From the Departments of Medicine, Physiology and
Human Genetics, McGill University and Royal Victoria Hospital,
Montreal, Quebec H3A 1A1, the ¶ Department of Pharmacology,
Faculty of Medicine, Université de Montréal and Centre de
Recherche, Centre Hospitalier de l'Université de Montréal,
Montréal, Québec H2X 1P1, and the
Department of
Medicine, University of Western Ontario and the Lawson Research
Institute, London, Ontario N6A 4V2, Canada
Received for publication, October 12, 2000
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ABSTRACT |
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Liver cells respond to changes in
Ca2+o. The hepatic functions affected include
bile secretion, metabolic activity, liver regeneration, and the
response to xenobiotics. In the present study, we demonstrate the
presence, in the liver, of the extracellular calcium-sensing receptor
(CASR), described previously in the parathyroid and thyroid glands and
kidney. CASR mRNA was specifically expressed in hepatocytes and was
absent in nonparenchymal liver cells (stellate, endothelial, and
Kupffer cells). Western blot analysis using a specific CASR antibody
showed staining in both whole liver and hepatocyte extracts.
Immunohistochemistry and in situ hybridization of rat liver
sections showed expression of CASR protein and mRNA by a subset of
hepatocytes. The known agonists of the CASR, gadolinium (Gd3+; 0.5-3.0 mM) and spermine (1.25-20
mM), in the absence of Ca2+o, elicited
dose-related increases in Ca2+i in isolated rat
hepatocytes loaded with Fura-2/acetoxymethyl ester. There was a
greatly attenuated response to a second challenge with either agonist.
The response was also abrogated when inositol 1,4,5-trisphosphate
(IP3)-sensitive calcium pools had been depleted by
pretreatment with either thapsigargin or phenylephrine, an A calcium-sensing receptor
(CASR)1 expressed on the cell
surface of parathyroid, thyroid (calcitonin-secreting C cells), and kidney tubule cells senses changes in the extracellular calcium level
([Ca2+]o) within the normal circulating range
in vivo (1). The modulation of cellular signaling pathways
thereby brought about affects hormone secretion and renal electrolyte
handling. The CASR plays a critical role in coordinating hormonally
regulated systemic calcium homeostasis, and this is emphasized by the
fact that naturally occurring loss or gain of function mutations in the
CASR gene on human chromosome 3q13-21 (2) cause inherited hypercalcemic or hypocalcemic disorders, respectively (3-8). In
addition, there is an association between serum calcium concentrations in the normal Caucasian population and a common polymorphism in the
C-terminal tail of the CASR (9). The CASR is a G protein-coupled receptor, and its activation can lead to cell signaling via more than
one pathway. Coupling to a Gq/11 type protein activates
phospholipase C effecting membrane phosphatidyl inositol bisphosphate
turnover with production of inositol 1,4,5-trisphosphate
(IP3) and subsequent mobilization of Ca2+ from
intracellular stores, as well as production of diacylglycerol and
activation of protein kinase C (10, 11). Activation of the CASR leads
to reduced production of intracellular cAMP in parathyroid cells (12)
and cells of the nephron, for example, distal convoluted tubule cells
(13). This might occur via coupling to the pertussis toxin-sensitive
Gi-protein, which inhibits the adenylyl cyclase enzyme, or
via an indirect mechanism involving arachidonic acid metabolites
(14).
With respect to ligand specificity the CASR is promiscuous, being
responsive not only to divalent cations Ca2+ and
Mg2+ but also trivalent cations such as the transition
metal, gadolinium (Gd3+) (15) and polycationic molecules
like spermine (16). It has been proposed that the CASR could function
as the physiological receptor for Mg2+ and polycations,
such as spermine, at certain sites in the body. Changes in ionic
strength are also able to modulate CASR activation (17). In addition,
certain phenylalkylamine compounds and their deschloro derivatives
selectively potentiate the responsiveness of the CASR to its ligands by
acting as positive allosteric modulators (18). Such compounds, so
called calcimimetics, if of sufficient selectivity and potency, have
enormous potential for the pharmacological modulation of PTH secretion
and the medical management of hyperparathyroid states (19-21).
Although the CASR is highly expressed in tissues important for
regulating the [Ca2+]o, including parathyroid,
thyroid, and kidney, it is also found at lower levels in many other
tissues not known to play a role in calcium homeostasis (22). These
latter tissues include regions of the brain such as hippocampus and
pituitary, lung, and keratinocytes. For many of these, the
physiological role of the CASR is not yet clear, and it may be that the
CASR senses endogenous ligands other than Ca2+ at these
particular sites.
With respect to the gastrointestinal tract and associated organs, the
CASR is expressed in cells within the small and large intestines (23,
24), gastrin-secreting cells of the gastric antrum (25), different
epithelial cells of gastric mucosa and enteric nerve regions (26), and
acinar cells and interlobular ducts of the pancreas (27). However, CASR
expression has not been studied in the liver. With respect to calcium
homeostasis, the liver plays an important role in the uptake and
metabolism of intact PTH, and this occurs specifically in Kupffer cells
(28). PTH binding occurs on hepatocytes and sinusoidal cells and is linked in the former cells to adenylyl cyclase (29, 30). At one time
the hepatic metabolism of PTH was suggested to be
[Ca2+]o-regulated, although this is not now
thought to be the case (31). The liver is also the site of the
C-25-hydroxylation of vitamin D, which is a prerequisite step for the
final production of the hormonally active, 1- However, liver cells do respond in a variety of ways to changes in
[Ca2+]o, and certain liver functions may be
compromised when serum calcium levels rise or fall above or below the
normal range. These functions include bile secretion, metabolic
activity, liver regeneration, and resistance to xenobiotics. In the
present study, we demonstrate the presence of the CASR in the liver.
Its expression is diffuse throughout the hepatic acinus, the
functionally active receptor mobilizes [Ca2+]i
from IP3-sensitive pools, and CASR agonists stimulate bile
flow in the perfused rat liver.
Tissues--
Protocols for obtaining rat tissues were approved
by local animal ethics committees. Tissues for RNA and Western blot
analysis were flash frozen and stored at Reverse Transcription-Polymerase Chain Reaction Analysis of the
CASR--
Total RNA was extracted from tissues that were homogenized
using a Polytron and from cells using TRIzol (Life Technologies, Inc.).
RT-PCR was carried out as follows. First-strand cDNA was made by
reverse transcribing 3 µg of DNase-I- treated total RNA with a
recombinant superscript II RNase H (Life Technologies, Inc.) using
oligo(dT)15-18 (Amersham Pharmacia Biotech) in a total
volume of 20 µl as described (33). Five microliters of the RT
reaction were electrophoresed in ethidium bromide-stained 1%
agarose gels to check for quantity and quality of the cDNAs. Five
microliters were used for PCR amplification with a GeneAmp PCR System
thermocycler model 9600 (PerkinElmer Life Sciences). Portions of the
CASR cDNA were amplified using the primer pairs shown in Table I.
The PCR mixture contained 2.25 mM MgCl2, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.5 mM of each dNTP, 300 nM of forward and reverse
primers, and 2.5 units of Taq polymerase (Life Technologies,
Inc.) in 50 µl. The PCR amplification consisted of 30 (unless
otherwise stated) cycles of denaturation of 94 °C for 40 s,
annealing at 58 °C for 35 s, and polymerization at 72 °C for
1 min 30 s. Aliquots (10 µl) of the PCR reactions were electrophoresed through ethidium bromide-stained 1% agarose gels.
Nucleotide Sequence Analysis--
Gel purified PCR-amplified
CASR products were subcloned into the pCRII TA cloning vector
(Invitrogen, San Diego, CA). Semi-automated sequence analysis was
performed on three independent clones of each product using either Alf
Express (Amersham Pharmacia Biotech) or ABI-373 (Applied Biosystems
Inc.) sequencers located at the Sheldon Biotechnology Center of McGill University.
Western Blot Analysis of the CASR--
Tissues and cells were
lysed in triple detergent buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.02% NaN3, 0.1% SDS, 1 mM EDTA, 100 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotonin, 0.1% Nonidet P-40, 0.5%
sodium deoxycholate) for 5 min at 0 °C. The cell lysates were spun
at 1200 × g for 2 min at 4 °C, and the supernatants
were stored at Immunohistochemistry of the CASR--
12-week-old female Harlan
Sprague-Dawley rats (Charles River, St. Constance, Canada) were
sacrificed by CO2 inhalation, and tissues were removed and
fixed in 4% paraformaldehyde, 0.2% glutaraldehyde overnight at
4 °C. Specimens were dehydrated with an increasing concentration of
ethanol before paraffin embedding. 5-µm sections were mounted on
Superfrost slides (Fisher, Nepean, Canada). Sections were post-fixed in
4% paraformaldehyde for 15 min, followed by a 10-min incubation in 10 µg/ml proteinase K (Life Technologies, Inc.) at room temperature to
retrieve fixation-concealed antigens. After stabilization in 4%
paraformaldehyde for 15 min, sections were incubated with the CASR
monoclonal antibody (described above under Western blotting) at a
dilution of 1:750 in 1% bovine serum albumin in phosphate-buffered
saline overnight at 4 °C. After rinsing with phosphate-buffered
saline, staining was done with the Vectastain ABC kit (Vector Labs,
Burlington, Canada) according to the manufacturer's directions. As a
control, some sections were treated with primary antibody that had been
preadsorbed overnight at 4 °C with the peptide (10 µg/ml) against
which it had been raised.
In Situ Hybridization--
In situ hybridization was
carried out using biotin-labeled sense and antisense riboprobes and the
Genpoint CSA kit (Dako Diagnostics, Mississauga, Canada). Labeled
riboprobes were prepared from a plasmid containing a 509-bp insert
corresponding to the rat CASR (13) using T3 and T7 RNA polymerases
(Life Technologies, Inc.) and a nucleotide labeling mix containing
biotin-16-UTP (Roche Molecular Biochemicals). Tissue sections were
deparaffinized and rehydrated in a standard xylene and alcohol series
and in situ hybridization was performed according to the
manufacturer's directions. Briefly, slides were heated in target
retrieval solution and proteinase K to reveal hidden antigens.
Endogenous peroxidase activity was quenched in 0.3% hydrogen peroxide
in methanol. Sections were hybridized for 2 h at 50 °C with 5 ng/ml biotin-labeled riboprobe in the supplied RNA hybridization
buffer. After stringent washing at 55 °C, sections were treated to
successive incubations in primary streptavidin-horseradish peroxidase,
biotinyl-tyramide solution, and secondary streptavidin-horseradish
peroxidase (Dako) for 15 min each at room temperature. Color was
developed with the supplied diaminobenzidine diluted as directed
(DAKO), and sections were counterstained with Carazzi's hematoxylin
prior to dehydration and mounting.
Isolation of Nonparenchymal Liver Cells--
Sinusoidal cells
were isolated from livers of nonfasting rats by the method of Knook and
Sleyster (35) with the following modifications. After Nicodenz
density gradient, cells were washed in Gey's balanced salt solution
(GBSS), pH 7.4, at 4 °C, resuspended, and introduced in a
type J2-21M centrifuge (Beckman Instruments, Palo Alto, CA) equipped
with a JE-6B elutriation rotor and a Sanderson chamber. While being
centrifuged at 2500 rpm, cells were washed out at pump flows of 13, 23, and 42 ml/min to collect stellate, endothelial, and Kupffer cells
respectively using GBSS, pH 7.4 at 4 °C. Stellate, endothelial, and
Kupffer cells were centrifuged and counted, and viability was
evaluated. Sinusoidal cells had a viability greater than 95% and were
free of hepatocytes.
Isolation and Primary Culture of Hepatocytes--
Hepatocytes
were obtained from livers of nonfasting rats as described previously
(36). The freshly isolated cells were suspended in William E medium
containing 1% albumin, 0.5 mM glucose, sequentially filtered through 250- and 74-µm filters, and centrifuged at 250 rpm
for 3 min. The freshly isolated hepatocytes were then equilibrated in
William E medium (Life Technologies, Inc.). Cell viability was
evaluated by Trypan blue exclusion, and those cells showing greater
than 85% were taken for further study.
Rat Bile Duct and Mouse Biliary Cell Line--
Rat whole
bile duct was harvested. The mouse biliary cell line (BDC) was kindly
provided by Dr. Emile Levy (Hôpital Ste-Justine, Montreal,
Canada) and was cultured as described previously (37).
Intracellular Calcium Measurements--
Hepatocytes were
plated at a density of 3.5-5 × 105 cells/ml onto
collagen-coated coverslips in Williams E medium containing 25 mM bicarbonate, 1% bovine serum albumin, at pH 7.4, 37 °C in a 5% CO2 atmosphere. After incubation for 60 min cells were loaded for 30 min at 20 °C with the fluorescent probe
Fura-2/AM (2 µM) (Molecular Probes Inc., Eugene, OR) in
bicarbonate-free Williams E medium supplemented with 0.25% fetal
bovine serum and 1% bovine serum albumin. Dye-loaded cells were then
transferred in a 100-nl plastic chamber to the stage of an inverted
microscope (Diaphot, Nikon Corp., Tokyo, Japan) equipped for
epifluorescence measurement. Cells were superfused at a rate of 3.6 ml/min, pH 7.4, at 32 °C in a 10 mM Krebs buffer
containing 120 mM NaCl, 4.8 mM KCl, 5.5 mM glucose, 1.2 mM MgCl2, 0.2 mM EDTA for the gadolinium (0.5-3 mM) studies,
and 10 mM Hepes buffer containing 120 mM NaCl,
4.8 mM KCl, 5.5 mM glucose, 1.2 mM
MgSO4 0.2 mM EDTA for the spermine (1-20
mM) studies. Phenylephrine and thapsigargin were used at a
concentration of 5 nM.
Calcium measurements were made as described previously (38). Briefly,
fluorescence signals from single hepatocytes were obtained with an MCID
dual excitation spectrofluorometer system (Imaging Research Inc., St.
Catharines, Canada). Excitation wavelengths were 340 and 380 nm, and
fluorescence emission was measured at 505 nm. A refrigerated camera
(Hamamatsu Photonics C4880, Hamamatsu City, Japan) was used as the
imaging device. Output from the fluorometer was digitized, ratioed, and
computer analyzed. Intracellular dye calibration was performed
in situ by perfusion of 10 µM ionomycin in a
solution containing 4 mM EGTA (Rmin)
or 4 mM CaCl2 (Rmax). After correction for sample autofluorescence, signal ratios
(F340/F380) were
transformed into [Ca2+]i as previously reported
(39). The presence of nonhydrolyzable dye was periodically verified by
quenching with 2 mM MnCl2 and found to be
negligible when compared with autofluorescence.
Measurement of Bile Flow in Isolated Perfused Rat Liver--
To
investigate the involvement of the CASR in bile secretion, bile flow
was monitored in the isolated-perfused rat liver either (1) following a
period of cholestasis induced by calcium deprivation in
vitro or (2) under conditions in which a submaximal flow was
maintained, and the effects of additional Ca2+,
Gd3+ or NPS 467 compounds were tested. Rats were
anesthetized with forane, and the liver was perfused in situ
as described previously (40) using a perfusion apparatus (MX Ambec
Perfuser, MX International Inc., Aurora, CO). Briefly, the common bile
duct was cannulated using PE-10 tubing (Clay-Adams, Parsipanny, NJ),
and the bile was collected throughout the experiment. The perfusion
medium consisted of a 10 mM Hepes buffer, pH 7.4, saturated
with a mixture of O2/CO2 (95:5, v/v) that
contained 120 mM NaCl, 4.8 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4
H2O, 21.4 µM taurocholic acid, and 100 mg/100
ml glucose. Experiment 1 was done in Ca2+-free perfusate or
in perfusate containing 1.25 mM Ca2+ and 0, 1.25 or 2.5 mM spermine. Experiment 2 was done in perfusate containing 0.5 mM Ca2+ (base-line conditions)
throughout or at 20 min changed to 1.25 mM
Ca2+, 100 µM Gd3+, 0.5 mM Ca2+ + 1 µM NPS R-467, or 0.5 mM Ca2+ + 1 µM NPS S-467 (0.5 mM MgSO4 was used throughout). The liver remained in situ throughout the experiments, and its
temperature was maintained at 37 °C. The perfusate was recirculated
with a Masterflex pump (Cole Palmer Instrument Co., Chicago, IL)
through a stainless steel filter. A pressure regulator was used to damp down pulsatile flow. The perfusion rate through the liver was set at 30 ml/min. In each preparation, perfusate Ca2+ measurements
were done using a ICA2 ionized calcium analyzer (Radiometer,
Copenhagen, Denmark).
Statistical Analysis--
Data are presented as the means ± S.E. Significant differences between grouped means were
determined by Tukey's analysis of variance or by Student's
t test (41) where appropriate.
The CASR Gene Is Expressed in Liver--
Expression of CASR
mRNA was examined using semi-quantitative RT-PCR. A cDNA
fragment of 367 bp corresponding to the CASR mRNA sequence encoded
by part of exons 2 and 3 was amplified
using forward primer r2BF and r3AR (Fig. 1 and Table
I). Aliquots of the PCR reaction were
taken after 21, 24, 28, and 32 cycles for CASR and 18, 21, 24, and 28 cycles for GAPDH and electrophoresed through ethidium bromide-stained
gels. Fig. 2 shows that a low level of
CASR mRNA expression was observed in total liver and hepatocytes
compared with the parathyroid, thyroid, and kidney. The series of PCR
products 1, 2, 4, and 5, which together comprise the entire coding
region of rat CASR mRNA, were amplified from rat liver cDNA,
subcloned into plasmid vectors, and sequenced. The derived sequence was
identical to that of rat kidney (42) and brain (43) CASR cDNA.
Liver CASR mRNA Is in Hepatocytes but Not in Stellate,
Endothelial, and Kupffer Cells--
RNA extracted from rat liver
isolated stellate, endothelial, Kupffer, and hepatocyte cell
populations was subjected to RT-PCR using primer set r2AF and r4AR
(Fig. 1 and Table I), generating a 1318-bp fragment. Fig.
3 shows that hepatocytes were positive for CASR mRNA expression, whereas all other cell types were
negative.
Western Blot Analysis of the Liver CASR--
Mouse cell or rat
tissue extracts (10 µg of protein each) were subjected to
SDS-polyacrylamide gel electrophoresis on a 4-12% gradient gel. The
blot shown in Fig. 4 was stained with
CASR mouse monoclonal antibody (ADD, raised against a peptide
comprising CASR residues 214-236). The predominant species present in
rat kidney and parathyroid are the nonglycosylated and glycosylated forms ranging from 120 to 160 kDa, with some higher molecular weight
aggregates, which the rat liver lacks. A similar positive result was
also obtained by Western blot analysis of an extract of isolated rat
hepatocytes (data not shown). Extracts of mouse NIH3T3 fibroblasts were
negative for specific CASR staining (Fig. 4), as was a blot of liver
extract probed with the CASR antibody after it had been preadsorbed
with the peptide against which it had been raised (data not shown).
Immunohistochemistry of the CASR--
Strong immunostaining was
observed on the chief cells of the parathyroid gland and parafollicular
cells (C cells), but not the follicular cells, of the thyroid (Fig.
5A). In the kidney cortex,
whereas no staining of the glomeruli was apparent, staining of the
proximal and distal convoluted tubules was observed (Fig. 5C). In these controls, the pattern of staining by this
particular CASR antibody was similar to that reported previously using
different antibodies (44, 45). In the liver, although not all
hepatocytes were found to be positive for CASR, immunostaining was
observed throughout the hepatic acinus (Fig. 5E). Clear
immunostaining was present in both the perivenous and periportal areas
such as the hepatocytes neighboring or radiating from the central vein and those surrounding the portal triad (Fig. 5, E and
G). Lack of specific staining was demonstrated in control
sections treated with antibody that had been preadsorbed with CASR
peptide (Fig. 5, B, D, and H).
In Situ Hybridization of CASR mRNA--
The expression of CASR
transcripts was analyzed by in situ hybridization using a
biotin-labeled antisense probe. There was strong staining in endocrine
cells of the parathyroid gland and the C cells of the thyroid (Fig.
6A). The specificity of the
signal was demonstrated by lack of staining with the sense control
probe (Fig. 6B). Specific staining was observed in
hepatocytes (Fig. 6C) especially in the cells surrounding
the central and portal veins (Figs. 6, C and
E).
Bile Duct Cells Do Not Express the CASR--
RNA extracted from
dissected rat bile duct and a mouse bile duct cell line (BDC) was
subjected to RT-PCR using primer set r2AF and r4AR (Fig. 1 and Table
I). The control hepatocyte RNA was positive for the 1318-bp product,
but the bile duct cell and bile duct cell line RNAs were negative (Fig.
7a). Although clear immunostaining of hepatocytes was observed, bile duct cells (Fig. 7b) were negative.
Response of Isolated Hepatocytes to Gd3+--
To test
whether hepatocytes express a functional CASR, Fura-2-loaded
hepatocytes were stimulated with CASR agonists.
[Gd3+]o acts like [Ca2+]i
on PTH release, cyclic AMP levels, and IP3 production (46)
as well as [Ca2+]i (20) in dispersed parathyroid
cells, although at a much higher potency. It was also used, rather than
[Ca2+]o itself (which would have additionally
activated Ca2+ channels), to monitor the isolation of the
parathyroid CASR by expression cloning in Xenopus oocytes
(15). In the present study we utilized [Gd3+]o as
a well characterized ligand of the CASR. In Fura-2-loaded hepatocytes
bathed in a [Ca2+]-free medium, in response to 2 mM Gd3+, the [Ca2+]i was
increased and then decreased to a level about half-maximal at the end
of the 200-s period and thereafter to a basal level (Fig.
8, inset). This response is
not dissimilar to that observed in a subset of isolated rat pancreatic
acinar cells (27), although in that case there was a longer delay
before the increase in [Ca2+]i. In the
hepatocytes there was a linear increase in [Ca2+]i from 1 to 3 mM
Gd3+ (Fig. 8). A concentration of 500 µM
Gd3+ was without effect in stimulating
[Ca2+]i. A similar lack of
[Ca2+]i response to this Gd3+
concentration was noted in studies of pancreatic acinar cells (27).
Response of Isolated Hepatocytes to Spermine--
Polyamines such
as spermine can stimulate [Ca2+]i in HEK293 cells
expressing the CASR (16), and it is suggested that tissues such as
brain and intestine could use the CASR as a target for spermine and
other endogenous polycations. Increased extracellular concentrations of
spermine (1.25-10 mM) caused increased
[Ca2+]i mobilization in a dose-responsive manner
in Fura-2/AM-loaded hepatocytes (Fig. 9).
The effect plateaued at the 10 mM concentration with the
[Ca2+]i response to 20 mM spermine
being no different (Fig. 9).
Effect of Prior Emptying of IP3-sensitive
Ca2+ Pools on [Ca2+]i Mobilization by
Spermine--
When Fura-2-loaded hepatocytes, bathed in a
[Ca2+]o free medium, had their
IP3-sensitive Ca2+i pools depleted in
two different ways, there was no [Ca2+]i
transient in response to a subsequent challenge with 2.5 mM
spermine (Fig. 10). This was the case
whether the Ca2+i pool was depleted by
phenylephrine, an Calcimimetic NPS R-467 Enhances the [Ca2+]i
Response to Spermine in Hepatocytes--
Phenylalkylamine compounds,
NPS R-467 and NPS S-467, act as specific allosteric activators of the
CASR (21). They do not activate other G protein-coupled receptors,
including the related metabotropic glutamate receptors when tested at
the same concentrations. The phenylalkylamine compounds act in a
stereoselective manner with the R enantiomer being at least
10-fold more potent than the S enantiomer (21). In
Fura-2-loaded hepatocytes bathed in a
[Ca2+]o-free medium, addition of 1.25 mM spermine plus 10 µM NPS S-467 caused no
greater stimulation of [Ca2+]i than addition of
1.25 mM spermine alone (Fig.
11). However, addition of 1.25 mM spermine plus 10 µM NPS R-467 caused a
quadrupling of the [Ca2+]i response (Fig. 11).
This provided good evidence that the hepatocyte polyvalent cation
sensing receptor is, indeed, the CASR. It can be noted that the effect
of these agents is complex in that both compounds appear to induce a
delay in the response to spermine compared with the control. The
underlying mechanism for this is unknown.
Spermine Enhances Bile Flow Recovery in Isolated Perfused
Liver--
Bile flow remained constant throughout the 60-min
experimental period when normal [Ca2+]o was
present in the perfusate. Bile flow rapidly stopped, however, when the
liver was perfused in Ca2+-free medium despite the
continuous presence of taurocholic acid to ensure normal bile
acid-dependent bile flow. Normalization of
[Ca2+]o rapidly restored bile flow with, however,
a significantly more rapid bile flow recovery in the presence of the
CASR agonist spermine (Fig. 12). This
effect was dose-related, being significantly greater at 2.5 mM relative to 1.25 mM spermine (Fig. 12,
inset). The effectiveness of spermine in the presence of
normal [Ca2+]o may relate to cooperativity
between the two ions for activation of the CASR as noted previously
(16).
CASR Agonists Ca2+ and Gd3+ Stimulate and
NPS R-467 Selectively Enhances Bile Flow in Isolated Perfused
Liver--
To further assess the effect of CASR agonists on bile flow,
isolated livers were perfused with a 0.5 mM
Ca2+-containing medium. For the base-line condition, the
perfusate was changed at 20 min for one containing 0.5 mM
Ca2+ again. Note that this caused a transient increase in
bile flow because of the re-equilibration of the taurocholic acid (Fig. 13). Changing to a medium containing
1.25 mM Ca2+ (Fig. 13A) or 0.5 mM Ca2+ + 100 µM Gd3+
(Fig. 13B) provoked a significantly increased bile flow
relative to base line. Maintaining the perfusate at 0.5 mM
Ca2+ but adding 1 µM NPS R-467 caused a
marked stimulation of bile flow (Fig. 13C), whereas addition
of 1 µM NPS S-467 produced an effect little different
from base line (Fig. 13D). This provides strong evidence
that activation of the CASR is responsible for the increased bile
flow.
In this study we have demonstrated that liver cells do express the
CASR initially cloned and characterized from the parathyroid (15) and
kidney (42). This was shown by semi-quantitative RT-PCR of total liver
RNA in which low levels of CASR PCR product were observed in the whole
tissue and from isolated hepatocytes in which there was an enrichment
of the product. Isolated nonparenchymal stellate, sinusoidal
endothelial, and Kupffer cells were negative for CASR mRNA. In the
case of the Kupffer cell where PTH metabolism takes place, this result
would be consistent with the lack of modulation of PTH metabolism by
Ca2+. In our study virtually the entire liver CASR mRNA
protein coding region was amplified and confirmed as such by nucleotide
sequencing. By Western blot analysis, using a well characterized
anti-CASR monoclonal antibody, the presence of CASR protein was
demonstrated in whole liver extract as well as in isolated hepatocytes.
Throughout the hepatic acinus, some hepatocytes, but not all, stained
positively for the CASR. Specific immunostaining was present in
both the perivenous and periportal areas such as the hepatocytes
neighboring or radiating from the control vein and those surrounding
the portal triad. Likewise, by in situ hybridization CASR
mRNA was shown to be well expressed in cells surrounding the
central and portal veins. The detection of the CASR by
immunohistochemistry in only a subset of hepatocytes may relate to the
hepatocyte being a polarized cell. The histological preparation may
expose only one side of the cell, the sinusoidal membrane (the side
where the blood flows), or the canalicular membrane (the biliary side).
It is not known on which side of the cell the CASR resides, but if
its expression is localized this could, in part, explain the
patchy staining. Some support for this hypothesis comes from the fact
that the in situ hybridization signal measuring CASR
mRNA was more widespread.
The CASR has been well studied with respect to its ability to stimulate
inositol phosphate turnover and Ca2+ release from
intracellular stores. Here, we show in isolated rat hepatocytes loaded
with Fura-2/AM that when IP3-sensitive pools had been
depleted by pretreatment with either thapsigargin or phenylephrine,
responsiveness to the CASR ligand spermine was abrogated. The
pharmacological characteristics of the hepatic CASR appear to be
somewhat different from those of the CASR expressed in parathyroid
cells (46), distal convoluted tubule cells (13), and heterologous
systems such as Xenopus oocytes (15, 42), and mammalian
cells in culture (47). Thus higher [Gd3+]o was
required to stimulate [Ca2+]i in hepatocytes than
in these other cells and systems. However, similar to what we observed
in hepatocytes, high [Gd3+]o was required to
elicit [Ca2+]i increases in pancreatic acinar
cells (27). This may be due to the reduced density of the hepatic (and
pancreatic) CASRs in the isolated cells or indicate that the CASR is
coupled less efficiently to G proteins, perhaps by having its function modified by tissue-specific regulatory proteins. On the other hand, the
concentrations of spermine required to promote
[Ca2+]i responses in hepatocytes were not that
dissimilar from those used by Quinn et al. (16) in their
study of the responsiveness to polyamines of the transiently
transfected CASR in HEK293 cells, especially since their studies were
conducted in 0.5 mM Ca2+, 0.5 mM
Mg2+ and the present studies with hepatocytes were done in
a Ca2+-free medium.
Phenylalkylamine compounds that are positive allosteric modulators and
specifically increase the sensitivity of the CASR to its ligands have
been developed (18). They decrease the plasma PTH and calcium levels in
patients with primary hyperparathyroidism and in dialysis patients with
secondary hyperparathyroidism (21). These compounds are stereoselective
(the R enantiomer is much more active than the S
enantiomer) and are therefore useful in the pharmacological
identification of the CASR. In the present study the responsiveness of
the hepatocyte CASR to spermine was shown to be markedly enhanced in
the presence of NPS R-467, whereas NPS S-467 had no effect, providing
further evidence that the Ca2+ mobilization from
intracellular pools was indeed a result of activation of the CASR.
What could be the physiological significance of the hepatic CASR? There
is good evidence that a variety of liver functions are modulated by
Ca2+o (and, in some cases, polyamine)
concentrations. These functions include bile and lipoprotein secretion,
prevention of cholestasis, resistance to toxicity and injury,
regeneration and proliferation, and metabolism. In the present study,
we have focused on the regulation of bile secretion. The hepatocyte is
the site of bile acid synthesis and plays a key role in bile formation and secretion as well as the clearance of bile acids from the portal
circulation and their transport to the canalicular lumen. Calcium may
play an important role in the regulation of bile secretion. A minimum
perfusate Ca2+ concentration is required for normal bile
secretion from perfused rat liver (48, 49). Reduction of calcium in rat
liver perfusate leads to increased paracellular permeability and a
decrease of both biliary excretion and bile flow. At very low calcium
levels bile flow is virtually abolished (50). A similar decrease in bile secretion was found in the hypocalcemic thyroparathyroidectomized rat (51, 52). The present study indicates that in the rat liver, bile
flow is very sensitive to the prevailing extracellular Ca2+
concentration. The CASR agonist spermine enhanced the rate of restoration of bile flow in perfused liver preparations following a
period of cholestasis induced by calcium deprivation. This suggested that the CASR might play an important role in bile secretion. Further
evidence of this was provided by the finding that the bile secretion
rate obtained at reduced cation concentration (0.5 mM
CaCl2, 0.5 mM MgCl2) could be
stimulated by increasing the calcium concentration or perfusing another
CASR agonist, Gd3+. Importantly, addition of 1 µM NPS R-467 (but not the inactive enantiomer, NPS S-467)
markedly stimulated bile flow, providing strong evidence for the
involvement of the CASR.
The suggested role of the CASR in bile secretion is compatible with its
localization in hepatocytes rather than bile duct itself. It is known
that cells within the hepatic acinus display important functional
differences according to their location, although all hepatocytes
contribute to the formation of bile. However, the rate of extraction of
bile acids is highest in the periportal region, suggesting that
periportal cells contribute a greater proportion of the bile
acid-dependent flow, whereas perivenous hepatocytes
contribute a greater proportion of the bile acid-independent flow.
However, there is substantial overlap between these mechanisms (53). In
our perfused liver preparation, taurocholate was present to ensure both
bile acid-dependent as well as bile acid-independent bile
flow. Additional studies will be required to refine the precise
cellular localization of the CASR and elucidate its role in the
different bile flow mechanisms.
Familial (benign) hypocalciuria hypercalcemia (FHH) is an autosomal
dominant disorder caused by inactivating mutations in the CASR (see
Ref. 8 for review). Law and Heath (54) reported clinical findings on 21 families with FHH. With respect to clinical correlates of hepatic
expression of the CASR, they found a higher incidence of FHH-affected
family members with gall stones than in the general population. Some
reports (55-57) have suggested that FHH can be associated with
recurrent pancreatitis. Pancreatitis is most often secondary to
alcoholism or biliary tract disease and less often to other causes
including hyperparathyroidism. In the exocrine pancreas, the CASR is
present in acinar cells and interlobular ducts, and in the latter cells
activation of the CASR stimulates fluid secretion (27). It is suggested
that the CASR monitors the Ca2+ concentration in the
pancreatic juice and regulates the level of Ca2+ in the
lumen, normally preventing the occurrence of conditions leading to
pancreatic stone formation and pancreatitis. The CASR in hepatocytes
may subserve a similar function and monitor the Ca2+
concentration in the bile secreted from hepatocytes. There is the
potential for involvement of altered polyvalent cation sensing in the
etiology of hepatic and (associated) pancreatic disorders.
Finally, retention of biliary constituents such as bile salts during
cholestasis may result in hepatocyte damage and apoptosis (58). It
has been demonstrated in other cell models that elevated [Ca2+]o can act as a first messenger to prevent
apoptotic cell death and that the protective effects are mediated
by the CASR (59). Therefore, potent and tissue-specific allosteric modulators of the CASR may be useful in states in which apoptosis is
thought to contribute to liver damage such as alcohol-induced hepatitis, drug-induced liver diseases or during organ storage before
liver transplantation.
1-adrenergic receptor agonist known to mobilize
Ca2+i from IP3-sensitive pools.
Addition of the deschloro-phenylalkylamine compound, NPS R-467, but not
the S enantiomer, NPS S-467, increased the sensitivity of
the Ca2+i mobilization response to 1.25 mM spermine. Bile flow ceased after
Ca2+o withdrawal, and its recovery was enhanced by
spermine in isolated perfused liver preparations. The CASR agonists
Ca2+ and Gd3+ increased bile flow, and the
response to a submaximal Ca2+ concentration was enhanced by
NPS R-467 but not the S compound. Thus, the data indicate that rat
hepatocytes harbor a CASR capable of mobilizing
Ca2+i from IP3-sensitive stores and
that activation of the CASR stimulates bile flow.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-hydroxylated
metabolite. The 25-hydroxylase step has not yet been fully investigated
with respect to regulation by [Ca2+]o (32).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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80 °C.
80 °C. Aliquots were electrophoresed through
4-12% SDS-polyacrylamide gels and blotted onto polyvinylidene
difluoride membranes (Bio-Rad). Membranes were rinsed in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20, blocked with 5% dried milk powder in TBST for 3 h, and
incubated with a CASR antibody. The mouse monoclonal antibody (ADD) was raised against a peptide comprising residues 214-236 of the
extracellular domain of the CASR, which are completely conserved
between human, bovine and rat. This antibody was kindly provided by
Drs. P. K. Goldsmith and A. M. Spiegel (National Institutes
of Health, Bethesda, MD) and K. V. Rogers (NPS Pharmaceuticals,
Salt Lake City, UT). The specificity of this antibody for the
parathyroid/kidney CASR has been previously well documented (13, 34).
The antiserum was immunoaffinity purified to reduce the nonspecific
binding. As a control, immunoblotting was carried out as described
above with the antibody preadsorbed for 1 h with the peptide (10 µg/ml) against which it was raised. Antibody-antigen complexes were
detected by chemiluminescence using the Lumi-glo kit (Life
Technologies, Inc.).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Fig. 1.
Schematic diagram of RT-PCR analysis of rat
CASR mRNA. The bar represents the rat CASR cDNA
encoded by exons 2-7. Nucleotide numbers are according to
GenBankTM accession number U20289 and indicate the
initiation codon, ATG, the start of each exon, and the stop
codon, TAA. The positions of the primers used are indicated,
and their sequences are given in Table I. The PCR products (products
1-5) generated are shown.
Primer sets used to amplify hepatocytes mRNAs
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Fig. 2.
The CASR gene is expressed in rat liver.
Complementary DNA was synthesized from total RNA and used as template
for PCR amplification. A cDNA of 367 bp corresponding to CASR
mRNA encoded by parts of exons 2 and 3 was amplified using primers
r2BF and r3AR (Fig. 1 and Table I). A cDNA of 469 bp corresponding
to part of the GAPDH mRNA was also amplified. Aliquots were taken
at 21, 24, 28, and 32 cycles (CASR) and 18, 21, 24 and 28 cycles
(GAPDH) and electrophoresed through ethidium bromide-stained agarose
gels.
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Fig. 3.
RT-PCR analysis of CASR in isolated rat liver
cells. RNA extracted from stellate (Ito), endothelial,
Kupffer, and hepatocyte cell populations was subjected to RT-PCR using
primer set r2AF and r4AR (Fig. 1 and Table I) generating a 1318-bp
product. Aliquots of the PCR reactions were electrophoresed through
ethidium bromide-stained gels. RT, minus reverse
transcriptase control.
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Fig. 4.
Western analysis of the CASR. Rat tissue
or mouse cell extracts (10 µg of protein each) were subjected to
SDS-polyacrylamide gel electrophoresis on a 4-12% gradient gel. The
blot was stained with CASR mouse monoclonal antibody (ADD). Positive
staining was demonstrated in the liver as well as the parathyroid and
kidney positive controls but not in the NlH3T3 fibroblast negative
control. The bands were demonstrated to be specific by staining a
similar blot with the same antibody preincubated with the peptide
against which it was raised (data not shown).
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Fig. 5.
Rat hepatocytes express the CASR
protein. Immunohistochemistry was conducted on rat
parathyroid/thyroid (A and B), kidney
(C and D), and liver (E-H) sections
using a specific anti-CASR antibody (A, C,
E, and G) or the same antibody preadsorbed with
CASR peptide (B, D, F, and
H). Magnification: A, B,
E, and F, ×75; C, D,
G, and H, × 300.
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Fig. 6.
In situ hybridization
demonstrating expression of specific CASR transcripts in rat
hepatocytes. Light micrographs of parathyroid/thyroid
(A and B), and liver (C-F) sections
with either antisense (A, C, and E) or
sense (B, D, and F) CASR RNA probe are
shown. Magnification: A, B, E, and
F, ×75; C and D, × 300.
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Fig. 7.
Rodent bile duct cells do not express the
CASR. a, RNA extracted from rat bile duct, a mouse bile
duct cell line, and rat hepatocyte populations was subjected to RT-PCR
using primer set r2AF and r4AR (Fig. 1 and Table I). Aliquots of the
PCR reactions were electrophoresed through ethidium bromide-stained
gels. RT, minus reverse transcriptase control. Although
the CASR product of 1318 bp was successfully amplified from hepatocyte
RNA, the bile duct cells were negative. b,
immunohistochemistry was conducted on rat liver sections using a
specific anti-CASR antibody. Magnification: × 750. Arrow indicates the
bile duct.
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Fig. 8.
Extracellular gadolinium
[Gd3+]o increases cytosolic calcium
[Ca2+]i in rat hepatocytes. Rat
hepatocytes were loaded with Fura-2/AM and bathed in calcium-free
medium. The bathing solution was changed to one containing increasing
[Gd3+]o and [Ca2+]i
measured by microfluorescence. The inset shows a typical
response to 2 mM GdCl3.
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Fig. 9.
Extracellular spermine increases cytosolic
Ca2+ in hepatocytes. Rat hepatocytes loaded with
Fura-2/AM were bathed in a [Ca2+]o free medium,
the medium was replaced with one containing increasing concentrations
of spermine, and [Ca2+]i mobilization measured by
microfluorescence. The inset shows a typical response to 2.5 mM spermine.
1-receptor agonist (Fig. 10,
left panel), or by thapsigargin (Fig. 10,
right panel). This demonstrated that the source of the
[Ca2+]i mobilized by CASR ligands was localized
in the IP3-sensitive Ca2+ pools of the
endoplasmic reticulum.
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Fig. 10.
Prior emptying of IP3-sensitive
Ca2+ pools abrogates
[Ca2+]i mobilization by spermine.
Rat hepatocytes loaded with Fura-2/AM and bathed in a
[Ca2+]o-free medium and
IP3-sensitive [Ca2+]i pools
were depleted by either addition of phenylephrine (left
panel) or thapsigargin (right panel) after which
the bathing solution was changed to one containing 2.5 mM
spermine. [Ca2+]i mobilization was monitored by
microfluorescence.
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Fig. 11.
Calcimimetic NPS R-467 enhances the
[Ca2+]i response to spermine in
hepatocytes. Rat hepatocytes loaded with Fura-2/AM were bathed in
a [Ca2+]o-free medium. The medium was changed to
one containing 1.25 mM spermine either without or
with 10 µM NPS S-467, or with 10 µM NPS
R-467, and [Ca2+]i mobilization was monitored by
microfluorescence.
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Fig. 12.
CASR agonist spermine enhances bile flow
recovery in isolated perfused liver. Bile flow was measured in
isolated-perfused rat liver preparations. After an initial perfusion in
medium containing 1.25 mM Ca2+ for 15 min,
cessation of bile flow was provoked by perfusion in a
Ca2+-free medium for 10 min. At this time (time 0 of the
figure) bile flow recovery was stimulated by perfusion in 1.25 mM Ca2+ medium with either no, or 1.25 mM, or 2.5 mM spermine added (n = 3). Statistically significant differences in bile flow recovery were
analyzed by comparing the slopes of each condition by analysis of
variance (see inset). *, p < 0.01; **,
p < 0.001.
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Fig. 13.
CASR agonists Ca2+ and
Gd3+ stimulate and NPS R-467 selectively enhances bile flow
in isolated perfused liver. Bile flow was measured in isolated
perfused rat liver preparations and expressed as a percentage change
relative to base line. At 20 min base-line bile flow was 61.2 ± 9 (mean ± S.E.) µl/min. Control ( ), perfusate 0.5 mM Ca2+ throughout, changed to fresh medium at
20 min. Experimental (
), perfusate 0.5 mM
Ca2+ for the first 20 min and then changed to 1.25 mM Ca2+ (A); 0.5 mM
Ca2+ + 100 µM Gd3+
(B); 0.5 mM Ca2+ + 1 µM NPS R-467 (C); or 0.5 mM
Ca2+ + 1 µM NPS S-467 (D). Each
point is the mean ± S.E. of three or four determinations.
Asterisks indicate significant differences determined by analysis of
variance for experimental values relative to control (base line) values
obtained at the corresponding time. *, p < 0.05; **,
p < 0.01.
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ACKNOWLEDGEMENTS |
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We thank Drs. P. K. Goldsmith, A. M. Spiegel, K. V. Rogers, and E. F. Nemeth for anti-CASR antibody and calcimimetic compounds, Dr. E. Levy for the BDC cell line, Dr. E. M. Brown for helpful insights into the potential functions of the hepatic CASR, and Dr. J. Fox for advice on conducting the functional studies.
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FOOTNOTES |
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* This work was supported by Medical Research Council of Canada Grant MT-9315 (to G. N. H.) and Grants MT-6511 and MT-14578 (to M. G.-B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Studentship from the Medical Research Council of Canada.
** To whom address correspondence should be addressed: Calcium Research Laboratory, Rm. H4.67, Royal Victoria Hospital, 687 Pine Ave. West, Montréal, PQ H3A 1A1, Canada. Tel.: 514-843-1632; Fax: 514-843-1712; E-mail: gnhendy@med.mcgill.ca.
Published, JBC Papers in Press, November 18, 2000, DOI 10.1074/jbc.M009317200
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ABBREVIATIONS |
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The abbreviations used are: CASR, Ca2+-sensing receptor; RT, reverse transcriptase; PCR, polymerase chain reaction; IP3, inositol 1,4,5-trisphosphate; bp, base pair(s); AM, acetoxylmethyl ester; FHH, familial hypocalciuria hypercalcemia.
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