants Scho 466/1-3, Wa 757/1-1, SFB 280, and SFB 265.

Address for reprint requests: C. Schöfl, Abteilung Klinische Endokrinologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany.

Received 19 December 1997; accepted in final form 14 October 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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