Store-operated Ca(2+) channels and Stromal Interaction Molecule 1 (STIM1) are targets for the actions of bile acids on liver cells.
ABSTRACT Cholestasis is a significant contributor to liver pathology and can lead to primary sclerosis and liver failure. Cholestatic bile acids induce apoptosis and necrosis in hepatocytes but these effects can be partially alleviated by the pharmacological application of choleretic bile acids. These actions of bile acids on hepatocytes require changes in the release of Ca(2+) from intracellular stores and in Ca(2+) entry. However, the nature of the Ca(2+) entry pathway affected is not known. We show here using whole cell patch clamp experiments with H4-IIE liver cells that taurodeoxycholic acid (TDCA) and other choleretic bile acids reversibly activate an inwardly-rectifying current with characteristics similar to those of store-operated Ca(2+) channels (SOCs), while lithocholic acid (LCA) and other cholestatic bile acids inhibit SOCs. The activation of Ca(2+) entry was observed upon direct addition of the bile acid to the incubation medium, whereas the inhibition of SOCs required a 12 h pre-incubation. In cells loaded with fura-2, choleretic bile acids activated a Gd(3+)-inhibitable Ca(2+) entry, while cholestatic bile acids inhibited the release of Ca(2+) from intracellular stores and Ca(2+) entry induced by 2,5-di-(tert-butyl)-1,4-benzohydro-quinone (DBHQ). TDCA and LCA each caused a reversible redistribution of stromal interaction molecule 1 (STIM1, the endoplasmic reticulum Ca(2+) sensor required for the activation of Ca(2+) release-activated Ca(2+) channels and some other SOCs) to puncta, similar to that induced by thapsigargin. Knockdown of Stim1 using siRNA caused substantial inhibition of Ca(2+)-entry activated by choleretic bile acids. It is concluded that choleretic and cholestatic bile acids activate and inhibit, respectively, the previously well-characterised Ca(2+)-selective hepatocyte SOCs through mechanisms which involve the bile acid-induced redistribution of STIM1.
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ABSTRACT: Sphingosine kinases (SphKs) and their product sphingosine-1-phosphate (S1P) have been reported to regulate apoptosis and survival of liver cells. Cholestatic liver diseases are characterized by cytotoxic levels of bile salts inducing liver injury. It is unknown whether SphKs and/or S1P play a role in this pathogenic process. Here, we investigated the putative involvement of SphK1 and S1P in bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA) to induce apoptosis. GCDCA-exposed hepatocytes were co-treated with S1P, the SphK1 inhibitor Ski-II and/or specific antagonists of S1P receptors (S1PR1 and S1PR2). Apoptosis and necrosis were quantified. Ski-II significantly reduced GCDCA-induced apoptosis in hepatocytes (-70%, p < 0.05) without inducing necrosis. GCDCA increased the S1P levels in hepatocytes (p < 0.05). GCDCA induced [Ca(2+)] oscillations in hepatocytes and co-treatment with the [Ca(2+)] chelator BAPTA repressed GCDCA-induced apoptosis. Ski-II inhibited the GCDCA-induced intracellular [Ca(2+)] oscillations. Transcripts of all five S1P receptors were detected in hepatocytes, of which S1PR1 and S1PR2 appear most dominant. Inhibition of S1PR1, but not S1PR2, reduced GCDCA-induced apoptosis by 20%. Exogenous S1P also significantly reduced GCDCA-induced apoptosis (-50%, p < 0.05), however, in contrast to the GCDCA-induced (intracellular) SphK1 pathway, this was dependent on S1PR2 and not S1PR1. Our results indicate that SphK1 plays a pivotal role in mediating bile salt-induced apoptosis in hepatocytes in part by interfering with intracellular [Ca(2+)] signaling and activation of S1PR1.Biochimica et Biophysica Acta 06/2013; · 4.66 Impact Factor
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ABSTRACT: The major membrane lipid regulators of ion channel function include cholesterol, one of the main lipid components of the plasma membranes, phosphoinositides, a group of regulatory phospholipids that constitute a minor component of the membrane lipids but are known to play key roles in regulation of multiple proteins and sphingolipids, particularly sphingosine-1-phosphate, a signaling biolipid that is generated from ceramide and is known to regulate multiple cellular functions. Furthermore, specific effects of all the lipid modulators are highly heterogeneous varying significantly between different types of ion channels, as well as between different cell types. In terms of the mechanisms, three general mechanisms have been shown to underlie lipid regulation of ion channels: specific lipid-protein interactions, changes in the physical properties of the membrane, and facilitating the association of the channel proteins with other regulatory proteins within multiproteins signaling complexes termed membrane rafts. In this article, we present comprehensive analysis of the roles of several lipid modulators, including cholesterol, bile acids, phosphoinositides, and sphingolipids on ion channel function. © 2012 American Physiological Society. Compr Physiol 2:31-68, 2012.Comprehensive Physiology. 01/2012; 2(1):31-68.
Store-operated Ca2+channels and Stromal Interaction Molecule 1 (STIM1)
are targets for the actions of bile acids on liver cells
Edoardo C. Aromatarisa,1, Joel Castrob,1, Grigori Y. Rychkova, Greg J. Barrittb,⁎
aSchool of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, 5005, Australia
bDepartment of Medical Biochemistry, School of Medicine, Flinders University, GPO Box 2100, Adelaide SA 5001, Australia
Received 16 August 2007; received in revised form 7 February 2008; accepted 11 February 2008
Available online 23 February 2008
and necrosis in hepatocytes but these effects can be partially alleviated by the pharmacological application of choleretic bile acids. These actions of
bile acids on hepatocytes require changes in the release of Ca2+from intracellular stores and in Ca2+entry. However, the nature of the Ca2+entry
pathway affected is not known. We show here using whole cell patch clamp experiments with H4-IIE liver cells that taurodeoxycholic acid (TDCA)
and other choleretic bile acids reversibly activate an inwardly-rectifying current with characteristics similar to those of store-operated Ca2+channels
activated a Gd3+-inhibitable Ca2+entry, while cholestatic bile acids inhibited the release of Ca2+from intracellular stores and Ca2+entry induced by
2,5-di-(tert-butyl)-1,4-benzohydro-quinone (DBHQ). TDCA and LCA each caused a reversible redistribution of stromal interaction molecule 1
(STIM1, theendoplasmic reticulum Ca2+sensorrequired for theactivation ofCa2+release-activated Ca2+channels and some otherSOCs) topuncta,
similar to that induced by thapsigargin. Knockdown of STIM1 using siRNA caused substantial inhibition of Ca2+-entry activated by choleretic bile
acids. It is concluded that choleretic and cholestatic bile acids activate and inhibit, respectively, the previously well-characterised Ca2+-selective
hepatocyte SOCs through mechanisms which involve the bile acid-induced redistribution of STIM1.
© 2008 Elsevier B.V. All rights reserved.
Keywords: Liver cell; Cholestasis; Ca2+channel; STIM1; Bile acid; Patch clamp recording
Cholestasis arises from hepatocyte dysfunction, or intrahe-
patic or extrahepatic biliary obstruction, leading to impaired
secretion of bile into the bile canaliculus and impaired move-
ment of bile along the biliary tree. Causes of cholestasis include
primary sclerosing cholangitis, primary biliary cirrhosis, liver
injury, and genetic abnormalities in hepatocyte bile acid trans-
porters. If untreated cholestasis can ultimately progress to liver
failure (reviewed in [1–3]). Cholestasis is associated with de-
creases in the expression and/or activity of hepatocyte bile acid
transporters, and the accumulation of conjugated and unconju-
gated bile acids in hepatocytes and the blood . The more
hydrophobic bile acids, including taurolithocholic (TLCA),
lithocholic (LCA), cholic (CA) and taurocholic (TCA) acids,
inhibit bile flow (are cholestatic), while the less hydrophobic
bile acids, including taurodeoxycholic (TDCA), tauroursode-
oxycholic (TUDCA) and ursodeoxycholic (UDCA) acids, en-
hance bile blow (are choleretic) . TLCA is a potent inducer of
cholestasis [5,6], and cholestatic bile acids induce liver injury
leading to apoptosis and necrosis of hepatocytes [7–12]. The
choleretic bile acids TUDCA and UDCA have been used at
pharmacological doses to treat cholestasis [13–23].
Central to the actions of bile acids on hepatocytes are bile
reticulum (ER), and/or the activation of Ca2+entry. In hepato-
cytes 7and liver cell lines many bile acids increase [Ca2+]cyt,
Available online at www.sciencedirect.com
Biochimica et Biophysica Acta 1783 (2008) 874–885
⁎Corresponding author. Tel.: +61 8 8204 4260; fax: +61 8 8374 0139.
E-mail address: Greg.Barritt@flinders.edu.au (G.J. Barritt).
1Each author contributed equally to the work.
0167-4889/$ - see front matter © 2008 Elsevier B.V. All rights reserved.
release Ca2+from intracellular stores, and induce Ca2+entry,
while TLCA inhibitsCa2+entry [24–30].TUDCAmayenhance
bile flow by activating Ca2+entry and increasing hepatocyte
[Ca2+]cyt[26,27,29,30]. This, in turn, may activate Ca2+-depen-
and enhance contraction of the bile canaliculus . While the
effects of bile acids on the release of Ca2+from intracellular
stores in hepatocytes [25,26] and other cell types  have been
reasonably well described, little is known about the Ca2+entry
pathways modulated by bile acids.
The main Ca2+entry pathway activated by hormones and
growth factors in hepatocytes is the store-operated Ca2+channel
(SOC) [33–36]. While only one type of SOC has been detected
in hepatocytes and in hepatocyte cell lines [33,34], several other
types of Ca2+entry channels are known to be present, although
most have not been well-characterised . There is some
the maintenance of normal bile flow . Hepatocyte SOCs
exhibit a high selectivity for Ca2+and electrophysiological pro-
perties essentially indistinguishable from those of Ca2+release-
activated Ca2+channels in mast cells and lymphocytes [34,36].
Under physiological conditions, SOCs are activated by a de-
of IP3and Ca2+on IP3receptors (reviewed in ). SOCs
can also be activated pharmacologically by inhibiting the ER
(Ca2++Mg2+) ATP-ase (SERCA) (reviewed in ).
Recent studies with other cell types have shown that Orai
(CRACM) proteins constitute the pore of Ca2+-selective SOCs,
while stromal interaction molecule (STIM) proteins constitute
the Ca2+sensor which detects the decrease in Ca2+in the ER
and conveys this information to Orai1 in the plasma membrane
(reviewed in [40–42]). The release of Ca2+from the ER alters
the localisation of STIM1 in the ER, and in some studies STIM1
has been observed to move to locations adjacent to the plasma
membrane, the junctional ER [43–49]. STIM1 redistribution is
necessary for SOC activation in lymphocytes, mast cells, some
other cell types (reviewed in [41,42]) and liver cells .
The aim of the present experiments was to identify the Ca2+
entry channel(s) involved in the actions of choleretic and cho-
lestatic bile acids on hepatocyte [Ca2+]cyt. The experiments
have been conducted with the H4-IIE rat liver cell line and with
isolated rat hepatocytes. The results indicate that the main type
of hepatocyte plasma membrane Ca2+entry channel activated
by choleretic bile acids and inhibited by cholestatic bile acids is
the hepatocyte Ca2+-selective SOC, and provide evidence that
bile acids affect SOCs by modifying the intracellular localisa-
tion of STIM1. SOCs and STIM1 may provide a potential target
for pharmacological interventions in the treatment of cholestasis
and cholestasis-induced liver damage.
2. Materials and methods
Collagenase (Type IV) was obtained from Worthington Biochemical Corpo-
ration; Dulbecco's Modified Eagles Medium (DMEM), fura-2 (acetoxymethyl-
ester), goat anti-mouse Alexa-Fluor 488 antibody and pluronic acid F-127 from
(IP3), ionomycin, bile acids, and 2-aminoethyl diphenylborate (2-APB) from
Sigma Australia; thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydro-quinone
(DBHQ) from Sapphire Bioscience; mouse anti-GOK STIM1 monoclonal anti-
body from BD Biosciences Pharmingen, San Jose, CA, USA; FuGENE 6 from
Roche Pharmaceutical, Nutley, NJ, USA; the pDsRed2-ER plasmid from Clon-
tech, Mountain View, CA, USA; and glass coverslips from Menzel-Glaser
GmgH, Braunschweig, Germany. The GFP-STIM1 plasmid, in which cDNA
encoding GFP is located immediately downstream of the predicted signal se-
quenceof the STIM1 gene andinserted into the pApuro expressionvector (GFP-
STIM1/pApuro)  was kindly provided by Dr T Kurosaki, RIKEN Research
Centre for Allergy and Immunology, Kanagwa, Japan.
2.2. Cell culture
H4-IIE rat liver cells (ATCC CRL 1548)  were plated on No. 1 glass
coverslips, (12, 22 and 30 mm diameter for immunofluorescence, fura-2 and live
cell confocal imaging experiments, respectively) and cultured for 24–72 h in
DMEM supplemented with 10% (v/v) fetal calf serum, penicillin (100 U/ml),
as described previously [33,34]. Hepatocytes were isolated from rat liver by
perfusion with collagenase, as described previously . Animals received
humane care, and the experimental protocols were conducted according to the
criteria outlined in the “Australian Code of Practice for the Care and Use of
of Australia). Hepatocytes were plated on glass coverslips pre-treated with
concentrated HCl to facilitate their attachment, then cultured for 24–72 h in
DMEM/F12 supplemented with 10% (v/v) fetal calf serum, penicillin/strepto-
mycin and HEPES in 5% (v/v) CO2(pH 7.4) at 37 °C.
Whole-cell patch clamping was performed at room temperature using a
computer-based patch clamp amplifier (EPC-9 HEKA Electronics, Germany)
and PULSE software (HEKA Electronics) . The bath solution contained
(mM): NaCl, 140; CsCl, 4; CaCl2, 10; MgCl2, 2; glucose, 10; and HEPES, 10;
adjusted to pH 7.4 with NaOH. The internal solution contained (mM): Cs
glutamate, 120; CaCl2, 5; MgCl2, 5; MgATP, 1; EGTA, 10; and HEPES, 10;
adjusted to pH 7.2 with NaOH. Depletion of the intracellular Ca2+stores was
achieved by addition of 20 μM IP3to the internal solution. Patch pipettes were
pulled from borosilicate glass and fire-polished; pipette resistance ranged be-
tween 2 and 4 MΩ. In order to monitor the development of the SOC current
(ISOC), voltage ramps between −138 and +102 mV were applied every 2 s,
starting immediately after achieving the whole cell configuration. All voltages
shown have been corrected for the liquid junction potential of −18 mV between
the bath and electrode solutions (estimated by JPCalc ). The holding po-
tential was −18 mV throughout. Cell capacitance was compensated automa-
tically by the EPC9 amplifier.
2.4. Measurements of [Ca2+]cytusing fura-2
Cells attached to glass coverslips were incubated with 5 μM fura-2 aceto-
xymethylester in Krebs–Ringer–HEPES buffer containing 0.02% (v/v) pluronic
acid. The Krebs–Ringer–HEPES solution contained (mM): NaCl, 136; KCl,
4.7; CaCl2, 1.3; MgCl2, 1.25; glucose, 10; and HEPES, 10; adjusted to pH 7.4
with NaOH. Isolated hepatocytes were loaded for 1 h at 37 °C, while H4-IIE
cells were loaded for 30 min at room temperature. Fura-2 fluorescence was
measured at room temperature in cells incubated in Krebs–Ringer–HEPES in
the presence of the indicated concentrations of CaCl2, using a Nikon TE300
Eclipse microscope in conjunction with a Sutter DG-4/OF wavelength switcher,
Omega XF04 filter set for fura-2, Photonic Science ISIS-3 ICCD camera and
UIC Metafluor software. Fluorescence images at 340 nm and 380 nm excitation
were obtained every 20 s using a 40× objective.
Values of fluorescence ratio (340 nm:380 nm) were converted to [Ca]cyt
using the equation and Kdvalue derived by Grynkiewicz et al.  for the
binding of fura-2 to Ca2+, and ionomycin and EGTA to determine the maximum
(Rmax) and minimum (Rmin) ratios. The results are expressed as means±SEM
(between 10 and 20 cells for each experiment). Initial rates of Ca2+entry were
875E.C. Aromataris et al. / Biochimica et Biophysica Acta 1783 (2008) 874–885
determined using a Ca2+“add-back” protocol in which cells were initially in-
figuresandtext), and the increase in [Ca2+]cytwas measured as a function of time.
for the peak (maximum) [Ca2+]cytobserved after Caext
by calculating the difference between the maximum [Ca2+]cytobserved after Caext
re-addition and the value of [Ca2+]cytobtained immediately before Caext
addition. Amounts of Ca2+released were determined by measuring the height of
the peak of the DBHQ- or bile acid-induced increase in [Ca2+]cyt.
2+addition were determined
2.5. Cell transfection, immunofluorescence, and fluorescence
H4-IIE cells were transfected with GFP-STIM1/pApuro or pDsRed2-ER
DNA (3 μg) or co-transfected with both plasmids using FuGENE 6, according
to the manufacturer's instructions. The cells were then incubated at 37 °C for
48–72 h before imaging GFP (488 nm excitation/500–550 nm emission) or
pDsRed2 (561 nm excitation/550–620 nm emission) fluorescence using a Leica
SP5 scanning confocal microscope. For the measurement of STIM1 by immu-
nofluorescence, H4-IIE cells (attached to glass coverslips) were washed 3 times
with phosphate buffered saline (PBS) and then fixed in 3% (v/v) paraformalde-
hyde at room temperature for 10 min. The fixed cells were washed 5 times with
100 mM glycine in PBS and permeabilised with 0.1% (v/v) saponin in PBS for
10 min at room temperature. Cells were washed 5 times in PBS, then blocked
with blocking buffer (gelatin 0.2% (w/v) plus TWEEN 0.1% (w/v) in PBS) for
30 min at room temperature, incubated with mouse anti-GKO/STIM1 (1:100
dilution inblocking buffer) for3 h, andwashed 5 times with blocking buffer. The
cells were then incubated with Alexa-Fluor 488-conjugated goat anti-mouse IgG
(1:500 dilution in blocking buffer) for 1 h, washed 5 times with PBS and the
coverslips mounted on slides in IMMU-Mount medium (Thermo Shandon,
excitation/500–550 nm emission).
2.6. Transfection of cells with siRNA targeted against STIM1
The sequencesof siRNAtargeted against STIM1andcontrol siRNAandcell
transfection were as described by Litjens et al. .
2.7. Statistical analysis
Statistical significance was assessed using Student's t-test to compare two
samples. Multiple groups were compared using ANOVA followed by the Bon-
ferroni post hoc test. Differences between means were considered significant at
P≤0.05. Curves showing activation of the ISOCunder different conditions were
compared using 2-way ANOVA.
3.1. Choleretic bile acids activate Ca2+entry through SOCs in
H4-IIE liver cells and rat hepatocytes
To test whether bile acids can affect membrane conductance,
IIE liver cells. When applied to the bath at concentrations
between 100 and 300 μM, the choleretic bile acids TDCA,
TUDCA and UDCA each activated an inwardly-rectifying
as shown by the observation that the current induced by UDCA
was completely inactivated within 3–4 min upon washout of the
to deplete intracellular Ca2+stores and activate ISOC, the sub-
sequent application of choleretic bile acids had no effect on
Fig. 1. Choleretic bile acids activate a Ca2+current in H4-IIE liver cells with properties similar to those of ISOC. (A, B) Inward currents measured at −118 mV in the
presence of TDCA, UDCA and TUDCA plotted against time. The horizontal bar shows the time of the addition of a bile acid to the bath. The internal solution did not
contain IP3. (C) The effect of UDCA is reversible. (D) UDCA does not activatefurther current over and above that activated by IP3. The results are the means±SEM of
the number of experiments indicated.
876 E.C. Aromataris et al. / Biochimica et Biophysica Acta 1783 (2008) 874–885
membrane conductance (shown for UDCA in Fig. 1D), indi-
The I–V plot of the current activated by choleretic bile salts
showed marked inward rectification and a reversal potential
above 100 mV (Fig. 2A). The current recorded in response to
voltage steps negative to −100 mV showed fast inactivation,
similar to that of the ISOCpreviously characterised in H4-IIE
cells and rat hepatocytes (Fig. 2B). This current could be
blocked by 1 μM La3+and was abolished by the replacement of
the extracellular Ca2+by Mg2+(not shown). To confirm that the
current activated by choleretic bile acids is the hepatocyte ISOC,
medium containing 100 mM Ba2+in place of 10 mM Ca2+was
applied to the bath after the current induced by TUDCA had
developed. This produced a spike in the amplitude of the inward
current followed by inhibition (Fig. 2C), characteristic of the
properties of ISOCin H4-IIE cells [33,34]. Furthermore, current
activated by choleretic bile acids was inhibited by 2-APB
(Fig. 2D). Unlike choleretic bile acids, the cholestatic bile acids
(LCA and TLCA) did not activate ISOCin H4-IIE cells when
applied to the bath at similar concentrations (100–300 μM)
(results not shown).
To test whether the activation of Ca2+entry through SOCs
by choleretic bile acids is associated with the release of Ca2+
from intracellular stores and an increase in [Ca2+]cyt, changes in
[Ca2+]cytwere measured using fura-2. When added to H4-IIE
caused a substantial increase in [Ca2+]cyt(Fig. 3A). Further
experiments on bile acid-induced activation of Ca2+entry were
conducted at 10 mM Caext
that employed in the patch clamping experiments. At 10 mM
observed at 2.4 mM Caext
[Ca2+]cytdecreased to the basal level when [Ca2+]extwas re-
duced from 10 mM to zero (Fig. 3B). The ability of TDCA to
increase [Ca2+]cytis retained when Caext
out and then re-introduced to the perfusion medium (Fig. 3B).
The TDCA-induced increase in [Ca2+]cyt was substantially
reduced to near basal values by addition of Gd3+(10 μM)
(Fig. 3C). To confirm that TDCA induces Ca2+release from
intracellular stores under conditions employed to measure Ca2+
entry, cells were incubated in the presence of 2.4 mM Caext
10 μM Gd3+(to block Ca2+entry). Addition of TDCA caused a
transient increase in Cacyt
Ca2+from intracellular stores. No TDCA-induced release of
Ca2+from intracellular stores was observed when the bile acid
was added in the absence of extracellular Ca2+(results not
shown). The reason for this is not clear, however. It may be
because extracellular Ca2+affects the solubility of bile acids
and their ability to form micelles [54,55].
In cells incubated in the presence of 10 mM Caext
addition of TDCA following thapsigargin did not cause a fur-
ther increase in [Ca2+]cyt (Fig. 4A). The magnitude of the
plateau following TDCA addition was similar to that following
thapsigargin addition (Fig. 4B cf A and C). These results pro-
vide further evidence that TDCA activates the same Ca2+entry
pathway as that activated by SERCA inhibitors.
The choleretic bile acid TUDCA (300 μM) also caused an
increase in [Ca2+]cyt(Fig. 5). Consistent with previous obser-
vations  the TUDCA-induced increase in [Ca2+]cytwas
observed as oscillations in [Ca2+]cyt. The oscillatory increases in
[Ca2+]cytwere dependent on [Ca2+]extand were substantially
reduced by addition of Gd3+(Fig. 5), indicating a requirement
for Ca2+entry through SOCs in the maintenance of Cacyt
The effects of bile acids on Ca2+entry in rat hepatocytes
were also investigated. When added directly to the hepatocyte
incubation medium, TDCA increased [Ca2+]cytin the presence
of 10 mM Caext
Gd3+(results not shown).
2+, the same concentration of Caext
2+, TDCA-induced an increase in [Ca2+]cytsimilar to that
2+(Fig. 3B). In the presence of TDCA,
2+and TDCA are washed
2+(Fig. 3D) indicating the release of
2+(Fig. 3E). This increase was inhibited by 10 μM
3.2. Cholestatic bile acids inhibit Ca2+entry through SOCs in
H4-IIE cells and rat hepatocytes
To test whether bile acids inhibit Ca2+-entry through SOCs
in H4-IIE liver cells, whole cell patch clamping experiments
were performed using IP3(20 μM in the patch pipette) to induce
ISOC[34,36] (Fig. 6A, control curve). The IP3-initiated current
was inhibited by the cholestatic bile acids DCA (~25% inhi-
bition), CA (~40%), TLCA (~40%) and LCA (~80%) at a
concentration of 50 μM, when these were pre-incubated with
H4-IIE cells for 12 h before the measurement of ISOC(examples
of TLCA and LCA are shown in Fig. 6A). When tested at
Fig. 2. Properties of the current activated by choleretic bile acids. (A) I–V plot
and (B) current traces in response to −118 and −138 mV steps obtained for cells
incubated in the presence of 300 μM TDCA. (C) The effect of substitution of
Ba2+for Ca2+in the extracellular medium on the current induced by TUDCA.
The horizontal bars show the time of addition of TUDCA and of the application
of an extracellular medium composed of an iso-osmolar solution containing
100 mM Ba2+in place of the medium containing 10 mM Ca2+. (D) Inhibition of
the current induced by UDCA by 2-APB (50 μM). The horizontal bars show the
times of addition of UDCA and 2-APB. The results are representative of those
obtained from 5 to 7 cells.
877 E.C. Aromataris et al. / Biochimica et Biophysica Acta 1783 (2008) 874–885
10 μM, CA, LCA and TLCA each inhibited the current by 25,
27 and 20%, respectively (result for LCA shown in Fig. 6A).
Two-way ANOVA showed that at 10 μM concentration the
effect of these bile acids on the development of ISOC was
significant (Pb0.001). No inhibition of ISOC was observed
when the cholestatic bile acid was added directly to the bath
(results not shown). Unconjugated forms of LCA and CA
caused greater inhibition than their conjugated counterparts
(TLCA and TCA, respectively), possibly due to the greater
hydrophobicity of the unconjugated forms. DCA and TCA
(50 μM) had little effect on ISOC(not shown). In similar expe-
riments in which cells were incubated for 12 h with a choleretic
Fig. 3. The choleretic bile acid TDCA reversibly activates Ca2+entry, measured using fura-2, in H4-IIE liver cells and in rat hepatocytes. (A) TDCA induces an
increase in [Ca2+]cytin the presence of 2.4 mM Caext
presence of TDCA. A second addition of TDCA increases [Ca2+]cytin the presence of 10 mM [Ca2+]ext. This increase in [Ca2+]cytis abolished when TDCA is removed
from the extracellular medium (in the presence of 10 mM Caext
2.4 mM Caext
reversibly activates Ca2+entry in rat hepatocytes. The measurement of [Ca2+]cytas a function of time in cells loaded with fura-2 was conducted as described in
Materials and methods. The additions to the bath are indicated by the horizontal bars. The results shown are representative of those obtained for one of 3 or more
experiments which each gave similar results.
2+. (B) The TDCA-induced increase in [Ca2+]cytat 10 mM Caext
2+, is abolished when [Ca2+]extis reduced to zero in the
2+). (C) The increase in [Ca2+]cytinduced by TDCA is inhibited by 10 μM Gd3+. (D) In the presence of
2+and 10 μM Gd3+(to inhibit Ca2+entry), TDCA induces a transient increase in [Ca2+]cytindicating Ca2+release from intracellular stores. (E) TDCA
Fig. 4. The effects of TDCA and thapsigargin are not additive. (A) Effect of addition of TDCA following thapsigargin. (B) Effect of addition of TDCA. (C) The
magnitude of the plateaus following TDCA or thapsigargin addition. [Ca2+]cytwas measured as a function of time in H4-IIE cells loaded with fura-2, as described in
Materials and methods. The additions to the bath are indicated by the horizontal bars. The results shown in (A) and (B) are representative of those obtained for one of 3
experiments which each gave similar results. The values in (C) are the means±SEM of 3 experiments (PN0.05).
878E.C. Aromataris et al. / Biochimica et Biophysica Acta 1783 (2008) 874–885
bile acid (50 μM of TDCA, TUDCA or UDCA), no inhibition
of the IP3-initiated current was observed (Fig. 6B).
Of all cholestatic bile salts tested, LCA (50 μM) had the
strongest inhibitory effect (~80%) on the amplitude of ISOC
activated by intracellular IP3. However, when ISOCwas acti-
vated by application of 300 μM of UDCA to the bath, LCA had
much weaker inhibitory effect, reducing the current by ~50%
(Fig. 6C). In cells treated with LCA (50 μM) the ISOCactivated
by UDCA was approximately twice as large (Pb0.001, 2-way
ANOVA) than that activated by IP3(Fig. 6C (LCA) cf Fig. 6A
(IP3)). These results indicate that the choleretic bile acid UDCA
can counteract the inhibitory effects of cholestatic bile acids on
Ca2+entry through SOCs.
To test the effects of cholestatic bile acids on the release of
Ca2+from intracellular stores and Ca2+entry monitored by
measuring changes in [Ca2+]cyt, experiments were conducted
with cells loaded with fura-2. Pre-incubation of H4-IIE cells
with LCA (50 μM) for 12 hgreatly reduced the ability of DBHQ
to induce the release of Ca2+from intracellular stores, and
inhibited Ca2+entry (Fig. 7A, B and C). When the bile acid was
added directly to the incubation medium immediately before
measuring changes in [Ca2+]cytthere was no effect on DBHQ-
induced Ca2+release or Ca2+entry (results not shown). Pre-
incubation for 12 h with CA (50 μM) also caused a decrease in
DBHQ-induced Ca2+release and Ca2+entry (Fig. 7A, B and C).
These results confirm, using fura-2, that cholestatic bile acids
inhibit Ca2+entry through SOCs, and indicate that pre-incu-
bation with LCA or CA leads to a release of Ca2+in intracellular
stores. When the choleretic bile acid TDCA was added to cells
pre-incubated for 12 h with the cholestatic bile acid LCA, a
substantial increase in [Ca2+]cytwas observed (Fig. 7D), sug-
gesting that TDCA can counteract the inhibition of Ca2+entry
caused by pre-incubation with a cholestatic bile acid.
In rat hepatocytes, cholestatic bile acids also inhibited Ca2+
entry when pre-incubated with the cells for 12 h. LCA and CA
(50 μM) each inhibited both the initial rate and plateau of the
increase in [Ca2+]cytinduced by the addition of 1.3 mM Caext
hepatocytes previously incubated in the absence of added Caext
and in the presence of DBHQ (results not shown). The initial
rates of Ca2+entry were 121±11, 21±3.3 and 33±7.2 nM/min
and the values of the plateau reached after Caext
120±18, 51±8.2 and 70±2.4 nM for control, LCA, and CA,
respectively (means±SEM for 3–4 experiments). (The degrees
of significance, determined using ANOVA followed by the
Bonferroni post hoc test, were Pb0.01 for comparison of the
initial rate for the control with each of the initial rates for LCA
and CA, and Pb0.05 for comparison of the value of the plateau
for the control with that of the plateau for LCA.) These de-
creases in the rate and plateau of Ca2+entry were associated
with a substantial reduction in the DBHQ-induced release of
Ca2+from intracellular stores (results not shown). The amount
of Ca2+released was 39±7, 3.9±1.2 and 13.8±6.9 nM for
control, LCA and CA, respectively.(The degrees of significance
(ANOVA followed by the Bonferroni post hoc test) were Pb
0.01 and Pb0.05 for comparison of the amount of Ca2+re-
leased from the control and that for LCA and CA, respectively.)
Fig. 5. TUDCA activates Ca2+entry associated with oscillations in [Ca2+]cytin
H4-IIE liver cells. [Ca2+]cytwas measured as a function of time in H4-IIE cells
loaded with fura-2, as described in Materials and methods. The additions to the
bath are indicated by the horizontal bars. The results shown are representative of
those obtained for one of 3 experiments which each gave similar results.
Fig. 6. Inhibition of ISOCafter pre-incubation of H4-IIE liver cells for 12 h with
cholestatic bile acids. (A, B) Plots of IP3-induced current as a function of time in
H4-IIE cells pre-incubated with a given bile acid. (C) Plots of UDCA-induced
current as a function of time in control cells and in cells pre-incubated with
cholestatic bile acid LCA. H4-IIE cells were pre-incubated for 12 h with a given
bile acid as described in Materials and methods. The amplitude of ISOCactivated
by intracellular perfusion with IP3(20 μM in the patch pipette) was measured at
−118 mV and plotted against time. The results are the means±SEM of the
number of experiments indicated.
879E.C. Aromataris et al. / Biochimica et Biophysica Acta 1783 (2008) 874–885
3.3. Effects of bile acids on the intracellular distribution of
Since the redistribution of STIM1 in the ER and the loca-
lisation of some STIM1 at the plasma membrane are associated
with the activation of SOCs in lymphocytes, mast cells, some
other cell types [43–49], and liver cells , the effects of bile
acids on STIM1 distribution in liver cells were investigated. In
H4-IIE cells transfected with DNA encoding GFP-STIM1 and
incubated in the absence of agonist (control cells), GFP fluo-
rescence was distributed throughout the ER as indicated by co-
localisation of GFP-STIM1 with the ER marker pDsRed2-ER
(Fig. 8A). (Fig. 8A shows a confocal image of a cell in a Z-plane
in the middle of the cell (the nucleus, which does not contain
ER or STIM1 is evident).) TDCA caused a redistribution of
GFP fluorescence to give a punctate pattern (Fig. 8B, TDCA)
similar to that caused by thapsigargin (Fig. 8B, Tg). (Fig. 8B
shows a confocal image of a cell in a Z-plane near the plasma
membrane (no nucleus visible) and hence shows STIM1 distri-
bution close to the plasma membrane.) When the distribution of
endogenous STIM1 was monitored by immunofluorescence, it
was also found that TDCA altered the distribution of STIM1 to
give more concentrated localisations and a less dispersed pat-
tern (Fig. 8C, TDCA). This was similar to that observed for the
effects of thapsigargin (Fig. 8C, Tg). The effect of TDCA on
STIM1 distribution was reversible (Fig. 8D).
Incubation of cells for 12 h with the cholestatic bile acid
LCA also caused a redistribution of ectopically expressed GFP-
STIM1 similar to that caused by thapsigargin (Fig. 8B, LCA).
Incubation of cells with LCA (50 μM) for 5 min did not affect
the distribution of STIM1, as monitored by GFP fluorescence in
cells ectopically expressing GFP-STIM1 (results not shown).
To test the possibility that TDCA causes a re-organisation
of the ER , cells were transfected with DNA encoding
pDsRed2-ER. Neither TDCA (300 μM incubated for 15 min at
10 mM Caext
the absence of added Caext
structure of the ER (results not shown). This indicates that the
observed redistribution of STIM1 induced by TDCA and thap-
sigargin is unlikely to be due to a substantial rearrangement of
ER structure. Pre-incubation of cells for 12 h with the cho-
lestatic bile acid LCA (50 μM) also did not cause any detectable
change in the structure of the ER (results not shown).
We have shown previously that knockdown of STIM1 in
H4-IIE cells using siRNA inhibits development of the ISOC
in response to IP3or thapsigargin . Here we investigated
2+) nor thapsigargin (1 μM incubated for 10 min in
2+) caused any detectable change in the
Fig. 7. Inhibition of DBHQ-stimulated Ca2+entry after pre-incubation of H4-IIE liver cells for 12 h with the cholestatic bile acids LCA and CA. (A) Fura-2 loaded
cells were exposed to the SERCA inhibitor DBHQ (10 μM) in the absence of Caext
[Ca2+]cytwas reduced in cells which were pre-treated for 12 h with 50 μM LCA or CA. (B) Amounts of Ca2+released (peak height) by DBHQ in the presence and
absence of LCA or CA. (C) Initial rates of Ca2+entry and peak (plateau) values of [Ca2+]cytfollowing Caext
methods. (D) TDCA induces an increase in [Ca2+]cytin cells pre-incubated for 12 h with the cholestatic bile acid LCA. [Ca2+]cytwas measured as a function of time in
cells loaded with fura-2, as described in Materials and methods. (A, D) The additions to the bath are indicated by the horizontal bar. The results are representative
of those obtained for one of 3 experiments which each gave similar results. (B, C) The values are the means±SEM of 6–7 experiments similar to the one shown in
panel A. The degrees of significance between LCA and the control, and between CA and the control, determined using ANOVA followed by Bonferroni post hoc
test, are⁎Pb0.05 and⁎⁎Pb0.01.
2+. When Ca2+was added back to the bath the typical DBHQ-dependent increase in
2+addition, determined as described in Materials and
880 E.C. Aromataris et al. / Biochimica et Biophysica Acta 1783 (2008) 874–885
whether STIM1 is required for activation of ISOCby choleretic
bile salts. At 72 h post transfection with siRNA against STIM1
the amplitude of ISOCactivated by 300 μM UDCAwas reduced
by ~80% (Fig. 9A) and the initial rate of Ca2+entry measured
using fura-2 was inhibited by 50% (Fig. 9B). These results
indicate that STIM1 is required for the activation of Ca2+entry
by choleretic bile acids.
In this study with H4-IIE liver cells and rat hepatocytes we
have shown that TDCA and other choleretic bile acids, when
added directly to the bath, activate Ca2+entry through the SOCs
and cause a redistribution of STIM1 similar to the redistribution
caused by thapsigargin. By contrast, LCA and other cholestatic
bile acids, when pre-incubated with the cells for 12 h, inhibit
Ca2+entry through SOCs.
Evidence that the Ca2+entry pathway activated by choleretic
bile acids is the liver cell SOC is provided by the similarities
between the current induced by choleretic bile acids and the
current induced by IP3[34,36]. These include the Ca2+selec-
tivity, the time course of activation, the effects of substituting
Ba2+for Ca2+,activation and inhibition by 2-APB, inhibition by
La3+and Gd3+; and the observations that in the presence of IP3,
UDCA does not activate additional current and, in fura-2 loaded
cells incubated in the presence of thapsigargin, TDCA does not
further increase [Ca2+]cyt. Washout patch clamp and fura-2
experiments indicate that the ability of choleretic bile acid to
activate Ca2+entry is reversible. Several previous studies have
shown that exposure of liver cells to choleretic bile acids in-
duces an increase in [Ca2+]cytwhich subsequently affects di-
verse cellular process [7,26,57]. However the site(s) of action of
the choleretic bile acids on cellular Ca2+movements was not
clearly defined. Our results identify SOCs as a target for cho-
leretic bile acids in liver cells. While TDCA induced a sustained
increase in [Ca2+]cyt, TUDCA induced oscillations in [Ca2+]cyt.
These differences may relate to the degree of involvement of
ryanodine receptors in the bile acid Ca2+response .
Fig. 8. Incubation of H4-IIE cells with choleretic TDCA or pre-incubation with cholestatic LCA causes a redistribution of STIM1. (A) Representative confocal images
in a Z-plane in the middle (equatorial plane) of a cell co-expressing GFP-STIM1 and the ER marker psDsRed2-ER, and the merged image showing co-localisation of
STIM1 and the ER. (B) Representative confocal images in a Z-plane close to the plasma membrane of GFP fluorescence in cells transfected with GFP-STIM1 and
incubated with 1 μM thapsigargin for 10 min in the absence of added Caext
12 h at 1.3 mM Caext
incubated in the absence of agonist (Control), or in the presence of 1 μM thapsigargin for 10 min in the absence of added Caext
at 10 mM Caext
STIM1 before addition of TDCA (Before), after TDCA addition (TDCA), and after washing out TDCA (After TDCAwashout). The results are representative of those
obtained for 18 cells in 4 separate experiments. Transfection of cells with DNA encoding GFP-STIM1, psDsRed2-ER, immunofluorescence, and confocal microscopy
were conducted as described in Materials and methods. The images shown are representative of those obtained from at least 2 coverslips for each of 3 independent
experiments conducted on different days. The scale bar represents 10 μm.
2+(Tg); with 300 μM TDCA for 15 min at 10 mM Caext
2+(TDCA); and with 50 μM LCA for
2+(LCA). (C) Representative confocal immunofluorescence images in a Z-plane close to the plasma membrane of endogenous STIM1 in cells
2+(Tg), or with 300 μM TDCA for 10 min
2+(TDCA). (D) Representative confocal images in a Z-plane close to the plasma membrane of GFP fluorescence in a single cell transfected with GFP-
881E.C. Aromataris et al. / Biochimica et Biophysica Acta 1783 (2008) 874–885
Results obtained with GFP-STIM1, immunofluorescence and
fluorescence microscopy indicate that the activation of Ca2+
entry through SOCs by TDCA is associated with a redistribution
of STIM1 similar to that caused by the SERCA inhibitor thap-
sigargin. The redistribution of STIM1 by TDCAwas reversible,
indicating that TDCA does not act by inducing the covalent
modification of a protein, or as a tight-binding inhibitor, and is
unlikely to cause non-specific damage to the plasma membrane
or to intracellular membranes. Since TDCA caused a redistribu-
tion of STIM1 similar to that caused by thapsigargin, it is consi-
dered most likely that the mechanism by which TDCA activates
SOCs involves the release of Ca2+from ER, the movement and
relocalisation of STIM1, and the activation of SOCs by STIM1
through the interaction of STIM1 with a member of the Orai
family (reviewed in ) (shown schematically in Fig. 10A).
The pre-incubation of liver cells with a cholestatic bile acid
resulted in inhibition of Ca2+entry through SOCs and no de-
tectable release of Ca2+from intracellular stores when DBHQ
was subsequently added.The latter result suggeststhat eitherthe
intracellular Ca2+stores were depleted of Ca2+before DBHQ
addition, or that the interaction of cholestatic bile acids with the
ER leads to disruption of the action of DBHQ on SERCAs.
While the amount of Ca2+in intracellular stores was not directly
measured, it is considered that the most likelyexplanation is that
stores. This is consistent with previous findings that cholestatic
Since the release of Ca2+from the ER and a redistribution of
bile acids leads to depletion of the ER Ca2+stores and re-
distribution of STIM1, SOCs would be constitutively activated
(i.e. active in the absence of IP3or SERCA inhibitor). Hence, it
is, perhaps, surprising that the release of Ca2+from intracellular
stores and redistribution of STIM1 induced by pre-incubation
with cholestatic bile acids are associated with inhibition of Ca2+
Fig. 9. Knockdown of STIM1 expression using siRNA causes a substantial
reduction in UDCA-induced Ca2+entry. (A) Inward currents measured at
−118 mVin the presence of UDCA (indicated by the horizontal bar) plotted as a
function of time for cells treated with siRNA directed againstSTIM1 and control
siRNA. The results are the means±SEM of the number of experiments indi-
cated. (B) Initial rates of Ca2+entry measured in cells loaded with fura-2 using
the Ca2+add-back assay. The results in (B) are the means±SEM of 3 experi-
ments. The degree of significance, determined using Student's t-test for un-
paired samples is⁎Pb0.05.
Fig.10. Aschematicrepresentationof the proposedmechanisms bywhichcholereticTDCAactivatesSOCs(A) andcholestaticLCA inhibitsSOCs(B) inhepatocytes.
Activation (A) and inhibition (I) are indicated by the broken lines. (A) It is proposed that TDCA induces the release of Ca2+from the ER by enhancing Ca2+outflow
[26,28,29] or by inhibiting SERCA [59,60]. This, in turn, leads to the movement of STIM1 to the junctional ER close to the plasma membrane and the subsequent
activation of SOCs. (B) It is proposed that pre-incubation with LCA causes a sustained release of Ca2+from the ER, which in turn leads to relocalisation of STIM1, an
alteration in the fine structure of the junctional ER and/or inhibition of movement of Orai1 in the plasma membrane so there is no activation of SOCs.
882E.C. Aromataris et al. / Biochimica et Biophysica Acta 1783 (2008) 874–885
entry. Moreover, this inhibition could be partially counteracted
by a choleretic bile acid. It is clear that this can only occur if the
effects of bile acids are not restricted to the depletion of Ca2+
stores alone. There must be some other component(s) of store-
operated Ca2+entry that is affected by cholestatic and choleretic
bile acids in opposite ways. One possible explanation is that
cholestatic bile acids inhibit, while choleretic bile acids faci-
litate, a step in the pathway of activation of SOCs downstream
from the release of Ca2+from the ER and redistribution of
STIM1 . Cholestatic and choleretic bile acids might have
differential effects on the movement of Orai1 in the plasma
membrane towards junctional ER sites, the interaction between
STIM1 and Orai1, or the open probability of Orai1. It has been
shown previously that impairment of hepatobiliary exocytosis
and bile flow caused by TLCA is associated with activation of
phosphatidylinositol 3-kinase and the translocation of protein
kinase Cɛ to the plasma membrane . At the same time it was
also shown that TUDCA inhibits the activity of phosphatidy-
linositol 3-kinase-dependant protein kinase B and membrane
bindingofprotein kinase Cɛ,thus reversing theeffects of TLCA
. It is possible that differential regulation of protein kinase
Cɛ and phosphatidylinositol 3-kinase by TUDCA and TLCA
underlies their opposite effects on Ca2+entry through SOCs.
action of the cholestatic bile acid LCA are shown schematically
in Fig. 10B.
The results reported here may have implications for
understanding the progression of cholestasis and subsequent
damage to hepatocytes, and for the treatment of cholestasis.
Previous studies have provided some evidence that Ca2+entry
through SOCs is required for normal bile flow, most likely by
contributing to the increase in [Ca2+]cytwhich initiates con-
traction of the bile canaliculus . The inhibition of Ca2+
entry through SOCs by cholestatic bile acids would lead to
the disruption of intracellular Ca2+homeostasis and down-
stream Ca2+signaling pathways. This may contribute to the
further inhibition of bile flow, and altered hepatocyte growth,
apoptosis and necrosis which are consequences of cholestasis
The choleretic bile acids UDCA and TUDCA are used phar-
macologically to enhance bile flow in cholestatic conditions
primary biliary cirrhosis [18,19] and primary sclerosing cho-
langitis . Moreover, it has been shown in rat models of
cholestasis, that treatment with choleretic bile acids can stimu-
late bile flow . The present results, showing that choleretic
bile acids activate Ca2+entry through SOCs, and can activate
Ca2+entry in cells previously treated for 12 h with a cholestatic
bile acid, may provide a mechanism by which choleretic bile
acids exert beneficial effects on cholestasis.
It is concluded that SOCs and STIM1 are important targets
for the actions of bile acids on hepatocytes. Inhibition of Ca2+
entry through SOCs by cholestatic bile acids may be responsible
for enhancing the cholestatic condition, and activation of Ca2+
entry through SOCs may contribute to explaining the beneficial
pharmacological effects of choleretic bile acids.
We would like to thank Rachael Hughes and Yabin Zhou for
preparing isolated rat hepatocytes, Dr T Kurosaki, RIKEN
Research Centre for Allergy and Immunology, Kanagwa, Japan,
Bethesda for advice on cell transfections, and Dr Alan F Hof-
mann, University of California, for discussions on bile acid
solubility. This work was supported by the Australian Research
Council and the National Health and Medical Research Council
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