Investigations on the turnover of adrenocortical mitochondria. XII. Studies on the mechanism of the ACTH-induced elongation of the half-life of rat zona fasciculata mitochondria.

The half-life of rat adrenocortical mitochondria was determined by high resolution autoradiography and liquid scintillation spectroscopy as previously described (Mazzocchi et al., '76). The results obtained by the two approaches were in good agreement. In the normal rats the half-life of adrenocortical mitochondria averaged 11 days. ACTH significantly increased mitochondrial half-life to about 16 days, and chloramphenicol significantly decreased this parameter in both untreated and ACTH-administered rats to about seven days. It is suggested that the ACTH-induced maintenance and slowing down of the degradation rate of adrenocortical mitochondria requires continuous mitochondrial DNA-dependent protein synthesis.     

Retention of both parental mitochondrial DNA species in mouse-Chinese hamster somatic cell hybrids

Interspecific somatic cell hybrids were isolated following the fusion of an oligomy-cin-resistant derivative of LM(TK^–) mouse cells to a chloramphenicol-resistant derivative of AK412 Chinese hamster cells. Hybrids were selected in either HAT medium, HAT plus chloramphenicol (CAP), HAT plus oligomycin (OLI), or HAT plus chloramphenicol and oligomycin. Cytogenetic analysis of the hybrids indicated that their karyotype reflected the sum of the parents. Hybrids selected in HAT medium alone or HAT plus OLI retained primarily mouse mitochondrial DNA while those selected in HAT plus CAP, or HAT plus CAP plus OLI retained both species of mitochondrial DNA. There was no evidence for mitochondrial DNA recombination, despite the continued growth of these hybrids in CAP plus OLI. Hybrids that were removed from dual antibiotic selection for over three months retained both species of mitochondrial DNA in approximately equal amounts with no detectable loss or rearrangement.     

Anti-adrenal cellular hypersensitivity in Addison''s disease. IV. In vivo and in vitro investigations on the mitochondrial fraction

Nine patients with idiopathic Addison's disease and three patients with diabetes mellitus and circulating anti-adrenal antibody but without Addison's disease were studied.

The organ-specific, anti-adrenal cellular hypersensitivity previously demonstrated by means of the leucocyte migration test in patients with idiopathic Addison's disease was found to be directed against the mitochondrial fraction of normal, human, foetal adrenocortical cells as well as of human, benign hyperplastic adrenal glands. The mitochondrial fraction of human, foetal liver cells did not cause inhibition of migration.

In lymphocyte cultures from the patients with idiopathic Addison's disease the adrenocortical, mitochondrial fraction did not induce blast transformation. The two in vitro reactions therefore probably express different kinds of reactivity.

The adrenocortical mitochondrial fraction was able to elicit intracutaneous reactions of delayed type in patients with a positive leucocyte migration test to the same antigen. This observation confirms, that inhibition of leucocyte migration in vitro indicates a state of anti-adrenal cellular hypersensitivity in patients with idiopathic Addison's disease.

     

Mitochondrial protein synthesis in interspecific somatic cell hybrids

The fusion of an oligomycin (OLI) -resistant mutant of mouse LM(TK^–) cells to a chloramphenicol (CAP) -resistant mutant of AK412 Chinese hamster cells resulted in a series of interspecific somatic cell hybrids. Hybrids selected in HAT medium retained only mouse mitochondrial genomes while hybrids selected in HA T plus CAP and OLI retained both hamster and mouse mitochondrial genomes in approximately equal amounts. Nuclear-coded mitochondrial proteins from both parental species were incorporated into mitochondria in all of the hybrids. However, the mitochondrially coded proteins of three individually isolated hybrid cell lines were predominantly mouse-specific, with only trace amounts of hamster protein detected.     

Mitochondrial DNA analysis of mouse-rat hybrid cells: Effect of chloramphenicol selection on the relative amounts of parental mitochondrial DNAs

Several mouse-rat somatic hybrid cell lines were isolated by fusing chloramphenicol-resistant (CAP^R) and CAP-sensitive (CAP^S) parent cells, and propagation of the parent mitochondrial DNA (mtDNA) species in the hybrid cells was studied. The restriction endonucleases EcoRI, HpaII, and HaeIII were used for identification of mtDNA species. Both mouse and rat mtDNAs were propagated in all the hybrid cells examined and maintained during long-term cultivation and repeated cell division. Moreover, in CAP^R mouse-rat hybrid cells, selection and successive cultivation in the presence of CAP did not increase the relative amount of mtDNA species of CAP^R parent cell origin, and when CAP was removed from the culture medium, mtDNA species of CAP^R parent cell origin did not decrease appreciably. The amount of mouse mtDNAs was consistently 1–4 times that of rat mtDNAs in the mouse-rat hybrid cells regardless of the species of parent cells from which the CAP resistance was derived. Thus mouse-rat hybrid cells have a stable mtDNA population in which the amount of mouse mtDNAs is larger than that of rat mtDNAs without any influence of CAP selection.     

Production of transmitochondrial mouse cell lines by cybrid rescue of rhodamine-6G pre-treated L-cells

Treatment of mouse LMTK⁻ cells with the toxic mitochondrial dye rhodamine 6G (R-6G) at 2.5 μg/ml for 7 days prevented cell growth while maintaining viability, with less than 10⁻⁶ cells recovering to form colonies. Pre-treatment of LMTK⁻ cells with R-6G was followed by fusion with enucleated mouse 501–1 cells harboring a homoplasmic point mutation in the mitochondrial DNA (mtDNA) 16S rRNA gene conferring chloramphenicol resistance (CAP^R). Cybrids and any surviving unfused LMTK⁻ cells were selected in BrdU with or without CAP and their mtDNAs screened for the presence of the CAP^R marker. Approximately 1 colony per 2×10⁵ LMTK⁻ cells appeared in the fusion plates selected both with and without CAP. Most clones investigated were confirmed to be cybrids by showing the presence of the generally homoplasmic CAP^R mutation, whether or not CAP selection was used. Hence, R-6G pre-treatment permits construction of transmitochondrial cybrid cell lines carrying a variety of mtDNAs, without the need for ρ^o cell lines.     

Ultrastructural studies of cell-virus interaction in reptilian cell lines. IV. Effects of chloramphenicol and ethidium bromide on VSW cell mitochondria and associated virions.

Structural effects of chloramphenicol (CAP) and ethidium bromide (EB) on VSW cell mitochondria and intramitochondrial virions (IMV) have been studied on a comparative basis by thin-section electron microscopy. CAP-treated cells show a wide variety of mitochondrial alterations, frequently involving swelling of the organelle and loss of cristae orientation. IMV are generally severely disrupted, particularly in peripheral regions. In such configurations, strand-like material radiates in a spokelike fashion from the shell zone to adjacent cristae-matrix area. EB-treated cells also display considerable mitochondrial distortion evidenced primarily by the formation of small, localized multimembrane regions. IMV exposed to EB, however, are less structurally damaged than CAP-treated ones. The relative incidence of IMV production is enhanced approximately fourfold in EB-treated cells compared to CAP-treated ones, suggesting that virion synthesis may be under nuclear, rather than mitochondrial, control.     

Acute effects of ACTH on dissociated adrenocortical cells: quantitative changes in mitochondria and lipid droplets.

To study the role of certain organelles in steroidogenesis, dissociated rat adrenocortical cells were incubated for two hours with ACTH at a concentration that induces a high level of steroid production. Sections of ACTH treated and untreated cells were photographed in the electron microscope, and morphometric analysis was undertaken to assess possible ACTH-induced changes in total cell volume, volume density and numerical denisty of lipid droplets and mitochondria. There was no change in total cell volume. Lipid droplet volume density and numerical density decreased. Mitochondrial volume density did not change, but numerical density increased. The decrease in lipid droplet volume density indicates a rapid depletion of cholesterol for steroid production. This depletion is almost entirely due to the disappearance of lipid droplets, rather than to an overall diminution in their size, as shown by the decrease in lipid droplet numerical density. The mitochondrial data suggest that the adrenocortical cell has an adedquate mitochondrial apparatus to respond to acute ACTH stimulation with increased steroid output without an increase inmitochondrial volume.     

Effects of hypoxia and fasting on the cytochrome concentration in intestinal epithelial villous cell mitochondria: Role of changes in the life-span of the cells

The effects of hypoxia in vivo (40.8 kPa barometric pressure up to 120h) and fasting on the characteristics of intestinal epithelial villous cell mitochondria and the turnover of epithelial villous cells and mitochondria were studied in rats. Using cells and mitochondria isolated in the isotonic mannitol medium, it was found that 24-h hypoxia or fasting did not alter the mitochondrial cytochrome content, but 48-h hypoxia or fasting led to increases of 70% and 37% in the cytochrome aa₃ concentration in the hypoxic and fasting animals respectively. The turnover of intestinal epithelial cells was studied by observing the labelling kinetics of the cells with ³H-thymidine and the turnover of the cell and mitochondrial proteins with (guanido-¹⁴C)-arginine or ³H-leucine. The decay in thymidine radioactivity obeyed exponential kinetics from which half-lives of 1.15, 1.31 and 1.53 days were calculated in the control, fasting and hypoxic animals respectively. The half-lives for total cellular protein were 1.31, 1.54 and 1.54 days respectively when calculated from the (guanido-¹⁴C)-arginine experiments, or 0.69, 0.75 and 0.99 days when calculated from the leucine experiments. The labelling experiments with (guanido-¹⁴C)-arginine indicated that the turnover of mitochondrial proteins in intestinal epithelial cells is the same as that of the cells themselves. Since the turnover of mitochondrial proteins in other tissues is known to be a relatively slow process, the increase in the cytochrome concentration in the intestinal cells of the hypoxic rats must be due to the longer life of the cells, which allows for the synthesis of larger amounts of the mitochondrial components.     

Investigations on the turnover of adrenocortical mitochondria. XI. Effects of dexamethasone on the half-life of mitochondria from the rat zona fasciculata.

The effects of dexamethasone on the half-life of rat adrenocortical mitochondria were investigated by high resolution autoradiography and liquid scintillation spectroscopy. According to the method employed, the half-life averaged 11.27 and 10.46 days. Dexamethasone was found to decrease significantly this parameter (to about 5 days). Since dexamethasone-treated rats can be regarded as pharmacologically hypophysectomized animals, the data indicates that ACTH is involved in the maintenance of the half-life of adrenocortical mitochondria.     

Calcium uptake in skeletal muscle mitochondria

Summary In order to ascertain the effects of long-term exercise training and long-term exhaustive exercise on mitochondrial ⁴⁵Ca²⁺ uptake and related variables in rat skeletal muscle, female rats were randomly divided into three groups: sedentary-rested (SR), trained-rested (TR), and trained-exhausted (TE). The trained groups were exercised five times per week on a treadmill for 22 weeks. At the conclusion of the training period, the TE group was exercised to exhaustion following their daily 1 h run. The ⁴⁵Ca²⁺ uptake and endogenous mitochondrial Ca²⁺ content of skeletal muscle followed stepwise increases of approximately 25% and 50%, respectively, across the groups, suggesting that long-term exercise induces the mitochondria to play an important role as a Ca²⁺ ion buffer. A 75–83% reduction in ⁴⁵Ca²⁺ binding in the TE group suggests a selective loss and partial saturation of membrane phospholipids with exhaustive exercise. The TE group had a two-fold greater content of mitochondrial Mg²⁺ than did the rested groups. It is speculated that the mitochondria accumulate Mg²⁺ during acute exercise to maintain the functional integrity of the membrane, thus offsetting the deleterious effects of excessive Ca²⁺ uptake.     

Sequestration of 45Ca2+ by mitochondria from rabbit heart, liver, and kidney after doxorubicin or digoxin/doxorubicin treatment.

Doxorubicin, an antibiotic of the anthracycline group, has proven effective in treating a variety of malignant disorders. However, its use has been limited due to the cardiotoxic side effects which include myocardial necrosis that is characterized by mitochondrial calcification. The present studies were conducted to determine if treatment of rabbits with doxorubicin (an anthracycline) would affect the ability of mitochondria isolated from heart, liver, and kidney to retain ⁴⁵Ca²⁺. Increases in mitochondrial retention of ⁴⁵Ca²⁺ by all of the tissues studied were observed, although only that from the heart showed a significant increase. The changes in ⁴⁵Ca²⁺ retention and morphology (i.e., increased mitochondrial swelling and intra-mitochondrial calcium phosphate crystals) of heart mitochondria from doxorubicin-treated rabbits suggest that this anthracycline directly or indirectly affects mitochondrial flux of calcium. That liver and kidney (as compared to heart) mitochondria are relatively insensitive to the effects of doxorubicin suggests a chemical difference in the mitochondria isolated from these tissues. Digoxin/doxorubicin treatment of rabbits, however, leads to a decrease in mitochondrial retention of ⁴⁵CA²⁺, except for hear tissue, which again was significantly increased over the control.² The effects of this treatment on the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ activated ATPase of the heart, and on the accumulation of doxorubicin by the heart, were not significantly different from the control, suggesting that digoxin and doxorubicin do not compete for the same binding site.     

Human myocardial mitochondria: Size differences in parts of the cell

Mitochondrial size varies in different areas of the heart and in certain physiologic and pathologic processes. This study evaluated the mitochondrial size in three areas of the heart cell: (1) perinuclear, (2) intermyofibrillar, and (3) subsarcolemmal in health and disease. Group I was 14 patients with normal cardiovascular examinations (age = 47 ± 4, $\text{X}\text{±}\text{SE}$); Group II was 16 patients with nonischemic cardiomyopathy (age = 53 ± 5). All had an endomyocardial biopsy of the right side of the interventricular septum. Morphometric analysis of the electron micrographs (×31,200) from the three areas quantitated mitochondrial area and cristae to matrix ratio. The perinuclear mitochondria were significantly larger than the intermyofibrillar or subsarcolemmal mitochondrial in Group I (0.25± 0.1 μ² > 0.23 ± .01 μ²and 0.22 ± .01 μ², both P < 0.01, respectively). The same size differential was noted in Group II with the perinuclear mitochondria larger than the intermyofibrillar or subsarcolemmal mitochondria (0.26 ± .01 μ² > 0.25 ± .01 μ², and 0.23 ± .01 μ², both P < 0.05). In addition, the intermyofibrillar mitochondria were significantly larger than the subsarcolemmal mitochondria in Group II (P < 0.05). Group II mitochondria were not significantly larger than Group I. The cristae to matrix ratio was the same in all three areas. The reason for the mitochondrial size differential is not clear but may be related to increased metabolic demand.     

Acute effects of ACTH on dissociated adrenocortical cells: Quantitative changes in mitochondria and lipid droplets

To study the role of certain organelles in steroidogenesis, dissociated rat adrenocortical cells were incubated for two hours with ACTH at a concentration that induces a high level of steroid production. Sections of ACTH treated and untreated cells were photographed in the electron microscope, and morphometric analysis was undertaken to assess possible ACTH-induced changes in total cell volume, volume density and numerical density of lipid droplets and mitochondria. There was no change in total cell volume. Lipid droplet volume density and numerical density decreased. Mitochondrial volume density did not change, but numerical density increased. The decrease in lipid droplet volume density indicates a rapid depletion of cholesterol for steroid production. This depletion is almost entirely due to the disappearance of lipid droplets, rather than to an overall diminution in their size, as shown by the decrease in lipid droplet numerical density. The mitochondrial data suggest that the adrenocortical cell has an adequate mitochondrial apparatus to respond to acute ACTH stimulation with increased steroid output without an increase in mitochondrial volume.     

Investigations on the turnover of adrenocortical mitochondria. VI. An autoradiographic study of the effect of ACTH on the radioactivity decay in the mitochondrial compartment from the zona fasciculata of 3H-thymidine injected rats.

The radioactivity decay of the mitochondrial compartment from the zona fasciculata of the adrenal cortex of 3H-thymidine-injected rats was followed by high resolution autoradiography. The number of days in which the radioactivity of the mitochondrial compartment was reduced to a half was calculated from the semilogarithmic plots of radioactivity versus time. Since DNA is a very stable molecule, it was assumed that this parameter can be an estimate of the half-life of adrenocortical mitochondria. The half-life of mitochondria from the zona fasciculata of the normal rat averaged 11.17 days, and ACTH was found to increase significantly this figure to about 15 days. It is hypothesized that the ACTH-elicited stimulation of the growth of rat adrenal zona fasciculata mitochondria involves not only hypertrophy and proliferation of the organelles (Nussdorfer et al., '74b), but also the slowing down of the degeneration rate of mitochondria.     

Key role of mitochondria in apoptosis of lymphocytes

Changes in the mitochondrial potential, expression of phosphatidylserine, parameters of direct and lateral light scattering, and DNA fragmentation during spontaneous and induced apoptosis in peripheral blood lymphocytes were studied by flow cytofluorometry. Dexamethasone and Ca²⁺ ionophore A23187 served as inductors of apoptosis. A decrease in the mitochondrial potential is an early sign of spontaneous and induced apoptosis. Phosphatidylserine expression on the outer plasma membrane occurred later and inversely depended on the mitochondrial potential. Our results indicate that the involvement of mitochondria in spontaneous and induced apoptosis accompanied by a decrease in the mitochondrial potential is an early and key event of programmed lymphocyte death. The decrease in the mitochondrial potential of lymphocytes induced degradation of their nuclei (DNA fragmentation) and promoted elimination of apoptotic cells (phosphatidylserine expression).     

Ratiometric high-resolution imaging of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria

Using the mitochondrial potential (ΔΨ_m) marker JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) and high-resolution imaging, we functionally analyzed mitochondria in cultured rat hippocampal astrocytes. Ratiometric detection of JC-1 fluorescence identified mitochondria with high and low ΔΨ_m. Mitochondrial density was highest in the perinuclear region, whereas ΔΨ_m tended to be higher in peripheral mitochondria. Spontaneous ΔΨ_m fluctuations, representing episodes of increased energization, appeared in individual mitochondria or synchronized in mitochondrial clusters. They continued upon withdrawal of extracellular Ca²⁺, but were antagonized by dantrolene or 2-aminoethoxydiphenylborate (2-APB). Fluo-3 imaging revealed local cytosolic Ca²⁺ transients with similar kinetics that also were depressed by dantrolene and 2-APB. Massive cellular Ca²⁺ load or metabolic impairment abolished ΔΨ_m fluctuations, occasionally evoking heterogeneous mitochondrial depolarizations. The detected diversity and ΔΨ_m heterogeneity of mitochondria confirms that even in less structurally polarized cells, such as astrocytes, specialized mitochondrial subpopulations coexist. We conclude that ΔΨ_m fluctuations are an indication of mitochondrial viability and are triggered by local Ca²⁺ release from the endoplasmic reticulum. This spatially confined organelle crosstalk contributes to the functional heterogeneity of mitochondria and may serve to adapt the metabolism of glial cells to the activity and metabolic demand of complex neuronal networks. The established ratiometric JC-1 imaging—especially combined with two-photon microscopy—enables quantitative functional analyses of individual mitochondria as well as the comparison of mitochondrial heterogeneity in different preparations and/or treatment conditions.     

Effect of senescence on Ca2+—ion transport by heart mitochondria

Both the rate of uptake of Ca²⁺ into isolated rat heart mitochondria and the rate of release of Ca²⁺ by a separate, Na⁺-dependent, pathway were shown to be diminished in senescence (24-month relative to 6-month animal). These processes were studied at lower concentrations of Ca²⁺ and loads of Ca²⁺ per mg of mitochondrial protein than those generally employed, and it is argued that these are more appropriate physiologically. In addition, Ca²⁺ release was characterized in terms of the sum of the added Ca²⁺ and the endogenous Ca²⁺ of the mitochondrial preparation; the endogenous Ca²⁺ content was found to be unchanged with age. The decrements in rates of transport are not caused by altered rates of substrate oxidation and are inferred to reflect decreased carrier-protein content or activity. The buffering of extramitochondrial free Ca²⁺ concentration by heart mitochondria was studied and found to be less than complete at mitochondrial Ca²⁺ loads inferred to be physiological. No change was shown in senescence, in keeping with the essentially equal age-linked decrements in the activity of mitochondrial Ca²⁺ uptake and release.