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 |
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) (ATP
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 > ATP
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,
,
-methylene-ATP,
,
-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 |
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 |
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.
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 |
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.
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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)
(ATP
S), ADP, UDP, AMP, adenosine,
,
-methylene-ATP, and
,
-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, ATP
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 > ATP
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,
,
-methylene-ATP, and
,
-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. ATP 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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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+
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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).
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 |
DISCUSSION |
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, ATP
S, ADP, UDP,
,
-methylene-ATP,
,
-methylene-ATP, AMP, and adenosine. UTP and ATP
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 > ATP
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
,
-methylene-ATP and
,
-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
,
-methylene-ATP. Because not all the P2X receptor subtypes are equally sensitive to
,
-methylene-ATP or
,
-methylene-ATP (3, 5, 14), we cannot
rule out with certainty that a specific P2X receptor subtype
insensitive to
,
-methylene-ATP or
,
-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.
 |
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