Bcl-2 sensitive mitochondrial potassium accumulation and swelling in apoptosis

doi:10.1016/S1567-7249(01)00039-3

Mitochondrion

Volume 1, Issue 4, February 2002, Pages 361-370

https://doi.org/10.1016/S1567-7249(01)00039-3 Get rights and content

Abstract

During etoposide-induced apoptosis in HL-60 cells, cytochrome c release was associated with mitochondrial swelling caused by increased mitochondrial potassium uptake. The mitochondrial permeability transition was also observed; however, it was not the primary cause of mitochondrial swelling. Potassium uptake and swelling of mitochondria were blocked by bcl-2 overexpression. As a result, cytochrome c release was reduced, and apoptosis delayed. Residual cytochrome c release in the absence of swelling in bcl-2 expressing cells could be due to observed Bax translocation into mitochondria.

This study suggests several novel aspects of apoptotic signaling: (1) potassium related swelling of mitochondria; (2) inhibition of mitochondrial potassium uptake by bcl-2; (3) co-existence within one system of multiple mechanisms of cytochrome c release: mitochondrial swelling and swelling-independent permeabilization of the outer mitochondrial membrane.

Introduction

The mitochondrial role in apoptosis involves the release of different proteins from the intermembrane space, one of which is cytochrome c (CytC) (Liu et al., 1996). The mechanism of CytC release is still elusive. One current hypothesis suggests the non-specific leakage of CytC from the intermembrane space due to swelling of mitochondria followed by rupture of the outer membrane (Kroemer et al., 1995, Kroemer et al., 1997, Vander Heiden et al., 1997).

The most popular theory on the mechanism of mitochondrial swelling in apoptosis is the mitochondrial permeability transition (MPT). The MPT is caused by opening of a cyclosporin A (CsA) sensitive megapore in the inner mitochondrial membrane (Crompton et al., 1988, Gunter and Pfeiffer, 1990). This causes swelling of mitochondria, leading to rupture of the outer membrane and release of CytC (Kantrow and Piantadosi, 1997). The most common method of monitoring the MPT is the measurement of mitochondrial depolarization. There are controversial data on the behavior of the mitochondrial membrane potential (ΔΨm) during apoptosis. Some groups have detected the release of CytC prior to any substantial depolarization (Kluck et al., 1997, Manon et al., 1997, Yang et al., 1997), while others found CsA-sensitive depolarization before the release of CytC (Vander Heiden et al., 1997, Kroemer, 1997).

In the light of this controversy, other mitochondrial volume-regulating mechanisms should be considered as potential causes of swelling. Two such possible pathways, discovered earlier, have not been associated with apoptosis. Both these pathways could lead to accumulation of potassium in mitochondria and potassium-driven swelling of the organelles. First is the so-called ‘low permeability (or sub-conductance) channel’, which was described by several groups (Ichas and Mazat, 1998, Kushnareva and Sokolove, 2000). This low permeability channel has lower general permeability to most molecules than the MPT, but has significant permeability to cations, particularly to potassium. The channel is almost insensitive to CsA. If opening this low permeability channel induces significant potassium permeability of the inner membrane, this could cause swelling of the matrix and perhaps rupture of the outer membrane.

The second possible potassium-related mechanism is the activation of an inducible potassium uniporter of the inner mitochondrial membrane (Beavis et al., 1993) also called K-ATP channel. The uniporter is normally inactive, however, under the conditions of apoptosis, it may be activated. The activated potassium uniporter allows potassium to move down its electrochemical gradient into the matrix, which results in a volume increase.

Increased potassium uptake via either the low permeability channel or potassium uniporter could lead to mitochondrial swelling by a pathway which does not involve the MPT and has different properties to those of the MPT. This potassium-induced swelling, for example, would be accompanied by less dramatic depolarization, compared to the MPT. Potassium accumulation in mitochondria has been observed during some pathological situations (Zoeteweij et al., 1992, Zoeteweij et al., 1994, Szewczyk and Murban, 1999), but the possibility of its involvement in apoptosis has not been vigorously investigated. We hypothesized that the increase in mitochondrial potassium permeability during apoptosis could lead to mitochondrial potassium accumulation and swelling, resulting in CytC release.

An alternative to the swelling hypothesis suggests that CytC is released via a specific channel in the outer membrane either composed of or regulated by pro-apoptotic bcl-2 family proteins (Manon et al., 1997, MacGibbon et al., 1997, Reed, 1997), such as Bax. During apoptosis, Bax translocates into mitochondria and this is followed by CytC release without significant swelling or the MPT (Murphy et al., 1999, Gross et al., 1999, Shimizu et al., 2000). Anti-apoptotic members of this protein family, such as bcl-2 itself, inhibit the release of CytC (Reed, 1997, Kharbanda et al., 1997, Mignotte and Vayssiere, 1998). There may also be cross-talk between bcl-2 family proteins, MPT, and other volume-regulating mechanisms.

The apoptotic model, used here, involves etoposide treatment of HL-60 cells. In this model, CytC release was coincident with mitochondrial swelling. This mitochondrial swelling was associated with increased permeability of mitochondria to potassium, resulting in accumulation of potassium inside mitochondria, followed by opening of the MPT. Bcl-2 overexpression inhibited mitochondrial potassium uptake, MPT induction, and mitochondrial swelling. As a consequence, CytC release was greatly reduced, and apoptosis was delayed. Bax progressively translocated into mitochondria during apoptosis, and this could possibly be the cause of the observed swelling-independent residual release of CytC in bcl-2 expressing cells.

Section snippets

Materials

Most chemicals were obtained from Sigma unless otherwise indicated.

Cell culture and induction of apoptosis

Human acute leukemia HL-60 cells (ATCC) were grown in suspension to the density of 1×10⁶/ml in RPMI-1640 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml antimycin (media and all components were from Life Technologies). Apoptosis was induced by treatment of cells with 50 μM etoposide with or without 10 μM CsA (Calbiochem). Etoposide was chosen for its ability to rapidly induce apoptosis in HL-60

Overexpression of bcl-2

Cells transfected with pIRESneo/bcl-2 constitutively expressed elevated amounts of bcl-2 in mitochondria, as confirmed by western blotting (Fig. 1a). Cells transfected with pIRESneo only were not different from wild type in any measured parameters (data not shown).

Timing of CytC release and apoptosis

During etoposide-induced apoptosis in HL-60 cells, CytC was released to the cytosol between 1 and 2 h after the beginning of treatment (Fig. 1b). CsA could not reverse CytC release, however, bcl-2 overexpression greatly reduced and

Discussion

During etoposide-induced apoptosis in HL-60 cells, CytC release was primarily due to mitochondrial swelling associated with potassium uptake. This was followed by the MPT, however the MPT, which often causes mitochondrial swelling, was not essential for mitochondrial swelling in this case. Bcl-2 inhibited mitochondrial potassium uptake, the MPT and swelling and greatly reduced CytC release. At the same time, Bax translocated into mitochondria. This could possibly cause residual CytC release in

Acknowledgements

This work was supported by NIEHS Grant ES 10041, by NCI Grant CA 71603, by HEI Contract 99-11, by NIAMS Grant AR 40325 and by the University of Rochester Medical Center.

We thank Dr Randy Rosier for stimulating discussions, Dr Claire Gavin, Dr Mark Dumont, and Dr Robert Freeman for reading and critiquing the manuscript, and Karen Jensen, M.S. for preparation of electron micrographs.

References (34)

A Beavis et al.
On the regulation of K⁺ uniport in intact mitochondria by adenine nucleotides and nucleotide analogs
J. Biol. Chem.
(1993)
M Bradford
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding
Anal. Biochem.
(1976)
H Fearnhead et al.
An interleukin-1 beta-converting enzyme-like protease is a common mediator of apoptosis in thymocytes
FEBS Lett.
(1995)
A Gross et al.
Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death
J. Biol. Chem.
(1999)
F Ichas et al.
From calcium signaling to cell death: two conformations of the permeability transition pore
Biochem. Biophys. Acta
(1998)
U Igbavboa et al.
Release of mitochondrial matrix proteins through a Ca²⁺-requiring, cyclosporin a-sensitive pathway
Biochem. Biophys. Res. Commun.
(1989)
S.P Kantrow et al.
Release of cytochrome c from liver mitochondria during permeability transition
Biochem. Biophys. Res. Commun.
(1997)
G Kroemer et al.
Mitochondrial control of apoptosis
Immunol. Today
(1997)
Y Kushnareva et al.
Prooxidants open both the permeability transition pore and a low-conductance channel in the inner mitochondrial membrane
Arch. Biochem. Biophys.
(2000)
X Liu et al.
Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c
Cell
(1996)

G.A MacGibbon et al.

Bax expressions in mammalian neurons undergoing apoptosis, and in Alzheimer disease hippocampus

Brain Res.

(1997)

S Manon et al.

Release of cytochrome c and decrease in cytochrome c oxidase in Bax-expressing yeast cells, and prevention of these effects by coexpression of Bcl-2

FEBS Lett.

(1997)

R Moreadith et al.

Isolation of mitochondria from ascites tumor cells permeabilized with digitonin

Anal. Biochem.

(1984)

E O'Gorman et al.

Differential effects of creatine depletion on the regulation of enzyme activities and on creatine stimulated mitochondrial respiration in skeletal muscle, heart and brain

Biochem. Biophys. Acta

(1996)

J Robertson et al.

Distinct pathways for stimulation of cytochrome c release by etoposide

J. Biol. Chem.

(2000)

S Shimizu et al.

Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c

J. Biol. Chem.

(2000)

G Terstappen

Functional analysis of native and recombinant ion channels using a high-capacity nonradioactive rubidium efflux assay

Anal. Biochem.

(1999)

Cited by (25)

3-Methyladenine prevents energy stress-induced necrotic death of melanoma cells through autophagy-independent mechanisms
2021, Journal of Pharmacological Sciences
We investigated the effect of 3-methyladenine (3MA), a class III phosphatidylinositol 3-kinase (PI3K)-blocking autophagy inhibitor, on cancer cell death induced by simultaneous inhibition of glycolysis by 2-deoxyglucose (2DG) and mitochondrial respiration by rotenone. 2DG/rotenone reduced ATP levels and increased mitochondrial superoxide production, causing mitochondrial swelling and necrotic death in various cancer cell lines. 2DG/rotenone failed to increase proautophagic beclin-1 and autophagic flux in melanoma cells despite the activation of AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin complex 1 (mTORC1). 3MA, but not autophagy inhibition with other PI3K and lysosomal inhibitors, attenuated 2DG/rotenone-induced mitochondrial damage, oxidative stress, ATP depletion, and cell death, while antioxidant treatment mimicked its protective action. The protection was not mediated by autophagy upregulation via class I PI3K/Akt inhibition, as it was preserved in cells with genetically inhibited autophagy. 3MA increased AMPK and mTORC1 activation in energy-stressed cells, but neither AMPK nor mTORC1 inhibition reduced its cytoprotective effect. 3MA reduced JNK activation, and JNK pharmacological/genetic suppression mimicked its mitochondria-preserving and cytoprotective activity. Therefore, 3MA prevents energy stress-triggered cancer cell death through autophagy-independent mechanisms possibly involving JNK suppression and decrease of oxidative stress. Our results warrant caution when using 3MA as an autophagy inhibitor.
Different approaches to modeling analysis of mitochondrial swelling
2018, Mitochondrion
Citation Excerpt :
As mentioned above, PTP opening induces mitochondrial swelling. However, inhibition of the PTP does not prevent mitochondrial swelling completely, suggesting a role of PTP-independent mechanisms are involved in the swelling (Eliseev et al., 2002; Gogvadze et al., 2004). The mechanisms of swelling are not clear, though ions, pHi, and membrane potential (ΔΨm) are the major players that regulate matrix swelling.
Mitochondria are critical players involved in both cell life and death through multiple pathways. Structural integrity, metabolism and function of mitochondria are regulated by matrix volume due to physiological changes of ion homeostasis in cellular cytoplasm and mitochondria. Ca²⁺ and K⁺ presumably play a critical role in physiological and pathological swelling of mitochondria when increased uptake (influx)/decreased release (efflux) of these ions enhances osmotic pressure accompanied by high water accumulation in the matrix. Changes in the matrix volume in the physiological range have a stimulatory effect on electron transfer chain and oxidative phosphorylation to satisfy metabolic requirements of the cell. However, excessive matrix swelling associated with the sustained opening of mitochondrial permeability transition pores (PTP) and other PTP-independent mechanisms compromises mitochondrial function and integrity leading to cell death. The mechanisms of transition from reversible (physiological) to irreversible (pathological) swelling of mitochondria remain unknown. Mitochondrial swelling is involved in the pathogenesis of many human diseases such as neurodegenerative and cardiovascular diseases. Therefore, modeling analysis of the swelling process is important for understanding the mechanisms of cell dysfunction. This review attempts to describe the role of mitochondrial swelling in cell life and death and the main mechanisms involved in the maintenance of ion homeostasis and swelling. The review also summarizes and discusses different kinetic models and approaches that can be useful for the development of new models for better simulation and prediction of in vivo mitochondrial swelling.
Modulation of cadmium-induced mitochondrial dysfunction and volume changes by temperature in rainbow trout (Oncorhynchus mykiss)
2015, Aquatic Toxicology
We investigated how temperature modulates cadmium (Cd)-induced mitochondrial bioenergetic disturbances, metal accumulation and volume changes in rainbow trout (Oncorhynchus mykiss). In the first set of experiments, rainbow trout liver mitochondrial function and Cd content were measured in the presence of complex I substrates, malate and glutamate, following exposure to Cd (0–100 μM) at three (5, 13 and 25 °C) temperatures. The second set of experiments assessed the effect of temperature on Cd-induced mitochondrial volume changes, including the underlying mechanisms, at 15 and 25 °C. Although temperature stimulated both state 3 and 4 rates of respiration, the coupling efficiency was reduced at temperature extremes due to greater inhibition of state 3 at low temperature and greater stimulation of state 4 at the high temperature. Cadmium exposure reduced the stimulatory effect of temperature on state 3 respiration but increased that on state 4, consequently exacerbating mitochondrial uncoupling. The interaction of Cd and temperature yielded different responses on thermal sensitivity of state 3 and 4 respiration; the Q₁₀ values for state 3 respiration increased at low temperature (5–13 °C) while those for state 4 increased at high temperature (13–25 °C). Importantly, the mitochondria accumulated more Cd at high temperature suggesting that the observed greater impairment of oxidative phosphorylation with temperature was due, at least in part, to a higher metal burden. Cadmium-induced mitochondrial volume changes were characterized by an early phase of contraction followed by swelling, with temperature changing the kinetics and intensifying the effects. Lastly, using specific modulators of mitochondrial ion channels, we demonstrated that the mitochondrial volume changes were associated with Cd uptake via the mitochondrial calcium uniporter (MCU) without significant contribution of the permeability transition pore and/or potassium channels. Overall, it appears that high temperature exacerbates Cd-induced mitochondrial dysfunction and volume changes in part by increasing metal uptake through the MCU.
14-3-3γ protein attenuates lipopolysaccharide-induced cardiomyocytes injury through the Bcl-2 family/mitochondria pathway
2014, International Immunopharmacology
Citation Excerpt :
However, after pFLAG-14-3-3γ transfection, Bcl-2 shifted from the cytosol to the mitochondria, Bax was sequestered in the cytosol, and the cytochrome c release to cytosol was significantly inhibited, indicating that 14-3-3γ may block apoptosis through a mitochondrial pathway mediated by the translocation of Bcl-2 and Bad (Fig. 6). Members of the Bcl-2 family of proteins associate with mitochondrial membranes and regulate mitochondrial pathophysiology [29]. To elucidate the direct effect of pFLAG-14-3-3γ on the mitochondria permeability transition, cardiomyocyte mitochondria were isolated as described in Materials and methods.
Previous studies have indicated that 14-3-3γ is upregulated by stress in LPS-induced cardiovascular injury. In this study, we investigated the interaction of 14-3-3γ and Bcl-2 family members in the control of the mitochondrial permeability transition (MPT) to test the hypothesis that abundant levels of 14-3-3γ can protect against LPS-induced injury via a Bcl-2 family/mitochondria pathway. The cardiomyocytes were treated with LPS (1 mg l^− 1) for 6 h; the interaction between 14-3-3γ and phospho-Bad(S112) was detected by co-immunoprecipitation (co-IP); the levels of Bcl-2 family members in the cytosolic and mitochondrial fractions were examined by Western blot; the apoptosis and mitochondrial membrane potential (ΔΨm) were detected by flow cytometry; and the mitochondrial permeability transition pore (mPTP) opening was tested by mitochondrial swelling. Our results revealed that LPS treatment results in cardiomyocyte injury, and these effects were significantly attenuated by pFLAG-14-3-3γ. Moreover, LPS treatment induced Bax translocation to the mitochondria, ΔΨm loss, mitochondrial swelling, and cytochrome c release, and pFLAG-14-3-3γ reversed these effects induced by LPS. Moreover, overexpressed 14-3-3γ protein could assist Bad(S112) phosphorylation and interact with it to form a complex, which might result in the disassociation of Bcl-2 from the Bad/Bcl-2 complex and its translocation from the cytosol to the mitochondria. Our data firstly confirmed that a high level of 14-3-3γ protects against LPS-induced cardiomyocyte injury likely through a pathway associated with the regulation of the subcellular localizations of Bcl-2 and Bad that results in the prevention of mPTP opening, the maintenance of ΔΨm, and ultimately the inhibition of apoptosis.
The mitochondrial permeability transition pore (PTP) - An example of multiple molecular exaptation?
2012, Biochimica et Biophysica Acta - Bioenergetics
Citation Excerpt :
In human tissues, three different K+ channels have been detected: i) the ATP-regulated K+ channel (mito K+ATP) [139–141]; ii) the mitochondrial large conductance calcium-activated K+ channel (mito BKCa) [142]; the voltage-regulated K+ channel (mito Kv1.3) [143]. These K+ channels are potentially involved in the prevention of ischemic heart disease [144,145], in cytoprotection [146] and, at least for mito K+ATP [147,148] and mito Kv1.3 [143], in PCD signal transduction. These effects, similarly to those observed in Tracheophytes, Amoebozoa and Euglenozoa (see Sections 2.4 and 2.5), might be associated to PT, linked to matrix swelling and moderate membrane depolarization [148].
The mitochondrial permeability transition (PT) is a well-recognized phenomenon that allows mitochondria to undergo a sudden increase of permeability to solutes with molecular mass ≤ 1500 Da, leading to organelle swelling and structural modifications. The relevance of PT relies on its master role in the manifestation of programmed cell death (PCD). This function is performed by a mega-channel (in some cases inhibited by cyclosporin A) named permeability transition pore (PTP), whose function could derive from the assembly of different mitochondrial proteins.
In this paper we examine the distribution and characteristics of PTP in mitochondria of eukaryotic organisms so far investigated in order to draw a hypothesis on the mechanism of its evolution. As a result, we suggest that PTP may have arisen as a new function linked to a multiple molecular exaptation of different mitochondrial proteins, even though they could nevertheless still play their original role.
Furthermore, we suggest that the early appearance of PTP could have had a crucial role in the establishment of endosymbiosis in eukaryotic cells, by the coordinated balancing of ATP production by glycolysis (performed by the primary phagocyte) and oxidative phosphorylation (accomplished by the endosymbiont). Indeed, we argue on the possibility that this new energetic equilibrium could have opened the way to the subsequent evolution toward metazoans.
Mitochondrial dysfunction in cancer cells due to aberrant mitochondrial replication
2011, Journal of Biological Chemistry
Warburg effect is a hallmark of cancer manifested by continuous prevalence of glycolysis and dysregulation of oxidative metabolism. Glycolysis provides survival advantage to cancer cells. To investigate molecular mechanisms underlying the Warburg effect, we first compared oxygen consumption among hFOB osteoblasts, benign osteosarcoma cells, Saos2, and aggressive osteosarcoma cells, 143B. We demonstrate that, as both proliferation and invasiveness increase in osteosarcoma, cells utilize significantly less oxygen. We proceeded to evaluate mitochondrial morphology and function. Electron microscopy showed that in 143B cells, mitochondria are enlarged and increase in number. Quantitative PCR revealed an increase in mtDNA in 143B cells when compared with hFOB and Saos2 cells. Gene expression studies showed that mitochondrial single-strand DNA-binding protein (mtSSB), a key catalyst of mitochondrial replication, was significantly up-regulated in 143B cells. In addition, increased levels of the mitochondrial respiratory complexes were accompanied by significant reduction of their activities. These changes indicate hyperactive mitochondrial replication in 143B cells. Forced overexpression of mtSSB in Saos2 cells caused an increase in mtDNA and a decrease in oxygen consumption. In contrast, knockdown of mtSSB in 143B cells was accompanied by a decrease in mtDNA, increase in oxygen consumption, and retardation of cell growth in vitro and in vivo. In summary, we have found that mitochondrial dysfunction in cancer cells correlates with abnormally increased mitochondrial replication, which according to our gain- and loss-of-function experiments, may be due to overexpression of mtSSB. Our study provides insight into mechanisms of mitochondrial dysfunction in cancer and may offer potential therapeutic targets.

View all citing articles on Scopus

View full text

Bcl-2 sensitive mitochondrial potassium accumulation and swelling in apoptosis

Abstract

Introduction

Section snippets

Materials

Cell culture and induction of apoptosis

Overexpression of bcl-2

Timing of CytC release and apoptosis

Discussion

Acknowledgements

J. Biol. Chem.

Anal. Biochem.

FEBS Lett.

J. Biol. Chem.

Biochem. Biophys. Acta

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Immunol. Today

Arch. Biochem. Biophys.

Cell

Brain Res.

FEBS Lett.

Anal. Biochem.

Biochem. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.