Knockout of cyclophilin D in Ppif⁻/⁻ mice increases stability of brain mitochondria against Ca²âº stress.

The mitochondrial peptidyl prolyl isomerase cyclophilin D (CypD) activates permeability transition (PT). To study the role of CypD in this process we compared the functions of brain mitochondria isolated from wild type (BMWT) and CypD knockout (Ppif(-/-)) mice (BMKO) with and without CypD inhibitor Cyclosporin A (CsA) under normal and Ca(2+) stress conditions. Our data demonstrate that BMKO are characterized by higher rates of glutamate/malate-dependent oxidative phosphorylation, higher membrane potential and higher resistance to detrimental Ca(2+) effects than BMWT. Under the elevated Ca(2+) and correspondingly decreased membrane potential the dose response in BMKO shifts to higher Ca(2+) concentrations as compared to BMWT. However, significantly high Ca(2+) levels result in complete loss of membrane potential in BMKO, too. CsA diminishes the loss of membrane potential in BMWT but has no protecting effect in BMKO. The results are in line with the assumption that PT is regulated by CypD under the control of matrix Ca(2+). Due to missing of CypD the BMKO can favor PT only at high Ca(2+) concentrations. It is concluded that CypD sensitizes the brain mitochondria to PT, and its inhibition by CsA or CypD absence improves the complex I-related mitochondrial function and increases mitochondria stability against Ca(2+) stress.

Molecular and biochemical investigations in fumarase deficiency.

Fumarase (FH) deficiency is a rare autosomal recessive disease of the Krebs cycle causing severe neurological impairment in early childhood, characterized by encephalopathy with seizures and muscular hypotonia. Only a handful of patients with various recessive mutations in the FH gene have been described so far. Interestingly, autosomal dominant mutations in the same gene are associated with hereditary leiomyomatosis and renal cell cancer (HLRCC). We investigated a boy with developmental and growth delay, microcephaly, and muscular hypotonia recognized at the age of 3 months. No leiomyomatosis or renal cancer is known in the parents. Investigation of the patient's urine revealed massive fumarate excretion. FH activity was severely reduced in muscle and fibroblasts. Respirometric investigation of fibroblasts showed only modest changes indicating that fumarate mediated inhibition of enzymatic pathways other than oxidative phosphorylation might be more relevant in pathophysiology of FH deficiency. Molecular analysis revealed a known 435insK mutation on the paternal allele and a novel H275L mutation due to an A to T transversion of nucleotide 824 on the maternal allele. This mutation affects the same codon as a C to T transition of nucleotide 823, resulting in a H275Y mutation that was found in two families with HLRCC.

Effect of thyroxine on calcium distribution in rat thymocytes in vitro.

Chlorotetracycline (CTC) was used as a fluorescent Ca2+-sensitive probe to study the redistribution of intracellular membrane-bound Ca2+ in thyroxine (T4)-treated rat thymocytes. Incubation of thymocytes in the Ca2+-supplemented medium in the presence of 1-100 nM T4 for 30 min resulted in a twofold increase in the amount of EGTA-accessible plasma membrane-bound Ca2+ as compared to that in the Ca2+-free medium. The induced decrease in CTC fluorescence was more pronounced with the occurrence of respiration and oxidative phosphorylation in inhibitors. The mitochondrial Ca2+ pool was shown to increase. The nonmitochondrial Ca2+ pool decreased after a 30-min incubation in the presence of 1 nM T4 and increased when 100 nM T4 was used under the same conditions. Without incubation, different concentrations of T4 stimulated the decrease in the Ca2+ pool of the endoplasmic reticulum (ER) compared to the control cells, which was demonstrated using inhibitors of the ER Ca2+-ATPase (vanadate, BHQ). Calmodulin blockers (triftazin and R24) caused a significant decrease (over 50%) in CTC fluorescence in the T4-treated thymocytes. This suggests that T4 can act as an in vitro stimulator of calmodulin-dependent Ca2+ accumulation in thymocyte membranes. The results of our experiments with AlF4- suggest that T4 stimulates the activity of G-proteins by a receptor-mediated mechanism.

In vitro effect of hydrocortisone on calcium distribution in rat thymocytes.

Chlorotetracycline (CTC) was used as a fluorescence indicator to investigate the intracellular membrane-bound Ca2+ redistribution in the hydrocortisone (HC)-treated rat thymocytes. The effect of HC (0.1-1 microM) on the thymocytes incubated in Ca2+ containing media for 60-120 min increased 1.5-fold the quantity of membrane-bound Ca2+ in the plasma membrane. When thymocytes were incubated in Ca(2+)-free media, no HC-induced changes in the Ca2+ pool of plasma membrane were revealed. In the presence of inhibitors of respiration and oxidative phosphorylation, the CTC fluorescence intensity in the HC-treated cells decreased to a greater extent than in the control, thereby indicating an increase in the mitochondrial Ca2+ pool. Using inhibitors of endoplasmic reticulum Ca(2+)-ATPase (vanadate, BHQ). HC was shown to decrease the non-mitochondrial Ca2+ pool. Calmodulin blockers (triphthasine and R24) slightly decreased the CTC fluorescence intensity in the HC-treated cells as compared to the control. HC was found to inhibit the calmodulin-mediated Ca2+ accumulation in the thymocyte membrane. Based on the experiments using AlF4, we concluded that HC stimulates the activity of G-proteins through the receptor-mediated mechanism. A possible role of the observed Ca2+ redistribution between the thymocyte intracellular compartments, specifically between mitochondria and reticulum, is discussed.

[Inhibition of gluconeogenesis in the kidneys by a cytoplasmic regulator of mitochondrial membrane permeability]. / Tormozhenie gliukoneogeneza v pochkakh tsitoplazmaticheskim reguliatorom pronitsaemosti membrany mitokhondrii.

Cytoplasmatic glycoprotein previously shown to be an inhibitor of mitochondrial membrane permeability for oxidation substrates inhibits in-vitro gluconeogenesis from pyruvate, lactate, succinate, alpha-ketoglutarate and glutamate in slices of the renal cortical layer. The inhibition was reduced with Ca2+ concentration increase in the incubation medium from 0.02 to 2 mM.

[Dose-dependent character of the effect of hydrocortisone on energy metabolism of rat thymocytes]. / Dozozavisimyi kharakter vliianiia gidrokortizona na énergeticheskii metabolizm timotsitov krys.

The influence of the hydrocortisone (GC) different doses on rat's thymocytes energetic metabolism was investigated. It has been demonstrated that as the prolonged action of GC in vivo (20 micrograms/kg of mass during 6 d. daily) as the preincubation of thymocytes with 10 microM GC in vitro are cytotoxic, leading to absolute uncoupling of oxidative phosphorylation (OP) and decreasing the rate of DNP-stimulated respiration speed. The injection of GC (20 micrograms/kg mass) at once before 72 hours to killing resulted the OP uncoupling almost on 2,5 time. Short-time action of GC at the same dose before 3 hours to drilling and so the thymocytes preincubation with 1 microM GC stimulated the transport processes on the level of substrate oxidation without the OP uncoupling. Short-time action of GC in vivo caused the inhibition of respiration, if we used glucose as the oxidative substrate, and stimulated the respiration in a presence of pyruvate Na in incubation media.

[Regulation of mitochondrial membrane permeability by a factor from mitochondrial interaction]. / Reguliatsiia pronitsaemosti mitokhondrial'noi membrany faktorom vzaimodeistviia mitokhondrii

It was shown that a factor from the thyroxine-injured mitochondria caused inhibition of mitochondrial swelling in isotonic KNO3 and NH4NO3 solutions in the presence of rotenone. These data were interpreted as permeability inhibition for K+ and H+ ions.

[The action of defoliants on the energetics of rat liver mitochondria in vitro]. / Deistvie defoliantov na énergetiku mitokhondrii pecheni krys in vitro.

The effect of defoliants butyphos (I), dropp (II), butylcaptacs (III), hinazopin (IV), syhat (V), tetra-n-butylammonium bromide (VI), etrel (VII), gemetrel (VIII), allyl-4-methylpyridinium bromide (IX), 1-amino-cyclopropan-1-carbonate (ACPC) (X) at various concentrations (1 x 10(-5)-2 x 10(-4) M) on respiration, oxidative phosphorylation (OP) and permeability of the inner mitochondrial membrane from rat liver has been studied. It has been established that some of the compounds uncouple OP by increasing the inner mitochondrial membrane permeability for H+ (II) inhibit the respiration in V3 condition and induce less selective permeability for a number of ions (I, III). The other defoliants either induce respiration generally in metabolic states 3 and 4 (IV, VI, IX) or have no effect on the respiration and OP (V, VII, VIII, X). On the whole a good correlation between the common toxicity of the studied preparation (LD50) and their mitochondrial effect has been revealed, therefore the latter can be considered as intracellular "targets" involved in the realization of pesticide action.

Disturbances in oxidative phosphorylation in the liver of rats with heliotrine-induced hepatitis and restoration by phosphatidylcholine and ATP.

Acute hepatitis induced by heliotrine is accompanied by uncoupling of oxidative phosphorylation in liver mitochondria. The rate of oxygen uptake during succinate oxidation increased in all metabolic states, while the respiratory control index decreased by 45% because of the greater increase in the respiration rate in state 4 by comparison with that in state 3. Heliotrine poisoning also halved the rate of oxygen uptake in rat liver homogenates in the presence of ascorbate and tetramethylene-p-phenylenediamine. This is indicative of a lowering of cytochrome oxidase activity and of energy metabolism disturbances in rat liver. Preparations of cotton phosphatidylcholine (PC), both purified and as ATP-containing complexes (PC+ATP), as well as ATP alone, reduced the metabolic disorders in liver mitochondria of rats with acute heliotrine-induced hepatitis. The therapeutic effect of these preparations consisted in the restoration of oxidative phosphorylation coupling and of the cytochrome oxidase activity. The effect of PC+ATP was much greater than either PC or ATP alone. In contrast, the commercial preparation, Essential, had no beneficial effect.