Department of Biomedical Sciences and Italian Research Council (CNR) Center for the Study of Biomembranes, University of Padova, 35121 Padova, Italy
The functional characteristics of a nonacidic, inositol 1,4,5-trisphosphate- and thapsigargin-insensitive Ca2+ pool have been characterized in mammalian cells derived from the rat pituitary gland (GH3, GC, and GH3B6), the adrenal tissue (PC12), and mast cells (RBL-1). This Ca2+ pool is released into the cytoplasm by the Ca2+ ionophores ionomycin or A23187 after the discharge of the inositol 1,4,5-trisphosphate-sensitive store with an agonist coupled to phospholipase C activation and/or thapsigargin. The amount of Ca2+ trapped within this pool increased significantly after a prolonged elevation of intracellular Ca2+ concentration elicited by activation of Ca2+ influx. This pool was affected neither by caffeine-ryanodine nor by mitochondrial uncouplers. Probing mitochondrial Ca2+ with recombinant aequorin confirmed that this pool did not coincide with mitochondria, whereas its homogeneous distribution across the cytosol, as revealed by confocal microscopy, and its insensitivity to brefeldin A make localization within the Golgi complex unlikely. A proton gradient as the driving mechanism for Ca2+ uptake was excluded since ionomycin is inefficient in releasing Ca2+ from acidic pools and Ca2+ accumulation/release in/from this store was unaffected by monensin or NH4Cl, drugs known to collapse organelle acidic pH gradients. Ca2+ sequestration inside this pool, thus, may occur through a low-affinity, high-capacity Ca2+-ATPase system, which is, however, distinct from classical endosarcoplasmic reticulum Ca2+-ATPases. The cytological nature and functional role of this Ca2+ storage compartment are discussed.
The cytosolic free Ca2+ concentration ([Ca2+]i)1 of
eukaryotic cells rests in the range of 50-200 nM, i.e.,
at a very low level, if compared to the Ca2+ concentration of physiological media (2 mM). However, the total cellular Ca2+ content is closer to this latter value (1-3
mmol/l of cell water). In other words, eukaryotic cells sequester large amounts of Ca2+ mainly by uptake inside intracellular Ca2+ stores (~90%) (for reviews see Pozzan et al.,
1994 The complexity of intracellular Ca2+ stores has been intensively investigated in recent years (for reviews see Meldolesi et al., 1990 Other drugs, such as 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBHQ) and cyclopiazonic acid (CA), also block
SERCAs, albeit with a significantly lower affinity (Mason
et al., 1991 In the vast majority of cell types so far investigated, the
InsP3- (and/or the ryanodine-) sensitive stores almost completely overlap with those sensitive to Tg (Zacchetti et al.,
1991 The complexity of the relationships between the InsP3-
and ryanodine/caffeine-sensitive stores does not cover the
entire issue of intracellular Ca2+ pool heterogeneity. Other
types of Ca2+ pools are known to exist, the size of which
varies considerably among different cell types. These latter Ca2+ stores account for roughly half of all sequestered
Ca2+ (Chandra et al., 1991 Here we demonstrate that a nonacidic, InsP3- and Tg-
insensitive Ca2+ pool rapidly accumulates large amounts
of Ca2+ when high and sustained increases of [Ca2+]i are
induced by opening of voltage- or store-operated Ca2+
channels. This Ca2+ storage compartment is insensitive to
mitochondrial uncouplers and appears diffusely distributed in the cell cytosol. The possibility is discussed that
this low-affinity, high-capacity Ca2+ pool represents a previously unidentified subcompartment of the ER expressing a Tg-insensitive Ca2+-ATPase.
Materials
Tissue culture medium and complements were purchased from Technogenetics (Milan, Italy); indo-1 and indo-1/AM were from Molecular Probes
(Eugene, OR); S202791 was a gift of Dr. D. Pietrobon (University of Padova); and all other reagents were from Sigma Chemical Co. (St. Louis,
MO).
Cell Culture and Transfection
Rat pituitary GH3 cells (from Dr. Hescheler, University of Berlin, Germany) were grown in Ham's F-10 medium supplemented with 15% horse
serum, 2.5% FCS, nonessential amino acids, and penicillin/streptomycin. The clonal cell lines GC and GH3B6 (from Dr. Argenton, University of
Padova), derived from GH3 cells, were maintained in the same conditions
(with the addition of 10 Transient transfection with recombinant cytosolic or mitochondrial aequorin (cytAEQ or mtAEQ) (Rizzuto et al., 1992 Ca2+ Measurements
Cells, grown on coverslips (24-mm-diam), were loaded with indo-1 by incubation with 5 µM indo-1/AM at 37°C for ~30 min in Ham's F-10 medium containing 3% FCS and 0.04% pluronic acid. After washing the cells
with a modified Krebs-Ringer medium (mKRB: in mM, 125 NaCl, 5 KCl,
1 MgSO4, 1 Na3PO4, 1 CaCl2, 20 Hepes, 5.5 glucose, pH 7.4), the coverslips
were mounted in a chamber and placed on the stage of an inverted microscope (model Diaphot 300; Nikon Europe B.V., Badhoevedorp, The
Netherlands), equipped with a 40× water immersion objective (NA=1.1;
Nikon) and connected with a real-time UV confocal system (model RCM8000; Nikon). The 351-nm band of the argon ion laser was used for excitation and the emitted light, separated into its two components (405 and 485 nm) by a dichroic mirror, was collected by two photomultipliers. The ratio
of the intensity of the light emitted at the two wavelengths (F405/F485), a
function of [Ca2+]i, was displayed as a pseudocolor scale. Unless otherwise
indicated, all experiments were performed at room temperature and time
series were acquired with a frame interval of 2 s, averaging 16 images for
each frame. Online analysis of the ratio was obtained from the signals of
individual cells. For presentation, ratios were off-line averaged and normalized to the average value obtained in Ca2+-free medium before ionomycin addition.
For measurements of cytosolic or mitochondrial free Ca2+ concentration ([Ca2+]m) with recombinant aequorins, cells were transiently transfected with cytAEQ or mtAEQ and seeded onto 13-mm-diam poly-l-lysine- coated glass coverslips (106 cells/coverslip) 2 d before the experiment. Aequorin was reconstituted by adding 5 µM coelenterazine to the culture
medium 1-2 h before the experiment. During the experiment, cells were
continuously perfused with mKRB containing different stimuli. At the
end of each experiment, cells were lysed by perfusion with an ipoosmotic solution containing only digitonin (100 µM) and 10 mM CaCl2 to expose
all the cellular aequorin to a high [Ca2+]. Light emission was measured by
a purpose-built luminometer and calibrated in terms of [Ca2+]i or [Ca2+]m
as described (Rizzuto et al., 1994 The total content of cellular Ca2+ was assayed by atomic absorption
spectrophotometry. Cells were suspended in mKRB (107 cells/ml) and
challenged with 30 mM KCl (GH3 cells) or 1 µM Tg (RBL-1 cells) for 3 min before addition of 4 mM EGTA. Control experiments were carried
out under the same conditions in Ca2+-free mKRB containing 1 mM
EGTA. After centrifugation, cells were resuspended in Ca2+-free mKRH
containing 1 mM EGTA and centrifuged for 1 min at 14,000 rpm in Eppendorf tubes containing 100 µl sucrose 12.5% and 400 µl silicon oil. The
pellet was resuspended in 0.05% Triton plus 0.2 N NaOH before measurement.
Immunolocalization of TGN38
GH3 cells were fixed and immunostained as described previously (Brini et al.,
1995 The Ionomycin-sensitive Ca2+ Pool in GH3 Cells
Increases after Depolarization, and Its Size in the Cell
Population Is Heterogeneous
The existence of different Ca2+ storage compartments whose
content can be discharged in the cytoplasm and/or in the
extracellular medium by treatment with a variety of agents
appears to be a widespread characteristic of eukaryotic
cells. Initially in the neuroendocrine cell line PC12 (Fasolato et al., 1991 Fig. 1, a and b, shows the typical protocol used to ascertain the existence of the different Ca2+ pools in GH3 cells.
Cells, loaded with the fluorescent Ca2+ indicator indo-1,
were incubated in mKRB medium and analyzed by confocal microscopy. The cells were treated in sequence with EGTA, to chelate extracellular Ca2+; thyrotropin-
releasing hormone (TRH), an agonist coupled to InsP3
generation; a SERCA inhibitor, such as Tg; ionomycin, a Ca2+ ionophore; and monensin, a Na+-K+/H+ ionophore.
Addition of TRH induced a rapid and transient increase in
[Ca2+]i, while Tg, after TRH, caused a barely detectable
rise. The subsequent challenge with ionomycin resulted in
a further small increase of [Ca2+]i, and finally monensin
discharged the acidic pool.
The kinetics of the [Ca2+]i peaks reflect the balance between Ca2+ efflux from the stores, pumping out of the cell
and reuptake into the stores themselves. However, the integral of the curve underlying the [Ca2+]i rise, induced by
each stimulus, mirrors, to a reasonable level of approximation, the Ca2+ content of the different stores as estimated
by 45Ca2+ measurements (Fasolato et al., 1991 Table I.
Analysis of Releasable Ca2+ in GH3 Cells
; Clapham, 1995
).
; Pozzan et al., 1994
; Simpson et al.,
1995
). Attention has been focused mainly on Ca2+ stores
that are highly dynamic because of their ability to rapidly take up and release Ca2+. Ca2+ sequestration into these
pools depends on Ca2+-ATPases, known as sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) (Burk et al.,
1989
; Bobe et al., 1994
; Wuytack et al., 1994
). All the
SERCA isoforms share the property of being selectively
inhibited by thapsigargin (Tg), a tumor-promoting sesquiterpene lactone (Lytton et al., 1991
). Tg acts with both high affinity, at nanomolar concentrations, and high specificity, with virtually no effect on the Ca2+- or Na+/K+-
ATPase of the plasmalemma.
). Ca2+ release, on the other hand, depends
mainly on two types of Ca2+ release channels named inositol 1,4,5-trisphosphate (InsP3) and ryanodine receptors
(for reviews see Mikoshiba, 1993
; Sorrentino and Volpe,
1993
; Ehrlich, 1995
). These channels are expressed in variable proportions in different cell types and couple extracellular stimuli to the release of Ca2+, with possible ensuing generation of Ca2+ waves and spikes (for reviews see
Amundson and Clapham, 1993
; Taylor, 1994
; Bootman
and Berridge, 1995
). The relationship between these types
of Ca2+-release channels is still largely debated. The ryanodine-sensitive channel is also activated by caffeine, and
ryanodine- and caffeine-sensitive stores are generally regarded to comprise the same pool (Zacchetti et al., 1991
;
Barry and Cheek, 1994
; but also see Giannini et al., 1992
;
McNulty and Taylor, 1993
).
; Gamberucci et al., 1995
) and are thus referred to
also as Tg-sensitive Ca2+ pools. From the cytological point
of view, the InsP3-/Tg-sensitive Ca2+ pool is identified with
the ER or with a subfraction of it (Hashimoto et al., 1988
).
; Fasolato et al., 1991
; Shorte et
al., 1991
; Bastianutto et al., 1995
; Mery et al., 1996
). They
have been identified through the increase in [Ca2+]i upon
application of Ca2+ ionophores, after depletion of the Tgsensitive pool with a combination, or a sequence, of InsP3generating agonists, Tg, and caffeine. These residual Tginsensitive pools appear rather heterogeneous in terms of
cytological identity and pharmacological sensitivity. Part
of these pools shows an acidic lumenal pH and is discharged only by a combination of a Ca2+ ionophore and of
agents that collapse internal acidic pH gradients (such as
monensin and NH4Cl). 45Ca2+ labeling of Tg-insensitive
pools is slower than that of the Tg-sensitive store, and, for
this reason, they have been generally indicated as slowly
exchanging Ca2+ pools (Fasolato et al., 1991
). As far as
their identification is concerned, the acidic pool seems
largely identifiable with secretory compartments and lysosomes, while very little is known yet about the rest of the
Tg-insensitive store.
Materials and Methods
7 M estradiol for the latter cell type). The pheochromocytoma cell line PC12 was grown in RPMI supplemented with
12.5% horse serum, 2.5% FCS, and gentamycin. The rat basophilic leukemia cell line RBL-1 (from Dr. Penner, Max-Planck-Institut, Göttingen,
Germany) was cultured in DME supplemented with 10% FCS and penicillin/streptomycin. For Ca2+ measurements, all cells were allowed to attach to poly-l-lysine-coated glass coverslips and grow for 1 d.
) was made by electroporation (Kodak, IBI, Rochester, NY). Cells were harvested and resuspended in fresh medium in 4-mm cuvettes in the presence of 10 µg of
cytAEQ/VR1012 or mtAEQ/VR1012 plasmid/106 cells. Cells were subjected to a single pulse characterized by an electric field of 300 V, 1,500 µF.
Transfected cells were transferred to 13-mm-diam poly-l-lysine-coated
glass coverslips (106 cells/coverslip), and after an overnight incubation, the
medium was changed and the incubation continued under the same conditions. After 2 d, the cells were used for Ca2+ measurements, according to
the procedure described in detail elsewhere (Rizzuto et al., 1994
).
; Brini et al., 1995
).
). The antibody, used at 1:200 dilution (kind gift of Dr. O. Rossetto,
University of Padova), was against the type I membrane protein TGN38, a
marker of the trans-Golgi network (Reaves et al., 1996
). Binding of the
antibody was revealed with an FITC-labeled anti-rabbit IgG antibody.
Fluorescence was analyzed with a microscope (model RCM 8000; Nikon)
and photographed (Technical Pan film; Kodak) after digital subtraction of
the background.
Results
; Zacchetti et al., 1991
), and later in several
other cell types, we have functionally distinguished three
types of intracellular Ca2+ pools: (a) an InsP3-sensitive
pool, largely identified with the ER and endowed with a
Tg-sensitive Ca2+-ATPase; (b) an acidic pool, whose Ca2+
content could be released into the cytoplasm by Ca2+ ionophores (A23187 or ionomycin), but only after neutralization of the luminal acidic pH with monensin or NH4Cl; and
(c) a Ca2+ pool that could be discharged by treatment only
with Ca2+ ionophores. This last Ca2+ store, presumably
heterogeneous, is the most mysterious in terms of cytological nature, mechanism of loading, and physiological role.
The experiments presented below were aimed at the characterization of this elusive Ca2+ compartment, using as a
model system primarily the rat pituitary cell line GH3.
Fig. 1.
Dynamic properties of Ca2+ pools in GH3
cells. Experiments were carried out on monolayers of
GH3 cells, loaded with indo-1, as described in Materials and
Methods. The traces in a and
c are an average of 30 cells,
while in b and d, four typical
single cell traces of the same
experiment are presented. In
this and the following figures,
representative of at least
three experiments carried
out in different cell batches,
the normalized ratio of the
intensity of the light emitted
at the two wavelengths
(F405/F485), a function of
[Ca2+]i, is displayed on the
left-hand side. Where indicated, KCl (30 mM), EGTA
(4 mM), TRH (1 µM), Tg, (1 µM), ionomycin (Ion; 1 µM) and monensin (Mon; 1 µM)
were added.
[View Larger Version of this Image (14K GIF file)]
; Bastianutto
et al., 1995
; Mery et al., 1996
) and can be used to calculate
the size of the different pools. Accordingly (see Table I),
the TRH- and Tg-releasable pools represent altogether
38%, the ionomycin-pool 17%, and the monensin-pool
45% of total mobilizable Ca2+. In Fig. 1 a, the trace is the
mean of 30 cells, while in b the kinetic changes of [Ca2+]i
in four typical cells are presented. As previously reported, in Ca2+-containing medium, GH3 cells underwent spontaneous oscillations that ceased immediately upon addition
of EGTA (Schlegel et al., 1987
). Noteworthily, the size of
the various Ca2+ pools was quite homogeneous in the cell
population.
No [Ca2+]i increase was detected upon application of
10 mM caffeine (not shown), although in another pituitary
cell line (GH4C1), a caffeine-sensitive store has been identified (Law et al., 1990; Tanaka and Tashjian, 1993
).
Fig. 1, c and d, shows that 1-min depolarization with 30 mM KCl in Ca2+-containing medium affected the size of the different pools. In particular, c, mean of 30 cells, shows that addition of KCl induced a sharp increase of [Ca2+]i that rapidly subsided upon chelation of extracellular Ca2+ with EGTA (or addition of an L channel blocker such as nifedipine; not shown). The kinetics and amplitude of TRH- and Tg-induced [Ca2+]i rise were hardly different from those of control cells (Fig. 1 a and Table I), while a massive increase occurred in the ionomycin-releasable pool. When the other Ca2+ ionophore A23187 was used instead of ionomycin, the results were indistinguishable (see below). This Ca2+ ionophore-sensitive pool induced by KCl stimulation will be referred to, for simplicity, as stimulus-induced-calcium (SIC) pool. Finally, the monensin pool appeared decreased by KCl-induced depolarization, as expected if it is accounted for, at least in part, by Ca2+ sequestered within secretory compartments. Similar results for both controls and treated cells have been obtained when the experiments were carried out at 37°C (not shown).
Fig. 1 d shows the behavior of four typical cells. While all the examined cells reacted to KCl stimulation with high and sustained [Ca2+]i rises, the increase of the SIC pool was consistently observed in >50% of the cells. In particular, out of the 30 cells analyzed in this experiment, in a fraction of them (10%) the ionomycin peak and the integrated area were indistinguishable from those of controls, while in ~30% the increase in the integrated area was two to sixfold higher than in controls, and the increase in the remaining 60% was between 6- and 15-fold. On average, in this experiment, there was a 10-fold increase in the area of the SIC pool (Fig. 1, a and c). In a series of similar experiments (n = 33), in 16 trials the averaged area of this pool was between 6-10-fold the control value, in 15 trials the area was three to fivefold, and the area hardly increased in the remaining two trials.
The possibility was considered that the increase of the SIC pool was only apparent and due to an impairment of the Ca2+ extrusion mechanisms. Two types of evidence argue against this possibility: (a) The peaks and kinetics of decay of the TRH-induced [Ca2+]i increase were similar in cells that have been treated with KCl when compared to controls (1.90 ± 0.17 and 1.88 ± 0.04 normalized ratio U for the peak, and 14.9 ± 0.9 and 9.8 ± 0.3 s for the half time of decay in control and KCl-treated cells, respectively [mean ± SD]) and (b) the substitution of Na+ with N-methyl- glucamine+ in the medium (to inhibit any contribution of the Na+/Ca2+ exchanger) resulted in no alteration of the kinetics and amplitude of the [Ca2+]i transients caused by the different stimuli (not shown). These results suggest that, overall, extrusion of Ca2+ into the medium or reaccumulation into the stores is not grossly affected by depolarization. In addition, these experiments indicate that, in GH3 cells, the Na+/Ca2+ exchanger has minor relevance in Ca2+ extrusion, at least under our experimental conditions.
Ca2+ indicators, such as indo-1, when loaded as acetoxymethyl-esters, are not only found in the cytosol but also
trapped within intracellular organelles. In addition, given
that their final cytosolic concentration is in the order of
20-100 µM, they substantially increase the cytosolic Ca2+
buffering capacity. To determine whether the appearance
of the SIC pool was at least in part an artifact of the methodology used, the [Ca2+] in the cytosol was measured with
recombinant aequorin. In this latter case, the increase in
Ca2+ buffering capacity is negligible and the transfected
protein is excluded from organelles (Brini et al., 1995).
Fig. 2 shows that, while in control cells, A23187 added after TRH and tBHQ caused a marginal increase in [Ca2+]i
(Fig. 2 b), a very large rise was caused by the ionophore in cells pretreated with KCl (Fig. 2 a). In the experiments
with recombinant aequorin (see also below), tBHQ and
A23187 were used instead of Tg and ionomycin because
the latter compounds tend to adhere strongly to the tubes
of the perfusion system (Rizzuto et al., 1994
).
To further confirm that the increase in the ionomycin response was indeed due to Ca2+ accumulation and not to impairment of the extrusion mechanisms, the total cellular Ca2+ content was measured by atomic absorption (see Materials and Methods). After 3 min of exposure to 30 mM KCl in mKRB medium, the total Ca2+ content increased by 43% compared to controls (5.65 ± 0.74 and 3.94 ± 0.4 nmol/mg protein, respectively; n = 5 for each condition, P < 0.002, Student's t test).
The SIC Pool Is Not Identifiable with Known Ca2+ Stores
One of the key features of the SIC pool, besides its InsP3
insensitivity, is its resistance to unloading by classical
SERCA inhibitors. Fig. 3 a demonstrates that Tg, when
added before depolarization, even at doses as high as 10 µM,
while inhibiting the TRH-induced [Ca2+]i changes, did not
significantly decrease the amplitude and kinetics of the
Ca2+ rise induced by ionomycin. Other SERCA inhibitors
such as tBHQ (30 µM) and CA (10 µM) were similarly ineffective (not shown). Thus, not only are SERCA inhibitors unable to deplete this pool, but they are also ineffective in preventing its loading.
The SIC pool was also unaffected by caffeine (10 mM) or ryanodine (10 µM), whether added before or after KCl depolarization (not shown). Last but not least, preincubation with monensin resulted in an increase in the ionomycin-dependent [Ca2+]i rise of both controls and KCltreated cells. The integrals of the curves, however, were equivalent to the sum of the individual ionomycin- and monensin-induced [Ca2+]i increases. Thus, alkalinization of luminal pH does not prevent loading of the SIC pool.
The insensitivity to SERCA inhibitors and caffeine/ryanodine, as well as the ability to load efficiently only as a
consequence of large [Ca2+]i increases, appear compatible
with the identification of the SIC pool with mitochondria
(for reviews see Pietrobon et al., 1990; Gunter et al., 1994
;
Pozzan et al., 1994
). Two lines of evidence, however, argue
against this hypothesis. Fig. 3 b shows that in cells subjected to the same protocol of Fig. 1 c (i.e., KCl for 1 min
followed by EGTA, TRH, and Tg), the addition of the mitochondrial uncoupler p-(trifluoro-methoxy)phenylhydrazone (FCCP, 10 µM) caused no increase of [Ca2+]i per se
and, more importantly, hardly affected the amplitude and
kinetics of the ionomycin-induced [Ca2+]i rise. The area of
the SIC pool was 57.1 ± 12.3 and 60.5 ± 24.8 normalized
ratio U (mean ± SD, n = 3) in the presence and absence of FCCP, respectively. At these concentrations, FCCP
completely collapses the mitochondrial membrane potential and, in isolated organelles, causes rapid release of accumulated Ca2+. Accordingly, the inefficacy of the uncoupler in reducing the size of the SIC pool strongly argues
against the possibility that mitochondria represent a significant part of this store. Similar results were obtained when
the mitochondrial membrane potential was collapsed by a
combination of the ATP-synthase inhibitor oligomycin (2 µM) and the mitochondrial complex-I inhibitor rotenone
(4 µM) (not shown).
The role of mitochondria was directly tested in the experiment presented in Fig. 4. GH3 cells were transiently
transfected with the cDNA coding for recombinant mtAEQ
(Rizzuto et al., 1992). Fig. 4 shows that the mean resting
[Ca2+]m of GH3 cells was somewhat higher than that reported previously in other cell types, such as HeLa or endothelial cells (~500 vs. ~200 nM) (Rizzuto et al., 1994
;
Lawrie et al., 1996
). This, however, was not unexpected
since, as shown in Fig. 1 b, GH3 cells undergo spontaneous
[Ca2+]i oscillations. Addition of KCl resulted in a very
sharp and large increase of [Ca2+]m, with a peak around or
above 10 µM. After the peak, [Ca2+]m rapidly returned toward basal and addition of EGTA caused a further decrease to, and below, resting level. While perfused with
Ca2+-free, EGTA-containing medium, addition of TRH + tBHQ resulted in an increase of [Ca2+]m to ~3 µM, and finally the Ca2+ ionophore A23187 caused a further slow
and reproducible increase. Thus, the ionophore caused an
increase in [Ca2+]m (presumably by equilibrating the Ca2+
concentration of the cytosol with that of the mitochondrial
matrix) and not a decrease, as expected if mitochondria
represented a significant part of the SIC pool.
Loading and Unloading Properties of the SIC Store
The experiments shown in Fig. 5 were undertaken to define the kinetics of loading and unloading of the SIC pool.
A 10-s exposure to 30 mM KCl (Fig. 5 a, continuous trace)
failed to induce a reproducible increase, compared to controls, of the ionomycin-induced rise of [Ca2+]i, while a 20-s
depolarization caused, in 50% of the trials (n = 13), a significant increase in the size of this store (Fig. 5 a, dotted
trace). After 60 s of depolarization, the increase of [Ca2+]i
caused by the Ca2+ ionophore was similar to that observed
after 5 min of KCl exposure (Fig. 5 a, long- and shortdashed traces, respectively). In addition, the SIC pool
loaded faster and was larger if the depolarization was carried out in high external [Ca2+] (5 mM) (compare Fig. 5 b
vs. a). In nondepolarizing conditions, the size of the pool
was not appreciably affected by the increase in [Ca2+]out
(not shown).
In the experiments presented in Figs. 1-4, loading of the
SIC pool was caused by a prolonged depolarization with
KCl. The question then arises as to whether other stimuli
causing Ca2+ influx can similarly induce an increase in the
size of this Ca2+ store. The amplitude and kinetics of the
ionomycin-sensitive pool were thus investigated in cells
pretreated for 3 min with TRH in Ca2+-containing medium, a condition that discharges the InsP3-sensitive store
and activates voltage-operated Ca2+ channels (VOCCs)
(Gollasch et al., 1991; Mantegazza et al., 1995
) and, presumably, store-dependent Ca2+ influx. Fig. 5 c shows that
TRH addition in Ca2+-containing medium resulted in a biphasic Ca2+ transient, a sharp rise followed by a sustained
plateau that is much smaller, however, than that induced
by KCl. Upon chelation of extracellular Ca2+ with EGTA,
the addition of ionomycin, after Tg, resulted in a transient
[Ca2+]i increase, which was, however, indistinguishable
from that of cells treated with TRH and Tg in Ca2+-free,
EGTA-containing medium (Fig. 5 c, continuous and
dashed traces, respectively). Thus, the influx of Ca2+ induced by TRH is insufficient to induce a significant increase in the size of the SIC pool.
The kinetics of the unloading of the SIC pool were next investigated. The protocol was the following: the pool was first loaded with Ca2+ through a 1-min exposure to KCl depolarization, followed by EGTA addition, to block Ca2+ influx through VOCCs. The cells were then washed and kept, for different times, in Ca2+-containing medium. Finally, after readdition of EGTA, the size of the pools was tested, as before, by addition of TRH + Tg followed by ionomycin. Fig. 5 d shows that complete unloading of the SIC pool required a total of 20-30 min between the end of KCl to the ionomycin treatment.
The insensitivity of the SIC pool to Tg and monensin indicates that Ca2+ uptake in this pool is not driven by H+ or
Na+ gradients and does not use classical SERCAs. We
considered the possibility that Ca2+ accumulation in the
SIC pool occurred by a mechanism of vesicle endocytosis
after KCl-induced secretion. However, the lack of a significant uptake of Lucifer yellow, a marker of fluid phase endocytosis, in KCl-treated cells (not shown) makes this possibility unlikely. If it occurred through endocytosis, the
amount of Ca2+ increase (0.4 mmol/l of cell water)
caused by depolarization would have, in fact, implied an
accumulation of extracellular medium that was not only
rapid but also exceedingly vast, i.e., corresponding to
about 1/4 of the cell volume.
Characteristics of the SIC Pool in Other Cell Types
A Ca2+ pool with characteristics similar to those identified
in GH3 cells was found also in other neuroendocrine cell
lines, i.e., two pituitary cell lines, GC and GH3B6 (not
shown), and in PC12 cells, derived from a rat pheochromocytoma. In this latter cell line, however, the SIC pool
was detectable only when depolarization with KCl was
carried out in the presence of an agonist of L type VOCCs,
S202791 (1 µM) (Fig. 6, a and b). In the absence of this drug, in fact, KCl-induced depolarization resulted in a plateau level of [Ca2+]i consistently lower than in GH3 cells.
Thus, considerable Ca2+ accumulation in the SIC pool requires a [Ca2+]i rise of large amplitude and long duration.
A low-affinity, high-capacity SIC pool was also found in
nonexcitable cells, such as the rat basophilic leukemia cell
line, RBL-1. In these cells, large loading of this pool could
not be achieved by membrane potential depolarization,
since they do not possess VOCCs, but rather through activation of another Ca2+-selective current, named ICRAC, for
Ca2+ release-activated Ca2+ current (Hoth and Penner,
1992; Fasolato et al., 1993
), maximally activated by Tg (for
review see Fasolato et al., 1994
). Fig. 6 d shows that Tg induced a long-lasting elevation of [Ca2+]i that rapidly returned to basal level upon EGTA addition. Under these
conditions, ionomycin induced a large, transient increase in [Ca2+]i, severalfold higher and more prolonged than
that caused by the ionophore in cells treated with Tg in
EGTA-containing medium (c). Similarly to GH3 cells, the
increase in total cellular Ca2+ content, as estimated by
atomic absorption, accounted for ~33% (n = 3). Moreover,
in these cells addition of monensin (1 µM) after ionomycin
never elicited a [Ca2+]i rise, either in control or stimulated
cells (not shown). Finally, also in the RBL-1 cells the SIC
pool was insensitive to caffeine (10 mM) and ryanodine
(10 µM), added either before or after Tg stimulation, and to
the mitochondrial uncoupler FCCP (10 µM) (not shown).
Intracellular Localization
A final property of the SIC pool here investigated is its
intracellular distribution. In both GH3 and RBL-1 cells,
high-resolution (67 ms/ratio frame) analysis by confocal
microscopy failed to reveal a localized origin of the ionomycin-induced response. In fact, Fig. 7 shows that, upon
ionomycin addition in both GH3 (a) and RBL-1 cells (b),
Ca2+ increased rather homogeneously in the cytosol and
quickly diffused to the central nuclear region, with no evidence for a localized origin of the [Ca2+]i rise. These observations would tend to exclude the Golgi complex, which has been previously suggested to accumulate Ca2+
(Virk et al., 1985; Chandra et al., 1991
; Wahl et al., 1992
) as the major source of the ionomycin-releasable Ca2+. As
confirmed by immunofluorescence, the Golgi network is,
in fact, specifically localized to a distinct perinuclear region (Fig. 8 c). It may be argued, however, that the increase in [Ca2+]i caused by the ionophore is relatively
slow, i.e., 8.1 ± 2.9 s to the peak (mean ± SD, in 12 similar
experiments with GH3 cells), and thus the localization of
the increase may be masked by diffusion of Ca2+ and/or of
the indicator.
To obtain further insights into the possible involvement of the Golgi complex, induction of the SIC pool was monitored in the continuous presence of brefeldin A (BFA) (10 µg/ml, after a pretreatment of 15 min at 37°C). BFA is known to disassemble the Golgi network (LippincottSchwartz et al., 1989) and to cause a major intermixing of membrane and lumenal Golgi components with the ER. Thus, the prediction will be that if the SIC pool was largely identifiable with the Golgi, BFA treatment should make it largely sensitive to Tg. Fig. 8 shows that in both RBL-1 (a) and GH3 cells (b), BFA did not significantly affect the size of the SIC pool, although it caused the known fragmentation of the Golgi complex (c).
The Ca2+ concentration in the cytoplasm controls a variety
of cellular functions and is in dynamic equilibrium with
that of intracellular organelles. Within the lumen of different compartments, Ca2+ plays different roles, such as that
of a reservoir to be mobilized upon cell activation (e.g., in
the ER and its subcompartments), of a modulator of enzymatic reactions (e.g., in the mitochondria), and of a cofactor in the packaging of secretory products (e.g., in the
Golgi complex and secretory vesicles). With the exception of the ER and mitochondria, however, the mechanism of
uptake into and release from other organelles is still
largely obscure. In the cell lines PC12, HeLa, mouse fibroblasts, and human lymphocytes, three major pools (or
groups of pools) have been functionally characterized on
the basis of their loading and release properties: the first
released by InsP3 and Tg, the second insensitive to the
above agents and released by Ca2+ ionophores, and the
third released by the combination of the latter compounds
with a drug that dissipates H+ gradients, such as monensin
and NH4Cl (Fasolato et al., 1991; Clementi et al., 1994
;
Bastianutto et al., 1995
; Mery et al., 1996
).
The Ca2+ pool released by ionomycin in the presence of
monensin was undetectable in RBL-1 cells, while it was
consistently observed in the neurosecretory cell types. In
GH3 cells and, as previously observed, in PC12 cells (Fasolato et al., 1991), the size of the acidic compartment is
reduced after stimulation of secretion and may suggest its
identification, at least in part, with secretory compartments.
The key observation of this contribution is that only a fraction of the nonacidic, InsP3- and Tg-insensitive pool, which in unstimulated cells is rather heterogeneous (including mitochondria, Golgi, etc.), is highly dynamic and dramatically increases its Ca2+ content upon prolonged rises of [Ca2+]i.
The two major questions generated by our data concern,
on the one hand, the cytological nature of the SIC pool,
and on the other hand, the physiological role of this compartment. As to the first question, our approach has been
that of comparing the pharmacological sensitivity and subcellular distribution of the Ca2+ uptake and release into
and from this pool with that of known organelles. The first
compartment that we considered as a candidate was the
mitochondrion, since some of the characteristics of the SIC pool, i.e., low affinity and high capacity, resemble
those traditionally attributed to this organelle. In particular, several authors have suggested that mitochondria play
a key role in buffering large and prolonged increases in
[Ca2+]i (Carafoli and Crompton, 1976; Bygrave, 1978
;
Brinley et al., 1979
; Friel and Tsien, 1994
; Werth and
Thayer, 1994
; Budd and Nicholls, 1996
; Herrington et al.,
1996
;). However: (a) The Ca2+ accumulated in the SIC
pool cannot be released into the cytosol by mitochondrial
uncouplers; and (b) direct monitoring of [Ca2+]m with targeted recombinant aequorin demonstrates that Ca2+ ionophores cause an increase and not a decrease in [Ca2+]m.
Thus, though mitochondria may well contribute to the ionomycin-sensitive pool of resting cells, they do not represent a major part of the pool that accumulates Ca2+ after
prolonged [Ca2+]i increases. Rather than Ca2+ sinks, mitochondria appear therefore to be in dynamic equilibrium with the changes in [Ca2+]i, with dramatic transient increases of their matrix [Ca2+] during cell activation.
Similarly, identification of the SIC pool with acidic compartments of the cells, i.e., secretory vesicles, granules, lysosomes, endosomes, etc., appears unlikely since the kinetics, amplitude, and duration of the [Ca2+]i increases
induced by ionophores are unaffected by agents such as
monensin or NH4Cl, which collapse internal pH gradients.
Moreover, Ca2+ ionophores, such as A23187 and even
more ionomycin, are highly inefficient at releasing Ca2+
from acidic compartments (Liu and Hermann, 1978; Fasolato and Pozzan, 1989
).
Another possible candidate is the Golgi complex, given
that several lines of evidence, though mainly indirect, suggest that this compartment may contain large amounts of
Ca2+ (Chandra et al., 1991; Cui et al., 1995
). Since neither
the mechanism of Ca2+ uptake nor that of release from the
Golgi complex are known to date, we have tested this idea
by taking advantage of the high spatial and temporal resolution of the confocal microscope. In fact, if the Ca2+ released by ionomycin was largely coming from the Golgi,
the response was expected to get started in a region of
high Ca2+ close to the nucleus, where the Golgi complex is
known to be localized. On the contrary, the [Ca2+]i increase caused by the ionophore appeared from the very
beginning to distribute in the whole cytosol. In addition,
the increase in [Ca2+]i caused by ionomycin was unaffected by BFA pretreatment. This drug is known to cause
dissolution of the Golgi complex and intermixing of its
membrane and content with the ER (Lippincott-Schwartz
et al., 1989
).
Among the other known organelles, peroxisomes were
considered unlikely candidates since they represent at the
most 1% of the cell volume (Lazarow, 1989), while the increase in total Ca2+ in GH3 cells treated with KCl is about
0.4 mmol/l of cell water. Thus, if peroxisomes accounted
for the majority of the SIC pool, their Ca2+ content should
exceed 40 mM, a value in the order of that estimated for
the cell organelle with the highest Ca2+ content reported
so far: the terminal cysternae of sarcoplasmic reticulum
(Volpe and Simon, 1991
; Grohovaz et al., 1996
).
As far as the ER is concerned, the insensitivity to
SERCA inhibitors, InsP3, caffeine, and ryanodine would
tend to exclude also this compartment as the candidate organelle. SERCAs are in fact believed to be widespread in
this endomembrane system. However, several lines of evidence indicate not only that the ER itself may be heterogeneous (for review see Sitia and Meldolesi, 1992), but
also that Ca2+ pumps other than those sensitive to the classical inhibitors (Tg, CA, and tBHQ) may be present in this
organelle (Bian et al., 1991
, Chen et al., 1993
). Ca2+-ATPases
with molecular weight different from those of classical SERCAs have been described by Burgoyne et al. (1989)
in
adrenal chromaffin cells (but see also Poulsen et al., 1995
)
and by Rooney and Meldolesi (1996)
in PC12 cells. Finally, in subcellular fractionation experiments, the existence of a microsomal fraction whose Ca2+ content is insensitive to InsP3, caffeine-ryanodine, and also to SERCA inhibitors has been repetitively reported (Nori et al., 1993
,
1996; Hussain et al., 1995
). If indeed the SIC pool represents a subcompartment of the ER, it must be concluded,
however, that this part of the organelle equilibrates very
slowly, or not at all, with the rest of the membrane network. Until now, its existence might have gone unnoticed
given that under resting conditions the Ca2+ content of
this subcompartment is relatively small and cannot be released into the cytosol by known physiological stimuli. It should be mentioned that a low-affinity Ca2+ storage compartment, which loaded only upon large and prolonged increases in [Ca2+]i caused by KCl depolarization, had been
previously described in a clone of PC12 cells by Reuter's
group. Unlike the experiments reported here, however,
this compartment was sensitive to caffeine, while its Tg
sensitivity was not tested (Reber and Reuter, 1991
; Reber
et al., 1993
).
As to the physiological role of the SIC pool, the interest
depends not only on its large capacity (it can account, on
average, for >40% of total cell Ca2+) but also on the fact
that it is inducible (Clementi et al., 1994; Hussain et al.,
1995
), and its expression and loading properties appear
widespread, if not ubiquitous, as documented by its presence in numerous cell types of different origin (GH3, GC,
GH3B6, PC12, RBL-1, and lymphocytes; Clementi et al.,
1994
; Pizzo, P., C. Fasolato, and T. Pozzan, unpublished).
Admittedly, the conditions needed for loading of this
Ca2+ pool may appear extreme. However, long-lasting increases in [Ca2+]i can be elicited by high agonist doses or
under pathological conditions (Choi, 1989). Furthermore,
the observation that loading and unloading of this Ca2+
pool can be relatively fast suggests the possibility that this store plays a role also under physiological conditions, but
its high-capacity is unraveled by large and long-lasting
[Ca2+]i increases, when its size overwhelms that of the
other Ca2+ storage compartments. In other words, it can
be suggested that a Ca2+ pool, endowed with a Ca2+ accumulation mechanism distinct from that of mitochondria
and acidic compartments and independent from classical
SERCAs, is a normal component of the Ca2+ homeostatic
machinery of the cell. The role of this previously unrecognized compartment could be twofold: on the one hand, by accumulating part of the Ca2+ coming from the extracellular medium or released from other stores, it becomes the
most relevant Ca2+ sink under conditions of stress, a function traditionally attributed to mitochondria; on the other
hand, by slowly releasing its Ca2+ content into the cytosol
after a prolonged stimulation, it may also participate in
signaling integration.
Received for publication 16 July 1996 and in revised form 28 October 1996.
We would like to acknowledge the involvement of Dr. W.J.J.M. Scheenen in the preliminary stages of this study and thank Dr. A.M. Hofer for critical discussion and comments on the manuscript, Dr. R. Rizzuto for kindly providing recombinant aequorins, M. Mancon for total Ca2+ measurements, and G. Ronconi, M. Santato, and B. Zavan for skilfull assistance.This work was supported by grants from the Italian Research Council (CNR) Special Project "Oncology," from the Italian University Ministry, from "Telethon" (grant N.495), from the EU programs "Biomed2," "Human Capital and Mobility," and "Copernicus," and from the "Human Frontier Science Program" to T. Pozzan.
BFA, brefeldin A; CA, cyclopiazonic acid; [Ca2+]i, intracellular free Ca2+ concentration; [Ca2+]m, intramitochondrial free Ca2+ concentration; cytAEQ, cytosolic aequorin; FCCP, p-(trifluoro-methoxy) phenylhydrazone; InsP3, inositol 1,4,5-trisphosphate; mKRB, modified Krebs Ringer Buffer; mtAEQ, mitochondrial aequorin; SERCAs, sarco/endoplasmic reticulum Ca2+-ATPases; SIC, stimulus-induced-calcium; tBHQ, 2,5-di(tert-butyl)-1,4-benzohydroquinone; Tg, thapsigargin; TRH, thyrotropin-releasing hormone; VOCCs, voltageoperated Ca2+ channels.