1Department of Physiology and Pathophysiology, Faculty of Medicine, University of Witten/Herdecke, Witten, Germany; and 2Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
Submitted 7 February 2005 ; accepted in final form 12 April 2005
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ABSTRACT |
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bongkrekic acid; cyclosporin A; lanthanum; Ru360; ruthenium red
When free Cd2+ reaches the cytosol of PT cells, it induces the formation of reactive oxygen species (ROS) and subsequent damage to critical organelles (49, 57). If ROS-mediated stress events are not sufficiently balanced by protective and repair processes, PT cells affected by Cd2+ are induced to undergo cell death via apoptosis or necrosis in vivo (34, 48) and in vitro (50, 51). After a toxic stimulus such as Cd2+, a number of proapoptotic proteins are liberated from the intermembrane space (IMS) of mitochondria into the surrounding cytosol, and these proteins activate downstream pathways (41, 49). Factors that are normally sequestered in the mitochondria but are released upon apoptotic signals include cytochrome c (35), apoptosis-inducing factor (AIF) (47), and Smac/Diablo (12, 52), as well as a number of procaspases, which appear to be key elements in the cascade of biochemical events leading to apoptosis.
It has been hypothesized that temporary or permanent alterations of the integrity of the mitochondrial inner and/or outer membrane induce the release of these proapoptotic proteins (6, 32, 36). Various mechanisms have been implicated in alterations of the mitochondrial membrane permeability and release of cytochrome c that either require Ca2+ or are Ca2+ independent. The latter mechanism may involve members of the Bcl-2 protein family in the release of cytochrome c but is not necessarily associated with changes in mitochondrial volume (44, 45, 53). Alternatively, the opening of a permeability transition pore (PTP) is activated by Ca2+, resulting in swelling and rupture of the outer membrane followed by the release of proapoptotic mitochondrial proteins (9, 22). Characteristic properties of PTP are its activation by Ca2+ and inhibition by cyclosporin A (CsA) (10). However, there is additional evidence from studies in intact cells and isolated mitochondria that changes of mitochondrial membrane permeability and/or release of cytochrome c may also occur by processes that are independent of these canonical mechanisms (2, 3, 19, 20, 23, 40). We recently demonstrated that Cd2+ induces swelling and release of cytochrome c from energized kidney cortex mitochondria suspended in mannitol-sucrose-HEPES (MSH) buffer independently of PTP opening (33). Rather, Cd2+ entered the matrix space through the mitochondrial Ca2+ uniporter (MCU) to activate aquaporin H2O channels and H2O influx resulted in osmotic swelling and release of cytochrome c (33).
In mitochondria suspended in KCl buffer, a K+ cycle has been proposed to contribute to the maintenance of the structural and functional integrity of mitochondria. It involves K+ influx by activation of K+ uniporters or channels in the inner mitochondrial membrane (IMM) and K+/H+ exchange, which regulates physiological mitochondrial volume homeostasis (17). Mitochondria possess several K+ channels or uniporters in the IMM, such as the K+- and Na+-permeable mitochondrial ATP-sensitive channel (KATP channel) (11, 25) or the Ca2+-stimulated, voltage-dependent, and exclusively K+-selective channel (KCa channel) (46, 56). It also has been suggested that activation of both channels may contribute to cytoprotection against the mitochondrial death pathway induced by hypoxia in cardiac cells (1, 56). However, Eliseev and colleagues (13, 14) described CsA-independent swelling and cytochrome c release in mitochondria isolated from etoposide-treated HL-60 cells and suspended in KCl buffer that were caused by increased K+ influx through a K+ uniporter.
In the present study, we have investigated the effect of Cd2+ on rat kidney cortex mitochondria suspended in isotonic KCl buffer. Cd2+ (550 µM) induced K+-dependent osmotic swelling of nonenergized or energized kidney cortex mitochondria by a process involving a K+ uniporter and the MCU, but not the PTP. In energized mitochondria only, Cd2+ induces a biphasic mitochondrial osmotic swelling-contraction response. Evidence is provided that the underlying process is Cd2+-induced K+ cycling involving K+ influx through the K+ uniporter and K+ efflux through a quinine-sensitive K+/H+ antiporter, which is driven by the mitochondrial pH gradient (pHm).
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EXPERIMENTAL PROCEDURES |
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CdCl2 was obtained from Merck (Darmstadt, Germany). Alamethicin, bongkrekic acid (BKA), carbonyl cyanide m-chlorophenylhydrazone (CCCP), CsA, diazoxide, 5-hydroxydecanoate (5-HD), nigericin, quinine hydrochloride, rhodamine 123+ (Rh123+), rotenone, and ruthenium red (RR) were all purchased from Sigma (St. Louis, MO). Ru360 was obtained from Calbiochem (San Diego, CA). 2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was purchased from Molecular Probes (Eugene, OR). 4,5,6,7-Tetrachloro-2-trifluoromethylbenzimidazole (TTFB) was a kind gift from Dr. R. B. Beechey (University of Wales, Aberystwyth, UK). All other reagents were purchased at the highest purity grade possible. Inhibitors and drugs were dissolved in water, ethanol, or dimethyl sulfoxide. In control experiments, solvents were added to isolated mitochondria at concentrations not exceeding 0.2%.
Animals
All experiments conducted were in accordance with the National Institutes of Healths Guiding Principles for Research Involving Animals and Human Beings. Male Sprague Dawley rats (250300 g) were obtained from Charles River Laboratories (Sulzfeld, Germany) and were allowed access to food and water ad libitum.
Methods
Isolation of mitochondria and measurements of mitochondrial O2 consumption.
Rat kidney cortex mitochondria were isolated by performing differential centrifugation essentially as described by Ott et al. (39), with minor modifications (33). Protein concentration was determined using the Bradford method (7). Only mitochondria with a respiratory control ratio 4 were used for experiments.
Osmotic swelling assay of mitochondria.
Mitochondria were used for osmotic swelling experiments within 4 h of isolation. Mitochondria (0.71.1 mg/ml) were incubated in 2 ml of KCl buffer (140 mM KCl, 3 mM HEPES, pH 7.4, with Tris and without or with 10 mM sodium succinate and 1 µM rotenone to energize mitochondria). In some experiments, KCl was replaced by chloride salts of the monovalent cations tested. Inhibitors, when used, were added before mitochondria unless otherwise stated. Changes in the volume of mitochondria due to colloid osmotic effects of solute flux into and out of the mitochondrial matrix after addition of salts and modulators were monitored by the change in absorbance at 540 nm (4). Kinetic measurements were performed at 25°C in a Beckman DU-640 spectrophotometer equipped with a Peltier constant temperature chamber and an automatic 6-U sampler (Beckman Instruments, Fullerton, CA). Data were captured and converted using DU-WinConnection Suite software and analyzed using Microsoft Excel and SigmaPlot 8.0 (SPSS, Chicago, IL). Rates of swelling (
absorbance540 nm/min) were calculated on the basis of the initial linear portion of mitochondrial absorbance curves and the rates of contraction (
absorbance540 nm/min) from the initial portion of the contraction phase. Swelling curves were normalized to maximal mitochondrial swelling. To determine maximal swelling, mitochondria were suspended in KCl buffer and the maximal absorbance changes induced by the monovalent cation ionophore alamethicin (12.5 µg per mg/ml protein) were defined as 100% swelling. Alamethicin-induced absorbance changes amounted to 53 ± 1.4% of initial absorbance. To simplify calculations, maximal swelling was assumed to be 50% of initial absorbance. Normalized values were obtained from the formula {1 [
absorbance/(initial absorbance/2)]} x 100 and expressed as %swelling, which means swelling increase relative to the alamethacin-induced maximal signal.
Determination of mitochondrial membrane potential.
As an indication of the changes of mitochondrial membrane potential (m), the ability of the mitochondria to sequester the cationic and lipophilic green fluorescent dye Rh123+ (
excitation = 454 nm,
emission = 550 nm) was monitored using an LS50B luminescence spectrophotometer (PerkinElmer, Wellesley, MA) and the data were captured using a FL Data Manager program (PerkinElmer). In energized mitochondria, Rh123+ accumulation into the mitochondrial matrix is driven by the inside negative
m of the IMM and the fluorescence becomes quenched. Upon depolarization of the IMM, the dye is released into the surrounding medium, and Rh123+ quenching decreases, causing an increase in fluorescence intensity (15). Mitochondria (0.3 mg/ml) were added to 3 ml of a buffer (in mM: 110 mannitol, 60 Tris, and 60 KCl, pH 7.4, with HCl) containing 2.6 µM Rh123+ and 5 mM MgCl2. The mitochondria were energized with 10 mM sodium succinate and 1 µM rotenone. The protonophore CCCP (0.3 µM) was used as a positive control to dissipate
m. In preliminary experiments, nonspecific signals and/or false-positive results due to interference of the tested compounds with Rh123+ were excluded.
Measurement of pHm.
The protocol described by Kapus et al. (28) was used with slight modifications. Mitochondria (4050 mg/ml) were preloaded with 10 µM BCECF-AM for 30 min at room temperature. BCECF-loaded mitochondria were centrifuged at 10,000 g for 5 min at 4°C, and the supernatant was discarded. The pellet was resuspended in mitochondria isolation buffer (in mM: 210 mannitol, 70 sucrose, and 5 HEPES, pH 7.4, with Tris) and then further diluted 20-fold with the same buffer. Mitochondria were centrifuged again as described above. Supernatants were discarded and pellets were resuspended to the original starting concentration with mitochondria isolation buffer. Mitochondria (1 mg/ml) were added to 3 ml of experimental buffer (140 mM KCl, 10 mM MOPS, 2.5 µg/ml oligomycin, 1 µM rotenone, pH 7, with KOH). BCECF fluorescence intensity was measured at
excitation 503 nm and
emission 528 nm on a PerkinElmer LS50B luminescence spectrophotometer, and data were captured with the FL Data Manager program. Mitochondria were energized with 10 mM sodium succinate (titrated to pH 7), and nigericin (0.4 µM) and CCCP (1 µM) were used as a positive control to dissipate
pHm. In preliminary experiments, nonspecific signals and/or false-positive results due to interference of the tested compounds with BCECF were excluded.
Statistical Analyses
All experiments were repeated at least three times with different preparations of mitochondria. Representative data or means ± SE are shown. Statistical analysis using an unpaired Students t-test was performed with the SigmaPlot 8.0 spreadsheet software program. For more than two groups, statistical differences were compared using one-way ANOVA assuming equality of variance with Levenes test and Tukeys post hoc test for pairwise comparison. Statistical analysis was performed with the SPSS 11.0 software program. Results with P 0.05 were considered to be statistically significant. To obtain EC50 values, dose-response curves of Cd2+-induced mitochondrial swelling were fitted using the SigmaPlot 8.0 spreadsheet software program assuming a sigmoidal dose-response curve (variable slope). Dose-response curves of Cd2+ effects on mitochondrial membrane potential (
m) were curve fitted assuming a hyperbolic function according to the equation for one-site saturation plus nonspecific binding using SigmaPlot 8.0, and EC50 values were determined.
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RESULTS |
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Addition of Cd2+ (550 µM) induced swelling of nonenergized rat kidney cortex mitochondria suspended in KCl buffer (Fig. 1A). The kinetics of swelling during a period of up to 12 min had a characteristic pattern showing an initial swelling phase that reached a plateau within 12 min. The initial rate of swelling during the first minute increased as a function of the Cd2+ concentration and saturated at 100 µM Cd2+ (EC50 = 18.8 ± 2.4 µM; n = 4). The magnitude of the plateau showed a similar dependence on the Cd2+ concentration (data not shown). When K+ was replaced by other monovalent cations, the initial rates of swelling induced by 10 or 50 µM Cd2+ were reduced according to the sequence K+ = Na+ > Li+ >> choline+ independently of the Cd2+ concentration tested (Fig. 1B). Replacement of K+ by the bulky cation choline+ decreased swelling by
70%, which indicates that Cd2+ activates K+- and Na+-permeable influx pathways, which cause osmotic swelling of the mitochondrial matrix.
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Cd2+-Induced Swelling of Energized Mitochondria in KCl Buffer Is Biphasic
Cd2+ also caused swelling of energized rat kidney cortex mitochondria suspended in KCl buffer, but the kinetics of swelling were more complex (Fig. 2). Swelling occurred more rapidly, but the amplitude of swelling was smaller. More important, the initial phase of swelling was immediately followed by a phase of mitochondrial contraction to control levels at concentrations >2 µM Cd2+ (Fig. 2). The initial rate of swelling during the first minute was also a function of the Cd2+ concentration and saturated at 20 µM Cd2+ (EC50 = 8.4 ± 0.7 µM; n = 8). Once mitochondrial contraction had reached control absorbance values, it was maintained for at least 68 min after addition of Cd2+ (see, e.g., Fig. 4A). Contraction was observed in Na+ buffers as well but was abolished in the presence of choline+ (data not shown). These data indicate that the mechanism underlying the initial rate of swelling is K+ dependent but does not require energization of mitochondria, whereas the contraction phase is also K+ dependent but requires the generation of m and/or
pHm to operate.
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We have previously shown in kidney cortex mitochondria suspended in MSH buffer that swelling induced by Cd2+ is independent of opening of the PTP but requires Cd2+ influx into the mitochondrial matrix through the MCU to occur (33). CsA (1 µM), a potent inhibitor of the PTP (10) or 5 µM BKA, another drug that blocks the PTP by binding to the adenine nucleotide translocator (ANT) (21), did not significantly affect initial rates of swelling of nonenergized or energized mitochondria induced by 2050 µM Cd2+ (Table 1; see also Ref. 33), which makes the involvement of the PTP in the process of swelling of mitochondria suspended in KCl buffer very unlikely.
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Cd2+ Influx Through the MCU Induces Rapid Dissipation of m
Figure 3A shows that addition of Cd2+ results in dissipation of m in energized kidney cortex mitochondria with an EC50 of
11.5 µM Cd2+ (Fig. 3A, inset). A rapid breakdown of
m was observed within 1 min at Cd2+ concentrations >5 µM. Consequently, breakdown of
m occurred at time points when mitochondrial contraction was still operative (see Figs. 2 and 4A), which indicates that
m is not necessary for mitochondrial contraction to occur. Ru360 also reduced Cd2+-induced dissipation of
m by
50% (Fig. 3B), which suggests that Cd2+-induced electrogenic K+ influx through K+ permeability is responsible for dissipation of
m.
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Whereas mitochondrial contraction of energized mitochondria was still observed while m was completely dissipated (Fig. 3A), dissipation of
pHm by the addition of the electroneutral K+/H+ exchanger nigericin (0.4 µM) at the peak of mitochondrial swelling (when K+ in buffer and matrix space are at equilibrium) abolished mitochondrial contraction induced by 10 µM Cd2+ (Fig. 4, A and B). The electrogenic ionophore CCCP (0.3 µM) had the same effect (data not shown), but it cannot be used as a valid tool to determine the role of
pHm, because it simultaneously affected
pHm and
m (Fig. 3A). These experiments suggest that a pH gradient across the IMM is required for mitochondrial contraction to occur.
Activation of the Mitochondrial K+/H+ Exchanger Mediates the Contraction Phase of Mitochondrial Swelling
As shown in Fig. 5, A and B, the contraction phase of osmotic swelling induced by 10 µM Cd2+ was abolished when an inhibitor of the K+/H+ exchanger of the IMM (quinine; 1 mM) (38) was added at the peak of mitochondrial swelling. Moreover, the addition of quinine to control mitochondria also enhanced swelling, suggesting that basal activity of the mitochondrial K+/H+ exchanger contributes to the stability of energized kidney mitochondria suspended in KCl buffer.
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DISCUSSION |
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CsA and BKA did not inhibit Cd2+-induced swelling of kidney cortex mitochondria suspended in KCl buffer (Table 1), which confirms the findings of our previous study (33). This demonstrates that Cd2+-induced swelling of kidney cortex mitochondria is not involved in the activation of the PTP. Other studies also have reported mitochondrial swelling and cytochrome c release in isolated mitochondria induced by stimuli such as Hg2+ (13, 24), etoposide (13, 14), valinomycin (19), or long-chain fatty acids (43) that were not inhibited by CsA, suggesting that the classical PTP was not involved in these processes. For instance, Eliseev and colleagues (13, 14) described CsA-independent swelling and cytochrome c release in mitochondria isolated from etoposide-treated HL-60 cells that were caused by increased K+ influx through the K+ uniporter. These effects also have been mimicked with the electrogenic K+ ionophore valinomycin (19).
K+-dependent swelling induced by Cd2+ was observed in nonenergized and energized rat kidney cortex mitochondria (Figs. 1 and 2). Whereas K+ influx into nonenergized mitochondria is driven by the concentration gradient between the medium and the matrix space, in energized mitochondria, m in addition drives K+ influx. Indeed,
m may play a significant role in the initiation of K+ influx, because swelling occurred more rapidly in energized mitochondria than in nonenergized ones (compare Figs. 1 and 2). This may also have an effect on the EC50 values used to describe the effectiveness of Cd2+ (
19 µM for nonenergized and
8 µM for energized mitochondria). The different values may appear to suggest that Cd2+ is more effective in energized mitochondria, which may be misleading. However, these differences likely reflect differences in the driving forces for K+ (and Cd2+), because the measured indirect parameter "swelling" reflects Cd2+-dependent K+ influx rather than direct binding affinity of Cd2+ to the Ca2+ uniporter. In contrast, the amplitude of swelling was smaller in energized mitochondria, which indicates that the electrochemical gradient across the IMM may counteract the process of swelling.
We also found that Cd2+-activated, K+-mediated swelling was abolished by inhibition of the MCU with RR or Ru360 (Table 1). This suggests that Cd2+ must enter the matrix space to induce K+ influx. Swelling could occur by activation of KCa conductance in the IMM. Halestrap et al. (23) previously showed that Ca2+-activated K+ influx into energized liver mitochondria is prevented by RR and concluded that Ca2+ must enter the mitochondria to activate Ca2+-sensitive, K+-dependent swelling. Moreover, in patch-clamp experiments, Siemen et al. (46) found the Ca2+ binding site of the mitochondrial K+-selective KCa channel to be on the matrix side. In our hands, Cd2+-induced mitochondrial swelling was not K+ selective and followed a selectivity sequence of K+ = Na+ > Li+ >> choline+ in nonenergized mitochondria (Fig. 1). Our data are also similar to those associated with swelling induced by long-chain fatty acids, which does not show any K+ selectivity (42). In HL-60 cells, K+-dependent mitochondrial swelling and cytochrome c release are associated with the activation of a KATP channel (14). However, neither the specific inhibitor of the mitochondrial KATP channel, 5-HD (26), nor the specific opener of KATP channels, diazoxide (18), affected Cd2+-induced mitochondrial swelling (data not shown). This suggests that a mitochondrial KATP channel does not contribute to K+-dependent swelling.
We could also exclude with certainty the alternative possibility of Cd2+ cycling through the MCU and a Na+/Ca2+ antiporter being responsible for swelling. Apart from the fact that the osmotic load generated by micromolar Cd2+ concentrations is too low to account for swelling and that swelling is clearly K+ dependent (Fig. 1), 2050 µM concentrations of Sr2+, which permeate the MCU (29) and the Na+/Ca2+ antiporter (37) equally as well as Ca2+, did not induce any swelling of mitochondria suspended in KCl buffer (data not shown).
The observation that the replacement of K+ by the bulky cation choline+ did not completely abolish Cd2+-induced swelling (Fig. 1B) indicates that several IMM pathways must contribute to Cd2+-induced mitochondrial swelling. It is noteworthy that Cd2+-induced swelling with choline+ had approximately the same magnitude (25% of swelling in KCl buffer) because Cd2+ induced swelling in MSH buffer, where H2O flow through Cd2+-activated aquaporin-8 H2O channels accounts for osmotic swelling (33). Thus it is likely that Cd2+ activates one or several more or less K+-selective channels or uniporters, such as a nonselective K+ channel (5), which results in K+ influx that is driven by the electrochemical potential across the IMM. Because Cd2+ also activates H2O channels (33), H2O follows the osmotic K+ load into the matrix space.
The extent of swelling induced by Cd2+ was quite different in energized and nonenergized mitochondria (compare Figs. 1A and 2). Hence it would have been interesting to determine the functional consequences of mitochondrial swelling under the different energization statuses, particularly regarding the impact on cytochrome c release as described in our previous publication regarding the use of mitochondria suspended in low ionic strength buffer (33). However, data obtained in KCl buffer consistently showed high basal cytochrome c release in controls that was marginally increased by addition of Cd2+ (data not shown; see also Ref. 33). The increase of cytochrome c release in KCl buffer may seem worrisome, because it reflects the physiological situation. First of all, it must be stated that intact mitochondria suspended in KCl buffer do not swell (and hence do not release cytochrome c), so that it can be concluded that KCl per se does not damage mitochondria (see Fig. 2). The damaging effect may result from centrifugation of mitochondria at 10,000 g for 5 min in high ionic strength buffer, which is performed after the swelling experiment to separate the mitochondrial pellet from the supernatant. Moreover, salt promotes the release of cytochrome c that is attached to the inner mitochondrial membrane by electrostatic interaction (8). The nature of this cytochrome c pool is not well defined, and it is unclear how it is prevented from being released in vivo.
What Is the Nature of Cd2+-Induced Mitochondrial Contraction?
Three lines of evidence suggest that a K+/H+ exchanger is responsible for mitochondrial contraction: 1) contraction does not operate in nonenergized mitochondria (Fig. 1), 2) contraction is abolished by addition of the electroneutral protonophore nigericin at the peak of mitochondrial swelling (Fig. 4), and 3) contraction is abolished by the inhibitor of K+/H+ exchanger quinine (Fig. 5, A and B). Therefore, the driving force for K+/H+ exchange must be pHm generated by the respiratory chain. Moreover, pH measurements with BCECF after the addition of 10 µM Cd2+ showed dissipation of
pHm that was significantly slowed by 1 mM quinine (Fig. 6C). Cd2+ indirectly contributes to dissipation of
m by inducing K+ influx through a K+ uniporter, which in turn initiates K+/H+ exchange through the quinine-sensitive K+/H+ antiporter, with the action of both transporters resulting in K+ cycle and mitochondrial contraction.
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Other studies have shown that not all changes in mitochondrial function triggered by inducers cause irreversible mitochondrial damage. For instance, Schönfeld et al. (42) showed a decrease of m upon induction of K+ influx by long-chain fatty acids but a recovery of
m due to K+ efflux and mitochondrial contraction. Also, temporary changes in mitochondrial volume induced by moderate hypotonicity may lead to cytochrome c release while mitochondria remain intact and functionally active (19). In contrast, the results of this study show that the changes in mitochondrial function induced by Cd2+ are detrimental. Cd2+ at concentrations
5 µM induced a permanent breakdown of
m (Fig. 3A). This differs from the findings in the study by Schönfeld et al. (42), in which a decrease of
m upon induction of K+ influx by long-chain fatty acids was followed by a recovery of
m during the contraction phase. Indeed, Cd2+ concentrations
3 µM induced a block of O2 consumption within 12 min. States 4 and 3 respiration were irreversibly inhibited 23 min after addition of Cd2+ concentrations
3 µM (data not shown), which is in accordance with a recent study demonstrating that 520 µM Cd2+ inhibited mitochondrial complexes II and III of the electron transfer chain (54).
We therefore propose the following working hypothesis to explain the Cd2+-induced osmotic swelling-contraction response, changes in K+ permeability pathways, and mitochondrial dysfunction in energized mitochondria (Fig. 6). Cd2+ enters the matrix space through the MCU. Both the chemical gradient for K+ as well as m drive K+ influx through a Cd2+-activated K+ uniporter. The osmotic load of the mitochondrial space by K+ is directly reflected by the changes in mitochondrial volume over time. The breakdown of
m induced by K+ influx and the inhibition of the respiratory chain by Cd2+ finally limit K+ influx and lead to a K+ equilibrium across the IMM. K+ in the matrix space triggers activation of a K+/H+ exchanger in the IMM that dissipates
pHm and decreases the K+ concentration and hence the osmotic load of the matrix space, resulting in mitochondrial contraction. Thus the K+ load of the matrix space determines the rate and magnitude of dissipation of
pHm via the K+/H+ exchanger. Consequently, apart from Cd2+ inhibition of the electron transfer chain (54), Cd2+-induced activation of a K+ cycle also affects mitochondrial damage by contributing to the dissipation of the mitochondrial protonmotive force.
Independently of the complexity of these alterations of mitochondrial permeability and function induced by Cd2+, a major event associated with Cd2+-induced mitochondrial swelling remains the release of proapoptotic factors such as cytochrome c (33). Once released into the cytosol, cytochrome c typically forms an apoptosome with other cytosolic components and triggers activation of a cellular cascade of proteases that ultimately leads to apoptosis. It is likely that these processes are operative in Cd2+-induced apoptosis of PT cells (4951), and these processes are currently under investigation.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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