Inhibition of mitochondrial membrane bound-glutathione transferase by mitochondrial permeability transition inhibitors including cyclosporin A.

AIMS: Effect of mitochondrial permeability transition (MPT) inhibitors on mitochondrial membrane-bound glutathione transferase (mtMGST1) activity in rat liver was investigated in vitro. MAIN METHODS: When mitochondria were incubated with MPT inhibitors, mtMGST1 activity was decreased dose dependently and their 50% inhibition concentration (IC(50)) were 1.2 microM (cyclosporin A; CsA), 31 microM (bongkrekic acid; BKA), 1.8 mM (ADP), and 3.2 mM (ATP). The decrease of mtMGST1 activity by the MPT inhibitors was not observed in the presence of detergent Triton X-100. On the contrary, mtMGST1 inhibition by GST inhibitors such as cibacron blue (IC(50), 4.2 microM) and S-hexylglutathione (IC(50), 480 microM) was not affected in the presence of detergent. Although mtMGST1 resides in both the inner (IMM) and outer mitochondrial membranes (OMM), only mtMGST1 in the IMM was inhibited by the MPT inhibitors in the absence of detergent. GST inhibitors decreased mtMGST1 activity both in the IMM and OMM regardless of the presence or absence of detergent. Cytosolic GSTs and microsomal MGST1 were not inhibited by the MPT inhibitors. KEY FINDINGS: These results indicate that mtMGST1 is inhibited by MPT inhibitors through membrane components, not directly by the inhibitors. SIGNIFICANCE: Since CsA binds to cyclophilin D (Cyp-D) in the mitochondrial matrix whereas BKA or ADP binds to adenine nucleotide translocator (ANT) in the IMM, it was suggested that mtMGST1 in the IMM interacts with Cyp-D/ANT and the binding of MPT inhibitors to Cyp-D or ANT causes their conformational change followed by an alteration of mtMGST1 conformation, resulting in decreasing mtMGST1 activity.

Contribution of liver mitochondrial membrane-bound glutathione transferase to mitochondrial permeability transition pores.

We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated with GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca(2+)-mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1.