Institute for Biodiagnostics, National Research Council, Winnipeg, Manitoba R3B 1Y6, Canada
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The rubidium efflux from hypothermic rat hearts perfused by the
Langendorff method at 20°C was studied. At this
temperature 87Rb-NMR efflux experiments showed the
existence of two 87Rb pools: cytoplasmic and mitochondrial.
Rat heart mitochondria showed a very slow exchange of mitochondrial
Rb+ for cytoplasmic K+. After washout of
cytosolic Rb+, mitochondria kept a stable Rb+
level for >30 min. Rb+ efflux from mitochondria was
stimulated with 0.1 mM 2,4-dinitrophenol (DNP), by sarcolemmal
permeabilization and concomitant cellular energy depletion by saponin
(0.01 mg/ml for 4 min) in the presence of a perfusate mimicking
intracellular conditions, or by ATP-sensitive K (KATP)
channel openers. DNP, a mitochondrial uncoupler, caused the onset of
mitochondrial Rb+ exchange; however, the washout was not
complete (80 vs. 56% in control). Energy deprivation by saponin, which
permeabilizes the sarcolemma, resulted in a rapid and complete
Rb+ efflux. The mitochondrial Rb+ efflux rate
constant (k) decreased in the presence of glibenclamide, a
KATP channel inhibitor (5 µM;
k = 0.204 ± 0.065 min1; n = 8),
or in the presence of ATP plus phosphocreatine (1.0 and 5.0 mM,
respectively; k = 0.134 ± 0.021 min
1;
n = 4) in the saponin experiments (saponin only;
k = 0.321 ± 0.079 min
1; n = 3),
indicating the inhibition of mitochondrial KATP channels. Thus hypothermia in combination with 87Rb-NMR allowed the
probing of the mitochondrial K+ pool in whole hearts
without mitochondrial isolation.
rubidium ion permeability; hypothermia; mitochondria; ATP-sensitive potassium channels; energy depletion; nuclear magnetic resonance
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TO ACHIEVE A HIGH TRANSPLANTATION success rate, organs are often preserved under conditions that significantly limit metabolism or decrease their metabolic rates. One of the standard preservation procedures involves hypothermia (25), whereas other techniques rely on reducing the ion gradients across the cell membrane (13). Since the discovery of sarcolemmal ATP-sensitive K+ (KATP) channels by Noma (23), research has focused on the regulation of K+ fluxes by means of these channels (9, 10, 12). Modulation of KATP channel activity could be beneficial during organ transplantation or ischemic events. With the discovery of mitochondrial KATP channels in the inner mitochondrial membrane, regulation of K+ fluxes across the mitochondrial membrane could be manipulated without the use of ionophores (15). Although K+ channels and their activity in both the sarcolemma and the inner mitochondrial membrane have been studied, not much is known about the regulation of K+ fluxes between the cytoplasm and the mitochondrial matrix, especially in intact tissues. The study of transmitochondrial K+ fluxes relies on the separation of these fluxes from those occurring across the sarcolemma. The separate detection of these fluxes has not been achieved.
Recently we observed a K+ efflux anomaly in rat hearts cooled to 20°C by using 87Rb as a K+ congener (11). Our 87Rb-NMR measurements showed incomplete Rb+ efflux with a dramatically (5-fold) increased rate constant at 20°C compared with that for efflux observed at 36°C (11). The Rb+ plateau reached after this initial rapid washout remained stable for >30 min. This observation led to the hypothesis that at 20°C residual Rb+ is of mitochondrial origin. Additional evidence for this phenomenon was obtained from 31P-NMR spectra. These measurements revealed the appearance of Pi resonance in a compartment with a pH higher than that of the cytoplasm. This additional resonance could originate from an alkaline mitochondrial pool.
Kinetic studies of 42K+ efflux from hearts have failed to disclose an ionic fraction that might reflect the loss of K+ from a large mitochondrial compartment with a relatively low permeability (20). However, Altschuld et al. (2) reported that a relatively constant amount of mitochondrial K+ was produced when isolated rat heart cells were washed with cold medium. Conditions that produce low levels of ATP, e.g., uncoupling of the mitochondria, also promoted a loss of mitochondrial K+. The authors concluded that mitochondrial K+ is not in equilibrium with that in the cytosol, even after long periods of incubation.
This work presents data from studies of hypothermic rat hearts perfused by the Langendorff method at 20°C. K+ transport was monitored by using its biological congener, 87Rb (1, 18). In addition to the study of Rb+ fluxes, intracellular H+ concentration ([H+]) was measured by 31P-NMR, which also provided information on the energy status of the heart during the experiments. The measurements indicate the existence of at least two intracellular Rb+ pools at 20°C. One of these pools was attributed to mitochondrial Rb+. This pool showed efflux kinetics much slower than that of the cytosol. Rb+ efflux from the mitochondrial pool was stimulated by a variety of methods to exchange Rb+ for K+ in the cytosol, including cellular energy depletion and a method involving KATP channel openers.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experiments performed in this study were carried out in accordance with the Guide for the Care and Use of Experimental Animals, published by the Canadian Council on Animal Care (2nd ed., Ottawa, ON, Canada, 1993).
Solutions. 87Rb- and 31P-NMR experiments were performed on hearts perfused with a phosphate-free Krebs-Henseleit (KH)-K buffer containing (in mM) 118 NaCl, 25 NaHCO3, 4.7 KCl, 1.75 CaCl2, 1.2 MgSO4, 0.5 EDTA (Sigma, St. Louis, MO), and 11 glucose. The perfusate was equilibrated with a mixture of O2 and CO2 gas, the flow ratio of which was manually adjusted to maintain a buffer pH of 7.4 at all temperatures. Perfusate pH was continuously monitored throughout the experiments with a pH meter. For 87Rb-NMR loading studies the perfusate contained (in mM) 3.76 KCl and 2.14 RbCl (KH-Rb) (Sigma) instead of 4.7 mM KCl, resulting in a 36% substitution of K+ with Rb+. Extracellular K+ replacement by 20, 36, or 100% Rb+ results in only a slight decrease in heart rate (~10 and ~20% for 36 and 100% Rb+ substitution, respectively) and no changes in the systolic and diastolic pressures or in the 31P-NMR spectrum (17-19). In addition, in vivo, in the chronic case, experiments showed that substitution of 30-40% of the intracellular K+ with Rb+ is not toxic (22). As the present work focuses on the properties of a second Rb+ pool (possibly mitochondrial), changes in intracellular water volume in response to Rb+ loading and washout were not measured. Switching from Rb+ loading with KH-Rb to Rb+ washout with a medium containing no Rb+ results in a change in total [K+] plus [Rb+] from 5.9 to 4.7 mM. This results in a return of the slightly depolarized membrane potential during Rb+ loading to that characteristic of control conditions. Changes in membrane potential are fast, and a new equilibrium will be established during the first few minutes of the washout. Because of the nonvanishing exchange time of perfusates in the extracardiac space (see below), the equilibration time of the membrane potential is easily absorbed by the protocol. In a recent publication (19) describing the opposite experiment, an increased (from 5.9 to 21 mM) extracellular [K+] was shown not to change the Rb+ washout kinetics, compared with that of the control. Extracellular [K+] is thus not critical for Rb+ efflux kinetics. Rb+ efflux studies were performed with several perfusates. The control efflux was studied with Rb+-free KH, whereas two other media were used after the induction of Rb+ efflux by saponin (Sigma). After saponin treatment, the perfusion medium was switched to a solution mimicking an intracellular composition of metabolites. Two different media were used for this purpose. The first intracellular medium (ICM1) contained (in mM) 25 NaHCO2, 25 KCl, 100 C6H11O7K (potassium gluconate; Sigma), 20 taurine (Sigma), 2 C3H3O3Na (sodium pyruvate; Sigma), 1.2 MgSO4, and 1.0 EGTA (Sigma). The second medium (ICM2) was the same as ICM1, except that it contained 2.2 mM MgSO4, 5 mM disodium phosphocreatine (PCr), and 1.0 mM ATP (the last 2 from Sigma). All reagents were purchased from BDH (Toronto, ON, Canada) unless mentioned otherwise. Saponin, glibenclamide, bumetanide, and 2,4-dinitrophenol (DNP; all from Sigma) were added to the perfusion buffer as aqueous solutions through separate infusion lines. The concentrations in the stock solutions were 0.6 mg/ml for saponin, 0.6 mM for both glibenclamide and bumetanide, and 6 mM for DNP. The final concentrations in the perfusate for these four agents were 0.01 mg/ml, 5 µM, 10 µM, and 0.1 mM, respectively.
Heart perfusion.
Male Sprague-Dawley rats with an average body weight of 350 ± 28 g
(n = 34) and an average heart wet weight of 1.56 ± 0.24 g
were used. The rats were anesthetized with pentobarbital (120 mg/kg
body wt ip). After anesthesia, the heart was quickly removed and
retrogradely perfused via the aorta at a constant flow of 10-12
ml · min1 · g wet wt
1. After
placement of a left ventricular apical drain, a water-filled balloon
was inserted through the mitral valve into the left ventricle. The
balloon was connected to a Statham P23Db pressure transducer and a
Digi-Med model 210 heart performance analyzer (Micro-Med, Louisville,
KY), which allowed monitoring of the heart rate (HR) and left
ventricular pressure (LVP). Functional parameters such as HR, LVP,
end-diastolic pressure (EDP; set to 5-10 mmHg), and systolic
pressure were recorded continuously. An ultrasonic flowmeter (Transonic
Systems, Ithaca, NY) in the aortic inflow line allowed the coronary
flow to be monitored. Perfusion pressure was measured continuously
through the catheter connecting the aortic line and the pressure transducer.
NMR spectroscopy.
All NMR experiments were performed on a Bruker 360 AM spectrometer.
87Rb spectra were acquired at 117.8 MHz by using a 20-mm
broad band dedicated probe from Morris Instruments (Gloucester, ON,
Canada). 31P spectra were obtained with a dedicated Bruker
20-mm 13C/31P probe tuned to 145.8 MHz. The
signal of 23Na present in the heart and perfusate was used
to shim the field. By using a spectral width of 18 kHz, a recycle time
of 10 ms, and a pulse length of 55 µs (90° pulse length),
87Rb-NMR spectra were acquired in 2-min blocks containing
256 data points zero-filled to 512 points (18). 87Rb spin
lattice relaxation times (T1) in rat hearts typically are 2-3 ms
(1). With a recycle time of 10 ms the 87Rb resonance was
not saturated. As a standard, a capillary containing 10 µl of a
solution composed of 1 M RbCl plus 5 M KI was included in the setup.
I served as a shift reagent to shift the
87Rb resonance of the standard away from that of the heart
(1). To minimize the signal from extracellular 87Rb during
signal acquisition, the hearts were perfused in the "dry mode."
In this approach, the superfusion line placed at the bottom of the NMR
tube was used as a suction line to remove fluid surrounding the heart.
As a result, the contribution from the extracellular component was
reduced to ~16% of the total equilibrium Rb+ signal
after loading at 36°C. 31P spectra were obtained by using
a spectral width of 10 kHz and a 24-µs pulse length (60° flip
angle), and 4,096 data points per scan were collected. The acquisition
of 360 scans with a recycle time of 2.0 s per scan took 12 min. In some
experiments, after saponin infusion, 31P-NMR spectra were
collected continuously every 3.25 min in 96 scans, with a recycle delay
of 2.0 s. During the experiments the temperature of the heart was
monitored continuously by means of a copper-constantan thermocouple
(Omega Engineering, Stamford, CT) placed in the right ventricle. At the
bore of the magnet the thermocouple was connected to a low-pass filter
to minimize the noise picked up by the wires outside of the magnet.
Experimental protocols. After stabilization of mechanical function (~10 min) the hearts were subjected to the following protocols. Rb+ accumulation and washout were measured by using the two protocols shown in Fig. 1. Protocol A started with a 60-min Rb+ accumulation at 36°C. After Rb+ loading the temperature was changed to 20°C, and this was followed by a 30-min equilibration period during which the hearts were still perfused with KH-Rb. After this equilibration period 87Rb-NMR spectra were obtained for a 10-min period. Subsequently, the perfusate was switched to a (Rb+-free) KH-K buffer, and Rb+ efflux was monitored during the next 40 min. A second protocol (schematics are shown in Fig. 1B) began with hearts preequilibrated to 20°C. This protocol facilitated the measurement of Rb+ influx and efflux at 20°C. The perfusion time with KH-Rb at 20°C was extended to 90 min to allow equilibration of the hearts with Rb+ at this temperature.
|
Measurement of Rb+ fluxes: kinetics. To measure Rb+ uptake, the perfusate was switched from KH to KH-Rb and the experimental apparatus was changed to the "dry-mode," which reduced the contribution of Rb+ present in the bath to the observed NMR signal. With a coronary flow of 15 ml/min, the perfusate reached the heart in 3 min, after which NMR data acquisition was started. The duration of KH-Rb perfusion was determined by the protocol (Fig. 1). To measure Rb+ efflux, perfusion was switched to the KH solution. After a 3-min delay (flow of 12-15 ml/min, depending on temperature) NMR data acquisition was started. Analysis of the data from the Rb+ influx and efflux experiments did not include data acquired during the first 4 min. During the first 4 min multiple kinetics resulting from the rapid equilibration of the bath and the slower kinetics of ion washout or uptake from the heart are observed (18).
Before Fourier transformation, exponential multiplication with a line broadening of 50 Hz was applied to the NMR data. All NMR data were processed by using UXNMR software (Bruker, Rheinstetten, Germany). Resonance area and amplitude reliably represent the amount of intracellular Rb+ in the heart. The time dependence of the resonance amplitude was then fitted to a three-parameter equation as described below. A similar approach was used by Allis et al. (1). The Rb+ washout curves obtained from the experiments were fitted to a three-parameter expression
![]() |
(1) |
Statistical analysis. Data are represented as means ± SD. The number of experiments averaged is indicated by n. To compare data between different groups, Student's t-test was performed and P < 0.05 was taken as a significant difference between the groups.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rb+ compartmentation caused by hypothermia. Cooling the hearts from 36 to 20°C resulted in a change in Rb+ efflux kinetics. In a control experiment (Fig. 2A), washout of Rb+ with Rb+-free KH-K was incomplete after the heart was loaded with Rb+ according to protocol B. Only 44 ± 2% of the Rb+ present after loading could be washed out, leaving a Rb+ plateau (11). This part of the experiment is referred to as the first phase of the washout. The remaining cellular pool of Rb+ (the 2nd pool) remained stable for >30 min of perfusion with Rb+-free KH. The second Rb+ pool and its size were independent of the loading temperature (36°C for protocol A and 20°C for protocol B). To investigate the properties of this second Rb+ pool, the effects of agents that increase sarcolemmal permeability were tested. After washout of the first Rb+ pool, the washout of the second Rb+ pool could be initiated through a 4-min infusion of 0.01 mg/ml saponin. Saponin is known to permeabilize the sarcolemma (10). Upon the infusion of saponin, the perfusion medium was changed to a medium mimicking intracellular conditions (ICM1). This led to the disappearance of the second Rb+ pool and resulted in a complete washout of Rb+ (Fig. 2B).
|
|
Changes in phosphates and cytosolic pH during hypothermia and
saponin treatment.
31P-NMR spectra were acquired at 36 and 20°C. The spectra
did not show any large changes in high-energy phosphates during the transition from 36 to 20°C. The cytosolic pH was estimated from the
chemical shift of the Pi resonance relative to that of PCr (11). At 36 and at 20°C, pH values of 7.05 ± 0.04 and
7.21 ± 0.05, respectively, were measured. Figure
4 shows the time dependence of the
high-energy phosphate signal intensities after switching to ICM1
perfusate and saponin infusion (n = 3). After saponin infusion the Pi resonance vanished rapidly while the
resonance amplitudes of ATP and the PCr decayed more slowly. Figure 4
shows that both - and
-ATP diminish significantly in amplitude
but do not vanish during the 30-min observation period. During this time a large portion of the second Rb+ pool was washed out
(see Fig. 1). The data on the decay of the PCr and
-ATP signal
amplitudes (Fig. 4) were fitted to a single-exponential function as
given by Eq. 1. For PCr and
-ATP, rate
constants of 0.164 ± 0.018 and 0.128 ± 0.027 min
1,
respectively, were obtained. Although Pi was washed out,
both PCr and ATP signals remained after 30 min of ICM1 perfusion and had relative amplitudes of 34 and 31%, respectively, of their initial
values. Figure 4, inset, shows the relative decay of the total
integrated 31P-NMR signal intensity as a function of time.
This integral was set at 100% before saponin infusion. During the
course of the experiment the total integral decayed to ~40% of the
initial amplitude with a rate constant of 0.223 ± 0.082 min
1. Throughout the experiment the ratio of the PCr
signal to the ATP signal remained
1.
|
Heart function.
At 36°C, before Rb+ loading, the hearts showed an average
pressure-rate product (PRP) of 27.4 ± 2.8 mHg · beats · min1
(n = 18). In agreement with previous
observations (18), Rb+ loading slightly decreased (~10%)
the HR and did not affect systolic pressure and EDP. After
equilibration to 20°C, PRP was 6.4 ± 1.5 mHg · beats · min
1 (n = 5) as a
consequence of the decrease in HR from 245 ± 30 beats/min at 36°C
to 52 ± 16 beats/min, whereas left ventricular developed pressure
remained constant, from 112 ± 10 to 120 ± 27 mmHg,
due to an increase in systolic pressure. As the hearts were cooled from 36 to 20°C, EDP increased by a factor of
three to four from 10.2 ± 1.7 to 36.8 ± 5.6 mmHg (n = 8).
A simultaneous increase in systolic pressure from 106 ± 7 to 147 ± 5 mmHg was observed, in agreement with previous
observations (20). The parallel increase in EDP and systolic pressure
upon cooling the hearts to 20°C may reflect an increase in
intracellular [Ca2+] or sensitivity of the myofibrils to
Ca2+. Saponin treatment resulted in a complete loss of
heart function due to permeabilization of the myocyte sarcolemma. After
saponin infusion, perfusion pressure increased from 129 ± 6 to 165 ± 25 mmHg (n = 5), whereas EDP further increased to 45 ± 12 mmHg (n = 5), indicating vasoconstriction. After saponin
treatment the hearts appeared white, probably because of a loss of myoglobin.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
87Rb-NMR has been shown to be a useful tool in the study of
K+ fluxes in rat hearts and has been shown to accurately
reflect K+ movement across the sarcolemma with 100%
visibility (3, 18). Sarcolemmal Rb+ rate constants obtained
during normothermic experiments are in excellent agreement with rate
constants previously published (18). Neither 87Rb
visibility data nor rate constants have been published for mitochondria; however, measurements of cardiomyocytes (cytoplasm plus
organelles) indicate that 87Rb is also visible in
mitochondria (3, 18). A recent study of the temperature dependence of
monovalent cation fluxes in rat hearts showed incomplete washout of
Rb+ from hearts equilibrated at 20°C (Fig. 2A)
(11). These results were interpreted in terms of a large difference in
Rb+ permeability between the sarcolemma and the inner
mitochondrial membrane. Other cell compartments are less able to
furnish an explanation for these observations because they have a
negligibly small volume fraction and do not maintain a large membrane
potential or pH gradient. Thus, at 20°C, the sarcolemmal permeability
was assumed to be much larger than that observed for the inner
mitochondrial membrane. It was also shown that at this temperature the
size of the mitochondrial Rb+ pool was comparable to that
of the cytoplasmic pool, 56 and 44% of the total observed
Rb+, respectively. Mitochondria are known to swell upon
hypothermia and occupy ~30% of the total cell volume in rat
ventricular myocytes and hepatocytes at 37°C (5, 14, 16). In
addition, mitochondria are known to have a high K+ content,
corresponding to a [K+] 180 mM (7). NMR measures the
number of nuclei present and not their direct
concentration. Mitochondria thus represent the perfect compartment to
contain this nonremovable Rb+ pool observed in the 20°C
Rb+ efflux experiments. Research of Altschuld et al. (2)
has shown that the number of K+ in rat heart
mitochondria remains relatively constant when cellular K+
is depleted by washing the cells with cold perfusate. This observation is in agreement with our observations.
To study the pool of Rb+ that remains in myocytes at 20°C, the experiments described in this work were performed. Two different approaches are discussed. In one approach mitochondrial ion metabolism was manipulated directly with DNP or a KATP channel opener. In the second approach the myocardial sarcolemma was permeabilized with saponin, which led to cytoplasmic energy deprivation. This depletion of PCr and ATP was expected to stimulate the mitochondrial KATP channels and consequently enable Rb+ efflux in the second phase of the washout experiments.
The experiment shown in Fig. 2C displays Rb+ efflux stimulated by 0.1 mM DNP at 20°C; the results indicate a specific role for mitochondria in this process. DNP is known to prevent ATP synthesis through uncoupling of the protonmotive force, resulting in a collapse of the H+ gradient. Arresting mitochondrial ATP production should result in a decrease in [ATP] in both the matrix and the cytoplasm and could result in an opening of mitochondrial KATP channels. These channels are known to have ATP regulatory sites facing the cytosol (7). The K+/H+ antiporter could also increase its activity due to the uncoupling of the protonmotive force. These two effects could allow previously trapped mitochondrial 87Rb to exchange with cytosolic K+ and then with extracellular K+ during the efflux experiment. This is schematically represented by Fig. 5, A and B. Direct stimulation of KATP channels, without the disruption of the protonmotive force, by using the opener diazoxide resulted in only a slow release of Rb+ from the mitochondrial pool. However, in experiments with reconstituted channels, diazoxide has recently been reported to be ~3 orders of magnitude more effective in opening mitochondrial KATP channels than in opening sarcolemmal channels (7). Our measurements at 20°C did not show a large effect of 20-60 µM diazoxide on Rb+ efflux. Similar observations were made by Szewczyk et al. (26), who also reported diazoxide to be less effective in opening rat liver mitochondrial KATP channels. A similar mild stimulation was observed with the antibiotic nigericin, which promotes K+/H+ exchange across the inner mitochondrial membrane (24). At 20°C, however, nigericin might not be distributed effectively across the inner mitochondrial membrane. Our experimental results are consistent with those of Gamble (6), who first showed that the level of K+ exchange in nonrespiring mitochondria is low and that exchange can be stimulated by respiration. Stimulation of respiration results in increased activity of the protonmotive force and the K+/H+ antiporter. The net result of this increase in activity is a decrease in the average residence time of mitochondrial K+. However, a reduction in respiration alone cannot explain all our observations because at 10°C no 87Rb plateau was observed during the washout (11).
|
Another way of opening KATP channels is through depletion of cellular ATP by saponin, schematically shown in Fig. 5C. Saponin forms large holes in cholesterol-rich membranes through the formation of large molecular complexes with cholesterol (8). The sizes of these holes, which can be as large as 80 Å in diameter, allows the exchange of many metabolites, such as ions and high-energy phosphates, between the compartments separated by the membrane. A 4-min infusion with a low saponin concentration of 0.01 mg/ml after reaching the Rb+ plateau already resulted in the onset of an additional Rb+ efflux in the second phase. That saponin indeed caused large holes in the sarcolemma was supported by the color of the hearts after the final washout, which was white, the hearts likely having lost most of their myoglobin. From Fig. 4 it is clear that saponin treatment results in a rapid loss of all cell phosphates as revealed by the disappearance of Pi, in addition to the decay in ATP and PCr levels. The inner mitochondrial membrane does not contain any cholesterol; this membrane therefore presumably remains intact during the experiment, allowing the mitochondria to maintain the synthesis of ATP under appropriate conditions. To maintain intact mitochondria, the perfusate used for Rb+ efflux studies was switched to ICM1 upon saponin infusion. This ensured that the mitochondria were submerged in a medium mimicking the cytosol and energized by 2 mM pyruvate present in ICM1.
The washout of PCr and ATP (Fig. 4) occurred at similar rates, indicating enzymatic coupling between the two high-energy phosphates in addition to sustained mitochondrial ATP production after saponin permeabilization of the sarcolemma. There was a residual amount of PCr and ATP, and the 31P-NMR spectrum integral contains contributions of two pools. The first pool comprises contributions from the cytoplasm and mitochondria in intact cells that were not affected by saponin. The second pool comprises mitochondrial ATP in permeabilized cells. It is uncertain if this last pool is visible by NMR. If mitochondrial ATP were not visible by NMR, the percentage of permeabilized cells would be equal to the depletion in the total phosphate signal intensity (60%; Fig. 4, inset). However, if mitochondrial ATP were visible by NMR, permeabilization by saponin would exceed 60% because a fraction of the total phosphate signal is mitochondrial and not removed by saponin treatment. In reality we have a spectrum of cells with different degrees of permeabilization and different degrees of phosphate loss. It is not possible that ~40% of the cells remain normal and nonpermeabilized, because saponin was successful in the complete removal of residual Rb+. In other words, varying degrees of high-energy phosphate loss must have taken place in the entire cell population, causing complete Rb+ washout from the mitochondria.
Figure 2B shows that saponin treatment of the second Rb+ pool results in complete washout of this mitochondrial Rb+ in the second phase. Part of this efflux is apparently regulated by mitochondrial KATP channels because 5 µM glibenclamide reduced the efflux rate constant by ~40% (Fig. 3). Other evidence for ATP-regulated Rb+ efflux was obtained from perfusion experiments with ICM2, which partially restored cytoplasmic ATP levels (Fig. 3). When ICM2 was used, the observed Rb+ efflux rate constant was significantly smaller (by ~60%) than that obtained with ICM as the perfusate. However, perfusion with ICM1 plus 5 µM glibenclamide and ICM2 did not result in significantly different Rb+ efflux rate constants. These experiments show that a considerable part of the final Rb+ washout in the second phase is regulated by mitochondrial KATP channels. Inasmuch as the sarcolemmal K+ permeability was significantly increased by saponin treatment, these KATP channels must reside in the inner mitochondrial membrane. The remainder of the Rb+ efflux could be provided through the K+/H+ exchanger pathway (7).
A possible explanation for the observed Rb+ plateau in efflux experiments carried out at 20°C may be related to the thermodynamics of lipid bilayers at this temperature. The sarcolemma contains a large cholesterol fraction, whereas the inner mitochondrial membrane shows no presence of cholesterol. Cholesterol is known to smoothen or remove phase transitions in the bilayer system (4). McMurchie et al. (21) observed a discontinuity in the Arrhenius behavior of the state 3 and 4 rates of respiration in isolated rabbit heart mitochondria at ~20°C. This discontinuity was explained in terms of a membrane phase transition. Such a phase transition could reduce the chemical exchange of ions between the cytoplasm and the mitochondrion, resulting in an alkalinization of the latter. It is likely that the observation of an incomplete Rb+ efflux at 20°C is related to the observations of McMurchie et al. (21). An additional 87Rb-NMR experiment showed that this Rb+ plateau could also be removed by either cooling or warming the heart (11).
Conclusion. Using 87Rb as a biological K+ congener, we monitored mitochondrial K+ efflux in the perfused rat heart at 20°C without the superposition of transsarcolemmal fluxes. Myocardial hypothermia at 20°C caused mitochondria to retain previously accumulated Rb+ for >30 min. This phenomenon allowed the complete exchange of cytosolic Rb+, leaving the mitochondrial Rb+ pool unchanged. Rb+ efflux from this pool could be stimulated by cellular energy depletion or direct manipulation of mitochondrial ion metabolism. The addition of ATP and PCr to the perfusate or the addition of glibenclamide reduced the Rb+ efflux rate constant relative to that resulting from perfusion without ATP and PCr or glibenclamide. This reduction in rate constant indicates the inhibition of mitochondrial KATP channels.
These experiments allow a more detailed study of mitochondrial K+ fluxes. By using hypothermia, mitochondria can be prepared in a Rb+-loaded state, whereas the cytoplasmic pool can be exchanged for K+. Fast temperature switching would then allow the study of mitochondrial K+ fluxes in intact cell systems under physiological conditions. ![]() |
ACKNOWLEDGEMENTS |
---|
This study was funded in part by a grant from the Heart and Stroke Foundation of Manitoba, Canada.
![]() |
FOOTNOTES |
---|
Present address of M. L. H. Gruwel: Plant Biotechnology Institute, National Research Council, Saskatoon, SK S7N 0W9, Canada. E-mail: marco.gruwel{at}nrc.ca.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: V. V. Kupriyanov, National Research Council, Institute for Biodiagnostics, 435 Ellice Ave., Winnipeg, MB R3B 1Y6, Canada.
Received 17 February 1998; accepted in final form 29 September 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Allis, J. L.,
C. D. Snaith,
A.-M. L. Seymour,
and
G. K. Radda.
87Rb NMR studies of the perfused rat heart.
FEBS Lett.
242:
212-217,
1989.
2.
Altschuld, R.,
C. Hohl,
A. Ansel,
and
G. P. Brierley.
Compartmentation of K+ in isolated adult rat heart cells.
Arch. Biochem. Biophys.
209:
175-184,
1981[Medline].
3.
Cross, H. R.,
G. K. Radda,
and
K. Clarke.
The role of Na+-K+-ATPase activity during low flow ischemia in preventing myocardial injury: a 31P, 23Na and 87Rb NMR spectroscopic study.
Magn. Reson. Med.
34:
673-685,
1995[Medline].
4.
Davis, J. H.,
M. Bloom,
K. W. Butler,
and
I. C. P. Smith.
The temperature dependence of molecular order and the influence of cholesterol in Acholeplasma laidlawii membranes.
Biochim. Biophys. Acta
597:
477-491,
1980[Medline].
5.
Dufour, S.,
E. Thiaudière,
G. Vidal,
J.-L. Gallis,
N. Rousse,
and
P. Canioni.
Temperature dependence of NMR relaxation times of nucleoside triphosphates and inorganic phosphate in the isolated perfused rat liver. Effect on Pi compartmentation.
J. Magn. Reson.
B113:
125-135,
1996.
6.
Gamble, J. L.
Potassium binding and oxidative phosphorylation in mitochondria and mitochondrial fragments.
J. Biol. Chem.
228:
955-971,
1957
7.
Garlid, K. D.
Cation transport in mitochondriathe potassium cycle.
Biochim. Biophys. Acta
1275:
123-126,
1996[Medline].
8.
Glauert, A. M.,
J. T. Dingle,
and
J. A. Lucy.
Nature
196:
953-955,
1962.
9.
Gross, G. J.,
and
J. A. Auchampach.
Role of ATP dependent potassium channels in myocardial ischemia.
Cardiovasc. Res.
26:
1011-1016,
1992[Medline].
10.
Grover, G. J.
Protective effects of ATP-sensitive potassium-channel openers in experimental myocardial ischemia.
J. Cardiovasc. Pharmacol.
24:
S18-S27,
1994[Medline].
11.
Gruwel, M. L. H., B. Kuzio, B. Xiang, R. Deslauriers, and V. V. Kupriyanov. Temperature dependence of monovalent cation
fluxes in isolated rat hearts: a magnetic resonance study. Biochim.
Biophys. Acta. In press.
12.
Hearse, D. J.
Activation of ATP-sensitive potassium channels: a novel pharmacological approach to myocardial protection?
Cardiovasc. Res.
30:
1-17,
1995[Medline].
13.
Hearse, D. J.,
M. V. Braimbridge,
and
P. Jynge.
Protection of the Ischemic Myocardium: Cardioplegia. New York: Raven, 1981.
14.
Hoek, J. B.,
D. G. Nicholls,
and
J. R. Williamson.
Determination of the mitochondrial protonmotive force in isolated hepatocytes.
J. Biol. Chem.
255:
1458-1464,
1980
15.
Inoue, I.,
H. Nagase,
K. Kishi,
and
T. Higuti.
ATP-sensitive K+ channel in the mitochondrial inner membrane.
Nature
352:
244-245,
1991[Medline].
16.
Kim, H. D.,
C. H. Kim,
B. J. Rah,
H. I. Chung,
and
T. S. Shim.
Quantitative study on the relation between structural and functional properties of the hearts from three different mammals.
Anat. Rec.
238:
199-206,
1994[Medline].
17.
Kupriyanov, V. V.,
J. Shen,
B. Xiang,
B. Kuzio,
J. Sun,
and
R. Deslauriers.
Three-dimensional 87Rb imaging of isolated pig hearts: effects of regional ischemia.
Magn. Reson. Med.
40:
175-179,
1998[Medline].
18.
Kupriyanov, V. V.,
L. C. Stewart,
B. Xiang,
J. Kwak,
and
R. Deslauriers.
Pathways of Rb+ influx and their relation to intracellular [Na+] in the perfused rat heart: a 87Rb+ and 23Na NMR study.
Circ. Res.
76:
839-851,
1995
19.
Kupriyanov, V. V.,
E. Yushmanov,
B. Xiang,
and
R. Deslauriers.
Kinetics of ATP-sensitive K+-channels in isolated rat hearts assessed by 87Rb NMR spectroscopy.
NMR Biomed.
11:
131-140,
1998[Medline].
20.
Langer, G. A.,
and
A. J. Brady.
The effects of temperature upon contraction and ionic exchange in rabbit ventricular myocardium.
J. Gen. Physiol.
52:
682-713,
1968
21.
McMurchie, E. J.,
J. K. Raison,
and
K. D. Cairncross.
Temperature-induced phase changes in membranes of heart: a contrast between the thermal response of poikilotherms and homeotherms.
Comp. Biochem. Physiol.
44B:
1017-1026,
1973.
22.
Meltzer, H. L.
A pharmacokinetic analysis of long-term administration of rubidium chloride.
J. Clin. Pharmacol.
31:
179-184,
1991
23.
Noma, A.
ATP-regulated K+ channels in cardiac muscle.
Nature
305:
147-148,
1983[Medline].
24.
Rottenberg, H.,
and
A. Scarpa.
Calcium uptake and membrane potential in mitochondria.
Biochemistry
13:
4811-4817,
1974[Medline].
25.
Southard, J. H.,
and
F. O. Belzer.
Organ preservation.
In: Principles of Organ Transplantation, edited by M. W. Flye. Philadelphia, PA: Saunders, 1989, p. 194-215.
26.
Szewczyk, A.,
B. Mikolajec,
S. Pikula,
and
M. Nalecz.
Potassium channel openers induce mitochondrial matrix volume changes via activation of ATP-sensitive K+-channel.
Pol. J. Pharmacol.
45:
437-443,
1993[Medline].
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |