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Combined Effect of G3139 and TSPO Ligands on Ca(2+)-induced Permeability Transition in Rat Brain Mitochondria

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Combined Effect of G3139 and TSPO Ligands on Ca(2+)-induced Permeability Transition in Rat Brain Mitochondria

T Azarashvili et al. Arch Biochem Biophys.

Abstract

Permeability of the mitochondrial outer membrane is determined by the activity of voltage-dependent anion channels (VDAC) which are regulated by many factors and proteins. One of the main partner-regulator of VDAC is the 18 kDa translocator protein (TSPO), whose role in the regulation of membrane permeability is not completely understood. We show that TSPO ligands, 1 μM PPIX and PK11195 at concentrations of 50 μM, accelerate opening of permeability transition pores (mPTP) in Ca(2+)-overloaded rat brain mitochondria (RBM). By contrast, PK11195 at 100 nM and anti-TSPO antibodies suppressed pore opening. Participation of VDAC in these processes was demonstrated by blocking VDAC with G3139, an 18-mer phosphorothioate oligonucleotides, which sensitized mitochondria to Ca(2+)-induced mPTP opening. Despite the inhibitory effect of 100 nM PK11195 and anti-TSPO antibodies alone, their combination with G3139 considerably stimulated the mPTP opening. Thus, 100 nM PK11195 and anti-TSPO antibody can modify permeability of the VDAC channel and mPTP. When VDAC channels are closed and TSPO is blocked, permeability of the VDAC for calcium seems to be the highest, which leads to accelerated pore opening.

Keywords: G3139; Mitochondria; PK11195; Permeability transition pore; TSPO; VDAC.

Conflict of interest statement

Conflicts of interest

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
Combined effect of 100 nM PK11195 and G3139 on Ca2+-induced mPTP opening in purified RBM. Mitochondrial parameters were measured as described in Materials and methods. (A) Control RBM, (B) 100 nM PK11195-treated RBM, (C) 5 μM G3139-treated RBM, (D) 100 nM PK11195 and 5 μM G3139-treated RBM. The arrows indicate where CaCl2 (150 and 220 μM) was added to the mitochondrial suspension.
Fig. 2
Fig. 2
Combined effect of anti-TSPO antibody and G3139 on Ca2+-induced mPTP opening in RBM. Mitochondrial parameters were measured as described in Materials and methods. (A) Control RBM, (B) anti-TSPO antibody (1:1500) treated RBM, (C) RBM pre-treated with anti-TSPO antibody and then incubated with 5 μM G3139, (D) RBM pre-treated with 5 μM G3139 and then incubated with anti-TSPO antibody. The arrows indicate where CaCl2 (150 and 220 μM) was added to the mitochondrial suspension.
Fig. 3
Fig. 3
Effect of 50 μM PK11195 in the presence of 5 μM G3139 on Ca2+-induced mPTP opening in RBM. Mitochondrial parameters were measured as described in Materials and methods. (A) Control RBM, (B) 50 μM PK11195 treated RBM, (C) 50 μM PK11195 and 5 μM G3139 treated RBM. The arrows indicate where CaCl2 (150 and 220 μM) was added to the mitochondrial suspension.
Fig. 4
Fig. 4
Effect of 1 μM protoporphyrin IX and G3139 on Ca2+-induced mPTP opening in RBM. Mitochondrial parameters were measured as described in Materials and methods. (A) Control RBM, (B) 1 μM protoporphyrin IX treated RBM, (C) 5 μM G3139 treated RBM, (D) 1 μM protoporphyrin IX and 5 μM G3139 treated RBM. The arrows indicate where CaCl2 (150 and 220 μM) was added to the mitochondrial suspension.
Fig. 5
Fig. 5
Ca2+-capacity of RBM in the presence of TSPO ligands and G3139. Ca2+-capacity was measured with Ca2+-sensitive electrode as described in Materials and methods in control RBM (trace a) and in RBM incubated in the presence of 5 μM G3139 alone (trace b) and in its combination with 1 μM protoporphyrin IX (trace c) and 100 nM PK11195 (trace d). The arrows indicate where CaCl2 (60 μM) was added to the mitochondrial suspension.
Fig. 6
Fig. 6
Mitochondrial parameters in the presence of 100 nM PK11195, 1 μM PPIX, 5 μM G3139 alone and in combination. Mitochondrial parameters (rate of Ca2+-influx (vCa2+in), lag time before Ca2+ release and Ca2+-capacity) were calculated as described in [29]. Column 1: mitochondrial parameters in the presence of 5 μM G3139, column 2: in the presence of 100 nM PK11195, column 3: in the presence of 5 μM G3139 and 100 nM PK11195, column 4: in the presence of 1 μM protoporphyrin IX and column 5: in the presence of 5 μM G3139 and 1 μM protoporphyrin IX. All parameters of RBM under experimental conditions were normalized to those of control RBM without any additions. Values represent means ± standard deviation from four independent experiments. *P < 0.05 versus control, as determined by Student’s t-test.

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