Ca Homeostasis in the Agonist-sensitive Internal Store: Functional Interactions Between Mitochondria and the ER Measured In Situ in Intact Cells

Abstract

Mitochondria have a well-established capacity to detect cytoplasmic Ca2+ signals resulting from the discharge of ER Ca2+ stores. Conversely, both the buffering of released Ca2+ and ATP production by mitochondria are predicted to influence ER Ca2+ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca2+] directly using trapped fluorescent low-affinity Ca2+ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca2+ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca2+ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca2+ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca2+ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca2+ signals.

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The Journal of Cell Biology, Volume 142, Number 5, September 7, 1998 1235–1243
http://www.jcb.org 1235
Ca
2
1
Homeostasis in the Agonist-sensitive Internal Store:
Functional Interactions Between Mitochondria
and the ER Measured In Situ in Intact Cells
Barbara Landolfi,* Silvana Curci,* Lucantonio Debellis,* Tullio Pozzan,
‡
and Aldebaran M. Hofer
‡
*Istituto di Fisiologia Generale, Università degli Studi di Bari, Via Amendola 165/A, I-70126 Bari, Italy; and
‡
University of
Padova, Consiglio Nazionale delle Ricerche Center for Biomembranes, Viale G. Colombo 3, I-35121 Padova, Italy
Abstract.
Mitochondria have a well-established capac-
ity to detect cytoplasmic Ca
2
1
signals resulting from the
discharge of ER Ca
2
1
stores. Conversely, both the buf-
fering of released Ca
2
1
and ATP production by mito-
chondria are predicted to influence ER Ca
2
1
handling,
but this complex exchange has been difficult to assess
in situ using conventional measurement techniques.
Here we have examined this interaction in single intact
BHK-21 cells by monitoring intraluminal ER [Ca
2
1
]
directly using trapped fluorescent low-affinity Ca
2
1
indicators. Treatment with mitochondrial inhibitors
(FCCP, antimycin A, oligomycin, and rotenone) dra-
matically prolonged the refilling of stores after release
with bradykinin. This effect was largely due to inhibi-
tion of Ca
2
1
entry pathways at the plasma membrane,
but a significant component appears to arise from re-
duction of SERCA-mediated Ca
2
1
uptake, possibly as
a consequence of ATP depletions in a localized sub-
cellular domain. The rate of bradykinin-induced Ca
2
1
release was reduced to 51% of control by FCCP. This
effect was largely overcome by loading cells with
BAPTA-AM, highlighting the importance of mitochon-
drial Ca
2
1
buffering in shaping the release kinetics.
However, mitochondria-specific ATP production was
also a significant determinant of the release dynamic.
Our data emphasize the localized nature of the interac-
tion between these organelles, and show that compe-
tent mitochondria are essential for generating explosive
Ca
2
1
signals.
Key words: metabolic microdomains • calcium ho-
meostasis • organelle interactions • mitochondrial un-
couplers • signal transduction
C
ellular
Ca
2
1
homeostasis reflects an intricate bal-
ance of many factors (pumps, leaks, release chan-
nels, and buffer systems; Berridge, 1993; Clapham,
1995) that are expected to directly or indirectly influence
Ca
2
1
handling by internal stores. Among these are interac-
tions with other organelles, including mitochondria. By
now it has been firmly established using a variety of tech-
niques (for example, using targeted recombinant ae-
quorin; Rizzuto et al., 1992) that mitochondria sense Ca
2
1
released into the cytoplasm during Ca
2
1
signaling events
(Rizzuto et al., 1993; Sparagna et al., 1995; Babcock et al.,
1997), and that this uptake of Ca
2
1
constitutes a physiolog-
ically important mechanism for activating mitochondrial
metabolism (Denton and McCormack, 1990; Rutter et al.,
1996; Hajnóczky et al., 1995).
Thus far this problem has been analyzed from the point
of view of the mitochondria or of the cytoplasm. But how
do mitochondrial metabolism and Ca
2
1
uptake influence
Ca
2
1
release from the ER? In fact, interactions between
these two organelles may be expected to occur on multiple
levels.
First, with respect to the refilling of internal stores with
Ca
2
1
, it is obvious that ATP (derived from mitochondrial
and glycolytic sources) is required to fuel accumulation of
Ca
2
1
by SERCAs, the Ca
2
1
-ATPases resident on the ER
membrane. Second, previous studies have demonstrated
that store-operated Ca
2
1
entry pathways (those that are
activated by store depletion; Putney, 1990) are influenced
by mitochondrial metabolism (Gamberucci et al., 1994;
Hoth et al., 1997; Marriot and Mason, 1995). Since the re-
filling of internal stores after agonist-induced Ca
2
1
release
relies principally on this pathway (see for example Hofer
et al., 1998), mitochondrial inhibition will also be expected
to impede the recharging of internal stores.
Regarding the mobilization of Ca
2
1
from stores, ATP
is known to modulate Ca
2
1
release channels in the ER,
Address all correspondence to Aldebaran M. Hofer, University of Padova,
CNR Center for Biomembranes, Viale G. Colombo 3, I-35121 Padova,
Italy. Tel.: +39-49-827-6065. Fax: +39-80-5443388. E-mail: wim@civ.bio.
unipd.it

The Journal of Cell Biology, Volume 142, 1998 1236
including inositol 1,4,5-trisphosphate (InsP3) receptors
(Bezprozvanny and Ehrlich, 1995; Ferris et al., 1990; Smith
et al., 1985), as well as Ca
2
1
leaks (Hofer et al., 1996). The
allosteric modulation of InsP3 receptors by ATP shows a
bell-shaped dependence on [ATP], with progressive re-
duction in open probability of the channel at ATP concen-
trations below 500
m
M and above 4 mM (Bezprozvanny
and Ehrlich, 1993). It has been questioned in the past
whether local [ATP] can vary sufficiently under physiolog-
ical conditions to effect any sort of regulation on the re-
lease channels in situ.
The ability of mitochondria to buffer cytoplasmic Ca
2
1
transients is expected to influence InsP3 receptor opening
further, since both this release channel and the ryanodine
receptor (that mediates Ca
2
1
-induced Ca
2
1
release) have
also been shown to exhibit a bell-shaped dependence of
open probability on cytosolic [Ca
2
1
] (Bezprozvanny et al.,
1991; Finch et al., 1991). In support of this view is the re-
cent demonstration by Jouaville et al. (1995) that mito-
chondrial substrates alter the propagation of Ca
2
1
waves
in oocytes, a finding confirmed by another study using
blockers of mitochondrial respiration in cultured oligo-
dendrocytes (Simpson and Russell, 1996). These phenom-
ena have been attributed to local alterations in Ca
2
1
buf-
fering as a consequence of Ca
2
1
uptake into energized
mitochondria, resulting in modulation of InsP3 receptor
opening.
Here we have addressed the integrated actions of the
above factors from the perspective of the ER, using a fluo-
rescence technique that permits selective measurement of
free [Ca
2
1
] changes in the agonist-sensitive store of single
intact cells. Our work demonstrates that not only is the
ability of mitochondria to buffer Ca
2
1
transients impor-
tant, but also that mitochondrial ATP production alters
the dynamics of Ca
2
1
release, the two factors acting in con-
cert to permit explosive discharge of Ca
2
1
from the store
under conditions where mitochondria are actively respir-
ing. Our results also indicate that the effects of mitochon-
drial inhibitors on the Ca
2
1
entry pathways at the plasma
membrane result in considerable inhibition of store refill-
ing. At the same time, SERCAs in BHK-21 cells appear to
depend on mitochondrial ATP production for Ca
2
1
se-
questration to a surprising extent, a result best explained
by the existence of subcellular metabolic microdomains.
Our findings suggest that there is an intimate functional
(and possibly physical) relationship between these two or-
ganelles.
Materials and Methods
Cell Culture and Dye Loading
BHK-21 cells (purchased from Consorzio. Gest. Biotec. Avanzate, Ge-
nova, Italy) were grown in Earle’s MEM medium containing 10% FBS,
and were maintained in a humidified incubator at 37
8
C in the presence of
5% CO2/95% air. Cells were seeded at low density on glass coverslips and
used the following day for microspectrofluorimetric or ratio imaging mea-
surements of cytoplasmic or intraorganellar free [Ca
2
1
] with fura-2-AM
(Grynkiewicz et al., 1985), or mag-fura-2-AM (Raju et al., 1989), respec-
tively. Cells were loaded in tissue culture medium for 20–30 min at room
temperature with fura-2-AM (5
m
M) or at 37
8
C for 45–60 min with mag-
fura-2-AM (5–10
m
M). Subcultures were prepared by trypsinization, and
the cells used for not more than 10 passages after receipt from the distrib-
utor.
Microspectrofluorimetric Measurements
Coverslips with dye-loaded cells were mounted into a heated metal flow-
through perfusion chamber described previously (Negulescu and Machen,
1990) placed on the stage of an inverted Zeiss IM 35 microscope and per-
fused by gravity feed at a rate of 1.5–2 ml/min. Change of solutions was
made by a remote-controlled electronic manifold. Emitted fluorescence
from single cells was measured in response to alternate pulses of excita-
tion light (5-msec duration) at 340 nm and 380 nm using a computer-con-
trolled four-place sliding filter holder manufactured in-house. The emitted
fluorescence (510 nm) was focused on a photomultiplier tube, amplified,
digitally converted, and sampled on an IBM-compatible computer. The
filter exchange system and data sampling software were designed by
Giuseppe and Antonio Troccoli (Bari, Italy). All measurements were au-
tomatically corrected for background. The ratio of emitted light from the
two excitation wavelengths (340/380) of fura-2 or mag-fura-2 provide a
measure of ionized cytoplasmic [Ca
2
1
] (Grynkiewicz et al., 1985) or intra-
store [Ca
2
1
] (Hofer and Machen, 1993; Hofer et al., 1998), respectively.
Mag-fura-2 data are presented as uncalibrated ratio changes and not free
[Ca
2
1
] due to uncertainties in the calibration procedure as described pre-
viously by Hofer and Schulz (1996).
Ratio Imaging Experiments
Some measurements in this study were made using a commercial imaging
system (Georgia Instruments, Roswell, Georgia) described previously in
more detail (Gamberucci et al., 1994). The 345/375 nm excitation ratio
(emission 450 nm) was acquired from individual cells within the micro-
scope field every 4 s. Cells were superfused continuously on the heated
microscope stage in an open Leiden chamber equipped with gravity feed
inlets and vacuum outlets for solution changes.
Solutions and Materials
Unless otherwise stated, all chemicals were purchased from Farmitalia
Carlo Erba (Milano, Italy), Fluka AG (Buchs, Switzerland) or Sigma
Chemical Co. (St. Louis, MO). Experiments were performed with a
Ringer’s solution containing (in mM) 121 NaCl, 2.4 K
2
HPO
4
, 0.4 KH
2
PO
4
,
1.2 CaCl
2
, 1.2 MgCl
2
, 5.5 glucose, 10 Hepes/NaOH, pH 7.20. Bradykinin
and ionomycin were from Calbiochem-Novabiochem Corp. (La Jolla,
CA); fura-2-AM and mag-fura-2-AM were obtained from Molecular
Probes (Eugene, OR); InsP3 was from L.C. Services (Woburn, MA).
When DMSO or ethanol were used as a solvent, their final concentration
never exceeded 0.01 or 0.1%, respectively. Where applicable, data are ex-
pressed as means
6
SEM, with
n
equal to the number of experimental
runs.
Cell Permeabilization
As described previously (Scheenen et al., 1998; Hofer et al., 1995), dye-
loaded cells were rinsed briefly in a high K
1
solution (in mM: 125 KCl, 25
NaCl, 10 Hepes, pH 7.25, 0.1 MgCl
2
), and then exposed for 2–3 min to an
intracellular buffer at 37
8
C (the same solution supplemented with 0.5 mM
MgATP, pH 7.25, and Ca
2
1
/EGTA buffers, 0.1 total [EGTA], 200 nM free
Ca
2
1
, calculated according to the computer program described in Bers et
al., 1994) also containing 5 mg/ml digitonin. After plasma membrane per-
meabilization, cells were continuously superfused with intracellular buffer
(without digitonin). Imaging measurements were performed as above for
intact cells.
Results
Mitochondria are known to take up Ca
2
1
released into the
cytoplasm during agonist stimulation via an electrogenic
Ca
2
1
uniporter, a process dependent on the membrane po-
tential across the inner membrane (Rizzuto et al. 1992,
1993; Pozzan et al., 1994). Carbonyl cyanide p-trifluo-
romethoxy-phenylhydrazone (FCCP)
1
is a protonophore
1.
Abbreviations used in this paper
: BK, bradykinin; FCCP, carbonyl cya-
nide p-trifluoromethoxy-phenylhydrazone; InsP3, inositol 1,4,5-trisphos-
phate; tBHQ, 2, 5-Di(tert-butyl)hydroquinone.

Landolfi et al.
Internal Store [Ca
2
1
] Dynamics in Intact Cells
1237
that uncouples mitochondrial respiration and ATP pro-
duction by dissipating the proton gradient across the inner
mitochondrial membrane. As it abolishes the membrane
potential normally maintained by oxidative phosphoryla-
tion, FCCP also completely prevents mitochondrial Ca
2
1
uptake (Gunter and Pfeiffer, 1990; Gunter et al., 1994;
Rizzuto et al., 1994).
Mitochondrial Inhibitors Impede Ca
2
1
Uptake into
Internal Stores by at Least Two Mechanisms
We tested the effects of FCCP and other mitochondrial in-
hibitors on Ca
2
1
handling by the ER of BHK-21 cells
loaded with the AM-ester of the fluorescent Ca
2
1
dye
mag-fura-2 (Fig. 1). We have shown previously in this cell
type that prolonged loading with this indicator results in
preferential accumulation of dye into organelles (Hofer et al.,
1998; see also Golovina and Blaustein, 1997). The low af-
finity of the dye for Ca
2
1
allows for sensitive measure-
ments almost exclusively in organelles where Ca
2
1
is high,
i.e., the ER (Hofer and Schulz, 1996; Hofer et al., 1995).
Adding 1
m
M of FCCP to resting cells (in the presence of
glucose) frequently caused a slow, persistent, and revers-
ible decrease in the ratio (Fig. 1
A
;
n
5
8 out of 13 cells)
comparable to that elicited by treatments that block
SERCA-mediated Ca
2
1
uptake (Hofer et al., 1995; Hofer
et al., 1996). A similar, although poorly reversible de-
crease in the ratio was observed upon acute treatment
with 1
m
M oligomycin, which blocks the ATP synthetase
responsible for coupling the proton gradient to ATP pro-
duction (
n
5
3; not shown).
As further illustrated by Fig. 1
A
, when intact cells were
stimulated in the presence of FCCP with 100 nM bradyki-
nin (BK) to release stored Ca
2
1
, the rapid recovery of
Ca
2
1
that was normally seen when the agonist was re-
moved (see for example Fig. 3) was not observed until
FCCP was washed out. Adding FCCP during the recovery
phase in BK-stimulated cells prevented store refilling
(Fig. 1
B
). When SERCA activity was abolished with 2,
5-Di(tert-butyl)hydroquinone (tBHQ), however, (result-
ing in a relatively slow leak of Ca
2
1
out of the store) FCCP
did not further reduce the mag-fura-2 ratio, showing that
tBHQ and FCCP act on the same Ca
2
1
pool (Fig. 1
B
;
n
5
6). In fact, FCCP caused a slight reversal of the downward
trend in the signal, a result we attribute to effects of ATP
depletion on the passive Ca
2+
leak of internal Ca
2+
stores
(Hofer et al. 1996).
Collectively, the data of Fig. 1 strongly suggest that un-
coupling mitochondria grossly affects Ca
2
1
uptake into the
ER. However, mitochondrial inhibitors, particularly pro-
tonophores such as FCCP, are subject to several nonspe-
cific effects (e.g., membrane potential and pH changes)
that could influence our measurements. It should be
stressed that in previous experiments on permeabilized
BHK-21 cells where [ATP] in the superfusion was kept
constant at 3 mM, there was no effect of 1
m
M FCCP on
the mag-fura-2 ratio (Hofer et al., 1995), nor was there any
consequence of adding ruthenium red, valinomycin, or oli-
gomycin
1
azide (Hofer et al., 1995), agents that either
prevent mitochondrial Ca21 uptake and/or induce Ca21 re-
lease from that organelle. Thus, these drugs have no effect
per se on the fluorescence of mag-fura-2, and do not cause
release of Ca21 from mitochondria that can be detected by
this low-affinity fluorophore in permeabilized BHK-21
cells. Furthermore, as shown in Fig. 2, InsP3-induced re-
lease and reloading of internal stores of permeabilized
cells was unaffected by FCCP. The response to a supra-
maximal dose of InsP3 (6 mM) was similar before and after
treatment with 1 mM FCCP under conditions where [Ca21]
and [ATP] of the intracellular-like buffer were clamped to
170 nM and 0.5 mM, respectively. When FCCP was given
to cells during the recovery phase after InsP3 washout,
there was no consequence on the mag-fura-2 ratio (Fig. 2).
In contrast to the data of Fig. 2, measurements of store
[Ca21] in intact cells revealed that metabolic poisons dra-
matically prolonged the time course of store refilling (Fig.
3). Direct comparison of the rates of recovery after BK
stimulation between control and FCCP-treated cells (Fig.
3 A) showed that refilling rates were on average only 18.3 6
2.5% of control values in the same cell (n 5 23), with some
Figure 1. FCCP inhibits ER Ca21 uptake. Mag-fura-2 measure-
ments of [Ca21] in agonist-sensitive internal stores in intact cells.
(A) Resting intact cell treated with FCCP (1 mM) followed by
FCCP 1 BK (100 nM). (B) FCCP administered during store refill-
ing subsequent to BK-induced release inhibited Ca21 sequestra-
tion, but had little effect on the mag-fura-2 ratio when given after
treatment with a SERCA blocker, tBHQ (10 mM).

The Journal of Cell Biology, Volume 142, 1998 1238
variability at the single-cell level. Similar experiments us-
ing oligomycin to block ATP synthesis revealed that the
inhibitor reduced the rate of recovery to 25 6 4% of con-
trol (Fig. 3 B). Comparable results were obtained using
4 mM rotenone (not shown; blocks oxidative phosphoryla-
tion at complex I; n 5 3), oligomycin plus rotenone (n 5
3), or combinations of rotenone, oligomycin, and 1 mM
atractiloside (inhibits ATP/ADP exchange across the in-
ner mitochondrial membrane; n 5 4). Taken together,
these data indicate that a specific effect on mitochondrial
function accounts for the inhibition of store refilling in the
intact cell.
How do mitochondrial inhibitors impede internal store
refilling? While the simplest explanation for the effects of
such pharmacologically diverse compounds as FCCP, oli-
gomycin, rotenone, and atractiloside in the intact cell
(Figs. 1 and 3) is that the energy supply for Ca21 uptake
had been rapidly compromised upon mitochondrial poi-
soning, another important consideration is the effect of
these agents on capacitative Ca21 entry at the plasma
membrane. Since store refilling depends strictly on Ca21
supplied from the extracellular milieu in many cell types,
including BHK-21 cells (Hofer et al., 1998), inhibition of
this pathway will have important consequences on Ca21
reuptake. To bypass the requirement for Ca21 entry, we
took advantage of a phenomenon that we have described
recently, permitting internal store refilling in Ca21-free ex-
ternal medium, provided that the cells have been pre-
loaded with BAPTA-AM (Hofer et al., 1998). As seen in
Fig. 3 C (typical of n 5 14), the rate of recovery in the
presence of FCCP after agonist washout was considerably
faster in the Ca21-free solution/BAPTA-AM pretreated
condition, where Ca21 entry pathways had been bypassed
(FCCP alone was 71 6 7% of the BAPTA/FCCP recov-
ery rate). However, the rate of refilling was even faster in
zero Ca21/BAPTA conditions in the absence of FCCP
(147 6 6% of the FCCP 1 BAPTA recovery rate).
A similar protocol using BAPTA preloading to circum-
vent the plasma membrane Ca21 entry pathway showed
that oligomycin reduced the rate of recovery in BAPTA/0
Ca21 to 47 6 7% of that of the control (n 5 19; Fig. 3 D).
It should be noted that somewhat higher concentrations of
oligomycin have been reported to inhibit store-operated
channels by a means independent of cellular ATP deple-
tion (Cho et al., 1997).
These data imply that a major action of mitochondrial
inhibitors on store refilling operates via an indirect mecha-
nism as a consequence of inhibition of capacitative Ca21
entry pathways at the plasma membrane. However, Fig. 3,
C and D also indicates that mitochondrial ATP production
(or alterations in the ATP/ADP ratio) likely influence
Ca21 reaccumulation by the ER to a significant degree.
This result was quite surprising, as the available literature
suggests that rather dramatic reductions in [ATP] would
be required to block SERCA-mediated Ca21 uptake (see
Discussion). We therefore checked this point in permeabi-
lized BHK-21 cells by comparing the rates of Ca21 reac-
cumulation after InsP3-induced release while clamping
[ATP] (and [ADP]) in the perfusate to various levels. As
shown in Fig. 4 A, rapid recovery was observed even when
[ATP] in the bath was reduced to 50 mM. Stores reaccu-
mulated Ca21 at near maximal speed with [ATP] fixed at
200 mM (not shown). Thus, profound cellular [ATP] de-
pletions are necessary to impede Ca21 reuptake.
A further consideration, however, is that metabolic inhi-
bition may alter not only [ATP], but also the ATP/ADP
ratio (which is the true index of the driving force for Ca21
reaccumulation). There was very little effect on the recov-
ery when the ATP/ADP ratio was set to one (Fig. 4 B; 0.5
mM ATP, and 0.5 mM ADP or 0.05 ATP and 0.05 ADP;
n 5 6). In contrast, the recovery after InsP3-induced Ca21
release was almost completely blocked when [ATP] was
0.5 mM and [ADP] was 4 mM (n 5 4; Fig. 4 C).
Mitochondrial Inhibitors Impede Ca21 Release from
Internal Stores by at Least Two Mechanisms
In addition to interfering with the refilling process, mito-
chondrial inhibitors also affect the kinetics of Ca21 release
in intact cells. These results can be better appreciated
when the time scale of the data presented in the inset of
Fig. 3 is expanded, as shown in Fig. 5.
As seen in Fig. 5 A after a 1-min pretreatment with
FCCP, cells were stimulated briefly (to minimize receptor
desensitization), consistently resulting in a marked slowing
of the rate and magnitude of Ca21 release compared with
the control BK response in the same cell (rate 51.6 1 4.5%
of control; n 5 23). This result was observed irrespective
of whether the control stimulation was performed before
or after the stimulation plus FCCP. On the other hand, the
rates of release for two successive control challenges with
BK were not significantly different from one another (not
shown; n 5 16; see also additional data in Hofer et al.,
1998).
Using the same experimental protocol, oligomycin was
also shown to alter the kinetics of Ca21 release (Fig. 5 B),
although to a lesser extent (73 6 4% of control, n 5 23)
Figure 2. Effects of FCCP on Ca21 homeostasis in internal stores
of digitonin-permeabilized mag-fura-2-loaded BHK-21 cells.
Cells were continuously superfused with intracellular buffer.
[Ca21] was clamped with EGTA buffers to 170 nM, and a con-
stant [ATP] of 0.5 mM was maintained. FCCP had no effect on
the mag-fura-2 ratio or InsP3-induced release and reloading of in-
ternal stores under these conditions.

Landolfi et al. Internal Store [Ca21] Dynamics in Intact Cells 1239
Figure 3. FCCP and oligomycin impede store refilling after BK-induced Ca21 release in intact mag-fura-2-loaded cells. (A) Control re-
covery and response in the same cell to hormone after brief pretreatment with FCCP. (Inset) Entire experimental record. (Bottom) En-
larged overlay emphasizing the recovery phases with and without FCCP. (B) As above, profile of store reloading before and after treat-
ment with 1 mM oligomycin. (C) Comparison of store refilling after three separate stimulations with 100 nM BK: first in the presence of
1 mM FCCP in Ca21-containing medium; second after a 15-min incubation with 40 mM BAPTA-AM (added at arrow); cells were
washed with normal Ringer’s for 5 min, and were then stimulated with BK in Ca21-free medium containing 1 mM FCCP. Finally, cells
were stimulated in the absence of FCCP, still in Ca21-free external medium. (D) Cells were loaded with 40 mM BAPTA-AM for 15 min
before the start of experiment. (Inset) Responses to 100 nM BK in Ca21-free medium before and after treatment with 1 mM oligomycin.
(Bottom) Enlarged overlay of intraluminal [Ca21] changes, oligomycin vs. control.

The Journal of Cell Biology, Volume 142, 1998 1240
than FCCP. Direct comparison in the same cells further
confirmed that the uncoupler was more effective in slow-
ing the release of Ca21 than was oligomycin (the rate in
FCCP was 68 6 6% of that in oligomycin; n 5 22).
The question then arises as to whether the effects of
FCCP or oligomycin in intact cells are due to impairment
of ATP production, inhibition of Ca21 uptake by mito-
chondria, or both. It should be stressed that oligomycin
does not block Ca21 uptake by mitochondria, but only in-
hibits ATP synthesis. Thus, the most plausible explanation
for inhibition of Ca21 release by oligomycin alone is that in
the absence of mitochondrial ATP production, the release
channels in the store become susceptible to reductions in
[ATP] (possibly in a localized domain), and to an extent
that can compromise the Ca21 release process. The fact
that FCCP was more effective in slowing the discharge
from internal stores (Fig. 3, A and C) could therefore be
explained by the fact that FCCP produces a more pro-
found depletion of cytoplasmic [ATP] than does oligomy-
cin (see for example Budd and Nicholls, 1996). Alterna-
tively, there may be an additional effect of FCCP because
of the failure of mitochondria to buffer cytoplasmic Ca21
changes near the InsP3 receptor, resulting in a reduced
rate of Ca21 release from the stores. To distinguish be-
tween these possibilities, we compared the rates of release
after FCCP alone with those after treatment with a combi-
nation of FCCP and oligomycin (not shown). The ratio-
nale for these experiments is as follows: with oligomycin
present, the ATP consumption by the mitochondrial F0-F1
ATPase that normally occurs upon FCCP addition will be
prevented so that the degree of ATP depletion with the
combination of FCCP and oligomycin will be the same as
with oligomycin alone. Therefore, if the effect that we ob-
serve on Ca21 release is due only to ATP depletion, then
FCCP alone should slow down the response to BK much
more than the combination of FCCP and oligomycin.
However, we observed that the rate of Ca21 release under
these two conditions was essentially identical in paired ex-
periments conducted on the same cells (n 5 44 cells). This
result indirectly suggests that Ca21 uptake by mitochon-
dria (abolished by FCCP) also plays a role in modulating
Ca21 release from the stores.
We next undertook a series of experiments to more di-
rectly assess the role of mitochondrial Ca21 buffering in
Figure 4.(A) Effects of changing [ATP] on rates of refilling in
digitonin-permeabilized BHK-21 cells loaded with mag-fura-2.
Rates of refilling after stimulation with 6 mM InsP3 in the pres-
ence of 50 mM ATP is compared with that in 0.5 mM. Maximal
recovery velocity was obtained when [ATP] was 200 mM (not
shown). (B) Rates of recovery after release with 6 mM InsP3 were
barely affected by adding 0.5 mM ADP during the recovery
phase. (C) 4 mM ADP (in the presence of 0.5 mM ATP) blocked
refilling.
Figure 5. Effects of mitochondrial inhibitors on rates of release
in intact mag-fura-2–loaded cells. Data are taken from the
records shown in Fig. 3, and are represented on an expanded time
scale to allow better appreciation of the release kinetics. (A)
FCCP vs. control, (B) oligomycin vs. control, (C) as in Fig. 3 C,
comparison in the same cell between rates of release: (a) in the
presence of 1 mM FCCP (FCCP); (b) after a 15-min incubation
with 40 mM BAPTA-AM, after which cells were washed with
normal Ringer’s for 5 min and then stimulated with BK in Ca21-
free medium containing 1 mM FCCP (BAPTA/ FCCP); (c) cells
stimulated in the absence of FCCP, still in Ca21-free external me-
dium (BAPTA control). (D) Data from Fig. 3 D: rates of release
after BAPTA-AM pretreatment in the presence and absence of
oligomycin.

Landolfi et al. Internal Store [Ca21] Dynamics in Intact Cells 1241
shaping the profile of Ca21 discharge by comparing the
rates of release in the presence of FCCP before and after
loading of the cytoplasm with the rapid Ca21 buffer
BAPTA-AM. The exogenous Ca21 buffer should prevent
local increases in [Ca21]cyt near the InsP3 receptor, in ef-
fect rescuing the physiological Ca21-buffering activity that
we predict to be furnished by the mitochondria. Fig. 5 C
shows that the inhibitory effect of FCCP on BK-induced
Ca21 release was largely overcome when the same cells
were loaded on the microscope stage for 15 min with 40 mM
BAPTA-AM. The rates of release for the first stimulation
in the presence of FCCP alone were 59 6 7% (n 5 51) of
those with FCCP and BAPTA. This rate was in turn gen-
erally slightly slower than the BK-induced Ca21 release
measured in the BAPTA-loaded cells in the absence of
FCCP, and as illustrated by Fig. 5 C, with an altered ki-
netic profile.
Fig. 5 D shows that the rates of release were also attenu-
ated by oligomycin under these conditions; noteworthy
were the kinetic differences between the two conditions.
The results of Fig. 5 C attest to the importance of mito-
chondrial Ca21 buffering in modulating the kinetics of ER
Ca21 discharge. However, the data of Fig. 5, B and D also
strongly suggest that [ATP] depletion alters the dynamics
of Ca21 mobilization, possibly through a direct interaction
with the InsP3 receptor. We again checked this point in
permeabilized cells. Alterations in [ATP] over a wide
range of concentrations had clear effects on the kinetics of
Ca21 release. As shown in Figs. 4 and 6 A, while the initial
rates of release were somewhat slower when [ATP] was
reduced from 5 to 0.5 to 0 mM, the most striking action
was on the later phases of the response (n 5 18). The drop
in luminal [Ca21] was consistently less abrupt (less quan-
tal; Meyer and Stryer, 1990) as [ATP] was lowered, as fur-
ther illustrated by Fig. 6 B. Our results in BHK-21 cells
differ in part from those of Bezprozvanny and Ehrlich
(1993) regarding the type I InsP3 receptor, which was
found to have a reduced open probability at [ATP] above
4 mM. No effect of ADP (used at concentrations of 0.5 to
4 mM) was seen (not shown; n 5 6). These results in per-
meabilized cells confirm that alterations in intracellular
[ATP] can potentially influence the profile of Ca21 release
after agonist activation.
Discussion
A large literature exists concerning the effects of mito-
chondrial inhibitors on Ca21 homeostasis in intact cells
(see for example Mohr and Fewtrell, 1990; Babcock et al.,
1997; Drummond and Fay, 1996; Friel and Tsien, 1994).
These studies relied principally on measurements of Ca21
changes in the cytoplasm (for example, using fluorescent
indicators), or used 45Ca21 to make population measure-
ments of the total cellular Ca21 content. While some inves-
tigators concluded that poisoning of mitochondrial metab-
olism could compromise Ca21 pumping by the SERCAs
(resulting in loss of Ca21 from the ER; Fasolato et al.,
1991; al-Baldawi et al., 1993), many other studies have
promoted the idea that the mitochondria themselves serve
as a large reservoir of Ca21 that can be released into the
cytoplasm after treatment with inhibitors (Babcock et al.,
1979; Fulceri et al., 1991).
Here we have reexamined this complex issue by measur-
ing Ca21 in the ER directly using a low-affinity fluorescent
indicator in intact cells. Our results show that brief pre-
treatment with a wide range of mitochondrial inhibitors
(atractiloside, rotenone, oligomycin, and FCCP) all re-
sulted in a dramatic inhibition in store refilling after re-
lease of stored Ca21 with agonists (Fig. 2). In addition,
FCCP or oligomycin added acutely to resting cells fre-
quently caused a slow but measurable loss of stored Ca21
that cannot be attributed to release of the cation from the
mitochondria themselves, as the effect was not additive to
that obtained by blocking SERCAs with tBHQ. Further-
more, the lack of effect of FCCP in the permeabilized cell
preparation, where the [Ca21] (buffered by EGTA) and
the [ATP] of the extraorganellar milieu are fixed (Fig. 2),
are consistent with the idea that physiological interactions
between mitochondria and other cellular components (the
ER and plasma membrane) account for the actions of
these drugs in the intact cell.
The data of Fig. 3 C, in which stores were refilled in the
intact BAPTA-loaded cells in the absence of external
Figure 6. ATP modulates InsP3-induced Ca21 release in digito-
nin-permeablized cells. (A) Comparison of two sequential stimu-
lations with 6 mM InsP3 in the absence of ATP and in the pres-
ence of 5 mM ATP. (B) Increasing [ATP] to 5 mM during the
release phase enhances the rate of InsP3-induced Ca21 discharge.

The Journal of Cell Biology, Volume 142, 1998 1242
Ca21, indicate that a significant mechanism by which mito-
chondrial inhibitors block store refilling is through an indi-
rect effect on plasma membrane Ca21 entry pathways.
Consistent with this idea is the finding that FCCP signifi-
cantly inhibited the plateau phase of Ca21 entry as mea-
sured by fura-2 (data not shown; see also Hoth et al., 1997;
Marriot and Mason, 1995; Gamberucci et al., 1994). These
data illustrate that the entry pathway was potently inhib-
ited in spite of the fact that stores remained relatively
empty after treatment with the uncoupler (Fig. 3 A).
The results of Fig. 3, C and D also show, however, that
mitochondrial inhibitors influence Ca21 reuptake by an
additional mechanism besides that at the plasma mem-
brane, as the rate of refilling in BAPTA-pretreated cells
was significantly slower in the presence of oligomycin or
FCCP compared with the control condition. We attribute
this difference to effects on mitochondrial ATP produc-
tion. Interpreted in this way, however, these results are
somewhat surprising. In the presence of glucose in the
bathing medium (such as was the case here), mitochon-
drial inhibitors are known to cause only a relatively slow
and partial (50%) depletion of intracellular ATP in BHK-
21 cells (Burkhardt and Argon, 1989), and in a variety of
other cell types as well (see for example Mohr and Few-
trell, 1990; Fulceri et al., 1991; Luo et al., 1997). Mean-
while, SERCA pumps, like other ion-motive ATPases, are
known to function even when [ATP] falls to quite low lev-
els. In fact, our data from permeabilized BHK-21 cells
(Fig. 6) show that stores are able to recover Ca21 at maxi-
mal speed when the [ATP] in the bath is clamped to just
200 mM. Reducing the [ATP] in the bath to 50 mM only
slowed the rate of refilling to z65% (n 5 37 cells), and still
allowed complete recovery. Our data may therefore sug-
gest that local ATP depletions (and/or extreme local alter-
ations in ATP/ADP) near the SERCAs that result from
the increased pumping activity after agonist activation oc-
cur before global changes in cellular [ATP] (or the ATP/
ADP ratio) are evident, making the Ca21 uptake process
more susceptible to mitochondrial inhibition than might
be expected from measurements of total cellular adenine
nucleotide content. A wide variety of morphological evi-
dence showing close physical apposition between mito-
chondria and ER lends support to this idea (McGraw et
al., 1980; Nixon et al., 1994; Takei et al., 1992; Rizeuto et al.,
1998).
Ca21 buffers have well-established effects on the dy-
namics of Ca21 release from internal stores (Hajnóczky
and Thomas, 1997; Montero et al., 1997). A number of
other studies have stressed the importance of mitochon-
drial Ca21 uptake and release in modulating cytosolic Ca21
signals (Mohr and Fewtrell, 1990; Babcock et al., 1997;
Drummond and Fay, 1996; Loew et al., 1994; Jouaville et al.,
1995; Simpson and Russel, 1996; Hoth et al., 1997), and
our direct measurements of intraluminal ER Ca21 dynam-
ics (Fig. 5, A and C) also support this view. In addition,
however, our data using oligomycin alone to inhibit ATP
synthesis also indicates that local ATP availability can sub-
tly alter the responsiveness of stores to agonists. This ef-
fect is most readily explained by the ATP dependence of
the InsP3 receptor (Smith et al., 1985; Ferris et al., 1990;
Bezprozvanny and Ehrlich, 1993), although the possibility
that ATP depletion can impede InsP3 production must
also be seriously considered. The data of Figs. 4 and 6
show that altering [ATP] in the intracellular buffer in per-
meabilized BHK-21 cells had clear effects on the kinetics
of InsP3-induced release, which were largely compatible
with the observed effects of mitochondrial inhibitors on
ER [Ca21] in intact cells. Again, it is possible that local
[ATP] changes may be responsible for the observed differ-
ences.
In any event, our results clearly demonstrate that active
mitochondrial respiration modulates the dynamics of Ca21
release, allowing for explosive efflux of Ca21 from internal
stores. How these kinetic differences influence cytosolic or
intraluminal Ca21-binding effectors, thereby influencing
the signal transduction potential of intracellular [Ca21]
changes, remains to be established. These findings prompt
the interesting possibility that subtle alterations in the re-
lease dynamics may somehow provide a signal to the rest
of the cell, conveying information about the cellular meta-
bolic status.
The authors thank Dr. Paolo Pinton for help with some preliminary mea-
surements, and Dr. Rosario Rizzuto for helpful comments on the manu-
script. B. Landolfi was a recipient of a fellowship from the E.E.C.
This work was supported by a grant from Ministero Universitá
Ricerche Scientifica e Tecnologica (40% fund) to S. Curci and by grants to
T. Pozzan from Telethon (N845), Consiglio Nazionale delle Ricerche Bio-
technology, Human Science Frontiers, and the Armenise-Harvard Foun-
dation. A.M. Hofer was supported by Human Science Frontiers.
Received for publication 4 May 1998 and in revised form 19 June 1998.
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