Department of Physiology & Biophysics, University of Washington, Seattle, Washington 98195-7290; and * Image Analysis Laboratory, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104
Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca2+ uptake and extrusion modulate free cytosolic [Ca2+] (Cac) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca2+ entry, and to monitor Ca2+-activated currents that report near-surface [Ca2+]. We show that rapid recovery from elevations of Cac requires both the mitochondrial Ca2+ uniporter and the mitochondrial energization that drives Ca2+ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca2+] (Cam). The indicated resting Cam of 100-200 nM is similar to the resting Cac reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Cam and Cac at high temporal resolution shows that, although Cam increases less than Cac, mitochondrial sequestration of Ca2+ is fast and has high capacity. We find that mitochondrial Ca2+ uptake limits the rise and underlies the rapid decay of Cac excursions produced by Ca2+ entry or by mobilization of reticular stores. We also find that subsequent export of Ca2+ from mitochondria, seen as declining Cam, prolongs complete Cac recovery and that suppressing export of Ca2+, by inhibition of the mitochondrial Na+/ Ca2+ exchanger, reversibly hastens final recovery of Cac. We conclude that mitochondria are active participants in cellular Ca2+ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca2+.
The Ca2+ content of mitochondria within resting
cells is believed to be low, yet isolated mitochondria
can accumulate large quantities of Ca2+ when it is
provided to them (Lehninger et al., 1967 Here we introduce two fluorescent Ca2+ probes, one into
the cytosol and the other into mitochondria of single rat
chromaffin cells. Simultaneous optical monitoring demonstrates that even modest elevations of Cac produce rapid
transient redistributions of Ca2+ from the cytosol into mitochondria. The mitochondrial Ca2+ uptake shapes and
dampens the elevations of Cac during stimulation, and
subsequent Ca2+ export from mitochondria delays complete recovery of Cac and may facilitate refilling of intracellular stores. Cellular Ca2+ signaling may best be considered as an interactive, multicompartmental network in which
mitochondria are active participants.
All experiments were performed at a room temperature of 24-27°C. Results are reported as mean ± SEM.
Chemicals
CGP-37157 (7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-on)
was the generous gift of Dr. Alain DePover, CIBA-GEIGY (Basel). Dyes
were from Molecular Probes (Eugene, OR); ionomycin and BHQ (2,5 di(t-butyl)-1,4-hydroxyquinone) from Calbiochem (La Jolla, CA); CCCP
(carbonyl cyanide m-chlorophenylhydrazone) and all other reagents from
Sigma Chem. Co. (St. Louis, MO).
Cells and Media
Adrenal chromaffin cells were prepared as described previously (Herrington et al., 1996 Patch Clamp Procedures
Voltage clamp recordings used an Axopatch 1C amplifier (Axon Instruments, Foster City, CA) and the BASIC-FASTLAB analysis system (Indec Systems, Capitola, CA). The standard voltage protocol was a 0.05-1 s
step to +10 mV from a holding potential of For the whole-cell configuration of the patch clamp technique (Hamill
et al., 1981 The standard external solution used during seal formation contained:
150 NaCl, 2.5 KCl, 2 mM CaCl2, 1 MgCl2, 10 Hepes, and 20 mM glucose,
pH 7.4. For perforated-patch recordings external solutions also contained
2.5 mM Na pyruvate and 2.5 mM malic acid to optimize mitochondrial
function (Villalba et al., 1994 Activation of voltage-dependent Ca2+ channels by depolarization provides precisely timed, but imperfectly predictable control of Ca2+ entry, as
judged by the peak Cac produced. The degree of buffering by introduced
dyes and chelators, and the extent of time-dependent channel `rundown'
(more severe in whole-cell- than in perforated-patch-recordings), and previous stimulus history all contribute to this variability. For these reasons,
the durations of depolarizing stimuli often were adjusted upwards during
the course of an experiment in an attempt to produce similar Cac challenges. In the interpretation of these experiments, differences in peak Cac
reflect mainly imperfect compensation for this rundown.
Dye Loading and Photometry
Rhod-2-AM (0.2 mM) and Calcium GreenTM-AM (10 mM) were dispensed from DMSO stocks, dispersed in 10% Pluronic 147, and diluted to
1 and 12 µM in 0.25 ml standard external solution. A single coverslip of
cells was immediately added, incubated 35-50 min at 22-25°C in the dark,
then transferred to an examination chamber containing 2-4 ml fresh buffer. Indo-1 (100 µM), or either rhod-2 or Calcium Green dextran (3,000 Mr)
at 10 µM together with 90 µM BAPTA, were introduced by inclusion in
the whole-cell pipette solution. All photometric measurements were performed on an inverted microscope using a 1.3 NA 40× oil objective (Nikon),
an attenuated (1.0 NDF) 100 W Hg source, and paired photon-counting
detectors (Hamamatsu). An electronic shutter (Uniblitz) restricted excitation to the sampling interval (50-100 ms; duty cycles of 0.5% in normal and 50% in fast mode). Pinhole diaphragms limited the excitation and emission fields to ~25 µm diameter regions. Indo-1 was excited at 365 ± 10 nm
and detected at 405 ± 35 and 500 ± 40 nm. A filter cube with dual band
excitation (490 ± 15 and 560 ± 15 nm) and dual band emission (530 ± 15 and 650 ± 75 nm) (XF-52; Omega Optical) allowed simultaneous excitation of Calcium Green and rhod-2. Detection at 525 ± 10 and 580 ± 30 nm
was accomplished with additional interference filters separated by a 540nm dichroic mirror.
Calibration of Indo-1, Rhod-2, and Calcium
Green Signals
The ratiometric probe indo-1 was calibrated as detailed previously (Herrington et al., 1996 [Ca2+ ]=K*(R where Rmin (0.47), Rmax (7.15), and K* (2540 nM) were determined empirically from cells dialyzed with pipette solutions containing 50 mM EGTA,
15 mM CaCl2, or 20 mM EGTA and 15 mM CaCl2 (calculated free [Ca2+]
of 251 nM), respectively.
Fluorescence signals from the single-wavelength indicators rhod-2 and
Calcium Green were corrected first for autofluorescence observed with
nonloaded cells (<5% of total signal), then for the spillover of fluorescence from Calcium Green (22-48%) or its dextran conjugate (10-15%)
into the long-wavelength (rhod-2) detector channel, as determined from
cells loaded only with these probes. The extent of spillover increased with
age of the dual pass excitation filter set, presumably as a consequence of
deterioration. Spillover of fluorescence from rhod-2 into the short wavelength (Calcium Green) channel was negligible. Compensation also was
applied for time-dependent loss of signal (primarily photobleaching) as
determined from exponential fits of resting-state fluorescence before and after recovery from stimulus (mean rate constants: rhod-2, 4.89 ± 0.10 × 10 [Ca2+ ]=Kd(F where the Kd determined in vitro for rhod-2 (500 nM) or Calcium Green
(240 nM; Eberhard and Erne, 1991 These treatments elicited fluorescence in an average ratio (FMnSat/FCaSat)
of 0.75 ± 0.05 (n = 11) and 0.78 ± 0.04 (n = 9) for Calcium Green and its
dextran conjugate, indistinguishable from FMnSat/FCaSat ratios determined in
vitro in a 150 mM KCl, 1 mM MgCl2, pH 7.4 medium, containing 1 µM dye
and 10 mM CaCl2 or MnCl2. Compared with dye in the same medium containing 10 mM EGTA, Ca2+-free:Ca2+-saturated fluorescence (Ffree/FCaSat)
was 0.091. Assuming this ratio also applies in vivo, cellular Ffree was calculated from the observed FCaSat elicited by ionophore.
For the K+ salt of rhod-2, Ffree/FCaSat determined in vitro was 0.068. When the rhod-2 salt was applied to the cytosol by whole cell dialysis, the
FMnSat/FCaSat ratio (0.29 ± 0.01; n = 3) agreed well with that determined
fluorimetrically for rhod-2 in vitro (0.24). FMnSat/FCaSat also measured in
vitro was unchanged from pH 7-8.2 and altered little or not at all by variation of ionic strength (0.1-0.2 M KCl), viscosity (0-2 M sucrose), or dielectric constant (0-10% ethanol) that might be encountered intramitochondrially. However, for rhod-2 introduced into mitochondria by AM-loading, FMnSat/FCaSat was much larger either before (0.50 ± 0.13; n = 14) or after
(0.68 ± 0.11; n = 4) whole cell dialysis, presumably indicating that the
much higher affinity (>100-fold) of rhod-2 for Mn2+ than for Ca2+ and the
greater efficacy of the ionophore in Mn2+ transport (Erdahl et al., 1996 Imaging
Images were collected on a Deltavision SA3.1 wide-field deconvolution
microscope system (Applied Precision, Inc., Issaquah, WA) using a Nikon
PlanApo 60× 1.4 NA objective on an Olympus IMT-2 inverted microscope with a Photometrics PXL-2 cooled CCD camera containing a
Kodak KAF1400 chip. Images were collected with an intermediate magnification of 1.5×. Binning was used to increase signal-to-noise. For every 2-s
time point, four optical sections were collected at 0.2 µm spacing each with
0.3-s exposure. Final voxel resolution was 0.157 µm (planar) × 0.200 µm
(axial). Images of each z-axis series were corrected for photobleaching
and variations in lamp intensity; only intensity corrections were applied
between time points. The deconvolution reconstruction algorithms of
Agard and Sedat (Agard et al., 1989 We begin with the Ca2+-sensitive dye rhod-2 as a probe of
Cam. Extrusion of protons during electron transport creates a large inside-negative potential difference ( Rhod-2 Localizes to Mitochondria and Responds to
Ca2+ Entry
To verify compartmentalization of dye, cells loaded with
rhod-2AM were visualized by fluorescence deconvolution
imaging. In Fig. 1, rhod-2 fluorescence was found primarily in punctate and filamentous extranuclear regions, consistent with a mitochondrial localization, and in 1-3 small
(~1 µm) intranuclear bodies, tentatively identified as nucleoli. With long exposure images, the characteristic vermiform mitochondrial morphology (Loew et al., 1995
Calcium entry induced by KCl depolarization increased
the rhod-2 fluorescence within the presumptive mitochondria (Fig. 1 B). The fluorescence returned to baseline levels within a minute after the depolarization (Fig. 1, C and
D). No fluorescence increase occurred during application
of KCl in the nominal absence of external Ca2+ (not
shown). Hence, rhod-2 reports a transient rise of mitochondrial calcium following depolarization-induced Ca2+
entry. This is the phenomenon we wish to study.
Rapid Clearance of Cytosolic Ca2+ Requires Energized
Mitochondria and the Ca2+ Uniporter
Uptake of Ca2+ by isolated mitochondria involves electrophoretic transport through a Ca2+ uniporter driven by the
mitochondrial membrane potential
The first of these experiments (Fig. 3) showed that
CCCP and ruthenium red block rapid Ca2+ clearance.
Ca2+ entry into the cell was induced by a step depolarization of the plasma membrane, and removal of cytosolic
Ca2+ was monitored with the Ca2+ probe indo-1 introduced directly into the cytoplasm from a whole-cell pipette. In Fig. 3 A, spatially averaged Cac rose to >4 µM
during a brief depolarizing voltage step (first arrow) and
then decayed within a few seconds to submicromolar levels. As reported previously (Herrington et al., 1996
The second set of experiments (Fig. 3, C and D) tested
the ability of oligomycin and cyanide to block Ca2+ clearance. Ca2+ entry was again induced by a brief depolarization, but here clearance of submembrane cytosolic Ca2+ was
monitored electrophysiologically by the decay (tail) of a Ca2+-activated K+ current that is prominent in rat chromaffin cells (SK current: Neely and Lingle, 1992a Monitoring of the CCCP-sensitive
Mitochondrial Compartment
Using compartmentalized dyes, we can now verify the assumption that CCCP slows Cac clearance by preventing
uptake of Ca2+ into mitochondria. We have seen that coloading of chromaffin cells by coincubation with AM esters compartmentalizes rhod-2 into mitochondria and disperses Calcium Green throughout the cell. Signals from
the two dyes were followed at high time resolution using
photometry and a filter cube designed for simultaneous
dual-wavelength excitation and dual-wavelength detection.
After sequential corrections for autofluorescence, signal
cross-talk, and photobleaching, Cam and global free [Ca2+]
were calculated from rhod-2 and Calcium Green fluorescence by application of calibration parameters determined
for each cell examined. Voltage-clamp depolarizations controlled Ca2+ entry, and inhibitors were applied and removed rapidly by fast, local perfusion. We applied CCCP
in the presence of oligomycin to prevent any accelerated
consumption of cellular ATP by reverse-mode operation
of the ATP synthase in proton-permeant mitochondria (Budd and Nichols, 1996
The rapid rise and decline of [Ca2+] calculated from the
Calcium Green signal during and following Ca2+ entry
(Fig. 4, lower traces) resemble the Cac responses reported from indo-1 applied directly in the cytosol (Fig. 3 A).
Other common features include a delayed final return to
the resting level (Figs. 4, 6, and 8) and a small [Ca2+] increase when CCCP was applied late in the recovery from
brief Ca2+ entry (Fig. 4). Calcium Green and indo-1 signals also both report that CCCP slows the initial clearance
of Cac yet hastens the final recovery to resting levels.
These similarities in the signals from the two probes confirm the usefulness of AM-loaded Calcium Green to measure Cac. We note, however, that in the experiments of
Fig. 3, A and B and in previous studies using pipetteloaded indo-1 (Herrington et al., 1996
In comparison to the quick changes of Cac during and
after brief plasma membrane depolarizations, rhod-2 reported a more limited but far more sustained elevation of
Cam (Fig. 4, insets). Subsequent addition of CCCP caused
a simultaneous fall in Cam and rise in Cac (Fig. 4; n = 4),
presumably reflecting release of Ca2+ from depolarized
mitochondria. CCCP also strongly attenuated the rise of
Cam in response to elevation of Cac. The small residual signal may result from incomplete inhibition by CCCP, from
incomplete compensation for crossover of the Calcium
Green signal, or from rhod-2 reporting from a nonmitochondrial compartment.
Redistribution of Ca2+ during and after
Ca2+ Entry
We further refined the compartmentation of the two dyes
by using the patch pipette in whole-cell rather than perforated patch mode to allow introduction of the Calcium Green
as a dextran conjugate in the pipette solution. This strategy should restrict Calcium Green to the cytosol and also
remove any cytosolic rhod-2 and remaining ester by dialysis. Little or no loss of rhod-2 fluorescence occurred during
entry of Calcium Green (not shown), confirming the extensive compartmentation of this dye indicated by imaging in Fig. 1. Subsequent responses of the two dye signals to
Ca2+ entry evoked by depolarizations in the presence and
absence (Fig. 5) of CCCP were qualitatively similar to those
in Fig. 4. Because removal of CCCP restored Cam responses to Ca2+ entry (not shown), mitochondrial depolarization apparently does not result in artifactual release and
diffusional loss of rhod-2.
Under these more stringent conditions of dye compartmentation, simultaneous recording of Cac and Cam at high
temporal resolution suggests that three phases of Ca2+
traffic are initiated by Ca2+ entry. Fig. 5 A shows typical
(n = 4) time courses of rhod-2 and Calcium Green dextran
responses to a 500-ms depolarization, and Fig. 5 B shows
overlaid initial segments of these records (sampled every
100 ms) on an expanded time scale. The three phases are
best seen by plotting each Cam value against Cac (Fig. 5 C). During the 500-ms depolarization-induced Ca2+ entry,
Cam rose without delay and in direct proportion to the rise
in Cac. In the following 5 s, Cam continued to rise as Ca2+
was cleared from the cytosol, and Cac fell proportionately.
In the final >50 s, Cam fell slowly during the final return of
Cac to its resting value.
Our previous work suggested that mitochondria sequester the majority of the Ca2+ that enters the cell in response
to depolarization (Herrington et al., 1996 Ca2+ Is Extruded from Mitochondria by
Na+-Ca2+ Exchange
In the simplest interpretation, the fall in Cam that occurs
when Cac returns to near resting values would represent
export of mitochondrial Ca2+. Alternatively, Cam might
fall as a consequence of conversion of Ca2+ to an inaccessible form within the mitochondrial matrix (Lehninger, 1970 The benzothiazepine CGP-37157 selectively and potently inhibits the Na+-Ca2+ exchanger that exports Ca2+
from isolated mitochondria (Cox and Matlib, 1993 Cam Responds to Release of Ca2+ from
Intracellular Stores
Having established that mitochondria take up Ca2+ that
enters through voltage-gated Ca2+ channels of the plasma
membrane, we now consider if they also take up Ca2+ released from intracellular stores. Chromaffin cells have IP3sensitive Ca2+ stores that can be mobilized by agonists acting at G protein-coupled receptors (O'Sullivan et al.,
1989 Cells were first briefly perfused with bradykinin (n = 5)
or muscarine (n = 3) and 3-4 min later given a brief depolarizing step (Fig. 7). The Ca2+-mobilizing agonists and
voltage steps raised Cac to similar, but not identical, levels
(see Materials and Methods), and the amplitudes of the
corresponding Cam signals also were similar. Hence, mitochondria appear to sequester cytoplasmic Ca2+ ions,
whether entering from outside or released from intracellular stores. The initial recovery of Cac after stimulation by
agonist was consistently slower than after depolarizationinduced Ca2+ entry, very likely because IP3-dependent
mobilization continues for some seconds after the agonist
is washed away.
Chromaffin cells also contain a Ca2+ store that can be
mobilized by caffeine or by Ca2+ entry during perforatedpatch recording (Park et al., 1996 To substantiate the interpretation that mitochondria sequester Ca2+ released from CICR stores, we coapplied
caffeine and the reticular Ca2+-ATPase inhibitor BHQ to
deplete CICR stores and prevent their refilling (Fig. 9).
When caffeine and BHQ were added, Cam and Cac increased transiently (Fig. 9 A; n = 4), showing that when caffeine induces release of Ca2+ from stores, mitochondria
will take it up. For the preparation of cells used in these
experiments, channel rundown (see Materials and Methods) was more severe than for those of Fig. 8. A control stimulus of 500 ms was needed to produce a Cac challenge
similar to that found with a 250-ms pulse in the previous
experiments (compare the trajectories marked with circles
in Figs. 8 B and 9 B). Lengthening of the depolarization to
1 and 1.5 s then was required for subsequent stimuli applied in the presence of inhibitors.
Consider the trajectories of Cac and Cam that follow the
depolarizing pulses (Fig. 9 B). Under the control conditions, there was a full second during which Cac was relatively constant while Cam continued to rise. In the presence of caffeine and BHQ, subsequent depolarizations still
gave large averaged peak Cac responses but the rise of Cam
after the end of the depolarization was greatly reduced
(Fig. 9 B), confirming that much of the late rise of Cam represents uptake of Ca2+ released by CICR.
The initial rate of rise of Cam during Ca2+ entry is faster
in these perforated-patch experiments than it was with the
whole-cell recordings of Fig. 5. Using perforated-patch it
was 674 ± 150 nM s The original description of the synthesis and characterization of rhod-2 noted that its AM-ester might be selectively
accumulated, hydrolyzed, and trapped in mitochondria
(Minta et al., 1989 Previous work using blockers of mitochondrial function
implicated mitochondria in the recovery of neurons and
chromaffin cells from imposed Ca2+ entry (Thayer and
Miller, 1990 What Is the Free Calcium Level in Mitochondria?
In the next sections we show that the Ca2+-handling properties of mitochondria within cells are similar to those
found in detailed studies of isolated mitochondria. Some
previous work used permeant precursors to load the fluorescent Ca2+ probes indo-1 and fura-2 into isolated mitochondria (Lukács et al., 1987; Gunter et al., 1988 When cytoplasmic or extramitochondrial [Ca2+] is
raised to 1-2 µM, all studies with dyes report elevations in
Cam to 250-1,000 nM, again as we have found. The general
agreement supports the calibration protocols we have
used for the nonratiometric rhod-2 dye. On the other
hand, work with cells expressing mitochondrially targeted
aequorin reports that Cam increases (for only a few seconds) to >5 µM upon Ca2+ entry into excitable cells or
mobilization of Ca2+ stores in nonexcitable cells (Rizzuto
et al., 1992 What is the State of Mitochondrial Calcium?
Mitochondria can accumulate enormous quantities of
Ca2+. Classical experiments showed that isolated mitochondria exposed to modest Ca2+ concentrations, socalled "limited loading," accumulate up to 100 nmol Ca2+
(mg protein) We now calculate how much the mitochondrial Ca2+
content should rise in our experiments if, for example,
70% (Herrington et al., 1996 Why is the rise of Cam several thousand times less than
expected? Like the cytoplasm, the mitochondrial matrix
must contain Ca2+ buffers. These could include membrane
phospholipids, Ca2+-binding proteins, and phosphate compounds. The inner leaflet of the inner mitochondrial membrane contributes 6-20 mM of phospholipids (Carafoli,
1979 We would suggest that the Ca2+ taken up in our experiments binds quickly and reversibly to sites in the mitochondrion and does not precipitate as insoluble compounds. If this were not true, the full Ca2+ load would not
be able to re-enter the cytoplasm within seconds after application of CCCP (e.g., Figs. 1 D and 5 B of Herrington et
al., 1996 How Fast Can Mitochondria Transport Ca2+?
Rates of Ca2+ transport for isolated mitochondria have
been extensively studied. Taking typical reported values,
the Ca2+ uniporter is half maximally activated around 10-
20 µM Ca2+ and has a maximum velocity at room temperature ~10-30 nmol (mg protein) What would be the effect of a uniporter operating near
its maximum velocity, at 20 nmol Ca2+ (mg protein) We have previously measured the rates of Cac decay in
chromaffin cells attributable to mitochondrial uptake after
depolarization-induced Ca2+ entry (Herrington et al., 1996 Exploring the Cellular Ca2+ Network
We propose that mitochondria have a unique role in the
cytoplasmic Ca2+ network. The plasma membrane and reticular stores deliver Ca2+ rapidly to the cytoplasm, by
opening ion channels, and then remove it slowly by primary and secondary active transport. Conversely, and consistent with their origins as a cellular symbiont within eukaryotic cytoplasm, mitochondria rapidly remove Ca2+ by
a uniporter that acts like a channel, then return it more slowly by secondary active transport. Hence, the plasma
membrane and internal stores create pulses of Cac with
microdomains of high concentration suitable for initiating
exocytosis or rapid muscle contraction. Mitochondria, on
the other hand, act in an apparently constitutive manner,
capturing Ca2+ pulses and then returning the Ca2+ slowly
to the cytoplasm where it can prolong activation of highaffinity, Ca2+-dependent processes and perhaps feed refilling of stores. In some cells this transient sequestration into
mitochondria is important in generation of Ca 2+ waves
and oscillations (Jouaville et al., 1995; Carafoli, 1979
;
Gunter and Pfeiffer, 1990
). During the 1970s and 1980s, however, uptake of Ca2+ into mitochondria was often considered to require pathological levels of cytoplasmic Ca2+.
Because elevated [Ca2+] increases the activity of key metabolic enzymes of mitochondria, reversible uptake of Ca2+
into mitochondria is proposed to coordinate energy production to cellular needs (McCormack et al., 1990
; Gunter
et al., 1994
; Hajnóczky et al., 1995
). Recently, the availability of new optical indicators for Ca2+ ions has enabled
studies of mitochondrial Ca2+ uptake in living cells and tests
of possible roles for it. Signals from populations of cells expressing mitochondrially targeted aequorin reported rapid
and large transient increases of free intramitochondrial
[Ca2+] (Cam)1 and suggested that mitochondria sequester
Ca2+ from locally high microdomains of cytosolic free
[Ca2+] (Cac) associated with Ca2+ entry or with release
from intracellular stores (Rizzuto et al., 1992
, 1993, 1994;
Rutter et al., 1993
; Lawrie et al., 1996
). In complementary
studies, inhibitors that should block mitochondrial Ca2+
uptake severely compromised clearance of cytoplasmic
Ca2+ after an imposed elevation, indicating that accumulation by mitochondria also is important for cellular Ca2+
homeostasis (Thayer and Miller, 1990
; Friel and Tsien,
1994
; Werth and Thayer, 1994
; White and Reynolds, 1995
;
Herrington et al., 1996
; Park et al., 1996
). Comparing the
rates of Cac decay after selective blockade of known mechanisms for cellular Ca2+ sequestration and extrusion
showed that mitochondrial uptake (defined pharmacologically) accounts for as much as 70% of cytosolic Ca2+ removed during the initial rapid phase of recovery from
large imposed Ca2+ loads in chromaffin cells (Herrington
et al., 1996
).
Materials and Methods
; Park et al., 1996
) from ~300 g male rats. Briefly, adrenal medullae were dissected and digested 50-70 min at 37°C with (mg/ml
in modified Hanks' solution): 1.2 collagenase D; 0.4 trypsin; 0.1 DNase
type I. Dispersed cells were plated on ~5-mm square coverglass slips
coated with poly-l-lysine and laminin, then maintained 1-4 d as primary
cultures in a fortified DMEM medium.
80 mV. Tail currents in SKtype Ca2+-activated K-channels were recorded at a holding potential of
60 mV (Park et al., 1996
).
), we usually used a standard pipette solution (mM): 75 Cs2SO4;
15 CsCl; 6.5 NaCl; 2.5 Na pyruvate; 2.5 malic acid; 1 NaH2PO4; 1 MgSO4;
50 Hepes; 5 MgATP; 0.3 Tris GTP; 0.5 Tris cAMP; 0.1 leupeptin; pH to
7.3 with CsOH. In some cases, 78 K2SO4 and 10 KCl replaced 75 Cs2SO4
and 15 CsCl. Perforated-patch recording (Horn and Marty, 1988
) also
used methods detailed previously (Park et al., 1996
), and a simplified pipette solution: 111 Cs2SO4; 6 CsCl; 10 NaCl; 5 MgSO4; 20 Hepes; pH to 7.4 with CsOH (where noted, Cs+ salts were replaced with K+). Briefly, the
tips of patch pipettes were filled by dipping for 1-3 s in simplified pipette
solution, then backfilled with the same solution supplemented with 480 µg
amphotericin/ml.
). For depolarization-evoked Ca2+ entry, the
external CaCl2 was increased to 10 mM.
; Park et al., 1996
). Briefly, the ratio R (F405/F500) of
background-corrected signals was applied to the standard calibration
equation (Grynkiewicz et al., 1985
):
Rmin)/(Rmax
R)
4 s
1; Calcium Green, 5.80 ± 0.17 × 10
4 s
1). The corrected signals
(F) were converted to [Ca2+] using the simplified calibration equation:
Ffree)/(FCaSat
F)
) was assumed to apply, and Ffree and
FCaSat were calculated from postexperimental responses of each cell as follows. Cells were sequentially perfused with a Mg2+-free saline containing
10 mM CaCl2 and 10 µM ionomycin, then with the same solution in which
2 mM MnCl2 replaced CaCl2.
)
allowed full saturation of the compartmentalized probe with Mn2+ but not
with Ca2+. Therefore, cellular Ffree and FCaSat were both calculated from the FMnSat observed for rhod-2AM loaded, ionophore-treated cells, using
the FMnSat/FCaSat and Ffree/FCaSat ratios observed in vitro for rhod-2. These
determined ratios (and the Kd for Ca2+ used here) agree with the specifications now provided by the manufacturer, but differ from those reported
in the original description of rhod-2 (Minta et al., 1989
), presumably due
to removal of a Ca2+-insensitive contaminant present in the initial preparations of this probe.
; Hiraoka et al., 1990
, 1991; Chen et
al., 1995
) were applied to each z-series using an optical transform function
determined empirically from a 0.1-µm bead.
Results
m) across
the inner mitochondrial membrane that is subsequently
harnessed by the ATP synthase in the production of ATP.
This negative
m has been exploited in the past to label
energized mitochondria with membrane-permeant cations
like Rhodamine 123 (Johnson et al., 1981
). Similarly, the
permeant cation rhod-2 acetoxymethyl ester (rhod-2AM)
should also accumulate preferentially in mitochondria
where it should become trapped when subsequent esterolysis generates zwitterionic rhod-2 (Minta et al., 1989
). Unlike the membrane permeant Rhodamine 123, the trapped
rhod-2 should not be sensitive to subsequent changes in
mitochondrial membrane potential. The following experiments confirm these predictions for rat chromaffin cells.
) was
more apparent (Fig. 1 E). Pharmacological validation of
the mitochondrial localization of rhod-2 is found in the following sections below. When the neutral ester of Calcium
Green was coloaded in the same cell together with cationic
rhod-2AM, the Calcium Green fluorescence showed a
more pancellular distribution (Fig. 1 F), consistent with a
localization in the cytosol and nucleus as well as some additional association with mitochondria.
Fig. 1.
Rhod-2 images of chromaffin cell responses to depolarization and coimages of rhod-2 with Calcium Green. (A-D) Fluorescence deconvolution images of a single cell after loading with
AM ester of rhod-2. Images were collected before, during, ~4 s
after, and ~1 min after a 10-s, fast local perfusion with a depolarizing (70 mM KCl) medium. (E-F) After coloading with AM esters of rhod-2 and Calcium Green, images were collected from a
central plane of a single cell not subjected to stimulus, using excitation and emission filters for rhod-2 in E and then for Calcium
Green in F. Scale bar represents 4 µm.
[View Larger Version of this Image (92K GIF file)]
m (Gunter and Pfeiffer, 1990
; Gunter et al., 1994
; but also see Sparagna et al.,
1995
). We now show that Ca2+ uptake by mitochondria
within the cell also depends on the negative
m and the
uniporter. We perturb mitochondrial function with four
agents, oligomycin, cyanide, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and ruthenium red as illustrated in Fig. 2. Oligomycin blocks mitochondrial ATP
production by direct inhibition of the ATP synthase but
should not decrease
m by itself. Cyanide blocks electron
transport but should allow maintenance of
m by an alternate route, the reversal of the ATP synthase fueled by
cytoplasmic ATP. However, oligomycin and cyanide in
combination block both routes for maintaining
m and
should depolarize mitochondria. Likewise, the ionophore
CCCP collapses
m by making membranes permeable to
protons. Finally, ruthenium red blocks the Ca2+ uniporter
directly.
Fig. 2.
Pathways of Ca2+ and H+ transport in the inner mitochondrial membrane and diagnostic inhibitors. Abbreviations:
CU, Ca2+ uniporter inhibited by ruthenium red, RR; F1F0, ATP
synthase inhibited by oligomycin; NCE, Na+-Ca2+ exchanger inhibited by CGP-37157, CGP; e.t.c., electron transport chain inhibited by cyanide; CCCP, a protonophore that collapses the mitochondrial membrane potential, m.
[View Larger Version of this Image (24K GIF file)]
), addition of CCCP produced a small transient Cac increase of
variable duration and size apparently dependent on mitochondrial Ca2+ content at the time of addition. CCCP also
greatly slowed the recovery of Cac after a second depolarization. When this experiment was repeated with other
cells internally dialyzed with ruthenium red to block the
uniporter, the initial decay was already slowed even without the addition of CCCP (Fig. 3 B). Subsequent treatment with CCCP had no apparent further effect on recovery from Ca2+ entry (n = 4). These experiments show that
mitochondria cannot clear Ca2+ rapidly from the cytoplasm when the
m is collapsed or the Ca2+ uniporter is
blocked.
Fig. 3.
Prolongation of
Ca2+ clearance by blockade
of mitochondrial energization or of the mitochondrial
uniporter. (A-B) Time
course of cytoplasmic [Ca2+]
monitored with indo-1 (100 µM) introduced into the cytosol by inclusion in the pipette solution alone (A) or,
in a different cell, together
with 2 µM RR (B). Beginning 3-5 min after the wholecell patch-clamp configuration was established, dual
emission fluorescence signals were recorded. One or
two 500-600-ms depolarizing pulses were applied (arrows) while each cell was
perfused with control medium alone. Another one or
two pulses were applied after
2 µM CCCP was included in
the perfusion medium. (C-
D) Effect of mitochondrial inhibitors on clearance of
submembrane Cac as measured by the decay (tail) of a
Ca2+-activated K+ current. Tail currents were recorded in perforated-patch configuration in response to 1-s depolarizations to 0 mV applied every 2 min (Park, 1996). After 9.5 min, the perfusion medium was supplemented with 3 µM oligomycin or 5 mM NaCN and at
11.5 min, with both. (D) Peak amplitude and half-decay times of currents observed after treatment were normalized to those obtained
in the preceding control stimulus applied to the same cell. The numbers of cells examined are shown in parentheses.
[View Larger Version of this Image (22K GIF file)]
; Park,
1994
). This method requires neither cell dialysis nor introduction of dyes into the cytoplasm and thus provides a useful, non-invasive indication of [Ca2+] at the plasma membrane. As in previous work (Park et al., 1996
), the K+
current that was activated by Ca2+ entry during a 1-s depolarization then declined with a half-time of 2-7 s as the
[Ca2+] fell and the SK channels closed. The decay was not
slowed by oligomycin or cyanide alone but was prolonged
greatly by the combination of these agents. Blockade of both
available routes for mitochondrial energization increased
the half-time of current decay 5.4 ± 1.8-fold (n = 4), without appreciably affecting the peak amplitude. These experiments without cell dialysis show again that rapid clearance of cytoplasmic Ca2+ depends on the existence of a
negative
m and not, in the short term, on the ongoing
oxidative production of ATP.
; Park et al., 1996
). As expected,
oligomycin alone did not affect Cam or Cac of resting cells
(Fig. 4).
Fig. 4.
Reciprocal actions of CCCP on mitochondrial and cytosolic Ca2+. Fluorescence signals from
rhod-2 and Calcium Green, coloaded as AM esters
in a preliminary incubation, were recorded simultaneously from a single cell, beginning 3-5 min after
the perforated patch clamp configuration was established. The perfusion medium then was supplemented with 10 mM Ca2+ and 3 µM oligomycin and
then with 2 µM CCCP as indicated. At the arrows
500-ms depolarizations were applied. A postexperimental calibration procedure (see Materials and
Methods) provided parameters for conversion of
rhod-2 and Calcium Green fluorescence to the indicated mitochondrial and cytosolic free Ca2+ concentrations (Cam and Cac, respectively). The results
shown are representative of four similar experiments. (Insets) The initial portions of Cam and Cac
responses are aligned in time to the depolarizing stimulus.
[View Larger Version of this Image (21K GIF file)]
), resting Cac was
lower (by 100-200 nM) and peak Cac evoked by Ca2+ entry usually was higher than the values reported here by
Calcium Green loaded as its AM ester and examined in
perforated patch protocols. It is not yet clear whether
these differences reflect alterations in the extent of Ca2+
entry, in cytosolic buffering or clearance mechanisms (all
of which may be affected by the dye loading and patch
clamp procedures employed), or by differences in dye localization and inaccuracies in calibration.
Fig. 6.
Reversible elimination of the sustained phase of
Cac recovery by blockade of
mitochondrial Ca2+ extrusion. Cells were coloaded
with rhod-2 and Calcium
Green esters, examined in
the perforated patch clamp
configuration, and the data
analyzed as in Fig. 4. Responses are representative of five similar experiments.
(A) Arrows mark the application of sequential depolarizations of 1.5, 2.0, and 2.5 s
duration, applied to a single
cell in the presence or absence of perfusion with 10 µM CGP-37157 as indicated.
(B) Another cell received 0.5, 1.0, and 1.5 s stimuli before,
during, and after application
of inhibitor.
[View Larger Version of this Image (23K GIF file)]
Fig. 8.
Calcium-induced release of Ca2+ and its mitochondrial
sequestration. Cells were coloaded with rhod-2 and Calcium Green
and examined in perforated patch as in Fig. 4. (A) Calibrated
(left) and aligned (right) Cam and Cac records are from a single
cell subjected to sequential depolarizations of 50 (diamond), 100 (box), 250 (circle), and 500 (triangle) ms depolarizations, applied
at the arrows. Results are representative of four similar experiments. (B) Averaged responses from four cells treated exactly as
above. The enlarged, closed symbols mark the observed or interpolated Cam and Cac values at the termination of stimulus.
[View Larger Version of this Image (27K GIF file)]
Fig. 5.
Cam increases during and following Ca2+ entry and decreases during late clearance. Rhod-2 was loaded as its AM ester and
Calcium Green Dextran by whole cell dialysis. Calcium Green fluorescence observed in the on-cell patch clamp configuration was subtracted before calibration of signals simultaneously recorded from a single cell 5 min after the whole-cell configuration was established. (A) Cam and Cac responses to a 500-ms depolarization applied at the arrow. The final portion of Cac recovery is shown (dots) on a 20×
expanded scale. (B) The initial portions of Cam (open circle) and Cac (open triangle) traces from A are aligned in time to the depolarizing stimulus. (C) The relationship of Cam to Cac during this response to stimulus and subsequent recovery; a closed diamond marks the
Cam and Cac values at the termination of stimulus. The curved arrow indicates the approximate nonlinear time course; the lines divide
elapsed time into segments of ~0.5, 5, and 50 s duration.
[View Larger Version of this Image (22K GIF file)]
; Park et al.,
1996
). As was expected, we now see directly that Cam increases as Cac decreases. It now also is clear that half of
mitochondrial uptake is accomplished while entry is still in
progress and that uptake thus must attenuate the peak Cac
reached.
; Friel and Tsien, 1994
). The following experiments
distinguish between these possibilities.
). Fig. 6,
A and B show that this agent has no effect on the rise of
Cam evoked by Ca2+ entry into chromaffin cells but reversibly slows the Cam fall (n = 5), providing good evidence
that extrusion of Ca2+ by the mitochondrial Na+-Ca2+ exchanger mediates the normal fall in Cam. While Cam is elevated and Ca2+ is being extruded from mitochondria, Cac
often remains slightly above its resting value (Figs. 3-5).
This small elevation has been attributed to a steady flux of
Ca2+ into the cytoplasm from mitochondria (Freil and Tsien,
1994; Werth and Thayer, 1994
; Herrington et al., 1996
).
The reversible reduction of this persistent Cac elevation by
the exchange inhibitor CGP-37157 (Fig. 6) confirms this
hypothesis. Unexpectedly, the fall in Cam that followed removal of CGP-37157 was accompanied by no elevation
(Fig. 6 A) or only minor elevation (Fig. 6 B) of Cac. It
would be useful to repeat these experiments in cells where
the other mechanisms of Ca2+ clearance are inhibited.
; Malgaroli et al., 1990
; Neely and Lingle, 1992b
). We
tested the selectivity of mitochondrial Ca2+ uptake for this
source of Ca2+ by comparing responses of Cam and Cac to
depolarizing steps and to agonists. For these experiments
we used the perforated-patch configuration for recording
since intracellular stores tend to run down quickly in wholecell recording so responses to agonists vanish (Herrington,
J., Y.B. Park, D.F. Babcock, and B. Hille, unpublished observations).
Fig. 7.
Similar mitochondrial responses to calcium
entry and to calcium mobilization by agonists. Cells were
coloaded with rhod-2 and
Calcium Green esters, examined in perforated patch, and
the data analyzed, all as in
Fig. 4. Each record is representative of 3-5 similar experiments. (A) At the indicated times a single cell was
perfused with 100 nM bradykinin (BK) for 5 s. After recovery, a 250-ms depolarization (V) was applied. (B)
Cam and Cac responses of another cell to a 5-s perfusion
with 50 µM muscarine
(musc) and subsequent depolarization.
[View Larger Version of this Image (19K GIF file)]
). This Ca2+-induced
Ca2+ release (CICR) is especially apparent in cells with
impaired mitochondrial Ca2+ uptake (as in Figs. 3 C and 4)
and can be recognized in experiments like that in Fig. 8 A
where depolarizations of increasing duration were applied
to a cell in perforated-patch mode. The plots of averaged
Cam vs Cac from four such experiments (Fig. 8 B) show that Cac continued to rise for several hundred milliseconds
after the Ca2+ entry evoked by depolarization had ceased
(large filled symbols). These plots can be compared with
the similar plot for whole-cell recording (Fig. 5 C) where
Cac began to fall as soon as the membrane depolarization
ended. In the perforated patch experiments (Fig. 8 B), Cam
also rose more steeply during the period of CICR, as if mitochondria were taking up the additional Ca2+ released
from stores. While spatially averaged Cac was still highly
elevated, Cam then began to fall, as if reuptake into the depleted stores was creating a local sink for Ca2+ fed from
mitochondria.
Fig. 9.
Mitochondrial sequestration of Ca2+ released from
caffeine-sensitive stores. Cells were coloaded with rhod-2 and
Calcium Green and examined in perforated patch as in Fig. 4. (A)
Cam and Cac responses from another cell subjected to sequential
depolarizations of 0.5 s applied before (circle), and of 1.0 or 1.5 s
(box) applied after perfusion with 10 mM caffeine and 10 µM
BHQ. (B) Averaged responses from three cells treated exactly as
above. The enlarged, closed symbols mark the observed Cam and
Cac values at the termination of stimulus. The intermediate response to the first depolarization applied after treatment is not
shown, to avoid excessive overlap of records.
[View Larger Version of this Image (21K GIF file)]
1 (n = 9), and in whole-cell mode it
was 79 ± 30 nM s
1 (n = 6). We suspect that the faster rise
reflects a combination of CICR and greater proximity of
mitochondria to locally high microdomains of Cac around
Ca2+ channels (Neher and Augustine, 1992
; Robinson et
al., 1995
) in the undialyzed cells. Dialysis may disrupt cytoskeletal elements that establish a mitochondrial localization near the plasma membrane and other organelles. In
the first second after entry ceased, when such microdomains should be dispersed, Cam continued to rise at ~40
nM s
1 in the experiment of Fig. 5 B.
Discussion
), and recent confocal imaging has shown
compartmentation of this probe (Burnier et al., 1994
;
Tsien and Bacskai, 1995
). It has also been shown qualitatively that Ca2+ entry (Tsien and Bacskai, 1995
) and Ca2+
mobilization (Hajnóczky et al., 1995
, Rutter et al., 1996
)
increase the signal from compartmentalized rhod-2. Our
imaging of rhod-2 by fluorescence deconvolution methods
and our quantitative photometric observations during application of pharmacological agents further validate and
extend the use of rhod-2 as a probe of mitochondrial [Ca2+] in living cells.
; Friel and Tsien, 1994
; Werth and Thayer,
1994
; White and Reynolds, 1995
; Park et al., 1996
; Herrington et al., 1996
). For chromaffin cells we had found
that blockers of mitochondrial Ca2+ uptake but not blockers of other cellular Ca2+ transporters greatly slow the initial clearance of Cac yet shorten the total time to return to
the resting state (Park et al., 1996
; Herrington et al., 1996
).
Now our simultaneous monitoring of Cam and Cac clearly
establishes that Ca2+ uptake through the mitochondrial
uniporter underlies rapid removal of cytosolic Ca2+. It
shows further that subsequent Ca2+ export from mitochondria requires the mitochondrial Na+-Ca2+ exchanger
and accounts for the slowness of the final decrease in Cac.
With the less-invasive perforated-patch method we find that mitochondria accumulate Ca2+ mobilized from either
IP3- or caffeine-sensitive internal stores, and speculate that
stores may be refilled with Ca2+ exported from mitochondria. A picture emerges of a highly interactive network of
cellular Ca2+ signaling that involves mitochondria and at
least two mobilizable pools. The rapid rise of Cam to even
modest physiological Cac loads presumably also means
that mitochondrial energy metabolism adjusts itself to cellular activities from moment to moment (McCormack et
al., 1990
; Gunter et al., 1994
; Hajnóczky et al., 1995
; Rutter et al., 1996
).
; Reers et al.,
1989
; McCormack et al., 1989
; Leisey et al., 1993
). Mitochondria within cells have also been loaded nonselectively
with these dyes and studied after quench of cytosolic dye
with Mn2+ (Miyata et al., 1991
) or by imaging of mitochondria-rich regions (Sheu and Jou, 1994
) to estimate a compartmentalized signal. For both isolated and cellular mitochondria, these studies with ratiometric dyes obtained low
resting values of Cam in the range of 80-200 nM, much as
for nonratiometric rhod-2 in our work. Thus it is agreed
that there is hardly any gradient of free [Ca2+] across the
resting mitochondrial membrane, despite a large, insidenegative
m. Presumably the uptake via the uniporter is in balance with extrusion via exchange and other mechanisms, and perhaps the uniporter is largely shut down at
rest (Gunter and Pfeiffer, 1990
; Gunter et al., 1994
).
, 1993, 1994; Rutter et al., 1993
). This higher
level could be reconciled with the dye results if heterogeneity among dye-containing compartments allowed some
to rise briefly to very high Ca2+ levels, which would saturate dyes and give high aequorin emission, while others
had only moderate and longer lasting [Ca2+] elevations,
keeping the spatially averaged dye signal below saturation.
Alternatively, there may be subtle difficulties in calibrating aequorin signals. For single CHO cells responding to a
Ca2+ mobilizing agonist, Rutter et al. (1996)
found that
rhod-2 fluorescence transiently increased 2-3-fold. Assuming a resting Cam of 100 nM and using the spectral
properties and affinities of rhod-2 that we observe in vitro,
we estimate that this corresponds to a peak Cam of 400-
800 nM, much like the responses found in the present
study, but much smaller than the >10 µM peak Cam estimated from the responses of the same CHO cells transfected with mitochondrial aequorin.
1 in half a min without damage (Lehninger
et al., 1967
; Gunter and Pfeiffer, 1990
). Exposure to millimolar Ca2+ ("massive loading") can lead to accumulations
up to 2,600 nmol Ca2+ (mg protein)
1, accompanied by irreversible damage, morphological changes, and precipitation of hydroxyapatite crystals. These values would be equivalent to Ca2+ contents of 36 and >900 mM within the
mitochondria, respectively!
) of the Ca2+ in a single 1.5-µM
cytoplasmic challenge were taken up into the mitochondria. The mitochondria would actually remove much more than the 1.5-µM free Ca2+ because almost all the cytoplasmic Ca2+ is reversibly bound to proteins and other Ca2+
buffers. In rat chromaffin cells (see Figs. 2 and 3 of Park et al.,
1996
) and in other excitable cells (Neher, 1995
), typical values for cytoplasmic Ca2+-binding ratios are near 100. Thus, mitochondria would have to remove 0.7 × 100 × 1.5 µM = 105 µM total Ca2+. Since the mitochondrial volume
is only 6% of the combined cytosolic and nuclear volumes
in rat chromaffin cells (Tomlinson et al., 1987
), this uptake
should raise the mitochondrial Ca2+ content by 1.75 mM.
These numbers emphasize how remarkable it is that compartmentalized dyes report Cam rises of only 250-450 nM
during such a challenge.
; Schwerzmann et al., 1986), and the matrix contains tens of millimolar of phosphate compounds. The kind and
concentration of Ca2+-binding proteins is not well known.
). We conclude therefore that the mitochondrial
matrix buffers Ca2+ reversibly with an effective Ca2+ binding ratio of ~4,000 (see also Coll et al., 1982
; Lukács and
Kapus, 1987
). This is 40 times the Ca2+-binding ratio of cytoplasm.
1 s
1 in energized cardiac
and liver mitochondria (Carafoli, 1979
; Gunter and Pfeiffer, 1990
). The maximum velocity of the mitochondrial Na+-Ca2+ exchanger is 30-400 times slower. Although the
maximum velocity may be significantly affected by the
bathing phosphate, Mg2+, pH, and other conditions, we
will use it to compare with the uptake rates we measure in
cells.
1 s
1?
Since 1 nmol (mg protein)
1 corresponds to 385 µmol/liter
of mitochondria (Schwerzmann et al., 1986), we would expect that a liter of mitochondria could take up 7,700 µmol
Ca2+ s
1. Once again we apply the mitochondrial volume
(6%) and the cytoplasmic Ca2+-binding ratio (100) and
predict that Cac (the free Ca2+) should fall at 4.6 µM s
1
when the uniporter is working at full velocity.
).
At 1.5 µM Cac the mean rate was 0.8 µM s
1. In Park et al.
(1996)
, we gave evidence for appreciable uptake during a
200-ms depolarization, and in the present paper, we find that Cam rises more rapidly during the Ca2+ entry than afterward; indeed more than half of the rise in Cam occurs
during a 500-ms depolarizing pulse (Figs. 5, 8, and 9). Thus, we presume that early uptake proceeds at rates
equivalent to a decay of Cac > 0.8 µM s
1. These velocities
for Ca2+ import by mitochondria in living cells seem quite
compatible with the 4.6 µM s
1 maximum velocity predicted from uptake by isolated mitochondria; however,
they are higher than would be expected at 1.5 µM Ca2+ if
we take into account the reported Ca2+ affinity and cooperativity of uptake. A model in which a subset of mitochondria lies so close to the plasma membrane that they transiently see elevated Cac values well above those reported by the cytoplasmic dye (Lawrie et al., 1996
) could
explain these results. Perhaps also Ca2+ transport is more
effective for mitochondria bathed in cytoplasm than for
isolated mitochondria.
; Hehl et al., 1996
). In
addition, the transiently elevated Cam probably stimulates mitochondrial ATP production in anticipation of cellular
needs. Such a major role for mitochondria in Ca2+ clearance holds for chromaffin cells and neurons but not for skeletal and cardiac muscles with a highly elaborated sarcoplasmic reticulum. We need now to examine the importance of mitochondria in other cell types and to determine
if mitochondrial Ca2+ uptake is a regulated process.
Received for publication 26 July 1996 and in revised form 2 December 1996.
Please address all correspondence to B. Hille, Department of Physiology & Biophysics, G424 Health Sciences Building, University of Washington, Box 357290, Seattle, WA 98195-7290. Tel.: (206) 543-8639. Fax: (206) 6850619. E-Mail: hille{at}u.washington.eduWe thank Applied Precision Inc. (Issaquah, WA) for generous use of their Deltavision image analysis system. The authors also thank Drs. P.B. Detwiler, J. Howard, J.S. Isaacson, and W.W. Parson for critical evaluation of this manuscript and Lea Miller, Don Andersen, and Paulette Brunner for technical assistance.
This work was supported by National Institutes of Health grants AR17803 and HD07878, the W.M. Keck Foundation, and the Royalty Research Fund of the University of Washington.
Cac, free cytosolic [Ca2+]; Cam, free intramitochondrial [Ca2+]; CGP-37157, 7-chloro-3,5-dihydro-5-phenyl-1H4,1-benzothiazepine-2-on; BHQ, 2,5 di(t-butyl)-1,4-hydroxyquinone; CCCP, carbonyl cyanide m-chlorophenylhydrazone.