From the
The calcitonin receptor has been proposed to function as an
extracellular Ca
Characterization of a calcitonin receptor (CTR)
The ligand for the CTR, calcitonin
(CT), is a 32-amino acid peptide hormone secreted by the thyroid C
cells in response to elevated serum Ca
A recent
report by Stroop et al.(15) has suggested that the CTR
functions as a [Ca
The
[Ca
The present studies were undertaken to more
rigorously analyze the CTR for Ca
PC
Basal [Ca
Fig. 1A and show events
elicited in LLC-PK
Fig. 1B and show events elicited by addition of 1 and 10 mM Ca
Cultured LLC-PK
This study provides evidence that the sCT-induced Ca
Our
results indicate that activation of the CTR in LLC-PK
According to these results, we hypothesize that the CTR does not
function as a [Ca
In
conclusion, our study provides evidence indicating that a
Ca
We thank Drs. Elio Ziparo, Mario Molinaro, and Mario
Stefanini who kindly made available to us the equipment of the
Institute of Histology and General Embryology, University ``La
Sapienza'' of Rome. We also acknowledge the excellent technical
support of Giancarlo Sciortino.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
concentration
([Ca
]
) sensor (Stroop,
S. D., Thompson, D. L., Kuestner, R. E., and Moore, E. E.(1993) J.
Biol. Chem. 268, 19927-19930). To test this hypothesis we
studied the LLC-PK
renal tubular cells and the PC
cells, a cell line stably transfected with the cloned porcine
calcitonin receptor. [Ca
]
was measured by fura-2 single cell microfluorometry.
Addition to the cells equilibrated in 1.25 mM Ca
-containing media of 1-10 mM extracellular Ca
did not result in a significant
increase of [Ca
]
.
Treatment with 10
M salmon calcitonin (sCT)
elicited a rapid, persistent elevation of
[Ca
]
. Addition of
1-10 mM extracellular Ca
in the
presence of sCT induced a significant
[Ca
]
elevation, about
10-fold that observed in the absence of the hormone. Ca
influx was inhibited by lanthanum. The rise of
[Ca
]
at elevated
[Ca
]
was not due to a
Ca
sensing mechanism with release of Ca
from intracellular stores, since it was prolonged, and was not
abolished by prior depletion of Ca
stores with
10
M thapsigargin. On the contrary, this
agent potentiated Ca
influx after addition of
1-10 mM Ca
by 13-fold versus control. Prior stimulation of
[Ca
]
with
10
M arginine-vasopressin had similar
effects, enhancing the subsequent Ca
influx.
Enhancement of Ca
influx by sCT was confirmed by
increased Mn
quenching of fura-2 fluorescence. In
conclusion, arginine-vasopressin or calcitonin enhance Ca
influx in LLC-PK
cells via a Ca
release-activated conductance, probably dependent upon
capacitative Ca
entry. Thus, these effects are not
unique to the calcitonin receptor and argue against the receptor
functioning as a [Ca
]
sensor.
(
)cloned from a porcine renal epithelial cell line,
LLC-PK
, predicts a 482-amino acid binding protein with high
affinity for salmon calcitonin (sCT) (dissociation constant K
6 nM)(1) . This CTR
belongs to a subfamily of G-protein-linked receptors which, based on
similarity in amino acid sequence, includes, for example, the
parathyroid hormone/parathyroid hormone related peptide receptor (32%
amino acid identity and 56% similarity) (2) and the secretin
receptor (30% identity, 58% similarity)(3) . These receptors are
likely to represent a new family of G-protein-coupled receptors,
associated with, among other activities, the regulation of
Ca
homeostasis.
levels. CT
administration results in a reduction of the Ca
concentration in the extracellular fluid
([Ca
]
)(4) .
This is accomplished via inhibition of the bone resorbing activity of
the osteoclast (5, 6) and enhanced renal calcium
excretion(7, 8) . Most cellular effects of CT are
associated with activation of the adenylyl cyclase
pathway(9, 10) . However, recently it has become
apparent that the CTR is also associated with the phospholipase C
enzyme pathway(11, 12) , which induces breakdown of
membrane phosphoinositol lipids to yield inositol 1,4,5-triphosphate
(InsP
) and diacylglycerol. The two second messenger
molecules, in turn, stimulate Ca
release into the
cytoplasm from intracellular pools, and activate the serine/threonine
protein kinase C, respectively(13, 14) .
]
sensor. A [Ca
]
sensor has been proposed as a mechanism that allows the cell
to sense increases of [Ca
]
in the millimolar range, and to respond with significant
elevations of the cell signal cytosolic free Ca
concentration
([Ca
]
)(16, 17) .
Should the CTR operating as a
[Ca
]
sensor be
confirmed, the observation would be of great interest, in view of the
fact that only selected cell types, including the CTR-expressing
osteoclasts(16, 17, 18) , retain this unique
Ca
sensing activity. The secretion of CT in
hypercalcemic conditions (4) would thus be predicted to activate
the calcium sensor activity of the CTR resulting in modulation of
osteoclast function. A synergistic role of the two factors, CT and
elevated [Ca
]
, would
reciprocally potentiate the cellular responses.
]
sensing receptor,
recently cloned and characterized from bovine parathyroid
glands(18) , predicts a
120-kDa seven-trans membrane domain
receptor, which shares limited similarity to the metabotropic glutamate
receptor(19) . Its extracellular domain contains clusters of
acidic amino acid residues, which are likely to represent a
Ca
binding sequence, whereas the intracellular domain
shows sequences similar to those of other G-protein-coupled receptors.
The predicted structure of the CTR is unrelated to that of the
[Ca
]
sensing receptor,
since it lacks similarity in amino acid sequence and does not possess
extracellular putative Ca
-binding consensus
sequences(1) . This observation argues against the CTR
functioning as a [Ca
]
sensor.
sensing function.
We have performed single cell
[Ca
]
studies, by
fura-2 microfluorometry, in CTR-positive cells in vitro employing the LLC-PK
cell line(1) , and the
PC
cells, a line derived from MC-3T3-E
cells
stably transfected with the CTR cloned from LLC-PK
cells(15) . In nonstimulated conditions, both cell types
were insensitive to an elevated
[Ca
]
, whereas upon
stimulation with sCT they acquired the capability of responding to an
increase of [Ca
]
with
a [Ca
]
elevation.
Characterization of the mechanism inducing the
[Ca
]
-dependent
[Ca
]
elevation,
however, demonstrated that this was exclusively due to Ca
influx across the plasma membrane, induced by a nonspecific
post-receptor event that was not unique for the CTR but was shared by
arginine-vasopressin (AVP), another Ca
-mobilizing
hormone.
Materials
Dulbecco's modified and
modified minimum essential media, fetal bovine serum, reagents, and
sterile plasticware for cell culture were from Flow Laboratory (Irvine,
CA). Fura-2 acetoxymethyl ester (fura-2/AM) was from Molecular Probes
(Eugene, OR). Ionomycin and fatty acid-free bovine serum albumin were
from Calbiochem (La Jolla, CA). Salmon calcitonin was kindly donated by
Drs. Francesco Bartucci and Vera Calcagno, Sandoz Prodotti Farmaceutici
S.p.A. (Milan, Italy). All other reagents were from Sigma.
Cell Cultures
LLC-PK cells are a
porcine kidney epithelial cell line previously characterized in our
laboratory (9). Cells were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum and
antibiotics and fed once a week. At confluence, cells were split 1:20
by standard trypsin procedures.
cells were obtained
by stable transfection of MC-3T3-E
cell line as described
previously(11) . Cells were grown in
modified minimum
essential medium supplemented with 2.5% fetal bovine serum, fed once a
week, and split 1:3 every 7-14 days. MC-3T3-E
cells
were grown in the same condition as PC
cells.
Buffer
Bathing medium through all experiments was
a modified Krebs-Henseleit solution (KHH) with or without 1.25 mM Ca, buffered with 20 mM HEPES, and
containing 0.2% bovine serum albumin.
Measurement of
[Ca
[Ca]
]
of nonconfluent monolayers of LLC-PK
, PC
,
and MC-3T3-E
cell lines was measured by dual wavelength
fluorescence of cells loaded with the Ca
-sensitive
intracellular probe fura-2(16, 20) . Cells, seeded on
glass coverslips at a density of 100,000/3.5-cm diameter dish, were
loaded with 3 µM fura-2/AM in serum-free medium, at 37
°C for 60 min. Measurements were performed in single cells, at 340
and 380 nm excitation wavelengths, with an AR-CM fluorometer (Spex
Industries, Inc., Edison, NJ) connected with a Diaphot TMD inverted
microscope (Nikon Corp., Tokyo, Japan) equipped with a Nikon CF X40
objective. Emissions were collected by a photomultiplier carrying a
510-nm cut-off filter and recorded by an ASEM Desk 2010 computer (ASEM
S.p.A, Buia, Italy), which automatically calculated real-time 340/380
ratios. Calibration of the signal was obtained at the end of each
observation by adding 5 µM ionomycin to saturate the dye
to maximal fluorescence, followed by 7.5 mM EGTA plus 60
mM Tris-HCl, pH 10.5, to release Ca
from
fura-2 and obtain minimal fluorescence.
[Ca
]
was calculated
according to previously described formulas(20) .
Statistics
Data are presented as average ±
S.E. Statistical analysis was performed by analysis of variance
(ANOVA). p < 0.05 was conventionally considered to indicate
statistical significance. For the dose-response data, when ANOVA
revealed a statistical significance, Student's t test
was used.
]
in LLC-PK
, PC
and MC-3T3-E
cells is indicated in . In all cell types
[Ca
]
remained stable
over a period of at least 30 min. No spontaneous fluctuations were
observed.
, PC
, and MC-3T3-E
cells by treatment with 1 and 10 mM Ca
. Unless otherwise specified, Ca
was added to the 1.25 mM Ca
-containing
KHH buffer; therefore, the final
[Ca
]
was 2.25 and
11.25, respectively. This elevation of the
[Ca
]
did not show
ability to elicit a [Ca
]
response in the cells. No significant differences were
observed in high Ca
-treated cells versus control [Ca
]
levels.
Figure 1:
Effect of 1 and 10 mM Ca on [Ca
] of single
LLC-PK
cells. A, traces representing real-time
fura-2 fluorescence ratio (excitation wavelength 340 and 380 nm,
emission 510 nm) in a single LLC-PK
cell challenged with 1
and 10 mM Ca
. Initial
[Ca
] in the medium = 1.25
mM. Calibration of fluorescence as
[Ca
] has been obtained upon addition of 5
µM ionomycin, followed by 7.5 mM EGTA plus 60
mM Tris (pH 10.5). B, a single LLC-PK
cell was treated with 10
M sCT, which
elicited a [Ca
] increase. When
[Ca
] stabilized, the cell was sequentially
treated with 1 and 10 mM Ca
, which further
increased [Ca
]. Initial
[Ca
] in the medium = 1.25
mM.
LLC-PK and PC
cells were
similarly sensitive to sCT, whereas MC-3T3-E
cells failed
to show sensitivity to the hormone. illustrates the effect
of 10
M sCT on
[Ca
]
. The hormone
induced a severalfold increase of
[Ca
]
over basal levels
in the two CT-sensitive cell types. Responses were transient, with a
[Ca
]
peak followed by
a decrease to lower levels.
in LLC-PK
, PC
and
MC-3T3-E
cells pretreated with 10
M sCT. Ca
was added to the cells during the
sustained [Ca
]
elevation induced by sCT. Addition of Ca
resulted in a significant
[Ca
]
elevation in
LLC-PK
and PC
, but not in MC-3T3-E
cells ().
Mechanism of Ca
LLC-PK-induced
[Ca
]
increase in sCT treated
LLC-PK
cells
cells were
utilized to investigate the mechanisms responsible for the
Ca
-dependent
[Ca
]
transients
induced by sCT. Fig. 2shows concentration-dependent curves
obtained in LLC-PK
cells treated with the doses of
[Ca
]
as indicated on
the abscissa. These curves were constructed computing peak
[Ca
]
in cells
equilibrated in Ca
-free KHH, to which Ca
was added to the final desired concentration from 1 M
CaCl
stock solution. In the absence of sCT, cells showed
low sensitivity to elevated
[Ca
]
, even though
responses were approximately 2-fold higher compared to those observed
when the same doses of CaCl
were added to cells
equilibrated in the 1.25 mM Ca
-containing
KHH. Treatment with 10
M sCT significantly
enhanced the
[Ca
]
-induced
[Ca
]
increase. Maximal
response was observed at 10 mM Ca
, with
EC
= 6 mM Ca
.
Figure 2:
Concentration-dependent curves of
[Ca]-induced
[Ca
] increases in single LLC-PK
cells. Cells were challenged with the doses of Ca
indicated on the abscissa, in the presence or in the
absence of 10
M sCT. Each point shows mean
± S.E. of at least six independent experiments. Curves were
significantly different (p < 0.01), as assessed by ANOVA
followed by Student's t test.
In Fig. 3A the effect of chelation of extracellular
Ca by EGTA is shown. Addition of 1.25 mM EGTA slightly reduced
[Ca
]
. In this
circumstance, cells responded to sCT with a rapid transient increase of
[Ca
]
, which peaked at
133 ± 27 nM (n = 6), a level
approximately 4-fold lower than that observed in the presence of
extracellular Ca
. Then,
[Ca
]
rapidly returned
toward base line. No sustained phase was observed. Subsequent addition
of extracellular Ca
resulted in a rapid, sustained
[Ca
]
elevation. This
indicates that sCT treatment has two effects. First, it is capable of
stimulating Ca
release from intracellular storing
organelles, even in Ca
-free conditions. Second, sCT
stimulates a large inward Ca
flux from the
extracellular environment, abolished by removal of extracellular
Ca
by EGTA. This is further confirmed by the
experiment shown in Fig. 3B. A single LLC-PK
cell has been stimulated by sCT to obtain the
[Ca
]
increase. During
the sustained phase, the cell was treated with LaCl
, an
agent known to block all Ca
influx mechanisms across
the plasma membrane(21) . In the presence of
La
, [Ca
]
rapidly dropped to near basal levels, indicating once again
the sustained phase to be dependent upon Ca
influx.
Addition of 1-10 mM Ca
in the presence
of La
failed to induce a significant
[Ca
]
rise compared to
untreated cells (Fig. 3B, I). This
indicates that Ca
-dependent
[Ca
]
increase
stimulated by sCT largely depends on gating of Ca
channels of the plasma membrane.
Figure 3:
Effect of EGTA and La on the Ca
influx in single LLC-PK
cells. A, [Ca
] in the KHH was
reduced by addition of the Ca
-chelating agent EGTA.
The cell was then treated with 10
M sCT,
followed by 1 mM Ca
. Initial
[Ca
] in the medium = 1.25
mM. Predicted [Ca
] upon addition
of EGTA = 0 mM. B, a single LLC-PK
cell was treated with 10
M sCT.
During the sustained [Ca
] increase phase,
10
M La
was added to
abolish Ca
influx. Further addition of 1 and 10
mM Ca
in the presence of La
failed to stimulate a significant
[Ca
] rise. Initial
[Ca
] in the medium = 1.25
mM.
To rule out the potential
confounding effects of modifications in Ca efflux,
and to confirm that actual Ca
influx was responsible
for the changes in
[Ca
]
, we performed
experiments with Mn
quenching of the fura-2 dye to
monitor influx of another extracellular divalent cation. Mn
permeates the cell through the same channels employed by
Ca
, rapidly quenching fura-2 fluorescence by
irreversible binding(22) . In Fig. 4A, addition
of sCT in the presence of Mn
resulted in a rapid
increase of fura-2 fluorescence, followed by a slow progressive
reduction. Similar fura-2 quenching was observed in cells pretreated
with sCT and then with 1 and 10 mM Ca
prior
to addition of Mn
(Fig. 4B). This
further indicates that both the sustained phase observed during
stimulation with sCT and the [Ca
]
increase observed in cells further challenged with high
Ca
are due to massive influx of divalent cations
across the plasma membrane.
Figure 4:
Mn quenching of fura-2
fluorescence. In A, a single LLC-PK
cell was
treated with 10
M Mn
,
then with 10
M sCT. In B, a single
LLC-PK
cell was treated with 10
M sCT, then with 1 and 10 mM Ca
prior to
addition of Mn
. Initial
[Ca
] in the medium = 1.25
mM. The drop of fura-2 fluorescence, measured at 340 nm
excitation wavelength, indicates quenching of the dye by the
Mn
that permeates the cell.
The
[Ca]
-dependent
elevation of [Ca
]
could not be explained by Ca
release from
intracellular stores, as it was persistent (Fig. 1B).
However, to further examine the role of Ca
stores in
the sCT-induced Ca
influx, we performed experiments
involving Ca
depletion by thapsigargin, a specific
inhibitor of the Ca
ATPase of the endoplasmic
reticulum(23) . Fig. 5A shows the effect of
thapsigargin on the signal induced by sCT. Thapsigargin transiently
increased [Ca
]
to 558
± 39 (n = 4) and completely abolished the
response of the cell to sCT, indicating that Ca
release from the stores is involved not only in the sCT-induced
peak increase of [Ca
]
,
but also in the secondary Ca
influx across the plasma
membrane. This is further demonstrated in experiments (not shown) in
which Ca
depletion was obtained by treatment with 1
µM of the Ca
ionophore ionomycin, in
which we observed similar complete inhibition of the response to sCT.
We next examined whether depletion of intracellular Ca
stores modulated the response of the cells to elevated
extracellular Ca
. To accomplish this, we treated the
LLC-PK
cells with thapsigargin and then added 1-10
mM Ca
(Fig. 5B). In this
circumstance, thapsigargin not only failed to prevent, but, similar to
sCT, significantly stimulated Ca
entry into the cells (I). Finally, we depleted Ca
stores with
a physiologic stimulus. LLC-PK
cells are known to express
Ca
mobilizing receptors for AVP. Therefore, we
pretreated the cells with AVP and observed a rapid
[Ca
]
transient with a
peak and a sustained phase (Fig. 6). Similar to sCT, addition of
1-10 mM Ca
in the presence of AVP
greatly stimulated [Ca
]
increases with a similar pattern (Fig. 6, I). This indicates that the effect of sCT on
Ca
influx in LLC-PK
cells is not unique
for the CTR but is shared by other Ca
mobilizing
receptors, such as the V
-type AVP receptor.
Figure 5:
Effect of thapsigargin on the
[Ca] in single LLC-PK
cells. A, trace representing [Ca
] in a
single LLC-PK
cell treated with the endoplasmic reticulum
Ca
ATPase inhibitor thapsigargin (TPS).
Thapsigargin depleted the intracellular Ca
storing
organelles, inducing a transient [Ca
]
increase and preventing the response of the cell to 10
M sCT. B, a single cell was pretreated with
10
M thapsigargin (TPS) prior to
addition of 1 and 10 mM Ca
. Note that
Ca
store depletion by thapsigargin stimulated
[Ca
]-induced
[Ca
] increases.
Figure 6:
Effect of AVP on
[Ca] of a single LLC-PK
cell. A
single cell was first challenged with 10
M AVP, which elicited a [Ca
] transient,
then with 1 and 10 mM Ca
. This maneuver
stimulated Ca
influx similar to that observed in
sCT-treated cells.
and PC
cells express
abundant CTRs (1, 11) and therefore represent an
excellent model for studying the signal transduction pathways activated
by sCT. The two cell lines respond to sCT with typical, biphasic
[Ca
]
transients, a
feature of the signaling pathway involving phospholipase C(13) .
The initial rise has been demonstrated to result from a direct effect
of InsP
on InsP
-activated Ca
channels in intracellular Ca
storing
organelles(11) . The sustained elevation that follows the
initial peak is due to persistent Ca
entry across the
plasma membrane(11) . The amplitude of the sustained phase,
which is responsible for the maintenance of
[Ca
]
higher than basal
for several minutes, suggests that the predicted Ca
influx mechanism shows a large conductance, determined by an
extensive Ca
movement across the plasma membrane.
conductance is significantly stimulated when the Ca
gradient on the two sides of the plasma membrane is increased.
This causes a [Ca
]
increase, which is prolonged in nature and lasts several
minutes. Such a feature argues against the activation of a
[Ca
]
sensing, since
this should result in a transient
[Ca
]
rise that would
be abolished by agents that deplete intracellular Ca
stores(16, 17, 24, 25) .
cells
induces a nonspecific Ca
release-activated
Ca
influx. This is demonstrated by several
observations. First, the release of Ca
is produced
not only by sCT, but also by activation of another Ca
mobilizing receptor, such as the V
-type AVP receptor.
Second, agents that nonspecifically stimulate Ca
release from intracellular stores, such as thapsigargin, promote
Ca
internalization similar to that induced by sCT and
AVP. As a result of these observations, we can expect that activation
of the Ca
conductance does not require receptor
occupancy. This rules out the involvement of InsP
as a
direct second messenger necessary for determining the sCT-induced
Ca
-dependent
[Ca
]
rise. The
relevance of the activity of membrane Ca
channels is
further demonstrated by experiments involving blockade of
Ca
influx from the extracellular environment.
La
, a nonspecific inhibitor of all Ca
entry mechanisms, is significantly active in blocking both the
sustained phase induced by sCT and the additional
[Ca
]
increase due to
addition of Ca
to the bathing buffer. Experiments
performed with Mn
further confirm that influx of
divalent cations is already occurring upon stimulation with sCT, and
that it proceeds when an elevated
[Ca
]
is created.
]
sensor. This argues against the data reported by Stroop et al.(15) , who studied a recombinant human CTR
transfected in baby hamster kidney (BHK) cells. In our hands, the
mechanism stimulated by sCT resembles that described first by Casteels
and Droogmans (26) and then by Takemura and Putney (27), which
has been termed ``capacitative Ca
entry,''
or ``store-operated Ca
entry
pathway''(28) . According to Putney and Bird(29) , a
capacitative Ca
entry is stimulated by Ca
mobilizing signals, through activation of PLC-coupled receptors.
It is largely accepted that InsP
, produced by phospholipase
C-dependent membrane phosphoinositol lipid breakdown, is responsible
for such Ca
influx via a secondary, indirect
mechanism(30) . The most compelling evidence for this hypothesis
comes from the observation that inhibitors of the microsomal
Ca
ATPase, such as thapsigargin, which cause depletion
of intracellular stores without InsP
production, mimic the
effect of surface membrane InsP
-linked agonists to activate
Ca
entry (29). Regardless of the primary process that
produces intracellular Ca
pool depletion,
capacitative Ca
entry allows the emptied stores to be
rapidly refilled, in order to restore the resting
conditions(26, 27, 28, 29, 30) .
The mechanism producing the capacitative Ca
influx
has not yet been fully elucidated. It implies a system for
communication between the Ca
pool and the plasma
membrane, such that the permeability of the plasma membrane would be
increased when the intracellular pool is empty(27) . To date two
fundamental mechanisms for the retrograde signal for capacitative
Ca
entry have been considered; (i) a diffusible
messenger is produced or released when the intracellular stores are
depleted, diffuses to the plasma membrane and activates Ca
entry(31, 32, 33) , or (ii) the emptying
of the intracellular Ca
stores causes a
conformational change in the organelle and/or its surface proteins, and
this information is delivered to the plasma membrane either by direct
coupling (34) or via the cytoskeleton(35) .
release Ca
influx mechanism is
operating in the CTR-expressing LLC-PK
and in the PC
cells stably transfected with the CTR. This mechanism resembles
the recently described capacitative Ca
entry observed
in cells challenged with Ca
mobilizing agents. Such
Ca
influx is not due to Ca
release
from intracellular stores, but rather is stimulated by Ca
store depletion. These results seem to rule out the CTR
functioning as a [Ca
]
sensor.
Table: Effect of sCT on
[Ca]
of
single LLC-PK
, PC
, and
MC-3T3-E
cells
Table: Effect of 1 and 10
mM Ca on
[Ca
]
of
LLC-PK
, PC
, and
MC-3T3-E
cells
Table: Effect of
La, thapsigargin, and AVP on
Ca
influx in LLC-PK
cells
, inositol 1,4,5-triphosphate; AVP,
arginine-vasopressin; ANOVA, analysis of variance; KHH, Krebs-Henseleit
solution.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.