1 Dipartimento di Fisiologia Generale ed Ambientale, Universitá di Bari,
Via Amendola 165/A, I-70126 Bari, Italy
2 West Roxbury VAMC and the Department of Surgery, Harvard Medical School, West
Roxbury, MA 02132, USA
* Author for correspondence (e-mail: ahofer{at}rics.bwh.harvard.edu)
Accepted 14 January 2003
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Summary |
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Key words: Calcium oscillations, Extracellular signals, Intercellular communication
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Introduction |
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It is important to note that CaR is also stimulated by Mg2+ and
certain polycations [including some endogenous polyamines such as spermine and
spermidine (Quinn et al.,
1997)]. Synthetic small molecule agonists of the receptor, such as
the allosteric modulator NPS R-467 (Nemeth
et al., 1998
), have also been prepared. Brown and colleagues
recently showed that the CaR is activated by physiological concentrations of
amino acids [particularly aromatic and small aliphatic L-amino acids
(Conigrave et al., 2000b
)],
and that it is modulated by ionic strength
(Quinn et al., 1998
). It is
therefore likely that multiple agonists act in concert to stimulate the
receptor physiologically.
We noted previously that stimulation of CaR with maximal doses of CaR
agonists was frequently manifested by an oscillatory Ca2+ signal in
HEK293 cells transfected with the human extracellular Ca2+-sensing
receptor (HEK CaR cells) (Hofer et al.,
2000). The same observation was made in CaR-transfected HEK cells
by Breitwieser and Gama (Breitwieser and
Gama, 2001
), who also reported that Ca2+ spiking was
acutely sensitive to small incremental increases in extracellular
[Ca2+]. Such oscillations were sustained for up to 40 minutes,
until they gradually diminished in amplitude. Young and Rozengurt recently
extended these findings, showing that aromatic amino acids can sensitize the
receptor to small incremental increases in external [Ca2+] in HEK
CaR cells, producing distinctive patterns of oscillations
(Young and Rozengurt, 2002
).
Native tissues that express CaR endogenously, including parathyroid cells
(Miki et al., 1995
), thyroid
follicular cells (McGehee et al.,
1997
) and pancreatic acinar cells
(Bruce et al., 1999
), have
also been observed to oscillate following stimulation with maximal doses of
CaR activators, hinting that this mode of signaling may be a fundamental
characteristic of the receptor.
The mechanisms underlying Ca2+ oscillations have been the
subject of intense investigation ever since the first direct observations of
this phenomenon were made by Cuthbertson and Cobbold in aequorin-loaded mouse
oocytes (Cuthbertson and Cobbold,
1985). Numerous models have been put forth to account for
Ca2+ oscillations (Berridge et
al., 2000
; Bootman et al.,
2001
; Thomas et al.,
1996
; Thorn et al.,
1993
). Prominent among these are themes involving sensitization of
intracellular Ca2+ release channels by Ca2+ ions
(Patel et al., 1999
;
Yule, 2001
). Certain inositol
(1,4,5)-trisphosphate (InsP3) receptor subtypes are in fact known
to have a bell-shaped dependency of open probability on cytoplasmic
[Ca2+], leading to alternate activation and inhibition of
Ca2+ release as cellular [Ca2+] rises
(Bezprozvanny et al., 1991
;
Mak et al., 1998
;
Moraru et al., 1999
).
Oscillations in internal store [Ca2+] have been observed directly
in permeabilized cells, indicating a minimal requirement for intact functional
stores and InsP3 receptors
(Hajnoczky and Thomas, 1997
).
These and other data (Miyakawa et al.,
2001
) would suggest that oscillations are an intrinsic property of
InsP3-sensitive internal Ca2+ stores.
Other mechanisms for Ca2+ oscillations have been described that
rely on negative feedback exerted on PLC by PKC
(Bird et al., 1993;
Taylor and Thorn, 2001
).
Following activation of G-protein-coupled cell surface receptors, PLC
hydrolyzes phosphatidylinositol (4,5)-bisphosphate (PIP2) into DAG
and InsP3. DAG activates PKC, which, according to several models,
exerts negative effects on PLC, resulting in oscillatory InsP3
production. Confirmatory evidence for this model has been provided recently by
experiments in which PIP2 hydrolysis was measured in real-time in
single living cells, using genetically encoded GFP-based fluorescent sensors.
Meyer and colleagues (Codazzi et al.,
2001
) and Ferguson and colleagues
(Dale et al., 2001
) recently
showed that InsP3 levels in the cell, as well as PKC translocation
to the plasma membrane do indeed oscillate, and with a time course that
closely parallels that of Ca2+ spiking. These experiments confirm
that InsP3 is produced cyclically and may indicate that negative
feedback loops involving PKC contribute to shaping this particular type of
oscillatory behavior. Using similar techniques, Nash et al., also showed that
stimulation of metabotropic glutamate receptor subtype 5a (mGluR5a) resulted
in Ca2+ oscillations concurrent with oscillations in intracellular
[InsP3] (Nash et al.,
2001b
). However, oscillations in [InsP3] were not noted
when the same cells were stimulated with a different Ca2+
mobilizing agonist, methacholine, acting through muscarinic M3
receptors, even though oscillations in intracellular [Ca2+] were
observed under these conditions. Thus it appears that different mechanisms for
generating Ca2+ oscillations can co-exist even in the same
cell.
We previously reported that heterotypic assemblages of cells may
communicate with each other through extracellular [Ca2+] increases
that result from the extrusion of cellular Ca2+ across the plasma
membrane during Ca2+ signaling events
(Hofer et al., 2000;
Thomas, 2000
). These external
[Ca2+] increases are detected by CaR on adjacent cells, activating
signaling cascades (including intracellular Ca2+ signals) in those
cells. Moreover, using Ca2+-selective microelectrodes, we recently
performed direct measurements of extracellular [Ca2+] changes in
external microdomains of intact polarized gastric epithelium following
stimulation with a Ca2+-mobilizing agonist
(Caroppo et al., 2001
). We
found that substantial increases in external [Ca2+] (up to 0.5 mM)
occurred at the apical face of the tissue in restricted microdomains near the
membrane. Our data indicated that these changes were a consequence of
PMCA-mediated Ca2+ extrusion from stimulated cells. These findings
open up the possibility that the PMCA may exert feedback effects on CaR
located on the same or adjacent cells.
In the present study we provide evidence for a novel autocrine mechanism that contributes to sustained Ca2+ signaling in CaR-expressing HEK-293 cells. Our data indicate that Ca2+ extruded to the extracellular space by the plasma membrane Ca2+ ATPase (PMCA) following intracellular Ca2+ spikes exerts a positive feedback effect on CaR by increasing local external [Ca2+]. This additional stimulus enhances the likelihood of generating subsequent spikes in Ca2+. Although it is probable that other `classical' mechanisms are responsible for the genesis of Ca2+ oscillations in these cells, our data would suggest that cycling of Ca2+ across the plasma membrane following an intracellular Ca2+ transient reinforces CaR stimulation, and may modulate the periodicity of rhythmic spiking.
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Materials and Methods |
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Measurement of intracellular [Ca2+]
CaR-transfected HEK cells were plated onto specially prepared glass
coverslips as described previously (Hofer
et al., 2000). Briefly, randomly arranged polypropylene fibers
were melted onto the coverslip, resulting in the formation of small pockets
between the fibers and the glass. Following plating, cells grew into these
restricted spaces, and were thus partially shielded from the bulk solution.
Only cells growing under such polypropylene pockets were selected for
Ca2+ imaging experiments. Cells were cultured in this way for 48
hours and loaded with 2 µM fura-2 AM in DMEM for 40 minutes at 37°C.
Coverslips were then mounted in an open-topped perfusion chamber (Series 20,
Warner Instrument, Hamden CT) and placed on the stage of an inverted
microscope (Olympus IMT-2). Test agents (diluted to final concentration in
Ringer's) were often added directly to the chamber while the perfusion was
stopped, resulting in complete exchange of the chamber volume (1 ml). In
control experiments, addition of Ringer's solution alone to the chamber did
not affect Ca2+ oscillations (not shown).
During the measurements cells were bathed with a Ringer's solution at
33°C containing 145 mM NaCl, 4 mM KCl, 0.5 mM MgCl2, 10 mM
HEPES, pH 7.45 and CaCl2 at the concentrations indicated in the
figures. Selected clusters of cells were excited alternately at 345 nm and 375
nm by a xenon arc lamp. Emitted fura-2 fluorescence at 510 nm was recorded
every 5 seconds (frame averaging=16) by an intensified charge-coupled device
camera (model IC-100; Photon Technology International, South Brunswick, NJ)
and converted to pseudocolor images using ImageMaster 1.4 software (Photon
Technology International). The data were expressed as the ratio of the
fluorescence emitted at the two fura-2 excitation wavelengths
(F340/F380). In a few imaging studies, fura-2
measurements were carried out using an alternate ratio imaging set-up running
MetaFluor software (Universal Imaging, West Chester, PA). `n' refers
to the number of independent experimental runs; data from 5 to 50 individual
cells in the microscope field was collected and analyzed for each experiment.
Statistical analyses were performed using InStat 2.03 (GraphPad software)
using a paired t-test. Data are expressed as ±s.e.m., and
differences considered highly significant when P<0.005; a
P-value calculated by the program is provided for many of the data
sets if very different from P=0.005. Representative tracings from
single cells are shown. Calibrations were performed for some experiments
according to the method of Grynkiewicz et al., assuming a
Kd for fura-2 of 224 nM
(Grynkiewicz et al.,
1985).
Measurement of extracellular near-membrane [Ca2+] with
Fura-C18-HEK CaR cells plated on glass coverslips without
polypropylene mesh were incubated with 10 µM fura-C18
(Etter et al., 1994) in normal
Ringer's for 3 minutes on the microscope stage and then rinsed in dye-free
Ringer's. The fura-C18 ratio (F340/F380) was
measured as described above for fura-2. The extracellular location of the dye
was confirmed by quenching with 5 mM NiCl2 in Ca2+-free
solution, which very rapidly reduced fluorescence intensity at both
wavelengths to that of cellular autofluorescence (determined in the same cells
prior to loading with dye). Treatment of fura-2-AM loaded cells with 5 mM
NiCl2 had no effect whatsoever on intracellular fura-2
fluorescence, confirming that Ni2+ is indeed impermeant to HEK293
cells.
Immunofluorescence staining
CaR-expressing HEK293 cells were washed in PBS (137 mM NaCl, 2.6 mM KCl, 10
mM Na2HPO4, 1.8 mM KH2PO4) and
fixed in methanol for 10 minutes at 20°C. After rinsing in PBS at
room temperature (25°C) cells were pretreated for 1 hour in PBS containing
1% BSA and 1% normal goat serum (used as a blocking solution to reduce
nonspecific antibody binding). Samples were then co-incubated with a rabbit
polyclonal anti-CaR antibody directed against amino acid residues 344-358 of
the human CaR receptor diluted 1:100 in blocking solution, and with a mouse
monoclonal anti-PMCA antibody, diluted 1:200 (Affinity BioReagents, Golden,
CO), overnight at 4°C in a humidified chamber. Following five washes with
1% BSA in PBS, slides were incubated with goat anti-rabbit
Alexa-Fluor-488-conjugated IgG (Molecular Probes, Eugene, OR) and goat
anti-mouse affinity-purified Cy5-conjugated IgG (Jackson ImmunoResearch
Laboratories, West Grove, PA), each diluted 1:500, for 1 hour at room
temperature (25°C). After washing three times in PBS and once in distilled
water, coverslips were mounted onto microscope slides with Pro-Long Antifade
Kit (Molecular Probes, Eugene, OR). Controls for background and for
nonspecific binding of the secondary antibody for PMCA were performed by
omitting the anti-PMCA antibody. Controls for the nonspecific binding of the
CaR antibody entailed co-incubation of samples with the CaR antibody and the
immunizing peptide against which the antibody was made.
Confocal microscopy
Confocal fluorescence images (1 µm sections) were obtained using a
Bio-Rad (Hercules, CA) MRC1024ES multi-photon/confocal system with a
krypton/argon ion laser excitation source. Cells were viewed with a Zeiss
(Oberkochen, Germany) Axiovert S100 inverted microscope equipped with a
high-quality c-apochromat water immersion objective (40x; 1.2 numerical
aperture) in epifluorescence mode. The 512x512 pixel images were
collected in a direct detection configuration at a pixel resolution of 0.484
µm with a Kalman 5 collection filter. Multiple labeled images were acquired
in separate channels using narrow bandpass filters to restrict the emission
wavelengths. Merged images of immunofluorescence staining were reconstructed
using Metamorph software (Universal Imaging, West Chester, PA).
Confocal imaging of intercellular spaces
Cells were bathed in 10,000 Mr fluorescein-dextran (1
mg/ml in Ringer's solution), and a z-series (1 µm) of 25 to 40 confocal
sections through a given cell cluster were acquired as described above using
the 488 nm line of the krypton-argon laser (1% power; iris setting 1.4). The
resulting stacks of images were processed and reconstructed into a side view
profile (3 pixels or 1.452 µm in depth) using the `kymograph' function of
the Metamorph program.
Chemicals
NPS R-467 was a generous gift of E. Nemeth (NPS Pharmaceuticals, Toronto,
Ontario, Canada). Fetal bovine serum was obtained from Life Technologies
(Grand Island, NY). BAPTA tetrasodium salt, fura-2 acetoxymethyl ester,
fura-C18 (pentapotassium salt), and fluorescein dextran (10,000 MW)
were purchased from Molecular Probes (Eugene, OR). A stock solution of
fura-C18 (1 mM) was prepared in Ringer's solution containing 1 mM
CaCl2. Caloxin 2A1 peptide (VSNSNWPSFPSSGGG-NH2) was
prepared by custom synthesis from Dalton Chemical Laboratories, Toronto ON,
Canada. All other reagents were all purchased from Sigma (St Louis, MO).
Preparation of BAPTA-Ca2+ buffer-A buffered Ringer's solution
was prepared containing 1 mM BAPTA free acid and 0.49 mM CaCl2 to
yield an estimated free [Ca2+] of approximately 180 nM. Free
[Ca2+] was calculated with the MaxChelator program (`WEBMAXC
v2.10';
http://www.stanford.edu/~cpatton/maxc.html).
For the unbuffered solution, 10 µM BAPTA free acid was added to a nominally
Ca2+-free Ringer's solution (containing approximately 5 µM
contaminating Ca2+) to yield a calculated free [Ca2+] of
175 nM. To confirm that the actual free [Ca2+] was matched in each
of the solutions, HEK293 cells loaded with fura-C18 were exposed
sequentially to the two buffers during continuous measurement of the
fura-C18 ratio. These measurements revealed that the free
[Ca2+] in the buffered solution (1 mM BAPTA/0.49 mM
CaCl2) was approximately 200 nM, whereas the unbuffered solution
(10 µM BAPTA) had a lower ratio corresponding to a free [Ca2+]
that was approximately 150 nM. The fura-C18 ratio was converted
into rough approximations of free [Ca2+] using calibration
techniques described previously (Etter et
al., 1994).
Preparation of citrate-Ca2+ buffers
A modified Ringer's solution was prepared using 20 mM sodium citrate (NaCl
substitution), pH 7.45. Ringer's solutions containing 1 mM or 0.65 mM added
CaCl2 were used as the control (unbuffered Ringer's) against which
the free [Ca2+] was matched in the buffered (+ citrate) solution.
Free [Ca2+] was matched precisely using a Ca2+-selective
electrode (model 97-20 ionplus, Orion Research, Beverly, MA) for each
solution. The Ca2+-buffering power of the citrate Ringer's solution
is illustrated in Fig. 1E.
Addition of CaCl2 >600 µM was necessary to start detecting an
increase in the measured free [Ca2+] in the buffered solution. The
Ca2+-buffering power of the buffered Ringer's solution was at least
twice as great as that of the control solution at 2 mM Ca2+, since
the free [Ca2+] was elevated by less than half as much in the
citrate-containing solution compared to control.
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Results |
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We considered the possibility that a component of the oscillatory phenomenon observed in the present study might result from a particular form of autocrine and/or paracrine signaling involving CaR and the PMCA. According to our hypothesis, Ca2+ spiking, once initiated, would result in Ca2+ extrusion via the PMCA that activates CaRs on the same cell (or adjacent cell) through local increases in [Ca2+]out. This would reinforce the initial stimulus, promoting the initiation of a second spike in intracellular Ca2+.
In order to test this hypothesis, cells were seeded onto special glass
coverslips, as described previously (Hofer
et al., 2000), and allowed to grow underneath small lacunae
created between fused polypropylene microfibers and the glass of the coverslip
(see Materials and Methods for details). In this way cells were maintained in
a restricted extracellular volume shielded from the bulk solution, as an
approximation of the limited extracellular volumes found in intact
tissues.
We first tested the effects of extracellular Ca2+ buffers on CaR-mediated Ca2+ signaling. HEK CaR-expressing cells were stimulated with 1 mM spermine in nominally extracellular free Ca2+ to which BAPTA free acid (used at concentrations ranging from 10 to 60 µM) was added to the bath to reduce [Ca2+] to very low levels (from 175 nM to 17 nM, respectively). Under these conditions spermine was still able to induce repetitive intracellular Ca2+ spikes (Fig. 1A). The intracellular [Ca2+] at the peak of the initial spike was estimated to be 410.7±21.5 nM, starting from a resting value that averaged 73.8±6.6 nM (n=5 calibrated experiments; data from 93 cells). Oscillations were sustained for 5-10 minutes until they gradually dampened out (typical of n=19 independent experiments; 347 cells), presumably due to the depletion of intracellular stores that could not be refilled by calcium entry mechanisms (i.e. store-operated channels, or SOCs). This demonstrates that oscillations mediated by CaR resulted from the release of Ca2+ from intracellular stores triggered by CaR and did not depend, at least over the short-term, on entry of external Ca2+. However, as seen in Fig. 1B, increasing BAPTA to 1 mM (thereby greatly increasing the external buffering capacity for Ca2+, while still maintaining free Ca2+ at a little less than 2 nM) reversibly blocked or attenuated intracellular spiking (n=7; 65 cells). In other experiments (not shown), the presence of 1 mM BAPTA did not significantly alter the speed or magnitude of the initial spike of Ca2+ following spermine addition as compared to the spike in the same cells in the presence of 50 µM BAPTA (n=5 experiments; 85 cells). Thus it does not appear that BAPTA interferes with the binding of the agonist to CaR. To ensure that the difference in the oscillatory response was not due to the fact that [Ca2+] was slightly lower in the solution containing 1 mM BAPTA as compared to the solutions containing 10-60 µM BAPTA, experiments were also performed in solutions where free [Ca2+] was matched but the total [BAPTA] was varied (see Materials and Methods for details). Fig. 1C compares a control stimulation to spermine in nominally Ca2+-free Ringer's containing 10 µM BAPTA (measured free [Ca2+] approximately 150 nM) to the response in a solution containing 1 mM BAPTA and 0.49 mM CaCl2 (measured free [Ca2+] approx. 200 nM). Oscillations were consistently attenuated in the highly buffered solution, even though the free [Ca2+] was actually slightly elevated in that solution (typical of n=4 experiments; 93 cells).
A variation of this experiment in the presence of higher external [Ca2+] is shown in Fig. 1D,E, in which 20 mM citrate was used as the Ca2+ buffer, allowing an external [Ca2+] that more closely matches physiological concentrations (i.e. around 1 mM Ca2+). Ca2+ signals stimulated by spermine were compared in the same cells under control conditions (1 mM added Ca2+ in the bath) with those elicited in the presence of the Ca2+ buffer (with the free Ca2+ matched to the control solution as described in Materials and Methods). As seen in Fig. 1D, the presence of the buffer dramatically reduced the frequency of the oscillations (n=2, 56 cells). As shown in Fig. 1E, this effect was reversible (n=4, 85 cells). Cells were initially stimulated with spermine in a control Ringer's that contained 0.65 mM added Ca2+, and the solution switched between buffered and unbuffered during oscillatory spiking (the lower [Ca2+] was used in these later experiments because it more closely matches the Kd of citrate for Ca2+, but this did not significantly affect the Ca2+ buffering power of the solution). There was a marked effect of the buffer on both the amplitude and frequency of the oscillations. In contrast, stimulation of HEK CaR cells with carbachol and ATP (to activate endogenous muscarinic and purinergic receptors, respectively) in the presence of the citrate buffer yielded a response that was similar to control (Fig. 1F), demonstrating that the Ca2+ buffer itself did not have deleterious effects on cellular Ca2+ signaling machinery. Moreover, some cells were observed to oscillate (on top of an elevated plateau), showing that Ca2+ spiking elicited by carbachol/ATP was insensitive to the citrate buffer (typical of n=4 experiments, 70 cells). The buffering power of the citrate solution used in the experiment shown in Fig. 1D is shown in Fig. 1G.
In a second type of study, we explored the role of the PMCA in the
maintenance of intracellular oscillations. As shown in
Fig. 2A, 100 nM
HgCl2, a potent but highly nonspecific PMCA antagonist that does
not interact with fura-2, and is not a ligand of the CaR, was added acutely to
oscillating cells. HgCl2 blocked oscillations mediated by spermine
in Ca2+ free solutions (n=3 experiments, 42 cells), and
also blocked oscillations elicited by NPS-R-467 (5 µM) in the presence of 1
mM Ca2+ (not shown; n=2 experiments, 28 cells). As shown
in Fig. 2B, the compound did
not affect the initial release of Ca2+ from stores (typical of
n=4 experiments, 25 cells). In contrast to the actions of
HgCl2 on CaR-mediated oscillations, the agent did not appreciably
block repetitive spiking induced by carbachol/ATP in HEK CaR cells, as shown
in Fig. 2C. This record also
illustrates that HgCl2 is an effective inhibitor of the PMCA. The
initial peak following carbachol/ATP stimulation was significantly
(P<0.0001) greater in the presence of HgCl2 (120% of
control in the same cell; peak ratio 0.779±0.017 for control vs.
0.9435±0.0331 with HgCl2; n=3 experiments; data
from 35 cells), and the return to baseline following Ca2+ removal
in the continued presence of agonist (largely a measure of PMCA-mediated
extrusion) was likewise significantly retarded, by more than two-fold
(relative slope of 0.0020±0.0002 for control vs. 0.0009±0.001 in
the presence of HgCl2; P<0.0001). We also tested the
effect of HgCl2 on Ca2+ spiking induced by low
concentrations of carbachol (10 µM) in HEK293 WT cells. The phenomenon of
baseline spiking induced by low doses carbachol in HEK WT cells has been well
studied, and seems to be highly dependent on influx of Ca2+ through
entry pathways that continue to be characterized
(Luo et al., 2001;
Shuttleworth, 1996
). Thus 1.5
mM Ca2+ was present in the external medium for these control
experiments on WT cells, which showed that HgCl2 caused a slight
slowing of carbachol-induced oscillations
(Fig. 2D; n=8, 98
cells). This effect may be the consequence of inhibition of Ca2+
entry by HgCl2, which is known to block some Ca2+
channels. This effect was very different, however, from the fast inhibition
observed in the CaR-expressing cells shown in
Fig. 2A.
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The actions of a different PMCA inhibitor, sodium orthovanadate
(Qi et al., 2000), are shown
in Fig. 2E. Since this compound
acts on a cytoplasmic site of the pump, relatively high concentrations (2 mM)
were used to insure penetration into cells. Ca2+ oscillations in
HEK CaR cells elicited by 1 mM spermine in Ca2+-free solutions were
significantly blocked by addition of VO43 to the
bath (n=4, 46 cells). In contrast, in control experiments
(Fig. 2F), Ca2+
spiking induced in non-transfected HEK293 WT cells by 10 µM carbachol was
completely unaffected in 42% of the cells by addition of vanadate to the
external solutions. In the remaining 58% of cells, however, an elevated
plateau phase was observed as illustrated in
Fig. 2G (n=6, 153
cells total). This latter effect might be partially explained by inhibitory
actions of vanadate on the SERCA (Sarco-Endoplasmic Reticulum Calcium ATPase),
which would prevent Ca2+ clearance from the cytoplasm into the ER,
and lead to greater entry of Ca2+ though SOCs by preventing store
refilling. Overall it appears that PMCA inhibition does not have a dramatic
effect on oscillations in WT cells. This conclusion is consistent with
previous reports of Morgan and Jacob
(Morgan and Jacob, 1998
) who
found that inhibiting the PMCA with La3+ (which could not be used
in the present study because it is an agonist of CaR) had little effect on
Ca2+ oscillations in endothelial cells. Moreover, Green et al.,
also showed that another PMCA inhibitor, carboxyeosin (which also could not be
used here due to its intense fluorescence) did not block Ca2+
oscillations in rat hepatocytes as measured by the luminescent photoprotein
aequorin (Green et al.,
1997
).
The PMCA inhibitors HgCl2 and VO43
are not ideal. HgCl2 unfortunately has many well-recognized
cytotoxic effects, including induction of free radical formation, lipid
peroxidation, and other actions resulting from its ability to form strong
bonds with SH groups of proteins. Likewise,
VO43 inhibits all P-type ATPases, including the
SERCAs. Breitwieser and Gama (Breitwieser
and Gama, 2001) demonstrated that exposure of HEK293
CaR-expressing cells to thapsigargin (1 µM), a specific blocker of SERCAs,
completely eliminated Ca2+ oscillations induced by increasing
[Ca2+] in extracellular bath from 2 to 3.5 mM (although the
presence of elevated Ca2+ in the bath might obscure the effects on
oscillations due to the increased entry of Ca2+). Nevertheless the
experiments with VO43 must be interpreted with
caution, due to the inhibitory effects of this agent on SERCAs. We therefore
tested a more specific PMCA antagonist, Caloxin 2A1, a recently described
peptide inhibitor comprised of 12-amino acids
(Chaudhary et al., 2001
).
Caloxin 2A1 is active at an extracellular site, so the peptide can simply
be added exogenously to inhibit the PMCA. Caloxin has a relatively
low-affinity for the pump (Ki=0.4 mM; complete inhibition at 2 mM)
(Chaudhary et al., 2001).
Therefore cells were pretreated for one minute with the peptide prior to
stimulating the cells. As seen in Fig.
2H, under control conditions, activation of CaR with 5 µM NPS
R-467 in the presence of 1 mM Ca2+ produced an initial transient in
Ca2+ followed by repetitive Ca2+ spikes superimposed on
a sustained plateau. The spiking did not diminish during agonist exposure. In
contrast, following Caloxin 2A1 pretreatment, NPS R-467 elicited a similar
initial spike, and then a few feeble oscillations that gradually decayed
towards the baseline. Spiking was sometimes restored upon removal of the
peptide inhibitor (not shown), but more frequently there was a modest increase
in the plateau without oscillations. Oscillatory behavior was recovered
following a third (control) stimulation (n=5; 41 cells). Acute
addition of 2 mM Caloxin 2A1 during oscillations stimulated by 1 mM spermine
in Ca2+-free Ringer's (20 µM BAPTA) also attenuated spiking
(n=3 experiments, 51 cells; not shown).
Thus far our data suggest a role for PMCA-mediated Ca2+ extrusion in providing an additional extracellular stimulus to CaR that results in intracellular Ca2+ spiking. As shown in Fig. 3A, the physical architecture of these cells may also contribute to this phenomenon. Optical sectioning through HEK-CaR cell clusters bathed with membrane-impermeant fluorescein-dextran (10,000 MW) showed that the junctions between cells consisted of very limited aqueous spaces. These convoluted clefts extended to the bottom of the cell clusters, as illustrated by the reconstructed side-view image, where immunostaining for CaR and PMCA showed that these two membrane proteins were also present. As seen in the confocal section in Fig. 3B, there was substantial overlap between PMCA and CaR (shown by yellow color in the merged image) in these junctional areas. The PMCA, in particular, was frequently observed to be absent from the free edges of the cell clusters.
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We attempted to evaluate whether co-localization of PMCA and CaR in membrane microdomains such as caveolae might be important for generating Ca2+ oscillations. HEK CaR cells were therefore pre-incubated with ß-cyclodextrin (see Materials and Methods) to in order to disrupt caveolae. While this treatment inhibited spermine- and NPS-R-467-induced oscillations in most cells, these experiments were inconclusive because ß-cyclodextrin also abolished the initial spike of Ca2+ release in 52% of the cells following addition of the CaR agonists (n=4 experiments; 134 cells; data not shown). In the same cells the response to carbachol/ATP was also absent.
Additional support for our model, however, derives from the time course for
extracellular [Ca2+] changes following agonist activation, as
measured directly using the near-membrane Ca2+ probe
fura-C18 (Etter et al.,
1994). When incubated with intact cells, near-membrane indicators
become anchored to the external leaflet of the plasma membrane via a lipid
tail, and have been used previously to measure Ca2+ extrusion from
cells (Blatter and Niggli,
1998
; Nitschke et al.,
1997
). The fluorescence of dye-loaded cells was quenched
completely by addition of 5 mM NiCl2 (which is cell impermeant),
confirming the extracellular location of the fluorophore (see Materials and
Methods for details). Because fura-C18 is a high affinity
Ca2+ probe (Kd=150 nM)
(Etter et al., 1994
), we were
obliged to conduct our experiments in nominally Ca2+-free solutions
containing 25 µM BAPTA free acid.
Stimulation of HEK CaR cells with 1 mM spermine resulted in a relatively
slow peak in extracellular [Ca2+] that originated from the
junctional regions in the center of cell clusters
(Fig. 4A). Several different
patterns of extracellular [Ca2+] change were apparent. In 6 out of
19 experiments, there was a small initial undershoot in the
Fura-C18 ratio (illustrated in
Fig. 4B), followed by a larger
elevation that peaked 34.8±1.3 seconds after stimulation (data from 6
experiments; junctional regions between 64 cells). This was significantly
slower than the time to attain the peak in cytoplasmic Ca2+
following spermine stimulation, as measured by fura-2 under the same
experimental conditions (10.0±0.4 seconds; data from 73 cells). The
time course of a typical cytoplasmic spike is overlaid on the
fura-C18 trace at an expanded scale in the inset of
Fig. 4B to illustrate this
point. In five (out of 19) other experiments the initial undershoot was absent
leaving just the slow transient elevation. Treatment of cells with 1 mM
VO43 (to block the PMCA) prevented the
spermine-induced peak, unmasking only the undershoot (typical of n=6
experiments; not shown). In some experiments (8 out of 19) oscillatory
fluctuations in extracellular [Ca2+] were detected following
spermine addition, as shown in Fig.
4C. Significantly larger elevations in extracellular
[Ca2+] (which were also sometimes oscillatory) were observed in
response to other Ca2+-mobilizing agonists, carbachol and/or ATP
(Fig. 4D). ATP and other
agonists have been reported previously to activate the PMCA in HEK293 cells
(Qi et al., 2000) and other
cell types (Usachev et al.,
2002
).
|
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Discussion |
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---|
|
Our immunolocalization data showed that in multi-cellular clusters of HEK
CaR cells, the PMCA was largely restricted to junctures between cells
(Fig. 3B), where it
co-localized with CaR. Confocal sectioning of living cell clusters incubated
with membrane-impermeant fluorescent dextrans revealed that an aqueous cleft
occupies these junctions between cells, as the fluorescent marker was able to
penetrate into these spaces (Fig.
3A). Thus it would appear that Ca2+ is expelled from
the cell by the PMCA into highly restricted extracellular domains, as
confirmed by our direct measurements of extracellular [Ca2+]. This
may help to explain why single cells oscillated less vigorously than those
growing in clusters. In parathyroid cells (which express CaR very abundantly)
the receptor is known to localize to caveolae
(Kifor et al., 1998), small
invaginations in the plasma membrane that have been considered to be cellular
`signaling centers'. The PMCA has also been localized to caveolae
(Fujimoto, 1993
;
Ogi et al., 2000
) in other
cell types. Unfortunately, our attempts to assess the role of caveolae in
Ca2+ oscillations using ß-cyclodextrin were inconclusive
because the compound frequently altered the initial spike of Ca2+
in response to agonists. Thus it remains to be determined whether CaR and PMCA
may reside together in the same specialized membrane domain, and how this may
influence the signaling properties of the cell.
It is noteworthy that parathyroid cells have been observed to oscillate
`spontaneously' in the presence of elevated extracellular Ca2+
(Miki et al., 1995).
`Spontaneous' Ca2+ oscillations have also been observed in number
of other diverse cell types, including human osteoblast-like cells (hOB cells)
(Tsai et al., 1999
), the
pancreatic glucagon-secreting cell line INR1 G9
(Bode et al., 1994
), mouse
mammary epithelial cells, both in primary culture and in an established cell
line MMT060562 (Furuya et al.,
1993
), isolated secretory ciliary epithelium
(Giovanelli et al., 1996
), and
primary rat mammotropes (Shorte et al.,
2000
). CaR is a widely expressed receptor, but is not known
whether it may play any role in activating Ca2+ spiking in these
latter cell types. Moreover, additional studies will be needed to determine
whether the mechanism described here in the HEK CaR model will be functional
in cell types where CaR is expressed endogenously.
Of particular interest, however, are `spontaneous' Ca2+
oscillations in the developing nervous system that have been shown to be
mediated by metabotropic glutamate receptors
(Flint et al., 1999). Certain
members of the group I mGluR family, specifically subtypes mGluR1 and mGluR5,
are linked to PLC/InsP3/Ca2+ signaling pathways
(Hermans and Challiss, 2001
).
CaR in fact bears significant molecular homology with mGluRs
(Brown et al., 1993
).
Moreover, mGluR1a and mGluR5a have been suggested to have
Ca2+-sensing capacity (EC50 for Ca2+ approx.
1 mM), and can apparently utilize Ca2+ as a co-agonist
(Kubo et al., 1998
;
Nash et al., 2001a
).
Considering the putative Ca2+-sensing properties of these receptors
(particularly mGluR5a), it is conceivable that cycling of Ca2+
across the plasma membrane may also contribute to stimulation of the mGluR in
the presence of an agonist, subtly amplifying the stimulus for oscillatory
signaling.
The role of PKC in mGluR-mediated oscillations has been investigated
extensively. Meyer and colleagues (Codazzi
et al., 2001) recently re-examined this complex phenomenon in
astrocytes (which express mGluR5 endogenously) using total internal reflection
fluorescence imaging of GFP-tagged PKC
and GFP-tagged C1 domains of
PKC
. The latter construct binds DAG, and can be used as a reporter of
[DAG] as well as to buffer its concentration. DAG was found to oscillate along
with Ca2+ following glutamate stimulation, and over-expression of
the DAG binding domain altered the ability of the cell to return to baseline
during the downstroke of the Ca2+ signal. PKC may therefore be
important for the termination of the Ca2+ spike. However the
various factors contributing to the initiation of the oscillatory spike have
not been fully elucidated.
While it is tempting to speculate that Ca2+ cycling across the
plasma membrane is the primary driving force behind the oscillatory signal,
our data and those of others suggest that additional mechanism (s) apart from
PMCA-mediated cycling of Ca2+ likely contribute to sustaining
oscillations in HEK CaR cells. In spite of treatments that would be expected
to inhibit the PMCA or buffer external Ca2+ completely, repetitive
Ca2+ spiking was not always fully abolished (e.g.
Fig. 2E,H). Moreover, our
direct measurements of extracellular [Ca2+] using
fura-C18 showed that the extracellular [Ca2+] increase
gradually dissipated at a time point well before intracellular oscillations
were expected to cease under Ca2+-free conditions. Mutational
analyses of CaR have shown that consensus phosphorylation sites for PKC are
important for shaping the intracellular Ca2+ signal arising from
CaR stimulation (Bai et al.,
1998; Chang et al.,
1999
). In a study using CaR expressing oocytes Chang et al.,
showed that 45Ca2+ efflux (used by the authors as a measure of CaR
activation) was susceptible to PMA treatment
(Chang et al., 1999
).
Meanwhile, interpretation of these studies is complicated by the fact that PKC
may also modulate some isoforms of the PMCA
(Enyedi et al., 1997
;
Penniston et al., 1997
;
Qu et al., 1992
;
Zylinska et al., 1998
). A role
for PKC in CaR-mediated Ca2+ oscillations has been suggested by
several studies. For example, Breitwieser and Gama
(Breitwieser and Gama, 2001
)
found that PKC inhibition (e.g. with staurosporine) caused cells to be more
likely to oscillate (although the inter-spike frequency was similar to
control). In our own unpublished studies, stimulation of PKC using a brief
application of PMA (4ß-phorbol 12 myristate 13-acetate; 100 nM) abolished
CaR-dependent Ca2+ oscillations in HEK CaR cells (Y. Jiang, A.M.H.,
B. W. Lau and M. Bai, unpublished). Thus, for CaR-expressing cells, it is
possible that activated PKC is somehow involved in terminating individual
Ca2+ spikes, as was suggested for mGluR-expressing cells.
CaR desensitizes relatively slowly
(Breitwieser and Gama, 2001;
Gama and Breitwieser, 1998
),
yet physiological conditions may exist in which the receptor is chronically
exposed to elevated levels of CaR activators. Ca2+ spiking is a
mode of signaling that allows continuous encoding of information while
avoiding the potentially deleterious effects of persistent elevations in
cytoplasmic [Ca2+], as well as the harmful effects of chronic
depletion of Ca2+ in the endoplasmic reticulum. We report here that
appropriately timed cycling of Ca2+ across the plasma membrane by
the PMCA may assist in the initiation of the oscillatory spike by amplifying
the actions of other CaR agonists (including extracellular Ca2+).
We speculate that in CaR expressing cells (and possibly also in cells
harboring other Ca2+ sensors such as mGluRs) several mechanisms may
coexist, converging to reinforce oscillatory behavior. These seemingly
redundant mechanisms may be important to ensure that extracellular signals are
translated into Ca2+ oscillations, even in the face of prolonged
exposure to maximal agonist concentrations.
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Acknowledgments |
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