(Received for publication, July 28, 1995; and in revised form, September 13, 1995)
From the
A controversy in the field of bioenergetics has been whether
mitochondria are capable of sequestering enough Ca from cytosolic Ca
pulses to raise their
intramitochondrial free Ca
level
([Ca
]
). This is
significant because an increase in
[Ca
]
has been linked
to an increase in cellular metabolic rate through various mechanisms.
To resolve this question, we exposed isolated liver mitochondria to
physiological type pulses of Ca
produced using a
pulse-generating system (Sparagna, G. C., Gunter, K. K., and Gunter, T.
E.(1994) Anal. Biochem. 219, 96-103). We then measured
the resulting mitochondrial Ca
uptake. The uniporter
was previously thought to be the only specific Ca
uptake mechanism in mitochondria. Our studies have uncovered an
additional uptake mechanism, the rapid mode of uptake or RaM,
which functions at the beginning of each pulse and allows mitochondria
to sequester a considerable amount of Ca
from short
pulses. We have shown that the RaM is reset by decreasing the
[Ca
] between pulses for a very short time,
making this uptake mode ideally suited for Ca
sequestration from Ca
pulse sequences. With
rapid Ca
uptake occurring at the beginning of each
pulse, liver mitochondria may be able to sequester sufficient
Ca
from a short sequence of pulses to activate the
cellular metabolic rate.
It has been suggested that the intramitochondrial free calcium
concentration ([Ca]
)
can function as a metabolic mediator, acting at several loci to control
the cellular metabolic
rate(1, 2, 3, 4) . Key steps within
several important metabolic processes are activated by
[Ca
]
. At least two
dehydrogenases important in the control of the Krebs cycle, pyruvate
dehydrogenase and
-ketoglutarate dehydrogenase, can be controlled
by [Ca
]
changes of a
few nanomoles per milligram of protein (1, 5) (equivalent to
[Ca
]
values of a
micromolar or less). A third dehydrogenase, isocitrate dehydrogenase,
while also activated by
[Ca
]
, requires a
matrix [Ca
]
above 3
µM for significant activation and therefore may or may not
be involved in physiological responses(6) .
The activities
of other important metabolic steps have also been suggested to be
controlled by matrix
[Ca]
. Halestrap and
Griffiths (7) have suggested that the rate of electron
transport in liver mitochondria is enhanced through a
Ca
-induced swelling process mediated by accumulation
of pyrophosphate. Both the F
ATPase (8, 9) and the adenine nucleotide translocase (10, 11, 12, 13) have also been
identified as sites for Ca
modulation of metabolic
rate.
The primary controversy has been whether or not mitochondria
are capable of sequestering a sufficient amount of Ca from physiological pulses to mediate these
Ca
-activated metabolic steps. Most of the results of
electron probe microanalysis studies used in conjunction with rapid
freezing of the tissue have suggested that little if any Ca
is sequestered by mitochondria from physiological
pulses(14, 15, 16, 17, 18) .
Extrapolation of existing data (19) on Ca
uptake by mitochondria to the short times relevant to rapid
pulses of Ca
has also suggested that mitochondrial
uptake of Ca
from these pulses would probably be
insufficient for [Ca
]
to function as an intramitochondrial metabolic mediator.
Conversely, other experiments have suggested that significant
Ca may in fact be sequestered by mitochondria from
physiological pulses. One of these experiments involves electron probe
microanalysis(20) , and the two described below utilize
fluorescence techniques. In the first set of experiments, increases in
intramitochondrial [Ca
]
were measured following an increase in the frequency of
Ca
pulses in cardiac myocytes using the fura-2
fluorescence technique by using Mn
to selectively
quench the fluorescence of cytosolic fura-2(21) . In the second
set of experiments, Rizzuto et al.(22, 23, 24) fused the
Ca
-sensitive photoprotein aequorin in frame with a
mitochondrially directed leader sequence from subunit VIII of human
cytochrome oxidase. This hybrid DNA was transfected into bovine
endothelial cells, obtaining stable clones that express variable
amounts of intramitochondrial aequorin. Activation of these cells with
external ATP caused an increase in the cytosolic free calcium
concentration ([Ca
]
)
(measured using fura-2 techniques) from 100 to 500 nM and in
[Ca
]
(measured using
aequorin luminescence) from around 200 nM to over 5
µM. This observed mitochondrial response was very rapid
and returned to base line in about 10 s. It was also drastically
reduced by exposure to the uncoupler FCCP, which, by dissipating the
electrochemical proton gradient,
µ
, would greatly
reduce the driving force for mitochondrial Ca
accumulation via the uniporter.
With few
exceptions(25, 26) , studies of Ca
uptake via the mitochondrial uniporter have at best utilized steady
buffered levels of free calcium ([Ca
]) (see (19) and (27) ), while in many cell types the exposure
of the mitochondria within a cell to significant
[Ca
] occurs because of cytosolic
Ca
pulses. In view of the controversy as to
whether mitochondria can actually sequester significant Ca
from physiological pulses, it would seem appropriate to
reinvestigate Ca
sequestration by isolated
mitochondria, focusing on uptake of Ca
from pulses.
The use of isolated mitochondria would greatly increase the accuracy
with which this uptake data could be obtained. Furthermore, on the
basis of extrapolations of data in the existing literature, it is
difficult to understand how the
[Ca
]
levels could
increase so rapidly upon exposure to a pulse of cytosolic
[Ca
]
as was observed
by Rizzuto et al.(22, 23, 24) .
We have set up an apparatus that allows us to create and measure
Ca pulses in a cylindrical cuvette similar to those
observed in vivo in many types of tissue. At the same time,
this apparatus permits us to measure mitochondrial Ca
uptake accurately using a dual label isotope technique. A
detailed description of this apparatus and of the results of
preliminary experiments studying Ca
sequestration by
mitochondria has been published(26) . We report here the
results of a more extensive study of sequestration of Ca
from pulses by isolated liver mitochondria. Our studies indicate
that mitochondria can sequester Ca
very rapidly for a
short period of time via a process unsuspected prior to the initiation
of these studies(26) , the rapid mode of Ca
uptake (RaM). (
)
Rat liver mitochondria were prepared as described in Sparagna et al.(26) and Wingrove and Gunter (28) using
Sprague-Dawley specific pathogen-free rats weighing 180-200 g.
Experiments were carried out in either medium A or B. Medium A
contained (in mM) 150 KCl, 24 potassium-HEPES, 5 potassium
succinate, 0.1 sucrose, and 0.1 potassium P at pH 7.2.
Medium B was medium A plus 3 µM fura-2 FA (Texas
Fluorescence Labs). Where appropriate, components of these solutions at
high concentration were passed through a Chelex-100 column to remove
divalent cations. Ca
-HEDTA and EGTA solutions were
made using separate standard solutions (in mM) of 100
CaCl
, 500 EGTA, and 100 HEDTA diluted with medium A. The
standard solutions had been titrated as described (29) to check
the accuracy of their concentrations. The Ca
-HEDTA
solution contained 3.05 mM
CaCl
(20
nCi/ml) and 20 mM HEDTA at pH 7.2. The EGTA solution contained
either 2.5 or 100 mM EGTA (see figure captions) at pH 7.2. The K
of fura-2 FA was determined to be 275
nM(29) . A dual label technique was used to determine
mitochondrial Ca
uptake as described (30) .
In addition to the
Ca
in the
Ca
-HEDTA solution, a [
H]sucrose
solution was used for uptake studies containing 1 µCi/ml
[
H]sucrose (DuPont NEN) in 70% ethanol diluted
with double distilled water to a specific activity of 0.25 µCi/ml.
All solutions used are Na
-free. For each experiment, 3
ml of medium B, 0.5 mg/ml isolated mitochondria, and 8 µl of
[
H]sucrose were used.
The pulse-generating and monitoring system used contains a dual syringe automatic pipettor (Microlab 940, Hamilton) and an MS-III fluorimeter (Photon Technology International) controlled by IBM-compatible 286 and 486 computers, respectively. The details of the experimental set up and characteristics of the pulse apparatus are as described previously (26) .
Fig. 1is a diagram of a sequence of two
Ca pulses showing the parameters that were varied in
our experiments. The pulses we generated are trapezoidal-shaped, and
the width (w) is therefore taken as the width at halfway
between the initial Ca
level before the pulse (h
for the first pulse, h
for
the second) and the height of the pulse (h
). The
interpulse period (i) is also measured at halfway between
these two Ca
levels.
Figure 1:
Schematic diagram of our Ca pulses. The letters in the diagram represent the following: w, pulse width; i, interpulse period; h
, initial
[Ca
] level of the medium before the pulse; h
, [Ca
]
height of the pulse; h
,
[Ca
] level during the interpulse
period.
Fig. 2is a graph
showing Ca uptake from a single calcium pulse as the
width (w) is varied. Each curve represents a series of single
pulses of a single height (h
). For clarity, curves
having only three different heights and one to which ruthenium red (a
potent inhibitor of the uniporter) was added are shown, but we have
carried out this experiment with pulses of many heights (data not
shown). All pulses have an initial [Ca
] (h
) of between 30 and 60 nM and an ending
[Ca
] of less than 10 nM.
Figure 2:
Mitochondrial rapid and slower
Ca uptake. Mitochondria were exposed to single
Ca
pulses with widths between 1 and 10 s, and the
resulting amount of Ca
uptake was determined for
pulses of various heights. All pulses were made by adding 2.5 mM EGTA and the Ca
-HEDTA solution as defined under
``Materials and Methods.'' Mitochondria were suspended at 0.5
mg of protein/ml in 3 ml of medium B, to which 33 µM EGTA
(final concentration) had been added before pulses were made. The (closed circles) data represent pulses with an average height
of 484 ± 38 nM [Ca
], which
were made using 17 µl of Ca
-HEDTA and 100 µl
EGTA. The (closed triangles) data represent pulses with an
average height of 281 ± 15 nM [Ca
], which were made using 10 µl
of Ca
-HEDTA and 60 µl of EGTA. The (closed
squares) data represent pulses with an average height of 171
± 5 nM [Ca
], which were
made using 5 µl of Ca
-HEDTA and 40 µl of
EGTA. In the control pulses (
), ruthenium red (4
nmol/mg of mitochondrial protein) was added before the pulses were
made. The control pulses had an average height of 329 ± 64
nM [Ca
] and were made using 10
µl of Ca
-HEDTA and 60 µl of EGTA. Error
bars represent one standard deviation of three
repetitions.
Clearly, a mitochondrial suspension that is exposed to a
Ca pulse for zero time can sequester no
Ca
from that pulse. Therefore, the observation that
an extrapolation of all uptake curves of the type shown in Fig. 2invariably intercepts the zero time line at a value of
sequestered Ca
significantly above zero must be
interpreted as an indication that a brief period of very rapid
Ca
uptake preceded the earliest time point
represented. Uptake during this period is described as being a result
of the high conductivity or rapid mode (RaM) of mitochondrial
Ca
uptake. The slower uptake, whose rate is
proportional to the slope of the lines fit to each set of data in Fig. 2, is referred to as the lower conductivity or uniporter
mode. The rates of uptake observed for the lower conductivity mode are
consistent with those reported earlier as Ca
sequestration via the uniporter(19, 31) . The
amount of rapid uptake in mitochondria seems to be relatively
independent of pulse height (h
) in pulses with
heights above 500 nM [Ca
] (data
not shown), whereas the amount of slower uptake increases rapidly as
pulse heights increase at pulse heights above 400 nM
[Ca
].
To determine precisely how ruthenium
red effects both the rapid (RaM) and slower modes of mitochondrial
Ca uptake, we performed titration studies using this
inhibitor. Fig. 3shows the percent inhibition for the RaM (closed circles) and slower uptake (open circles)
with increasing ruthenium red concentration. To determine the ruthenium
red inhibition of the RaM and slower uptake, we calculated the y intercept (RaM uptake) and slope (slower uptake) from curves like
those shown in Fig. 2with pulses having an average height (h
) of 534 nM for a series of ruthenium red
concentrations. The percent inhibition was based on Ca
uptake with no ruthenium red added. From Fig. 3, we can
clearly see that at a concentration of 0.1 nmol of ruthenium red/mg of
mitochondrial protein, the slower uptake is inhibited, whereas the RaM
is not. At concentrations below 0.1 nmol/mg of mitochondrial protein,
we consistently find that ruthenium red activates Ca
uptake via the RaM. At concentrations below 0.003 nmol/mg of
protein, it may also activate the slower mode of Ca
uptake, but that is less certain. What is clear from this data is
that at higher concentrations, ruthenium red inhibits both modes of
mitochondrial Ca
uptake and that the amount of
ruthenium red necessary to inhibit the slower uptake is over an order
of magnitude less than that required to inhibit the RaM.
Figure 3:
Ruthenium red titration curve. Ruthenium
red of various concentrations was added to 3 ml of medium B plus 33
µM EGTA (final concentration) in the cuvette. Mitochondria
at a concentration of 0.5 mg of protein/ml were then added, and pulses
were made. Curves similar to those shown in Fig. 2having an
average height of 534 ± 31 nM
[Ca] were made for each ruthenium red
concentration. The slope and y intercept,
corresponding to slower and rapid mitochondrial uptake, respectively,
were determined using linear regression, and the percent inhibition for
rapid uptake (closed circles) and slower uptake (open
circles) was calculated using the curve with 0 nmol of ruthenium
red/mg of mitochondrial protein as a standard for 0% inhibition. All
pulses were made by adding 100 mM EGTA and the
Ca
-HEDTA solution as defined under ``Materials
and Methods.'' The pulses were made using 10 µl of
Ca
-HEDTA and 12 µl of EGTA. Error bars represent 95% confidence limits on the slope and y intercept converted to percentages.
Cyclosporin
A has been shown to inhibit the mitochondrial permeability
transition(32, 33) . To rule out the possibility that
calcium uptake during the RaM is related to the mitochondrial
permeability transition, we added 1 µM cyclosporin A to
our medium before making the pulses. The Ca uptake
resulting from these pulses was identical to uptake from pulses made
without cyclosporin A (data not shown).
The first experiment we performed was to
expose the mitochondria to two 5-s-wide Ca pulses
under conditions in which the [Ca
] between
pulses (h
) was approximately 100 nM and
the time between the pulses (i) was varied from 0 to 10 s. The
zero time point was actually a 10-s-wide pulse where both the
Ca
and the EGTA used to create other pulses in this
set were added simultaneously after 5 s so as to produce a pulse whose
total Ca
and EGTA concentrations were identical to
that experienced by the mitochondria in other experiments in this set.
We also made single 5-s-wide pulses, which were the same height as the
double pulses. The Ca
uptake during the second pulse
was then determined by subtracting the Ca
uptake due
to a single pulse only (the first pulse) from the uptake resulting from
both pulses. The results can be seen in Fig. 4where the closed circles represent the uptake from the second pulse
only. We can see from Fig. 4that the uptake from the second
5-s-wide pulse when the two pulses are separated by as little as 0.75 s
is twice as great as the uptake from the last 5 s of a single 10-s-wide
pulse. This indicates that the RaM is occurring at the beginning of the
second pulse as well as the first.
Figure 4:
Variation of the time between pulses. Two
pulses of average heights 435 ± 15 and 421 ± 15 nM [Ca], respectively, and widths of 5 s
were made, and the interpulse period was varied from 0.75 to 10 s. The
control pulse (interpulse period = 0 s) was a 10-s-wide pulse
where a mixture of buffered Ca
and EGTA was added in
the middle of the pulse to make the total additions identical to those
of the separated pulses. Mitochondria were suspended at 0.5 mg of
protein/ml in 3 ml of medium B. All pulses were made by adding 2.5
mM EGTA and the Ca
-HEDTA solution as defined
under ``Materials and Methods.'' The (open squares)
data represent uptake from both pulses made with 15 µl of EGTA, 9
µl of Ca
-HEDTA, 35 µl of EGTA, 23.5 µl of
Ca
-HEDTA, and 70 µl of EGTA. The (open
triangles) data represent uptake from one pulse made with 15
µl of EGTA, 9 µl of Ca
-HEDTA, and 35 µl
of EGTA, and the (closed circles) data represent uptake from
the second pulse only by subtracting the uptake from one pulse from the
uptake from both pulses. Error bars represent one standard
deviation of four repetitions.
We next set out to determine the
minimum level that the [Ca] between pulses
must be dropped to reset the RaM. To do this, the
[Ca
] level (h
) between
two 10-s-wide pulses separated by an interpulse period (i) of
1 s was varied over the range from 118 nM up to the 402
nM height of the pulses. This is shown in Fig. 5where
the x axis indicates the amount that the Ca
level between pulses was lowered from the pulse height
of 402 nM.
Figure 5:
Variation of the
[Ca] level between pulses. Two pulses of
average heights 397 ± 18 and 408 ± 24 nM
[Ca
], respectively, and widths of 10 s were
made, and the interpulse period was fixed at 1 s. The interpulse
[Ca
] was varied between 118 and 407
nM. Mitochondria were suspended at 0.5 mg of protein/ml in 3
ml of medium B. All pulses were made by adding 2.5 mM EGTA and
the Ca
-HEDTA solution as defined under
``Materials and Methods.'' The (open triangles) data
represent uptake from both pulses made with 15 µl of EGTA, 9 µl
of Ca
-HEDTA, 0.5-25 µl of EGTA, 1-18
µl of Ca
-HEDTA, and 25-60 µl of EGTA.
The (open squares) data represent uptake from either one
10-s-wide or one 11-s-wide pulse made with 15 µl of EGTA, 9 µl
of Ca
-HEDTA, and 30 µl of EGTA. The (closed
circles) data represent uptake from the second pulse only by
subtracting the uptake from one pulse from the uptake from both pulses. Error bars represent one standard deviation of five
repetitions.
Fig. 5, again, shows the amount of
calcium uptake from the second pulse only (closed circles) by
subtracting the uptake of one pulse from the uptake from both pulses.
If the [Ca] between pulses is not lowered
and the pulse is, in effect, a 21-s-wide pulse instead of two 10-s-wide
pulses separated by 1 s, the Ca
uptake from the
second pulse is due to uptake during the lower conductivity mode only
and is therefore less than uptake from the second pulse when h
is lowered by 200 nM or more.
Therefore, decreasing the interpulse [Ca
]
level (h
) by more than 200 nM appears to
completely reset the high conductivity phase of Ca
uptake (RaM) for a 400 nM high Ca
pulse.
These results clearly show that the RaM is reset very
rapidly (within a time of less than 0.75 s) when the
[Ca] falls to a level of less than 200
nM between pulses for pulses with heights of 400 nM.
It appears then that the high conductivity phase can easily be reset
under conditions similar to those encountered physiologically during
the period between Ca
pulses.
Figure 6:
Uptake from steady state versus pulsed [Ca]. Uptake from pulses with
widths of 5-25 s and heights of 412 ± 22 nM
[Ca
] (open circles) are compared
with uptake from one to five 5-s-wide pulses with an interpulse period
of 2 s and average heights of 437 ± 35 nM [Ca
] (closed triangles).
Mitochondria were suspended at 0.5 mg of protein/ml in 3 ml of medium
B, to which 33 µM EGTA (final concentration) had been
added before pulses were made. All pulses were made by adding 2.5
mM EGTA and the Ca
-HEDTA solution as defined
under ``Materials and Methods.'' The single (steady state)
pulses were made with 2.5 µl of Ca
-HEDTA and 7
µl of EGTA. The multiple pulses were made with all or part of 2.5
µl of Ca
-HEDTA, 7 µl of EGTA, 2.5 µl of
Ca
-HEDTA, 9 µl of EGTA, 3 µl of
Ca
-HEDTA, 14 µl of EGTA, 4 µl of
Ca
-HEDTA, 20 µl of EGTA, 8 µl of
Ca
-HEDTA, and 28 µl of EGTA. Error bars represent one standard deviation of three
repetitions.
The uptake observed during the continual exposure (open
circles) corresponds to high conductivity plus low conductivity
contributions for one pulse period followed by an addition of one low
conductivity contribution for each succeeding 5-s pulse period. The
uptake observed from the multiple pulse exposure (closed
triangles), on the other hand, corresponds to both a high
conductivity plus a low conductivity contribution from each 5-s pulse
encountered, resulting in more total calcium uptake from separate
pulses than from a prolonged exposure to an elevated Ca level.
The uniporter is known
to be activated by spermine, which is present in many types of cells at
a concentration of around 1 mM. Fig. 7shows the effect
of spermine concentrations ranging from 100 nM to 1 mM on both the RaM and slower uptake mode. At a concentration of 0.1
mM and higher, spermine appears to increase the RaM by
approximately a factor of 6 and the lower conductivity uptake by about
a factor of 2. The great increase in Ca uptake during
even a single pulse, mediated by this endogenous activating agent,
brings the levels of intramitochondrial
[Ca
] into the range in which it could
significantly increase the metabolic rate as considered in the
Introduction.
Figure 7:
Spermine titration. Various concentrations
of spermine ranging from 100 nM to 1 mM were added to
3 ml of medium B plus 33 µM EGTA (final concentration) in
the cuvette. Mitochondria at a concentration of 0.5 mg of protein/ml
were then added, and pulses were made. Curves of Ca
uptake versus pulse width (not shown) were made for each
spermine concentration, and the slope and y intercept
for each curve was calculated using linear regression. The y intercept (left axis) corresponding to rapid
mitochondrial uptake is shown with the closed circles, and the slope (right axis) corresponding to slower
mitochondrial uptake is shown with open circles. All pulses
were made by adding 2.5 mM EGTA and the
Ca
-HEDTA solution as defined under ``Materials
and Methods.'' Pulses had heights of 925 ± 14 nM
[Ca
] and were made using 5 µl of
Ca
-HEDTA and 25 µl of EGTA. Error bars represent 95% confidence limits.
Uptake during RaM was identified as net uptake as opposed to
either external binding or rapid exchange of labeled external
Ca for unlabeled intramitochondrial Ca
in a series of experiments described earlier (26) .
External binding was ruled out by 1) showing that the addition of small
amounts of the inhibitor ruthenium red added prior to the
Ca
pulse eliminated this uptake and 2) showing that
ruthenium red added subsequent to the Ca
pulse had no
effect on the uptake and that, furthermore, adding large amounts of
external unlabeled Ca
could not compete away the
Ca
previously taken up by the mitochondria. Rapid
exchange was ruled out by 1) showing that depletion of endogenous
unlabeled Ca
, using the procedure of Wingrove and
Gunter(28) , prior to exposure of the mitochondria to the
Ca
pulse and measurement of Ca
uptake had no effect on the magnitude of Ca
uptake and 2) showing that exchanging endogenous unlabeled
Ca
for labeled Ca
followed by
depleting the mitochondria of internal Ca
as
described (28) had no effect on the magnitude of Ca
uptake. In this latter case, the amount of labeled Ca
present subsequent to the Ca
depletion
procedure and prior to the uptake experiment (approximately 1-2
nmol/mg of mitochondrial protein) was measured and subtracted from the
uptake of labeled Ca
during the pulse to obtain net
uptake.
The observation that ruthenium red may activate
Ca uptake at lower concentrations and inhibit at
higher concentrations suggests that there may be two ruthenium red
binding sites involved. Binding of ruthenium red to the higher affinity
site would lead to activation of uptake, while binding of ruthenium red
to the lower affinity site would lead to inhibition. The similarity of
the effects of ruthenium red on the behavior of the RaM and the slower
mechanism despite its lower affinity for inhibition of the RaM and the
similarity of the effects of spermine on the behavior of these two
modes of transport suggest, but do not prove, that both these modes may
be mediated by the same transport complex. If this is true, then it is
possible that the binding of a Ca
ion to the
cytosolic side of the RaM complex induces a conformational change,
resulting in the slower mode of transport. This view is consistent with
the data of Fig. 4and Fig. 5, which show that RaM
transport can be reset by simply lowering the
[Ca
].
The observation that the RaM is
reset by simply dropping the [Ca] into the
range usually observed after or between pulses for a very short period
(<0.75 s) strongly suggests that this high conductivity uptake may
have physiological relevance since it implies that rapid Ca
uptake could occur at the beginning of each pulse. Even though
our efforts to produce pulses short enough to allow us to measure the
duration of the RaM were unsuccessful, data such as that shown in Fig. 6are reassuring. This is because these results demonstrate
that even if our techniques are not fast enough to permit us to
directly measure conductivity during the high conductivity mode, this
type of uptake is real and reproducible, and it occurs at the
initiation of each pulse as the data of Fig. 2, Fig. 4,
and Fig. 5suggest that it should. The data of Fig. 7demonstrate that the amount of uptake through this
mechanism can be sufficient for very significant activation of the
Ca
-activated metabolic reactions in the mitochondrial
matrix and is consistent with the high levels of
[Ca
]
observed following
physiological activation of Ca
pulses by Rizzuto et al.(22, 23, 24) . The current
data along with considerable data already in the literature (see (4) for a review) provide very strong support for the
hypothesis that [Ca
]
can
function as a metabolic mediator and probably is a primary mechanism
controlling the metabolism of some cells.
As shown in Fig. 7,
the maximum amount of transport observed via the high conductivity mode
during only a single pulse can be between 1 and 2 nmol of
Ca/mg of mitochondrial protein and in the range where
Ca
modulation of the Krebs cycle
Ca
-sensitive dehydrogenases, pyruvate dehydrogenase
and
-ketoglutarate dehydrogenase, and other
Ca
-sensitive metabolic processes occurs(4) .
The observations described above clearly possess the power to reconcile
recent observations describing relatively high levels of
[Ca
]
following Ca
pulses in cells (21, 22, 23, 24) and older
measurements of Ca
sequestration by isolated
mitochondria using buffered
Ca
(4, 19, 27) .
We have
found that most of the Ca sequestered into liver
mitochondria from pulses with heights below 400 nM or from
narrow pulses like those seen in the cytosol of heart cells is taken up
via the RaM. In many cell types, pulse intensities fall within a
limited range(4) . It is possible that, under some conditions,
the total amount of Ca
sequestered within a given
period and consequently the level of metabolic stimulation could depend
more on the pulse frequency than on pulse intensity. In other words,
metabolic signaling to the mitochondria could show some of the
properties of frequency modulation. Mitochondria would be expected to
sequester Ca
more effectively from a pulse that
reaches its peak very rapidly than one that may reach the same level
more slowly, since the duration of the RaM is clearly short. This may
explain the very rapid mitochondrial [Ca
]
uptake observed by Rizzuto et al.(23) . In some
respects the possible frequency-modulated behavior of mitochondrial
Ca
uptake seems akin to the relationship between the
strength of a stimulus and the frequency of action potential spikes, i.e. the stronger the signal, the higher the frequency of the
action potential and the stronger the response.
The reason why the
system might function in this way could be simple. The buffering power
of the cytosol for Ca is not constant as a function
of [Ca
]
but increases
significantly as [Ca
]
increases
above 1 µM. This is because there are more low affinity
than high affinity Ca
binding sites present in the
cytosol; consequently, it requires much more than twice the
Ca
to produce a 1 µM Ca
pulse than one of 500 nM. The energetic costs of ion
pumping are not negligible. It surely requires less energy to produce
two 500 nM Ca
pulses than one 1 µM pulse; it may require less to produce even three or four 500
nM pulses. With a significant fraction of the uptake during a
500 nM pulse being from the RaM, the uptake from two narrow
500 nM pulses could be as large as the uptake from one 1
µM pulse. The uptake from three or four 500 nM pulses would probably be significantly greater. Furthermore,
increasing the strength of the stimulus by increasing the pulse
frequency rather than pulse height has the additional advantage of
causing a smaller perturbation on the mechanisms maintaining
Ca
homeostasis within the cell by allowing them to
function over a smaller range of
[Ca
]
.
Interestingly, if one
considers the types of Ca pulses observed in various
types of cells, the primary variation is in pulse duration, number of
pulses in a sequence, and pulse frequency but not in pulse
intensity(4) . For example, in considering pulses in cells that
have a high relative metabolic rate, such as cardiac myocytes, cells
that have an intermediate metabolic rate, such as hepatocytes, and
cells that have a low metabolic rate, such as chondrocytes(4) ,
the Ca
pulse intensity varies by only a
factor of two from around 600 nM to above 1 µM.
On the other hand, the pulse duration varies by a factor of
200, from less than 0.5 s to around 2 min, the number of pulses in
a sequence varies from an indeterminately large number with
cardiac myocytes to one with chondrocytes, and the pulse frequency varies from around 1 Hz with cardiac myocytes to near zero with
chondrocytes. The pulse intensities observed physiologically in the
cytosol of various types of cells (approximately 1 µM) are
large enough to be unambiguously interpreted as ``pulse on''
against a background (approximately 100 nM), which is
``pulse off'' while not large enough to pose a significant
danger to the cell due to activation of proteases etc. Ca
signaling at the cellular level could well have evolved toward a
``frequency modulated'' mode of action both for reasons of
economy and safety. With the inclusion of the rapid uptake mode,
described here, the mitochondrial Ca
transport system
seems well adapted to utilize this Ca
signaling
system.
It is clearly important to determine the duration of the RaM
and to determine whether activation such as that by spermine represents
an increase in the conductivity of the mechanism or an increase in the
duration of the high conductivity mode or both. We have been unable to
resolve this problem with the shortest Ca pulses,
which we have produced to date with our pulse-generating system.
Further experiments have been planned to address this question.
Particularly, if it should turn out that the RaM is a separate
functional mode of the uniporter, this would represent a very unusual
transporter. The slower conductivity mode of the uniporter has been
recognized as a fast mechanism in its own right(31) . The high
conductivity mode is considerably faster, particularly at low
[Ca]. One possibility is that the gate of a
gated pore moves out of the transport path as the high conductivity
mode is reset, leaving a higher conductivity channel open during RaM
uptake. While this view is currently very speculative, this is clearly
both an interesting and functionally important mechanism.