Chieko Kurono

Free radical-induced megamitochondria formation and apoptosis.

Pathophysiological meaning and the mechanism of the formation of megamitochondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for free radical-mediated apoptosis. MG were detected in primary cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidney cell line Cos-1 treated for 22 h with various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydrazone, indomethacin, H2O2, and erythromycin using a fluorescent dye Mito Tracker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive oxygen species (ROS), monitored by staining of cells with a fluorescent dye carboxy-H2-DCFDA, were detected before MG were formed. Prolongation of the incubation time with various chemicals, specified above, for 36 h or longer has induced distinct structural changes of the cell, which characterize apoptosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G1 phase, and apoptotic sub-G1 populations then became gradually increased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mitochondria in control cells. Rates of the generation of H2O2 and O2- from MG isolated from the liver of rats treated with chloramphenicol or hydrazine were found to be lower than those of mitochondria of the liver of control animals. We suggest, based on the present results together with our previous findings, that the formation of MG may be an adaptive process at a subcellular level to unfavorable environments: when cells are exposed to excess amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may result in decreases in the rate of ROS production as shown in the present study. This will at the same time result in decreases in ATP production from MG. If cells are exposed to a large amount of free radicals beyond a certain period of time, lowered intracellular levels of ATP may result in apoptotic changes of the cell.

Folliculo‐stellate cells and intercellular communication within the rat anterior pituitary gland

Folliculo‐stellate (FS) cell are agranular and arranged around a follicle. They contain the S‐100 protein and β‐adrenergic receptors. It has been suggested that they can act as stem cells, since they show mitotic figures, and could transform into granular or chromophilic cells according to the concept of a “cell renewal system.” Cell‐to‐cell interactions among pituitary cells have been described, and recent progress with freeze‐fracture electron microscopy has provided novel observations of the cell surface and gap junctions within the rat or teleost fish pituitary gland, or in cultured rat pituitary cells. In adult rats, the anterior pituitary was composed of lobules incompletely separated by a basement membrane. Follicles consisted exclusively of FS cells. Gap junctions were observed only between adjacent FS cells, in rare cases on the tips of their cytoplasmic processes. Thus, the FS cells, connected by gap junctions, made up a dense cellular network throughout the pituitary. Gap and tight junctions were absent on granular cells. Elongated follicles with columnar FS cells were observed in 10‐day‐old rats and were separated into smaller units. The number of gap junctions rapidly increased with age until 40–45 days of age. Few S‐100 protein positive cells were observed on day 10, along the marginal cell layer and near the so‐called postero‐lateral wing. The frequency of positive cells increased with age and by day 40; numerous cells were observed throughout the anterior lobe.

Induction of megamitochondria by some chemicals inducing oxidative stress in primary cultured rat hepatocytes

Effects of hydrazine, hydrogen peroxide and bromobenzene, inducers of free radicals, and those of erythromycin and cycloheximide, inhibitors of protein synthesis on structural changes of mitochondria in primary monolayer culture of rat hepatocytes were examined using laser confocal microscope and electron microscope. After 22 h of incubation of hepatocytes with 0.2 mM hydrogen peroxide or 10 microg ml-1 of erythromycin, mitochondria became extremely enlarged. Mitochondria of hepatocytes isolated from control rats became slightly to moderately enlarged in the presence of 2 mM hydrazine, while those of hepatocytes isolated from phenobarbital-pretreated animals became extremely enlarged in the presence of 2 mM hydrazine. Cycloheximide (0.5-10.0 microg ml-1) and bromobenzene (0.1-1.0 mM) failed to induce structural changes of mitochondria. The level of cytochrome P-450 in freshly prepared hepatocytes from phenobarbital-treated rats was 2.5 times higher than that from the control rats, and remained about three times higher than the latter after 22 h of incubation with 2 mM hydrazine. The level of malondialdehyde was invariably elevated when megamitochondria were induced. These results may suggest that oxidative stress is intimately related to the mechanism of the formation of megamitochondria and that the inhibition of cytoplasmic protein synthesis seems not to contribute the phenomenon. However, the detailed mechanism by which free radicals may induce megamitochondria remains to be elucidated.

Immunohistochemical study of the post-natal development of the folliculo-stellate cells in the rat anterior pituitary gland

We investigated the post-natal development of cell-to-cell communication within the rat anterior pituitary gland cells using immunohistochemistry of the S-100 protein. Tissues of animals from 10 to 60 days of age were analyzed. At 10 days of age, S-100 protein-containing cells were rarely observed. With age, the population of S-100 immunostained cells increased until day 40 when they were found to be quite numerous. No further changes were noted from day 40 through day 60. From our previous studies, we conclude that the cells which reacted with the S-100 antiserum were folliculo-stellate cells and their developmental pattern parallels that of the hypophyseal-gonadal axis.

Cycloheximide and 4-OH-TEMPO suppress chloramphenicol-induced apoptosis in RL-34 cells via the suppression of the formation of megamitochondria

Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6, 6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2, 6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.

MECHANISM OF THE FORMATION OF MEGAMITOCHONDRIA INDUCED BY COPPER-CHELATING AGENTS

Processes of the formation of cuprizone‐induced megamitochondria in mouse liver have been studied in detail by electron microscopy. The earliest change observed was the presence of large intramitochondrial granules. The next stage was the formation of myelin flgures by which mitochondria were apparently connected. The third stage was characterized by megamitochondria connected with each other by their outer membranes. Continuity of mitochondria were further examined by serial sections, and megamitochondria were proved to be connected to each other far more frequently than expected on one plane of section. A model for the mechanism of megamitochondrial formation is proposed based on electron microscopic evidences, involving the fusion of mitochondrial membranes. Possibillty is also discussed that cuprizone‐induced megamitochondria may fuse to one single branching mitochondrion.

Functional aspects of megamitochondria isolated from hydrazine- and ethanol-treated rat livers.

It is essential to analyze functions of megamitochondria (MG) to elucidate the mechanism of the formation of MG induced under various pathological conditions. The MG fraction obtained by a routine isolation procedure for normal mitochondria always consists of a mixed population of mitochondria enlarged to various degrees and also normal‐sized ones. The purpose of the present study is to answer the question of whether or not data obtained from the MG fraction consisting of such a heterogeneous population of mitochondria with respect to their sizes really reflect functions of MG. In the present study mitochondria were obtained from the livers of rats treated with a 1% hydrazine diet for 8 days and those given 32% ethanol in drinking water for up to 2 months using various isolation procedures. Results obtained are summarized as follows: (i) mitochondria enlarged to various degrees and normal‐sized ones are sometimes connected with each other by a narrow stalk in the hepatocyte of hydrazine‐treated animals, and such connections are maintained to some extent when mitochondria are isolated; and (ii) mitochondria obtained from experimental animals by a routine isolation procedure for mitochondria ((700–7000)gR2′′′) and those obtained by alternative isolation procedure yielding the heavy ((500–2000)gR2′′′) and light ((2000–7000)gR2′′′) fractions show some functional similarities: decreases in the content of cytochrome a+a3; decreases in oxygen consumptions and phosphorylating abilities; decreases in monoamine oxidase and cytochrome c oxidase activities; lowered membrane potential of mitochondria; decreases in the rate of the generation of reactive oxygen species. These results may suggest that mitochondria enlarged to various degrees and normal‐sized ones are functionally similar to each other and that the MG fraction obtained by a routine isolation procedure for normal mitochondria can be applied to the study of the function of MG.

Intercellular communication within the rat anterior pituitary gland: VI. Development of gap junctions between folliculo-stellate cells under the influence of ovariectomy and sex steroids in the female rat

Farquhar (1957) initially described the folliculo‐stellate cells in the rat anterior pituitary gland and found them to be located in groups around follicles throughout the anterior lobe. Soji and his co‐workers have published a series of reports concerning cell‐to‐cell communication in the male rat hypophysis involving folliculo‐stellate cells as mediated through a gap junctional network and recorded a reduction in junctional number following castration that was reversed by the administration of testosterone (Soji and Herbert, 1990, Anat. Rec., 226:342–346; Soji et al., 1990, Anat. Rec., 226:337–341).

Septate-like junctions in the normal male rat pituitary gland

Septate-like junctions were observed in the rat anterior pituitary gland of the adult male solely between adjacent folliculo-stellate cells. Considering their location, it is presumed that their function is cellular adhesion and mechanical support for the hypophyseal follicles.

Steroidogenesis in the zona glomerulosa of the adrenal cortex II. Distribution of cytochrome P-450 in the zona glomerulosa of the bovine adrenal cortex

Microsomes were obtained from the zona glomerulosa of the bovine adrenal cortex. Contamination of microsomes with other cellular organelles was examined using various marker enzymes and the electron microscope. Distribution of cytochrome P-450 in the zona glomerulosa was studied using various fractions including microsomes, described above, and mitochondria. The amount of cytochrome P-450 in mitochondria and in microsomes was determined to be 0.73 and 0.32 nmol/mg protein, respectively. The CO difference spectrum was affected not only by the concentration of added deoxycholate but also by the incubation time after addition. Approximately 40–50% of cytochrome P-450 in the samples was converted to cytochrome P-420 within 20–30 sec of incubation with deoxycholate.The content of RNA, phospholipids, and cytochromeb5 in microsomes obtained from the zona glomerulosa is also evaluated in comparison to that in microsomes obtained from the zona fasciculoreticularis.