Department of Physiology, Limburgs Universitair Centrum/Transnationale Universiteit Limburg, Biomedisch Onderzoeksinstituut, B-3590 Diepenbeek, Belgium
Submitted 13 August 2003 ; accepted in final form 30 November 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
renal epithelial cells; intramitochondrial calcium; rhod 2; CGP-37157
Mitochondria take up Ca2+ primarily through a uniporter system (4, 28, 61). The influx of Ca2+ into the matrix by this route is dependent on the electrochemical gradient for Ca2+. This gradient is developed and maintained by the mitochondrial membrane potential (m), generally estimated to be on the order of 150200 mV negative to the cytosol and by a low resting [Ca2+]m, maintained primarily by the mitochondrial NCE. The second mode of inward Ca2+ transport is referred to as the "rapid mode" or RaM, because it is at least 300 times more rapid than uptake via the uniporter under the same conditions (27, 68). This RaM transports Ca2+ only for a brief period (a fraction of a second) during the initial phase of the cytosolic Ca2+ pulse. The driving force for Ca2+ uptake via the RaM is the Ca2+ electrochemical gradient, as it is for the uniporter. Consequently, the collapse of
m in response to pathological conditions will limit mitochondrial Ca2+ uptake and may contribute to cellular pathophysiology (17, 61). Moreover, ischemia-induced cellular ATP depletion will hamper the other previously mentioned ATPases responsible for cytosolic Ca2+ clearance.
In this study, we examined the alterations in cellular and mitochondrial Ca2+ homeostasis during metabolic inhibition (MI) in Madin-Darby canine kidney (MDCK) cells, a cell line of distal tubular origin that exhibits many similarities to mammalian cortical collecting tubular cells (74). Inhibition of cellular metabolism was used as an experimental model to simulate ischemic cell injury. It was realized by inhibiting both cellular glycolysis [with 2-deoxyglucose (2-DG)] and oxidative phosphorylation [with cyanide (CN)]. The aim of the present study was to investigate whether ATP-depleted MDCK cells were able to clear the cytosolic Ca2+ overload during MI and to determine the underlying Ca2+-transporting mechanism.
This report shows that MI induces a transient increase in [Ca2+]i in MDCK cells. Our results suggest that the second-phase clearance of [Ca2+]i during MI is not due to either Ca2+ extrusion out of the cells nor Ca2+ storage into the ER but rather to Ca2+ uptake into the mitochondrial matrix. Furthermore, our findings strongly suggest that the mitochondrial Ca2+-importing mechanism is not the uniporter or the RaM but rather the mitochondrial NCE acting in the reverse mode.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
MDCK cells (low passage number, 2230) were kindly donated by Dr. H. De Smedt (Laboratory of Physiology, Leuven, Belgium). Cells were cultured in a 1:1 mixture of DMEM and Ham's F-12, supplemented with 10% fetal calf serum, 14 mM L-glutamine, 25 mM NaHCO3, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were maintained in a humidified 5% CO2 atmosphere at 37°C. The medium was renewed every 34 days. When cultured on permeable supports, the cells formed confluent monolayers with a resistance of 400500 ·cm2. For all experiments, 12 x 105 cells were seeded onto round glass coverslips with a diameter of 24 mm. After 36 days of culture, confluent monolayers were used.
Cellular ATP Content
Confluent monolayers of MDCK cells were washed with normal saline solution and incubated with a solution containing metabolic inhibitors (see below for details) for various time intervals ranging from 5 to 60 min. Control cells were incubated in normal saline solution. ATP measurements were performed with a luciferin-luciferase-based assay kit (Molecular Probes, Eugene, OR). The reaction buffer contained 150 µg/ml luciferin, 1.25 µg/ml luciferase, 5 mM MgSO4, 1 mM dithiothreitol, 25 mM Tricine, 0.1 mM EDTA, and 0.1 mM azide, pH 7.8. The cells were solubilized in 450 µl of somatic cell ATP-releasing agent (Sigma, St. Louis, MO) for 30 s. Fifty microliters of cell extract was added to 450 µl of reaction buffer. ATP levels were measured with a luminometer (model 1250, Wallac, Turku, Finland). Calibration was performed with several standard ATP solutions in the concentration range 10-8-10-5 M. The results are expressed as percent change compared with control.
Fluorescence Imaging Microscopy
The coverslips with MDCK cells were mounted into a homebuilt holder and placed on the stage of an inverted epifluorescence microscope (Zeiss Axiovert 100, Jena, Germany). A thermostatic heating chamber (set at 37°C) enclosed the microscope stage. After measurement of the background signal, the cells were loaded with an appropriate fluorescent indicator. Both before and after dye loading, cells were gently washed several times with normal saline solution. Fluorescence was elicited by illumination with an XBO 75 W/2 OFR xenon lamp (Osram, Berlin-Munich, Germany). The excitation filters were inserted into a computer-controlled shuttered filter wheel (Lambda 102, Sutter Instrument, Novato, CA), which allows fast alternation between different excitation filters. All optical filters and dichroic mirrors were obtained from Chroma Technology (Brattleboro, VT). The excitation light was directed to the sample by a dichroic mirror and a Zeiss LD Achroplan objective (x40/0.6 corr.). Fluorescence collected by the objective was transmitted through the dichroic mirror and a band-pass emission filter to a Quantix CCD camera (Photometrix, Tucson, AZ), which is equipped with a Kodak KAF 1400 charge-coupled device (grade 2, MPP) with 1,317 x 1,035 pixels and cooled to -25°C by a thermoelectric cooler. Image-sequence acquisition (or pairs of images, in the case of ratiometric indicators) is controlled by a homemade program based on V for Windows software (Digital Optics, Auckland, New Zealand). To avoid bleaching of the probe and photodamage of the cells, the shuttered illumination was restricted to the periods when the images were taken. Signals were obtained by whole-image spatial integration of pixels over the confluent cells. The background image is automatically subtracted, pixel by pixel, from the loaded cell images. The value of the detected fluorescence was increased by applying 3 x 3 binning and a gain of 3.
Determination of [Ca2+]i. [Ca2+]i was monitored using the fluorescent probe fura 2. The cells were loaded with fura 2 by incubation with the membrane-permeant acetoxymethyl (AM) ester form of the dye (2 µM from a 5 mM stock solution in DMSO) for 1 h at 37°C in the presence of 0.05% wt/vol Pluronic F-127. For excitation, 340/10- and 380/10-nm band-pass filters were used. Emission was recorded using a dichroic mirror type 72100 (>500-nm long-pass filter) and a 535/50-nm emission band-pass filter. Data collection time for an image was 5 s. Fura 2 was calibrated in vivo at the end of each experiment, according to the equation derived by Grynkiewicz et al. (26)
![]() | (1) |
Determination of m.
m was evaluated using the potentiometric indicator 5,5',6,6'-tetrachloro-1,1',3,3'-tetra-ethylbenzimidazolyl-carbocyanine iodide (JC-1) (12, 23, 60, 64, 67). MDCK cells were loaded with JC-1 (10 µM from a 10 mM stock solution in DMSO) for 30 min at 37°C. JC-1 in its monomeric form emits fluorescence at 530 nm (green fluorescence) when excited at 490 nm, whereas the aggregates emit fluorescence at 590 nm (red fluorescence). Dual-emission ratiometric measurements were performed by manually changing the emission cube. The excitation was done through a 10-nm band-pass filter centred at 495 nm. JC-1 monomer fluorescence was collected through a >500-nm long-pass dichroic mirror and a 535/50-nm band-pass emission filter. The emission cube used to detect the J aggregates consisted of a >560-nm long-pass dichroic mirror and a 590/55-nm emission filter. Data collection time for an image was 5 s, and a neutral-density filter of 1.0 OD (Newport, Irvine, CA) was inserted in the excitation pathway. The mitochondrial uncoupler FCCP (10 µM) was added at the end of each experiment to determine the JC-1 emission ratio associated with a collapsed
m. 2-DG (10 mM) was administered simultaneously with FCCP to rule out glycolytic ATP provision for the reverse action of the mitochondrial F1F0-ATPase. Results are presented in terms of a normalized ratio (Rnorm)
![]() | (2) |
Determination of intracellular Na+ concentration. Intracellular Na+ concentration ([Na+]i) was monitored using the fluorescent probe sodium-binding benzofuran isophthalate (SBFI). The cells were loaded with SBFI for 2 h at 37°C (2 µM from a 5 mM stock solution in DMSO, in the presence of 0.05% wt/vol Pluronic F-127). SBFI was used in dual-excitation ratiometric mode. For excitation, 340/10- and 380/10-nm band-pass filters were used. Emission was recorded using a dichroic mirror type 72100 (>500-nm long-pass filter) and a 535/50-nm band-pass emission filter. Data collection time for an image was 5 s. The calibration of the fluorescence signals of SBFI was accomplished by exposing the cells to various extracellular [Na+] in the presence of the ionophore gramicidin D (10 µM from a 2 mM stock solution in ethanol). A calibration curve was derived according to the procedure described by Zahler et al. (79). The ratio r of the fluorescence signal of SBFI due to excitation at 340 nm (F340) over that at 380 nm (F380) was normalized as follows
![]() | (3) |
![]() | (4) |
Laser-Scanning Confocal Microscopy
The coverslips with MDCK cells were mounted into a holder and placed on the stage of the Zeiss LSM 510 META laser-scanning confocal microscopic (LSCM) system attached to an Axiovert 200 (motorized) frame (Zeiss). The microscope stage was equipped with a PeCon (model P, Erbach-Bach, Germany) heated specimen holder with an S-type incubator, all set at 37°C. To minimize temperature differences between sample and objective, a x63 oil model objective heater (PeCon) was used. Cells were loaded with two fluorescent probes: Mito Tracker Green (MTG), a fluorescent probe that selectively stains mitochondria, and rhod 2, a Ca2+-sensitive probe. Loading of MDCK cells with MTG and rhod 2 occurred in two subsequent steps. At first, MTG (200 nM) was loaded into the cells for 30 min at 37°C. This was followed by incubation of the cells for 30 min at 37°C in a loading solution containing rhod 2-AM (4 µM), MTG (200 nM), and pluronic acid (0.025% wt/vol). Rhod 2-AM is a cell-permeant Ca2+ indicator that carries a delocalized positive charge. Therefore, it is taken up preferentially into polarized mitochondria. On hydrolysis of the ester moieties, the rhod 2-free acid remains trapped inside the mitochondria, where it reports increased [Ca2+]m by an increase in its fluorescence intensity. After being loaded, the cells were gently washed three times with normal saline solution.
Fluorescence measurements were performed with a x63/1.4 Plan-Apochromat oil-immersion objective (Zeiss). MTG was excited by the Ar laser (488-nm line) and rhod 2 by the Green HeNe laser (543-nm line). Laser intensity was set at only 0.5% of the maximum level to minimize dye bleaching and to protect the cells against possible photodamage (44). Fluorescence emissions of MTG and rhod 2 were collected, respectively, via NFT490 and NFT545 dichroic mirrors and 525/25-nm and 590/25-nm barrier filters that came with the confocal microscope. The scanning speed was set to a pixel dwell time of 25.6 µs. Each 512 x 512-pixel image was averaged twice via software-selected repeated line scan mode to ameliorate the signal-to-noise ratio. The effective frame collection time was 33 s. All images were collected with a digital zoom factor of 2. The thickness of the optical slices was <1.4 µm in all experiments.
To monitor only the changes in [Ca2+]m during MI, rhod 2 fluorescence had to be corrected for nonmitochondrial rhod 2 signals, including the weak cytosolic rhod 2 staining and the pronounced staining of structures within the nuclei, presumably nucleoli (see Fig. 8C). Within the histogram of the MTG image, a threshold was chosen to retain only the high-end tail, excluding any saturated pixels. On visual inspection, resulting MTG intensities represented "well-delineated" mitochondrial locations. The intensities of the selected mitochondrial pixels were set at one, whereas all other pixel intensities in the MTG image were set at zero. Multiplication of this "mask" image with the rhod 2 image yielded an image consisting of mitochondrial rhod 2 fluorescence intensities (Frhod 2, mito; see Fig. 8D). These Frhod 2, mito values were normalized with regard to the number of pixels with a mask value of one in the mask images to compensate for differences in mitochondrial density. Furthermore, because the mitochondria continuously moved in the cytosol (see electronic file of mitochondrial movement in MDCK cells at http://ajprenal.physiology.org/cgi/content/full/00284.2003/DC1), at each time point both an MTG image and a rhod 2 image were collected. In the experimental protocol, one set of images was taken at the start under control conditions. Throughout the MI treatment, the number of image collection events was restricted to three (after 20, 40, and 60 min of MI, respectively) to minimize bleaching of rhod 2 fluorescence. For image transfer, conversion, and processing, a Zeiss LSM Image Browser and ImageJ Java-based freeware (Research Services Branch, National Institute of Mental Health/National Institute of Neurological Disorders and Stroke, Bethesda, MD) plugin routines were used. Deconvolution of the confocal images via Huygens Essential (SVI, Hilversum, The Netherlands) did not improve image quality.
|
Solutions and Chemicals
MDCK cells were bathed in a normal saline solution containing (in mM) 140 NaCl, 5 KCl, 1.5 CaCl2, 1 MgSO4, 10 HEPES, and 5.5 glucose, pH adjusted to 7.4 with Tris. MI was accomplished with a solution containing (in mM) 135 NaCl, 5 KCl, 1.5 CaCl2 1 MgSO4,10 HEPES, 10 2-DG, and 2.5 NaCN (pH 7.4). N-methyl D-glucamine (NMDG) replaced Na+ in the Na+-free solutions. CN- was applied as KCN whenever the effect of MI was studied in the absence of external Na+. Fura 2-AM, JC-1, SBFI-AM, BAPTA-AM, MTG, rhod 2-AM, and Pluronic F-127 were obtained from Molecular Probes. FCCP, ionomycin, and gramicidin D were bought from Sigma. CGP-37157 was obtained from Tocris (Bristol, UK) and LaCl3 from Janssen Chimica (Beerse, Belgium). All other chemicals were of analytic grade.
Statistics
Values from n different monolayers are given as means ± SE. All experiments described in this paper were performed at 37°C.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
To investigate whether MI (for details, see MATERIALS AND METHODS) had an effect on cytosolic Ca2+ levels, fura 2 fluorescence was monitored during a 60-min incubation period with metabolic inhibitors. In the first 20 min of MI, a profound increase in [Ca2+]i from 48 ± 2 to 631 ± 78 nM (n = 12) was seen (Fig. 1). This increase in cytosolic Ca2+ was due to an influx of extracellular Ca2+, because exposure to metabolic inhibitors in Ca2+-free medium did not evoke a significant increase in [Ca2+]i (n = 3; data not shown). Subsequently, a second-phase drop in [Ca2+]i occurred to a level of 118 ± 9 nM (n = 12) after 60 min of MI (Fig. 1). To unravel the mechanism underlying this second-phase decrease in cytosolic Ca2+, different Ca2+ efflux routes were checked: Ca2+ extrusion out of the cell and Ca2+ uptake into the intracellular organelles such as the ER and mitochondria.
|
Second-Phase Decrease in Cytosolic Ca2+ Is Not Due to Ca2+ Extrusion Out of the Cell
MDCK cells possess two different types of Ca2+ extruders in their plasma membrane: Ca2+-ATPases and NCEs (7, 47). Luciferine-luciferase-based ATP experiments revealed that intracellular ATP levels rapidly drop to only 9 ± 2% of control levels in 5 min of exposure to metabolic inhibitors. After a 20-min incubation period, only 1.8 ± 0.2% of the initial ATP content is left (n = 6, Fig. 2A). Furthermore, experiments (Fig. 2B) were performed with La3+, a known inhibitor of plasma membrane Ca2+ pumps in different cell types (8, 9, 22, 33) including MDCK cells (43). Addition of La3+ did not change the typical cytosolic Ca2+ clearance after 20 min of MI. Therefore, it is unlikely that the ATP-dependent Ca2+ pumps account for the decrease in [Ca2+]i seen after 20 min of MI. To investigate whether the NCEs play a role in Ca2+ extrusion after 20 min of MI, SBFI experiments were performed to monitor [Na+]i in MDCK cells during MI (Fig. 3). [Na+]i rapidly increased during MI from 23 ± 3 mM in control conditions to 91 ± 4 mM (n = 7) after 60 min of MI. Because [Na+]i already increased by a factor greater than thrree after 20 min of MI, the ability of NCEs to extrude Ca2+ is strongly attenuated at that moment.
|
|
Mitochondria Take Up Cytosolic Ca2+ During MI
Buffering of high cytosolic Ca2+ levels can occur via Ca2+ uptake into the ER via the thapsigargin-sensitive Ca2+-ATPases and/or into the mitochondria via uniporter systems (4, 18, 27, 28, 30, 46). Because the presence of thapsigargin (1 µM), a well-known inhibitor of the ATPase responsible for Ca2+ uptake into the ER or SR, did not alter the typical biphasic behavior of [Ca2+]i during MI (Fig. 4), the possibility of mitochondrial uptake of cytosolic Ca2+ was further explored. Mitochondria take up Ca2+ via uniporter systems. These Ca2+ uniporters are driven by the Ca2+ electrochemical gradient, whose dominant component in mitochondria is the highly negative m (18, 27, 29).
m is generated mainly by H+ extrusion in the electron transport chain (ETC). However, when CN, a specific inhibitor of the cytochrome c oxidase complex, is used, the ETC is blocked and
m is expected to collapse.
m was evaluated with the fluorescent indicator JC-1. As a parameter for
m the emission ratio F590/F535 was used. To allow comparison of experiments with different control ratios, JC-1 ratios were normalized (see MATERIALS AND METHODS). Figure 5 illustrates the time course of the drop in
m in response to CN and 2-DG incubation.
m depolarized steadily to 12 ± 4% of control in 12 min. Thereafter,
m declined further, but more slowly, to <1% of control after 30 min of MI (n = 8). Because of this rapid loss of
m, it is rather unlikely that the mitochondrial uniporter is responsible for the second-phase cytosolic Ca2+ clearance (4, 39). To rule out any possible contribution of the mitochondrial uniporter, the protonophore FCCP (10 µM) was added in addition to the metabolic inhibitors to fully depolarize
m (n = 3; data not shown). The second-phase decrease in [Ca2+]i persisted.
|
|
Mitochondrial Ca2+ Uptake During MI in MDCK Cells Occurs via the Mitochondrial Na+/Ca2+ Exchanger Acting in Reverse Mode
In metabolically inhibited rat cardiomyocytes, it was proposed that the mitochondrial NCE might reverse (24). We hypothesized that the observed second-phase clearance of cytosolic Ca2+ in MDCK cells might be ascribed to the mitochondrial NCE acting in reverse mode. To test this hypothesis, MDCK cells were preincubated during 15 min in a Na+-free solution (see MATERIALS AND METHODS) to deplete both mitochondria and the cytosol and thus to block the mitochondrial NCE. Subsequently, cells were exposed to MI under Na+-free conditions. As depicted in Fig. 6A, [Na+]i dropped to 6 ± 2 mM in the 15-min preincubation period in Na+-free Ringer. After a subsequent 20-min period of Na+-free MI, [Na+]i values were as low as 1 ± 1 mM. This observation suggests that, after 20 min of MI, intracellular Na+ and presumably mitochondrial Na+ has decreased to levels where mitochondrial Na+/Ca2+ exchange would be unlikely. The typical second-phase decrease in [Ca2+]i seen after 20 min of MI was absent under these conditions (Fig. 6B), indicating the presumable Na+ dependence of the transport mechanism responsible for the removal of cytosolic Ca2+. Long-term exposure to Na+-free solutions as such did not evoke any significant changes in [Ca2+]i, as indicated by the control trace in Fig. 6B. To further explore the hypothesis of mitochondrial NCE reversal, changes in [Ca2+]i during MI were evaluated in the presence of CGP-37157, a specific inhibitor of the mitochondrial NCE (1, 3, 11, 13, 14, 54, 65). When CGP-37157 was administered during a 30-min preincubation period and subsequent exposure during 60 min in the presence of metabolic inhibitors, the second-phase drop in [Ca2+]i was completely abolished (n = 8, Fig. 7).
|
|
Imaging Mitochondrial Ca2+ During MI with Confocal Microscopy
To evaluate changes in mitochondrial Ca2+ content during MI, experiments were performed in MDCK cells loaded with both the mitochondrion-specific dye MTG and the Ca2+-sensitive probe rhod 2. Wirelike mitochondria were observed in MTG-loaded cells in control conditions (Fig. 8A). Incubation with metabolic inhibitors induced conformational changes, resulting in the vesicle shape of the majority of the mitochondria (Fig. 8B). The changes in mitochondrial shape are related to MI rather than resulting from a time effect, because incubation in normal saline solution still revealed wirelike mitochondria after a 60-min incubation period (images not shown). Comparison of Fig. 8, B and C, illustrates that rhod 2 is mainly localized in mitochondria when loaded in MDCK cells with a weak staining of the cytosol. Moreover, a pronounced staining of structures within the nuclei of the cells, presumably nucleoli, was seen, as reported earlier by several other groups (3, 36, 57, 63). To resolve mitochondrial rhod 2 fluorescence (Frhod 2, mito) from nonmitochondrial rhod 2 contributions, a "mask" procedure was used based on mitochondrial localization in the MTG image (for details, see MATERIALS AND METHODS). The resulting "mitochondrial" rhod 2 image (Fig. 8D) confirms colocalization of MTG and rhod 2 in mitochondria, because only rhod 2 intensities are retained that correspond to MTG-positive pixels in the MTG mask. Figure 9 clearly depicts that mitochondria indeed buffer cytosolic Ca2+ during MI in MDCK cells. During 60 min of MI, Frhod 2, mito increased steadily to 346 ± 23% of the control level determined just before exposure to metabolic inhibitors (n = 5). The observed increase in Frhod 2, mito during MI is underestimated, because open bars In Fig. 9 indicate that some mitochondrial rhod 2 fluorescence is lost throughout the 60-min protocol performed (n = 3). This loss of signal might be ascribed to either photobleaching of the probe or leakage of the rhod 2 dye out of the mitochondrial matrix. When MI was applied under Na+-free conditions (after a preincubation period of 15 min in Na+-free solution), only a minimal increase in Frhod 2, mito was observed (n = 6), pointing to the Na+ dependence of mitochondrial Ca2+ uptake. Furthermore, when MDCK cells were exposed to metabolic inhibitors in the presence of the specific inhibitor CGP-37157 (25 µM) of the mitochondrial NCE, the mitochondrial accumulation of cytosolic Ca2+ was substantially reduced (n = 5). The fact that Frhod 2, mito is steadily increasing during MI and can be modulated by a specific mitochondrial agent, CG-P37157, tends to confirm the mitochondrial origin of the rhod 2 fluorescence intensities obtained via the mask procedure applied.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
MI Induces a Transient Increase in Cytosolic Ca2+ in MDCK Cells
Our data show biphasic behavior of [Ca2+]i during MI in MDCK cells. The peak value of 631 ± 78 nM was reached after 20 min of MI. Similarly, a previous study in confluent monolayers of MDCK cells reported that application of 5 mM CN and 5 mM 2-DG resulted in an increase in [Ca2+]i from 112 ± 11 to 649 ± 99 nM in 15 min of MI, which was the maximum time interval investigated (52). We concluded that the increase in cytosolic Ca2+ was due to an influx of extracellular Ca2+ because Ca2+-free MI did not evoke a substantial increase in [Ca2+]i (data not shown). Moreover, thapsigargin experiments revealed that release of Ca2+ from the ER did not underlie the observed increase in [Ca2+]i in the first phase of MI. When the ER was depleted for Ca2+ before MI during a preincubation period of 6 min with thapsigargin (1 µM), a well-known inhibitor of the ATPase responsible for Ca2+ uptake into ER, the typical biphasic behavior of [Ca2+]i during subsequent MI was not altered (Fig. 4). Furthermore, the possibility of release of Ca2+ from mitochondrial stores in the first phase of MI seems unlikely, because the rhod 2 measurements in this study clearly demonstrate that mitochondrial Ca2+ levels increase during the first 20 min of MI (Fig. 9). Because the MI-induced rise in [Ca2+]i was comparable in the absence (Fig. 6B) or presence (Fig. 1) of extracellular Na+, Ca2+ influx probably did not occur via the plasma membrane NCE. Ca2+ influx might be mediated via epithelial Ca2+ channels, e.g., via TRPV5 channels known to be present in the Ca2+-transporting distal part of renal tubules (34). However, whether these Ca2+ channels are present in MDCK cells is unknown.
To our knowledge, the only previous study that reported a decline in [Ca2+]i after an initial increase in renal cells exposed to ischemic conditions was performed in primary cultures of rat proximal tubular cells (10). In that study, 25% of cells exposed to glucose-free, anoxia [Ca2+]i showed values that peaked to >1 µM and then dropped rapidly to near 500 nM in
10 min. The observed partial recovery was associated with an extended period of survival.
In the present study, [Ca2+]i was monitored by fura 2 ratio imaging. The Kd of fura 2 is known to increase with intracellular acidification (40), and a given fluorescence ratio R will then correspond to a higher [Ca2+]i value (Eq. 1). BCECF fluorescence experiments revealed that the intracellular pH value (pHi) dropped in metabolically inhibited MDCK cells from 7.35 ± 0.03 to 7 ± 0.03 in 6 min (n = 7; data not shown). Because a constant Kd was used in our calculations, the reported [Ca2+]i values might underestimate real [Ca2+]i values.
Second-Phase Ca2+ Clearance During MI Is Not Due to Ca2+ Extrusion Out of the Cell
The direction of Ca2+ movement by the plasma membrane NCE is determined by several variables, including membrane potential as well as intracellular and extracellular concentrations of both Na+ and Ca2+. Both membrane depolarization and an increase in [Na+]i are expected to occur during ischemia, and both of these factors favour exchanger-mediated cellular Ca2+ entry rather than exit. We found that [Na+]i steadily increased more than three times times during 20 min of MI, which does not favor Ca2+ extrusion out of the cells after that time. Moreover, the NCE may reverse in ATP-depleted cells, as already reported in several cell types, including cardiac myocytes (31), neurons (66), and human keratinocytes (42).
In this study, incubation with metabolic inhibitors rapidly induced depletion of cellular ATP levels. After only 5 min of MI, cellular ATP levels dropped to <10% of control ATP levels. This result is consistent with previous reports on MDCK cells (16, 21, 72). Earlier reports indicate that the affinity for ATP of isolated plasma membrane Ca2+-ATPases (PMCA) is very high (55, 56): a high-affinity site, normally assumed to be the catalytic site, has a Michaelis constant (Km) as low as 12 µM, and the low-affinity site has a Km between 150 and 400 µM (9). However, when Km values for ATP were determined in membrane vesicles derived from rabbit proximal tubules (76) and rat kidney cortex (73), much higher values were obtained: 0.6 and 0.2 mM, respectively. Assuming that the cellular ATP concentration ([ATP]i) in MDCK cells in control conditions was near 2.4 mM as determined by Lynch and Balaban (50) for MDCK cells, a drop to 2% of the initial ATP content after 20 min of MI (Fig. 2A) yields a residual [ATP]i value near 0.048 mM. This value is still considerably lower than the above-mentioned Km values for ATP of renal PMCAs. Moreover, because ADP has been found to inhibit PMCA activity in membrane vesicles derived from rabbit proximal tubules (76), and cellular ADP as well as AMP levels are known to increase in ischemia (49, 77), the activity of PMCAs in metabolically inhibited MDCK cells might be additionally reduced. Furthermore, addition of La3+, a known inhibitor of plasma membrane Ca2+ pumps (8, 9, 33), did not change the typical cytosolic Ca2+ clearance after 20 min of MI (Fig. 2B). Therefore, Ca2+ extrusion via the PMCAs is unlikely after 20 min of MI.
Ca2+ Uptake in Depolarized Mitochondria via Na+/Ca2+ Exchanger Acting in Reverse Mode
Mitochondria can accumulate enormous quantities of Ca2+ (3, 17, 61). In renal mammalian cells, the mean mitochondrial volume is 25% of the combined cytosolic and nuclear volumes (20). Mitochondria are key players in clearing large loads of cytosolic Ca2+ by their fast, high capacity and reversible Ca2+ sequestration properties (3, 18, 58, 70).
The normal route for mitochondrial Ca2+ uptake in mitochondria is the m-dependent Ca2+ uniporter, as highlighted by the fact that experimental collapse of
m by mitochondrial inhibitors or uncouplers prevents mitochondrial Ca2+ accumulation (32, 35, 41, 59, 62). In the present study, mitochondrial Ca2+ uptake via the
m-dependent uniporter is limited to the first 2530 min of MI, because Rnorm decreased to <10% of control in 15 min and dropped to near-zero values after 30 min of MI (Fig. 5). However, our data show that in metabolically inhibited renal epithelial MDCK cells, the mitochondrial Ca2+ content steadily increased during the 60-min incubation period with metabolic inhibitors (Fig. 9). Because the second-phase decrease in [Ca2+]i was abolished and the increase in mitochondrial Ca2+ content was nearly prevented when cells were depleted of Na+ before and during induction of MI, it was concluded that mitochondria take up cytosolic Ca2+ via an Na+-dependent transport mechanism. Furthermore, the pronounced inhibition of the increase in [Ca2+]m when MI was applied in the presence of CGP-37157 provided evidence that the route of mitochondrial Ca2+ entry during MI is the NCE. The idea of mitochondrial Ca2+ loading by reversal of the NCE was proposed earlier in studies in isolated beef heart mitochondria (38) and in intact cardiomyocytes exposed to 2.5 mM KCN in glucose-free medium (24, 25). Jung et al. (38) treated mitochondria with rotenone and oligomycin to partially abolish
m, applied the blocker ruthenium red to inhibit the Ca2+ uniporter, and challenged subsequently with Ca2+ (13 µM). Only a minimal mitochondrial Ca2+ uptake was observed. To dissipate the remaining
m completely, the uncoupler FCCP was applied subsequently, leading to a 4.6-fold increase in the Ca2+ influx rate. This FCCP-stimulated entry of Ca2+ was 98% inhibited by the omission of Na+, which is consistent with the abolition of mitochondrial Ca2+ uptake in our Na+-free MI experiments.
To understand the observed reversal of the mitochondrial NCE, the following speculative conceptual model can be suggested. In this model, electrical as well as chemical driving forces for both Ca2+ and Na+ are considered. Because some investigators proposed that the stoichiometry of the mitochondrial NCE may be closer to 3Na+/1Ca2+ than 2Na+/1Ca2+ (4, 27, 38), elimination of m during MI removes the electrical driving force for Ca2+ efflux via the putative electrogenic NCE. The loss of
m allows for a reverse action of the NCEs. In general, resting values of [Ca2+]m are comparable to those in the cytoplasm (3, 18) or are lower, as demonstrated in cardiomyocytes (5, 53). The observed increase in [Ca2+]i in our experiments, from
50 nM to values of >600 nM, induced a chemical driving force for Ca2+ entry into the mitochondrial matrix. Therefore, the mitochondrial NCE in reverse mode is thought to be driven by the Ca2+ gradient in the initial phase of the [Ca2+]i decline. Despite the large [Na+]i increase, the mitochondrial Na+ gradient ([Na+]i/[Na+]m) may be surpassed by the larger Ca2+ gradient (in the first 20 min of MI, [Ca2+]i increased >10-fold, whereas [Ca2+]m only doubled). Moreover, in isolated heart mitochondria, it was demonstrated that [Na+]m increases with [Na+]i and that the Na+ gradient across the internal mitochondrial membrane is significantly lower for nonrespiring vs. respiring mitochondria (37). However, the more Ca2+ is taken up by the mitochondria, the more the chemical driving force for Ca2+ weakens. To allow the observed decrease in [Ca2+]i to values near 120 nM after 60 min of MI (Fig. 1), whereas Frhod 2, mito signals increased almost to a factor of four (Fig. 9), one should hypothesize that the Na+ gradient has to control Ca2+ uptake via the NCE. This is only possible if the intramitochondrial Na+ content ([Na+]m) was augmented sufficiently to provide abundant Na+ for exchange with entering cytosolic Ca2+. However, the exact mechanism for the reverse action of the mitochondrial NCE remains to be elucidated.
Bernardi (4) described the existence of Mg2+-modulated inner membrane channels with selectivity for Na+. Whether these Na+ channels contribute to mitochondrial Na+ loading during MI seems unlikely, because these channels are inhibited by nanomolar concentrations of Mg2+ and by acidification, which is generally seen in ischemic conditions. Moreover, as expected, inward Na+ fluxes through these channels are known to decrease with mitochondrial depolarization (4).
Apart from the NCE, mitochondria in kidney tissue have a Na+-independent Ca2+ exchanger as the primary Ca2+ efflux pathway (27, 61). It is unlikely to assume a contribution of this exchanger (in reverse mode) in the mitochondrial Ca2+ accumulation seen during MI, because of 1) its Na+ independency, in contrast to the Na+ dependency of mitochondrial Ca2+ uptake in our experiments; and 2) the inhibition of this transport mechanism by CN- (30, 78).
Is Mitochondrial Ca2+ Uptake Beneficial for Cells in Ischemic Conditions?
Because mitochondria have the capacity to take up huge amounts of Ca2+, they can remove toxic levels of Ca2+ from the cytosol. However, in some circumstances, mitochondrial Ca2+ uptake can switch from a useful physiological regulatory mechanism to a potentially harmful process that can initiate the progression toward cell death (18). Indeed, accumulating evidence suggests that [Ca2+]m may play a critical role in ischemia-reperfusion injury (59). For example, [Ca2+]m rises significantly during heart ischemia (2), and the magnitude of this rise determines the outcome of ischemia-reoxygenation (53). Mitochondrial Ca2+ overload can initiate opening of the mitochondrial permeability transition pore (MPTP) and cause cell death by either energetic collapse, ATP depletion, and necrotic cell death or by initiating mitochondrial swelling, cytochrome c release, and apoptotic cascades (1, 46, 48, 48). The MPTP is a large-conductance pore that forms under pathological conditions in the inner mitochondrial membrane. The channel is opened by a combination of high [Ca2+]m, oxidative stress, ATP depletion, high inorganic phosphate, and mitochondrial depolarization (15, 69). MPTP might play a major role in reperfusion injury rather than during the ischemic period, because the conditions needed for opening of the MPTP are exactly those that occur during reperfusion (69). Moreover, ischemia-induced intracellular acidification might prevent opening of the MPTP because opening is greatly inhibited at intracellular pH values <7 (69). In the present study, opening of the MPTP during mitochondrial Ca2+ accumulation in metabolically inhibited MDCK cells probably did not occur within the time course of our experimental protocol of 60 min, because MI induced a substantial intracellular acidification in MDCK cells (pHi dropped from 7.35 ± 0.03 to 7 ± 0.03, n = 7; data not shown). Furthermore, no substantial rhod 2 release was seen, although rhod 2 is known to be released through the MPTP in isolated mitochondria (5).
Summary
The above results demonstrate that 1) mitochondria take up Ca2+ during MI despite their fully collapsed m; and 2) Ca2+ entry into mitochondria occurs via the NCE, whereas the
m-dependent uniporter is inactive. Up to now, the proposed mechanism of Ca2+ uptake into deenergized mitochondria via the reverse action of the mitochondrial NCE was only described for cardiomyocytes (24, 38). Our observations demonstrate that important alterations occur in mitochondrial Ca2+ transport pathways of metabolically inhibited distal renal epithelial cells: Ca2+ entry occurs via the NCE (the normal Ca2+ efflux pathway), whereas the Ca2+ uniporter (the normal influx route) is inactive.
![]() |
ACKNOWLEDGMENTS |
---|
GRANTS
Part of this work was supported via bilateral projects of Flanders (Belgium) with Romania and Hungary.
![]() |
FOOTNOTES |
---|
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.
* I. Smets, A. Caplanusi, and S. Despa contributed equally to this report. Present addresses of coauthors: A. Caplanusi, Dept. of Medical Biochemistry, Carol Davila University of Medicine and Pharmacy, R-050474 Bucharest, Romania; S. Despa, Dept. of Physiology, Loyola University Chicago, Maywood, IL 60153; Z. Molnar, Dept. of Medical Chemistry, University of Szeged, H-6701 Szeged, Hungary; and M. Radu, Dept. of Health and Environmental Physics, Horia Hulubei National Institute for Physics and Nuclear Engineering, R-76900 Bucharest, Romania.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|