Cyclosporin A protects hepatocytes subjected to high Ca2+ and oxidative stress.

Hepatocytes incubated with 0.8 mM t-butylhydroperoxide are protected by cyclosporin A when the medium Ca2+ concentration is 10 mM, but not when it is 2.5 mM. The highest Ca2+ level is associated with an inhibition of t-butylhydroperoxide-dependent malondialdehyde accumulation and with mitochondrial Ca2+ loading within the cells. These findings are new evidence that t-butylhydroperoxide can kill cells by peroxidation-dependent and -independent mechanisms, and suggest that the mitochondrial permeability transition and the resultant de-energization are components of the peroxidation-independent mechanism. Cyclosporin A may have considerable utility for the protection of cells subjected to oxidative stress.

Inhibition of the mitochondrial permeability transition by cyclosporin A during long time frame experiments: relationship between pore opening and the activity of mitochondrial phospholipases.

Inhibition of the mitochondrial permeability transition pore by cyclosporin A or trifluoperazine is transient on the time scale of cell injury studies (hours). However, these agents act synergistically and produce long-lasting inhibition when used in combination. The cause of this synergism has been investigated from the perspective of the known action of trifluoperazine as an inhibitor of mitochondrial phospholipase A2. Free fatty acids, which are phospholipase reaction products, facilitate pore opening in a concentration-dependent manner (I50 approximately 2 nmol/mg of mitochondrial protein). Endogenous and exogenous fatty acids are similarly effective. Fatty acids of differing structure are also similarly effective, but long-chain alcohols and alkanes are ineffective. Free fatty acids accumulate in cyclosporin A-treated mitochondria when Ca2+ plus tert-butyl hydroperoxide or Ca2+ plus N-ethylmaleimide is present, but do not accumulate when Ca2+ plus inorganic phosphate is present. In the presence of cyclosporin A, bovine serum albumin markedly delays pore opening induced by tert-butyl hydroperoxide or N-ethylmaleimide, but has little effect on pore opening induced by inorganic phosphate, which is subject to long-lasting inhibition by cyclosporin A without trifluoperazine. Free fatty acid accumulation is thus a factor which limits pore inhibition by cyclosporin A. However, trifluoperazine has no effect on free fatty acid accumulation in intact, cyclosporin-inhibited mitochondria and thus does not act by inhibiting phospholipases. Comparing the actions of free fatty acids, trifluoperazine, long-chain acyl cations, and other effectors on the pore suggests that a more negative membrane surface potential favors pore opening and a more positive potential favors a closed pore. Expected surface potential effects of trifluoperazine can explain the synergism between this compound and cyclosporin A as pore inhibitors. Surface potential may influence the pore through the voltage-sensing element which responds to transmembrane potential. The present data also suggest that long-lived, solute-selective forms of the pore exist when it is opened in the presence of inhibitors. The implications of these findings for pore regulation and for the use of cyclosporin A to identify pore opening as a component of cell injury mechanisms are discussed.

Cyclosporin A-sensitive and insensitive mechanisms produce the permeability transition in mitochondria.

Cyclosporin A is a potent inhibitor of the mitochondrial permeability transition, possibly by blocking an inner membrane pore through which solute movements occur [Broekemeier et al. (1989) J. Biol. Chem. 264, 7826-7830]. The inhibitory effect of cyclosporin, however, is transient. Trifluoperazine, at concentrations which inhibit the mitochondrial phospholipase A2, also produces a transient inhibition. When both inhibitors are used together, the inhibitory effect is long lasting. These findings suggest that the transition can be caused by two overlapping and/or interactive mechanisms, one dependent on an inner membrane pore and the other on phospholipase A2.

Recent progress on regulation of the mitochondrial permeability transition pore; a cyclosporin-sensitive pore in the inner mitochondrial membrane.

The mitochondrial permeability transition pore allows solutes with a m.w. approximately less than 1500 to equilibrate across the inner membrane. A closed pore is favored by cyclosporin A acting at a high-affinity site, which may be the matrix space cylophilin isozyme. Early results obtained with cyclosporin A analogs and metabolites support this hypothesis. Inhibition by cyclosporin does not appear to require inhibition of calcineurin activity; however, it may relate to inhibition of cyclophilin peptide bond isomerase activity. The permeability transition pore is strongly regulated by both the membrane potential (delta psi) and delta pH components of the mitochondrial protonmotive force. A voltage sensor which is influenced by the disulfide/sulhydryl state of vicinal sulfhydryls is proposed to render pore opening sensitive to delta psi. Early results indicate that this sensor is also responsive to membrane surface potential and/or to surface potential gradients. Histidine residues located on the matrix side of the inner membrane render the pore responsive to delta pH. The pore is also regulated by several ions and metabolites which act at sites that are interactive. There are many analogies between the systems which regulate the permeability transition pore and the NMDA receptor channel. These suggest structural similarities and that the permeability transition pore belongs to the family of ligand gated ion channels.

Proton selective substate of the mitochondrial permeability transition pore: regulation by the redox state of the electron transport chain.

The permeability transition pore of rat liver mitochondria can be closed by chelating free Ca2+, with respect to the passage of large molecules such as mannitol and sucrose. However, an apparent H+-conducting substate remains open under these conditions, as indicated by the persistence of maximal O2 consumption rates and by the failure to recover a membrane potential. Agents which favor a closed pore, such as cyclosporin A, ADP, Mg2+, or bovine serum albumin, do not close the H+-conducting substate, but it closes spontaneously when respiration becomes limited by the availability of O2. Closure provoked by an O2 limitation requires free Mg2+ in the sub-micromolar concentration range and becomes less efficient with increasing time spent in the presence of free Ca2+. The H+-conducting substate is apparently regulated by the redox status of the electron transport chain, with a reduced form favoring closure. A physical association (or equivalence) between the pore and one of the respiratory chain complexes is supported. These characteristics suggest that the transition is irreversible in vivo, if it involves a small fraction of total mitochondria, and would lead to their elimination and/or replacement by the cell. The implications of this proposal are considered, as they relate to a possible role for the transition in cellular apoptosis and the elimination of mitochondria containing mutated DNA.

Inhibition of the mitochondrial Ca2+ uniporter by pure and impure ruthenium red.

Commercial ruthenium red is often purified by a single recrystallization as described by Luft, J.H. (1971) Anat Rec 171, 347-368, which yields small amounts of material having an apparent molar extinction coefficient of approximately 67,400 at 533 nm. A simple modification to the procedure dramatically improves the yield, allowing crystallization to be repeated. Three times recrystallized ruthenium red has an apparent extinction coefficient of approximately 85,900, the highest value reported to date. Both crude and highly purified ruthenium red can be shown to inhibit reverse activity of the mitochondrial Ca2+ uniporter (uncoupled mitochondria), provided that care is taken to minimize and account for Ca2+ release through the permeability transition pore. Crude ruthenium red is 7-10 fold more potent than the highly purified material in this regard, on an actual ruthenium red concentration basis. The same relative potency is seen against forward uniport (coupled mitochondria), however, the I50 values are 10 fold lower for both the crude and purified preparations. These data demonstrate unambiguously that the energy state of mitochondria affects the sensitivity of the Ca2+ uniporter to ruthenium red preparations, and that both the forward and reverse reactions are subject to complete inhibition. The data suggest, however, that the active inhibitor may not be ruthenium red per se, but one or more of the other ruthenium complexes which are present in ruthenium red preparations.

Cyclosporin A is a potent inhibitor of the inner membrane permeability transition in liver mitochondria.

The immunosuppressive peptide cyclosporin A is a powerful inhibitor of the Ca2+-dependent permeability transition in rat liver mitochondria. When swelling is used to monitor the transition, the inhibitor is effective regardless of whether N-ethylmaleimide, Hg2+, WY-14643, t-butyl hydroperoxide, oxalacetate, rhein, phosphate, phosphoenolpyruvate, or ruthenium red plus uncoupler is used as the inducing agent. Twenty-five to fifty pmol/mg protein of cyclosporin A reduces the swelling response by 50% with complete inhibition obtained at about 150 pmol/mg protein. The compound, which does not inhibit Ca2+ uptake or mitochondrial phospholipase A2, is effective when added before or after the transition promoting agent. These findings, together with the shape of the inhibition dose-response curve, suggest that cyclosporin A essentially titrates a mitochondrial component which is present at 80-90 pmol/mg protein. It is proposed that this component is a solute unselective, regulated pore or a factor involved in controlling such a structure.

Generation of the mitochondrial permeability transition does not involve inhibition of lysophospholipid acylation. Acyl-coenzyme A:1-acyllysophospholipid acyltransferase activity is not found in rat liver mitochondria.

The possible involvement of acyl-coenzyme A:1-acyllysophospholipid acyltransferase activity and phospholipid acylation-deacylation cycles in regulating the mitochondrial permeability transition have been examined by direct methods. 1-Acyllysophospholipid acyltransferase activity found in mitochondrial preparations obtained by differential centrifugation is inhibited by several transition-inducing agents and by glutathione disulfide. However, marker enzyme analysis employing mitochondria prepared by Percoll density gradient centrifugation or fractionated by a shear force-dependent method indicate that this activity is associated with contaminating microsomes and not with mitochondria. The absence of phospholipid acylation-deacylation cycles in isolated mitochondria is demonstrated by the absence of 18O incorporation from H2(18)O into phospholipid acylester carbonyl groups, confirming conclusions arrived at from marker enzyme data by a definitive independent approach. Mitochondria prepared by differential centrifugation and Percoll density gradient centrifugation are shown to be equivalent in requirements for induction of the permeability transition and the apparent rate of this process. It is concluded that 1-acyllysophospholipid acyltransferase activity and phospholipid acylation-deacylation cycles are not factors regulating the transition in isolated mitochondria. However, mitochondrial phospholipase A2 activity remains as a potential regulating factor, whereas the action of transition-inducing agents on microsomal 1-acyllysophospholipid acyltransferase may be important in mechanisms of cell injury.

Mitochondrial metabolism of 12- and 15-hydroxyeicosatetraenoic acids.

We have previously demonstrated that peroxisomal-deficient human skin fibroblasts and mutant Chinese hamster ovary cells do not convert 12- and 15-hydroxyeicosatetraenoic acids (HETEs) to chain-shortened, polar metabolites, suggesting that peroxisomes are the intracellular location for beta-oxidation of these compounds. This implies that mitochondria do not beta-oxidize HETEs. To test this hypothesis we incubated highly purified rat liver mitochondria with [3H]12-(S)- and [3H]15-(S)-HETE in the presence of carnitine and an acylcoenzyme A-generating system. Extracts obtained from these incubations were analyzed for radiolabeled polar metabolites. Both HETEs were converted to apparent products of beta-oxidation, although the 12-HETE compound was a markedly better substrate. The presence of 50 microM 2-tetradecyloxirane carboxylate, a potent inhibitor of carnitine palmitoyl transferase, completely blocked 12- and 15-HETE conversion to these metabolites as did omission of carnitine from the medium. These data demonstrate carnitine-dependent beta-oxidation of HETEs in isolated mitochondria and suggest that mitochondria are competent to carry out this metabolic process in eukaryotic cells. Prevailing metabolic conditions in subcellular compartments may have precluded observation of mitochondrial activity in our earlier work with cultured cells. Alternatively, transport mechanisms may exist in the cell types studied that distribute 12-(S)- and 15-(S)-HETEs specifically to peroxisomes.

Release of Ca2+ from mitochondria via the saturable mechanisms and the permeability transition.

The literature, reviewed in the previous article, supports three physiological roles for sequestration of calcium by mitochondria: 1) control of the rate of ATP production, 2) activation of the Ca2+-induced mitochondrial permeability transition (PT), and 3) modulation of cytosolic Ca2+ transients. Removal of Ca2+ from mitochondria permits rapid and efficient changes in the rate of ATP production to adapt to changing demands and can reverse the process of PT induction. Two separate, saturable mechanisms for facilitating Ca2+ efflux from mitochondria exist. In addition, the permeability transition or PT, which may also remove Ca2+ from the mitochondrial matrix, is intimately involved in other important functions such as apoptosis. Here we briefly review what is known about these important mitochondrial mechanisms and from their behavior speculate on their possible and probable functions.

Effects of phospholipase A2 inhibitors on ruthenium red-induced Ca2+ release from mitochondria.

The pharmacologic agents verapamil, nifedipine, diltiazem, prenylamine, N-oleoylethanolamine, R 24571, trifluoperazine, dibucaine, and quinacrine are examined as potential inhibitors of rat liver mitochondrial phospholipase A2 acting on endogenous phospholipid. Their potency as inhibitors of the enzyme is compared to their activities as inhibitors of phospholipase A2-dependent swelling and ruthenium red-induced Ca2+ release in intact mitochondria. For verapamil, diltiazem, trifluoperazine, dibucaine, and quinacrine, there is complete agreement between the relative potencies as inhibitors of phospholipase A2 and the two other processes. Nifedipine and prenylamine, which are weak inhibitors of phospholipase A2, produce a permeable inner membrane, provided that the mitochondrial have accumulated Ca2+. R 24571, which strongly inhibits the enzyme, disrupts mitochondria by a Ca2+-independent mechanism. N-Oleoylethanolamine, which is an effective inhibitor of swelling, does not inhibit phospholipase A2 or ruthenium red-induced Ca2+ release. The results support a proposed scheme wherein ruthenium red-induced Ca2+ release is viewed as reverse activity of the Ca2+-uptake uniporter occurring subsequent to decline in the proton motive force. The latter effect is proposed to arise from a specific phospholipase A2-dependent increase in inner-membrane H+ conductance of mitochondrial subpopulations. It is further shown that mitochondrial membranes display cyclic oscillations in free fatty acid content which are not dependent on the presence of Ca2+ or on the capacity to generate acylcoenzyme A.