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1.
Biomed Khim ; 69(1): 62-71, 2023 Feb.
Article in Russian | MEDLINE | ID: mdl-36857428

ABSTRACT

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.


Subject(s)
Alcoholism , Mitochondria, Liver , Animals , Rats , Mitochondria , Ethanol
2.
Arch Biochem Biophys ; 587: 70-7, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26498031

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.


Subject(s)
Calcium/metabolism , Carrier Proteins/metabolism , Isoquinolines/pharmacology , Mitochondria/drug effects , Mitochondrial Membrane Transport Proteins/metabolism , Receptors, GABA-A/metabolism , Thionucleotides/pharmacology , Voltage-Dependent Anion Channels/antagonists & inhibitors , Animals , Brain/drug effects , Brain/metabolism , Cations, Divalent/metabolism , Ligands , Mitochondria/metabolism , Mitochondrial Permeability Transition Pore , Permeability/drug effects , Rats
3.
Biochemistry (Mosc) ; 79(6): 555-65, 2014 Jun.
Article in English | MEDLINE | ID: mdl-25100014

ABSTRACT

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.


Subject(s)
2',3'-Cyclic-Nucleotide Phosphodiesterases/metabolism , Mitochondria/metabolism , Myelin Basic Protein/metabolism , 2',3'-Cyclic-Nucleotide Phosphodiesterases/chemistry , Animals , Brain/metabolism , Calcium/metabolism , Male , Mitochondria, Liver/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition Pore , Myelin Basic Protein/chemistry , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Structure, Tertiary , Rats , Rats, Wistar
4.
Biochemistry (Mosc) ; 74(4): 421-9, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19463096

ABSTRACT

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.


Subject(s)
Brain/metabolism , Calcium/metabolism , Carrier Proteins/metabolism , Mitochondria/metabolism , Peptides/metabolism , Receptors, GABA-A/metabolism , Voltage-Dependent Anion Channels/metabolism , Animals , Ligands , Membrane Potential, Mitochondrial , Phosphorylation , Protein Binding , Rats
5.
Gut ; 58(3): 431-42, 2009 Mar.
Article in English | MEDLINE | ID: mdl-18596195

ABSTRACT

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.


Subject(s)
Cytochromes c/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Pancreas/metabolism , Pancreatitis/metabolism , Reactive Oxygen Species/metabolism , Animals , Apoptosis/physiology , Calcium/metabolism , Calcium Signaling , Cell Death/physiology , Membrane Potential, Mitochondrial/physiology , Pancreas/physiology , Pancreatitis/physiopathology , Rats
6.
Cell Calcium ; 34(3): 253-9, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12887972

ABSTRACT

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.


Subject(s)
Brain/physiology , Calcium Signaling/physiology , Ion Channels/physiology , Mitochondrial Proteins/metabolism , Animals , Calcium/metabolism , Calcium/pharmacology , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Cyclosporine/pharmacology , Egtazic Acid/pharmacology , Imidazoles/pharmacology , Intracellular Membranes/drug effects , Intracellular Membranes/physiology , Ion Channels/antagonists & inhibitors , Isoquinolines/pharmacology , Membrane Potentials/drug effects , Mitochondria/drug effects , Mitochondria/physiology , Mitochondrial Membrane Transport Proteins , Mitochondrial Permeability Transition Pore , Phosphorylation/drug effects , Rats , Rats, Wistar
7.
IUBMB Life ; 50(1): 75-80, 2000 Jul.
Article in English | MEDLINE | ID: mdl-11087125

ABSTRACT

Suppression of the mitochondrial permeability transition pore (PTP) and induction of lymphoma P388 cell death were studied in the presence of cyclosporin A (CsA) and its derivatives. In experiments with permeabilized P388 cells, CsA and its nonimmunosuppressive derivative N-methyl-Val-4-CsA, but not cyclosporin H (CsH), enhanced Ca2+ accumulation in mitochondria and suppressed PTP opening. Moreover, CsA was able either itself to induce or to enhance a prooxidant-induced P388 programmed cell death. Blebbing and formation of apoptotic bodies were among the observed CsA effects. N-Methyl-Val-4-CsA showed similar effects, but CsH had no effect on P388 cell death. These results show that initial-stage P388 tumour cell death is not related to PTP opening but can be the result of PTP closing with a corresponding increase in the formation of reactive oxygen species.


Subject(s)
Cyclosporine/pharmacology , Immunosuppressive Agents/pharmacology , Lymphoma/drug therapy , Lymphoma/pathology , Mitochondria/drug effects , Animals , Cell Cycle/drug effects , Cell Death/drug effects , Cell Membrane Structures/drug effects , Intracellular Membranes/drug effects , Lymphoma/metabolism , Mice , Mitochondria/metabolism , Mitochondria/ultrastructure , Permeability , Reactive Oxygen Species/metabolism , Vitamin K/pharmacology
8.
Biochemistry (Mosc) ; 65(9): 1023-6, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11042493

ABSTRACT

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.


Subject(s)
Calcium/metabolism , Intracellular Membranes/metabolism , Mitochondria, Liver/metabolism , Oxidative Phosphorylation , Adenosine Triphosphate/biosynthesis , Animals , Calcium/pharmacology , Dose-Response Relationship, Drug , Hydrolysis , Ions , Kinetics , Peptides/metabolism , Proton-Translocating ATPases/metabolism , Rats , Rats, Wistar , Time Factors
9.
Biochemistry (Mosc) ; 64(5): 556-60, 1999 May.
Article in English | MEDLINE | ID: mdl-10381617

ABSTRACT

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.


Subject(s)
Mitochondria, Liver/metabolism , Peptides/metabolism , Adenosine Triphosphate/metabolism , Animals , Mitochondria, Liver/physiology , Molecular Weight , Oxidative Phosphorylation , Peptides/chemistry , Phosphorus Radioisotopes , Rats , Rats, Wistar
10.
Biochem Mol Biol Int ; 45(3): 501-10, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9679650

ABSTRACT

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.


Subject(s)
Calcium/metabolism , Cell Death , Intracellular Membranes/metabolism , Mitochondria/metabolism , Oxidants/pharmacology , Animals , Benzene Derivatives/pharmacology , Carcinoma, Ehrlich Tumor , Cell Survival , Membrane Potentials , Mice , Oxidative Stress , Permeability , Peroxides/pharmacology , Tumor Cells, Cultured , Vitamin K/pharmacology , tert-Butylhydroperoxide
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