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Pivotal Role of Mitochondrial Ca2+ in Microcystin-Induced Mitochondrial Permeability Transition in Rat Hepatocytes
Biochemical and Biophysical Research Communications 285, 1155–1161 (2001) doi:10.1006/bbrc.2001.5309, available online at http://www.idealibrary.com on
Pivotal Role of Mitochondrial Ca 2⫹ in Microcystin-Induced Mitochondrial Permeability Transition in Rat Hepatocytes Wen-Xing Ding, Han-Ming Shen, and Choon-Nam Ong 1 Department of Community, Occupational, and Family Medicine, Faculty of Medicine (MD3), National University of Singapore, 16 Medical Drive, Singapore 117597, Singapore
Received June 26, 2001
We have shown earlier that microcystin-LR (MLR), a specific hepatotoxin, induced onset of mitochondrial permeability transition (MPT) and apoptosis in rat hepatocytes. Here we attempted to investigate the role of mitochondrial Ca 2ⴙ in MLR-induced onset of MPT and cell death. Using confocal microscopy, we found that MLR caused an early surge of mitochondrial Ca 2ⴙ prior to the onset of MPT and cell death. Pretreatment with 1,2-bis(O-aminophenoxyl)ethane-N,N,Nⴕ,Nⴕtetracetic acid tetra(acetoxymethyl)ester (an intracellular Ca 2ⴙ chelator) or ruthenium red (an inhibitor of mitochondrial Ca 2ⴙ uniporter) prevented the early mitochondrial Ca 2ⴙ surge and attenuated the subsequent onset of MPT and cell death. On the other hand, a mitochondrial uncoupler, CCCP, rapidly disrupted the mitochondrial membrane potential and also prevented the mitochondrial Ca 2ⴙ surge, onset of MPT, and cell death. We thus conclude that mitochondrial Ca 2ⴙ plays an important role in the onset of MPT and cell death in MLR-treated rat hepatocytes. © 2001 Academic Press
Key Words: cyanobacteria; mitochondrial membrane potential; confocal microscopy; apoptosis; oxidative stress.
Microcystins produced by cyanobacteria (blue– green algae) have profound adverse health effect both on animals and human beings (1–3). Among the family of microcystins, microcystin-LR (MLR) has been found to be the most common one with potent hepatotoxicity and tumor promotion activity (3, 4). However, the exact Abbreviations used: BAPTA 1,2-bis(O-aminophenoxyl)ethaneN,N,N⬘,N⬘-tetracetic acid tetra(acetoxymethyl)ester; CCCP, carbonyl cyanide m-chlorophenyl-hydrazone; DCF, dichlorofluorescein; DCFH-DA, dichlorofluorescein diacetate; KRH, Krebs–Ringer– Hepes buffer; MCE, microcystic cyanobacteria extract; MLR, microcystin-LR; MMP, mitochondrial membrane potential; MPT, mitochondrial permeability transition; ROS, reactive oxygen species; RR, ruthenium red; TMRM, tetramethyrhodamine methyl ester. 1 To whom correspondence and reprint requests should be addressed. Fax: (65)-7791489. E-mail: [email protected].
mechanisms of microcystin-induced hepatotoxicity and tumor promotion activity have not been fully elucidated. Recent data have shown that MLR is capable of inducing rapid apoptosis as evidenced by characteristic apoptotic changes, including cell membrane blebbing, cell shrinkage, phosphatidylserine externalization and chromatin condensation in primary cultured rat hepatocytes (5, 6). Moreover, mitochondria have been found to be the major target in MLR-induced apoptotic process, and the onset of mitochondrial permeability transition (MPT) well precedes the characteristic signs of apoptosis (5). Nevertheless, the mechanisms how MLR mediates the onset of MPT remain largely elusive. MPT is executed by a dynamic multiprotein complex which is referred to as a permeability pore and is located at the inner/outer membrane contact sites of mitochondria (7). Although the exact molecular structure of this pore is not completely clear, it has been suggested that the pore is composed in part of the adenine nucleotide translocator proteins in the inner membrane, cyclophilin-D in the matrix, the voltagedependent anion channel in the outer membrane, and other possible proteins at the contact sites between the inner and outer membranes (7, 8). The onset of MPT represents an abrupt increase of permeability of the inner mitochondrial membrane to solutes with a molecular mass of less than 1500 Da (7, 9, 10). Following the onset of MPT, there are at least three important cellular events, including (i) elevation of ROS formation, (ii) lose of mitochondrial membrane potential (MMP), and (iii) release of apoptotic factors from mitochondria, such as cytochrome c, which all favor the execution of apoptosis (10). Various agents or conditions have been shown to cause onset of MPT in isolated mitochondria, and most notably is the high Ca 2⫹ loading (11). Mitochondria maintain a steady level of free Ca 2⫹ concentration through the balance of MMP-driven Ca 2⫹ uptake and Ca 2⫹ efflux by Na ⫹-dependent or independent exchange driven by both MMP and pH gradient (⌬pH) (9, 11). Ca 2⫹ enters mitochondria electrophoretically via
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the Ca 2⫹ uniporter and the influx rate increases with increasing MMP. This suggests that MMP is the main driving force of mitochondria Ca 2⫹ uptake (7). On the other hand, it has been suggested that mitochondrial Ca 2⫹ overloading would lead to onset of MPT, and MPT may serve as a mechanism in regulating the mitochondrial Ca 2⫹ homeostasis (11). However, such a hypothesis was rather difficult to prove due to the technical difficulties in evaluating these two events simultaneously in live cells (7, 9, 12). Hence we sought to study the regulatory role of mitochondria Ca 2⫹ in MLRinduced onset of MPT and apoptosis in cultured rat hepatocytes. Our results demonstrate that MLR causes an early surge of mitochondrial Ca 2⫹ that triggers the onset of MPT and eventually leads to cell death. The main driving force for the mitochondrial Ca 2⫹ surge could be MMP. MATERIALS AND METHODS Materials. MLR, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), ruthenium red (RR), Hepes, type IV collagenase, and Williams’ Medium E were all purchased from Sigma (St. Louis, MO). Rhod-2, calcein-AM, tetramethyrhodamine methyl ester (TMRM), 1,2-bis(O-aminophenoxyl)ethane-N,N,N⬘,N⬘-tetracetic acid tetra(acetoxymethyl)ester (BAPTA), and dichlorofluorescein diacetate (DCFH-DA) were all from Molecular Probes (Eugene, OR). Other common chemicals were all in analytical grade and from Merck (Darmstadt, Germany). Liver perfusion and primary rat hepatocytes culture. Male Sprague–Dawley rats with an average body weight of 180 to 200 g were used for cell preparation. Animals were handled in line with the “International guiding principles for animal research” adapted by the University. Liver perfusion and primary rat hepatocyte culture were carried out as reported earlier (5). Cell viability was always ⬎90% after isolation as determined by trypan blue exclusion test. For confocal microscopy studies, cells were plated at a density of 1 ⫻ 10 5 cells/well onto a four-well chamber (Nunc, Napervalle, IL) in 1 ml complete Williams’ Medium E (supplemented with 2 mM L-glutamine, 0.02 IU insulin/ml and 10% FBS). For other studies, a total of 2 ⫻ 10 6 cells were plated in 25-cm 2 flasks in 5 ml of complete Williams’ Medium E. After 2 h preincubation, the cells were washed with prewarmed Krebs–Ringer–Hepes buffer (KRH, containing 25 mM Na-Hepes, 115 mM NaCl, 5 mM KCl, 1 mM KH 2PO 4, 1.2 mM MgSO 4, and 0.5 mM CaCl 2, pH 7.4) to remove the detached dead cells. The hepatocytes were then incubated in serum-free Williams’ Medium E for subsequent treatments. 2⫹
Mitochondrial Ca measurement. The fluorescence dye, Rhod-2, can be drawn electrophoretically into mitochondria due to its net positive charge, where it is hydrolyzed to its free calcium indicating form (12). Cultured rat hepatocytes were first incubated with Rhod-2 (0.8 M) in cold medium (4°C) and left at room temperature for 15 min, the cells were then returned to 37°C incubator for 30 min. To observe MPT simultaneously, the cells were further loaded with 1 M calcein for another 15 min at 37°C. After washed with KRH buffer, the chamber was mounted onto the microscope stage (preheated to 37°C) for MLR treatment. In some experiments, before loading Rhod-2 and calcein, the cells were preincubated with 4 M BAPTA or 125 M RR for 30 min in culture medium at 37°C, respectively. In those experiments with CCCP, the cells were treated with 2 M CCCP simultaneously with MLR after loading with respective dyes.
Detection of MPT and MMP. To monitor onset of MPT and MMP changes, cultured hepatocytes were loaded in culture medium at 37°C with 0.5 M TMRM for 15 min, followed by 1 M calcein for another 15 min in the chamber as described earlier (5). TMRM is a membrane-permeable cationic fluorophore that accumulates electrophoretically into mitochondria in response to their negative potential. Whereas calcein (623 Da) is unable to enter mitochondria due to its impermeability of the mitochondrial inner membrane and thus it is distributed exclusively in the cytosolic space (13). Redistribution of the green fluorescence of calcein from cytosol into the mitochondria signifies the onset of MPT, and the decrease of TMRM red fluorescence in mitochondria indicates the disruption of MMP. After loading, the cells were then washed twice with KRH buffer, and incubated at 37°C in KRH buffer before mounted on the microscope stage for MLR treatment. Measuring of intracellular ROS formation using flow cytometry. The method to measure intracellular ROS formation using flow cytometry was modified from Tan et al. (14). Briefly, the cells were collected after the designated treatments and washed with PBS for 3 times, then incubated with 10 M DCFH-DA for 30 min in 1 mL PBS at 37°C. The cells were finally resuspended in PBS for flow cytometry analysis (Coulter Epics Elite ESP, Miami, FL) and a total of 10,000 cells for each group were counted. Dichlorofluorescein (DCF) data were collected with the following excitation and emission wavelengths: ex ⫽ 475 nm, em ⫽ 525 nm. The data obtained from flow cytometry were analyzed using WinMDI 2.7 software for subtracting the histogram and calculating the percentage of oxidative stressed cells (cells with higher level of ROS) in each group. Determination of cytotoxicity. Previous study has shown that MLR could induce rapid apoptosis and significant lactate dehydrogenase (LDH) leakage in cultured hepatocytes (5). Therefore, in the present study LDH leakage was used to monitor the cytotoxicity of MLR which was determined by the percentage of LDH leakage from cells into the medium, as described earlier (15). Laser-scanning confocal microscopy. Laser confocal microscopy was performed with a Zeiss LSM 410 confocal microscopy. The red fluorescence of Rhod-2, TMRM and the green fluorescence of calcein were excited simultaneously with the 568- and 488-nm lines of an argon-krypton laser. Fluorescence was split by a 560-nm emission dichroic reflector and collected by separate photomultipliers through 515- to 565-nm band-pass and 590-nm-long pass barrier filters. Statistical analysis. The morphological images were representatives of four to six sets of independent experiments. The numerical data were presented as means ⫾ SE from at least three independent experiments. The differences between different groups and control were examined using Student’s t test. A P value less than 0.05 was considered statistically significant.
RESULTS MLR Caused a Surge of Mitochondrial Ca 2⫹ before Onset of MPT We have shown earlier that MLR-induced onset of MPT and apoptosis in hepatocytes (5). Since increased mitochondrial Ca 2⫹ predisposes isolated mitochondria to MPT (7), we therefore decided to examine whether MLR causes an increase of mitochondrial Ca 2⫹, and in particular whether such an increase is critical to the onset of MPT in cultured hepatocytes. As shown in Fig. 1A, for untreated cells, the green calcein mainly distributed in both the cytosol and nucleus, leaving round dark voids indicating the mitochondria. In contrast, the red Rhod-2 fluorescence specifically localized in
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FIG. 1. Increase of mitochondrial Ca 2⫹ and onset of MPT in MLR-treated rat hepatocytes. Cultured rat hepatocytes were loaded with Rhod-2 and calcein, as described under Materials and Methods. Images of green calcein fluorescence (A–D) and red Rhod-2 fluorescence (E–H) were collected simultaneously by confocal microscopy. In the baseline images, calcein fluorescence was localized to the cytosol and nucleus, leaving mitochondria as dark voids (A), whereas Rhod-2 fluorescence was specifically in the mitochondrial area (E). After 10 min exposure to MLR (1 M), mitochondrial Rhod-2 fluorescence increased significantly (F), but onset of MPT did not occur as the mitochondrial dark voids were still evident in the calcein images (B). At 20 min, onset of MPT started to occur as mitochondria began to be filled with calcein (C), and mitochondrial Rhod-2 fluorescence started to decrease (G). At 30 min, onset of MPT was almost completed as indicated by the green calcein redistributed from cytosol to the mitochondria (D), and mitochondrial Rhod-2 fluorescence further decreased (H).
areas corresponding to mitochondria (Fig. 1E). After 10 min exposure to 1 M MLR, a surge of mitochondrial Ca 2⫹ was observed, as indicated by the increase of Rhod-2 fluorescence (Fig. 1F). However, there was no onset of MPT at the same time point, as calcein did not enter mitochondria and the dark voids were still evident (Fig. 1B). The onset of MPT was observed after 20 min of MLR exposure, as indicated by the presence of calcein in most of the mitochondria and the reduction of the dark voids (Fig. 1C). Concurrently, Rhod-2 fluorescence in most of the mitochondria started to decline (Fig. 1G). At 30 min, onset of MPT was almost completed (Fig. 1D) while there was a further lost of mitochondrial Rhod-2 fluorescence (Fig. 1H). Results presented here clearly demonstrate that there is a surge of mitochondrial Ca 2⫹ occurs prior to the onset of MPT in MLR-treated hepatocytes.
calcium chelator) or RR (an inhibitor of mitochondrial Ca 2⫹ uniporter) for 30 min, the cells were then exposed to MLR for a prolonged period for up to 50 min. In the cells pretreated with BAPTA, there were no significant changes of Rhod-2 and calcein fluorescence after exposure to MLR for 10 min (Figs. 2B and 2E) comparing with their respective basal images of cells loaded with Rhod-2 and calcein (Figs. 2A and 2D). Furthermore, no significant alterations of Rhod-2 and calcein fluorescence were observed even after a prolonged exposure to MLR for up to 50 min (Figs. 2C and 2F). Similar results were also found when cells were pretreated with RR (Figs. 2G–2L). The results from this part of experiments thus demonstrate that BAPTA or RR could inhibit the MLR-induced mitochondrial Ca 2⫹ surge and the onset of MPT. Protection by BAPTA or RR against MLR-Induced ROS Formation and Cell Death
Effects of BAPTA or RR on the Mitochondrial Ca 2⫹ Surge and Onset of MPT by MLR To assess the regulatory role of mitochondrial Ca 2⫹ on MLR-induced onset of MPT and cell death, the following experiments were carried out. Hepatocytes were first pretreated with BAPTA (an intracellular
We have shown previously that MLR could induce ROS formation in cultured rat hepatocytes (5). Here we tried to examine whether prevention of mitochondrial Ca 2⫹ surge could have any effects on ROS formation. In this set of experiments, the ROS level was measured
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FIG. 2. Effects of BAPTA or RR on mitochondrial Ca 2⫹ changes and onset of MPT in MLR-treated rat hepatocytes. Cultured hepatocytes were first pretreated with 4 M BAPTA or 125 M RR for 30 min, as described under Materials and Methods. A and D are the basal images of Rhod-2 and calcein after the pretreatment with BAPTA. Mitochondrial Ca 2⫹ did not rise and onset of MPT did not occur after 10 min exposure to MLR with the presence of BAPTA (B, E), and even after a longer exposure time for up to 50 min (C, F). Similarly, there were no mitochondrial Ca 2⫹ surge and onset of MPT in the cells pretreated with RR (G–L).
using flow cytometry with DCFH-DA as the fluorescence probe. As can be seen in Fig. 3A, for the cells exposed to 1 M MLR for 50 min, the percentage of oxidative stressed cells (cells with higher DCF fluorescence intensity) was nearly 4 times higher than that of
control cells. Interestingly, pretreatment with BAPTA or RR was able to reduce MLR-induced ROS production significantly. Moreover, pretreatment of BAPTA or RR also markedly reduced the LDH leakage (Fig. 3B). These results suggest that mitochondrial Ca 2⫹
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the presence of CCCP, mitochondria began to lose TMRM fluorescence within 1 min (data not shown) and nearly lost all TMRM fluorescence after 30 min exposure to MLR (Fig. 4B), suggesting that CCCP rapidly and efficiently disrupted MMP. In contrast, the dark voids representing mitochondria in the calcein images after MLR and CCCP exposure for 30 min were still evident (Fig. 4D). These results indicate that mitochondrial depolarization by CCCP is not sufficient to induce MPT, but rather protects against MLR-induced MPT. On the other hand, in the presence of CCCP, there is no mitochondrial Ca 2⫹ surge in MLR-treated hepatocytes. Conversely, nearly all the mitochondrial Ca 2⫹ was released as evidenced by the significant reduction of Rhod-2 fluorescence level (Fig. 4F). Furthermore, CCCP completely protected hepatocytes against MLRinduced cell death (Fig. 4G). DISCUSSION
FIG. 3. Effects of BAPTA or RR on MLR-induced ROS formation and cell death. The inhibitory effects on MLR-induced ROS formation was shown in A. ROS formation was detected by flow cytometry as described under Materials and Methods. After BAPTA or RR pretreatment, hepatocytes were further exposed to MLR for 50 min. The percentages of oxidative stressed cells (cells with higher DCF fluorescence intensity) are shown in each treatment. The inhibitory effects of BAPTA or RR pretreatment against MLR-induced LDH leakage are shown in B. Results are presented as means ⫾ SE from three independent experiments. *P ⬍ 0.05 versus control group and #P ⬍ 0.05 versus MLR group (Student’s t test).
surge may play an important role in MLR-induced ROS formation and cell death. Effects of CCCP on MLR-Induced MMP Changes, Onset of MPT, Mitochondrial Ca 2⫹ Surge, and Cell Death CCCP is a potent mitochondrial uncoupler which dissipates the H ⫹ gradient across the inner mitochondrial membrane and subsequently disrupts MMP (16). To further determine whether the MMP is the main driving force for mitochondrial Ca 2⫹ surge, the following experiments were conducted. Cells were either loaded with TMRM and calcein, or Rhod-2 and calcein before treated with 2 M CCCP and 1 M MLR simultaneously. Figures 4A, 4C, and 4E show the basal images of TMRM, calcein and Rhod-2, respectively. In
Our previous work has indicated that microcystin-LR could induce onset of MPT and lead to rapid apoptosis in hepatocytes (5). MPT has been well recognized as a critical event in the assembly of the apoptotic machinery. Although the regulatory mechanisms of MPT are not completely understood, accumulating evidence suggests that mitochondrial Ca 2⫹ may play a key role in this event (7, 10). Therefore, the main objective of this study is to examine the pivotal role of mitochondrial Ca 2⫹ in MLR-induced onset of MPT and apoptosis. First, we provide clear evidence that MLR causes an early mitochondrial Ca 2⫹ surge prior to onset of MPT (Fig. 1). Secondly, the pretreatment with BAPTA (an intracellular Ca 2⫹ chelator) or RR (an inhibitor of mitochondrial Ca 2⫹ uptake uniporter) prevented the early mitochondrial Ca 2⫹ surge, the onset of MPT and cell death (Fig. 2). These observations thus indicate that the mitochondrial Ca 2⫹ surge plays a key role in the onset of MPT and the subsequent cell death in MLRtreated hepatocytes. It has been firmly established that the rapid uptake of Ca 2⫹ by mitochondria is mediated by a uniporter. The driving force comes from the Ca 2⫹ concentration gradient as well as the MMP gradient (11). Using TMRM to measure the MMP, we have previously shown that MLR caused a transient mitochondrial hyperpolarization which preceded the onset of MPT (5). To examine the possible causative relationship of such a transient increase of MMP with the mitochondrial Ca 2⫹ surge, we used rhodamine-123 (a green fluorescence dye for measuring MMP) and red Rhod-2 to monitor both MMP and mitochondrial Ca 2⫹ changes simultaneously. We further confirmed that the transient mitochondrial hyperpolarization occurred concurrently with the mitochondrial Ca 2⫹ surge (data not shown). It is thus believed that such transient MMP changes may
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FIG. 4. Effects of CCCP on MMP, mitochondrial Ca 2⫹ changes, onset of MPT, and cell death. Hepatocytes were loaded with TMRM and calcein, or Rhod-2 and calcein respectively, then exposed to 2 M CCCP and 1 M MLR simultaneously as described under Materials and Methods. A, C, and E are basal TMRM, calcein, and Rhod-2 fluorescence images. After 30 min, red TMRM fluorescence was completely lost (B) but most green calcein did not redistribute to mitochondria (D). Red Rhod-2 fluorescence was significantly decreased after 10 min (F). The effect of CCCP against MLR-induced LDH leakage is shown in G. Results are presented as means ⫾ SE from three independent experiments. *P ⬍ 0.05 versus control group and #P ⬍ 0.05 versus MLR group (Student’s t test).
play a critical role in MLR-induced mitochondrial Ca 2⫹ surge. In the present study, we found that CCCP, a mitochondrial uncoupler, disrupted the MMP and also prevented the mitochondrial Ca 2⫹ surge (Fig. 4), which further supports that MMP may be the main driving force for the mitochondrial Ca 2⫹ surge in MLR-treated hepatocytes. At present, it is still controversial regarding the temporal events of onset of MPT and MMP changes. Bernardi et al. have suggested that onset of MPT is the consequence of mitochondrial depolarization (disruption of MMP) as evidenced by the studies in isolated mitochondria (17, 18). In contrast, our results showed that the depolarization of MMP by CCCP was not enough to induce onset of MPT, but rather to prevent MLR-induced onset of MPT in primary cultured rat hepatocytes (Figs. 4B and 4D). Similar results were also reported by Niemien et al. that CCCP could depolarize mitochondria within 20 s, however the onset of MPT did not occur even after exposing to CCCP for 19 min in rat hepatocytes (19). These paradoxical findings may partially be explained by the difference in experimental systems, i.e., isolated mitochondria and live cells. Thus the exact inter-relationship between MMP and MPT remains to be further elucidated.
It has long been hypothesized that MPT serves to regulate Ca 2⫹ in mitochondria by allowing it to efflux (11). However, this hypothesis has recently been challenged, based on the observation that hormoneinduced mitochondrial Ca 2⫹ efflux was independent of MPT (20). In the present study, it is interesting to note that in the presence of CCCP, mitochondrial Ca 2⫹ was almost completely depleted without the onset of MPT (Fig. 4). Since the onset of MPT always leads to loss of MMP, it is rather difficult to distinguish whether the efflux of mitochondrial Ca 2⫹ is due to the MPT per se or due to the loss of driving force of MMP. In the present study, we found that loss of MMP was sufficient to deplete mitochondrial Ca 2⫹ without the onset of MPT (Fig. 4). These results thus support the notion that mitochondrial Ca 2⫹ efflux may occur independent of MPT. At present, it is still not completely clear how elevated mitochondrial Ca 2⫹ triggers the onset of MPT. Petronilli et al. have proposed a model for the role of mitochondrial Ca 2⫹ on the onset of MPT (18). According to that model, there is an external Me 2⫹ (metal ion) binding site and an internal Me 2⫹ binding site that regulate the onset of MPT. Binding of any Me 2⫹ ion to the external site will decrease the probability of onset
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of MPT. On the other hand, the internal site is normally occupied by H ⫹, and the binding of Ca 2⫹ to this site will increase the probability of onset of MPT. Thus it is possible that MLR causes the mitochondrial Ca 2⫹ surge, which in turn occupies the internal regulatory site and eventually facilitates the onset of MPT. It is also interesting to note that pretreatment with calcium chelator (BAPTA) and the inhibitor of mitochondrial Ca 2⫹ uniporter (RR) significantly inhibited the MLR-induced ROS formation (Fig. 3). This finding is not surprising as it has been suggested that the increase of mitochondrial Ca 2⫹ contribute to ROS formation (12). On the other hand, onset of MPT per se could also lead to production of ROS (10). Thus it is possible that prevention of either the mitochondrial Ca2⫹ surge or the subsequent onset of MPT significantly reduced ROS formation in MLR-treated hepatocytes. In conclusion, our data clearly demonstrate that MLR causes an early mitochondrial Ca 2⫹ surge in primary cultured rat hepatocytes, which leads to onset of MPT and subsequent cell death. The protective effects of BAPTA (an intracellular Ca 2⫹ chelator) and RR (an inhibitor of mitochondrial Ca 2⫹ uniporter) against MLR-induced mitochondrial Ca 2⫹ surge, onset of MPT and cell death further support that mitochondrial Ca 2⫹ is one of the underlying mechanisms in MLR-induced onset of MPT and cell death. ACKNOWLEDGMENTS
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The authors thank Ms. C. Er, Z. Y. Han, B. L. Ng, and Mr. B. Qu for their technical assistance in confocal microscopy and flow cytometry. W. X. Ding is supported by a research scholarship from the National University of Singapore. This project is partly sponsored by a grant from the National Medical Research Council in Singapore.
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Pivotal Role of Mitochondrial Ca 2⫹ in Microcystin-Induced Mitochondrial Permeability Transition in Rat Hepatocytes Wen-Xing Ding, Han-Ming Shen, and Choon-Nam Ong 1 Department of Community, Occupational, and Family Medicine, Faculty of Medicine (MD3), National University of Singapore, 16 Medical Drive, Singapore 117597, Singapore
Received June 26, 2001
We have shown earlier that microcystin-LR (MLR), a specific hepatotoxin, induced onset of mitochondrial permeability transition (MPT) and apoptosis in rat hepatocytes. Here we attempted to investigate the role of mitochondrial Ca 2ⴙ in MLR-induced onset of MPT and cell death. Using confocal microscopy, we found that MLR caused an early surge of mitochondrial Ca 2ⴙ prior to the onset of MPT and cell death. Pretreatment with 1,2-bis(O-aminophenoxyl)ethane-N,N,Nⴕ,Nⴕtetracetic acid tetra(acetoxymethyl)ester (an intracellular Ca 2ⴙ chelator) or ruthenium red (an inhibitor of mitochondrial Ca 2ⴙ uniporter) prevented the early mitochondrial Ca 2ⴙ surge and attenuated the subsequent onset of MPT and cell death. On the other hand, a mitochondrial uncoupler, CCCP, rapidly disrupted the mitochondrial membrane potential and also prevented the mitochondrial Ca 2ⴙ surge, onset of MPT, and cell death. We thus conclude that mitochondrial Ca 2ⴙ plays an important role in the onset of MPT and cell death in MLR-treated rat hepatocytes. © 2001 Academic Press
Key Words: cyanobacteria; mitochondrial membrane potential; confocal microscopy; apoptosis; oxidative stress.
Microcystins produced by cyanobacteria (blue– green algae) have profound adverse health effect both on animals and human beings (1–3). Among the family of microcystins, microcystin-LR (MLR) has been found to be the most common one with potent hepatotoxicity and tumor promotion activity (3, 4). However, the exact Abbreviations used: BAPTA 1,2-bis(O-aminophenoxyl)ethaneN,N,N⬘,N⬘-tetracetic acid tetra(acetoxymethyl)ester; CCCP, carbonyl cyanide m-chlorophenyl-hydrazone; DCF, dichlorofluorescein; DCFH-DA, dichlorofluorescein diacetate; KRH, Krebs–Ringer– Hepes buffer; MCE, microcystic cyanobacteria extract; MLR, microcystin-LR; MMP, mitochondrial membrane potential; MPT, mitochondrial permeability transition; ROS, reactive oxygen species; RR, ruthenium red; TMRM, tetramethyrhodamine methyl ester. 1 To whom correspondence and reprint requests should be addressed. Fax: (65)-7791489. E-mail: [email protected].
mechanisms of microcystin-induced hepatotoxicity and tumor promotion activity have not been fully elucidated. Recent data have shown that MLR is capable of inducing rapid apoptosis as evidenced by characteristic apoptotic changes, including cell membrane blebbing, cell shrinkage, phosphatidylserine externalization and chromatin condensation in primary cultured rat hepatocytes (5, 6). Moreover, mitochondria have been found to be the major target in MLR-induced apoptotic process, and the onset of mitochondrial permeability transition (MPT) well precedes the characteristic signs of apoptosis (5). Nevertheless, the mechanisms how MLR mediates the onset of MPT remain largely elusive. MPT is executed by a dynamic multiprotein complex which is referred to as a permeability pore and is located at the inner/outer membrane contact sites of mitochondria (7). Although the exact molecular structure of this pore is not completely clear, it has been suggested that the pore is composed in part of the adenine nucleotide translocator proteins in the inner membrane, cyclophilin-D in the matrix, the voltagedependent anion channel in the outer membrane, and other possible proteins at the contact sites between the inner and outer membranes (7, 8). The onset of MPT represents an abrupt increase of permeability of the inner mitochondrial membrane to solutes with a molecular mass of less than 1500 Da (7, 9, 10). Following the onset of MPT, there are at least three important cellular events, including (i) elevation of ROS formation, (ii) lose of mitochondrial membrane potential (MMP), and (iii) release of apoptotic factors from mitochondria, such as cytochrome c, which all favor the execution of apoptosis (10). Various agents or conditions have been shown to cause onset of MPT in isolated mitochondria, and most notably is the high Ca 2⫹ loading (11). Mitochondria maintain a steady level of free Ca 2⫹ concentration through the balance of MMP-driven Ca 2⫹ uptake and Ca 2⫹ efflux by Na ⫹-dependent or independent exchange driven by both MMP and pH gradient (⌬pH) (9, 11). Ca 2⫹ enters mitochondria electrophoretically via
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the Ca 2⫹ uniporter and the influx rate increases with increasing MMP. This suggests that MMP is the main driving force of mitochondria Ca 2⫹ uptake (7). On the other hand, it has been suggested that mitochondrial Ca 2⫹ overloading would lead to onset of MPT, and MPT may serve as a mechanism in regulating the mitochondrial Ca 2⫹ homeostasis (11). However, such a hypothesis was rather difficult to prove due to the technical difficulties in evaluating these two events simultaneously in live cells (7, 9, 12). Hence we sought to study the regulatory role of mitochondria Ca 2⫹ in MLRinduced onset of MPT and apoptosis in cultured rat hepatocytes. Our results demonstrate that MLR causes an early surge of mitochondrial Ca 2⫹ that triggers the onset of MPT and eventually leads to cell death. The main driving force for the mitochondrial Ca 2⫹ surge could be MMP. MATERIALS AND METHODS Materials. MLR, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), ruthenium red (RR), Hepes, type IV collagenase, and Williams’ Medium E were all purchased from Sigma (St. Louis, MO). Rhod-2, calcein-AM, tetramethyrhodamine methyl ester (TMRM), 1,2-bis(O-aminophenoxyl)ethane-N,N,N⬘,N⬘-tetracetic acid tetra(acetoxymethyl)ester (BAPTA), and dichlorofluorescein diacetate (DCFH-DA) were all from Molecular Probes (Eugene, OR). Other common chemicals were all in analytical grade and from Merck (Darmstadt, Germany). Liver perfusion and primary rat hepatocytes culture. Male Sprague–Dawley rats with an average body weight of 180 to 200 g were used for cell preparation. Animals were handled in line with the “International guiding principles for animal research” adapted by the University. Liver perfusion and primary rat hepatocyte culture were carried out as reported earlier (5). Cell viability was always ⬎90% after isolation as determined by trypan blue exclusion test. For confocal microscopy studies, cells were plated at a density of 1 ⫻ 10 5 cells/well onto a four-well chamber (Nunc, Napervalle, IL) in 1 ml complete Williams’ Medium E (supplemented with 2 mM L-glutamine, 0.02 IU insulin/ml and 10% FBS). For other studies, a total of 2 ⫻ 10 6 cells were plated in 25-cm 2 flasks in 5 ml of complete Williams’ Medium E. After 2 h preincubation, the cells were washed with prewarmed Krebs–Ringer–Hepes buffer (KRH, containing 25 mM Na-Hepes, 115 mM NaCl, 5 mM KCl, 1 mM KH 2PO 4, 1.2 mM MgSO 4, and 0.5 mM CaCl 2, pH 7.4) to remove the detached dead cells. The hepatocytes were then incubated in serum-free Williams’ Medium E for subsequent treatments. 2⫹
Mitochondrial Ca measurement. The fluorescence dye, Rhod-2, can be drawn electrophoretically into mitochondria due to its net positive charge, where it is hydrolyzed to its free calcium indicating form (12). Cultured rat hepatocytes were first incubated with Rhod-2 (0.8 M) in cold medium (4°C) and left at room temperature for 15 min, the cells were then returned to 37°C incubator for 30 min. To observe MPT simultaneously, the cells were further loaded with 1 M calcein for another 15 min at 37°C. After washed with KRH buffer, the chamber was mounted onto the microscope stage (preheated to 37°C) for MLR treatment. In some experiments, before loading Rhod-2 and calcein, the cells were preincubated with 4 M BAPTA or 125 M RR for 30 min in culture medium at 37°C, respectively. In those experiments with CCCP, the cells were treated with 2 M CCCP simultaneously with MLR after loading with respective dyes.
Detection of MPT and MMP. To monitor onset of MPT and MMP changes, cultured hepatocytes were loaded in culture medium at 37°C with 0.5 M TMRM for 15 min, followed by 1 M calcein for another 15 min in the chamber as described earlier (5). TMRM is a membrane-permeable cationic fluorophore that accumulates electrophoretically into mitochondria in response to their negative potential. Whereas calcein (623 Da) is unable to enter mitochondria due to its impermeability of the mitochondrial inner membrane and thus it is distributed exclusively in the cytosolic space (13). Redistribution of the green fluorescence of calcein from cytosol into the mitochondria signifies the onset of MPT, and the decrease of TMRM red fluorescence in mitochondria indicates the disruption of MMP. After loading, the cells were then washed twice with KRH buffer, and incubated at 37°C in KRH buffer before mounted on the microscope stage for MLR treatment. Measuring of intracellular ROS formation using flow cytometry. The method to measure intracellular ROS formation using flow cytometry was modified from Tan et al. (14). Briefly, the cells were collected after the designated treatments and washed with PBS for 3 times, then incubated with 10 M DCFH-DA for 30 min in 1 mL PBS at 37°C. The cells were finally resuspended in PBS for flow cytometry analysis (Coulter Epics Elite ESP, Miami, FL) and a total of 10,000 cells for each group were counted. Dichlorofluorescein (DCF) data were collected with the following excitation and emission wavelengths: ex ⫽ 475 nm, em ⫽ 525 nm. The data obtained from flow cytometry were analyzed using WinMDI 2.7 software for subtracting the histogram and calculating the percentage of oxidative stressed cells (cells with higher level of ROS) in each group. Determination of cytotoxicity. Previous study has shown that MLR could induce rapid apoptosis and significant lactate dehydrogenase (LDH) leakage in cultured hepatocytes (5). Therefore, in the present study LDH leakage was used to monitor the cytotoxicity of MLR which was determined by the percentage of LDH leakage from cells into the medium, as described earlier (15). Laser-scanning confocal microscopy. Laser confocal microscopy was performed with a Zeiss LSM 410 confocal microscopy. The red fluorescence of Rhod-2, TMRM and the green fluorescence of calcein were excited simultaneously with the 568- and 488-nm lines of an argon-krypton laser. Fluorescence was split by a 560-nm emission dichroic reflector and collected by separate photomultipliers through 515- to 565-nm band-pass and 590-nm-long pass barrier filters. Statistical analysis. The morphological images were representatives of four to six sets of independent experiments. The numerical data were presented as means ⫾ SE from at least three independent experiments. The differences between different groups and control were examined using Student’s t test. A P value less than 0.05 was considered statistically significant.
RESULTS MLR Caused a Surge of Mitochondrial Ca 2⫹ before Onset of MPT We have shown earlier that MLR-induced onset of MPT and apoptosis in hepatocytes (5). Since increased mitochondrial Ca 2⫹ predisposes isolated mitochondria to MPT (7), we therefore decided to examine whether MLR causes an increase of mitochondrial Ca 2⫹, and in particular whether such an increase is critical to the onset of MPT in cultured hepatocytes. As shown in Fig. 1A, for untreated cells, the green calcein mainly distributed in both the cytosol and nucleus, leaving round dark voids indicating the mitochondria. In contrast, the red Rhod-2 fluorescence specifically localized in
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FIG. 1. Increase of mitochondrial Ca 2⫹ and onset of MPT in MLR-treated rat hepatocytes. Cultured rat hepatocytes were loaded with Rhod-2 and calcein, as described under Materials and Methods. Images of green calcein fluorescence (A–D) and red Rhod-2 fluorescence (E–H) were collected simultaneously by confocal microscopy. In the baseline images, calcein fluorescence was localized to the cytosol and nucleus, leaving mitochondria as dark voids (A), whereas Rhod-2 fluorescence was specifically in the mitochondrial area (E). After 10 min exposure to MLR (1 M), mitochondrial Rhod-2 fluorescence increased significantly (F), but onset of MPT did not occur as the mitochondrial dark voids were still evident in the calcein images (B). At 20 min, onset of MPT started to occur as mitochondria began to be filled with calcein (C), and mitochondrial Rhod-2 fluorescence started to decrease (G). At 30 min, onset of MPT was almost completed as indicated by the green calcein redistributed from cytosol to the mitochondria (D), and mitochondrial Rhod-2 fluorescence further decreased (H).
areas corresponding to mitochondria (Fig. 1E). After 10 min exposure to 1 M MLR, a surge of mitochondrial Ca 2⫹ was observed, as indicated by the increase of Rhod-2 fluorescence (Fig. 1F). However, there was no onset of MPT at the same time point, as calcein did not enter mitochondria and the dark voids were still evident (Fig. 1B). The onset of MPT was observed after 20 min of MLR exposure, as indicated by the presence of calcein in most of the mitochondria and the reduction of the dark voids (Fig. 1C). Concurrently, Rhod-2 fluorescence in most of the mitochondria started to decline (Fig. 1G). At 30 min, onset of MPT was almost completed (Fig. 1D) while there was a further lost of mitochondrial Rhod-2 fluorescence (Fig. 1H). Results presented here clearly demonstrate that there is a surge of mitochondrial Ca 2⫹ occurs prior to the onset of MPT in MLR-treated hepatocytes.
calcium chelator) or RR (an inhibitor of mitochondrial Ca 2⫹ uniporter) for 30 min, the cells were then exposed to MLR for a prolonged period for up to 50 min. In the cells pretreated with BAPTA, there were no significant changes of Rhod-2 and calcein fluorescence after exposure to MLR for 10 min (Figs. 2B and 2E) comparing with their respective basal images of cells loaded with Rhod-2 and calcein (Figs. 2A and 2D). Furthermore, no significant alterations of Rhod-2 and calcein fluorescence were observed even after a prolonged exposure to MLR for up to 50 min (Figs. 2C and 2F). Similar results were also found when cells were pretreated with RR (Figs. 2G–2L). The results from this part of experiments thus demonstrate that BAPTA or RR could inhibit the MLR-induced mitochondrial Ca 2⫹ surge and the onset of MPT. Protection by BAPTA or RR against MLR-Induced ROS Formation and Cell Death
Effects of BAPTA or RR on the Mitochondrial Ca 2⫹ Surge and Onset of MPT by MLR To assess the regulatory role of mitochondrial Ca 2⫹ on MLR-induced onset of MPT and cell death, the following experiments were carried out. Hepatocytes were first pretreated with BAPTA (an intracellular
We have shown previously that MLR could induce ROS formation in cultured rat hepatocytes (5). Here we tried to examine whether prevention of mitochondrial Ca 2⫹ surge could have any effects on ROS formation. In this set of experiments, the ROS level was measured
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FIG. 2. Effects of BAPTA or RR on mitochondrial Ca 2⫹ changes and onset of MPT in MLR-treated rat hepatocytes. Cultured hepatocytes were first pretreated with 4 M BAPTA or 125 M RR for 30 min, as described under Materials and Methods. A and D are the basal images of Rhod-2 and calcein after the pretreatment with BAPTA. Mitochondrial Ca 2⫹ did not rise and onset of MPT did not occur after 10 min exposure to MLR with the presence of BAPTA (B, E), and even after a longer exposure time for up to 50 min (C, F). Similarly, there were no mitochondrial Ca 2⫹ surge and onset of MPT in the cells pretreated with RR (G–L).
using flow cytometry with DCFH-DA as the fluorescence probe. As can be seen in Fig. 3A, for the cells exposed to 1 M MLR for 50 min, the percentage of oxidative stressed cells (cells with higher DCF fluorescence intensity) was nearly 4 times higher than that of
control cells. Interestingly, pretreatment with BAPTA or RR was able to reduce MLR-induced ROS production significantly. Moreover, pretreatment of BAPTA or RR also markedly reduced the LDH leakage (Fig. 3B). These results suggest that mitochondrial Ca 2⫹
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the presence of CCCP, mitochondria began to lose TMRM fluorescence within 1 min (data not shown) and nearly lost all TMRM fluorescence after 30 min exposure to MLR (Fig. 4B), suggesting that CCCP rapidly and efficiently disrupted MMP. In contrast, the dark voids representing mitochondria in the calcein images after MLR and CCCP exposure for 30 min were still evident (Fig. 4D). These results indicate that mitochondrial depolarization by CCCP is not sufficient to induce MPT, but rather protects against MLR-induced MPT. On the other hand, in the presence of CCCP, there is no mitochondrial Ca 2⫹ surge in MLR-treated hepatocytes. Conversely, nearly all the mitochondrial Ca 2⫹ was released as evidenced by the significant reduction of Rhod-2 fluorescence level (Fig. 4F). Furthermore, CCCP completely protected hepatocytes against MLRinduced cell death (Fig. 4G). DISCUSSION
FIG. 3. Effects of BAPTA or RR on MLR-induced ROS formation and cell death. The inhibitory effects on MLR-induced ROS formation was shown in A. ROS formation was detected by flow cytometry as described under Materials and Methods. After BAPTA or RR pretreatment, hepatocytes were further exposed to MLR for 50 min. The percentages of oxidative stressed cells (cells with higher DCF fluorescence intensity) are shown in each treatment. The inhibitory effects of BAPTA or RR pretreatment against MLR-induced LDH leakage are shown in B. Results are presented as means ⫾ SE from three independent experiments. *P ⬍ 0.05 versus control group and #P ⬍ 0.05 versus MLR group (Student’s t test).
surge may play an important role in MLR-induced ROS formation and cell death. Effects of CCCP on MLR-Induced MMP Changes, Onset of MPT, Mitochondrial Ca 2⫹ Surge, and Cell Death CCCP is a potent mitochondrial uncoupler which dissipates the H ⫹ gradient across the inner mitochondrial membrane and subsequently disrupts MMP (16). To further determine whether the MMP is the main driving force for mitochondrial Ca 2⫹ surge, the following experiments were conducted. Cells were either loaded with TMRM and calcein, or Rhod-2 and calcein before treated with 2 M CCCP and 1 M MLR simultaneously. Figures 4A, 4C, and 4E show the basal images of TMRM, calcein and Rhod-2, respectively. In
Our previous work has indicated that microcystin-LR could induce onset of MPT and lead to rapid apoptosis in hepatocytes (5). MPT has been well recognized as a critical event in the assembly of the apoptotic machinery. Although the regulatory mechanisms of MPT are not completely understood, accumulating evidence suggests that mitochondrial Ca 2⫹ may play a key role in this event (7, 10). Therefore, the main objective of this study is to examine the pivotal role of mitochondrial Ca 2⫹ in MLR-induced onset of MPT and apoptosis. First, we provide clear evidence that MLR causes an early mitochondrial Ca 2⫹ surge prior to onset of MPT (Fig. 1). Secondly, the pretreatment with BAPTA (an intracellular Ca 2⫹ chelator) or RR (an inhibitor of mitochondrial Ca 2⫹ uptake uniporter) prevented the early mitochondrial Ca 2⫹ surge, the onset of MPT and cell death (Fig. 2). These observations thus indicate that the mitochondrial Ca 2⫹ surge plays a key role in the onset of MPT and the subsequent cell death in MLRtreated hepatocytes. It has been firmly established that the rapid uptake of Ca 2⫹ by mitochondria is mediated by a uniporter. The driving force comes from the Ca 2⫹ concentration gradient as well as the MMP gradient (11). Using TMRM to measure the MMP, we have previously shown that MLR caused a transient mitochondrial hyperpolarization which preceded the onset of MPT (5). To examine the possible causative relationship of such a transient increase of MMP with the mitochondrial Ca 2⫹ surge, we used rhodamine-123 (a green fluorescence dye for measuring MMP) and red Rhod-2 to monitor both MMP and mitochondrial Ca 2⫹ changes simultaneously. We further confirmed that the transient mitochondrial hyperpolarization occurred concurrently with the mitochondrial Ca 2⫹ surge (data not shown). It is thus believed that such transient MMP changes may
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FIG. 4. Effects of CCCP on MMP, mitochondrial Ca 2⫹ changes, onset of MPT, and cell death. Hepatocytes were loaded with TMRM and calcein, or Rhod-2 and calcein respectively, then exposed to 2 M CCCP and 1 M MLR simultaneously as described under Materials and Methods. A, C, and E are basal TMRM, calcein, and Rhod-2 fluorescence images. After 30 min, red TMRM fluorescence was completely lost (B) but most green calcein did not redistribute to mitochondria (D). Red Rhod-2 fluorescence was significantly decreased after 10 min (F). The effect of CCCP against MLR-induced LDH leakage is shown in G. Results are presented as means ⫾ SE from three independent experiments. *P ⬍ 0.05 versus control group and #P ⬍ 0.05 versus MLR group (Student’s t test).
play a critical role in MLR-induced mitochondrial Ca 2⫹ surge. In the present study, we found that CCCP, a mitochondrial uncoupler, disrupted the MMP and also prevented the mitochondrial Ca 2⫹ surge (Fig. 4), which further supports that MMP may be the main driving force for the mitochondrial Ca 2⫹ surge in MLR-treated hepatocytes. At present, it is still controversial regarding the temporal events of onset of MPT and MMP changes. Bernardi et al. have suggested that onset of MPT is the consequence of mitochondrial depolarization (disruption of MMP) as evidenced by the studies in isolated mitochondria (17, 18). In contrast, our results showed that the depolarization of MMP by CCCP was not enough to induce onset of MPT, but rather to prevent MLR-induced onset of MPT in primary cultured rat hepatocytes (Figs. 4B and 4D). Similar results were also reported by Niemien et al. that CCCP could depolarize mitochondria within 20 s, however the onset of MPT did not occur even after exposing to CCCP for 19 min in rat hepatocytes (19). These paradoxical findings may partially be explained by the difference in experimental systems, i.e., isolated mitochondria and live cells. Thus the exact inter-relationship between MMP and MPT remains to be further elucidated.
It has long been hypothesized that MPT serves to regulate Ca 2⫹ in mitochondria by allowing it to efflux (11). However, this hypothesis has recently been challenged, based on the observation that hormoneinduced mitochondrial Ca 2⫹ efflux was independent of MPT (20). In the present study, it is interesting to note that in the presence of CCCP, mitochondrial Ca 2⫹ was almost completely depleted without the onset of MPT (Fig. 4). Since the onset of MPT always leads to loss of MMP, it is rather difficult to distinguish whether the efflux of mitochondrial Ca 2⫹ is due to the MPT per se or due to the loss of driving force of MMP. In the present study, we found that loss of MMP was sufficient to deplete mitochondrial Ca 2⫹ without the onset of MPT (Fig. 4). These results thus support the notion that mitochondrial Ca 2⫹ efflux may occur independent of MPT. At present, it is still not completely clear how elevated mitochondrial Ca 2⫹ triggers the onset of MPT. Petronilli et al. have proposed a model for the role of mitochondrial Ca 2⫹ on the onset of MPT (18). According to that model, there is an external Me 2⫹ (metal ion) binding site and an internal Me 2⫹ binding site that regulate the onset of MPT. Binding of any Me 2⫹ ion to the external site will decrease the probability of onset
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of MPT. On the other hand, the internal site is normally occupied by H ⫹, and the binding of Ca 2⫹ to this site will increase the probability of onset of MPT. Thus it is possible that MLR causes the mitochondrial Ca 2⫹ surge, which in turn occupies the internal regulatory site and eventually facilitates the onset of MPT. It is also interesting to note that pretreatment with calcium chelator (BAPTA) and the inhibitor of mitochondrial Ca 2⫹ uniporter (RR) significantly inhibited the MLR-induced ROS formation (Fig. 3). This finding is not surprising as it has been suggested that the increase of mitochondrial Ca 2⫹ contribute to ROS formation (12). On the other hand, onset of MPT per se could also lead to production of ROS (10). Thus it is possible that prevention of either the mitochondrial Ca2⫹ surge or the subsequent onset of MPT significantly reduced ROS formation in MLR-treated hepatocytes. In conclusion, our data clearly demonstrate that MLR causes an early mitochondrial Ca 2⫹ surge in primary cultured rat hepatocytes, which leads to onset of MPT and subsequent cell death. The protective effects of BAPTA (an intracellular Ca 2⫹ chelator) and RR (an inhibitor of mitochondrial Ca 2⫹ uniporter) against MLR-induced mitochondrial Ca 2⫹ surge, onset of MPT and cell death further support that mitochondrial Ca 2⫹ is one of the underlying mechanisms in MLR-induced onset of MPT and cell death. ACKNOWLEDGMENTS
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The authors thank Ms. C. Er, Z. Y. Han, B. L. Ng, and Mr. B. Qu for their technical assistance in confocal microscopy and flow cytometry. W. X. Ding is supported by a research scholarship from the National University of Singapore. This project is partly sponsored by a grant from the National Medical Research Council in Singapore.
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