Induction of apoptosis by valinomycin: mitochondrial permeability transition causes intracellular acidification.

In order to determine whether disruption of mitochondrial function could trigger apoptosis in murine haematopoietic cells, we used the potassium ionophore valinomycin. Valinomycin induces apoptosis in the murine pre-B cell line BAF3, which cannot be inhibited by interleukin-3 addition or Bcl-2 over-expression. Valinomycin triggers rapid loss of mitochondrial membrane potential. This precedes cytoplasmic acidification, which leads to cysteine-active-site protease activation, DNA fragmentation and cell death. Bongkrekic acid, an inhibitor of the mitochondrial permeability transition, prevents acidification and subsequent induction of apoptosis by valinomycin.

Thyroid hormones regulate the onset of osmotic activity of rat liver mitochondria after birth.

The effect of thyroid hormone deprivation on the osmotic activity of liver mitochondria from early newborn rats was studied. Experimentally induced hypothyroidism prevented the increase in the osmotic activity of mitochondria observed immediately after birth. Osmotic activity was restored by T4 and T3 treatment to hypothyroid newborns but not when this treatment was supplemented with cycloheximide. Under the same circumstances, streptomycin had no effect. Hypothyroidism abolished the change in the slope of the osmotic curve (plot of inverse absorbance of mitochondrial suspensions incubated in sucrose solutions vs. inverse sucrose concentration) observed in mitochondria from euthyroid newborns at 110-120 mOsm sucrose, suggesting that hypothyroidism prevents the formation of tight physical connections between mitochondrial outer and inner membranes. Thyroid hormone deprivation increased the passive permeability of the mitochondrial inner membrane to protons, resulting in a decreased respiratory control ratio. Hypothyroidism prevented the sharp decrease in the affinity of mitochondria for ATP observed in euthyroid newborns immediately after birth. These results corroborate our previous suggestion (Endocrinology, 1995, 136:4448) that, during the early neonatal period, thyroid hormones control the synthesis of some nucleus-coded protein(s) involved in the assembly of F0,F1-ATPase.

cAMP and Ca2+ involvement in the mitochondrial response of cultured fetal rat hepatocytes to adrenaline.

The effect of adrenaline on the control of respiratory activity of mitochondria from fetal hepatocytes in primary culture was studied. In the absence of adrenaline, the respiratory control ratio (RCR) of mitochondria increased during the first 3 days of culture due to a decrease in the rate of state 4 respiration. The presence of adrenaline in the incubation medium further increased the mitochondrial RCR through a decrease in the rate of respiration in state 4 and to an increase in the respiration rate in state 3. The effect of adrenaline was mimicked by dibutyryl-cAMP, forskolin, and isobutyl methyl xanthine. All these compounds increased cAMP concentrations, suggesting that cAMP may be involved in the effect of adrenaline. The increase in intracellular free Ca2+ concentrations caused by phenylephrine, vasopressin, or thapsigargin was also accompanied by an increase in the RCR, suggesting that both phenomena are associated. Dibutyryl-cAMP also increased free Ca2+ concentrations, suggesting that the effects of cAMP may be mediated by free Ca2+ concentrations. Adrenaline, dibutyryl-cAMP, phenylephrine, vasopressin, and thapsigargin promoted adenine nucleotide accumulation in mitochondria; this may be an intermediate step in the activation of mitochondrial respiratory function. These results suggest that the stimulatory effect of adrenaline on mitochondrial maturation in cultured fetal rat hepatocytes may be exerted through a mechanism in which both cAMP and Ca2+ act as second messengers. It is concluded that the effect of adrenaline on mitochondrial maturation is exerted by both alpha- and beta-adrenergic mechanisms and is mediated by the increase in adenine nucleotide contents of mitochondria.

Variations of muscle mitochondrial creatine kinase activity in mitochondrial diseases.

Mitochondrial creatine kinase (mtCK) activity has been measured in the mitochondria isolated from the muscle of 69 patients suspected of mitochondrial diseases. The isolated mitochondria did not contain significant amounts of the muscle isoform of creatine kinase, as checked by an immunoassay performed after electrophoretic separation of the various isoforms. Hence, the enzyme assay reliably represented the mtCK activity. Therefore, a simple measurement of CK activity in isolated mitochondria permitted the measurement of mtCK activity. An absence of mtCK activity in muscle was never observed. The lowest activities were not associated to defined mitochondrial diseases linked to defects of respiratory chain complexes or to defects of citric cycle enzymes. On the contrary, mtCK activity was significantly increased in the muscle of patients exhibiting ragged red fibers. This increase was generally associated to an increase of citrate synthase activity. Since ragged-red fibers and elevated mtCK activities were generally not found in children younger than 3 years, even in cases of characteristic oxidative phosphorylation deficiency, it is suggested that the increase in mtCK activity as well as the appearance of ragged-red fibers are not the first events which occur during the evolution of mitochondrial diseases but would rather be long-term secondary processes which slowly develop in deficient mitochondria.

Changes in adult rat liver mitochondrial populations at different energy states analyzed by flow cytometry.

The present work studies the changes in green fluorescence intensity after Rh-123 staining of the low (LFP) and the high fluorescence populations (HFP) in isolated mitochondria from rat liver. The results show that the HFP represents a mitochondrial compartment less sensitive to changes in energy states. In addition, it is concluded that the use of Rh-123 to monitor changes in mitochondrial membrane potential should be undertaken with caution because, under certain circumstances, there is no correlation between the Rh-123 intensity of fluorescence due to its uptake by mitochondria and previously reported changes in the mitochondrial membrane potential.

Hypothyroidism prevents postnatal changes in rat liver mitochondrial populations defined by rhodamine-123 staining.

The effect of hypothyroidism on the percentages of low fluorescence population (LFP) and high fluorescence population (HFP) rhodamine-123-stained mitochondria, respiratory parameters, and ATPase activity were studied in liver mitochondria from early newborn rats. Hypothyroidism prevented the decrease in the percentage of HFP and the subsequent increase in LFP that occurs immediately after birth. This effect coincides with the impairment of mitochondrial respiratory function, as shown by the low respiratory control ratio and the low activity of F0,F1-ATPase found in hypothyroid newborns. All of these changes were reversed by the administration of thyroid hormones. ATP in vitro promotes the conversion of HFP into LFP and increases the respiratory control ratio in hypothyroid newborns, although this effect was not observed after thyroid hormone treatment. The effect of thyroid hormones on both the postnatal changes in mitochondrial populations and in F0,F1-ATPase activity was prevented by cycloheximide, but not by streptomycin. Thus, the observed effects of thyroid hormones on neonatal mitochondria must be accomplished by the induction of the synthesis of some nuclei-coded protein, possibly involved in F0,F1-ATPase assembly.

Effect of ethanol consumption on adult rat liver mitochondrial populations analyzed by flow cytometry.

In the present study, the effects of administering ethanol to adult male rats on the distribution of the low fluorescence population (LFP) and high fluorescence population (HFP), and the rhodamine-123 fluorescence intensity of these groups of mitochondria are analyzed by flow cytometry. Our results show that ethanol administration to adult male rats induces a redistribution of the HFP and LFP mitochondrial populations leading to an increase of the less functional HFP mitochondria. In addition, ethanol induced an increase in the mean intensity of green fluorescence of the HFP that is probably related to an increased number of rhodamine-123 binding sites per mitochondria resulting from mitochondria enlargement.

Postnatal changes in rhodamine-123 stained mitochondrial populations are sensitive to protein synthesis inhibitors but mimicked in vitro by ATP.

The incubation of term fetus mitochondria with ATP mimicked in vitro the increase in the respiratory control index and in the percentage of the rhodamine-123-low fluorescence population that occurred in vivo immediately after birth, suggesting that both phenomena are closely associated. The administration of streptomycin inhibited the increase in the percentage of the low fluorescence population that occurred immediately after birth, while the administration of cycloheximide even reversed these changes. These results suggest that the in vivo interconversion between mitochondrial forms depends on both cytosolic and mitochondrial protein synthesis.

Changes in mitochondrial rhodamine-123-fluorescence populations of rat hepatocytes in primary culture.

Flow cytometry analysis of mitochondria isolated from rat hepatocytes at different stages of development revealed two different rhodamine-123-stained fluorescence populations distinguishable by their main fluorescence channel. The high-fluorescence population (HFP) was minor, accounting for about 32, 33, and 23% of the total mitochondrial fraction in hepatocytes from preterm, term, and early (1 h) newborn rats, respectively. The percentage of HFP decreased during the first 20 h of hepatocytes in culture from preterm and term fetuses but not those from early newborns, in which the decrease occurred during the second day of culture. However, after 20 h in culture more than 80% of hepatocytes reached the G0/G1 phase, whatever the stage of development in which they were sown. This suggests that the observed changes in the fluorescence populations are not associated with the cell cycle; instead these changes mimic those observed during postnatal development and are therefore presumably due to mitochondrial differentiation.

Docking the mitochondrial inhibitor protein IF1 to a membrane receptor different from the F1-ATPase beta subunit.

Monoclonal antibodies reacting with the inhibitor protein (IF1) of the mitochondrial ATPase/ATP synthase complex did not modify the IF1-induced inhibition of soluble F1 ATPase activity. On the contrary, they increased the ATPase activity of inverted electron-transport particles without inducing a significant release of IF1 from these particles. This suggested that IF1 could be linked to a membrane protein when it was not inhibiting the ATPase activity. IF1 antibodies have been used to show that IF1 can bind not only to the beta subunit of F1-ATPase [Klein, G., Satre, M., Dianoux, A. C. & Vignais, P. V. (1981) Biochemistry 20, 1339-1344] but also to a protein present in the inner-mitochondrial membrane. The cross-linking of IF1 to this membrane protein gave a product of M(r) 15000-16000 that migrated differently from IF1 and from the dimer of IF1 using SDS/PAGE. When the cross-linked product was obtained by using a cleavable cross-linking reagent, the complex between IF1 and the docking protein was partly dissociated and free IF1 was recovered. Considering the molecular mass of IF1, this docking protein for IF1 has apparent M(r) 5000-6000. The complex between IF1 and this receptor protein can be detected in low amounts by antibodies against IF1 in the absence of cross-linking reagent. Since this complex remained in the pellet after treatment of the membrane with Triton X-100, it should be a membrane protein. Therefore, IF1 can bind not only to its inhibitory-binding site at the beta subunit of F1, but also to a non inhibitory site which is a membrane protein of approximate M(r) 5000-6000.

2-Ketoglutarate dehydrogenase deficiency, a rare cause of primary hyperlactataemia: report of a new case.

Two new familial cases of 2-ketoglutarate dehydrogenase (2-KGD) deficiency are reported: a girl who died at 10 years and a boy, still alive at 4 years, born to consanguineous parents. The cases developed progressively severe encephalopathy with axial hypotonia, psychotic behaviour, pyramidal symptoms and failure to thrive. Both children exhibited permanent lactic acidosis with acute episodes during emotional stress and various infections, associated with elevated lactate/pyruvate (L/P) ratio and slightly decreased ketone body ratio in plasma. In fibroblasts, the L/P ratio was greatly increased in the boy. No respiratory chain complex deficiency could be demonstrated in cultured fibroblasts or in mitochondria isolated from a muscle biopsy performed on the boy. In muscle isolated mitochondria, a progressive decrease of the rate of glutamate oxidation was observed after ADP addition; the rate of 2-ketoglutarate oxidation was low in the absence of ADP and did not increase after ADP addition. 2-KGD deficiency was demonstrated in fibroblasts from both children and in the boy's muscle and myoblasts. The 2-KGD complex is composed of three separate enzymes: E1, E2 and E3. We could demonstrate in our patient that the E1 and E3 subunits were normal, suggesting that the E2 component could be responsible for the defect.

Developmental changes in rat liver mitochondrial populations analyzed by flow cytometry.

Isolated rat liver mitochondria were split into three density fractions when applied to a Percoll gradient. This phenomenon is observable in the fetus, in the early newborn (1 h), in the suckling newborn (7 days), and in the adult, suggesting that the three density fractions coexist regardless of the state of development. The medium-density fraction sharply decreased immediately after delivery, being replaced by the high-density fraction. Flow cytometry analysis of mitochondrial density fractions stained with rhodamine 123 showed the occurrence in each density fraction and in all developmental states studied of two distinct mitochondrial populations with different fluorescence intensities. Our results suggest that the high-fluorescence population might be an immature form of mitochondria that decreases with the progression of development, coinciding with the postnatal enhancement of mitochondrial respiratory efficiency.

Identification by flow cytometry of two distinct rhodamine-123-stained mitochondrial populations in rat liver.

Isolated rat liver mitochondria were split into three fractions of increasing density when applied to a Percoll gradient. NADH-ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome-c oxidase but not F1-ATPase activities increased with density as well as respiratory rate in state 3 and the respiratory control index. Flow cytometry of mitochondrial density fractions stained with rhodamine-123 revealed the occurrence in each density fraction of two distinct mitochondrial populations with different fluorescence intensity. The high fluorescence population was minor and its proportion decreased with density. The extent of high fluorescence population staining depended on the deenergized state of the mitochondria suggesting that this population represents an immature form of the mitochondria which may develop into a fully functional organelle by the incorporation of structural and/or functional proteins.