Effect of peripheral benzodiazepine receptor (PBR/TSPO) ligands on opening of Ca2+-induced pore and phosphorylation of 3.5-kDa polypeptide in rat brain mitochondria.

The effect of nanomolar concentrations of PBR/TSPO ligands--Ro 5-4864, PK11195, and PPIX--on Ca2+-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria was investigated. PBR/TSPO agonist Ro 5-4864 (100 nM) and endogenous ligand PPIX (1 microM) were shown to stimulate PTP opening, while antagonist PK11195 (100 nM) suppressed this process. Correlation between PBR ligand action on PTP opening and phosphorylation of a 3.5 kDa polypeptide was investigated. In intact brain mitochondria, incorporation of [gamma-(32)P]ATP into 3.5 kDa peptide was decreased in the presence of Ro 5-4864 and PPIX and increased in the presence of PK11195. At threshold Ca2+ concentrations leading to PTP opening, PBR/TSPO ligands were found to stimulate dephosphorylation of the 3.5 kDa peptide. Specific anti-PBR/TSPO antibody prevented both PTP opening and dephosphorylation of the 3.5-kDa peptide. The peptide was identified as subunit c of F(o)F(1)-ATPase by Western blot using specific anti-subunit c antibody. The results suggest that subunit c of F(o)F(1)-ATPase could be an additional target for PBR/TSPO ligands action, is subjected to Ca2+- and TSPO-dependent phosphorylation/dephosphorylation, and is involved in PTP operation in mitochondria.

Physiological Ca2+ level and Ca2+-induced Permeability Transition Pore control protein phosphorylation in rat brain mitochondria.

Phosphorylation of several low molecular mass proteins (3.5, 17, 23 and 29kDa) was observed in rat brain mitochondria (RBM) at ATP concentration close to that in the mitochondrial matrix. Furthermore, regulatory effects of Ca2+ on phosphorylation of these proteins were investigated. Protein phosphorylation was found to be modulated by Ca2+ in the physiological concentration range (10(-8) to 10(-6)M free Ca2+). Incorporation of 32P from [gamma-32P]ATP into the 17kDa protein was dramatically increased within the 10(-7) to 10(-6)M free Ca2+ range, whereas an opposite effect was observed for the 3.5kDa polypeptide. Strong de-phosphorylation of the 3.5kDa polypeptide and enhanced 32P-incorporation into the 17 and 23kDa proteins were found with supra-threshold Ca2+ loads and these effects were eliminated or reduced in the presence of cyclosporin A, an inhibitor of Permeability Transition Pore (PTP) opening. In the presence of calmidazolium (Cmz), a calmodulin antagonist, enhanced levels of phosphorylation of the 17 and 3.5kDa polypeptides were observed and the 17kDa protein phosphorylation was suppressed by H-8, a protein kinase A inhibitor. It is concluded that Ca2+ in physiological concentrations, as a second messenger, can control phosphorylation of the low molecular mass phospoproteins in RBM, in addition to well known regulation of some Krebs cycle dehydrogenases by Ca2+. The protein phosphorylation was strongly dependent on the Ca2+-induced PTP opening.

Mechanism of superoxide anion generation in intact mitochondria in the presence of lucigenin and cyanide.

In the presence of cyanide and various respiratory substrates (succinate or pyruvate + malate) addition of high concentrations of lucigenin (400 microM; Luc2+) to rat liver mitochondria can induce a short-term flash of high amplitude lucigenin-dependent chemiluminescence (LDCL). Under conditions of cytochrome oxidase inhibition by cyanide the lucigenin-induced cyanide-resistant respiration (with succinate as substrate) was not inhibited by uncouplers (FCCP) and oligomycin. Increase in transmembrane potential (Deltaphi) value by stimulating F0F1-ATPase functioning (induced by addition of MgATP to the incubation medium) caused potent stimulation of the rate of cyanide-resistant respiration. At high Deltaphi values (in the presence of MgATP) cyanide resistant respiration of mitochondria in the presence of succinate or malate with pyruvate was insensitive to tenoyltrifluoroacetone (TTFA) or rotenone, respectively. However, in both cases respiration was effectively inhibited by myxothiazol or antimycin A. Mechanisms responsible for induction of LDCL and cyanide resistant mitochondrial respiration differ. In contrast to cyanide-resistant respiration, generation of LDCL signal, that was suppressed only by combined addition of Complex III inhibitors, antimycin A and myxothiazol, is a strictly potential-dependent process. It is observed only under conditions of high Deltaphi value generated by F0F1-ATPase functioning. The data suggest lucigenin-induced intensive generation of superoxide anion in mitochondria. Based on results of inhibitor analysis of cyanide-resistant respiration and LDCL, a two-stage mechanism of autooxidizable lucigenin cation-radical (Luc*+) formation in the respiratory chain is proposed. The first stage involves two-electron Luc2+ reduction by Complexes I and II. The second stage includes one-electron oxidation of reduced lucigenin (Luc(2e)). Reactions of Luc(2e) oxidation involve coenzyme Q-binding sites of Complex III. This results in formation of autooxidizable Luc*+ and superoxide anion generation. A new scheme for lucigenin-dependent electron pathways is proposed. It includes formation of fully reduced form of lucigenin and two-electron-transferring shunts of the respiratory chain. Lucigenin-induced activation of superoxide anion formation in mitochondria is accompanied by increase in ion permeability of the inner mitochondrial membrane.

Lucigenin-derived chemiluminescence in intact isolated mitochondria.

There are many data both in favor and against the use of lucigenin as a probe for superoxide anion (SA) in mitochondria, cells, and simple enzymatic systems. In the present work high concentrations (50-400 micro M) of lucigenin were used for continuous recording of rapid and reversible changes in the SA level in intact isolated mitochondria. The SA level in the presence of lucigenin rapidly and reversibly changed during the transition of the mitochondria from one functional state to another: under conditions of ATP synthesis from ADP and Pi, of Ca2+ accumulation, and of reverse electron transfer. Induction of a Ca2+,cyclosporin A-sensitive pore in mitochondria completely suppressed the lucigenin-derived chemiluminescence (LDC). The electron transfer in the Q-cycle of the respiratory chain complex III and high electric potential difference across the inner membrane of mitochondria were obligatory conditions for generation of a SA-dependent chemiluminescent signal. Based on our own and literature data, a scheme of LDC generation is suggested. The origin of superoxide anion detected in intact mitochondria with lucigenin is discussed.