High-affinity peripheral benzodiazepine receptor ligand, PK11195, regulates protein phosphorylation in rat brain mitochondria under control of Ca(2+).

The effects of PK11195, a high-affinity peripheral benzodiazepine receptor (PBR) ligand, on protein phosphorylation in isolated purified rat brain mitochondria were investigated. The isoquinoline carboxamide ligand of PBR, PK11195, but not the benzodiazepine ligand Ro5-4864, in the nanomolar concentration range strongly increased the phosphorylation of 3.5 and 17 kDa polypeptides. The effect of PK11195 was seen in the presence of elevated Ca(2+) levels (3 x 10(-7) to 10(-6) m), but not at very low Ca(2+) levels (10(-8) to 3 x 10(-8) m). This indicates that PBR involves Ca(2+) as a second messenger in the regulation of protein phosphorylation. Staurosporine, an inhibitor of protein kinase activity was able to suppress the PK11195-promoted protein phosphorylation. When the permeability transition pore (PTP) was opened by threshold Ca(2+) load, phosphorylation of the 3.5-kDa polypeptide was diminished, but strong phosphorylation of the 43-kDa protein was revealed. The 43-kDa protein appears to be a PTP-specific phosphoprotein. If PTP was opened, PK11195 did not increase the phosphorylation of the 3.5 and 17-kDa proteins but suppressed the phosphorylation of the PTP-specific 43-kDa phosphoprotein. The ability of PK11195 to increase the protein phosphorylation, which was lost under Ca(2+)-induced PTP opening, was restored again in the presence of calmidazolium, an antagonist of calmodulin and inhibitor of protein phosphatase PP2B. These results show a tight interaction of PBR with the PTP complex in rat brain mitochondria. In conclusion, a novel function of PBR in brain mitochondria has been revealed, and the PBR-mediated protein phosphorylation has to be considered an important element of the PBR-associated signal transducing cascades in mitochondria and cells.

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.

Intracellular distribution and characteristics of Ca(2+)-transporting systems in cells of ciliate protozoan Tetrahymena pyriformis.

A significant digitonin-sensitive calcium pool has been shown to exist in Tetrahymena pyriformis cells. In the presence of exogenic energy sources, calcium released from the digitonin-sensitive pool can be accumulated by mitochondria or endoplasmic reticulum. The kinetic characteristics of the mitochondrial and reticulum systems of Ca2+ transport and their sensitivity to various inhibitors were studied.

Direct participation of phospholipids in transmembrane K(+)-transport.

The correlation between the characteristics of K(+)-transport across the mitochondrial and bilayer lipid membranes formed form mitochondrial lipids has been demonstrated. It has been shown that different modes of K(+)-efflux activation in mitochondria result in the appearance of K(+)-transporting phospholipid forms. The experiments described in this work suggest that current fluctuations similar to those observed for biological channels can be registered in unmodified bilayer lipid membranes containing no protein components.