 |
INTRODUCTION |
The maintenance of a steady state energy metabolism, by the
appropriate orchestration of mitochondrial energy conversion with work,
is a common process in many cells (1-4). The cytosolic transduction
systems involved in this balance of work with mitochondrial oxidative
phosphorylation has been proposed to involve at least four putative
cytosolic signaling systems. These include cytosolic concentrations of
ADP and Pi (5), creatine (6-9), and/or Ca2+
(10-15). The ADP and Pi levels have been proposed to serve
as cytosolic signals to the mitochondria, changing in proportion to
work (i.e. ATPase activity). Therefore, ADP and
Pi, the key substrates for ATP production in the
mitochondria, are believed to control ATP production by limiting the
substrate for the synthesis reaction (5). Creatine, another putative
cytosolic feedback metabolite related to the free ADP levels, is
generated by the rapid cytosolic ATPase and creatine kinase reactions
(6-9, 16-23) in the cytosol. The localization of creatine
phosphokinase (CPK)1 at the
myofilaments and the inner mitochondria space to generate creatine in
the cytosol from ADP and re-derive ADP in the inner membrane space
provided a logical "facilitated diffusion" of ADP to the
mitochondrion. Due to its higher concentration, creatine (~10
mM) in cytosol (24, 25) is thought to generate a higher "net" diffusional flux when compared with micromolar [ADP] in cytosol. This creatine kinase shuttle has been proposed in various forms over the years (for review see Ref. 26). Finally,
Ca2+ has also been proposed as another transducer between
cytosolic work and mitochondrial metabolism; however, the mechanism of
this activation is thought to occur differently than the proposed
feedback models for ADP, Pi, and creatine. Work at the
myofilaments and ion transport by the sarcoplasmic reticulum and
sarcolemmal membrane pumps are thought to be stimulated by
Ca2+ in parallel with mitochondrial ATP production. The
activation of the contractile apparatus of the heart by
Ca2+ has been well characterized (for review see Ref. 27)
as well as its effects on Ca2+ ion transport (for review
see Ref. 28). In the mitochondria, Ca2+ has long been
suggested to modulate Ca2+-sensitive dehydrogenase (10, 11)
as well as F0F1-ATPase activity (13-15). This
parallel activation scheme relies on Ca2+ having rapid, as
well as similar, kinetics for modulating work and mitochondrial metabolism.
In the intact cell it is likely that all of these regulatory processes
may be playing some role in the energy homeostasis. The relative
importance of these different pathways might be related to the relative
speed at which metabolism can respond to these cytosolic signals that
could conceivably be changing simultaneously in the cell. Despite the
fact that these models of mitochondrial regulation have been in the
literature for many years, there is very little information on the
rapid kinetics of these putative signaling molecules on oxidative
phosphorylation. The purpose of this study was to determine the rapid
kinetics of heart mitochondria to extramitochondrial concentrations of
these putative cytosolic signaling markers. This was accomplished using
isolated porcine heart mitochondria and rapid optical techniques to
follow NADH, light scattering (volume), and oxygen consumption in
response to step increases in these potential cytosolic signaling molecules.
 |
MATERIALS AND METHODS |
Mitochondria Isolation--
Heart mitochondria were isolated
from anesthetized pig according to Ref. 15 and were within the
guidelines listed in the Animal Care and Welfare Act (7 United
States Congress 2142, section 13). Buffers use in the isolation,
washing, and experimentation are presented in Table I. The mitochondria
were isolated and washed in solution A, and the final preparation was
suspended in solution B. To ensure that chloride, at the concentrations used in buffer B (Table I), did not detach creatine kinase from the
inner mitochondrial membrane (29), mitochondria were in some cases
resuspended in buffer A (Table I).
Cytochrome aa3 Determination--
Cytochrome
aa3 (Cyta) content was determined
spectrophotometrically as described previously (30), using a molar
extinction coefficient of 12 mM
1·cm
1.
On average the yield was 400.6 ± 32.1 nmol
Cyta·heart
1 (n = 31).
Fluorescence Dye Loading--
Approximately 90% of the
resuspended mitochondria were loaded with
5-(6)-carboxy-2'-7'-dichlorofluorescein diacetate, succinimidyl ester
(CF; Molecular Probes, Eugene OR) at 1 nmol of CF·nmol of Cyta
1 to provide an optical
correction for primary and secondary optical filters in the sample
(31).
To monitor mitochondrial matrix free [Ca2+], mitochondria
were loaded with RHOD 2 at 7.66 ± 1.07 nmol of RHOD 2·nmol of
Cyta
1 (n = 9) by
incubation at 0 °C for 10 min in a 30 mM solution of
RHOD 2-AM (Molecular Probes Inc, R-1244) dissolved in
Me2SO with trace amounts of Pluronic F-127
(Molecular Probes, P-6867). In all cases, loaded mitochondria were
washed and repelleted three times, with the third pellet resuspended in
plain buffer B, or A where appropriate, at 26.8 ± 1.85 nmol of
Cyta·ml
1 (n = 31). The intra- and extramitochondrial RHOD 2 content was determined in
mitochondria preincubated at 37 °C for 6 min. RHOD 2-loaded
mitochondria were pelleted at 4000 × g in an Ependorff centrifuge (model 5415C) for 2 min, and the supernatant was collected as the extramitochondrial pool. The pelleted mitochondria were resuspended in buffer C containing 2% Triton X to minimize sample light scattering. Total RHOD 2 in the Triton X-treated pellet and
supernatant were determined spectrophotometrically (Lambda 3B,
PerkinElmer Life Sciences) at the peak absorbance (545 nm), with two
reference wavelengths (i.e. 520 and 590 nm) to correct for
nonspecific absorbance in the samples. The absorbance of RHOD 2 at 545 nm was confirmed to be [Ca2+]-insensitive and independent
of esterase cleavage (data not shown). The extinction coefficient for
RHOD 2 determined from this three-point method was 40.12 mM
1·cm
1
at the peak absorbance, which was ~2-fold lower than reported extinction at this wavelength (Molecular Probes Inc.). Based on this,
the [RHOD 2] of the mitochondria was 3.01 ± 0.11 nmol of RHOD
2·nmol of Cyta
1 compared with
161.7 ± 0.009 pmol of RHOD 2·ml
1 in
the extramitochondrial space. At the concentration of mitochondria used
in these studies (4 nmol of
Cyta·ml
1), this corresponds to
a 5.06 ± 0.21% (n = 9) contamination of RHOD 2 in the non-mitochondrial space.
To determine the role of the extramitochondrial dye to the
Ca2+ effects observed, the fraction of dye cleaved in the
two compartments must be determined. Since the acetoxymethyl ester form
of RHOD 2 is Ca2+-insensitive and would not contribute to
any Ca2+-dependent fluorescence signal changes,
the cleaved form of [RHOD 2] was determined from standard curves
between the total RHOD 2 content (measured by absorbance) and
fluorescence enhancement in the presence and absence of
Ca2+. Samples from the supernatant and resuspended pellet,
with and without Ca2+, were then compared with standard
curves, determined for the potassium salt form of the dye (Molecular
Probes Inc., R14220), to estimate the cleaved fraction. If 100%
cleavage had occurred, then the ratio of fluorescence to absorbance in
the presence of Ca2+ should be the same in the samples as
in the standard curve. The fractional decrease in the
Ca2+-dependent fluorescence normalized to the
total [RHOD 2] was taken as the portion of uncleaved dye. By using
this approach, the fractional cleavage of the dye was 58.4 ± 4.6 and 99.4 ± 58.4% (n = 22) in the mitochondrial
and extramitochondrial space, respectively. Taking the relative
distribution of the dye content between the mitochondria and
extracellular space, along with the relative cleavage of the dye, the
total contribution of the extramitochondrial space to the
Ca2+-sensitive fluorescence signal is less than 8.05 ± 0.34% (n = 22) in
OD.
Preparation Quality and Incubation Conditions--
The quality
of each preparation was determined from the respiratory control ratio
(RCR) of m
O2.
ADP-driven respiration was stimulated with a single addition of 1.6 mM ADP, and the subsequent State 4 rate was determined
using a polarographic O2 electrode in buffer B. This
approach permitted direct comparison of RCR values with our previous
work and provided a mechanism for production of optical standards,
required for NADH determinations (see below).
To deplete mitochondria of endogenous Ca2+, all
preparations were incubated for 6 min in buffer C (Table I) at
37 °C. This procedure resulted in a matrix free [Ca2+]
of 1.26 ± 0.17 nM (n = 61), as
determined by matrix RHOD 2 fluorescence (see "NADH and RHOD 2 Fluorescence"), and was similar to previous reports for
Ca2+-depleted mitochondrial preparations (32). To estimate
the rate and response times for inorganic phosphate (Pi),
mitochondria were incubated in buffer D or H (Table I), where
Pi was incremented after G/M (5 mM) and ATPase
(1.6 IU·ml
1).
In an effort to optimize creatine (Cr)-driven respiration, a series of
experiments were performed with five different buffers (C, E-G, and
G0). Initial studies were performed in buffer C, in the
absence of ADP, and were driven with a single bolus of Cr, which match physiological levels (10 mM final) in cardiac tissue (33).
Reports from Saks and co-workers (35) have suggested that excess
chloride (>50 mM) and auto-oxidation may result in low
creatine kinase turnover rates (34-36) in isolated mitochondria (29).
To test these hypothesizes several incubation buffers were used; buffer E (Table I) attempted to reduce the level of auto-oxidation with the
addition of 0.5 mM dithiothreitol, whereas buffers F and G were formulated to lower the level of free chloride to near-physiologic levels (37). Since, under normal isolation procedures, mitochondria are
resuspended in buffer B which contains >140 mM chloride, a subpopulation was prepared and reconstituted in buffer A, and the
experiment was performed in buffer G (buffer G0), therefore
testing this hypothesis. Under these conditions (buffers E-G0), incubations and experimental substrate additions
were performed as described for buffer C; however, since ADP has been
shown to lower the Km for creatine kinase enzyme
(38), 0.4 IU·ml
1 ATPase was added to
provide a constant ADP background. Finally, in an effort to compare the
maximal creatine-stimulated rates in buffer G with ADP and
Pi, experiments were performed in buffers G and H (Table
I), respectively. Postincubation, carbon substrates, ATPase, and
Ca2+ were added to the suspension in series while optical
data were collected (see below).
Calculated free [Ca2+] in each buffer was based on
affinity constants previously reported (39). Where appropriate, data
are presented as calculated free concentrations. In those cases where 0 nM or "nominally zero" Ca2+ are indicated,
no additional Ca2+ was added to the media. In general,
mitochondria experiments were performed at 1 nmol of
Cyta·ml
1; however, RHOD 2 experiments were performed at higher concentrations of mitochondria (4 nmol of Cyta·ml
1) to increase
the RHOD 2 signal to noise ratio.
Chamber Mixing Kinetics--
The kinetic effects of metabolites
and Ca2+ were the primary interest of this work. Thus, the
determination of the inherent mixing rates in the experimental chamber
was critical for the interpretation of the data. The chamber mixing
kinetics was determined optically by monitoring the absorption of Hb
during its injection into the chamber, as described previously (40). A
single bolus of Hb was injected into the chamber containing deactivated
mitochondria while monitoring the Hb optical absorbance. This permitted
the determination of system-mixing times at the appropriate
temperature, ionic strength, and viscosity. These studies revealed a
response time of 0.184 ± 0.03 s (n = 15),
approximately double the spectral acquisition rate (100 ms·spectrum
1).
Oxygen Consumption
(m
O2)--
For the
determination of rapid changes in oxygen consumption, the
oxygen-sensitive optical absorption of extramitochondrial hemoglobin
was used as described previously (40). Standard hemoglobin dissociation curves were generated daily by monitoring Hb absorbance at
561 and 549 nm (an isosbestic reference) in a
mitochondria/Hb suspension oxidizing 800 µM glutamate + malate (G/M) at State 4 respiration while the O2 tension
was monitored with the polarographic electrode. By using these data, a
standard curve of Fsat and %O2 was
constructed and was constrained over the intervals 0
Fsat
1 and 0%
%O2
21%,
respectively. These data were then interpolated using a cubic spline to
approximate a continuous function and were then mathematically inverted
providing a lookup table where %O2 could be evaluated for
each Fsat obtained from experimental spectra
(see Ref. 40).
All mitochondrial respiratory measurements were carried out between 0 and 15% O2 saturation (0 to 0.982 Fsat) and anoxia (40). By using the standard
curve as a lookup table, soluble O2 in the chamber (nmol
O2) for each experimental spectra
(sO2(n)) was calculated as shown in
Equation 1,
|
(Eq. 1)
|
where %O2(n) is the percent oxygen in
the chamber for "n" spectra derived from the lookup
table;
is the solubility of oxygen in buffer for a given salt
content and temperature in nmol·ml
1, and
VC is the volume of the chamber in ml. The oxygen solubility used was 199 nmol·ml
1 at
37 °C (41). Similarly, given
Fsat(n) for each experimental spectrum, total O2 bound to Hb
(HbO2(n)), in nmol of O2, was
calculated as shown in Equation 2,
|
(Eq. 2)
|
where Fsat(n) is the
fractional saturation of Hb for n spectra; [Hb] is the
concentration of hemoglobin in the chamber in nmol·ml
1;
is the capacitance of
O2 per nmol of Hb in nmol, and VC is the
volume of the chamber in ml. Combining Equations 1 and 2, the total
oxygen content (nanomoles of O2) of the reaction chamber at
n spectra ([O2](n)) was calculated
as given in Equation 3.
|
(Eq. 3)
|
Since all spectra were acquired at fixed intervals (100 ms),
differentiating these with respect to time yields oxygen consumption rates in nmol of O2·nmol
Cyta
1·min
1
(see Equation 4),
|
(Eq. 4)
|
where
[O2] is the decrease in
[O2] in nmol of O2;
t is the
interval over which the decrease occurred in seconds; Cyta
is the cytochrome aa3 content in
nmol·ml
1, and Vc is the
chamber volume in ml.
NADH and RHOD 2 Fluorescence--
Reduced pyridine dinucleotides
(NADH) were monitored in parallel experiments using the same chamber
mixing system as for the m
O2; however,
excitation and light collection were provided on the same side of the
chamber (0o) with the RP400-7 UV-visible reflectance probe
(Ocean Optics Inc.). Excitation was provided with a 500-watt
Hg/Xe arc lamp (Oriel Inc., models 68811 and 66011) fitted with
a 360 nm band-pass filter (Edmund Scientific Inc. model 46085). The
result was a primary spectral line centered at 360 ± 10 nm, which
was coupled to the six fibers of the excitation arm of the reflectance
probe. Detection of NADH was provided with a linear CCD array (Ocean Optics Inc., model PC2000), described for
m
O2, coupled to the receive arm of the reflectance probe. Data were digitally sampled over
the spectral bandwidth of 335-1064 nm at 10 Hz with a 12 Bit A/D
converter as described for
m
O2.
To correct for potential non-linear amplitude response of the linear
diode array over the spectral bandwidth used in these studies, and to
calculated true irradiance, spectra were collected for a 3100 K
calibrated black body source (Ocean Optics Inc., model LS-1).
Theoretical calculations of black body irradiation for 3100 K were
performed using Plank's equation (Equation 5):
|
(Eq. 5)
|
where
is wavelength bound by 335 nm
1064 nm;
hc is Plank's constant 1240 eV·nm; Kb
is Boltzmann's constant for black body irradiation 8.617 × 10
5 eV·K
1, and
T is the source filament temperature in K. Given this,
detector quantum efficiency (Qeff) was
determined between theoretical (Irbb) estimates and the
calibrated source (Ircal) at 3100 K for all wavelengths
(
):
|
(Eq. 6)
|
Provided the function described in Equation 6, corrected
irradiance for each spectrum (Ircorr(n)) was
calculated as shown in Equation 7,
|
(Eq. 7)
|
where Irraw(
,n) is the raw data at the
"nth" spectrum collected for each wavelength (
);
Irdark is the dark current of the detector at each
, and
Qeff is the quantum efficiency of the detector
for each
.
By using the formulation in Equations 5-7, control spectra were
obtained in fully oxidized (0.067 mM ADP) and reduced (5 mM G/M) mitochondria in the absence and presence of CF.
Given these, model spectra for NADH (MNADH) and CF
(MCF) were constructed by difference. Spectra modeling
excitation light bleed through (MEBT) were obtained from
G-10 Sephadex beads (size 40-120 µm, Sigma) at 1 mg·ml
1. Model spectra (MNADH,
MCF, and MEBT) were fitted and compared with a
multiple linear regression to each experimental spectra, n,
yielding the coefficients for NADH (INADH), CF
(ICF), and EBT (IEBT) as described previously
(31). Coefficients for each experimental spectra (n) were
calculated as shown in Equation 8,
|
(Eq. 8)
|
For studies where matrix free Ca2+ was monitored,
additional difference spectra were constructed in the absence and
presence of Ca2+, permitting the construction of model
spectra for RHOD 2 (MRHOD). As with NADH, estimates of
matrix free [Ca2+] were determined from calculated model
coefficient described by Equation 9,
|
(Eq. 9)
|
By using the coefficients for NADH or RHOD 2 with CF, the
INADH·ICF
1 or
IRHOD·ICF
1 ratios
could be calculated, providing a method for eliminating both primary
and secondary inner filter effects on NADH and RHOD 2, respectively.
Estimates of free matrix [Ca2+] were determined using a
Kd of 570 nM as shown in Equation 10,
|
(Eq. 10)
|
where estimates of maximal RHOD 2 fluorescence
(Fmax) were determined at the end of each
experiment in the presence of excess Ca2+ (10 µM) plus 10 µM Br-A234187 (Sigma, B-7272).
Regression coefficients for RHOD 2 fluorescence
(IRHOD·ICF
1)
post-Ca2+ depletion approached zero and were taken to
represent minimal RHOD 2 fluorescence (Fmin).
In all cases, regressions were performed iteratively until the sum of
squares convergence was achieved using the Marquardt-Levenberg algorithm written in IDL (Research Systems Inc., version 5.2). The
algorithm provides the following: 1) the coefficients of the model
spectra; 2) standard deviation for the coefficients; 3) an F
test for fit between model and experimental data; and 4) multiple
linear correlation coefficients for the fitted spectra. The degree of
concordance between model and experimental spectra was high both within
(0.993 ± 0.0004, average n = 9355·study
1, p
0.0001)
and between (0.993 ± 0.0006, total n = 122050, p
0.0001) experimental studies.
Since NADH levels are in dynamic flux between CaDH production and
cytochrome consumption, calculations of changes in [NADH] to a new
steady state can be described by "jump" or transition kinetics (42)
using the following bimolecular Equation 11,
|
(Eq. 11)
|
where
(
x) and
t are the changes
in product x for a given change in time t.
k1 and k2 are the
reaction rate constants in the forward and reverse directions of the
initial step, whereas k3 is the forward reaction
rate constant in the formation of the product x.
so,
xo, t, and
are the initial [substrate], initial change in [product], time, and
time constants, respectively.
As the limit of t approaches 0 it yields Equation 11, thus
permitting estimates of NADH production rate from initial rates shown
in Equation 12,
|
(Eq. 12)
|
where the initial rates of product formation are described by
the rate constants k1,
k2, and k3 and the
initial concentrations of substrate and the change in product
formation, yielding a simple linear equation. Given the relations
described by Equations 11 and 12, the initial rate of NADH formation
can be described by a simple linear system and therefore is dominated
by the forward rate constant (k1) of the
reaction and the initial [substrate] (42).
By using the derivations from Equations 8 and 11-12, and the
acquisition rate per spectra (100 ms·spectra
1), NADH levels could be
differentiated with respect to time, yielding the initial rate of
change for NADH (m
NADH) in
INADH·ICF
1·Cyta
1·min
1
(see Equation 13),
|
(Eq. 13)
|
where
(INADH·ICF
1) is
the change in scattering corrected NADH levels, and
t is
the time interval in seconds over which the data were differentiated;
Cyta is the content of cytochrome
aa3 in the media in
nmol·ml
1, and Vc is the
chamber volume in ml.
Light Scattering--
Light scattering was used as an indirect
measure of mitochondrial volume changes. Mitochondrial volume was of
interest in this study since Ca2+ is known to modulate
matrix volume, and possibly mitochondrial metabolism (43). Therefore,
volume changes could provide possible insight into the mechanisms,
which underlie rapid mitochondrial kinetics. Illumination was provided
by a tungsten-halogen source (Titan Tool Inc., model F0-150) coupled to
the optical chamber via liquid light guide. The transmitted light
(180o from incident source) was detected using the receive
arm of the reflection probe (Ocean Optics Inc., model RP400-7
UV-visible), coupled to a linear CCD array, and was sampled as
described for Hb experiments. Mitochondria were placed in the chamber
at 1 nmol of Cyta·ml
1, and
incident light was adjusted via mechanical iris to permit collection of
the full dynamic range over the spectral bandwidth (360-1064 nm).
Additionally, a sample of the source light (S0) was collected, permitting calculations of light scattering
(
s) with the units of optical difference (
OD) as shown in
Equation 14,
|
(Eq. 14)
|
where S0 is the block average of 5 spectra obtained from the tungsten-halogen source, and
S1 is the optical intensity for the
mitochondrial suspension at n spectra. Since spectra were acquired at regular intervals, as with O2 and NADH,
differentiating
s with respect to time for discrete
wavelengths (
) yields Equation 15 in
OD·Cyta
1·min
1,
|
(Eq. 15)
|
where
s(
(n)) is the light scattering
for a given cytochrome isosbestic wavelength (455, 510, 520, 540, 575, and 630 nm) at n spectra;
t is the interval
over which the data was differentiated in seconds; Cyta is
the cytochrome aa3 content in
nmol·ml
1, and Vc is the
chamber volume in ml. m
s represents the
initial rates for light scattering in this system. Since the
relationship between mitochondrial volume and light scattering is
complex, this measure can only be considered an estimate of
mitochondrial swelling rates in response to these perturbations.
Optical Time Stamp (OTS)--
Addition of oxidizable carbon
substrates, ATPase, and experimental substrates (i.e.
Ca2+, ADP, creatine, G/M, ruthenium red, or EGTA) were
added serially and optically time stamped upon addition. The optical
time stamp was generated by exciting an external tungsten source
filtered with a 530 ± 20 nm band-pass filter (Fig. 1). The
resulting flash of light caused a brief (~100 ms) green shift in the
spectral line at the point of addition. This green shift in the spectra when post-processed using Equations 1-15 resulted in a downward
deflection in the signal intensity at the point where the addition was
made. In all cases the OTS was at least 2-fold greater than the noise at the point of addition, permitting precise determination of when the
addition was made.
IRT Determination--
The IRT of the mitochondria to the
experimental perturbation was calculated by selecting two ROI. The
first ROI placed just prior to the addition and the second just post to
the addition were used to demark the boundaries over which two least
squares linear regressions were performed. Based on the slopes and
intercepts from this analysis, the regression lines for each ROI were
interpolated over the entire data range selected by both ROIs. The
intersection of the two regression lines represented the initial point
where the transition to the new slope occurred. The IRT was then
calculated as the difference in time from the middle of the OTS to the
point of intersection. Provided this methodology, which was based on a
statistical framework, an unbiased estimate of the response time of the
system could be made. Analytical derivation of IRT, presented under the
"Appendix," can be shown to be essentially identical to the
conventionally used in exponential fits of time varying data. Given
this, it is reasoned that in the absence of exponentially time varying
functions, as observed for
m
O2, NADH, or
s, reliable estimates of "
" could be calculated by
IRT.
Electron Microscopy--
Porcine hearts were isolated from
anesthetized pigs according to Ref. 15 and were retrograde-perfused via
coronary arteries with normal saline, followed by fixative solution
containing 2.5% glutaraldehyde in 100 mM cacodylate buffer
(pH 7.2) and 250 mM CaCl2 at 25 °C. Tissue
blocks from the apex, epi-, and endocardium were further fixed in the
same fixative for 4 h (25 °C). Following fixation, tissue
blocks were washed three times in Sabatini's solution (100 mM cacodylate buffer with 6.8% sucrose). All samples were
then post-fixed with 1% osmium tetroxide in 100 mM
cacodylate buffer (pH 7.2) for an additional hour and washed three
times in Sabatini's solution. The samples were passed through a graded series of alcohols followed by treatment with propylene oxide, a 1:1
Epon/propylene oxide mix, and three changes in pure epon. Polymerization was performed at 60 °C overnight. Ultra-thin sections were cut with a Leica Ultracut UCT ultra-microtome, stained with uranyl
acetate and lead citrate, and examined with a transmission electron
microscope (JEOL 1000X) at accelerating voltage of 60 kV.
Data and Statistical Analysis--
Initial data processing for
O2, NADH, and light scattering were performed using
Equations 1-15 with custom programs written in IDL (Research Systems
Inc., version 5.2). Post-processing of initial rates before and after
"substrate" additions were performed by a least squares linear
regression, also in a custom program written in IDL (Research Systems
Inc., version 5.2). The resulting analysis determines the following: 1)
the regression coefficients; 2) the equation of the line describing the
relationship; and 3) the probability that the slope of the line is not
significantly different from zero. Individual slopes between treatments
for m
O2,
m
NADH, and
m
s, and IRTs were compared using a
multifactor-dependent variable t test
(Statistica version 5.0, Statsoft Inc.). Where appropriate, values are
expressed as mean ± 1 S.E. In all cases, the fiduciary level of
significance was taken at p
0.05.
 |
RESULTS |
Mitochondrial Characterization--
Oxygen consumption was tightly
coupled to ATP phosphorylation in this preparation with an average
respiratory control ratio (RCR) of 14.3 ± 0.41 (n = 31) and ranged between 10.5 and 18.7 while oxidizing G/M.
Mitochondria with RCR below 8 were not used in these studies. In all
cases, RCR estimates of mitochondrial integrity were performed in
buffer B (Table I) with 5 mM
G/M, 2 mM Pi, and a single addition of 1.3 mM ADP at 37 °C. This methodology permitted direct
comparison with our previous work (15, 44) and allowed for model
spectra for NADH and CF to be collected for fluorescence standards
(31).
View this table:
[in this window]
[in a new window]
|
Table I
Buffer composition, in mM, used in isolation, resuspension,
and experimental conditions
Buffer compositions are expressed in mM, where ATP, malate,
and Pi were added fresh daily, and pH was adjusted to listed
value just prior to use. Buffers made with the impermeant anion methane
sulfonate were derived from methanesulfonic acid solutions and were
titrated to the listed pH with KOH.
|
|
Calcium, ADP, Pi, and Creatine Activation of Oxygen
Consumption--
A typical
m
O2 time course for
evaluating the effects of Ca2+ is shown in Fig.
1A. The addition of carbon
substrates to Ca2+-depleted mitochondria in buffer C
resulted in an increase in m
O2, consistent
with a repolarization of 
post-depletion (15). Once at steady
state, exogenous ATPase was added to the media, providing a constant
generation of ADP. In all cases steady state was achieved for several
seconds before additional experimental perturbations were made. An OTS
was applied during the additions of "substrates," thus providing a
method for evaluating IRT as outlined under the "Materials and
Methods." The addition of Ca2+ resulted in an immediate
increase in respiration, and an expanded time course is presented in
Fig. 1B. Calculation of IRT for Ca2+ was
determined from regression analysis performed over two ROIs, just prior
to and after the substrate addition. IRT was evaluated as the time
difference between the OTS and point of intersection of the two slopes.
The IRT for Ca2+ was estimated to be 0.27 ± 0.02 s, which was virtually the same as the chamber mixing kinetics (Fig.
2B).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
Representative plot of [O2]
versus time for Ca2+-stimulated
mitochondria and determination of IRT. A, plot of
O2 content versus time for mitochondria
oxidizing 5 mM G/M. Mitochondria were initially
Ca2+ depleted for 6 min as described under "Materials and
Methods." ADP-driven respiration was initiated with a single addition
of ATPase (1.6 IU·ml 1 final), which was
followed upon steady state with a single addition of 535 nM
Ca2+. Two ROIs were delineated, and least squares linear
regression performed prior to (solid line) and
post-Ca2+ addition (dashed line). Rates and
statistical analyses
m O2 are presented
in Table II. B, a subset of data plotted in A
indicating the two ROIs over which the regression analysis was
performed. Regressions lines are as indicated in A. IRT was
determined as the time difference from the middle of the OTS (downward
deflection) to the intersection of the two regressions as described
under "Materials and Methods."
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 2.
IRT with [Ca2+] and treatment
for m O2.
A, the effects of [Ca2+] on IRT. Calculations
of IRT were as described under "Materials and Methods" and were
performed as paired studies from 0 to 1840 nM free
Ca2+ (n = 4 all [Ca2+],
n = 7 no Ca2+). Data are presented as
mean ± S.E., and asterisks indicate significant
differences from previous [Ca2+] (p 0.05-dependent variable t test). B,
effects of substrate additions on IRT. Substrate additions
(i.e. ADP, Pi, creatine, G/M, Ca2+,
and EGTA), OTS, and IRT were as indicated in the text. The
corresponding sample size for each treatment is indicated with
parentheses (n), and buffer conditions are
indicated by letters in parentheses (i.e.
(C) signifies buffer C) as described in both under
"Materials and Methods" and Table I. Pi experiments
were performed in buffers D and H. Creatine experiments indicated by
G0 signify mitochondria resuspended in buffer A and
respiring in buffer G. ADP IRT listed represent State 4 to ADP-driven
transition while oxidizing 5 mM G/M + 535 nM
Ca2+. Cr rates reported in buffer C are for mitochondria
oxidizing 5 mM G/M + 535 Ca2+. Creatine
experiments in buffers E-G0 were performed as in buffer C;
however, 0.4 IU·ml 1 ATPase was present to
lower the Km for creatine kinase (29). ATPase
additions denoted by were for mitochondria oxidizing 1.5 mM G/M + 535 nM Ca2+ prior to final
substrate addition. ATPase additions denoted by
- were for mitochondria oxidizing
-ketoglutarate, pyruvate, or malate, respectively. IRTs reported for
RuRed and EGTA were at 8 and 160 µM, respectively. In all
cases data are presented as means ± S.E., and significance is
indicated on the figure with square brackets indicating the
grouping.
|
|
By using the above setup, the effects of [Ca2+] on IRT
and m
O2 were
evaluated between nominally zero (0 nM) and 1835 nM free Ca2+ and are presented in Fig.
2A and Table II. The addition
of Ca2+ from 0 to 535 nM resulted in a
incremental decrease in IRT, with the largest difference from control
(~9-fold) occurring at the optimal [Ca2+] of 535 nM. Oxygen consumption increased in a
dose-dependent manor with [Ca2+] through 535 nM (Table II). However, [Ca2+] in excess of
1000 nM increased IRT by nearly 10-fold, and
m
O2 decreased by
approximately 20% indicating significant Ca2+ inhibition
and possible Ca2+ overload.
View this table:
[in this window]
[in a new window]
|
Table II
Oxygen consumption rates and regression statistics with
[Ca2+]
r2 and n are the combined regression
coefficient and number of observations. In all cases the level of
significance relative to a slope of zero for linear regressions used
for the IRT determination was p 0.0001. [Ca2+] are calculated free levels based on affinity constants
described previously (39).
|
|
To test the reversibility of the Ca2+ effects, EGTA was
added to the chamber to reduce [Ca2+] by ~150
nM, as calculated (39). The reduction in
[Ca2+] resulted in a decrease in
m
O2 by 17% in the
steady state from the full 535 nM Ca2+
stimulation. The IRT for this extraction of Ca2+ (1.55 ± 0.14 s) was much slower than the maximum rate of activation by
Ca2+ (0.27 ± 0.02 s) but did demonstrate
reversibility of the Ca2+ effect. The slow off-kinetic of
Ca2+ might be influenced by the fact that Ca2+
was not returned to nominally zero values with the EGTA addition.
The respiratory time response to Ca2+ was compared with
other putative cytosolic transducers including ADP, Pi,
creatine as well as carbon substrate additions. The results of these
studies are presented in Fig. 2B and Table
III. Several interesting
comparisons can be made from these data. First, the respiratory
response to Ca2+ (535 nM) was an order of
magnitude faster than ADP (2.36 ± 0.47 s), Pi
(2.32 ± 0.29 s), or carbon substrate additions (3.31 ± 0.42 s) in buffer C, and >25 and 10-fold higher than ADP
(8.90 ± 0.47 s) and Pi (3.20 ± 1.38 s) in buffers G and H, respectively. This implies that Ca2+
increases the efficiency of the reaction rate more effectively than ADP
or Pi additions alone. Creatine (10 mM) was
added to State 4 mitochondria oxidizing 5 mM G/M + 535 nM Ca2+ in buffer C. The creatine-stimulated
rate increased m
O2
by ~5-fold over State 4 rate (Table III); however, this rate was 3.6- and 5.3-fold lower than the ADP- and Ca2+-stimulated rates,
respectively. Similarly, IRTs for creatine were more than 6 and 50 times longer than for the ADP or Ca2+, respectively.
View this table:
[in this window]
[in a new window]
|
Table III
Oxygen consumption rates and regression statistics with calcium and
substrates
r2 and n are the combined regression
coefficient and number of observations. In all cases the level of
significance relative to a slope of zero for linear regressions used
for the IRT determination was p 0.0001. Data
represent mitochondria oxidizing 5 mM G/M as the oxidizable
carbon source and were Ca2+-depleted in experimental buffer
indicated by parentheses (i.e. (C) indicates buffer C) prior
to intervention as described under "Materials and Methods."
Creatine experiments indicated by (G0) signify mitochondria
resuspended in buffer A and respiring in buffer F. Oxygen consumption
rates 1 and 2 correspond to the first and second substrate additions,
respectively. ADP rates listed represent State 4 to ADP-driven
transition while oxidizing 5 mM G/M + 535 nM Ca2+. Creatine (Cr) rates reported in buffer C
are for mitochondria oxidizing 5 mM G/M + 535 nM Ca2+. Creatine experiments in buffers
D-F0 were performed as in buffer C; however, 0.4 IU · ml 1 ATPase was present to lower the Km
value for creatine kinase (38). Pi experiments were performed
in buffers D and H, which contained 0.25 mM Pi and
were maximally stimulated with 2 mM bolus. Rates reported
for Ruthenium Red (RuRed) and EGTA were at 8 and 160 µM,
respectively. In all cases, data are presented as means ± 1 S.E.
|
|
The fact that both
m
O2 and IRT were
substantially slower from creatine when compared with ADP or
Ca2+ suggested that creatine kinase activity may have been
reduced by either auto-oxidation (34-36) or excessive chloride (>50
mM) (29) in the media. To test this, creatine studies were
performed in buffers E-G0 with 0.4 IU·ml
1 ATPase and are presented in Fig.
2B and Table III. The addition of dithiothreitol alone
(i.e. buffer E, Table I), to minimize oxidation effects, did
not significantly alter the overall kinetics when compared with
experiments performed in buffer C. On the contrary, reducing
Cl
to physiological levels with the impermeant anion
methane sulfonate (buffer F, Table I) significantly shortened the IRT
by 1.6-fold; however, these changes were not accompanied by significant
changes in m
O2
(Table III). Interestingly, when both K+ and
Cl
were lowered in tandem by replacement with sucrose
(buffers G and G0, Table I), the IRTs for Cr-stimulated
respiration were indistinguishable from those obtained for ADP or
Pi in buffer C (Fig. 2B). These shorter IRT were
accompanied by a >2-fold increase in
m
O2 over those
obtained in either buffer C or E; however, these rates remained ~1.2-
(buffer G) and ~1.7-fold (buffer G0) lower than those
observed for ADP additions alone in buffer C and were on average
~2-fold lower than rates observed for Ca2+ (Table
III). Combined these data suggest that of all the putative cytosolic
transducers that could modify mitochondrial respiration, Ca2+ had the shortest response time with the greatest
dynamic range in
m
O2.
Extramitochondrial increase in [Ca2+] rapidly stimulates
m
O2 in a
dose-dependent manner when added to a previously
Ca2+-depleted preparation. However, it is unlikely that an
initial condition of nominally zero [Ca2+] is
physiological. Thus, serial additions of Ca2+ were
evaluated to establish whether the effects of [Ca2+] on
respiration were not dependent on the nominally zero initial conditions. In these studies, ATPase additions were made accompanied by
only 60 nM Ca2+, which was ~30% of the
Ca2+ K0.5 (Table V) for respiratory
effects. After reaching a new steady state, a final bolus of 475 nM Ca2+ was added, yielding a final
[Ca2+] of 535 nM. The final serial addition
of Ca2+ resulted in a short IRT (0.35 ± 0.02 s)
and was comparable to the single 535 nM bolus in steady
state rate (Table III) and the IRT (Fig. 2B). These data
suggest that activation by Ca2+ is not strongly dependent
on initial [Ca2+] conditions as long as the concentration
changes remain in the dose-dependent region.
To evaluate the matrix dependence of extramitochondrial
[Ca2+] perturbations, experiments were performed in the
presence of 8 µM Ruthenium Red (RuRed). Addition of RuRed
to the media prior to Ca2+ addition resulted in a
significantly longer IRT and was approximately 8-fold greater than that
observed for Ca2+ addition alone. The increase in IRT was
accompanied by a 1.2-fold lower maximal rate than for 535 nM Ca2+ alone, thus demonstrating partial
matrix Ca2+ inhibition at this concentration of RuRed.
Concentrations higher than 8 µM were attempted to inhibit
completely this activation by Ca2+; however, significant
optical artifacts prevented accurate rate determinations and therefore
are not reported.
Calcium Activation of NADH Production--
To compare with the
m
O2 results,
parallel kinetic experiments were performed for NADH production with
variable Ca2+ additions. A typical time course of scatter
corrected NADH is presented in Fig.
3A. Mitochondria were placed
in the chamber and allowed to Ca2+-deplete for 6 min prior
to substrate addition. Upon G/M addition, [NADH] rapidly increased
and reached steady state within ~10 s. Once at steady state,
respiration was initiated with a 1.6 IU·ml
1
addition of ATPase, which resulted in a rapid decline in [NADH]. Once
a constant NADH level was achieved, a single bolus of Ca2+
was added, which increased by 150% with 535 nM
Ca2+ addition. The IRT for NADH were calculated as the
difference in time between the OTS and the point of intersection of the
two regression lines as per previous (Fig. 3B). In addition,
initial rates of NADH production (m
NADH)
with [Ca2+] were determined as described under
"Materials and Methods." The results of the IRT and
m
NADH analyses with variable
[Ca2+] are presented in Fig.
4. NADH response times, like
m
O2, were non-linearly correlated with [Ca2+], with the apparent
optima occurring at 535 nM free Ca2+ (Fig.
4A). At concentrations greater than 1000 nM,
however, IRTs increases by more than 4-fold and were consistent with
trends observed for
m
O2. Calculations
of m
NADH with [Ca2+] showed
a similar non-linear relationship to that observed with IRT, with a
maximal rates increasing by 50-fold over base line at 535 nM Ca2+. At high [Ca2+] (>1000
nM) m
NADH decreased by
approximately 40% consistent with the decline in
m
O2 observed over
this same range (Table II).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Time course plot of mitochondrial NADH and
IRT determination. A, NADH time course with substrate
addition. In all cases mitochondria were Ca2+-depleted,
reduced with 5 mM G/M, and ADP-driven respiration was
initiated with a single addition of 1.6 IU·ml 1 (final) ATPase. At steady state
respiration, 535 nM Ca2+ was added. ROIs were
selected and least squares linear regressions performed as indicated
under "Materials and Methods." Downward deflections in the time
course correspond to OTS. B, a subset of data plotted in
A indicating the two ROIs over which the regression analysis
was performed. Regressions and IRT determination were as described
under "Materials and Methods."
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
NADH IRT and rates with
[Ca2+]. A, the effects of
[Ca2+] on IRT in mitochondria oxidizing 5 mM
G/M. In all cases, mitochondria were Ca2+-depleted where
substrates and exogenous ATPase (1.6 IU·ml 1
final) were added. Calculations of IRT were as described under
"Materials and Methods" and were performed as paired studies from 0 to 1840 nM free [Ca2+] (n = 6). B, the effects of [Ca2+] on
m NADH (NADH initial rates) in
mitochondria oxidizing 5 mM G/M. In all cases, mitochondria
were Ca2+-depleted, where substrates and exogenous ATPase
(1.6 IU·ml 1 final) were added. Data are
presented as mean ± S.E., where asterisks indicate
significant differences from nominally zero [Ca2+]
(p 0.05-dependent variable t
test), and sample sizes (n) are indicated by
parentheses.
|
|
Calcium and Light Scattering--
Light scattering, an indirect
measure of mitochondria swelling, was monitored to determine if changes
in mitochondrial volume were accompanying the metabolic changes
observed with m
O2
and m
NADH. A typical time course plot of
mitochondrial absorbance is illustrated in Fig.
5 for 6 cytochrome isosbestic
wavelengths. The addition of G/M to substrate and
Ca2+-depleted mitochondria caused an increase in light
scattering (decreased
OD) at all isosbestic wavelengths. This
decrease in transmitted light is consistent with a change in refractive
index of the suspension and is mediated via an increase in matrix water content, cristae unfolding, and mitochondrial swelling during substrate
accumulation. Upon steady state, ADP-driven respiration was initiated
with a single addition of 1.6 IU·ml
1
ATPase, resulting in an increase in
OD, consistent with
mitochondrial contraction. At steady state respiration, a single bolus
of Ca2+ was added to the suspension, resulting in a marked
increase in light scattering and/or mitochondrial swelling. As
described previously, ROIs were drawn prior to and post
Ca2+ addition, and from these estimates the rate of
OD
change and IRTs were determined (Fig. 5 and Table
IV). Interestingly, calculated IRTs at
all wavelengths showed little variation with [Ca2+] and
were on average 1.9 ± 0.5- and 4.9 ± 0.9-fold shorter than calculate IRTs for m
NADH and
m
O2,
respectively.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5.
Time course of mitochondrial light
scattering. A, light scattering for 6 cytochrome
isosbestic wavelengths (455, 510, 520, 540, 575, and 630 nm)
versus time. Mitochondria were Ca2+-depleted,
reduced with 5 mM G/M, and ADP-driven respiration initiated
with a single addition of 1.6 IU·ml 1
(final) ATPase. At steady state respiration 535 nM
Ca2+ was added. B, a subset of data plotted in
A indicating the ROIs over which the regression analysis was
performed prior to (solid line) and post (dashed
line)-Ca2+ addition. ROI selection, least squares
linear regressions, OTS, and IRT calculations were as described under
"Materials and Methods."
|
|
View this table:
[in this window]
[in a new window]
|
Table IV
Light scattering IRT and regression coefficients with [Ca2+]
IRT and n are the initial response time in seconds and the
number of observations, respectively. In all cases the level of
significance relative to a slope of zero for linear regressions used
for the IRT determination was p 0.0001. [Ca2+] are calculated free levels based on affinity constants
described previously (39). Data are presented as means ± 1 S.E.
In all cases data are from mitochondria oxidizing 5 mM G/M
as the carbon source and were Ca2+-depleted prior to treatment
as described under "Materials and Methods."
|
|
Analysis of m
s time course data with
[Ca2+] showed a strong non-linear dependence of
m
s with Ca2+ dose (Fig.
6A). Interestingly, at
[Ca2+] in excess of 500 nM the maximal
amplitude of light scattering, and therefore mitochondria swelling, was
achieved with little increase at progressively higher dose (Fig.
6A). A summary of this analysis for all studies is presented
in Fig. 6B, which illustrates saturation kinetics of
m
s with [Ca2+], and a
K0.5 of 175 nM (Table
V). Ca2+ above 1000 nM caused the highest rate of light scattering and showed
the greatest attenuation in m
NADH and
m
O2 (Figs.
4B and 5B and Table II) consistent with damage to
the mitochondria ATP production capacity.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 6.
Light scattering rates with
[Ca2+]. A, the effects of
[Ca2+] on OD in mitochondria oxidizing 5 mM G/M at ADP driven
m O2. Calculations
of m s were as described under "Materials
and Methods" and were performed as paired studies from 25 to 1840 nM free Ca2+ (n = 8) at 455 nm.
In all cases, data have been normalized in time to the point of
Ca2+ addition. Symbols corresponding to
[Ca2+] are as follows: 25 nM (filled
circles), 62 nM (open squares), 172 nM (filled triangles), 535 nM
(open circles), 1090 nM (filled
squares), and 1838 nM (open triangles).
B, the effects of [Ca2+] on average
m s (light scattering rate) in mitochondria
oxidizing 5 mM G/M. In all cases, mitochondria were
Ca2+-depleted, where substrates and exogenous ATPase (1.6 IU·ml 1 final) were added. Data are
presented as mean ± S.E., where asterisks indicate
significant differences from nominally zero [Ca2+]
(p 0.05 dependent variable t test), and
sample sizes (n) are indicated by parentheses.
IRTs for these preparations are presented in Table IV.
|
|
View this table:
[in this window]
[in a new window]
|
Table V
Regression analysis of ADP driven rate with [Ca2+]
Relationships for oxygen consumption and NADH production rates with
[Ca2+] are expressed as Y = Yo + a(1 e bx), where Y is oxygen consumption
(nmol O2 · nmol Cyta 1 · min 1) or NADH (INADH · ICF 1 · nmol Cyta 1 · min 1). Relationships for light scattering with
[Ca2+] are expressed as Y = Yo + a(e bx), where
Y is light scattering rate in OD · nmol
Cyta 1 · min 1. Yo,
a, b, r2,
K0.5, and p are DC offset, intercept,
slope, regression coefficient, half-saturation coefficient, sample
size, and level of significance compared to a zero slope, respectively.
In all cases K0.5 were calculated by mathematical
inversion of the above equations for half-maximal rates.
|
|
Kinetics of Mitochondrial Matrix [Ca2+]--
To
confirm further that the metabolic effects were due to matrix
Ca2+ accumulation, matrix Ca2+ was monitored
using RHOD 2 loaded in the mitochondrial matrix. Loading as described
under "Materials and Methods" resulted in a high degree of
compartmental specificity, with 92.0 ± 0.33% (n = 22) of the Ca2+-sensitive RHOD 2 signal originating from
the loaded mitochondrial matrix. A typical time course for RHOD
2-loaded mitochondria is presented in Fig.
7. The addition of carbon substrates and
ATPase resulted in no changes in RHOD 2 fluorescence signal as
determined by
IRHOD·ICF
1. The
addition of 535 nM Ca2+ resulted in a rapid
increase in matrix free Ca2+ with an IRT not significantly
different from chamber mixing times (Fig. 7A). To confirm
further the matrix dependence of these changes, the specific
Ca2+ uniport inhibitor Ru360 (Calbiochem, product 557440)
was added in excess (20 µM) to block Ca2+
transport via this mechanism. The steady state matrix Ca2+
level was reduced by ~50% after a Ca2+ bolus. The IRT
was also apparently lengthened by Ru360; however, the IRT was still
within the sampling rate of the system (50 ms·spectrum
1). Since the Nyquist could not
be properly satisfied, no estimates of IRT for Ca2+ uptake
could be made. By using Equations 9 and 10, estimates of matrix free
Ca2+ post-depletion were 1.26 ± 0.17 nM,
while after 535 nM Ca2+ addition, in the
presence and absence of excess Ru360, were 519.9 ± 54.17 (n = 32) and 273.6 ± 30.28 nM
(n = 29), respectively.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Time course plot of mitochondrial matrix RHOD
2. A, RHOD 2 time course with substrate addition in the
presence (open circles) and absence (filled
circles) of 20 µM Ru360. In all cases mitochondria
were Ca2+-depleted, reduced with 5 mM G/M, and
ADP-driven respiration initiated with a single addition of 1.6 IU·ml 1 (final) ATPase. At steady state
respiration, 535 nM Ca2+ was added. ROIs and
least squares linear regressions were not performed since the Nyquist
limit could not be satisfied. Downward deflections in the time course
correspond to OTS. B, a subset of data plotted in
A with expanded time scale are presented. In all cases
spectra were acquired at 50 ms·sample 1
where coefficients for CF and RHOD 2 were determined as outlined under
"Materials and Methods."
|
|
 |
DISCUSSION |
These data are consistent with the notion that extramitochondrial
Ca2+ can rapidly modulate mitochondrial ATP production
rates within ~200 ms. Determination of IRT for Ca2+
effects on m
O2,
NADH, light scattering, and RHOD 2 were on the order of the chamber
mixing times (Fig. 2B), suggesting that the kinetics could
even be more rapid.
The effects of Ca2+ on
m
O2 are assumed to
represent the effects of Ca2+ on ATP production rate and
may be caused by the matrix influx of Ca2+. These
conclusions are supported by our previous observations that
mitochondrial uncoupling by Ca2+ is minimal at
concentrations below 600 nM (15). Furthermore, a
correlation between
m
O2 and direct
measurements of ATP production has also been established in this system
(15). Finally, the metabolic effects observed with Ca2+,
increased m
O2, and
[NADH] are not consistent with an uncoupling of oxygen consumption
from ATP production by the influx of Ca2+ ions alone, which
should decrease [NADH] while increasing
m
O2.
Evidence that changes in matrix Ca2+ were responsible for
these metabolic effects of extramitochondrial Ca2+ were
obtained by the partial inhibition of these effects with RuRed, and the
kinetics of matrix [Ca2+] were detected with RHOD 2. The
partial inhibition of the metabolic effects of Ca2+ by
RuRed as well as the partial inhibition of Ca2+ uptake by
Ru360 suggests that Ruthenium Red-insensitive Ca2+ uptake
mechanisms exists in this preparation. These results are similar to the
recent results (45) demonstrating a partial inhibition of mitochondria
Ca2+ uptake by RuRed in intact heart cells. The nature of
this RuRed-insensitive transport mechanism is unknown but is consistent
with previous descriptions of the rapid uptake mode in liver
mitochondria (46, 47).
Matrix Ca2+ could effect ATP production at several levels
including an increase in [NADH] or 
driving force via CaDH
activation (11, 15, 48) or activation of the
F0F1-ATPase (15). The current data are
consistent with both of these mechanisms. The rapid increase in
[NADH] observed with Ca2+ is consistent with an
activation of CaDH. These data suggest that the CaDH are being
activated in ~200 ms, although taking seconds to reach maximum values
(Fig. 3). This is consistent with previous data on the activation of
pyruvate and 2-oxoglutarate dehydrogenases in mitochondria (49, 50).
These results suggest that the activation of CaDH and increase in
metabolic driving force through an increase in [NADH] was one of the
rapid metabolic response mechanisms initiated by Ca2+.
In previous work, it was shown that increases in
F0F1-ATPase activity were very significant in
Ca2+ activation of heart mitochondrial ATP production in
the steady state (15). Similar data have been recently obtained by
skeletal muscle mitochondria (51). Although the IRT for NADH and
m
O2 were similar
(Figs. 2 and 4), closer inspection of the NADH and m
O2 time courses
revealed a monotonic increase in [NADH] (Fig. 3B), whereas
the increase in m
O2
was almost a simple step function at the current temporal resolution
(Fig. 1B). This result was surprising since several other
studies have demonstrated a linear relationship between [NADH] and
m
O2 in mitochondria
(44, 52) and cells (53) in the steady state with varying substrate
delivery. These rapid kinetic effects of Ca2+ suggest that
the increase in NADH driving force alone was not linearly related to
m
O2, and some other
factors regulating ATP production must play a role. The likely
site of this activation is an increase in the
F0F1-ATPase synthetic flux in conjunction with the increase in CaDH as demonstrated previously (15).
The mechanism of Ca2+ activation of
F0F1-ATPase synthetic rate is unknown; however,
Ca2+ has been shown to modify cardiac mitochondrial volume
in vitro (31, 54). Halstrap and co-workers (55) have
suggested that mitochondrial volume changes observed in liver, and
stimulated by Ca2+, may play a role in activation of
oxidative phosphorylation through the pyrophosphate stimulation
pathway. Since matrix volume can be rapidly followed using light
scattering techniques (56), we used this approach to evaluate whether
matrix volume changes were kinetically associated with the observed
metabolic effects of Ca2+. Calcium additions resulted in a
rapid increase in light scattering consistent with an increase in
matrix volume and were concomitant with increases in [NADH] and
m
O2. Similar volume
effects of Ca2+ have been observed in liver (55, 57-62)
and heart (31, 54) mitochondria. It is unclear whether increases in
volume are due solely to matrix Ca2+ influx or to indirect
mechanisms such as the production of new matrix osmolytes, or changes
in other ion permeabilities. Since net Ca2+ entry into the
matrix in cardiac mitochondria is very low, with an estimated value of
~500 mOsm (see Fig. 7), it is therefore unlikely that it represents
the primary osmolyte which elicits these changes (63, 64). It is more
probable that the changes observed are reflective of either elevated
osmolyte production or an alteration in ion permeability of the inner
membrane, where Ca2+ serves as the second messenger. The
most likely ion permeability change would be for K+, as
this ion plays a key role in the regulation of mitochondrial volume.
Studies in liver mitochondria seem to support this contention showing
that changes in volume with Ca2+ are due to specific
alterations in K+ permeability (for review see Ref. 11).
The fact that the rate of light scattering
(m
s) was strongly dependent on [Ca2+], and IRT was not, may indicate that the effects of
Ca2+ on light scattering were occurring much faster than
the mixing time of the system resulting in the observed constant IRT
value as a function of Ca2+ concentration. Despite the
mechanism of Ca2+-induced volume changes, it is clear that
Ca2+ additions increased m
s,
with the largest magnitude change occurring between 172 and 535 nM, which was consistent with the calculated
K0.5 for [NADH] and
m
O2 effects with
Ca2+ (~175 nM; Table IV). Given this
correlation, it is reasonable to speculate that changes in matrix
volume could contribute to the stimulation of
m
O2 and
m
NADH observed and is consistent with
previous studies in rat liver mitochondria (see Ref. 55). Further
investigations are required to establish the role of matrix volume on
the Ca2+ metabolic effects in cardiac mitochondria.
For Ca2+ to serve effectively as a rapid modulator
(i.e. stimulate or inhibit) of mitochondrial oxidative
phosphorylation in the 25-600 nM concentration range, the
off-kinetics (i.e. removal of Ca2+) of the
process would need to be similar to the on-kinetics (i.e. addition of Ca2+) that were the focus of this study. We
attempted to estimate the off-kinetics with the addition of exogenous
EGTA to the chamber. This revealed that the metabolic effects of
Ca2+ were in fact reversible, but the kinetics was
significantly slower (1.55 ± 0.14 s, n = 6)
compared with the on kinetics (0.27 ± 0.02 s,
n = 43). The slower off kinetics might be due to the
incomplete removal of Ca2+ by the solubility limited
concentration of EGTA or the kinetics of EGTA Ca2+ binding.
A more rapid and complete trap of Ca2+ is required to
effectively quantitate the off-kinetics in this system. If the
off-kinetics for Ca2+ transport is slow, then this
phenomenon might contribute an "asymmetric" temporal response to
work or hypoxia transitions observed as metabolic overshoots in heart
(65, 66) and skeletal muscle (67).
It is interesting to compare the kinetics of Ca2+ with
Pi and ADP, the other putative cytosolic modulators (5) of
mitochondrial ATP production. The direct addition of near-saturating
levels of ADP or Pi alone resulted in a much slower
m
O2 response (>2 s) than Ca2+ additions (Fig. 2B). This implies
that the net effects of Ca2+ are occurring faster than the
initiation of serial transport of ADP or Pi into the matrix
and ADP phosphorylation by the F0F1-ATPase under near-optimal conditions. Similar kinetics for ADP activation of
ATP production (1-2.5 s) has been reported for rat liver mitochondria (68, 69). These data suggest that mitochondrial ATP production is more
responsive, kinetically, to changes in [Ca2+] than
[ADP] or [Pi]. The delay in ATP production after the
addition of ADP has been ascribed to the slow transport of ADP into the matrix space through the adenylate translocase. However, in heart mitochondria the translocase does not significantly contribute to the
overall rate limitation of ATP production since uncoupled rates of
respiration are similar to the State 3 rate (15, 44, 70). At State 3, even in the presence of extramitochondrial ATP, the ADP flux across the
inner membrane is ~580 nmol of ADP·nmol Cyta
1·min
1
(210 nmol of O2·nmol of
Cyta
1·min
1
and ADP·O
1 of 2.76). By assuming a
mitochondrial volume of 2.9 µl·nmol of Cyta
1 implies that the [ADP] is
increasing at the rate of 200 mM·s
1, approaching the ~0.1
mM Km for the
F0F1-ATPase (71) in less than ~2 ms. Thus,
the delay in ATP production with ADP additions is likely due to other
factors such as the buffering of ADP in the matrix (72) and the fact
that the ratio of ATP:ADP
1 is critical (73)
in the operation of cytochrome oxidase (74-77) as well as the
F0F1-ATPase (71, 78). Finally, since the
initial conditions these experiments had near-physiological levels of ATP (3.4 mM), the matrix ATP:ADP
1
ratio may have played a significant role in this delayed response to
ADP. If the matrix ATP was very high before the addition of ADP, the
large amounts of ADP would have to be exchanged before approaching the
free ATP:ADP
1 of ~2, as observed in intact
rat hearts (79) and human skeletal muscle (80). The time required to
reset the ATP:ADP
1 ratio could contribute to
the delay observed for the ADP effect on respiration.
Creatine has been suggested to be more efficient in delivering ADP to
the matrix than extramitochondrial ADP alone (19-21, 24, 81-83). In
this model, it has been suggested that local ADP synthesis, transfer to
adenine nucleotide translocase, and subsequent respiration was
preferential to simple exogenous bolus additions of ADP (81). Coupled
to cytosolic creatine kinase, this might lead to a faster cytosolic
transduction system than ADP alone. To test this hypothesis, creatine
in the presence of ATP was used to generate ADP via the endogenous CPK
in heart mitochondria. Using step increases of 10 mM
creatine in the presence of ATP and Pi resulted in very
slow IRT responses (15.6 ± 1.2 s) using our standard
conditions (buffer C). To ensure that the creatine kinase enzyme was
preserved in the mitochondrial membranes, several additional studies
were performed. A thiol (S-H)-reducing agent dithiothreitol (0.5 mM) was added (buffer E) to prevent auto-oxidation of the
CPK complex (34-36); however, no improvement in IRT was observed.
Alternatively, chloride in excess of (>50 mM) has been implicated in the loss of CPK function, which was reasoned to be due to
the detachment of CPK from the adenine nucleotide translocase complex in the inner mitochondrial membrane (29). To test this possibility, experiments were performed in only 32 mM
Cl
as the balanced anions to Na+,
Mg2+, and ATP. Under these conditions, the creatine IRT was
shorter (Fig. 2B). To investigate the effects of inorganic
ions further, experiments were performed in sucrose-based buffer,
i.e. buffers G and G0, after reconstitution in
either buffer B or buffer A, respectively. By using these systems,
results from our studies could then be directly compared with previous
findings for Cr respiration performed in similar media (20, 21, 24, 29,
38, 82, 84). Interestingly, the findings in buffers G and
G0 show a marked reduction in the creatine-driven IRT for
m
O2 values, which
were comparable to those observed for ADP in buffer C (Fig.
2B). Similarly,
m
O2 increased by
more than 100% for both preparations (Table III) in the sucrose
buffers and were comparable to previous work (20, 21, 29, 38, 82).
These findings are difficult to interpret, as the preparations are
absent of >120 mM K+ normally seen in the
cytosol, an ion known to play a major role in matrix volume regulation.
Interestingly, when one compares the Cr results from buffer F, which
contains physiologic levels of ions, with buffers G or G0,
it is clear that only under the extreme conditions of low
K+ and Cl
does one achieve elevated Cr rates
and faster IRT (Table III), and this suggests that great caution be
exercised when comparing results in dissimilar media. In any event,
under optimal conditions for Cr, the IRT for
m
O2 was still 2 orders of magnitude longer than for Ca2+ in our studies.
Combined, these data illustrate that the metabolic effects of
Ca2+ occur at least 2 orders of magnitude faster than all
other putative cytosolic signaling molecules tested and lend credence
to the hypothesis that Ca2+ may be capable of responding to
step changes in cytosolic work.
In the current study, the IRT was estimated for Ca2+
transport, and metabolic effects were found to occur well under 200 ms. Traditionally, matrix accumulation of Ca2+ has been thought
to occur too slowly to respond to cyclic changes in
[Ca2+]c (64, 85, 86). This discrepancy might in
part be due to the methodological differences between studies. One issue has been the temperature at which these studies are conducted. The current studies were performed at 37 °C, whereas much of the earlier work (87-91) was performed at significantly lower
temperatures. Temperature correcting these data, assuming a linear
dependence on temperature and a Q10 of 2, does
not resolve this issue, since these transport studies are still
5-7-fold slower. One possibility is that the Ca2+
transport process is not linearly dependent on temperature due to phase
transitions in the inner membrane lipid (92); however, this needs to be
experimentally evaluated. All of the Ca2+ observed effects
in the current study could only be observed if the mitochondria were
prepared with great care not to expose them to large concentrations of
Ca2+ (i.e. in situ perfusion of heart
with Ca2+ buffer solutions) as well as an ~6-min
Ca2+ depletion at 37 °C in the absence of carbon
substrates. This could result in much different initial conditions for
matrix Ca2+ levels when compared with previous work. Some
evidence for rapid Ca2+ uptake mechanisms has been found in
mitochondria both in vitro and in intact cells. Recent work
by Gunter and co-workers (46, 47) demonstrated mitochondrial
Ca2+ uptake mechanisms in liver with similar kinetics as
those described in the current study, and which showed reduced RuRed
sensitivity. Similarly, studies in RHOD 2-loaded intact rabbit myocytes
have also demonstrated RuRed insensitivity (45), suggesting this mechanism of transport is not an artifact of the isolation process. Moreover, studies in isolated astrocytes (89), neuroblastomas (93),
hepatocytes (87), and cardiac myocytes (45, 88, 94-97) have also shown
rapid matrix Ca2+ accumulation that is linked to phasic
cycling of cytosolic Ca2+ waves. Most interestingly,
Rizzuto et al. (98, 99), using a targeted
Ca2+-sensitive fluorescent protein to complex IV, have
shown that hormone-induced SR (ER) Ca2+ release propagates
directly into the mitochondria in approximately 1 s. Moreover,
work in SR and complex IV co-loaded HeLa cells illustrates the close
coupling of SR Ca2+ release and mitochondrial uptake (100).
These latter results suggest that more rapid mechanisms for
Ca2+ uptake may be present in cardiac mitochondria than
suggested by previous work. Further characterization of these rapid
Ca2+ transport mechanisms will be required to establish the
molecular mechanisms and pathways involved.
The current work is consistent with a rapid mechanism for the
modulation of matrix Ca2+ and mitochondrial oxidative
phosphorylation by extramitochondrial Ca2+. The rapid
metabolic effects of Ca2+ observed were consistent with
both the CaDH and F0F1-ATPase activation as
described previously (15) for steady state oxidative phosphorylation. The speed of this process has only been estimated to occur in less than
200 ms due to technical limitations of the current experimental approach; however, this time constant is more than adequate to have
extramitochondrial Ca2+ regulate oxidative phosphorylation
during work transitions in the heart (for example see Ref. 101). The
[Ca2+] dependence of this process is consistent with the
physiological variations in Ca2+ found in intact cells
assuming no intracellular compartmentation of SR calcium release around
the mitochondria. However, the morphological basis for a more direct
coupling between mitochondrial Ca2+ and SR release in the
heart is clearly present as previously proposed by Rizzuto
et al. (100) in other systems. Both scanning and
transmission electron micrographs have shown a close association of the
SR with mitochondria located between the Z lines of each myocyte
sarcomere. An example from one of these studies is presented in Fig.
8. This tight morphological association
of the SR with the mitochondria might further enhance the coupling of
matrix Ca2+ and associated metabolic effects with the
contraction and active ion transport processes also activated by the SR
Ca2+ release, resulting in a balanced activation of
metabolism with the mechanical and ion transport work in the
myocyte.

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 8.
Electron micrographs from heart tissue.
A, transmission electron micrograph of porcine heart tissue
fixed with 2.5% glutaraldehyde and thin-sectioned as described under
"Materials and Methods." Symbols indicate an individual
mitochondrion (M) and cross-sections through the
sarcoplasmic reticulum (SR), and bar represents
0.5 µm. B, scanning electron micrograph of longitudinal
section of canine cardiac muscle, adapted from Ref. 102.
Symbols indicate the thin filamentous SR that encompasses
the individual mitochondria (M) arranged longitudinally
along the myofibrils, and the bar represents 0.5 µm.
|
|