Martin Crompton

Mitochondrial intermembrane junctional complexes and their role in cell death

A mitochondrial complex comprising the voltage‐dependent anion channel (outer membrane), the adenine nucleotide translocase (inner membrane) and cyclophilin‐D (matrix) assembles at contact sites between the inner and outer membranes. Under pathological conditions associated with ischaemia and reperfusion the junctional complex ‘deforms’ into the permeability transition (PT) pore, which can open transiently, allowing free permeation of low Mr solutes across the inner membrane. This may be a critical step in the pathogenesis of lethal cell injury in ischaemia and reperfusion. Moreover, it is argued, the degree of pore opening may be an important determinant of the relative extent of apoptosis and necrosis under these conditions. In addition, mitochondria are the major sites of action of Bax and other apoptotic regulatory proteins of the Bcl‐2 family. These proteins control a mitochondrial amplificatory loop in the apoptotic signalling pathway in which cytochrome c and other apoptogenic proteins of the mitochondrial intermembrane space are released into the cytosol. There are indications that the junctional complex, or components of it, may also mediate the action of Bax, but in a way that does not involve PT pore formation.

Mitochondrial calcium transport

Knvestigati5ns over the last four years have revealed that m~to~hondr~a possess a system of considerable sophistication for the transport of Ca2* across their inner rnembrafles. In particular, it isnow apparent that a variety of mitochondria possess not only a uniport for the uptake of Ca” down its electrochemical gradient, but also a second Ca2* carrier which catalyzes a continuous Ca2* efflux from the matrix against its gradient of eI~~trochemica1 pote~t~a1. When A

Inhibition of anoxia-induced injury in heart myocytes by cyclosporin A

is in the physiological range, both carriers operate unidirectionall~ to permit eon~nuous recychng of Ca*” across the inner membrane, and thereby provide the basis for a kinetic regulation of the distribution of Ca2* between the cytosoi and the mitochondrial matrix. The purpose of this review is to examine the evidence for, and the consequences of, this cycling. Kinetic control carries with it three distinct advantages over any single carrier mechanism: (I) It enables the Ca’+ distribution to be controlled without changing either the A

Mitochondrial intermembrane junctional complexes and their involvement in cell death

component or the ApB component of the proton electrochemical potential, and therefore without disturbing ATP synthesis or the distribution of metabolites. (2) It allows for flexibility of control, in that the dis~r~but~on of Ca*’ may be regulated by a~t~ya~~n~ or inhibiting either or both of the pathways. (3) The system may be extremely sensitive, since relatively large changes in net flux of Ca’+ may resuit from relatively small changes in absolute carrier activity, 2. The nature of the uptake and efffux pa~wa~s

Bax. bid and the permeabilization of the mitochondrial outer membrane in apoptosis

Cyclosporin A is a potent immunosuppressant used to prevent graft rejection. The cellular target of cyclosporin A in T lymphocytes is believed to be cyclophilin, a ubiquitous protein with peptidyl prolyl cis trans isomerase activity located in both the cytosol and mitochondria. Recently, cyclosporin A-inhibition of mitochondrial cyclophilin has been implicated in the prevention of mitochondrial dysfunction induced in vitro by Ca2+ overload and other factors potentially relevant to ischaemic cell injury. This study investigates the effect of cyclosporin A on injury to cardiomyocytes induced by substrate-free anoxia. It is shown that cyclosporin A retards progression of the injury, most probably at a late step in the injury process.

The relationship between mitochondrial state, ATP hydrolysis, [Mg2+]i and [Ca2+]i studied in isolated rat cardiomyocytes.

Mitochondria establish contact sites between the inner and outer membranes. The contact sites are held together by junctional complexes of the adenine nucleotide translocase (ANT; inner membrane) and the voltage-dependent anion channel (VDAC; outer membrane). The junctional complexes act as multifunctional recruitment centres, binding a range of proteins according to the function to be executed. Some of these, involving kinases and enzymes of lipid transfer, are readily understood as ongoing functions in energy and lipid metabolism. But the roles of other proteins recruited to the junctional complexes are less well defined. Here, we focus on the complexes formed with Bax and with cyclophilin-D, and their possible roles in apoptotic and necrotic cell death. We have isolated both types of complexes using glutathione-S-transferase fusion proteins of Bax and of cyclophilin-D. The VDAC/ANT/cyclophilin-D complex reconstitutes Ca(2+)- and cyclosporin A-sensitive permeability transition pore activity when incorporated into proteoliposomes. The complex forms readily in the absence of factors required for pore opening in isolated mitochondria, suggesting that these factors act on the preexisting complex, rather than drive its assembly, and that the complex is a physiological entity in healthy cells.

Cyclophilin-D promotes the mitochondrial permeability transition but has opposite effects on apoptosis and necrosis

Mitochondria provide a key amplification step in the apoptotic pathway of many cells by releasing apoptogenic proteins into the cytosol. Recent studies have provided insights into how Bax and Bid may operate synergistically to recruit mitochondria into the pathway and how GD3 ganglioside, a metabolite of the sphingomyelin pathway, may also be used. In ischaemic disease, activation of the mitochondrial permeability transition pore may bypass the requirement for these factors.

Bax translocates to mitochondria of heart cells during simulated ischaemia: involvement of AMP-activated and p38 mitogen-activated protein kinases

1. As ATP has a higher affinity for Mg2+ than ADP, the cytosolic magnesium concentration rises upon ATP hydrolysis. We have therefore used the Mg(2+)‐sensitive fluorescent indicator Magnesium Green (MgG) to provide an index of changing ATP concentration in single rat cardiomyocytes in response to altered mitochondrial state. 2. In response to FCCP, [Mg2+]i rose towards a plateau coincident with the progression to rigor, which signals ATP depletion. Contamination of the MgG signal by changes in intracellular free Ca2+ concentration (the KD of MgG for Ca2+ is 4.7 microM) was excluded by simultaneous measurement of [Ca2+]i and [Mg2+]i in cells dual loaded with fura‐2 and MgG. The response to FCCP was independent of external Mg2+, confirming an intracellular source for the rise in [Mg2+]i. 3. Simultaneous measurements of mitochondrial NAD(P)H autofluorescence and mitochondrial potential (delta psi m; .‐1 fluorescence) and of autofluorescence and MgG allowed closer study of the relationship between [Mg2+]i and mitochondrial state. Oligomycin abolished the FCCP‐induced rise in [Mg2+]i without altering the change in autofluorescence. Thus, the rise in [Mg2+]i in response to FCCP is consistent with the release of intracellular Mg2+ following ATP hydrolysis by the mitochondrial F1F0‐ATPase. 4. The rise in [Mg2+]i was correlated with cell‐attached recordings of ATP‐sensitive K+ channel (KATP) activity. In response to FCCP, an increase in KATP channel activity was seen only as [Mg2+]i reached a plateau. In response to blockade of mitochondrial respiration and glycolysis with cyanide (CN‐) and 2‐deoxyglucose (DOG), [Mg2+]i rose more slowly but again KATP channel opening increased only when [Mg2+]i reached a plateau and the cells shortened. 5. Oligomycin decreased the rate of rise of [Mg2+]i delayed the onset of rigor and increased the rate of mitochondrial depolarization in response to CN‐_DOG. Thus, with blockade of mitochondrial respiration delta psi m is maintained by the mitochondrial F1F0‐ATPase at the expense of ATP reserves. 6. In response to CN‐_DOG, the initial rise in [Mg2+]i was accompanied by a small rise in [Ca2+]i. After [Mg2+]i reached a plateau and rigor developed, [Ca2+]i rose progressively. On reperfusion, in hypercontracted cells, [Ca2+]i recovered before [Mg2+]i and [ca2+]i oscillations were sustained while [Mg2+]i decreased. Thus on reperfusion, full recovery of [ATP]i is slow, but the activation of contractile elements and the restoration of [Ca2+]i does not require the re‐establishment of millimolar concentrations of ATP.

The Regulation of Mitochondrial Calcium Transport in Heart

Cyclophilin-D is a peptidylprolyl cis-trans isomerase of the mitochondrial matrix. It is involved in mitochondrial permeability transition, in which the adenine nucleotide translocase of the inner membrane is transformed from an antiporter to a non-selective pore. The permeability transition has been widely considered as a mechanism in both apoptosis and necrosis. The present study examines the effects of cyclophilin-D on the permeability transition and lethal cell injury, using a neuronal (B50) cell line stably overexpressing cyclophilin-D in mitochondria. Cyclophilin-D overexpression rendered isolated mitochondria far more susceptible to the permeability transition induced by Ca2+ and oxidative stress. Similarly, cyclophilin-D overexpression brought forward the onset of the permeability transition in intact cells subjected to oxidative stress. In addition, in the absence of stress, the mitochondria of cells overexpressing cyclophilin-D maintained a lower inner-membrane potential than those of normal cells. All these effects of cyclophilin-D overexpression were abolished by cyclosporin A. It is concluded that cyclophilin-D promotes the permeability transition in B50 cells. However, cyclophilin-D overexpression had opposite effects on apoptosis and necrosis; whereas NO-induced necrosis was promoted, NO- and staurosporine-induced apoptosis were inhibited. These findings indicate that the permeability transition leads to cell necrosis, but argue against its involvement in apoptosis.

Biphasic translocation of Bax to mitochondria.

The cytosolic protein Bax plays a key role in apoptosis by migrating to mitochondria and releasing proapoptotic proteins from the mitochondrial intermembrane space. The present study investigates the movement of Bax in isolated rat neonatal cardiomyocytes subjected to simulated ischaemia (minus glucose, plus cyanide), using green fluorescent protein-tagged Bax as a means of imaging Bax movements. Simulated ischaemia induced Bax translocation from the cytosol to mitochondria, commencing within 20 min of simulated ischaemia and progressing for several hours. Under the same conditions, there was an increase in the active, phosphorylated forms of p38 MAPK (mitogen-activated protein kinase) and AMPK (AMP-activated protein kinase). The AMPK activators AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) and metformin also stimulated Bax translocation. Inhibition of p38 MAPK with SB203580 attenuated the phosphorylation of the downstream substrates, MAPK-activated protein kinases 2 and 3, but not that of the upstream MAPK kinase 3, nor of AMPK. Under all conditions (ischaemia, AICAR and metformin), SB203580 blocked Bax translocation completely. It is concluded that Bax translocation to mitochondria is an early step in ischaemia and that it occurs in response to activation of p38 MAPK downstream of AMPK.