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INTRODUCTION |
Calcium-mobilizing agonists induce a rise in cytosolic
Ca2+ concentration
([Ca2+]c)1
with a defined spatio-temporal pattern. In most cases, the
[Ca2+]c rise is composed of an initial peak,
followed by repetitive [Ca2+]c spiking or a
sustained [Ca2+]c elevation (for a review, see
Ref. 1). Whereas the former is mostly contributed by the release of
Ca2+ from intracellular stores (the ER and Golgi
apparatus), the latter is sustained by Ca2+ entry from the
extracellular space, which may be directly receptor activated or
controlled by the filling state of the intracellular stores
(store-operated Ca2+ influx (SOC); for a review, see Refs.
2 and 3). Indeed, removal of Ca2+ from the extracellular
medium abolishes the sustained plateau phase but not the initial
Ca2+ release. However, the necessity of a continuous influx
from the extracellular medium for maintaining a prolonged
Ca2+ rise does not imply that Ca2+ directly
diffuses through the cytosol to the intracellular targets. Rather,
Ca2+ entry could serve the purpose of filling ER cisternae
in proximity of the plasma membrane; Ca2+ diffusion through
the ER would then provide the driving force for continuous
Ca2+ release in different cytosolic domains, including
ER/mitochondria contact sites. Two recent observations support this
scenario. First, examples of intracellular Ca2+ handling in
different cell types show that influx-dependent ER refilling from the subplasma membrane space, followed by rapid diffusion of Ca2+ in the ER lumen, and
IP3-dependent Ca2+ release at
distant sites may represent a general paradigm that allows the
maintenance of sustained [Ca2+] rises in the cell body
(4, 5) (for reviews, see Refs. 6 and 7). Second, some cytosolic
effectors appear to "sense" very efficiently the release of stored
Ca2+, of which an excellent example is provided by
mitochondria. Work from numerous laboratories has demonstrated that
when a Ca2+ signal is elicited in the cytosol by the
stimulation with IP3-generating agonists, the cytosolic
rise is always paralleled by Ca2+ uptake into the
mitochondrial matrix (for reviews, see Refs. 8 and 9). A major increase
in the mitochondrial matrix Ca2+ concentration
([Ca2+]m) is thus observed (ranging from 5 µM in neuronal cells to 500 µM in
chromaffin cells), which appears in contrast with the low affinity of
the mitochondrial uptake mechanisms (the electrogenic uniporter of the
inner membrane). The steep dependence of the mitochondrial
Ca2+ uptake machinery on the extramitochondrial
[Ca2+] has been well studied in isolated mitochondria
(for reviews, see Refs. 10 and 11) and intact or
digitonin-permeabilized cells (12-14). According to these studies, in
order to obtain the [Ca2+]m values observed in
intact cells (e.g. 10-100 µM in HeLa cells)
(15, 16), supramicromolar [Ca2+] should be generated at
the mitochondrial Ca2+ uptake sites. The apparent
discrepancy was reconciled by the concept that high
[Ca2+] microdomains generated at mouth of
IP3Rs during its activation are sensed by neighboring
mitochondria (17), which are thus exposed to [Ca2+], that
allow efficient Ca2+ uptake. Finally, the diffusion of
Ca2+ through the outer mitochondrial membrane creates a lag
time between the initial [Ca2+]c and
[Ca2+]m rises into mitochondria (14, 18, 19).
Thus, the properties of mitochondrial Ca2+ accumulation
suggest that these organelles may represent the prototype of a
cytosolic effector, which requires sustained release of
Ca2+ from the ER (and thus maintenance of a high
[Ca2+] microdomain at ER/mitochondria contacts) to be
actively recruited in the calcium signaling pathway.
To investigate the relationship between the kinetics of ER
Ca2+ release and mitochondrial Ca2+
accumulation, we carried out a study in the epithelial cell line HeLa,
utilizing organelle-specific probes and agonists that induce different
ER Ca2+ release patterns. These cells endogenously express
the H1 G-protein-coupled receptor coupled to phospholipase
C activation and consequent continuous IP3 production
without detectable desensitization (20). Thus, histamine generates a
typical biphasic cytosolic Ca2+ signal with sustained
[Ca2+]c elevation and parallel emptying of the ER
until the agonist is present (21). Activation of SOC following ER
emptying has been also shown after histamine stimulation (22, 23). In
contrast to the H1 receptor, the group I
(Ca2+-mobilizing) metabotropic glutamate receptors, such as
mGluR1 or mGluR5, undergo receptor desensitization in the continuous presence of glutamate (for a review, see Ref. 24). In several cell
types expressing either endogenous or recombinant mGluRs, the
desensitization of these receptors was shown to be mediated by PKC
(25). Feedback inhibition of the receptor by PKC phosphorylation results in inhibition of phosphoinositide hydrolysis; thus, application of glutamate induces only a transient IP3 production (26).
In the present study, we have taken advantage of the different
properties of H1 and mGluR1a receptors regarding the
kinetics of IP3 production for analyzing the role of
IP3-induced Ca2+ release in generating a
sustained cytosolic Ca2+ response and its efficacy in
inducing mitochondrial Ca2+ uptake.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transfection--
HeLa cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, in 75-cm2 Falcon flasks. For aequorin measurements,
cells were seeded onto 13-mm glass coverslips and were cotransfected
with 3 µg of mGluR1a/pcDM8 (27) and 2 µg of cytAEQ/VR1012,
erAEQmut/VR1012, or mtAEQmut/VR1012, as previously described (28).
Transient transfection was carried out using the Ca2+
phosphate precipitation technique. Luminescence analyses were carried
out 36-48 h after transfection.
Aequorin Measurements--
In the case of cytAEQ- or
mtAEQmut-expressing cells, the coverslips with the cells were incubated
with 5 µM wild type coelenterazine for 2 h in
Krebs-Ringer modified buffer (KRB; 135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 0.4 mM K2HPO4, 1 mM
CaCl2, 5.5 mM glucose, 20 mM HEPES,
pH 7.4) at 37 °C and then transferred to the perfusion chamber. In
erAEQmut-expressing cells, the luminal [Ca2+] of the ER
was reduced during aequorin reconstitution by incubating the cells for
1 h at 4 °C in KRB supplemented with 5 µM
coelenterazine n, the Ca2+ ionophore ionomycin (5 µM), and 600 µM EGTA. After this
incubation, cells were extensively washed with KRB without
CaCl2, supplemented with 2% bovine serum albumin and 1 mM EGTA. The ER Ca2+ store was refilled at the
beginning of the experiments by perfusing the cells with KRB
supplemented with 1 mM CaCl2.
All aequorin measurements were carried out in KRB containing either 1 mM CaCl2 (KRB/Ca2+) or 100 µM EGTA (KRB/EGTA). Histamine (100 µM) or
glutamate (100 µM) were added to the same medium. The
aequorin experiments were terminated by lysing the cells with 100 µM digitonin in a hypotonic Ca2+-rich
solution (10 mM CaCl2 in H2O), thus
discharging the remaining aequorin pool. The light signal was collected
in a purpose-built luminometer and calibrated into [Ca2+]
values as previously described (28). Chemicals and reagents were from
Sigma or from Merck except for coelenterazine and coelenterazine n,
which were from Molecular Probes, Inc. (Eugene, OR). Statistical data
are presented as mean ± S.E.
Imaging Measurement of Cytosolic [Ca2+]--
To
monitor [Ca2+] in the cytosol, mGluR1a-transfected HeLa
cells were placed on 24-mm coverslips and loaded with 2 µM fura-2/AM in KRB/Ca2+ for 30 min at
37 °C. Cells were then washed in the same solution, and
[Ca2+]c changes were determined using a high
speed, wide field digital imaging microscope. A Zeiss Axiovert 200 inverted microscope was used with a ×40 objective. Fura-2 was excited
at 340 and 380 nm using a random access monochromator (Photon
Technology International). Images were acquired by a Micromax 1300YHS
camera (Princeton Instruments) using 4× binning in both the horizontal
and vertical direction. Measurements were carried out at room
temperature. Images were analyzed using the MetaFluor software
(Universal Imaging Corp.).
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RESULTS |
Mitochondrial Ca2+ Uptake Depends on the Duration of ER
Ca2+ Release--
The start point of this work was to
determine the correlation between the kinetics of the changes in
cytosolic and mitochondrial Ca2+ concentration
([Ca2+]c and [Ca2+]m,
respectively) evoked in HeLa cells by the stimulation with a
Ca2+-mobilizing, IP3-coupled agonist. For this
purpose, HeLa cells were transfected with the appropriate targeted
aequorin chimera (cytAEQ and mtAEQmut, respectively) (28). 36-48 h
after transfection, functional aequorin was reconstituted by adding the
prosthetic group. The coverslip with the cells was then transferred to
the luminometer chamber and challenged with the agonist histamine. Light emission was collected and calibrated into [Ca2+]
values, as described under "Experimental Procedures" and references therein. HeLa cells endogenously express the H1
G-protein-coupled receptor, the stimulation of which leads to sustained
IP3 production. The results (Fig.
1) showed that agonist stimulation (100 µM histamine) evoked a cytosolic Ca2+ peak
(2.5 µM), followed by a plateau phase (Fig.
1A). The cytosolic response was followed by an efficient
mitochondrial Ca2+ uptake, reaching a peak level of ~70
µM (Fig. 1B). When the kinetics were compared,
it was apparent that [Ca2+]c peaked ~4 s after
histamine addition (i.e. when the [Ca2+]m rise was at <50% of the peak) and then
rapidly declined toward a sustained plateau that was maintained
throughout agonist stimulation. The [Ca2+]m peak
was reached when, through the activity of the SERCA and PMCA pumps,
[Ca2+]c was rapidly declining. As shown in Fig.
1, C and D, mitochondrial Ca2+ uptake
depended almost entirely on Ca2+ release from internal
stores, since stimulation of cells in Ca2+-free
extracellular medium led to the same mitochondrial Ca2+
uptake (64 ± 2.7 µM, n = 5 versus 66 ± 4.8 µM, n = 35 in control). We thus concluded that mitochondrial Ca2+
uptake depends on sustained release of Ca2+ from the
ER.

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Fig. 1.
Cytosolic (A and
C) and mitochondrial (B and
D) [Ca2+] responses of HeLa cells
stimulated with 100 µM
histamine. [Ca2+]c and
[Ca2+]m was measured in cytAEQ- or
mtAEQmut-expressing cells, respectively, 36 h after transfection.
Aequorin luminescence was collected and calibrated into
[Ca2+] values as described under "Experimental
Procedures." Where indicated, HeLa cells were stimulated with the
agonist, added to the perfusion medium (KRB in experiments shown in
A and B; Ca2+-free KRB plus 100 µM EGTA in C and D). The
dotted lines indicate the start of stimulation
and peak of cytosolic and mitochondrial Ca2+ signals,
respectively. The traces are representative of >30 trials.
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To test this possibility, we stimulated HeLa cells through a
G-protein-coupled receptor that rapidly undergoes
PKC-dependent inactivation and thus causes a transient
production of IP3 and Ca2+ release from the ER.
The receptor employed was mGluR1a (27), which was co-transfected with
the targeted aequorin probes. Fig. 2
shows the functional properties of the transfected mGluR1a receptor. First, we analyzed the kinetics of Ca2+ release from the ER
evoked by histamine and glutamate in parallel batches of
mGluR1a-expressing cells, cotransfected with erAEQmut (28) (Fig.
2A). The data obtained highlight the fundamental difference
between the two agonist responses: (i) stimulation of the endogenous
histamine receptor caused a rapid initial
[Ca2+]er drop followed by a continuous and slower
decrease reaching, ~2 min after the start of agonist stimulation, a
low [Ca2+]er steady-state value of ~50
µM, and (ii) in the case of glutamate, the initial rapid
[Ca2+]er decrease was followed by the refilling
of the Ca2+ store, which was almost complete in about 2 min
even in the continuous presence of glutamate.

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Fig. 2.
Transient Ca2+ release caused by
glutamate in mGluR1a-expressing HeLa cells. A, effect
of glutamate (100 µM; open circles)
and histamine (100 µM; filled
circles) on [Ca2+]er measured in HeLa
cells co-transfected with erAEQmut. B and C,
cytosolic Ca2+ transients measured in HeLa cells
cotransfected with cytAEQ in response to glutamate (100 µM; open circles) and histamine
(100 µM; filled circles) in
Ca2+-free medium (dotted line). The
agonists were applied as shown by the open (histamine) and
filled (glutamate) bars, respectively. The traces
are representative of >10 trials.
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Next, we applied another approach to explore the relationship between
the stores controlled by the two agonists. We perfused cells
co-transfected with cytAEQ and mGluR1a with a Ca2+-free
solution (KRB/EGTA; i.e. KRB supplemented with 100 µM EGTA instead of 1 mM CaCl2) in
order to prevent refilling of the stores, and we applied repetitive
histamine pulses in order to deplete its intracellular Ca2+
store and then glutamate. It is apparent, as shown in Fig.
2C, that glutamate evoked substantially lower further
release of Ca2+, compared with that observed when glutamate
was applied as first stimulus (see Fig. 2B). The reverse
protocol (i.e. applying histamine after depleting the
glutamate releasable pool) showed similar results (Fig. 2B).
These experiments demonstrated that most of the glutamate- and
histamine-releasable pools are overlapping; thus, the different kinetic
behavior of the Ca2+ signal evoked by the two agonists
cannot be ascribed to the use of separate intracellular stores.
We thus analyzed the amplitude and kinetics of
[Ca2+]m and [Ca2+]c rises
evoked by glutamate stimulation. Two major differences with the
histamine responses are apparent. The first relates to the
[Ca2+]c rise. After the initial peak, which
depends on the transient [Ca2+]er decrease and is
comparable in the two cases (2.1 ± 0.1 µM for
histamine versus 1.75 ± 0.1 µM for
glutamate; n = 18), there is no sustained plateau in
the case of glutamate stimulation (i.e.
[Ca2+]c returns to the basal values in ~30 s in
the continuous presence of the agonist) (Fig.
3A). The difference in
[Ca2+]m response was even more dramatic; the peak
rise evoked by glutamate stimulation was drastically reduced (16.5 ± 1.8 µM (n = 28) versus
66 ± 4.8 µM (n = 35) during
the histamine challenge) (Fig. 3B). Moreover, the
difference between the time to peak of [Ca2+]c and [Ca2+]m
responses in this case was reduced (4.1 s for histamine versus 2.4 s for glutamate). In order to show that the
same Ca2+ pools were used by both agonists to feed
mitochondrial Ca2+ uptake, we applied histamine after
30 s of glutamate-induced Ca2+ release, before
refilling of the ER (see Fig. 2A). In this case, the
histamine-induced mitochondrial Ca2+ uptake was markedly
reduced (23 ± 4.5 µM, n = 5) (Fig.
4A). Apparently, further
depletion of the ER Ca2+ pool by histamine is responsible
for the remaining [Ca2+]m increase after
glutamate stimulation as measured by erAEQmut (Fig. 4B).

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Fig. 3.
Cytosolic (A) and
mitochondrial (B) [Ca2+] responses of
HeLa cells co-transfected with mGluR1a stimulated with 100 µM glutamate.
[Ca2+]c and [Ca2+]m were
measured in cytAEQ- or mtAEQmut-expressing cells, respectively. Where
indicated, HeLa cells were stimulated with the agonist, added to KRB.
Dotted lines indicate the start of stimulation
and peak of cytosolic and mitochondrial Ca2+ signals,
respectively. The traces are representative of >10 trials.
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Fig. 4.
Ca2+ pools feeding mitochondrial
Ca2+ uptake in case of glutamate and histamine
stimulation. A, mitochondrial Ca2+
transients measured in HeLa cells cotransfected with mtAEQmut and
mGluR1a in response to 100 µM histamine alone
(open circles) and after glutamate stimulation
(filled circles). B, effect of 100 µM histamine on [Ca2+]er added
after 100 µM glutamate, as measured in HeLa cells
cotransfected with erAEQmut and mGluR1a. The agonists were applied as
shown by the bars below the traces.
The traces are representative of >5 trials.
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The Kinetics of Initial Ca2+ Release Induced by
Glutamate and Histamine Are Identical--
We thus investigated the
various possible reasons for the drastic reduction of the
[Ca2+]m rise in the glutamate response. The first
possibility is that the kinetics of Ca2+ release from the
ER is faster in histamine-stimulated cells, an effect that could be
overlooked by the aequorin measurements. Indeed, given that the high
rate of mitochondrial Ca2+ uptake depends on the exposure
of Ca2+ microdomains generated at the mouth of
IP3Rs, one would envision that a faster release through the
IP3Rs would have a direct impact on mitochondrial
Ca2+ accumulation. We thus evaluated the kinetics of the
upstroke of [Ca2+]c by fast digital imaging in
fura-2-loaded HeLa cells expressing mGluR1a. As shown on Fig.
5, the kinetics of
[Ca2+]c elevation evoked by the two agonists
appeared to be identical as analyzed by a time resolution of 200 ms
(t1/2 = 753 ± 47 ms for histamine,
t1/2 = 712 ± 86 ms for glutamate;
n = 12). Thus, the differences in mitochondrial
Ca2+ uptake do not originate from differences in the
velocity of the initial Ca2+ release. This conclusion is
compatible with the preceding observations in cell populations
transfected with the cytosolic and mitochondrial targeted aequorin
probes (see above), where the mitochondrial Ca2+ uptake
rate was slower than the cytosolic rise, and the mitochondrial uptake
continued even after the cytosolic peak (see Fig. 1).

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Fig. 5.
Cytosolic Ca2+ signal in a single
HeLa cell expressing mGluR1a in response to glutamate (100 µM; A) and histamine
(100 µM; B).
Cells were loaded with fura-2, and ratio images were acquired
every 200 ms, as described under "Experimental Procedures."
Conventional pseudocolor ratio images are shown every 200 ms in the
upper line of each panel and in each
second in the lower line. In case of histamine
stimulation, elevated ratio levels were observed even after 1 min of
stimulation (lower right image). Cells
were stimulated by the addition of the agonist into the bath, as
indicated by arrows. The series of images are
representative of >10 experiments.
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Alteration of the Duration of ER Ca2+ Release Modifies
Mitochondrial Ca2+ Uptake--
To further test the
hypothesis that the [Ca2+]m rise depends on a
sustained release of Ca2+ from the ER, we decided to modify
the kinetics of the cytosolic responses induced by the two agonists. As
to glutamate, we applied the PKC inhibitor staurosporine, which was
shown to prevent mGluR1a phosphorylation and to reduce the consequent
receptor desensitization, ensuring sustained IP3 production
during glutamate stimulation. Preincubation of the cells with 400 nM staurosporine reversed the kinetics of both the ER and
cytosolic Ca2+ signal; glutamate stimulation caused
continuous Ca2+ release from the ER (Fig.
6B), similarly to the
Ca2+ signal observed during histamine stimulation. As to
[Ca2+]c, no difference was observed in the peak,
but the [Ca2+]c decrease after the peak was
significantly slower, and a sustained plateau (maintained throughout
agonist stimulation) was reached (Fig. 6A). We have to note
that the addition of staurosporine does not prevent mGluR1a
desensitization completely (24), as indicated by the slower kinetics of
continuous ER Ca2+ release and the lower sustained
[Ca2+]c (see, for comparison, the sustained
plateau following histamine stimulation) (Fig. 1A). Still,
importantly, converting the glutamate-induced transient
Ca2+ release into a more continuous response by
staurosporine application, the [Ca2+]m peak was
markedly increased (25.6 ± 2.9 µM, compared with
16.5 ± 1.8 µM cells not treated with staurosporine;
n = 28) (Fig. 6C).

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Fig. 6.
Effect of staurosporine (400 nM,
added 5 min before cell stimulation) on Ca2+ signals
elicited by glutamate (100 µM) in
mGluR1a-expressing HeLa cells. Control traces are shown with
open circles, and traces of
staurosporine-pretreated cells are shown with filled
circles. [Ca2+]c (A),
[Ca2+]er (B), and
[Ca2+]m (C) were measured in cytAEQ,
erAEQmut, and mtAEQmut co-transfected cells, respectively. Glutamate
was applied continuously in perfusion from the time point indicated by
the arrows. The traces are representative of >10
trials.
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The opposite experiment was also performed (i.e. the
histamine-evoked release was made more transient) by reducing the
duration of the agonist challenge to 2 s (Fig.
7). Under those conditions, the peak
[Ca2+]c response was marginally reduced
(2.27 ± 0.1 versus 2.45 ± 0.1 µM
cells receiving a 2-min histamine challenge; n = 16 for
both groups) (Fig. 7A), but [Ca2+]c
rapidly returned to basal values, with disappearance of the sustained
[Ca2+]c plateau. Importantly, using the same
protocol for [Ca2+]m measurements, matching with
the reduction of Ca2+ release time, the
[Ca2+]m peak was substantially reduced (Fig.
7B, from 66 ± 4.8 µM, n = 35, for 2-min stimulation to 29.1 ± 2.9 µM,
n = 16, for 2-s stimulation), and
[Ca2+]m reached its peak earlier (9.6 ± 0.29 s control versus 4.5 ± 0.35 s for 2 s
stimulation) (Fig. 7B).

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Fig. 7.
Cytosolic (A) and
mitochondrial (B) [Ca2+] responses of
HeLa cells to sustained (filled circles)
and transient (2 s; open circles) histamine
stimulations (100 µM).
[Ca2+]c and [Ca2+]m were
measured in cytAEQ- or mtAEQmut-expressing cells, respectively. Where
indicated (filled bar for sustained,
open bar for transient stimulation) HeLa cells
were stimulated with the agonist, added to the perfusion medium.
Dotted lines indicate the start of stimulation
and the peak of cytosolic signals, respectively. The traces
are representative of >20 trials.
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Continuous Ca2+ Release from the Intracellular
Ca2+ Stores Is Balanced by SOC and Ca2+
Recycling by SERCA--
We thus concluded that prolonged release of
Ca2+ from the ER is necessary to achieve maximal
mitochondrial responses. But how can this release and the ensuing
microdomains at ER/mitochondria contacts be maintained, given that PMCA
efficiently reduces [Ca2+]c by extruding
Ca2+ in the extracellular space? To this end, it is
necessary that ER be continuously refilled by Ca2+ entry
and redistribute Ca2+ to the release sites, in keeping with
the pathway of Ca2+ studied in depth in pancreatic acinar
cells (29, 30). In other words, the steady-state phase of
agonist-stimulated Ca2+ release from the ER, which is
necessary for transferring the Ca2+ signal to mitochondria
and other cytosolic effectors, must be sustained by the process of
Ca2+ entry through the plasma membrane.
Thus, in the next set of experiments, we analyzed the contribution of
SOC to the generation of the sustained Ca2+ signal. For
this purpose, we released Ca2+ from the intracellular pools
by applying either histamine (Fig. 8A) or glutamate (Fig.
8B) in KRB/EGTA to cells transfected with cytAEQ and
mGluR1a. Then we evoked Ca2+ influx by changing the
perfusion medium from KRB/EGTA to KRB/Ca2+ (in the
continuous presence of the agonist). Ca2+ release from
intracellular Ca2+ stores produced a transient peak upon
stimulation with both agonists, which returned to the base line,
showing that the presence of extracellular Ca2+ is
essential to achieve sustained Ca2+ signal. Furthermore,
depletion of the stores by histamine activated SOC, as observed from
the increase of [Ca2+]c after the readdition of
external Ca2+ (see Fig. 8A). In contrast,
Ca2+ reintroduction into KRB caused only a slight elevation
in the presence of glutamate, probably due to smaller depletion of the stores caused by the transient IP3 production. However,
glutamate, after staurosporine preincubation, was able to activate SOC,
since the readdition of Ca2+ caused a
[Ca2+]c elevation comparable with that of caused
by histamine (see Fig. 8B). Thus, according to these data,
we confirmed that both Ca2+ influx and continuous
IP3 production are necessary for maintaining the cytosolic
Ca2+ signal.

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Fig. 8.
Dependence of Ca2+ signals evoked
by histamine (100 µM;
A, filled circles) and
glutamate (100 µM;
B, open circles) on the
presence of extracellular Ca2+. The extracellular
[Ca2+], either 0 (KRB plus 100 µM EGTA) or
1 mM, is shown on the bars above the
traces. B, effect of staurosporine (400 nM, added 5 min before cell stimulation; filled
circles) on glutamate-induced cytosolic Ca2+
signals in the absence and presence of extracellular Ca2+.
The starting points of the continuous stimulation by either agonist,
added to the perfusion medium, are shown by arrows. The
traces are representative of >10 trials.
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Then we tested the effect of Cd2+, a blocker of
Ca2+ entry pathways of the plasma membrane, including
store-operated channels, on [Ca2+]er at different
stages of cell stimulation (Fig. 9). As
expected, if Cd2+ is added after the stimulation with
histamine (i.e. when no Ca2+ release occurs and
the ER is actively reaccumulating Ca2+), the process of
refilling is blocked, and a [Ca2+]er steady state
lower than in unstimulated cells is reached. Conversely, if
Cd2+ is added in the presence of the agonist
(i.e. when the ER is largely depleted and a steady state
[Ca2+]er value of ~50 µM is
maintained), a rapid, further emptying of the ER is observed, leading
to almost complete emptying of the ER. Thus, importantly, an
equilibrium between refilling and Ca2+ release through
IP3Rs is maintained throughout the process of agonist
stimulation.

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Fig. 9.
Comparison of the effect on
[Ca2+]er of Cd2+ added at different
phases of the Ca2+ signal.
[Ca2+]er was measured in cells transfected with
erAEQmut as described under "Experimental Procedures." 100 µM histamine was added to the perfusion medium as
indicated by the bars below the
traces. The arrows mark the timing of
Cd2+ addition. The trace with filled
circles shows the effect of 1 mM
Cd2+ added at steady state [Ca2+]er
in the presence of histamine (see the filled
bar). The trace with open
circles demonstrates the immediate effect of 1 mM Cd2+ added during store refilling after the
removal of histamine from the perfusion medium (see white
bar). The traces are representatives of >6
trials.
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If this is the case, one would expect that both blocking
Ca2+ release through IP3Rs or the process of ER
refilling though store-operated channels should be equally effective in
reducing the sustained [Ca2+]c rise observed
during agonist stimulation. Moreover, the termination of the
[Ca2+]c signal in the former way should be more
rapid, whereas the effect of Ca2+ entry blockade should
occur only after the ER is depleted of the remaining Ca2+.
This was directly investigated in the experiment of Fig.
10, where we compared the kinetics of
terminating the sustained cytosolic signal after histamine stimulation
with two experimental protocols. In the first, we applied
Cd2+ to block Ca2+ influx still in the presence
of histamine and IP3-induced Ca2+ release. In
the second, we washed out histamine rapidly in order to terminate
IP3 production (i.e. to stop Ca2+
release). As shown in Fig. 9A, the half-decay time of the
cytosolic Ca2+ signal was about 2 times longer during
blockade of influx by 1 mM Cd2+
(t1/2 = 15.4 ± 0.98 s, n = 12) than in the case of histamine washout (t1/2 = 8.5 ± 0.49 s, n = 11, p < 0.01). These results show that Ca2+ release from the
internal stores has the major role in the generation of
[Ca2+]c elevation, whereas the Ca2+
entry maintains the state of filling of the Ca2+ store,
thus counteracting the forces of Ca2+ extrusion by
PMCA.

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Fig. 10.
Comparison of the kinetics of cytosolic
Ca2+ signal termination by washing out histamine
(traces with filled
circles) and by inhibiting Ca2+ entry with
Cd2+ (traces with open
circles). [Ca2+]c was
measured in cells transfected with cytAEQ as described under
"Experimental Procedures." 100 µM histamine was added
to the perfusion medium as indicated by the bars
below the traces (filled
bar, in the case of histamine washout; white
bar, in the case of the addition of 1 mM
Cd2+). The inset shows the magnification of the
decay phases (trace labeling as in the
main panel). The traces are
representative of >12 trials.
|
|
Finally, we compared [Ca2+]c changes during ER
refilling in the presence and in the absence of IP3. As
shown in Fig. 11A, after
emptying the intracellular Ca2+ pools by 100 µM histamine in the absence of extracellular
Ca2+, Ca2+ readdition to the medium exerted a
significant [Ca2+]c increase only if histamine
(i.e. IP3) was present. If histamine was washed
out 30 s before Ca2+ readdition, refilling of the
Ca2+ stores was accompanied by only a small
[Ca2+]c elevation. The efficiency of refilling in
this case was demonstrated by a second application of histamine, which
elicited a Ca2+ release comparable with the one exerted by
the first stimulation (Fig. 11A, filled
circles). Furthermore, prolonged histamine stimulation and
IP3-induced release caused further depletion of the
Ca2+ stores even after Ca2+ readdition
and following robust Ca2+ influx. This is illustrated by
the small amount of releasable Ca2+ remaining in the pools
even after a 20 s washout of histamine before its reapplication (Fig.
11A, open circles). Similarly, the readdition of 1 mM extracellular Ca2+ after
depleting the Ca2+ stores by the reversible SERCA inhibitor
2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBHQ; 10 µM) led to efficient store refilling without significant [Ca2+]c elevation (Fig. 11B,
filled circles). In contrast, in the continuous
presence of tBHQ, Ca2+ influx was conveyed to the cytosol,
as shown by the vast [Ca2+]c elevation following
the readdition of Ca2+ (Fig. 11B,
open circles).

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Fig. 11.
Analysis of Ca2+ reuptake by the
ER during store-operated Ca2+ influx after store depletion
by histamine stimulation (A) or by reversible
inhibition of SERCA by tBHQ (B).
[Ca2+]c was measured in cytAEQ-expressing HeLa
cells. Store-operated Ca2+ influx was evoked by emptying
the stores by perfusing 0 extracellular [Ca2+] (KRB + 100 µM EGTA) and then re-adding 1 mM
CaCl2, as shown on the bars above the
traces. A, histamine (100 µM) was
added to Ca2+-free KRB and either removed before
(filled bar, trace with
filled circles) or maintained during
(open bar, trace with open
circles) the readdition of extracellular Ca2+.
An additional histamine stimulus was added in both cases in 0 extracellular [Ca2+] to show the filling state of the
Ca2+ stores. B, tBHQ (10 µM) was
added to the Ca2+-free KRB and either removed before
(filled bar, trace with
filled circles) or maintained during
(open bar, trace with open
circles) the readdition of extracellular Ca2+.
The traces are representative of >6 trials.
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These data clearly indicate that Ca2+, after entering the
cells, is taken up robustly by the ER without significantly elevating [Ca2+]c, and then Ca2+ is distributed
by the ER to the entire cytosolic space. In this way, SOC is not
directly responsible for the generation of the sustained cytosolic
Ca2+ signal; nevertheless, it is necessary for its
maintenance, by ensuring the continuous refilling of the internal
Ca2+ stores. Furthermore, Ca2+ cycling at the
vicinity of Ca2+ release sites maintains an equilibrium on
both sides of the ER membrane (i.e. the steady state in the
[Ca2+]er and the sustained phase of the
[Ca2+]c elevation); thus, ER plays an important
role as a redistribution pathway between plasma membrane
Ca2+ entry and other intracellular organelles, such as
the mitochondria.
 |
DISCUSSION |
In recent years, much information has been acquired on how
specific spatio-temporal patterns of Ca2+ signaling can
control different cell functions (or deleterious effects in
pathological conditions). In particular, attention has been drawn to
the properties and functional significance of local gradients
(microdomains) and thus to the importance of the source and
intracellular route of the [Ca2+] rise. Subplasmalemmal
high [Ca2+] microdomains appear to regulate the activity
of plasma membrane (PM) ion channels (such as
voltage-dependent Ca2+ and Na+
channels, Ca2+-activated K+ channels, and SOC),
polarity and excitability of the PM (neurons, smooth muscle), secretory
responses, and neurotransmitter release. Ca2+ entry, by
means of voltage-, ligand-, or store-operated channels, in most
of the cases is necessary and sufficient to generate such a high
[Ca2+] microdomain below the PM, also for longer periods
of cell stimulation. Conversely, other long term processes regulated by
sustained Ca2+ signals take place deeper in the cell
interior. Among them, a well known example is the regulation of
mitochondrial enzymes involved in ATP production or steroid synthesis,
where Ca2+ taken up from microdomains generated at the
mouth of ER Ca2+ release channels plays a fundamental role
(15, 31). A transient [Ca2+]m peak was shown to
exert a long term effect at the level of ATP synthesis (32), and
continuous mitochondrial [Ca2+] elevation, even at
relatively lower extramitochondrial Ca2+ levels, was shown
to increase the activity of dehydrogenases of the Krebs cycle (13, 33,
34), thus elevating the NADH level and the activity of the electron
transport chain. At the same time, Ca2+ uptake by
mitochondria has been shown to be involved in a radically different
process (i.e. the release of proapoptotic factors and thus the induction of cell death) (35).
Mitochondria have thus recently emerged as key decoders of calcium
signals, and the mechanism and timing of their recruitment control key
decisions in cell life and death. In this contribution, we have
analyzed the correlation between the kinetics of
[Ca2+]c increase and its different components and
the [Ca2+]m rises occurring in agonist-stimulated
cells. For this purpose, we utilized a low affinity probe for
[Ca2+]m (that allows us to fully appreciate the
large [Ca2+]m swing) and two different agonist
stimulations, through the endogenous histamine receptor and through a
transfected metabotropic glutamate receptor, which undergoes rapid
desensitization and thus causes transient IP3 production.
The first observation is that, upon cell stimulation,
[Ca2+]m peaks well after
[Ca2+]c. As previously observed by various
groups, there is a short delay in the upstroke, possibly due to the
time needed for the diffusion of Ca2+ released by
IP3 receptors through the outer mitochondrial membrane, thus reaching the transport systems (uniporter) of the inner membrane (14, 18, 19). Then [Ca2+]m rises and reaches its
maximal value after ~10 s (i.e. when the
[Ca2+]c increase, through the activity of the
Ca2+ pumps, is rapidly declining).
How can the slow kinetics of mitochondrial Ca2+
accumulation be reconciled with the notion that the low affinity of the
mitochondrial uniporter requires, for rapid uptake, the high
[Ca2+] gradient generated upon cell stimulation by the
opening of IP3 receptors? The most logical explanation is
that, for maximal mitochondrial Ca2+ uptake, prolonged
Ca2+ release from the ER must occur. Different experiments
support this notion. Indeed, not only in the case of glutamate
stimulation (in which Ca2+ release from the ER is short
lived) is the mitochondrial Ca2+ response
drastically reduced, but the effect of the two stimuli (glutamate and
histamine) on mitochondria can be reversed by modifying the time course
of the Ca2+ release process. If desensitization of the
mGluR is prevented by PKC inhibitors, ER release becomes sustained
(with ensuing large amplitude emptying of the ER and activation of
store-dependent Ca2+ influx), and mitochondrial
responses are greatly enhanced. Conversely, a short (2-s) histamine
pulse causes transient emptying of the Ca2+ store and
drastically reduces the [Ca2+]m rise evoked by
the agonist. These data imply that in the late phases of agonist
stimulation (i.e. when the activity of the pumps (SERCA and
PMCA) counteracts the release of Ca2+ through the
IP3Rs), an equilibrium is attained between the two processes, as demonstrated by the direct measurement of
[Ca2+]er; blocking Ca2+ release
(e.g. by terminating cell stimulation) causes the rapid refilling of the store, while conversely interrupting the
reaccumulation of Ca2+ in the ER (e.g. by
blocking Ca2+ entry through SOC; see below) allows
IP3Rs to rapidly deplete the ER of Ca2+.
Mitochondria, and possibly other cytosolic effectors, appear thus to be
activated through the kinetic behavior of the ER release process.
Conversely, Ca2+ entry, which is invariably essential for
sustained Ca2+ signals, might not provide a direct supply
for these localized Ca2+ regulatory events. Two
considerations support this view. First, these processes rely on
specific patterns of IP3-dependent release of
Ca2+ from the ER store, as discussed above. Second,
Ca2+ entering the cytosol is strongly buffered by
Ca2+-binding proteins, such as parvalbumin, calbindin
D28K, and calretinin, rendering the diffusion rate of
Ca2+ in the cytosol rather low (36). Thus, Ca2+
coming from the extracellular medium would reach slowly if at all the
deeper regions of the cytosol. An ingenious solution for this challenge
has been recently shown in a polarized cell model, the pancreatic
acinar cells (30). At the initial phase of the physiological activation
of these cells, focal Ca2+ release occurs exclusively at
the secretory pole, serving as trigger for exocytosis and leading to
local emptying of the Ca2+ store (30). The presence of
Ca2+ signaling components and particularly TRP-like
channels at the vicinity of the apical pole has been suggested to
provide a straightforward route for local refilling of the depleted
stores (37). On the other hand, given that the ER in these cells forms
a continuous network (4), the resulting luminal [Ca2+]
gradient has been shown to cause rapid diffusion of Ca2+
from the basolateral part of the cells, in a model where SOC is
restricted to this area (38). The ground of this arrangement of
Ca2+ signaling is the relatively high Ca2+
mobility in the ER tunnel compared with the cytosol, depending on the
much lower binding capacity of the ER lumen (~20 versus ~2000 bound/free Ca2+ of the cytosol of mouse pancreatic
acinar cells (39)). Similarly, in neurons, which is still a highly
polarized cell type, it was proposed that subplasmalemmal ER cisternae
of the cell body may be responsible for Ca2+ refilling from
the extracellular space, and a continuous ER network would transport
Ca2+ to the site of release in dendritic spines (40). In
accordance with this idea, it has been shown that the cytosolic
buffering capacity of Purkinje neurons is as high (~2000) as that of
pancreatic acinar cells (41). On the other hand, in other cell types
such as chromaffin cells (42), lower values of binding capacity have been found (~40), but we should emphasize that even if
Ca2+ diffusion may occur at a similar velocity in the
cytosol and the ER, another important advantage in the use of ER for
distributing Ca2+ signals to the cell interior is that it
may ensure localized Ca2+ release. Thus, in smooth muscle
cells, even if the cytosolic buffering capacity is comparable with that
of the ER (~30-40) (43), a superficial layer of SR rapidly buffers
Ca2+ entering from the extracellular space, which is then
distributed into the cell interior, causing contraction after its
directed release (44).
Since our aim was to disclose the role of the ER in generating a
sustained Ca2+ signal and in recruiting cytosolic
effectors, in our work we did not characterize the exact nature of the
Ca2+ entry pathway. However, some information on
Ca2+ influx and store refilling can be obtained from the
experimental data. Recently, a receptor-activated, arachidonic
acid-mediated, noncapacitative mechanism for Ca2+ entry has
been demonstrated that appears to operate in a PM domain distinct from
that in which SOC operates in HEK293 cells (45). Two arguments suggest
that this pathway does not contribute to the Ca2+ signals
observed in our experiments. (i) We used maximal agonist concentrations
for prolonged periods, producing a substantial depletion of
Ca2+ stores; thus, we fully activated the SOC mechanism,
which inhibits arachidonate-regulated channels (46). (ii)
Ca2+ entry activated by store depletion in our system was
clearly necessary for store refilling, in contrast to Ca2+
entering the cells by arachidonate-activated Ca2+ entry,
which rather plays a role in potentiating the Ca2+ release
induced by IP3, thus increasing the frequency of
oscillations (47).
The other issue concerns the way Ca2+ refills the
Ca2+ stores. We demonstrated that in the case of transient
IP3 production (i.e. during glutamate
stimulation), ER refilling occurs without detectable rise of
[Ca2+]c (compare Figs. 2A and
3B). However, based on our data, we cannot distinguish
between refilling from the extracellular space through SOC channels and
direct Ca2+ reuptake from the cytosol by SERCA. It appears
that the buffering capacity of the cytosol determines the route by
which Ca2+ released from the ER is eliminated, since
increased buffering allows ER refilling by SERCA even in the absence of
extracellular Ca2+ (48). Thus, in cells with inherent high
cytosolic buffer capacity, such as the above mentioned pancreatic
acinar cells or neurons, the SERCA pumps appear to dominate over PMCAs
in rapidly reducing the [Ca2+]c peak (49, 50).
Thus, the oscillatory Ca2+ signals evoked by colecystokinin
in these cells were shown to occur in the absence of Ca2+
entry, given that Ca2+ is taken back almost entirely from
the cytosol after the Ca2+ spikes (see above). However, the
situation differs with other types of stimulation; the Ca2+
oscillations evoked by carbachol strongly depend on Ca2+
influx in the proximity of the Ca2+ release sites of the
apical pole (37). Up to now, there are no data concerning the cytosolic
buffering capacity of HeLa cells, but evidence from the similar Chinese
hamster ovary cell line shows that PMCA overexpression leads to larger
reduction and faster termination of cytosolic Ca2+ signal
compared with SERCA (51). Thus, it seems likely that the
glutamate-induced Ca2+ transient is rapidly extruded from
the cell, and the Ca2+ source of refilling in this case is
the extracellular space.
In conclusion, the data presented in this paper indicate that
mitochondria, important transducers of the Ca2+ signal,
depend on the process of ER Ca2+ release, which in turn is
sustained by continuous release through IP3 receptors and
refilling by SERCAs (with a primary role of Ca2+ influx in
counteracting the extrusion of Ca2+ by PMCAs). Altogether,
these data suggest that the ER provides a fast route for tunneling and
releasing Ca2+ in the deeper portions of the cytoplasm
(where mitochondria are only one of the numerous Ca2+
effectors) not only in the polarized pancreatic acinar cell (as proposed by Petersen et al. (30)) but also in
different cell types. This provides an additional mechanism by which
the selective placement and differential activation of Ca2+
channels in the ER and plasma membrane provide flexibility to the
Ca2+ transduction system, allowing this second messenger to
play a key role in the modulation of virtually all cellular processes.