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

Abstract

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.

Mitochondrial Iron Sequestration in Dopamine‐Challenged Astroglia: Role of Heme Oxygenase‐1 and the Permeability Transition Pore

“…When administered together, CSA (0.5 p M ) and TFP (30 pM) completely suppressed mitochondrial iron trapping in these preparations (Fig. 5), an observation consistent with other studies reporting synergistic effects of combined CSA/TFP treatment on MTP permeability (Broekemeier and Pfeiffer, 1995;Bernardi, 1996).…”

“…CSA is thought to inhibit pore opening by interacting with, and displacing, cyclophilin M. The latter is a peptidyl-propyl-cis-trans-isomerase localized to the mitochondrial matrix that binds to the MTP and increases the probability that the channel is maintained in the open configuration. TFP is an amphipathic molecule that promotes pore closure by inducing mitochondrial membrane hyperpolarization (Broekemeier and Pfeiffer, 1995;Bernardi, 1996). Incubation with CSA (0.06-0.5 pM) or TFP (1-30 pM) alone significantly inhibited mitochondrial iron sequestration elicited by DA exposure or HO-1 transfection in a dose-dependent fashion.…”

Oligomycin, inhibitor of the F0 part of H+-ATP-synthase, suppresses the TNF-induced apoptosis

“…It was found that CsA in combination with trifluoropyrasine (TFP), the inhibitor of phospholipase A 2 , inhibited the TNF-induced release of cytochrome c and apoptosis (Figures 2c and 3c). Neither CsA nor TFP were effective alone (this is in line with the temporary inhibition of PTP by CsA in isolated mitochondria and prolongation of the CsA effect by TFP (Broekemeier and Pfeiffer, 1995)). MeVal-CsA, a CsA analogue effective in PTP inhibition but ineffective in inhibition of calcineurin, also suppressed TNFinduced apoptosis (not shown) so a role of calcineurin, the major non-mitochondrial CsA target, could be excluded.…”

Singlet Oxygen Produced by Photodynamic Action Causes Inactivation of the Mitochondrial Permeability Transition Pore

“…Its lifetime is very short in biological materials, which restricts its action to the close vicinity (Ͻ0.1 m) of the photosensitizer (24). Although the membrane lipid phase is clearly involved in modulating the PT, possibly through surface potential effects (25,26), we tend to exclude that pore inactivation by HP photodynamic action is due to lipid peroxidation, which should rather promote the PT (1).…”