[Effect of erastin and G3139 on rat liver mitochondria in chronic alcoholic intoxication]. / Vliianie érastina i G3139 na mitokhondrii pecheni krys pri khronicheskoi alkogol'noi intoksikatsii.

The effect of modulators of VDAC channels - G3139 and erastin on the mitochondrial permeability transition pore (mPTP) functioning and changes in the content of proteins involved in regulation of mPTP (VDAC, CNPase, and TSPO) has been investigated in liver mitochondria of rats with chronic alcohol intoxication. It was shown that the mitochondria of rats treated with ethanol were more sensitive to mPTP induction. Moreover, ethanol induced changes in the expression of mPTP regulator proteins. G3139 and erastin were also able to influence the studied mitochondrial parameters, and they increased their effect in the liver mitochondria of rats treated with ethanol, as compared to the mitochondria of control rats. We hypothesize that the results of this study may help to elucidate the mechanisms of chronic action of ethanol on mitochondria and contribute to the development of new therapeutic strategies for treating the consequences of ethanol-related diseases.

Interaction of myelin basic protein and 2',3'-cyclic nucleotide phosphodiesterase with mitochondria.

The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and nonsynaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca2+ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.

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.

Mechanisms regulating cytochrome c release in pancreatic mitochondria.

BACKGROUND: Mechanisms of acinar cell death in pancreatitis are poorly understood. Cytochrome c release is a central event in apoptosis in pancreatitis. Here, we assessed the regulation of pancreatic cytochrome c release by Ca(2+), mitochondrial membrane potential (Delta Psi m), and reactive oxygen species (ROS), the signals involved in acute pancreatitis. We used both isolated rat pancreatic mitochondria and intact acinar cells hyperstimulated with cholecystokinin-8 (CCK-8; in vitro model of acute pancreatitis). RESULTS: Micromolar amounts of Ca(2+) depolarised isolated pancreatic mitochondria through a mechanism different from the "classical" (ie, liver) mitochondrial permeability transition pore (mPTP). In contrast with liver, Ca(2+)-induced mPTP opening caused a dramatic decrease in ROS and was not associated with pancreatic mitochondria swelling. Importantly, we found that Ca(2+)-induced depolarisation inhibited cytochrome c release from pancreatic mitochondria, due to blockade of ROS production. As a result, Ca(2+) exerted two opposite effects on cytochrome c release: Ca(2+) per se stimulated the release, whereas Ca(2+)-induced depolarisation inhibited it. This dual effect caused a non-monotonous dose-dependence of cytochrome c release on Ca(2+). In intact acinar cells, cytochrome c release, caspase activation and apoptosis were all stimulated by ROS and Ca(2+), and inhibited by depolarisation, corroborating the findings on isolated pancreatic mitochondria. CONCLUSIONS: These data implicate ROS as a key mediator of CCK-induced apoptotic responses. The results indicate a major role for mitochondria in the effects of Ca(2+ )and ROS on acinar cell death. They suggest that the extent of apoptosis in pancreatitis is regulated by the interplay between ROS, Delta Psi m and Ca(2+). Stabilising mitochondria against loss of Delta Psi m may represent a strategy to mitigate the severity of pancreatitis.

Regulation of oxidative phosphorylation in the inner membrane of rat liver mitochondria by calcium ions.

The effect of accumulation of Ca2+ at physiological concentrations (10(-8)-10(-6) M) on the rates of ATP synthesis and hydrolysis in rat liver mitochondria was studied. An addition of 5 x 10(-7) M Ca2+ resulted in the maximal rates of synthesis and hydrolysis of ATP. Decrease in the concentration of Ca2+ to 10-8 M or its increase to 5 x 10(-6) M inhibited oxidative phosphorylation and ATP hydrolysis. It was found that the rate of oxidative phosphorylation correlated with the phosphorylation level of a 3.5-kD peptide in the mitochondrial inner membrane on varying the Ca2+ concentration. The possible regulation of oxidative phosphorylation in mitochondria by Ca2+ is discussed.

Phosphorylation of a low-molecular-weight polypeptide in rat liver mitochondria and dependence of its phosphorylation on mitochondrial functional state.

We show that incubation of rat liver mitochondria in the presence of [gamma-32P]ATP results in cAMP-dependent phosphorylation of a low-molecular-weight (3.5-kD) polypeptide (LMWP). This component is tightly bound to the mitochondrial membrane. It is not released into solution after freezing and subsequent thawing of the mitochondrial suspension and does not incorporate 32P from [gamma-32P]ATP in the presence of uncouplers of oxidative phosphorylation. Inhibition of adenine nucleotide transport into the mitochondrial matrix by carboxyatractyloside suppresses phosphorylation of the LMWP. Moderate Ca2+ loading of mitochondria increases both phosphorylation and dephosphorylation of the LMWP. Chelation of Ca2+ by incubation in the presence of EGTA suppresses incorporation of 32P into the LMWP.

Effect of prooxidants on mitochondrial permeability transition and cell death in Ehrlich ascites tumour cells.

Ca2+ retention in mitochondria, opening of the Cysclosporin A- sensitive permeability transition pore and cell death were studied in Ehrlich ascites tumour cells in the presence of different prooxidants. Low concentrations (1-20 microM) of the prooxidants (menadione, cumenehydroperoxide, t-butylhydroperoxide) induced pore-opening in permeabilized cells at threshold Ca2+ load. Incubation of cells with low concentrations of prooxidants was able to induce cell cycle disturbance and cell death. Under the prooxidant effect, mitochondrial membrane potential drop and Ca2+ retention decrease in mitochondria were found to precede death of Ehrlich ascites tumour cells.