Mitochondrial permeability transition in rat hepatocytes after anoxia/reoxygenation: role of Ca2+-dependent mitochondrial formation of reactive oxygen species

Abstract

Onset of the mitochondrial permeability transition (MPT) is the penultimate event leading to lethal cellular ischemia-reperfusion injury, but the mechanisms precipitating the MPT after reperfusion remain unclear. Here, we investigated the role of mitochondrial free Ca(2+) and reactive oxygen species (ROS) in pH- and MPT-dependent reperfusion injury to hepatocytes. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 4 h and then reoxygenated at pH 7.4 to simulate ischemia-reperfusion. Some cells were loaded with the Ca(2+) chelators, BAPTA/AM and 2-[(2-bis-[carboxymethyl]aono-5-methoxyphenyl)-methyl-6-methoxy-8-bis[carboxymethyl]aminoquinoline, either by a cold loading protocol for intramitochondrial loading or by warm incubation for cytosolic loading. Cell death was assessed by propidium iodide fluorometry and immunoblotting. Mitochondrial Ca(2+), inner membrane permeability, membrane potential, and ROS formation were monitored with Rhod-2, calcein, tetramethylrhodamine methylester, and dihydrodichlorofluorescein, respectively. Necrotic cell death increased after reoxygenation. Necrosis was blocked by 1 μM cyclosporin A, an MPT inhibitor, and by reoxygenation at pH 6.2. Confocal imaging of Rhod-2, calcein, and dichlorofluorescein revealed that an increase of mitochondrial Ca(2+) and ROS preceded onset of the MPT after reoxygenation. Intramitochondrial Ca(2+) chelation, but not cytosolic Ca(2+) chelation, prevented ROS formation and subsequent necrotic and apoptotic cell death. Reoxygenation with the antioxidants, desferal or diphenylphenylenediamine, also suppressed MPT-mediated cell death. However, inhibition of cytosolic ROS by apocynin or diphenyleneiodonium chloride failed to prevent reoxygenation-induced cell death. In conclusion, Ca(2+)-dependent mitochondrial ROS formation is the molecular signal culminating in onset of the MPT after reoxygenation of anoxic hepatocytes, leading to cell death.

Fig. 1.

Protection by intramitochondrial Ca²⁺ chelation against reoxygenation injury to hepatocytes. A: hepatocytes cultured in 24-well plates were incubated in anaerobic Krebs-Ringer-HEPES (KRH) buffer at pH 6.2 with 10 μM BAPTA/AM or 2-[(2-bis-[carboxymethyl]aono-5-methoxyphenyl)-methyl-6-methoxy-8-bis[carboxymethyl]aminoquinoline] (Quin 2)/AM at 37°C for the first 20 min of anoxia to chelate cytosolic Ca²⁺, as described in materials and methods. After 4 h of anoxia, cells were reoxygenated in aerobic KRH at pH 7.4 or at pH 6.2. In some experiments, hepatocytes were treated with 1 μM cyclosporin A (CsA) 20 min before and continuously after reoxygenation. Necrotic cell death was evaluated by propidium iodide (PI) fluorometry. B: hepatocytes were loaded with 10 μM of BAPTA/AM or Quin 2/AM at 0–1°C for 20 min to chelate mitochondrial Ca²⁺ and then subjected to anoxia at 37°C for 4 h. Cell death was assessed after reoxygenation. Values for each treatment group are means ± SE from 3 separate hepatocyte isolations for each group. *P < 0.05 vs. sham loading.

Fig. 2.

Mitochondrial Ca²⁺ loading precedes onset of the mitochondrial permeability transition (MPT) in hepatocytes after anoxia/reoxygenation (A/R). A: hepatocytes cultured on glass coverslips were incubated with 10 μM Rhod-2 and 1 μM calcein by the warm loading protocol to monitor cytosolic Ca²⁺ and onset of the MPT, respectively, as described in materials and methods. Confocal images were collected after 5, 60, 120, 180, and 240 min of anoxia. Note that Rhod-2 fluorescence progressively increased in the cytosol. During 4 h of anoxia, mitochondrial dark voids remained unfilled by green calcein fluorescence, indicating a closed state of permeability transition (PT) pores. B: hepatocytes cultured on glass coverslips were incubated with 10 μM Rhod-2 by the cold loading protocol to monitor mitochondrial Ca²⁺. Calcein was coloaded by warm ester-loading to monitor onset of the MPT. Confocal images were collected after 0, 4, 8, 12, and 14 min of reoxygenation (Reoxy). Mitochondrial Rhod-2 fluorescence increased rapidly after reoxygenation. At the end of anoxia and after 4 min of reoxygenation, MPT onset did not occur. After 12 min, mitochondrial dark voids of calcein fluorescence disappeared due to onset of the MPT.

Fig. 3.

Suppression of reactive oxygen species (ROS) formation by intramitochondrial Ca²⁺ chelation. A: hepatocytes cultured on glass coverslips were subjected to 4 h of normoxia and then aerobically incubated in KRH for 40 min with 10 μM H₂DCF diacetate and 100 nM tetramethylrhodamine methylester (TMRM) to monitor ROS generation and ΔΨm, respectively. Confocal images of green dichlorofluorescein (DCF) and red TMRM were simultaneously collected. An inset at bottom represents the merged image of red and green channel. Normoxic hepatocytes maintained ΔΨm and cell viability, and mitochondrial DCF fluorescence increased progressively but more slowly than after reoxygenation. B: confocal images of DCF and TMRM were collected after 4 h of anoxia and 3, 7, 10, and 12 min of reoxygenation. Mitochondria repolarized at 3 min but depolarized after 12 min. Reoxygenation progressively increased DCF fluorescence over 10 min. Note that ROS formation was predominantly in mitochondria during the early phase of reoxygenation. After 12 min, hepatocytes lost viability, as shown by the release of DCF fluorescence. C: hepatocytes were loaded with 10 μM BAPTA/AM by the cold loading protocol, as described in Fig. 1B. Cold loading of BAPTA prevented the loss of ΔΨm and cell death and markedly suppressed both mitochondrial and cytosolic ROS increase after reoxygenation. D: warm loading of BAPTA suppressed the cytosolic ROS increase but failed to prevent mitochondrial ROS increase, ΔΨm depolarization, and cell death. E: hepatocytes cultured in 24-well microplates were anaerobically loaded with 10 μM of BAPTA/AM or Quin 2/AM by the cold ester loading, and then labeled with H₂DCF diacetate. DCF fluorescence was monitored after reoxygenation by using a multi-well fluorescence reader. As a normoxic control some hepatocytes were incubated in aerobic KRH at 37°C for 4 h. Values are means ± SE from 3 different cell preparations. *P < 0.05 vs. anoxia. F: hepatocytes were subjected to either 4 h of anoxia/22 h of reoxygenation (A/R) or normoxia. Some cells were treated with 10 μM BAPTA either by the warm (W) or by the cold (C) loading protocol. Apoptotic cell death was evaluated by immunoblotting of caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage and compared with the control (Con). AU, arbitrary units.

Fig. 4.

Lack of cytoprotection by NADPH oxidase inhibitors. Hepatocytes cultured on 24-well microplates were subjected to A/R and labeled with H₂DCF diacetate (A) and PI (B). Some hepatocytes were incubated with 0.6 mM desferal or 10 μM diphenylphenylenediamine (DPPD) beginning 20 min before and continuously after reoxygenation. ROS formation and necrotic cell death was evaluated by DCF and PI fluorometry, respectively, in the presence and absence of antioxidants. *P < 0.05 vs. no addition. C: hepatocytes cultured in 24-well microplates were subjected to 4 h of anoxia and incubated with 300 μM of apocynin or 10 μM of diphenyleneiodonium chloride (DPI). Cell death after reoxygenation was evaluated by PI fluorometry. Note the failure of cytoprotection by NADPH oxidase blockers. Values are means ± SE from 3 different cell preparations. D: hepatocytes were incubated with 20 ng/ml TNF-α for 24 h. Some cells were treated with 10 μM BAPTA either by the warm (W) or by the cold (C) loading protocol. Apoptotic cell death was determined by immunoblotting of caspase-3 and PARP cleavage. Control hepatocytes (Con) were incubated for 24 h without treatment.

Fig. 5.

Scheme of MPT induction and cell death after A/R. Reoxygenation of anoxic hepatocytes increases mitochondrial Ca²⁺ (mtCa²⁺), which in turn stimulates mitochondrial ROS (mtROS) generation. ROS generation subsequently promotes onset of the MPT, which is prevented by antioxidants like desferal and DPPD. CsA and acidotic pH also block pH-dependent onset of MPT after reoxygenation. After onset of the MPT, cells undergo either necrotic or apoptotic death, depending on the availability of glycolytic ATP. When cellular ATP is depleted, membrane failure and necrotic cell death ensues. However, when a glycolytic ATP is available after reoxygenation, necrosis is prevented and cytochrome c-dependent apoptotic death occurs instead.