Calcium-sensing receptor activation induces intracellular
calcium oscillations
Gerda E.
Breitwieser and
Lucio
Gama
Department of Physiology, Johns Hopkins University School of
Medicine, Baltimore, Maryland 21205
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ABSTRACT |
Parathyroid hormone secretion is
exquisitely sensitive to small changes in serum Ca2+
concentration, and these responses are transduced via the
Ca2+-sensing receptor (CaR). We utilized heterologous
expression in HEK-293 cells to determine the effects of small,
physiologically relevant perturbations in extracellular
Ca2+ on CaR signaling via
phosphatidylinositol-phospholipase C, using changes in fura 2 fluorescence to quantify intracellular Ca2+. Chronic
exposure of CaR-transfected cells to Ca2+ in the range from
0.5 to 3 mM modulated the resting intracellular Ca2+
concentration and the subsequent cellular responses to acute extracellular Ca2+ perturbations but had no effect on
thapsigargin-sensitive Ca2+ stores. Modest,
physiologically relevant increases in extracellular Ca2+
concentration (0.5 mM increments) caused sustained (30-40 min) low-frequency oscillations of intracellular Ca2+ (~45 s
peak to peak interval). Oscillations were eliminated by 1 µM
thapsigargin but were insensitive to protein kinase inhibitors (staurosporine, KN-93, or bisindolylmaleimide I). Staurosporine did
increase the fraction of cells oscillating at a given extracellular Ca2+ concentration. Serum Ca2+ concentrations
thus chronically regulate cells expressing CaR, and small perturbations
in extracellular Ca2+ alter both resting intracellular
Ca2+ as well as Ca2+ dynamics.
fura 2; intracellular calcium; calcium oscillations; thapsigargin-sensitive calcium stores; HEK-293 cells; protein kinase
inhibitors
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INTRODUCTION |
THE CALCIUM-SENSING
RECEPTOR (CaR) was hypothesized to exist based on the sensitivity
of parathyroid hormone secretion to serum calcium (5, 24).
Expression cloning utilizing RNA isolated from parathyroid cells
resulted in identification of the CaR (6), which is
activated by extracellular Ca2+ in the physiological range
(0.5-5 mM). A unique feature of CaR is the steep cooperativity of
Ca2+-dependent activation, which is observed both in vivo
(26) and when CaR is heterologously expressed (6,
15). In the parathyroid, CaR is chronically exposed to serum
Ca2+ concentrations that are within the steeply cooperative
range of its dose-response relation (EC50 of 2-3 mM,
and Hill coefficient n
2-3), providing tight
control over parathyroid hormone secretion. Although these qualitative
details of CaR-mediated modulation of parathyroid hormone secretion are
well characterized, the kinetics of single-cell responses to tonic,
physiological changes in extracellular Ca2+ have not been studied.
CaR couples changes in extracellular Ca2+ to a
variety of intracellular responses, including activation of
phospholipases, generation of inositol trisphosphate
(IP3) and diacylglycerol, increases in
intracellular Ca2+, changes in protein phosphorylation,
activation of ion channels, regulation of hormone secretion, and
modulation of gene expression (32). Chronic activation of
CaR resulting from constant exposure to serum Ca2+ would
necessarily contribute to regulation of cellular functions through many
of these pathways. Under conditions of constant agonist exposure,
however, many G protein-coupled receptors undergo desensitization and,
ultimately, downregulation of receptor and/or signal transduction pathway protein expression (7, 13). A key question with
respect to CaR function is therefore whether CaR desensitizes at a
fixed extracellular Ca2+ concentration and responds only to
acute changes in serum Ca2+ or whether CaR is chronically
activated under physiological conditions.
We have utilized heterologous expression of human CaR in HEK-293
cells to address two fundamental questions with respect to the in vivo
contributions of CaR to parathyroid cell function, namely, How do cells
expressing CaR adapt to chronic exposure to the concentrations of
extracellular Ca2+ that are present in normal serum? and
What are the kinetic features of CaR-mediated changes in intracellular
Ca2+ in response to small, acute perturbations of
extracellular Ca2+? We find that cells expressing CaR are
uniquely sensitive to alterations in extracellular Ca2+ and
that small perturbations in extracellular Ca2+ induce
low-frequency intracellular Ca2+ oscillations. These
results have implications for the mechanism(s) by which CaR activation
regulates parathyroid hormone secretion as well as diverse cellular
signal transduction pathways in many other cell types.
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METHODS |
Cell culture and transfection.
HEK-293 cells (American Type Culture Collection, Rockville, MD)
were grown in high-glucose DMEM (Life Technologies, Grand Island, NY)
supplemented with 10% BSA (Sigma, St. Louis, MO), 50 U/ml penicillin,
and 50 µg/ml streptomycin (37°C, 5% CO2). Cells were
transiently or stably transfected with the human CaR (obtained from Dr.
Klaus Seuwen, Novartis Pharma, Basel, Switzerland), which was subcloned
into pEGFPN3 (Clontech Laboratories, Palo Alto, CA) as previously
described (15). HEK-293 cells were transfected with 1 µg
of each CaR construct using Effectene (Quiagen, Valencia, CA) or
Novafector (VennNova, Pompano, FL), plated on collagen-coated coverslips, and kept in a 5% CO2 incubator at 37°C until
use (generally 72 h after transfection). HEK-293 cells stably
expressing human CaR were produced by linearization of the hCaR-pEGFPN3
plasmid, transfected as described above, and selected with Geneticin
(Life Technologies). Colonies were selected after 3 wk, and clones were confirmed by sequencing, CaR activity, and Western blotting with anti-green fluorescent protein (GFP) antibody (Molecular Probes, Eugene, OR).
Single-cell fluorescence measurements of intracellular
Ca2+.
Cells were loaded with 0.5 µM fura 2-AM (Calbiochem-Novabiochem, San
Diego, CA) for 30 min at 37°C in a solution containing variable
CaCl2 (0.5, 2, or 3 mM), 130 mM NaCl, 5 mM KCl, 1 mM MgSO4, 20 mM HEPES, 0.83 mM
Na2HPO4, 0.17 mM
NaH2PO4, 25 mM mannose, and 1 mg/ml BSA, pH
7.4. After the loading period, the coverslip was mounted in an imaging
chamber (Warner Instrument, Hamden, CT) having both a top and bottom
coverslip and washed with a bath solution having a CaCl2
concentration matched to the loading solution plus 140 mM NaCl, 5 mM
KCl, 0.55 mM MgCl2, 10 mM HEPES, pH 7.4. Selected regions
of the field were excited at 340/380 nm (emission wavelength 510 nm) at
8- or 10-s intervals (10 frame averaging) on a Universal imaging system
(Universal Imaging, West Chester, PA) based on the MetaFluor software
package. Background images at the same settings used during a
particular experiment were obtained on regions of the coverslip devoid
of cells. Calibration of intracellular Ca2+ utilized a
series of buffered Ca2+ standards (Molecular Probes),
assuming a dissociation constant of 145 nM (determined in vitro at
22°C; Molecular Probes Handbook, 7th edition). All solutions were
osmolality matched (290-300 mosM), measured on a Wescor 5500 vapor
pressure osmometer (Wescor, Salt Lake City, UT). Variations in
extracellular Ca2+ concentration were produced by isosmolar
substitution for NaCl. Thapsigargin and the protein kinase inhibitors
bisindolylmaleimide I, KN-93, and staurosporine
(Calbiochem-Novabiochem) were prepared as stock solutions in DMSO and
added to the appropriate extracellular solution. DMSO controls were
performed under identical experimental conditions and had no effect on
measured parameters. All experiments were performed at room temperature
(22-24°C). Multiple cells were analyzed from at least three
independent transfections or cell passages (for stably transfected cell
lines). Data obtained from stably and transiently transfected HEK-293
cells were comparable, and each result was verified for both
conditions. Data were normalized/averaged as described in the text and
are presented as means ± SE. Curves were fitted by least-squares
minimization using the Marquardt-Levenberg algorithm (NFIT; Island
Products, Galveston, TX). Statistical significance was determined by
unpaired t-test (SigmaPlot 2000 for Windows, version 6),
with significance ascribed at P < 0.01.
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RESULTS |
Contribution(s) of CaR expression to resting
intracellular Ca2+
concentration.
Activation of CaR is steeply dependent on extracellular
Ca2+. Figure 1A
illustrates a typical experiment in which extracellular Ca2+ is progressively increased from a baseline value of
0.5 to 30 mM, and intracellular Ca2+ is monitored with fura
2 fluorescence. Data compiled from 20 cells in this representative
experiment were fitted with the Hill equation (Fig. 1B) to
obtain the EC50 for extracellular Ca2+
(3.4 ± 0.4 mM) and the Hill coefficient (1.8 ± 0.4), as we
have previously reported (15). These results suggest that
exposure of cells expressing CaR to normal serum-containing media
(Ca2+ of ~1.8-2 mM) should partially activate the
receptor.

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Fig. 1.
Dose-dependent increase in intracellular Ca2+
on Ca2+-sensing receptor (CaR) activation. A:
HEK-293 cells expressing CaR-green fluorescent protein (GFP) were
exposed to incremental increases in extracellular Ca2+,
from a baseline of 0.5 mM. Responses from 20 cells (randomly chosen,
GFP positive) are illustrated. B: peak responses at each
extracellular Ca2+ concentration for cells in A
were averaged and fitted with the Hill equation (fit parameters:
maximum intracellular Ca2+ = 153 ± 10.7 nM;
EC50 = 3.39 ± 0.4 nM; n = 1.8 ± 0.4).
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The steady-state contributions of CaR activation on intracellular
Ca2+ in the presence of physiological levels of
extracellular Ca2+ were investigated with a pretreatment
protocol. HEK-293 cells expressing CaR were loaded with fura 2-AM at
various extracellular Ca2+ concentrations (0.5, 2, or 3 mM), and the Ca2+ imaging experiments were then begun in
the same concentration of extracellular Ca2+. Cells were
therefore maintained in a fixed concentration of extracellular
Ca2+ throughout loading and initiation of the
experiment (30-40 min). Intracellular Ca2+ was
determined at the start of the experiment (within the first minute of
recording). The averaged results of this type of experiment are
illustrated in Fig. 2A.
Culture, loading, and initiation of the experiment in elevated
extracellular Ca2+ (>0.5 mM) significantly increased both
intracellular Ca2+ and the range of intracellular
Ca2+ concentrations observed (Fig. 2A, 2 mM
Ca2+ > 0.5 mM Ca2+; 3 mM
Ca2+ > 2 mM Ca2+). Similar experiments
were performed on control HEK-293 cells (Fig. 2B); no
systematic effects on intracellular Ca2+ were observed,
i.e., intracellular Ca2+ concentration after 2 mM
preincubation was approximately equal to that after 3 mM preincubation
(although both had an intracellular Ca2+ concentration
higher than that observed after preincubation in 0.5 mM
Ca2+). Acute CaR-mediated changes in intracellular
Ca2+ were assessed by measuring the response to a decrease
in extracellular Ca2+ from 2 to 0.5 mM, followed by a
return to 2 mM extracellular Ca2+. The averaged responses
of 20 cells from a single experiment are illustrated in Fig.
2C (data representative of 3 independent experiments). There
was a statistically significant decrease in intracellular
Ca2+ in 0.5 mM extracellular Ca2+, with full
recovery on return to 2 mM Ca2+. Similar changes in
intracellular Ca2+ were not observed in untransfected
HEK-293 cells (Fig. 2D). Thus, by several criteria, cells
expressing CaR are sensitive to steady-state perturbations of
extracellular Ca2+ over the range of 0.5-3 mM and
respond with changes in the steady-state concentration of intracellular
Ca2+.

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Fig. 2.
Steady-state intracellular Ca2+ in cells expressing
CaR. A: HEK-293 cells transiently expressing CaR-GFP were
loaded with fura 2-AM in various Ca2+ concentrations (0.5, 2, and 3 mM), and experiments were begun in the same Ca2+
concentration. Intracellular Ca2+ concentrations were
measured during the first minute of recording. Data represent average
of >60 cells/condition from at least 3 independent transfections.
Significance was determined relative to 0.5 mM at P < 0.01 (*) or 2 mM at P < 0.01 (**). B:
untransfected HEK-293 cells were subjected to the same protocol as in
A, with significance determined relative to 0.5 mM at
P < 0.01 (*). C: 20 cells were exposed to 2 mM extracellular Ca2+, then to 0.5 mM, and then returned to
2 mM. Average intracellular Ca2+ at steady state in each
condition was measured. Significance is relative to 2 mM at
P < 0.01 (*). Data are representative of 3 independent
experiments. D: protocol similar to that of C
performed in untransfected HEK-293 cells.
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Contributions of CaR expression to
Ca2+ content of
thapsigargin-sensitive stores.
Expression of CaR has a significant effect on resting intracellular
Ca2+ concentrations when cells are exposed to extracellular
Ca2+ concentrations in the physiological range, i.e.,
0.5-3 mM (Fig. 2). A second compartment of intracellular
Ca2+ that might be affected by CaR expression (and chronic
activation) is the thapsigargin-sensitive store, which releases
Ca2+ on CaR activation (32). To assess the
content of thapsigargin-sensitive stores after exposure of
CaR-expressing HEK-293 cells to 0.5, 2, or 3 mM extracellular
Ca2+, experiments were begun in the loading
Ca2+ concentration to establish the resting intracellular
Ca2+ level, switched to a nominally zero Ca2+
bath solution, and then exposed to 1 µM thapsigargin in the continued absence of bath Ca2+. Figure
3, A-C, illustrates this
type of experiment in HEK-293 cells preincubated in 0.5, 2, and 3 mM
Ca2+, respectively, and Fig. 3, D-F,
illustrates the results obtained with CaR-expressing cells.

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Fig. 3.
Thapsigargin-sensitive Ca2+ stores in cells expressing
CaR. Intracellular Ca2+ was monitored in cells loaded with
fura 2-AM in 0.5 (A, D), 2 (B, E), or 3 (C,
F) mM extracellular Ca2+. Experiments were begun in
the same Ca2+ concentration. Extracellular Ca2+
was reduced to nominally zero, and then cells were exposed to 1 µM
thapsigargin. The entire time course of the thapsigargin-induced
intracellular Ca2+ transient was monitored. Experiments
illustrate the results from 20 untransfected HEK-293 cells
(A-C) or HEK-293 cells stably transfected with human
CaR (D-F). Experiments are representative of 3-5
independent experiments under each condition.
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To define the differences conferred by CaR expression, the total
thapsigargin-releasable Ca2+ content was determined by
measuring the area under the averaged Ca2+ transients of
experiments such as those illustrated in Fig. 3. As illustrated in Fig.
4A, there was no difference in
the magnitude of the thapsigargin-releasable Ca2+ store as
a function of extracellular Ca2+ concentration for either
HEK-293 cells or CaR-expressing cells. There was, however, a
significant difference between HEK-293 and CaR-expressing cells with
respect to the magnitude of the thapsigargin-releasable Ca2+ pool. The horizontal lines in Fig.
4A indicate an estimate of the thapsigargin-releasable
Ca2+ pool determined by averaging all data obtained, i.e.,
at 0.5, 2, and 3 mM Ca2+, for either HEK-293 cells
[11,744 ± 1,362 arbitrary units (AU)] or CaR-expressing
cells (9,325 ± 1,148 AU). Expression of CaR reduces the content
of the thapsigargin-sensitive Ca2+ store by ~20%, but
the content of this Ca2+ store is not altered by 30-40
min of incubation in extracellular Ca2+ concentration in
the range from 0.5 to 3 mM.

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Fig. 4.
Content but not kinetics of thapsigargin-sensitive
Ca2+ stores is altered by CaR expression. A:
experiments such as those illustrated in Fig. 3 were used to estimate
size of the thapsigargin-sensitive Ca2+ stores. Averages of
the thapsigargin-induced intracellular Ca2+ transients for
3-4 independent experiments from each condition (60-80 cells)
were transferred to Adobe Photoshop, and the pixel areas [in arbitrary
units (AU)] under the curves were calculated. There was a significant
difference in the average intracellular store content between HEK-293
cells and CaR-expressing cells (*P < 0.01).
B: half times for the rising (tR) or
declining (tD) phases of the
thapsigargin-induced intracellular Ca2+ transients for the
cells analyzed in A. There were no significant differences
between HEK-293 cells and CaR-expressing cells.
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Close inspection of the traces illustrated in Fig. 3 suggest that, in
addition to a significant decrease in the content of thapsigargin-releasable Ca2+ stores, expression of CaR also
increases the kinetic complexity of the intracellular Ca2+
transients. In particular, cells expressing CaR are more sensitive to
removal of bath Ca2+, e.g., HEK-293 cells (Fig.
3C) are relatively insensitive to switching from 3 mM to
zero bath Ca2+, whereas CaR-expressing cells (Fig.
3F) exhibit elevated intracellular Ca2+ (some
cells are undergoing intracellular Ca2+ oscillations) in 3 mM Ca2+, and this decreases significantly on removal of
extracellular Ca2+, confirming the results illustrated in
Fig. 2. Furthermore, the kinetics of the thapsigargin-induced
intracellular Ca2+ transient are more variable than those
exhibited by HEK-293 cells. To quantify the kinetic diversity, the half
times for the rising (tR) and declining
(tD) phases of the thapsigargin-induced
intracellular Ca2+ transient were determined for HEK-293
cells and CaR-expressing cells preincubated in various extracellular
Ca2+ concentrations (Fig. 4B). There were no
significant differences in half times for the thapsigargin response
when the averages of all cells in each condition were used to determine
half times (>60 cells/condition).
Small changes in extracellular
Ca2+ induce intracellular
Ca2+ oscillations.
While decreases in extracellular Ca2+ over the range from 3 to 0.5 mM cause CaR-mediated changes in the steady-state concentration of intracellular Ca2+ (Figs. 2 and 3), small incremental
increases in extracellular Ca2+ (0.5 mM) induce
oscillations of intracellular Ca2+. Illustrated in Fig.
5 is a typical experiment, in which
HEK-293 cells transiently transfected with CaR were loaded with fura
2-AM and the experiment was begun in 2 mM extracellular
Ca2+. Increases (0.5 mM increments) in extracellular
Ca2+ from 2 to 3.5 mM initiate oscillatory intracellular
Ca2+ changes, which are most pronounced at 3.5 mM
extracellular Ca2+. The oscillatory change in intracellular
Ca2+ ceased abruptly when extracellular Ca2+
was decreased to 2 mM. At the end of the experiment, the cells were
exposed to a concentration of extracellular Ca2+ that
maximally activates CaR (20 mM). This concentration of extracellular Ca2+ generated a rapidly activating peak of intracellular
Ca2+ (up to 200 nM), which decayed to an elevated plateau
for the duration of agonist exposure. This representative experiment
illustrates the range of responses to alterations in extracellular
Ca2+, from oscillatory changes in intracellular
Ca2+ at physiological levels of extracellular
Ca2+ to a maximal response that is typical of many G
protein-coupled receptors that activate the
phosphatidylinositol-phospholipase C (PI-PLC) pathway and
increase intracellular Ca2+.

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Fig. 5.
Small increases in extracellular Ca2+ induce
intracellular Ca2+ oscillations. HEK-293 cells transiently
transfected with CaR-GFP were loaded and the experiment was initiated
in 2 mM extracellular Ca2+. Extracellular Ca2+
was then increased in 0.5 mM increments up to 3.5 mM. Reducing
extracellular Ca2+ caused an immediate cessation of
oscillations. The experiment was ended by a step change in
extracellular Ca2+ to 20 mM, eliciting peak intracellular
Ca2+ release, which decayed to an elevated plateau.
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The threshold for CaR-mediated intracellular
Ca2+ oscillations.
The experiment illustrated in Fig. 5 suggests that the threshold for
intracellular Ca2+ oscillations is in the range of
2.5-3 mM extracellular Ca2+. To determine whether this
threshold is influenced by the starting extracellular Ca2+
concentration, HEK-293 cells expressing CaR were loaded with fura 2-AM
at fixed Ca2+ concentrations (0.5, 2, or 3 mM), and the
subsequent experiment was begun in the same extracellular
Ca2+ concentration. Extracellular Ca2+ was then
increased in 0.5 mM increments. Representative experiments begun in
0.5, 2, or 3 mM extracellular Ca2+ are illustrated in Fig.
6, A, B, and C,
respectively. Inspection of the plots reveals that cells do not
oscillate in the extracellular Ca2+ concentration in which
they have been loaded with fura 2-AM (although there are differences in
resting intracellular Ca2+ concentrations, as documented in
Figs. 2 and 3). Cells loaded in 2 or 3 mM extracellular
Ca2+ begin to oscillate after the first 0.5 mM increment in
extracellular Ca2+, with peak numbers of cells oscillating
after a 1 mM change from the initial Ca2+ concentration.
Cells loaded in 0.5 mM Ca2+ do not oscillate until
extracellular Ca2+ reaches 2 mM, with additional
recruitment of cells at concentrations up to 5 mM, the highest
concentration tested. The extracellular Ca2+ concentration
dependence of oscillation threshold, characterized as the percent of
cells oscillating in a given condition, was determined for three
independent experiments initiated at 0.5, 2, and 3 mM Ca2+,
and the results are illustrated in Fig. 6D.

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Fig. 6.
Threshold for intracellular Ca2+ oscillations.
A-C: HEK-293 cells transiently transfected with CaR-GFP
were loaded and experiments initiated in 0.5, 2, and 3 mM
Ca2+, respectively, and then exposed to 0.5 mM incremental
increases in extracellular Ca2+. Experiments were ended
with exposure to maximally activating concentrations of extracellular
Ca2+ (10-20 mM). D: threshold for
intracellular Ca2+ oscillations as a function of the
initial extracellular Ca2+ concentration. A total of 60 cells under each condition (20 cells/experiment, randomly chosen from
GFP-positive cells) were analyzed. E: peak to peak intervals
were measured for the cells illustrated in A-C and
plotted relative to the tested extracellular Ca2+
concentrations. Bars: solid, loaded in 0.5 mM Ca2+;
hatched, loaded in 2 mM; shaded, loaded in 3 mM. The dashed line
indicates the average of all data, independent of loading or test
Ca2+ concentration.
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Oscillation frequency is constant.
The dependence of the oscillation frequency on extracellular
Ca2+, characterized by the peak to peak duration, was
analyzed for the experiments in Fig. 6, A-C, plotted in
Fig. 6E. Regardless of the preconditioning extracellular
Ca2+, the peak to peak intervals of the oscillations
elicited over the range of extracellular Ca2+
concentrations from 2.5 to 5 mM were comparable; the average of all
conditions was 46.8 s (indicated by the dashed line in Fig.
6E).
Dependence of oscillations on intracellular
Ca2+.
Oscillations induced by small changes in extracellular Ca2+
were sustained for periods up to 30 min. An example is illustrated in
Fig. 7. In this experiment, the
oscillations were initiated by a step change in extracellular
Ca2+ from 2 to 3 mM, and intracellular Ca2+ was
monitored until oscillations dissipated. The insets in Fig. 7
illustrate the behavior of cells early (from 8 to 16 min) and late
(from 33 to 42 min) in the exposure to 3 mM Ca2+. This
experiment is representative of 12 experiments [3 independent transient transfections (n = 3 experiments each) plus 3 experiments in cells stably expressing CaR]. In all experiments,
oscillatory behavior ceased between 20 and 40 min after initiation by a
1 mM increase in extracellular Ca2+. Despite the cessation
of oscillations, the cells exhibited an elevated intracellular
Ca2+ in the presence of 3 mM extracellular
Ca2+, which decreased on return to 2 mM Ca2+,
e.g., Fig. 7.

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Fig. 7.
Intracellular Ca2+ oscillations are sustained for
30-40 min. Oscillations were initiated in cells stably expressing
CaR by a step from 2 to 3 mM extracellular Ca2+ and
followed until oscillations stopped. The experiment was ended by
exposure to 10 mM Ca2+ to assess the responsiveness of all
cells. Insets: the oscillatory behavior of the cells at
early (500-1,000 s) and late (2,000-2,500 s) times in the
experiment, depicting both the decrease in oscillating cells and the
rise in baseline intracellular Ca2+. Representative of >12
experiments in stably or transiently CaR-transfected HEK-293 cells.
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Figure 8A illustrates an
experiment designed to determine whether there is a relationship
between sustained oscillations and the level of Ca2+ in
intracellular stores. In this experiment, cells were exposed twice to
3.5 mM extracellular Ca2+, from a baseline extracellular
Ca2+ of 2 mM. Each time, there was an initial peak of
intracellular Ca2+ release, followed by oscillations that
were sustained for the duration of the exposure to 3.5 mM extracellular
Ca2+. The peak to peak intervals of the oscillations during
the first and second exposures to 3.5 mM Ca2+ were
comparable (Fig. 8B), while the initial peak release of intracellular Ca2+ on agonist addition decreased by 45%
and the baseline intracellular Ca2+ in 2 mM extracellular
Ca2+ decreased significantly during the experiment (Fig.
8C). Similar behavior was observed in >10 independent
experiments. These results suggest that neither the ability to support
sustained intracellular Ca2+ oscillations nor the
oscillation frequency is a simple function of the resting level of
intracellular Ca2+ or the degree of loading of
intracellular Ca2+ stores as inferred from the initial peak
response.

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Fig. 8.
Dependence of oscillations on intracellular
Ca2+. A: cells transiently expressing CaR-GFP
were twice exposed to 3.5 mM extracellular Ca2+ from a
baseline Ca2+ of 2 mM. B: oscillation intervals
were calculated for the first and second exposure to 3.5 mM
Ca2+ and were not significantly different. C:
peak intracellular Ca2+ was estimated during the first and
second exposures to 3.5 mM Ca2+, and baseline
Ca2+ was determined before, between, and after the 2 exposures to 3.5 mM Ca2+. In both cases, there was a
significant decrease in Ca2+ (*P < 0.01).
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Intracellular stores are, however, intimately involved in maintenance
of the intracellular Ca2+ oscillations, as indicated by the
experiments in Fig. 9. Figure 9A illustrates a control experiment in which oscillations
were induced by an increase in extracellular Ca2+ to 3.5 mM
from a baseline Ca2+ of 2 mM. Oscillations were sustained
until the experiment was ended with exposure of the cells to 10 mM
Ca2+ to elicit a maximal CaR-mediated response. The
companion experiment, illustrated in Fig. 9B, begins in the
same manner, but after 4 min of oscillations in 3.5 mM
Ca2+, 1 µM thapsigargin was added to the extracellular
solution. There was an immediate increase in intracellular
Ca2+ as uptake into the thapsigargin-sensitive stores was
blocked, followed by a slow decline in intracellular Ca2+.
Intracellular Ca2+ oscillations ceased abruptly on exposure
to thapsigargin, before the peak of the thapsigargin response. Thus
intracellular Ca2+ oscillations are obligatorily linked to
the integrity of the thapsigargin-sensitive intracellular
Ca2+ stores. Small step changes in extracellular
Ca2+ after the intracellular stores had been compromised by
thapsigargin application did not result in the establishment of
intracellular Ca2+ oscillations but, rather, generated a
monotonic increase in intracellular Ca2+ consistent with
influx of extracellular Ca2+ (data not shown).

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Fig. 9.
Effect of thapsigargin on intracellular Ca2+
oscillations. A: control experiment in which intracellular
Ca2+ oscillations were initiated by an increase in
extracellular Ca2+ from 2 to 3.5 mM. Experiment was ended
by exposure of the cells to 10 mM Ca2+. B:
experiment was begun as in A, but 1 µM thapsigargin was
added after 4 min in 3.5 mM Ca2+. Experiment was ended by
exposure of cells to 10 mM Ca2+. Note the reduced response
to 10 mM Ca2+, indicating compromised
thapsigargin-sensitive intracellular stores.
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Regulation of intracellular
Ca2+ oscillations.
Intracellular Ca2+ oscillations are observed in many cell
types, elicited by a wide variety of G protein-coupled receptors that activate the PI-PLC pathway, ultimately causing release of
Ca2+ from internal stores by activating intracellular
IP3 receptors, and often inducing influx of
Ca2+ from the extracellular medium (16). For
CaR-activated oscillations, the data in Fig. 9 indicate an integral
contribution of Ca2+ cycling into and out of intracellular
stores. A second widely observed mechanism regulating G protein-coupled
receptor activation is cyclical phosphorylation-dephosphorylation,
which has been shown to be responsible for mGluR5-mediated
intracellular Ca2+ oscillations in neurons (14,
22) and in heterologous expression systems (19).
The effects of several protein kinase inhibitors were therefore tested
for their ability to regulate CaR-mediated Ca2+
oscillations. Figure 10 illustrates the
results of typical experiments with staurosporine, a broad-specificity
protein kinase inhibitor that was applied at a concentration (100 nM)
that should inhibit protein kinases A (IC50
15 nM), C (IC50
7 nM), G (IC50
8.5 nM), myosin light chain kinase (IC50
1.3 nM),
and calmodulin kinase (IC50
20 nM). In the control
experiment, intracellular Ca2+ oscillations were elicited
by a step change in extracellular Ca2+ from 2 to 3.5 mM and
were sustained until a maximal response was elicited by 10 mM
extracellular Ca2+ at the end of the experiment (Fig.
10A). The companion experiment utilized the same
2.0-3.5 mM step in extracellular Ca2+ to elicit
oscillations, and then 100 nM staurosporine was applied during the
oscillations (Fig. 10B). In this and all other experiments of similar protocol, there was no effect on oscillation frequency on
addition of staurosporine, although, in some experiments, including that illustrated in Fig. 10B, there was a decrease in the
maximum intracellular Ca2+ response during oscillations. To
determine whether the lack of effect of staurosporine on oscillation
frequency was due to slow diffusion of the blocker into the cells, a
second protocol was used to assess the effect(s) of the protein kinase
inhibitor (Fig. 10C): oscillations were elicited by a step
from 2 to 3.5 mM Ca2+, followed by return to 2 mM
Ca2+, application of 100 nM staurosporine for 2 min, and
then a second step to 3.5 mM Ca2+ in the continued presence
of staurosporine. As with the protocol of Fig. 10B, there
was no effect of staurosporine on oscillation frequency despite the
2-min preexposure to the blocker. Experiments similar to that
illustrated in Fig. 10B were also performed with 1 µM
bisindolylmaleimide I and 5 µM KN-93, inhibitors of protein kinase C
(IC50
8-200 nM, depending on isoform) and
calmodulin kinase II (IC50
370 nM) , respectively.
Although the protein kinase inhibitors did induce a decrease in
baseline intracellular Ca2+ and the maximal peak
Ca2+ of the oscillations in some experiments, none of the
inhibitors blocked the oscillations or affected the peak to peak
interval of the oscillations (Fig. 10D). These results
suggest that protein kinases are not required for initiation or
maintenance of intracellular Ca2+ oscillations elicited by
CaR activation.

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|
Fig. 10.
Effect of protein kinase inhibitors on intracellular
Ca2+ oscillations. A: control experiment, as in
Fig. 9A. B: effect of staurosporine on
oscillations induced by 3.5 mM Ca2+. Cells were exposed to
3.5 mM Ca2+ for 4 min to permit determination of
oscillation interval, then 100 nM staurosporine was added in the
continued presence of 3.5 mM Ca2+. C: two-pulse
protocol, similar to that illustrated in Fig. 8A.
Immediately on washout of the first exposure to 3.5 mM, cells were
exposed to 100 nM staurosporine, and then, after 4 min, cells were
tested with a second exposure to 3.5 mM Ca2+ (in the
continued presence of staurosporine). D: summary of protein
kinase inhibitor studies on oscillation frequency. Data from 3 independent experiments (>60 cells) utilizing the protocol illustrated
in B were averaged for each inhibitor. Solid bars, no
blocker; open bars, protein kinase inhibitor. E: experiments
such as those in Fig. 6, A-C, were performed in the
continuous presence of 100 nM staurosporine, and the percent of
oscillating cells was determined at each Ca2+ concentration
>3 mM. Plotted are the average percent oscillating cells for 60 cells
from 3 independent experiments. The solid line is control data (absence
of staurosporine) redrawn from Fig. 6D. BIM,
bisindolylmaleimide I.
|
|
As a final test for the potential involvement of protein kinases in the
establishment or regulation of CaR-mediated intracellular Ca2+ oscillations, the ability of staurosporine to alter
the threshold for CaR-mediated oscillations was determined. Experiments
were performed as in Fig. 6C, with cells exposed to 3 mM
extracellular Ca2+ during fura 2-AM loading and experiment
initiation, with 100 nM staurosporine present throughout the
experiment. The percent of oscillating cells was determined over the
range of extracellular Ca2+ from 3 to 5 mM. The results are
illustrated in Fig. 10E, with the control data (absence of
100 nM staurosporine) redrawn from Fig. 6D. Staurosporine
induced an increase in the number of oscillating cells at every
extracellular Ca2+ concentration, although the oscillation
frequency was unaffected (data not shown, but see Fig. 10,
A-D). Protein kinase-dependent phosphorylation
therefore contributes to setting/resetting the threshold for
CaR-mediated intracellular Ca2+ oscillations.
 |
DISCUSSION |
This report delineates the complex intracellular Ca2+
responses resulting from activation of CaR by extracellular
Ca2+. Cells expressing CaR exhibit both modulation of
resting intracellular Ca2+ concentrations at extracellular
Ca2+ concentrations below the EC50 for CaR
activation and a decrease in net thapsigargin-sensitive
Ca2+ stores. Alterations in the resting intracellular
Ca2+ concentration may generate low-level activation of
Ca2+-sensitive signaling pathways, in particular those with
a high affinity for Ca2+. In addition, the low level of CaR
activation under these conditions may "sensitize" cells expressing
CaR to activation of other G protein-coupled receptors that also
utilize the PI-PLC pathway for cellular signaling. This sensitizing
effect has recently been seen in monocytes coactivated by extracellular
Ca2+ and the chemokine MCP-1 (25).
Increasing extracellular Ca2+ by as little as 0.5 mM in the
range around the EC50 for CaR activation generates
low-frequency (40- to 50-s peak to peak interval) intracellular
Ca2+ oscillations. The oscillation interval is independent
of the extracellular Ca2+ concentration, although the
number of cells responding to the perturbation of extracellular
Ca2+ peaks at ~1 mM above the ambient
extracellular Ca2+. Cells expressing CaR acclimate to the
ambient concentration of extracellular Ca2+ with a
cessation of oscillations after 30-40 min (Fig. 7). The threshold
for initiation of intracellular Ca2+ oscillations is thus
reset by the concentration of extracellular Ca2+ to which
the cells are chronically exposed. This "resetting" of the
threshold for initiation of oscillations is not due to alterations in
the Ca2+ content of intracellular stores (Fig. 4). In
parathyroid cells in vivo, increases in extracellular Ca2+
>1.8 mM induce oscillations with an interval of 42 s
(21). Our results suggest that this oscillatory behavior
(oscillation interval, threshold) can be conferred on a heterologous
cell type by expression of CaR and therefore must be inherent in either CaR or highly conserved elements of the PI-PLC signaling pathway.
Many G protein-coupled receptors that activate the PI-PLC pathway
generate intracellular Ca2+ oscillations over a limited
agonist concentration range. Oscillation frequencies range from very
rapid "pacemaker" activity (1 Hz) that signifies elemental
Ca2+ release events (4) to intermediate
(3-4 min
1) (2), to slow (<1
min
1) (12). Agonist concentration-dependent
alterations in oscillation frequency have also been noted (10,
12, 23, 27, 28). While we observed relatively slow intracellular
Ca2+ oscillations, there was no systematic increase in
oscillation frequency over the limited range of extracellular
Ca2+ (agonist) concentrations tested. It should be noted
that CaR is unique in that its agonist is Ca2+; therefore,
increases in extracellular Ca2+ mediate receptor activation
and increase the driving force for Ca2+ influx through a
variety of permeability pathways. This may limit the range of agonist
concentrations that can be explored, since increasing extracellular
Ca2+ from 2 to 5 mM is sufficient to induce a peak
Ca2+ response, which decays to a sustained plateau, largely
eliminating intracellular Ca2+ oscillations. Nevertheless,
small increases in extracellular Ca2+ "recruit" cells
to oscillate, and the net effect at the level of the parathyroid may be
to increase the fraction of cells in which parathyroid hormone
secretion is modulated.
A number of mechanisms have been proposed for initiation and
maintenance of intracellular Ca2+ oscillations, most
requiring cyclical alterations in the state of the Ca2+
release system (16), generally involving fluctuations in
either the cellular levels of IP3 or the activity of
IP3 receptors in the endoplasmic reticulum. Thapsigargin
rapidly disrupts CaR-initiated intracellular Ca2+
oscillations, implying that intracellular stores are intimately involved in their initiation and maintenance. In systems in which the
intracellular Ca2+ oscillations require plasma membrane
level Ca2+ influx, thapsigargin has no effect
(30). We cannot exclude a contribution of Ca2+
influx, since we cannot eliminate Ca2+ from the
extracellular solution and maintain CaR activation. We attempted to
address the question of Ca2+ influx during oscillations by
utilizing Mn2+ uptake and quench (29), but
found that this approach was not feasible since Mn2+ acted
as an agonist for CaR in the range required for quench of fluorescence
(data not shown). With respect to receptors having the closest homology
with CaR, namely group I metabotropic glutamate receptors,
mGluR5-induced intracellular Ca2+ oscillations have been
noted both in vivo (14, 22) and in vitro (19)
and result from the cyclical protein kinase C-mediated phosphorylation
and dephosphorylation of the receptor itself, at a threonine residue
within the carboxy terminus (19). Sequence alignment of
CaR with mGluR5 suggests that the critical threonine of mGluR5 is
present in CaR, although it is not part of a consensus sequence for
protein kinase C phosphorylation in CaR (1, 8). Furthermore, we have generated truncations of the carboxy terminus in
which all of the consensus sites for protein kinases A and C
phosphorylation were removed (15). These truncated forms
of human CaR were also observed to oscillate (data not shown).
In this report, we have tested the effects of a variety of protein
kinase inhibitors, with a particular focus on those that are regulated
by Ca2+. Neither staurosporine, bisindolylmaleimide I, nor
KN-93 altered the frequency of oscillations elicited by extracellular
Ca2+. Protein kinase C-mediated phosphorylation of CaR
causes a reduction in Ca2+ affinity of the receptor
(1, 8, 26). For the cholecystokinin receptor, a similar
protein kinase C-mediated decrease in agonist affinity of the
cholecystokinin 8 receptor is responsible for the cessation of
intracellular Ca2+ oscillations (31). It is
therefore possible that protein kinase C-mediated phosphorylation of
CaR at low agonist concentrations is responsible for the shift in
threshold for initiation of oscillations observed in the
"Ca2+ pretreatment" protocol, since staurosporine, a
broad-specificity protein kinase inhibitor, increased the number of
cells oscillating at each extracellular Ca2+ concentration
(Fig. 10E). Further work on protein kinase C-specific site
mutants of CaR will be needed to explicitly address this possibility.
Parathyroid hormone secretion is inversely controlled by extracellular
Ca2+, i.e., increases in extracellular Ca2+
result in decreases in parathyroid hormone secretion (32). Despite intensive efforts, a fundamental question with regard to CaR
regulation of parathyroid hormone secretion remains the locus of the
inversion signal, given the observation that CaR activation mediates an
increase in intracellular Ca2+. A confocal study that
followed subcellular changes in intracellular Ca2+ on a
step change in extracellular Ca2+ from 0.5 to 2 mM noted
significant time-dependent inhomogeneities in intracellular
Ca2+ (9). In this study, we have shown that
global intracellular Ca2+ also exhibits oscillations over
this concentration range. It is possible that the global and local
oscillatory changes in intracellular Ca2+ contribute to the
inversion of the secretory response. At a minimum, any model for
CaR-mediated regulation of parathyroid hormone secretion must
incorporate intracellular Ca2+ oscillations.
CaR contributes to the regulation of diverse physiological functions,
and there may be unique implications of CaR-mediated intracellular
Ca2+ oscillations that depend on cell type. It is
interesting to note, however, that intracellular Ca2+
oscillations have been recognized recently as an extremely potent and
specific means of increasing gene expression (11, 17, 20).
While direct contributions of CaR activation to changes in gene
expression have not been explicitly determined in most systems, there
is evidence in the Caco-2 intestinal epithelial cell line for rapid,
CaR-mediated increases in c-myc expression (18). In addition, CaR activation potently mediates
keratinocyte differentiation (3). Our results suggest that
another important locus for CaR-mediated signaling that should be
explored is the dynamic regulation of gene expression via intracellular
Ca2+ oscillations.
 |
ACKNOWLEDGEMENTS |
We sincerely thank Dr. John Carroll (Johns Hopkins Hospital,
Department of Pediatrics) for access to the single-cell calcium-imaging equipment and Dr. Michael Wasicko for advice on its use. Lynn Baxendale-Cox (1956-2000) was the inspiration for this work.
 |
FOOTNOTES |
This work was supported by National Institute of General Medical
Sciences Grant GM-58578 to G. E. Breitwieser.
Address for reprint requests and other correspondence: G. E. Breitwieser, Dept. of Physiology, Johns Hopkins Univ. School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205 (E-mail:
gbreitwi{at}pop.jhmi.edu).
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.
Received 28 June 2000; accepted in final form 3 January 2001.
 |
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