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J Biol Chem, Vol. 273, Issue 52, 34961-34969, December 25, 1998
From the Physiologisches Institut der
Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse
7, D-79104 Freiburg, Germany
Loading of HT29 cells with the
Ca2+ dye fura-2/AM resulted in an nonhomogeneous
intracellular distribution of the dye. Cellular compartments with high
fura-2 concentrations were identified by correlation with mitochondrial
markers, cellular autofluorescence induced by UV, and dynamic
measurement of autofluorescence after inhibition of oxidative
phosphorylation. Stimulation with carbachol (10 Initial observations with the AM form of the
Ca2+-sensitive dye quin-2 indicated that the free acid form
of the dye does not accumulate within intracellular compartments (1).
However, using microscopic methods with higher resolution, in numerous reports, it is now well accepted that most AM dyes will accumulate to a
variable extent in intracellular organelles such as endoplasmic reticulum (2), endocytotic vesicles (3), and mitochondria (4, 5). For
isolated mitochondria, it has been shown that they can convert the AM
form of fura-2 or indo-1 to the Ca2+-sensitive forms
(6-8). This has been used to measure the mitochondrial Ca2+ activity in isolated mitochondria, as well as in
intact cells by indirect methods (9), or after quenching the cytosolic
fluorescence of the dyes with Mn2+ (5). By using high
resolution fluorescence digital imaging (FDIM),1 initial attempts
were made to measure the Ca2+ activity of single
mitochondria ([Ca2+]m) (10-12). Due to limited
optical resolution in the x-y direction and to an even
greater extent in the z direction, further attempts were
made using confocal microscopy (13-15) or targeted aequorin (16-19).
However, these approaches have some limitations, as the single
wavelength Ca2+ dye fluo-3 was used. Similarly, the low
luminescence signals of aequorin did not allow for resolving signals
from single mitochondria. We have recently established confocal fura-2
ratio imaging (20) and further improved FDIM by using a high
sensitivity, high resolution, intensified CCD camera, which allows
simultaneous measurement of cytosolic, nuclear, and mitochondrial
Ca2+ signals in response to agonist stimulation in the
epithelial cell line HT29. Furthermore, we present data
showing high resolution two-photon fura-2 imaging using a prototype
microscope with a new turn-key pulsed IR laser source.
Cell Culture and Measurement of
[Ca2+]i--
HT29 cells were
cultured and prepared for measurements of [Ca2+]i
with fura-2 as described (21-23) previously. Experiments were
performed with three different experimental set-ups.
Camera-based System--
The set-up consisted of an inverted
fluorescence microscope equipped with a × 100 objective (Axiovert
100 TV plus an OPTAVAR × 1.6 lens, Fluar 100/1.3 oil, Zeiss), a
fast switching monochromator (T.I.L.L. Photonics, Planegg, Germany) for
changing the excitation wavelength, and a GEN3, intensified CCD camera
(ICCD 350, Videoscope, Sterling, VA) for fluorescence imaging. Images
were acquired using an average of 8-16 frames to increase the
signal:noise ratio. Control of the experiment, image acquisition, and
data analysis were done with the software package MetaFluor/MetaMorph
(Universal Imaging, West Chester, PA).
Confocal Microscope--
The set-up consisted of an inverted
confocal microscope with a × 63 objective (LSM 410, C-APO 63/1.2
water, Zeiss). The microscope was equipped with an argon UV laser (351 nm, 364 nm) and a second blue-enhanced argon laser (457, 488, and 514 nm). The confocal microscope (LSM) was modified as described recently
to allow fura-2 ratio imaging, as well as simultaneous parfocal UV and
visible excitation (20). For the Ca2+ ratio experiments,
the scanning speed was set to 1.08 s for a 512 × 512-pixel image,
the pinhole was set to achieve a full-width, half-maximum z
resolution of around 1 µm. In the double labeling experiments with
mitochondria-specific fluorescent markers DiOC(5)(3) or
rhodamine 6G, cells were first loaded with fura-2/AM as described above
and then the mitochondria specific dyes were directly added to the bath
chamber when the bath perfusion was stopped. The fluorescence staining
of the cells was followed directly by visualization at 488 nm
excitation with the LSM. Incubation was stopped by restarting the bath
perfusion after 20-40 s. This avoided dye overloading and unspecific
staining of other membranous cellular structures. Images were then
recorded at a scanning speed of 2 or 4 s per full-size image,
with a two-frame average. The fluorescence signal at 488 nm
excitation consisted of more than 95% from the mitochondrial markers,
the rest being cellular autofluorescence and the fura-2 emission signal.
Two-photon Microscope--
A prototype microscope system (LSM
560, Zeiss) with a turn-key fiber laser (780 nm; pulse-width, <180 fs;
maximum power, 7.4 mW) enabled us to image fura-2-loaded cells with
a × 40 objective (Zeiss, Plan-Neofluar 40/1.3 oil). A timed
series of fluorescence images (60-100 images of 512 × 512 pixels; 2-4-µs pixel time; measured mean laser power at the
objective plane reduced by gray filters, 1.2 mW) was recorded before
and during agonist stimulation. All fluorescence emission below 680 nm
was collected without using a pinhole in front of the fluorescence
detector. The two-photon excitation wavelength of 780 nm should result
in an excitation above the isosbestic point of fura-2. Therefore, one
expects a decrease of the fluorescence signal with increasing
[Ca2+]i.
Calibration of the fura-2 signal in terms of
[Ca2+]i was performed at the end of the
experiments using ionomycin as described (24). The problems of the
calibration are discussed below. The average of the pixel-by-pixel
ratios of user-selected areas marked inside each cell was used to
calculate [Ca2+]i according to Grynkiewicz
et al. (25). No calibrations were performed in the
experiment with the confocal or the two-photon system.
Microinjection of Oregon Green BAPTA-1 Dextran
10000--
Electrodes for microinjection were pulled with a laser
puller (P-2000, Sutter Instruments, Novato, CA) and the dye filled pipettes were beveled to a resistance of 20-30 M Solutions and Chemicals--
The standard solution contained 145 mmol/liter NaCl, 1.6 mmol/liter K2HPO4, 0.4 mmol/liter KH2PO4, 1.3 mmol/liter calcium gluconate, 1 mmol/liter MgCl2, 5 mmol/liter
D-glucose, pH 7.4. The detailed composition of the
solutions used in the experiments was described recently (21). The
injection solution contained 30 mmol/liter KCl, 4.8 mmol/liter
Na2HPO4, 1.2 mmol/liter
NaH2PO4, 95 mmol/liter potassium gluconate, 1 mmol/liter MgCl2, 5 mmol/liter D-glucose, 0.5 mmol/liter EGTA, pH 7.2. All fluorescent dyes were from Molecular
Probes (Eugene, OR). All other chemicals were of the highest grade of
purity available and were obtained from Sigma or Merck (Darmstadt, Germany).
Statistics--
Data are given as mean values ± S.E.
(n), where n refers to the number of experiments.
Paired and unpaired t tests were used to compare mean values
within one experimental series and different experimental series,
respectively. A P value of Intracellular Distribution of Fura-2 after Loading with
Fura-2/AM--
Loading of HT29 cells (4-7 days after
seeding) with fura-2/AM at room temperature resulted in an uneven
distribution of the dye inside the cell. Fig.
1A shows a typical confocal
image of such cells taken at 364 nm excitation, which is near the
isosbestic point of the fura-2 spectrum. Images taken at 351 nm
excitation show a similar dye distribution. Varying the loading
conditions did not alter the general distribution pattern of tubular
structures preferentially located around the nucleus. In younger cells
(2-3 days after seeding), the perinuclear tubular structures were not as marked and showed a more punctuate distribution of the spots of high
fluorescence. In earlier reports, it was described that fluorescent
dyes, such as fura-2 or fluo-3, when loaded in the AM form, accumulated
in the mitochondria (4, 26, 27). Therefore, we compared the
fluorescence distribution pattern of the fura-2/AM-loaded HT29 cells after co-staining with the mitochondrial markers
DiOC(5)(3) or rhodamine 6G. Fig. 1, A and
B, shows that the mitochondrial stain (here
DiOC(5)(3)) widely overlaps with the cellular structures that have accumulated high concentrations of fura-2. The differential interference contrast (DIC) image of the same area as depicted in Fig.
1C shows that the majority of the mitochondria in
HT29 cells are located around the nucleus. To prove the
specificity of the used mitochondrial marker, we compared the pattern
of the UV autofluorescence of HT29 cells with the stain
pattern obtained by DiOC(5)(3). In most cells, cellular
autofluorescence at 364 nm is related to NADH and NADPH fluorescence
mainly located in mitochondria (28). Fig. 1, D and
E, shows a high degree of co-localization of both
fluorescence signals, and again, most of the mitochondria are located
around the nucleus (Fig. 1F). Notably, when fura-2 was
loaded in the non-AM form via a patch pipette or microinjected, we did
not observe dye accumulation in the mitochondria (both types of
experiments, n = 3; data not shown).
To further characterize the mechanism of dye accumulation, we used the
spot bleach mode of the confocal microscope, which allows fast
wavelength and intensity changes from one scanned image to another by
using acousto-optical tunable filters. Fura-2 was equilibrated for a
period of 15 min at 37 °C and then bleached with a short pulse (0.2 s) of high laser intensity in the central, nuclear part of the cell.
This resulted in a strong decrease of the fluorescence in the cytosol
as well as in the nucleus. The remaining fluorescence was stable in the
mitochondria for at least 20-25 min (see Fig.
2A). In addition to the above
results, bleaching of a single mitochondrion with the spot bleach
method (0.02 s) after fura-2/AM loading and an equilibration period of
15 min at 37 °C (to allow for the cleavage of the AM-ester) did not
give a significant recovery of the fura-2 fluorescence signal (Fig. 2B). These results indicate that fura-2/AM is converted
inside the mitochondrion to the fura-2 free acid, which is then trapped inside the organelle. Localized excitation spectra of fura-2
(excitation, 320-400 nm; emission, >470 nm), recorded with the fast
monochromator by FDIM from single cells, showed no significant
excitation shift between the three compartments (cytosol, nucleus, and
mitochondria; data not shown; n = 3). Localized
emission measurements, using the confocal microscope and a CCD-based
spectrometer, showed no difference in the emission spectra in the
nucleus and the cytosol. Emission spectra from single mitochondria
could not been measured due to the limited sensitivity of the
spectrometer.
Effect of Agonist Stimulation on Mitochondrial Ca2+
Activity ([Ca2+]m)--
Fig.
3 shows that upon agonist stimulation, a
differential intracellular Ca2+ signal was observed by
confocal imaging of the fura-2 fluorescence. The top three
images show again the accumulation of fura-2 in discrete
organelles identified (see Fig. 1) mostly as mitochondria. The fura-2
emission signal is high, but almost equal at both excitation wavelengths. This results in a mitochondrial Ca2+ activity
([Ca2+]m) very similar to that of the cytosol or
the nucleus, as shown in the first 351/364 nm ratio image on the
second row of Fig. 3. Upon stimulation with a low
concentration of an agonist (here, CCH at 10
In order to address the problem of out of focus fluorescence and the
contribution of cytosolic dye signals to mitochondrial dye signals we
used three experimental approaches.
1) Cells were loaded with fura-2 as described under "Materials and
Methods" and then, in addition, microinjected with the high molecular
weight Ca2+ indicator dye Oregon green BAPTA-1 dextran
(OGD) 10,000. Fig. 4A shows
the typical fura-2 fluorescence image of a cell, and Fig. 4B
shows the OGD fluorescence image from the same cell. It is obvious from
the wide overlap of the darker intracellular structures in the OGD
image with the bright structures in the fura-2 image that OGD did not
enter the mitochondria. In addition, other intracellular structures,
such as the nuclear envelope, show within the limits of optical
resolution only a very low dye signal. To obtain a good overlap of the
two images, the focus of the microscope had to be changed by around 0.4 µm, in order to compensate for a chromatic aberration of the
microscope objective. Stimulation of the double-loaded cells with
neurotensin or CCH showed a response only from OGD at 488 nm
excitation, but almost no response in the fura-2 signals at 351 and 364 nm (data not shown, n = 5). This could be due to the
great overlap of the fura-2 emission signal with the OGD absorption spectrum. However, we did not examine this in more detail. The very
weak OGD signal from mitochondrial areas, which we interpret as a spill
over from the cytosol, showed small intensity changes (with a poor
signal:noise ratio), synchronous with the cytosolic or nuclear signal.
From the optical properties of a confocal microscope, it is more
difficult to exclude the surrounding bright cytosolic and nuclear
signal and resolve the small dark mitochondrial areas. Due to the focal
shift, the limited z resolution, and the poorly responsive
fura-2 signal, the measurements could not be used further.
2) In a second experimental approach, we tried to reduce the
contribution of nonmitochondrial fluorescence to the mitochondrial signal by using a prototype two-photon microscope system for single wavelength excitation of fura-2. We acquired simultaneously the fluorescence images and transmitted light DIC images. This enabled the
detection of changes in the focal plane due to movements of the
preparation or movements of intracellular organelles. Two-photon excitation gives a high z resolution (0.4-0.6 µm,
full-width, half-maximum) for fluorescence imaging. Fig.
5A shows a typical two-photon
fluorescence image of a single cell. Three cellular areas
(mitochondrion, nucleus, and cytosol) are marked inside the cell. Fig.
5B shows the fluorescence intensity changes in response to a
stimulation with ATP and CCH. The fluorescence signal dropped
reversibly in all compartments, with the mitochondrial area clearly
delayed compared with the signal change in the other compartments. This
type of experiment was successfully repeated four times. In other
experiments, the distinct mitochondrial structures disappeared,
probably due to changes in the the focal plane due to movements of the
whole preparation or to fast movements of the mitochondria in the
z image plane. The drop in the fura-2 signal in response to
a [Ca2+]i increase indicated that with a 780-nm
two-photon excitation, fura-2 is excited at a wavelength longer than
the isosbestic point.
3) A mitochondrial uncoupler, which would breakdown the mitochondrial
potential, was used in an attempt to prevent the
[Ca2+]m increase. A typical experiment, using
high resolution FDIM, is shown in Fig.
6A. FCCP reversibly inhibited
around 50% of the Ca2+ change in mitochondria. Fig.
6B shows a summary of the experimental series with FCCP
(n = 7); the mean inhibition was 66.7 ± 3.6%. We
could also separate the time courses of the mitochondrial
Ca2+ increase from the Ca2+ increase in the
cytosol and the nucleus by applying FCCP shortly before and during the
onset of a CCH-induced Ca2+ increase as shown in a typical
experiment in Fig. 7 (one out of four
similar experiments).
Agonist Dependence and Time Course of the Mitochondrial
Ca2+ Increase--
In one series of experiments, we
examined in more detail the agonist concentration dependence of the
mitochondrial [Ca2+]i increase. We recorded a
concentration response curve for [Ca2+]c,
[Ca2+]n, and [Ca2+]m with
carbachol, which stimulates M3 receptors in
HT29 cells (29). Carbachol was chosen because its
[Ca2+]i response is only slightly desensitizing
in comparison to neurotensin or ATP, as we have shown previously (22,
30). Fig. 8A shows a typical
measurement of [Ca2+]i in the cytosol, nucleus,
and a single mitochondrion in response to different carbachol
concentrations (0.5-100 µmol/liter). At low CCH concentrations, only
a small increase of [Ca2+]m could be measured,
and it stayed well below the value of [Ca2+]c and
[Ca2+]n. At a higher agonist concentration, a
biphasic increase of [Ca2+]m was observed (Fig.
8A). This mitochondrial transient was delayed in the second
phase, but in principle it followed [Ca2+]n and
[Ca2+]c. The peak response was slightly lower
than [Ca2+]c and [Ca2+]n.
Removal of the agonist led to a delayed decrease of
[Ca2+]m, which then stayed, in around 75% of the
experiments, for 1-3 min above the [Ca2+]c and
[Ca2+]n (see Fig. 8A (CCH,
10 Source of Ca2+ for the Mitochondrial Ca2+
Increase--
We further examined the mechanism of the
[Ca2+]m increase by using thapsigargin, which
inhibits the SERCA type Ca2+-ATPase of the intracellular
Ca2+ stores and thus increases
[Ca2+]c by emptying inositol
1,4,5-trisphosphate-sensitive Ca2+ stores. Thapsigargin, as
shown in Fig. 9, led to a
concentrationdependent, relatively slow increase of
[Ca2+]c, [Ca2+]n, and
[Ca2+]m (n = 7), very similar to
what was measured with CCH. Again, at a cytosolic 345/380 ratio of
around 4, a sharp increase of [Ca2+]m was
observed. Plotting the data for thapsigargin in the same way as
outlined for Fig. 8A resulted in a very similar relationship
between the cytosolic and the mitochondrial fura-2 ratios. This is
shown in Fig. 10B. Again,
the mean [Ca2+]c value, at which a the second,
strong and fast increase of [Ca2+]m also
occurred, was a ratio of around 5.
Fig. 11 shows the increase of
[Ca2+]i induced by neurotensin, when no
Ca2+ was present in the extracellular solution. Thus, after
emptying of the neurotensin-sensitive Ca2+ stores, no
Ca2+ influx could occur. Even then, a transient increase of
[Ca2+]m was measured, which implies that it must
have been due to release of Ca2+ from intracellular stores.
Fig. 12 shows the effect of removal of
Ca2+ from the external medium in the plateau phase of a
Ca2+ increase, when [Ca2+]c and
[Ca2+]n are dependent on the balance between
Ca2+ influx and Ca2+ efflux. Removal of
external Ca2+ in this phase of the transient led to a fast
decrease of whole cell [Ca2+]i, including
[Ca2+]m, [Ca2+]n, and
[Ca2+]c. However, it was obvious in these
experiments that the time course for the decrease was slower for
[Ca2+]m, and [Ca2+]m did
not decrease to the low values of [Ca2+]c or
[Ca2+]n within the 2 min of the low
Ca2+ solution in the bath. Readdition of a solution with
normal Ca2+ (1.3 mmol/liter), still in the presence of the
agonist, led to an increase of [Ca2+]i in all
three cellular compartments with a similar sequence, time course, and
shape compared with an initial agonist stimulation. After removal of
CCH (100 µmol/liter), [Ca2+]m remained elevated
in most experiments for more than 2 min (Figs. 8A,
6A, and 12). A similar observation could be made with high
concentrations of other agonists, such as ATP or neurotensin (each
n = 3; data not shown). From Figs. 11 and 12, one can
conclude that the [Ca2+]m transient has, similar
to the increase of [Ca2+]c and
[Ca2+]n, first an intracellular and then an
extracellular source.
Influence of Mitochondrial Ca2+ Increase on the Energy
State of Mitochondria--
To show the influence of agonist
stimulation on the mitochondrial energy state, we measured cellular
autofluorescence at 340 nm excitation and emission >500 nm, which, as
outlined in the first paragraph under "Results," is mostly due to
the mitochondrial NAD(P)H levels. As shown in Fig.
13, treatment of the cells with KCN and
NaN3 to inhibit oxidative phosphorylation and thus increase the level of NAD(P)H led to an increase of overall cellular
autofluores-cence. This effect was reversible and repeatable. A
subsequent, strong stimulation of the cells with a maximal dose of CCH
and ATP (both 10 The present experiments were performed to evaluate the role of
mitochondrial Ca2+ in response to agonist stimulation. By
using confocal fura-2 imaging, two-photon microscopy, and high
resolution FDIM, simultaneous measurement of the Ca2+
signals in at least three cellular compartments (cytosol, nucleus, and mitochondria) was possible.
Mitochondrial Localization of Fura-2--
Mitochondrial
localization of a substantial amount of fura-2 after AM loading was
evaluated by correlating the fura-2 distribution with the UV
autofluorescence and the pattern of mitochondrial markers. Our results
are comparable with reports on the fura-2 or fluo-3 distribution in
myocytes (12, 14), endothelial cells (31), or hepatocytes (17). In
addition, the bleaching experiments using confocal microscopy indicate
that fura-2 enters the mitochondria in the AM form, where it is trapped
or bound after cleavage. In addition, the bleaching of single
mitochondrial structures reveals that in HT29 cells
mitochondria do not form a continuous network throughout the cytosol.
This is in contrast to recent findings in HeLa cells, where recombinant
green fluorescent protein was used to label mitochondria (32). However,
measurements made in our laboratory on primary cultures of hepatocytes
also support the idea that mitochondria in these cells indeed can form
a network extending over 10-20
µm.2
Are the Properties of Intramitochondrial Fura-2 Comparable to Those
of Cytosolic Fura-2?--
The spectroscopic measurements, the
experiments with FCCP, and the bleaching experiments presented in this
paper strongly indicate that fura-2 inside the mitochondria is in a
Ca2+-sensing form. This has already been shown for fura-2
and indo-1 in isolated mitochondria (6, 8, 33). However, in these publications, no consensus was reached about how much fura-2 properties are changed within the mitochondrial environment. We tried to calibrate
the fura-2 Ca2+ signal in vivo inside the
mitochondrion using the usual calibration procedure of the fura-2
signal with ionomycin, as has been used for HT29 cells from
our laboratory extensively over the last years (22, 30, 34). Ionomycin
treatment gave almost equal fura-2 ratio values in the cytosol and the
nucleus, but the Rmax value in the mitochondria
was always slightly lower. In many cells, ionomycin is known to
increase [Ca2+]i, not directly by acting as a
Ca2+ ionophore in the outer cell membrane, but by first
emptying inositol 1,4,5-trisphosphate-sensitive intracellular stores
via an unknown mechanism and by the subsequent maximal opening of the
Ca2+ influx pathways usually named capacitative
Ca2+ entry (35, 36). This action of ionomycin can be
unmasked in HT29 cells by lowering the extracellular
Ca2+ to 10 Mitochondrial Ca2+ Response Is Dependent on Agonist
Concentration--
A number of other laboratories have described
changes of [Ca2+]m in response to agonist
stimulation in living cells using aequorin targeted to mitochondria
(17, 18), fluo-3 (31), or dihydro-rhod-2 (17, 18). However, none of
these studies reported the concentration response curve of
[Ca2+]c and [Ca2+]m to
agonists measured simultaneously and on the single cell level. This was
due to limitations in sensitivity, limited optical resolution, and/or
the dyes used.
With the higher concentrations of the agonists CCH, neurotensin, and
ATP (for ATP no data were shown), the response in
[Ca2+]m to [Ca2+]c was
biphasic. The initial rapid rise was only slightly slower than the
cytosolic response, followed by a slower rise in
[Ca2+]m when a threshold in
[Ca2+]c was exceeded. Surprisingly, the response
of [Ca2+]m to thapsigargin showed a similar
[Ca2+]c dependence. The
[Ca2+]c, corresponding to the threshold value for
agonists and thapsigargin, was estimated from the experiments shown in Fig. 10, A and B. A ratio of around 5 was
obtained, corresponding to around 1200 nmol/liter. Around this
cytosolic Ca2+ activity, a strong increase of
[Ca2+]m could be observed. A similar, biphasic
[Ca2+]m response has been described recently in
endothelial cells after stimulation with bradykinin (31) and in cardiac myocytes after electrical stimulation (5). Similar to the results of
these studies, we could also not rule out the possibility that the
initial, small and fast Ca2+ rise is the result of a
spill-over from the cytosolic signal due to a limited z
resolution in conventional confocal microscopy and/or the movement of
mitochondria. However, even when we used two-photon excitation of
fura-2 (the method currently giving the highest possible z
resolution for fast dynamic measurements) the results of the
Ca2+ measurements were very similar. Therefore, it seems
unlikely that the initial [Ca2+]m response is an
artifact, even if FCCP did not influence it. In addition, in some of
our experiments (for example, Figs. 9 and 11), we measured a
discrepancy in the time course for this early phase of the
[Ca2+]c and [Ca2+]m
increase. This would indicate that, indeed, different compartments were
contributing to the signal. It should also be pointed out that a
biphasic [Ca2+]m rise was described in an
isolated mitochondria preparation in response to Ca2+
pulses (38), which speaks against an optical artifact. The initial,
fast Ca2+ increase could be caused by the large
Ca2+ gradients postulated to exist at the beginning of a
Ca2+ signal at sites of close contact to
Ca2+-release stores and mitochondria (39). In these
so-called microdomains, very high Ca2+ activities of
10
All agonists could induce the biphasic increase of
[Ca2+]m. The time course of the
[Ca2+]m increase, as well as the observed
differences between the cytosolic/nuclear and the mitochondrial
concentration response curves (Figs. 3, 6, and 7), can be very well
explained by the characteristics of the mitochondrial Ca2+
uptake mechanisms. The major uptake mechanism for Ca2+ in
mitochondria is the Ca2+ uniporter (40), which is driven by
the mitochondrial potential. Isolated mitochondria show a biphasic mode
of Ca2+ uptake (38): the first rapid phase has a
KD of approximately 170 nmol/liter and inactivates
within 1 s, and the second mode is activated at a
[Ca2+]i starting > 400 nmol/liter. These
values fit quite nicely with our results (Fig. 8, A and
B) and with described relations of
[Ca2+]i and [Ca2+]m in
cardiac myocytes after electrical stimulation (5).
Physiological Role and Relevance of Changes of Mitochondrial
Ca2+ Activity--
The measurement of cellular UV
light-excited autofluorescence as a measurement of changes in the
mitochondrial NADH levels in correlation with changes of
[Ca2+]i has been used before by a number of
groups, for example in myocytes (41), HeLa cells (39), and hepatocytes
(17). The results of our measurement of autofluorescence (Fig. 10) are in good agreement with the measurements cited above. A number of
mitochondria-specific enzymes, such as the three dehydrogenases for
pyruvate, 2-oxyglutarate, and NAD+-isocitrate (18, 42), are
known to be stimulated by an increased [Ca2+]m.
These enzymes increase ATP production when cytosolic ATP consumption is
increased (40), and this helps the cell to keep pace with the higher
energy demand.
In conclusion, we have shown, 1) using fura-2AM loading, that fura-2
accumulates in the mitochondria of HT29 cells in a
Ca2+-sensitive form; 2) that intramitochondrial fura-2 can
be used to estimate the Ca2+ activity of mitochondria; 3)
that inositol 1,4,5-trisphosphate-coupled agonists (CCH, neurotensin,
and ATP) induce, in addition to their effect on
[Ca2+]c and [Ca2+]n, a
dose-dependent increase in the mitochondrial
Ca2+ signal (the differentiated mitochondrial
Ca2+ response may allow the cells to fine-tune their
energetic response to hormone stimulation); and 4) that mitochondria in
addition might serve as a sink or a modulator for
[Ca2+]c and [Ca2+]n.
We thank K. Zipfel, W. Rohm, M. Northemann,
and G. Kummer for their technical assistance and S. Pfitzinger for the
help with the experimental set-up. We thank Dr. I. Novak for carefully
reading and editing the manuscript.
*
Supported by the Deutsche Forschungsgemeinschaft Grants Gr
480/11-2 and Ni 451/1-1(2).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
FDIM, fluorescence
digital imaging; DiOC(5)(3), 3,3'-dipentyloxacarbocyanine
iodide; [Ca2+]i, intracellular free
Ca2+ activity; [Ca2+]n, nuclear free
Ca2+ activity; [Ca2+]m, mitochondrial
free Ca2+ activity; [Ca2+]c, cytosolic free Ca2+ activity; FCCP, carbonyl cyanide
p-trifluoromethoxyphenylhyrdrazone; CCH, carbachol; DIC, differential interference contrast; OGD, Oregon green BAPTA-1 dextran.
2
S. Ricken, R. Greger, and R. Nitschke,
unpublished data.
Simultaneous Measurements of Cytosolic and Mitochondrial
Ca2+ Transients in HT29 Cells*
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
4
mol/liter) increased cytosolic, nuclear, and mitochondrial
Ca2+ activity ([Ca2+]c,
[Ca2+]n, and [Ca2+]m,
respectively) measured by UV confocal and conventional imaging. Similar
results were obtained with a prototype two-photon microscope (Zeiss,
Jena, Germany) allowing for fura-2 excitation. The increase of
[Ca2+]m lagged behind that of
[Ca2+]c and [Ca2+]n by
10-20 s, and after removing the agonist, [Ca2+]m
also decreased with a delay. A strong increase of [Ca2+]m occurred only when a certain threshold of
[Ca2+]c (around 1 µmol/liter) was exceeded. In
a very similar way, ATP, neurotensin, and thapsigargin increased
[Ca2+]c and [Ca2+]m.
Carbonyl cyanide p-trifluoromethoxyphenylhyrdrazone reversibly reduced the increase of [Ca2+]m. The
source of the mitochondrial Ca2+ increase had intra- and
extracellular components, as revealed by experiments in low
extracellular Ca2+. We conclude that agonist-induced
Ca2+ signals are transduced into mitochondria. 1)
Mitochondria could serve as a Ca2+ sink, 2) mitochondria
could allow the modulation of [Ca2+]c and
[Ca2+]n signals, and 3)
[Ca2+]m may serve as a stimulatory metabolic
signal when a cell is highly stimulated.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
using a
microbeveler (BV10E, Sutter Instruments). Injections were performed by
using a piezo stepper (PMZ 20, Frankenberger, Gilching, Germany) and by
pressure injection of the dye (Injector 5242, Eppendorf, Hamburg, Germany). The injection solution contained 1 mmol/liter of the fluorescent dye.
0.05 was accepted to
indicate statistical significance.
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 1.
A, confocal images of HT29
cells at 364 nm excitation after loading with fura-2/AM at room
temperature. B, same cells as in A at 488 nm
excitation after additional staining with DiOC(5)(3) (1 µmol/liter for 50 s). C, DIC image of the cells shown
in A and B. D, autofluorescence image
of HT29 cells recorded at a high gain setting of the
confocal detector and light intensity 5 times higher than that used for
fura-2 imaging. E, same cells as in D at 488 nm
excitation after additional staining with DiOC(5)(3).
F, DIC image of the cells shown in D and
E.
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Fig. 2.
Spot bleaching of fura-2 fluorescence.
A, bleaching for 0.2 s in the boxed
area in the nucleus resulted in an immediate loss of most of
the cytoplasmic and nuclear fura-2 fluorescence (middle
image), whereas the mitochondrial fluorescence stayed stable for
more than 20-30 min (right image, after 5 min).
B, bleaching for 0.02 s in the marked mitochondrial
area resulted in the permanent loss of the fluorescence in that local
structure (middle image), whereas other surrounding
mitochondria did not loose their fluorescence, indicating noncontinuous
mitochondria in HT29 cells. The right image,
taken 5 min after bleaching, showed no recovery within the bleached
area.
6 mol/liter),
[Ca2+]i rises in the cytosol, as well as in the
nucleus, but only to a minor extent in the area of the mitochondria
(Fig. 3, second image of the second row). However,
increasing the agonist concentration (104 mol/liter) led
to a [Ca2+] response also in the mitochondrial area (Fig.
3, second image of the third row). A similar concentration
dependent [Ca2+]m response was observed for other
inositol 1,4,5-trisphosphate-coupled agonists, such as ATP and
neurotensin, and is examined in more detail under Agonist
Dependence and Time Course of the Mitochondrial Ca2+
Increase.
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Fig. 3.
Localized Ca2+ signals in
HT29 cells. The top row shows two confocal
fluorescence images acquired at 351 and 364 nm excitation and the
corresponding DIC image taken simultaneously with the 364 nm
excitation. The following images are all fura-2 ratio images in which
[Ca2+]i is coded in false color (blue
corresponds to low [Ca2+]i, and green
and red correspond to higher [Ca2+]i).
The middle row shows the increase of the fura-2 ratio after
stimulation with a low concentration of CCH (10 6
mol/liter). [Ca2+]i mostly occurs in the cytosol
and the nucleus and not in the area of mitochondria. The bottom
row shows an increase of [Ca2+]i after a
stronger stimulation with CCH (104 mol/liter). Here also
the mitochondria show a Ca2+ response. Typically, the
mitochondrial area still showed an elevated Ca2+ signal
sometime after the removal of the agonist, as can be seen in the last
image.
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Fig. 4.
Confocal images of a HT29 cell
loaded first with fura-AM and then in addition co-injected with the
Ca2+ dye Oregon green BAPTA-1 dextran 10,000 (OGD). In
A, the fura-2 image after excitation at 364 nm is shown
(emission, >505 nm). Note that the fluorescence signal from the
co-injected cell is much lower compared with the noninjected
neighboring cells. The cell still shows the typical fura-2 distribution
pattern. In B, the OGD image after excitation at 488 nm
excitation is shown (emission, >505 nm). The dark structures in the
image widely overlap with the bright structures (mostly mitochondria)
in the fura-2 image. In addition, the nuclear envelope is clearly
visible as a dark circular structure around the nucleus. As discussed
under "Results," recording of changes of the fura-2 fluorescence in
response to hormone stimulation was not possible due to an interference
of the fura-2 emission signal with OGD. Similar results were obtained
in four other experiments.
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Fig. 5.
Localized measurement of
[Ca2+]i using two-photon excitation of fura-2 at
780 nm. A, two-photon fura-2 fluorescence image. The
marked regions indicate areas were the fluorescence signals was
collected over time, as shown in (B). B,
two-photon excitation of fura-2 at 780 nm is comparable with the
one-photon excitation of fura-2 above the isosbestic point. This means
an increase of [Ca2+]i results in a decrease of
the emission signal. The approximate z resolution in these
measurements was around 0.6 µm, full-width, half-maximum. Stimulation
with neurotensin (NT) led to a reversible decrease of the
fluorescence in the cytosol, the nucleus, and the mitochondria. The
time course of the fluorescence change in the mitochondrial area was
quite similar to the recordings obtained with one-photon confocal or
FDIM measurements. Similar results were obtained in five other
experiments.
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Fig. 6.
A, localized measurement by FDIM of the
Ca2+ activity in three different cellular regions
identified as cytosol, nucleus, and mitochondria. Under control
conditions a maximal stimulation with carbachol (0.1 mmol/liter) led to
an increase in all three cellular areas. The mitochondrial uncoupler
FCCP (0.5-2.5 mol/liter) inhibited partially and reversibly the
increase in the mitochondrial region, whereas the rest of the cell was
not significantly influenced. B, summary of the mean values
for the three compartments from nine experiments.
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Fig. 7.
Partial inhibition of the increase of the
mitochondrial Ca2+ activity [Ca2+]m
after stimulation with CCH by preapplication of FCCP. After
removal of FCCP, the mitochondrial response fully recovered. This FDIM
experiment is representative of four similar experiments.
5 mol/liter) or Fig. 6A). Fig. 8B
summarizes the CCH concentration response curve from 18 paired
experiments. The response of [Ca2+]m was
significantly shifted to the right, with a 50% effect at around 0.3 µmol/liter, whereas the responses for the cytosol and the nucleus
were not significantly different up to a CCH concentration of
105 mol/liter. In Fig. 10A, the relationship
is plotted between [Ca2+]c and
[Ca2+]m from the experiments of the concentration
response curve for CCH. It is obvious that up to a cytosolic 345/380
ratio of around 4-5 only the small, fast changes of
[Ca2+]m occurred. When this threshold was
exceeded, a sudden increase of [Ca2+]m was
measured, and for higher cytosolic ratios, the mitochondrial ratio
followed more directly [Ca2+]c and
[Ca2+]n. Using the fura-2 calibration values for
the cytosol or the nucleus a ratio of 5 corresponds to Ca2+
activities of approximately 1200 nmol/liter.
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Fig. 8.
A, typical FDIM experiment showing the
dose dependent increase of the Ca2+ activity after
stimulation with different CCH concentrations in cellular areas
identified as cytosol, nucleus, and a single mitochondrion.
B, concentration response curve for the CCH-induced increase
in Ca2+ activity in cytosol, nucleus, and
mitochondria.
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Fig. 9.
Typical FDIM experiment showing the effect of
thapsigargin (20 nmol/liter) on the single cell Ca2+
activity in cellular areas identified as cytosol, nucleus, and
mitochondria. A strong increase in the mitochondrial ratio
occurred when the cytosolic ratio reached 4-5. These ratios are
equivalent to a Ca2+ activity of approximately 1200 nmol/liter in the cytosol.
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Fig. 10.
Relationship between the 345/380 nm ratio in
the cytosol and the mitochondria after stimulation with carbachol
(0.5-10 µmol/liter) (A) or thapsigargin (10-500
nmol/liter) (B). Separate linear correlations
can be drawn in both figures for the two data groups from 0.5 to 4 and
from 5 to 9. The switching point between the two data groups indicates
the starting value for the fast [Ca2+]m
increase. This again corresponds to a Ca2+ activity of
around 1200 nmol/liter in the cytosol.
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Fig. 11.
Original FDIM recording showing the effect
of neurotensin (1 nmol/liter) on [Ca2+]c,
[Ca2+]n, and [Ca2+]m in the
absence of extracellular Ca2+. The exclusive
release of Ca2+ from intracellular stores is
sufficient to increase [Ca2+]i in all areas,
including [Ca2+]m. The initial time course of
[Ca2+]m is comparable to the control conditions.
Similar results were obtained in four other experiments.
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Fig. 12.
Removal of extracellular Ca2+ (5 mmol/liter EGTA) led to decrease of the Ca2+ activity in
the whole cell. The ratio signal from areas inside the
nucleus and a single mitochondrion is shown. Generally, the traces had
a similar shape, but there was a difference in time courses and
absolute values. The mitochondrial Ca2+ signal
decreased more slowly in all experiments when Ca2+ was
removed and increased again more slowly after readmission of
extracellular Ca2+. The Ca2+ activity increased
to nearly pre-control values after readmission of extracellular
Ca2+ (n = 11; data not shown).
4 mol/liter), which will result in a
strong increase of [Ca2+]m as well as
[Ca2+]c and [Ca2+]n, also
led to a significant increase in NAD(P)H, as indicated by the increase
of autofluorescence.
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Fig. 13.
Microspectrofluorometric measurement of the
autofluorescence from a single cell at 340 nm excitation is an
indicator of the cellular NADH and NADPH level. The
fluorescence emission signal above 430 nm was measured in approximately
15-20 cells. When the cells were treated with NaN3
and KCN (both 5 mmol/liter) or in response to a strong stimulation with
a mixture of ATP and CCH (both 0.1 mmol/liter) the autofluorescence
increased, indicating that NAD decreased. The experiment is
representative of four other experiments.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
5 mol/liter after ionomycin
application, which results in a drop of [Ca2+]i
to or below the resting level of the cell (data not shown). From the
in vitro fura-2 calibration curve, this is unexpected, as
the fura-2 signal still should be nearly saturated at 105
mol/liter Ca2+. However, taking into the account the
described Ca2+ sensitivity of the capacitative
Ca2+ entry mechanism in HT29 cells (22, 37),
our data fit very well with the concept of an ionomycin-induced opening
of the capacitative Ca2+ entry-influx pathway (35). Raising
the ionomycin concentration to also enhance mitochondrial
Ca2+ permeability resulted in a fast loss of the dye from
the whole cell, including all cellular compartments. Other
permeabilizing nonspecific agents, such as Triton and digitonin, gave
similar results (data not shown; n = 5). Therefore, a
direct permeabilization of mitochondria for Ca2+ was not
possible in intact HT29 cells. Even the calibration of the
cytosolic and the nuclear fura-2 signal with ionomycin has to be
questioned in future studies, despite the fact that it is a well
accepted and frequently employed method. From our spectroscopic measurements and the results of others (6, 8, 33), we propose that the
fura-2 properties inside mitochondria are comparable to the cytosolic
form. In addition, the results of our measurements could hardly be
explained alone on the basis of changed fura-2 properties inside
mitochondria. Due to the problems with an adequate calibration of
[Ca2+]m, we preferred to use, throughout this
report, ratio values for [Ca2+]m, except for some
mean values, where we used the ionomycin calibration values from the
cytosol despite the observed limitations of the procedure.
4 mol/liter were postulated.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.:
49-761-2035195; Fax: 49-761-2035191; E-mail:
nitschro{at}sun2.ruf.uni-freiburg.de.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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