Eduardo Martínez-Abundis

Bax distribution into mitochondrial detergent‐resistant microdomains is related to ceramide and cholesterol content in postischemic hearts

Diverse changes have been described in mitochondria of apoptotic cells: the phospholipid content is modified, ceramide and GD3 concentrations increase, the cristae structure is modified, and nonresident proteins are recruited into the mitochondrial membranes. In particular, Bax, a Bcl‐2 family member protein, moves from the cytosol to the mitochondria, inducing cytochrome c release. Modifications of the content and distribution of specific lipids in the mitochondrial membranes, along with the well‐known participation of the mitochondrial permeability transition pore in triggering apoptosis, led us to propose that lipid microdomains in mitochondria could coexist as structural elements with some of the mitochondrial permeability transition pore‐forming proteins and with members of the Bcl‐2 family. In this work, we demonstrated that Bax was associated preferentially with mitochondrial detergent‐resistant membranes (mDRMs) in reperfused rat hearts, a well‐known apoptotic model. Bax insertion into mDRMs correlated with cytochrome c release from such mitochondria. Bax location in mDRMs was associated with both the voltage‐dependent anion channel and the adenine nucleotide translocator, two mitochondrial permeability transition pore‐forming proteins. Interestingly, the voltage‐dependent anion channel was more abundant in the mDRM fraction than in the Triton X‐100‐soluble fraction. Ceramide and cholesterol contents were higher in mDRMs from reperfused hearts. Our results suggest that membrane microenvironments enriched in cholesterol and ceramide in mitochondria favor Bax translocation to this organelle, fostering propagation of the apoptotic cascade.

Protective effect of sulforaphane against cisplatin-induced mitochondrial alterations and impairment in the activity of NAD(P)H: Quinone oxidoreductase 1 and γ glutamyl cysteine ligase: Studies in mitochondria isolated from rat kidney and in LLC-PK1 cells

This work was designed to further study the mechanism by which sulforaphane (SFN) exerts a renoprotective effect against cisplatin (CIS)-induced damage. It was evaluated whether SFN attenuates the CIS-induced mitochondrial alterations and the impairment in the activity of the cytoprotective enzymes NAD(P)H: quinone oxidoreductase 1 (NQO1) and γ glutamyl cysteine ligase (γGCL). Studies were performed in renal epithelial LLC-PK1 cells and in isolated renal mitochondria from CIS, SFN or CIS+SFN treated rats. SFN effectively prevented the CIS-induced increase in reactive oxygen species (ROS) production and the decrease in NQO1 and γGCL activities and in glutathione (GSH) content. The protective effect of SFN on ROS production and cell viability was prevented by buthionine sulfoximine (BSO), an inhibitor of γGCL, and by dicoumarol, an inhibitor of NQO1. SFN was also able to prevent the CIS-induced mitochondrial alterations both in LLC-PK1 cells (loss of membrane potential) and in isolated mitochondria (inhibition of mitochondrial calcium uptake, release of cytochrome c, and decrease in GSH content, aconitase activity, adenosine triphosphate (ATP) content and oxygen consumption). It is concluded that the protection exerted by SFN on mitochondrial alterations and NQO1 and γGCL enzymes may be involved in the renoprotection of SFN against CIS.

Relationship between oxidative stress and mitochondrial function in the post-conditioned heart

The pathways activated by post-conditioning may converge on the mitochondria, in particular on the mitochondrial permeability transition pore. We sought to characterize the inhibition status of the mitochondrial permeability transition early after the post-conditioning maneuver and before long reperfusion was established. We observed that post-conditioning maneuvers applied to isolated rat hearts, after a prolonged ischemia and before reperfusion, promoted cardiac mechanical function recovery and maintained mitochondrial integrity. These effects were evaluated by mitochondrial swelling, calcium transport, and NAD+ content measurements; the improvements were established before restoring a long lasting reperfusion period. Mitochondrial integrity was associated with a diminution in oxidative stress, since carbonylation of proteins was prevented and aconitase activity was preserved in the post-conditioned hearts, implying that ROS might mediate mitochondrial dysfunction and mPTP opening. In addition, we found that cytochrome release was significantly abolished in the post-conditioned heart, in contrast with conventionally reperfused hearts.

Changes in specific lipids regulate BAX‐induced mitochondrial permeability transition

Recent evidence suggests the existence of lipid microdomains in mitochondria, apparently coexisting as structural elements with some of the mitochondrial permeability transition pore‐forming proteins and members of the Bcl‐2 family. The aim of this study was to investigate the relevance of the main components of membrane microdomains (e.g. cholesterol and sphingolipids) in activation of the mitochondrial permeability transition pore (mPTP) by recombinant BAX (rBAX). For this purpose, we used chemically modified renal cortex mitochondria and renal cortex mitochondria from hypothyroid rats that show a modified mitochondrial lipid composition in vivo. Oligomeric rBAX induced an enhanced permeability conformation in the mPTP of control mitochondria. rBAX failed to induce mPTP opening when the cholesterol and ganglioside content of mitochondria were modified with the chelator methyl‐beta‐cyclodextrin. Accordingly, hypothyroid mitochondria, with endogenously lower cholesterol and ganglioside content, showed resistance to mPTP opening induced by rBAX. These observations suggest that enriched cholesterol and ganglioside domains in the mitochondrial membranes may determine BAX interaction with the mPTP. An intriguing observation was that chemical extraction of cholesterol and ganglioside in control mitochondria did not have an effect on rBAX insertion. Conversely, in hypothyroid mitochondria, rBAX insertion was diminished dramatically compared with control mitochondria. The membrane and protein changes associated with thyroid status and their possible role in rBAX docking into the membranes are discussed.

Bax induces cytochrome c release by multiple mechanisms in mitochondria from MCF7 cells

Bax, a pro-apoptotic member of the Bcl-2 family of proteins has the ability to form transmembrane pores large enough to allow cytochrome c (Cyt c) release, as well as to activate the mitochondrial permeability transition pore (mPTP); however, no differential study has been conducted to clarify which one of these mechanisms predominates over the other in the same system. In the present study, we treated isolated mitochondria from MCF7 cells with recombinant protein Bax and tested the efficacy of the mPTP inhibitor cyclosporin A (CsA) and of the Bax channel blocker (Bcb) to inhibit cytochrome c release. We also, induced apoptosis in MCF7 cell cultures with TNF-α plus cycloheximide to determine the effect of such compounds in apoptosis induction via mPTP or Bax oligomerization. Cytochrome c release was totally prevented by CsA and partially by Bcb when apoptosis was induced with recombinant Bax in isolated mitochondria from MCF7 cells. CsA increased the number of living cells in cell culture, as compared with the effect of Bax channel blocker. These results indicate that mPTP activation is the predominant pathway for Bax-induced cytochrome c release from MCF7 mitochondria and for apoptosis induction in the whole cell.

Copper induces permeability transition through its interaction with the adenine nucleotide translocase

In this work we examined the effect of low concentrations of Cu2+ on the opening of the mitochondrial non‐specific pore. The purpose was addressed to further contribute to the knowledge of the mechanisms that regulate the open/closed cycles of the permeability transition pore. Membrane leakage was established by measuring matrix Ca2+ efflux and mitochondrial swelling. The experimental results indicate that Cu2+ at very low concentrations promoted the release of accumulated Ca2+, as well as mitochondrial swelling, provided 1,10‐phenanthroline has been added. Carboxyatractyloside and Cu2+ exhibited additive effects on these parameters. After Cu2+ titration of membrane thiols, it might be assumed that the blockage of 5.9 nmol of SH/mg protein suffices to open the non‐specific pore. Taking into account the reinforcing effect of carboxyatractyloside, the increasing ADP concentrations, and that N‐ethylmaleimide inhibited the Cu2+‐induced Ca2+ efflux, it is proposed that the target site for Cu2+ is located in the ADP/ATP carrier.

Sexual hormones: Effects on cardiac and mitochondrial activity after ischemia-reperfusion in adult rats. Gender difference

In this work we studied the influence of sex hormones on heart and mitochondrial functions, from adult castrated female and male, and intact rats. Castration was performed at their third week of life and on the fourth month animals were subjected to heart ischemia and reperfusion. Electrocardiogram and blood pressure recordings were made, cytokines levels were measured, histopathological studies were performed and thiobarbituric acid reactive species were determined. At the mitochondrial level respiratory control, transmembranal potential and calcium management were determined; Western blot of some mitochondrial components was also performed. Alterations in cardiac function were worst in intact males and castrated females as compared with those found in intact females and castrated males, cytokine levels were modulated also by hormonal status. Regarding mitochondria, in those obtained from hearts from castrated females without ischemia-reperfusion, all evaluated parameters were similar to those observed in mitochondria after ischemia-reperfusion. The results show hormonal influences on the heart at functional and mitochondrial levels.

Titration of cardiolipin by either 10-N-nonyl acridine orange or acridine orange sensitizes the adenine nucleotide carrier to permeability transition.

Under the action of carboxyatractyloside or fatty acids, adenine nucleotide translocase switches its function from nucleotide carrier to modulator of the opening of a non-specific pore. In addition to the effect of these agents, this modification in activity is, in some way, dependent on the influence of the lipid milieu of the membrane. Cardiolipin is, among other membrane phospholipids, the one that interacts the most with the translocase. This work shows that 10-N-nonyl acridine orange and acridine orange, probes for this phospholipid, modify the sensitivity of the translocase to carboxyatractyloside, oleate, and palmitate to induce permeability transition. The results also show that these probes stimulate the release of mitochondrial cytochrome c, and increase labeling of the carrier by eosin 5-maleimide. Based on the aforementioned it is proposed that the increase in sensitivity is due to a conformational change in the translocase, induced by the binding of the probe to cardiolipin.

Cyclosporin a is unable to inhibit carboxyatractyloside-induced permeability transition in aged mitochondria

We studied the effect of mitochondrial ageing on membrane permeability transition. The results obtained indicate that aged mitochondria are neither able to retain Ca2+ nor to maintain a high transmembrane electric gradient. In addition, aged mitochondria undergo a large amplitude swelling. These dysfunctions were circumvented by the addition of cyclosporin A. Furthermore, it is shown that ageing-induced permeability transition causes oxidative damage on the matrix enzyme aconitase. The observed damage in aged mitochondria requires Ca2+ addition; therefore, it was not seen when Sr2+ replaced Ca2+. Two important findings in this work were the fact that despite of the presence of cyclosporin A, carboxyatractyloside was still able to induce permeability transition, and that ageing induced mitochondrial DNA disruption and release of cytochrome c. It is likely that the membranes increased permeability is due to the effect of fatty acids, since bovine serum albumin makes mitochondria able to retain Ca2+. However, the possibility that the damage might be the result of oxidative stress cannot be discarded.

A CRAC-like motif in BAX sequence: relationship with protein insertion and pore activity in liposomes.

Several proteins that interact with cholesterol have a highly conserved sequence, corresponding to the cholesterol recognition/interaction amino acid consensus. Since cholesterol has been proposed to modulate both oligomerization and insertion of the pro-apoptotic protein BAX, we investigated the existence of such a motif in the BAX sequence. Residues 113 to 119 of the recombinant BAX α5-helix, LFYFASK, correspond with the sequence motif described for the consensus pattern, -L/V-(X)(1-5)-Y-(X)(1-5)-R/K. Functional characterization of the point mutations, K119A, Y115F, and L113A in BAX, was performed in liposomes supplemented with cholesterol, comparing binding, integration, and pore forming activities. Our results show that the mutations Y115F and L113A changed the cholesterol-dependent insertion observed in the wild type protein. In addition, substitutions in the BAX sequence modified the concentration dependency of carboxyfluorescein release in liposomes, although neither pore activity of the wild type or of any of the mutants significantly increased in cholesterol-enriched liposomes. Thus, while it is likely that the putative CRAC motif in BAX accounts for its enhanced insertion in cholesterol-enriched liposomes; the pore forming properties of BAX did not depend on cholesterol content in the membranes, albeit those mutations changed the pore channeling activity of the protein.