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(Received for publication, September 25, 1995; and in revised form, February
26, 1996) From the
By using an endothelial cell line (ECV304), derived from human
umbilical vein and transfected with recombinant aequorin targeted to
the mitochondrial matrix, we find that stimulation with ATP evokes long
lasting increases in mitochondrial Ca In ECV304 cells <4% of mitochondria are
within 700 nm of the endoplasmic reticulum as opposed to 65% in HeLa
cells, whereas 14% are within 700 nm of the inner surface of the plasma
membrane, compared with <6% in HeLa cells. Following Ca A model is discussed in which the localization of
mitochondria with respect to Ca It has long been known that stimulation of nonexcitable cells
with phosphatidylinositol bisphosphate-coupled agonists leads to a rise
in the concentration of cytosolic calcium
([Ca Until recently the estimation of
[Ca ATP is known to be a
physiologically important stimulator of endothelial cells. Its
activation of the cells results chiefly in the production of the
vasodilators prostacyclin and endothelial-derived relaxing factor, now
known to be nitric oxide(14) , along with other factors
important in the control of vascular tone(15) . ATP binds
plasma membrane receptors of the purinergic P Previous studies, including those on HeLa
cells, have shown that the pattern of mitochondrial response to
phosphatidylinositol bisphosphate-coupled agonists in nonexcitable
cells is one of a rapid, large, and transient elevation in
[Ca In
particular, we show that the [Ca
Alternatively, [Ca
Figure 1:
a, mitochondrial calcium changes
([Ca
When cells were
treated with the mitochondrial uncoupling agent FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 1 µM), the
response was significantly attenuated.
Following an initial
stimulation of ECV304 cells with ATP, a brief wash followed by
subsequent applications of agonist resulted in comparable rises in
[Ca
Figure 2:
Comparison of the effect of readdition of
Ca
Replacement of
Ca An even more dramatic dissociation between
[Ca
Figure 3:
Comparison of the readdition of
Ca
Figure 4:
Electron micrographs of MH75 and ECV304
demonstrating the localization of mitochondria and membranous
organelles. a and b, ECV304 cells at 90,000 and
60,000
The micrographs in Fig. 4, a and b, show
the distribution of mitochondria in ECV304 cells, at 90,000 and 60,000
To
establish the localization of subcellular structures in respect to
mitochondria, the number of ``hits'' with each structure of
interest (plasma membrane, ER, or nuclear envelope) met along rings at
set distances (100 nm to 1 µm, step 100 nm) from the center of a
given mitochondrion was counted. The number of mitochondria included
for MH75 cells was 84 and 107 for ECV304 cells. The resulting
probability of meeting either endoplasmic reticulum (Fig. 5a) or plasma membrane (Fig. 5b)
at each distance has been plotted for both cell types in Fig. 5.
The size of mitochondria varied but was never less than 300 nm (i.e. <300 nm from center was still mitochondria). In
ECV304 cells, there was a much lower probability of meeting ER within a
short distance (<1 µm) from the mitochondria than in the MH75
cells (Fig. 5a). Fig. 5b shows that
mitochondria in the ECV304 cells were more likely to be close to the
plasma membrane than in the MH75 cells, but these probabilities are
still less than those for the MH75 mitochondrial relationship with ER (e.g. the probability of MH75 mitochondria being within 700 nm
of ER is >0.5, whereas the probability of ECV304 cell mitochondria
being within the same distance of the plasma membrane is <0.15).
Figure 5:
a shows the probability of meeting ER at
each fixed distance from the center of a mitochondrion and compares
ECV304 cells (dark bars) with MH75 cells (light
bars). b compares the probability of meeting plasma
membrane at each distance between ECV304 (dark bars) and MH75
cells (light bars). c compares the probability of
meeting ER (light bars) with the probability of meeting plasma
membrane (dark bars) in ECV304 cells. d shows a
similar comparison for MH75 cells. All probability values were
calculated by dividing the number of hits for each subcellular
structure with the total number of structures encountered at each fixed
distance from the center of the
mitochondrion.
Counting the total number of mitochondria situated with their
centers close to ER or plasma membrane reveals that only 3.8% of the
ECV304 cell mitochondria were within 700 nm of the ER, compared with
75% of MH75 cell mitochondria. In contrast, 14% of the ECV304 cell
mitochondria lie within the same distance of the plasma membrane
whereas only 5.6% were similarly located in MH75 cells. In Fig. 5, c and d, the probability of meeting ER
was compared with the probability of meeting plasma membrane for ECV304
cells (Fig. 5c) and MH75 cells (Fig. 5d). It can be seen from this that within 900 nm
of the mitochondrial center, in ECV304 cells, there is a much higher
chance of meeting the plasma membrane than any ER membrane (Fig. 5c), which is in contrast to the case in the MH75
cells (Fig. 5d). At 900 nm to 1 µm the likelihood
of meeting either organelle becomes equal (Fig. 5c). The probability of meeting the nuclear envelope was also measured
and was found to be the same for both cell types, i.e. very low at short distances (<600 nm) and
increasing to only 0.15 at 1 µm (not shown). From these data, it
would appear that although the mitochondria are distributed throughout
the cytosol in both cell types, the probability of their being close to
the ER is considerably lower in the ECV304 cells than in the MH75
cells. In addition, in the ECV304 cells there is a higher chance of the
mitochondria being situated close to the plasma membrane than to the
ER. The large, rapid increase in
[Ca The data presented
here for the endothelial cell line ECV304 are, at a first sight, in
contradiction with the microdomain hypothesis. In this cell type
addition of extracellular ATP in the presence of
Ca Because [Ca The ATP-stimulated slow-onset
sustained rise in [Ca It remains to be
established why in ECV304 cells a sustained increase in bulk
[Ca The
electron microscopy studies point out another difference between ECV304
cells and the HeLa MH75 clone (previously characterized in detail for
their [Ca Taken together the results described in this study lead to a few
general considerations as shown. (i) The kinetics and amplitude of the
[Ca
Volume 271,
Number 18,
Issue of May 3, 1996 pp. 10753-10759
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
([Ca]
) that
largely depend on Ca influx. In these cells, the
release of stored Ca is inefficient at elevating
[Ca]. Consequently it
appears that in ECV304 cells, bulk cytosolic Ca ([Ca]
) is the
main determinant of [Ca] changes. depletion, readdition of extracellular Ca evokes an increase in [Ca] but not in
[Ca]. Under these
conditions, microdomains of high
[Ca] may occur beneath
the plasma membrane of ECV304 cells resulting in the preferential
elevation of Ca in mitochondria located in this
region. sources is the main
determinant of their in situ Ca uptake
kinetics. Thus, in any given cell type mitochondria may be localized to
suit the energy and metabolic demands of their physiological actions.
]) (
)via the action of inositol 1,4,5-trisphosphate
(InsP
) on intracellular calcium (Ca)
stores and that this increase is important for the manifestation of
many cellular processes(1) . Agonist-evoked Ca release is associated with rises in the concentration of calcium
in the matrix of the mitochondria
([Ca])(2, 3, 4) .
High [Ca] has been
shown to stimulate the activity of key metabolic enzymes (5, 6) resulting in raised energy production for the
stimulated cell. Ca enters the mitochondria through a
uniporter, present in the inner membrane, and can be exported via the
stimulation of H
:Ca and
Na:Ca antiporters thus creating a
Ca cycling system for the control of
[Ca](7, 8, 9, 10) . ] changes in
vivo was only possible by following the activation of the
metabolic enzymes by Ca(5, 6) or
loading cells with high levels of a fluorescent indicator, followed by
quenching of the cytosolic dye(11) . Here, we have utilized
recombinant Ca-dependent luminescent photoprotein
aequorin specifically targeted to the mitochondria (mtAEQ) (2, 12) to monitor
[Ca] during agonist
activation. In particular we are interested in the changes in
[Ca] evoked by
stimulation of an endothelial cell line with the physiological agonist
ATP. For this purpose we have chosen a cell line, designated ECV304,
that was spontaneously immortalized from a primary culture of human
umbilical vein endothelial cells(13) .
classification.]. This kinetic
behavior was proposed to be due to the exposure of mitochondria to
microdomains of high [Ca] in the regions surrounding the InsP-sensitive
calcium release sites on the ER(3, 4) . This model was
recently supported by data obtained in freshly isolated hepatocytes
using dihydro-Rhod 2 to monitor
[Ca](16) . We
provide further evidence to support this hypothesis but demonstrate
that this proposal does not hold true for ECV304 cells.] increases seen in response to ATP in ECV304 cells are
slow-onset sustained signals that are quite insensitive to
Ca mobilization by InsP. This differing
pattern of [Ca] may be
due to the localization of the mitochondria, which is quite different
from other cells, e.g. HeLa cells. The ECV304 cell
mitochondria are not closely associated with any specific subcellular
structure or organelle, whereas in the case of MH75 cells over 50% of
the mitochondria lie within 700 nm of the ER. Thus in the ECV304 cells,
the increases in [Ca] seem to depend on global
[Ca] changes and thus
appear to be mainly affected by Ca influx across the
plasma membrane.
Reagents and Solutions
During all experiments cells were constantly bathed with
physiological saline, at 37 °C and pH 7.4, of the following
composition: 145 mM NaCl, 5 mM KCl, 1 mM Na
HPO
, 1 mM MgSO, 10
mM Hepes, 10 mMD-glucose, plus either 1
mM CaCl or 0.5 mM EGTA
(Ca-free). All reagents were added to this buffer as
indicated in the text and by the bars on the figures. All chemicals and
reagents were purchased from Merck Ltd. (Lutterworth, Leics., U.K.) or
Sigma (Poole, Dorset, U.K.) unless otherwise stated.Plasmids
Both cytoplasmic (untargeted, cytAEQ) and mitochondrially
targeted aequorin (mtAEQ) containing plasmids were obtained by the
method described previously(2, 12) . Extraction and
purification from bacterial suspensions were achieved using a Wizard
Maxiprep plasmid preparation kit (Promega Ltd., U.K.).Cell Culture
The human umbilical vein endothelial cell derived line,
ECV304, was maintained at 37 °C in a 5% CO humid
incubator, in 80-cm flasks containing Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum
and antibiotic, antimycotic solution (Sigma, Poole). A clone of the
HeLa cell line, stably transfected with mtAEQ and designated
MH75(3, 4) , was maintained in the same way. All
tissue culture reagents were purchased from Life Technologies
(Renfrewshire, U.K.).Transient Transfection of Endothelial Cells
One to two days prior to experimentation, ECV304 cells were
seeded onto 16-mm glass coverslips (Merck Ltd., Lutterworth), using
trypsin/EDTA in 12-well culture plates (Costar). On reaching
50-70% confluence, transient transfection was carried out by
calcium phosphate precipitation. Briefly, 4-6 µg of plasmid
per coverslip was further purified in ethanol with 23% TE (10 mM Tris-HCl + 1 mM EDTA) buffer, and 2% sodium acetate
(3 M, pH 8.0), in an ethanol dry ice bath held at -80
°C. After 30-40 min this mixture was spun at 14,000 rpm in an
Eppendorf microcentrifuge for 15 min. The supernatant was carefully
removed in a sterile laminar flow cabinet, and the plasmid pellet was
resuspended in TE buffer and 10% CaCl. This was then
transferred dropwise, while vortexing, to a tube contained
double-concentrated Hepes-buffered saline (2 
HBS, 280 mM NaCl, 50 mM Hepes, 1.5 mM NaHPO) at precisely pH 7.12. After exactly
30 min the precipitate was visible, and 100 µl of this was then
transferred dropwise to each coverslip. Dulbecco's modified
Eagle's medium was renewed the following morning, and cells were
at maximal expression state the subsequent day (36-48 h after
transfection).Measurement of Mitochondrial Calcium
([Ca
Cells transfected with mtAEQ and grown on 16-mm glass
coverslips were incubated with 5 µM coelenterazine
(Molecular Probes, Eugene, OR) at 37 °C for 1 h and then in a high
oxygen atmosphere at 37 °C for a further 1-2 h to favor the
regeneration reaction(17) . Following this incubation period,
coverslips were transferred to a superfusion chamber held in close
proximity to a photomultiplier tube, as described
previously(18) . The output from the photomultiplier tube was
then fed directly into a microcomputer and analyzed using OSCAR
software (Photon Technologies Inc. (PTI)). Calibration was then
performed using AEQASC software kindly provided by Javier
Alvarez(19) . The perfusion chamber was warmed to 37 °C
using a circulating water bath, and solutions were superfused over the
coverslip at a rate of 1 ml min]
)
.Measurement of Cytosolic Calcium
([Ca
Cells transfected with cytAEQ were reconstituted with
coelenterazine and monitored in the same way as described above. ]
)] was also monitored by loading cells, grown on 22-mm diameter
coverslips, with Fura-2AM (Molecular Probes). This was achieved by
replacing the growth medium with physiological saline containing 0.1%
bovine serum albumin, 2 µM Fura-2AM, and 0.0125% Pluronic
F127 detergent (Molecular Probes) and incubating coverslips at 37
°C for 45 min. Coverslips were placed in a purpose-built perfusion
chamber where they were constantly superfused, again at a rate of 1 ml
min. Changes in fluorescence were then monitored
using an intensified charge-coupled device camera (Photonic Science
Ltd.). The data were analyzed using a PTI digital imaging system
equipped with Image Master for Windows software (PTI). This method has
the advantage that single cells within a population can be monitored,
whereas data from whole coverslip populations are obtained when using
cytAEQ transfection.Electron Micrographs
Cells were prepared for fixation in one of two formats as
follows.Pellet Fixation
Cells from an 80-cm flask
were dislodged using trypsin/EDTA and placed in a sterile
microcentrifuge tube. The suspension was spun at 1000 rpm (80 g) for 2 min, after which they were fixed and stained
as indicated below.Cultured Cells
Cells were grown on small squares
(2 2 cm) of Mellinex 228 sheeting, this is softer than glass
coverslips and facilitates sectioning. Once 90-100% confluent the
sheets were fixed and stained as indicated below.Fixing and Staining
Cells were primarily fixed in
2.5% glutaraldehyde and 2.0% formaldehyde in 0.1 N sodium
cacodylate (NaCac) buffer at pH 7.3 and 3 °C, after which they were
washed twice in NaCac buffer at pH 7.3 and 3 °C. A postfixation was
then performed in 2.0% osmium tetroxide in NaCac buffer at pH 7.3 and 3
°C and then washed twice in 0.15 N sodium acetate (NaAc)
buffer at pH 7.3 and 3 °C. The preparation was then stained in 1.0%
uranyl acetate in NaAc buffer at pH 7.3, 25 °C to highlight
membranes. Dehydration in increasing concentrations of ethanol (10, 30,
50, 70, 90, and 100%) was then performed, including a final second step
in 100% ethanol. Samples were infiltrated with increasing
concentrations of LR White
resin (London Resin Co., U.K.)
(50, 75 and 100%) finishing with three applications of 100% LR
White for a total of 4 h. Samples were finally embedded in
LR White resin for 48 h at 60 °C. Secondary staining
was performed on the grid to highlight membranes further when
necessary. This was performed using 2% uranyl acetate in 30% methanol
for 1 min, followed by a wash in distilled water, then a 1-min
incubation in 10% lead citrate, and a final wash and dry.
Changes in Mitochondrial Calcium
([Ca
When the transfected human umbilical vein endothelial
cell-derived line ECV304 was stimulated with ATP (1-10
µM), a slow and sustained rise in
[Ca] ) in Response to
ATP] was observed (Fig. 1a). The time to half-maximal (t
) for the rise was 18.6 ± 1.3 s (x ± S.E.) (n = 11), and
[Ca] rose to between 700 nM and 1 µM, rarely exceeding 1 µM.
[Ca] remained elevated for the
duration of agonist presence, i.e. for up to 10 min
in some cases (Fig. 1a). The increase in
[Ca] was comparable with the
[Ca] response in these cells (Fig. 1, b and c) and contrasts dramatically
with the rapid and large increase in
[Ca] seen in hepatocytes and
several other cell lines(12, 16) .
]) were measured
in mtAEQ transiently transfected ECV304 endothelial cells. Applications
of 10 µM ATP for long periods resulted in sustained
[Ca] increases that
are comparable with changes in
[Ca] as shown in b and c. [Ca] changes in ECV304 cells in response to ATP were measured
using cytAEQ (b) (10 µM ATP) or Fura-2 (c) (1 µM ATP). For a and b,
points were obtained every 200 ms; for c, images were captured
every second resulting in ratio images every 2 s. a and b, the trace represents [Ca]
measured from the whole coverslip; c, the data are the mean of
20 single Fura-2-loaded cells. Bars represent the presence of
ATP in the perfusion medium; 1 mM Ca was
present throughout.
[Ca] for ATP alone was
1070 ± 136 nM as compared with a
[Ca] for ATP and FCCP of
only 185 ± 61 nM (p < 0.001, n = 6). In two of the six experiments, there was no
detectable ATP-evoked rise in [Ca] in the presence of FCCP. The effects seen with FCCP were
reversible following a short period of wash and readdition of ATP (n = 6). While FCCP had this dramatic effect on
[Ca], it was found to have no
effect of the ATP-evoked rise in [Ca] (n = 21). Examination of transfected cells by
immunofluorescent localization of the mitochondrially targeted aequorin
revealed a similar reticular pattern of fluorescence to that previously
seen for MH75 cells(19) . These observations confirm that the
detected rise in response to ATP is a true reflection of the rise in
[Ca].] with only a modest
reduction in magnitude (n = 11, not shown).Changes in Cytosolic Calcium
([Ca
The unusual kinetics of
[Ca]) in Response to
ATP] in ECV304 cells in
response to ATP prompted us to investigate whether this behavior was
due to some peculiarities of the [Ca] response in this cell type. This was monitored using either
untargeted aequorin (Fig. 1b, n = 9) or
Fura-2 (Fig. 1c, n = 20). In fact,
stimulation of ECV304 cells, with ATP (1-10 µM),
evoked a rise in [Ca] (n = 40), which was quite typical both in terms of kinetics,
with a t
of 2.12 ± 0.62 s for 1 µM (n = 20) and 1.97 ± 0.58 s for 10
µM (n = 20) (tfor 1
µM is not significantly different to that for 10
µM (p > 0.05)), and of amplitude (mean
[Ca] for 1 µM ATP
= 906.3 ± 27.2 nM (n = 20) and
for 10 µM = 933.6 ± 53.2 nM (n = 20) which are not significantly different from one
another (p > 0.05)). The typical biphasic nature is
revealed by stimulation in the absence of extracellular calcium
(following a 2-4-min prewash in 0.5 mM EGTA), which led
to a transient response (n = 11) (Fig. 2b, inset) with an equivalent t(1.61 ± 0.11 s) and maximal
[Ca] (955.9 ± 36.7
nM). In the presence of Ca
the
response is sustained for as long as the agonist is present (Fig. 1, b and c). Repetitive applications of
ATP each evoke a comparable response with only a small degree of
reduction, presumably due to receptor down-regulation, provided calcium
is present in the extracellular medium (not shown).
either during (a) or
following (b) stimulation with 10 µM ATP for
[Ca] and
[Ca](insets)
in ECV304 cells. Data points were obtained every 200 ms for
[Ca] traces and every
2 s for [Ca] traces. Bars represent the addition of reagents to the perfusion bath.
0.5 mM EGTA was present until 1 mM Ca was added where indicated.
The Effects of Calcium Influx on
[Ca
Because of the
sustained nature of the rise in [Ca]] in response to ATP, the dependence on Ca was investigated. Washing the cells in buffer supplemented with
0.5 mM EGTA (calcium-free) and subsequent stimulation with ATP
result in a significantly lower rise in
[Ca].
[Ca] for ATP + EGTA
= 280 ± 43.3 nM (Fig. 2, a and b), whereas control ATP responses are nearly three times as
large with a [Ca] of 763
± 63.6 nM (p < 0.001, n =
14). Two of the fourteen experiments in 0.5 mM EGTA resulted
in no rise above basal [Ca].
These small responses were extremely transient with a duration of only
62.2 ± 6 s (n = 12). during ATP stimulation, while
monitoring [Ca], in ECV304
cells (n = 11), led to a second rapid rise in
[Ca] (Fig. 2a),
which was significantly higher magnitude than the increase elicited by
agonist addition in Ca-free medium (315.8 ±
89% of initial increase n = 8, p < 0.001).
In contrast Fig. 2a, inset, shows results of the same
protocol while monitoring [Ca].
In 8 of the 11 experiments, the restoration of the response was of a
magnitude comparable with the plateau phase of a normal biphasic
sustained rise in [Ca] (Fig. 2a, inset). In less than 30% (3
experiments) of cells the rise in
[Ca] obtained upon
Ca readdition was equal to, or very slightly higher
than, the initial rise in response to ATP in EGTA-containing medium. ] and
[Ca] was observed using the
protocol employed in Fig. 2b. In this latter case, the
cells were first stimulated with ATP, in Ca-free,
EGTA-containing medium. The agonist was then washed away and the
Ca added back a few minutes later.
Ca readdition led to a large, transient rise in
[Ca] (Fig. 2b; n = 6), whereas a rise was not evident upon
Ca replacement when monitoring
[Ca] (n = 5, Fig. 2b, inset) or
[Ca] in the presence of 1
µM FCCP (n = 5, not shown). Following the
restoration of Ca, and a brief wash
period, subsequent addition of ATP produced the typical sustained rise
in [Ca], indicating that during
the experiment there had been no marked desensitization of the ATP
response (n = 14). In addition, application of the
receptor-mediated Ca entry blocker SKF96365 (50
µM) (20) completely abolished the rise in
[Ca] in all cases (n = 6).[Ca
Under identical experimental conditions, MH75 cells
stimulated with ATP show an increase in
[Ca] and
[Ca] Responses in MH75
Cells] in the order of 2-3
µM as opposed to 
1 µM in ECV304 cells.
However, the rise in [Ca] seen
in these cells was comparable with that seen in ECV304 cells (i.e. submicromolar). The tfor
[Ca] in MH75 cells in response
to ATP was 4.0 ± 0.5 s, and the duration was only 71.7 ±
10 s even though the agonist remained present in the perfusion medium
for several minutes. Such a short rise time is significantly different
from that seen in response to ATP in ECV304 cells (p <
0.005). These responses are similar to those previously reported for
histamine-stimulated MH75 cells(3, 4) , and there is
no significant difference between either duration (p =
0.5) or t(p = 0.5) for ATP or
histamine-evoked changes in [Ca] in MH75 cells. Stimulation of these cells in the absence of
Ca evokes a similar response but, in
contrast to the ECV304 cells, replacing Ca results in a secondary rise very much smaller than in the first (Fig. 3a, n = 6). The same protocol was
applied while monitoring [Ca] (Fig. 3b, n = 12) and revealed
comparable results to the ECV304 cell
[Ca] response in Fig. 2b.
during stimulation with 10
µM ATP between
[Ca] (a) and
[Ca] (using Fura-2) (b) for MH75 cells. 0.5 mM EGTA was present to start
until 1 mM Ca was applied as indicated by
the bars. The presence of ATP is also indicated by the bars. Points were obtained every 200 ms for a, and
images were captured every 2 s for b.
Localization of Mitochondria
Electron micrographs
were prepared of both MH75 and ECV304 cells from pellets of cells
harvested from tissue culture flasks ( Fig. 4a and c), and also from monolayers grown on Mellinex 228 sheeting (Fig. 4, b and d). Since there was no
difference in the observed distribution of structures using either
preparation method, the data from both sets of micrographs were pooled.
magnification, respectively. c and d,
MH75 cells at 90,000 and 40,000 magnification, respectively. a and c are from pelleted cells, and b and d are taken from cells grown on Mellinex 228 sheeting. M, mitochondria; P, plasma membrane; ER,
endoplasmic reticulum.
, respectively. It is evident that while mitochondria are
abundant, there is little ER in these cells. Fig. 4, c and d, shows the distribution of mitochondria in MH75
cells, at 90,000 and 40,000 , respectively. Abundant ER is
visible, which appears to be in close association with the mitochondria
even when near to the plasma membrane. Compare Fig. 4b for ECV304 with Fig. 4d for MH75 cells.
] evoked by
InsP-generating agonists was one of the most surprising new
pieces of information provided by the technique of recombinant,
organelle-targeted,
aequorins(2, 3, 4, 12) . For over a
decade the general consensus was that, under physiological conditions,
mitochondria should play a minor role in the control of Ca homeostasis(7) , given the low affinity of the uniporter
and the relatively small increases in
[Ca] elicited by receptor
stimulation. This apparent paradox has been explained by the hypothesis
that microdomains of [Ca] are
sensed by mitochondria localized in the vicinity of the Ca release sites(3, 4) . This hypothesis has been
further supported by recent data obtained in freshly isolated
hepatocytes using dihydro-Rhod 2 or the intrinsic fluorescence of
NAD(P)H(16) . Thus, in all cell types analyzed so far, the
kinetics of [Ca] changes in
response to agonist stimulation appear to be rapid and large increases
that decline to prestimulatory levels faster than
[Ca]. evokes a slow-onset but sustained rise
in [Ca] that can be reversibly
inhibited by treatment with the mitochondrial uncoupling agent FCCP.
Furthermore, in contrast to MH75 and other cell types, stimulation in
the absence of Ca results in a drastic
reduction, and in some cases even abolition, of the
[Ca] increases, suggesting that
the measured rise in [Ca] may
be critically dependent upon Ca influx rather than
the release of Ca from intracellular stores. ] responses are
comparable between ECV304 cells and MH75 cells, why does agonist
stimulation not evoke the large, rapid rise in
[Ca] characterized in other
cell types? This question may, in part, be answered by the electron
microscopic data. Here we show that a very high degree of association
exists between the mitochondria and the ER in the MH75 cells. However,
there is no such close association between these two organelles in the
ECV304 cells; indeed, <4% of the mitochondria in these cells are
within 700 nm of the ER. In ECV304 cells, therefore, the mitochondria
are not, as in the HeLa cells, located in sufficiently close proximity
to the ER to detect any [Ca] microdomains which would occur at the time of agonist-stimulated
Ca release.] is
2-3-fold smaller in magnitude than the
[Ca] responses seen in other
nonexcitable cells (1 µM as opposed to 2-3
µM in the MH75 cells)(3, 4) . A similar
type of small, slow-onset response can be obtained in MH75 cells and
hepatocytes when the ER Ca-ATPase inhibitors t-butyl-benzohydroquinone (21) or thapsigargin are
used to promote a sustained rise in
[Ca](4, 16, 19) via an
InsP-independent process involving Ca influx rather than the rapid release from intracellular stores.
Such responses lend support to the suggestion that the slow, sustained
[Ca] response in ECV304 cells
is maintained by Ca influx.] elicited by receptor
stimulation is more effective at maintaining a sustained increase in
[Ca] than in HeLa or
hepatocytes. Several not mutually exclusive alternatives can be
suggested. (i) The large, rapid rise in
[Ca] triggered by receptor
stimulation in HeLa and other cells (3, 4, 16) could, itself, be responsible for
the transient nature of their responses. The high
[Ca] may induce run-down of the
mitochondrial Ca uniporter by a process analogous to
the Ca-dependent run-down seen in voltage-gated
Ca channels(22, 23, 24) .
(ii) The H:Ca and
Na:Ca antiporters (in highly
responding cells) could undergo a prolonged activation by the large
increase in [Ca], in a manner
similar to the Ca activation of the plasma membrane
Na:Ca exchanger(25, 26) . The absence of a large
initial [Ca] spike in the
ECV304 cell mitochondria, and in the t-butyl-benzohydroquinone- or thapsigargin-stimulated cells,
would therefore favor the sustained elevation in
[Ca] that is observed.] responses) as far as
mitochondria localization is concerned. A small proportion of the
mitochondria in ECV304 cells is localized within a relatively small
distance (<800 nm) of the plasma membrane. Although the response to
the release of stored Ca is quite small, readdition
of Ca in ECV304 cells, either in the presence or
absence of ATP, causes a rapid and transient elevation of
[Ca] that is not reflected in
the [Ca]. It is tempting to
speculate that this association between mitochondria and plasma
membrane is at the basis of the fast and relatively large response of
these cells under conditions where the influx of Ca from the extracellular medium is maximized, i.e. by
first depleting the cells in EGTA medium followed by Ca readdition. A similar effect is not observed in
[Ca] or
[Ca] in MH75 cells. This
suggests that an influx of Ca may
generate a microdomain of high [Ca] close to the plasma membrane causing a rapid uptake of
Ca into the mitochondria located in this region. ] increases depend on the
cell type and are due to the structural relationships between
mitochondria and the source of [Ca] increases. (ii) Fast increases in
[Ca] are always dependent on
[Ca] microdomains; whether
these rapid increases are triggered by Ca release
from stores or by Ca influx depends on the
preferential vicinity of the mitochondria to the ER or to the plasma
membrane, respectively. Thus, in any cell type, different modes of
communication between cytosolic and mitochondrial Ca can exist. For example, in cell types where there is a close
association between mitochondria and ER, a sustained elevation of
[Ca], and thus a prolonged
activation of the dehydrogenases, can be elicited by high frequency
[Ca] oscillations, as shown by
Hajnóczky et al.(16) in
hepatocytes; alternatively, as in the case of ECV304 cells, the same
result can be obtained by a prolonged elevation of
[Ca]. (iii) Last, but not
least, the present data predict that in cell types where a major source
of [Ca] elevation is via a
large Ca influx current through plasma membrane
Ca channels, there may be a selective activation of
subpopulations of mitochondria in the proximity of the channels
themselves. Preliminary data from our laboratory ()indicate
that this is indeed the case. The possibility of different ways of
controlling mitochondrial Ca homeostasis offers a
unique flexibility to those signaling mechanisms that may have
important physiological consequences. For example, as far as
endothelial cells are concerned, since the regulation of blood vessel
tone is a long term physiological effect, the cells will be required to
release vasodilators such as nitric oxide for a prolonged period. This,
along with the maintenance of the
[Ca] signal, will require a
sustained supply of energy in the form of ATP and a large production of L-arginine for NO synthesis. Depending on the agonist and the
individual cell type this might be achieved by either high frequency
oscillations or long lasting increases of
[Ca], which we observed here.
)], cytosolic free
calcium concentration;
[Ca], mitochondrial
matrix free calcium concentration;
Ca, extracellular calcium;
ECV304, human umbilical vein-derived endothelial cell line; MH75, HeLa
cell clone stably transfected with mitochondrially targeted aequorin;
InsP, inositol 1,4,5-trisphosphate; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; ER, endoplasmic reticulum;
cytAEQ, cytoplasmic aequorin; mtAEQ, mitochondrial aequorin.)
We would like to thank Marisa Brini for her assistance
in the laboratory in Italy, Brian Boothroyd for his invaluable
expertise taking the electron micrographs, and Dave Bassi for
photography. We would also like to thank Tracie Walker and Alan Conant
for their help in preparing the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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