Status of mitochondria in living human fibroblasts during growth and senescence in vitro: use of the laser dye rhodamine 123

Rhodamine 123, a fluorescent laser dye that is selectively taken up into mitochondria of living cells, was used to examine mitochondrial morphology in early-passage (young), late-passage (old), and progeric human fibroblasts. Mitochondria were readily visualized in all cell types during growth (mid-log) and confluent stages. In all cell strains at confluence, mitochondria became shorter, more randomly aligned, and developed a higher proportion of bead-like forms. Treatment of cells for six days with Tevenel, a chloramphenicol analog that inhibits mitochondrial protein synthesis, brought about a marked depletion of mitochondria and a diffuse background fluorescence. Cyanide produced a rapid release of preloaded mitochondrial fluorescence followed by detachment and killing of cells. Colcemid caused a random coiling and fragmentation of mitochondria particularly in the confluent stage. No gross differences were discernible in mitochondria of the three cell strains in mid-log and confluent states or after these treatments. Butanol-extractable fluorescence after loading with rhodamine 123 was lower in all cell strains in confluent compared to mid-log stages. At confluence all three cell strains had similar rhodamine contents at zero-time and after washout up to 24 h. At the mid-log stage, young cells contained more rhodamine initially and lost it more rapidly than old or progeria cells, in that order. The data indicate no gross derangement in the morphology or number of mitochondria in old and progeria fibroblasts but there is a reduction of protonmotive force evident in these cells at the mid-log stage that may be growth limiting.     

Association of tyrosine protein kinase activity with mitochondria in human fibroblasts

A tyrosine protein kinase activity has been detected in the mitochondrial fraction purified from human fibroblasts. By enzymatic and sedimentation analysis this activity appeared to be localized in the mitochondrial outer membrane. Mitochondrial tyrosine phosphorylation was strictly dependent on the presence of Mn²⁺ ions. An inverse relationship between cell proliferation and mitochondrial protein phosphorylation on tyrosine residues has been found: a marked increase in the mitochondrial tyrosine kinase activity occurred when a significant reduction in the growth rate followed serum step-down. In mitochondria purified from resting cells, a protein band with apparent molecular weight of 50 kd appeared to be phosphorylated on tyrosine.     

Involvement of the mitochondrial compartment in human NCL fibroblasts

Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.     

Distinct Alterations in Mitochondrial Mass and Function Characterize Different Models of Apoptosis

Recent studies have shown that reduction in mitochondrial membrane potential (ΔΨ_m) and generation of reactive oxygen species are early events in apoptosis. In this study, we present two different models of apoptotic cell death, Chinese hamster ovary (CHO) cells treated with aphidicolin and dexamethasone-treated 2B4 T-cell hybridoma cells, which display opposing mitochondrial changes. CHO cells arrested at G1/S with aphidicolin have a progressive increase in mitochondria mass and number, assessed by flow cytometry and fluorescent microscopy with mitochondria-specific probes. The increase in mitochondrial mass was not accompanied by a gain in net cellular mitochondrial membrane potential, consistent with an accumulation of relatively depolarized mitochondria. Fluorescent microscopy demonstrated an increased content of low ΔΨ_mmitochondria in aphidicolin-treated CHO cells, but high ΔΨ_mmitochondria were also present and remained stable in number. Mitochondrial mass correlated with decreased clonogenicity of aphidicolin-treated CHO cells. Cycloheximide prevented both the proliferation of mitochondria and subsequent cell death. In contrast, dexamethasone treatment of 2B4 T-cell hybridoma cells caused a decrease in ΔΨ_mwithout mitochondrial proliferation. Cycloheximide and Bcl-2 overexpression inhibited the loss of ΔΨ_m, as well as apoptosis. In both models, cell death was associated with a decrease in mitochondrial potential relative to mitochondrial mass, suggesting that an accumulation of damaged or dysfunctional mitochondria had occurred.     

Mitochondrial growth and division during the cell cycle in HeLa cells

The growth and division of mitochondria during the cell cycle was investigated by a morphometric analysis of electron micrographs of synchronized HeLa cells. The ratio of total outer membrane contour length to cytoplasmic area did not vary significantly during the cell cycle, implying a continuous growth of the mitochondrial outer membrane. The mean fraction of cytoplasmic area occupied by mitochondrial profiles was likewise found to remain constant, indicating that the increase in total mitochondrial volume per cell occurs continuously during interphase, in such a way that the mitochondrial complement occupies a constant fraction( approximately 10-11(percent)) of the volume of the cytoplasm. The mean area, outer membrane contour length, and axis ratio of the mitochondrial profiles also did not vary appreciably during the cell cycle; furthermore, the close similarity of the frequency distributions of these parameters for the six experimental time-points suggested a stable mitochondrial shape distribution. The constancy of both the mean mitochondrial profile area and the number of mitochondrial profiles per unit of cytoplasmic area was interpreted to indicate the continuous division of mitochondria at the level of the cell population. Furthermore, no evidence was found for the occurrence of synchronous mitochondrial growth and division within individual cells. Thus, it appears that, in HeLa cells, there is no fixed temporal relationship between the growth and division of mitochondria and the events of the cell cycle. A number of statistical methods were developed for the purpose of making numerical estimates of certain three-dimensional cellular and mitochondrial parameters. Mean cellular and cytoplasmic volumes were calculated for the six time-points; both exhibited a nonlinear, approx. twofold increase. A comparison of the axis ratio distributions of the mitochondrial profiles with theoretical distributions expected from random sectioning of bodies of various three-dimensional shapes allowed the derivation of an "average" mitochondrial shape. This, in turn, permitted calculations to be made which expressed the two-dimensional results in three-dimensional terms. Thus, the estimated values for the number of mitochondria per unit of cytoplasmic volume and for the mean mitochondrial volume were found to remain constant during the cell cycle, while the estimated number of mitochondria per cell increase approx. twofold in an essentially continuous manner.     

Nicotinamide enhances mitochondria quality through autophagy activation in human cells

Nicotinamide (NAM) treatment causes a decrease in mitochondrial respiration and reactive oxygen species production in primary human fibroblasts and extends their replicative lifespan. In the current study, it is reported that NAM treatment induces a decrease in mitochondrial mass and an increase in membrane potential (ΔΨ m ) by accelerating autophagic degradation of mitochondria. In the NAM-treated cells, the level of LC3-II as well as the number of LC3 puncta and lysosomes co-localizing with mitochondria substantially increased. Furthermore, in the NAM-treated cells, the levels of Fis1, Drp1, and Mfn1, proteins that regulate mitochondrial fission and fusion, increased and mitochondria experienced dramatic changes in structure from filaments to dots or rings. This structural change is required for the decrease of mitochondrial mass indicating that NAM accelerates mitochondrial autophagy, at least in part, by inducing mitochondrial fragmentation. The decrease in mitochondria mass was attenuated by treatment with cyclosporine A, which prevents the loss of mitochondrial membrane potential by blocking the mitochondrial permeability transition, suggesting autophagic degradation selective for mitochondria with low ΔΨ m . All these changes were accompanied by and dependent on an increase in the levels of GAPDH, and are blocked by inhibition of the cellular conversion of NAM to NAD⁺. Taken together with our previous findings, these results suggest that up-regulation of GAPDH activity may prolong healthy lifespan of human cells through autophagy-mediated mitochondria quality maintenance.     

Impact of proliferative activity and tumorigenic conversion on mitochondrial function of fibroblasts in 2D and 3D culture

The purpose of the present study was to examine mitochondrial function in differently transformed cells relative to their tumorigenic state and proliferative activity in vitro. An established two-step carcinogenesis model consisting of immortal and tumorigenic rat embryo fibroblasts that can be cultured as monolayers and multicellular spheroids was investigated. Flow cytometric measurements were carried out using the two mitochondrial-specific fluorochromes rhodamine 123 (Rh123) and 10-N-nonyl acridine orange (NAO), in combination with the DNA dye Hoechst 33342 for simultaneous cell cycle analysis. Since the accumulation of Rh123 depends on mitochondrial membrane potential, Rh123 fluorescence intensity gives an estimate of mitochondrial activity per cell, as determined by both overall mitochondrial function and mass. In contrast, NAO uptake reflects mitochondrial mass only, as it binds to cardiolipin in the inner mitochondrial membrane independently of membrane potential. Aliquots of cell suspensions derived from exponential monolayer, confluent monolayer, and a range of sizes of multicellular spheroids were stained with either Rh123 or NAO and Hoechst 33342, then mitochondrial mass and activity per unit cell volume and cellular DNA content were measured by flow cytometry. Differences in the average mitochondrial activity per cell in different cell lines and culture conditions were primarily due to alterations in cell volume. Importantly, tumorigenic conversion by ras-transfection did not consistently change mitochondrial activity per unit cell volume. The mitochondrial mass per unit cell volume increased for all cells when cellular quiescence was induced, either in monolayers or spheroids. However, mitochondrial function (activity/mass) decreased when cells became quiescent, resulting in a positive correlation between mitochondrial function and S-phase fraction, independent of transformation status or culture condition. We conclude that mitochondrial function reflects proliferative activity rather than tumorigenic conversion.     

Changes in mitochondria density during oogenesis of loach]

The growth of loach oocytes is accompanied by an increase in the density of mitochondria. Simultaneously with the increase in density an acceleration of 14C-valine incorporation into mitochondrial proteins takes place. It is assumed that the increase in mitochondria density during oogenesis is due to an increase in the amount of membrane material per unit of mitochondria weight.     

Increased mitochondrial mass in cells with functionally compromised mitochondria after exposure to both direct gamma radiation and bystander factors

The bystander effect describes radiation-like damage in unirradiated cells either in the vicinity of irradiated cells or exposed to medium from irradiated cells. This study aimed to further characterize the poorly understood mitochondrial response to both direct irradiation and bystander factor(s) in human keratinocytes (HPV-G) and Chinese hamster ovarian cells (CHO-K1). Oxygen consumption rates were determined during periods of state 4, state 3 and uncoupled respiration. Mitochondrial mass was determined using MitoTracker FM. CHO-K1 cells showed significantly reduced oxygen consumption rates 4 h after exposure to 5 Gy direct radiation and irradiated cell conditioned medium (ICCM) and an apparent recovery 12-24 h later. The apparent recovery was likely due to the substantial increase in mitochondrial mass observed in these cells as soon as 4 h after exposure. HPV-G cells, on the other hand, showed a sustained increase in oxygen consumption rates after ICCM exposure and a transient increase 4 h after exposure to 5 Gy direct radiation. A significant increase in mitochondrial mass per HPV-G cell was observed after exposure to both direct radiation and ICCM. These findings are indicative of a stress response to mitochondrial dysfunction that increases the number of mitochondria per cell.     

Severe mitochondrial damage associated with low-dose radiation sensitivity in ATM- and NBS1-deficient cells

Low-dose radiation risks remain unclear owing to a lack of sufficient studies. We previously reported that low-dose, long-term fractionated radiation (FR) with 0.01 or 0.05 Gy/fraction for 31 d inflicts oxidative stress in human fibroblasts due to excess levels of mitochondrial reactive oxygen species (ROS). To identify the small effects of low-dose radiation, we investigated how mitochondria respond to low-dose radiation in radiosensitive human ataxia telangiectasia mutated (ATM)- and Nijmegen breakage syndrome (NBS)1-deficient cell lines compared with corresponding cell lines expressing ATM and NBS1. Consistent with previous results in normal fibroblasts, low-dose, long-term FR increased mitochondrial mass and caused accumulation of mitochondrial ROS in ATM- and NBS1-complemented cell lines. Excess mitochondrial ROS resulted in mitochondrial damage that was in turn recognized by Parkin, leading to mitochondrial autophagy (mitophagy). In contrast, ATM- and NBS1-deficient cells showed defective induction of mitophagy after low-dose, long-term FR, leading to accumulation of abnormal mitochondria; this was determined by mitochondrial fragmentation and decreased mitochondrial membrane potential. Consequently, apoptosis was induced in ATM- and NBS1-deficient cells after low-dose, long-term FR. Antioxidant N-acetyl-L-cysteine was effective as a radioprotective agent against mitochondrial damage induced by low-dose, long-term FR among all cell lines, including radiosensitive cell lines. In conclusion, we demonstrated that mitochondria are target organelles of low-dose radiation. Mitochondrial response influences radiation sensitivity in human cells. Our findings provide new insights into cancer risk estimation associated with low-dose radiation exposure.     

Changes of serum-induced ornithine decarboxylase activity and putrescine content during aging of IMR-90 human diploid fibroblasts

The roles of ornithine decarboxylase (ODC, EC 4.1.1.17) and polyamines in cellular aging were investigated by examining serum-induced changes of these parameters in quiescent IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL) and in human progeria fibroblasts. Serum stimulation caused increases of ODC and DNA synthesis in IMR-90 human diploid fibroblasts, with maximal values occurring, respectively, 10 hr and 22 hr after serum stimulation. Both serum-induced ODC activity and DNA synthesis in IMR-90 cells were found to be inversely related to their PDL. Maximal ODC activity and DNA synthesis in young cells (PDL = approximately 18-22) were, respectively, five-fold and six-fold greater than that in old cells (PDL = approximately 50-55), which in turn were comparable or slightly higher than that in progeria fibroblasts. Polyamine contents (putrescine, spermidine, and spermine) in quiescent IMR-90 cells did not show significant PDL-dependency. The putrescine and spermine contents in quiescent progeria cells were comparable to those in quiescent IMR-90 cells. The spermidine content in quiescent progeria cells, however, was extremely low, less than half of that in quiescent IMR-90 cells. Serum stimulation caused a marked increase in putrescine content in young cells but not in old cells or in progeria cells. The spermidine and the spermine content in IMR-90 cells, either young or old, and in progeria cells did not change significantly after serum stimulation. Our study indicated that aging of IMR-90 human diploid fibroblasts was accompanied by specific changes of polyamine metabolism, namely, the serum-induced ODC activity and putrescine accumulation. These changes were also observed in progeria fibroblasts derived from patients with Hutchinson-Gilford syndrome.     

Decreased uptake and retention of rhodamine 123 by mitochondria in feline sarcoma virus-transformed mink cells

A reduce uptake and retention of the mitochondria-specific membrane potential probe rhodamine 123 by feline sarcoma virus (FeSV)-transformed mink fibroblasts (64F3) has been detected. The decreased accumulation of rhodamine 123 by 64F3 mitochondria is not due to abnormal plasma membrane dye permeability, since after microinjection of the dye these cells are still unable to retain the dye at levels comparable to the untransformed parental cells, CCL 64. Nigericin, an ionophore that mediates an electrically neutral exchange of protons for potassium ions resulting the elimination of the pH gradient across the mitochondrial membrane and a compensatory increase in mitochondrial membrane potential with continued respiration, increases both the dye uptake and the retention time in transformed 64F3 cells. These results suggest that mitochondria in FeSV-transformed mink cells may have an abnormally low mitochondrial membrane potential accompanied by a relatively high pH gradient. Since anioic metabolites such as pyruvate and glutamate are accumulated by mitochondria in proportion to the delta pH across the mitochondrial membrane, the abnormal mitochondria described here may contribute to the abnormal metabolic state of FeSV-transformed cells.     

Mitochondrial c-Jun NH2-terminal kinase prevents the accumulation of reactive oxygen species and reduces necrotic damage in neural tumor cells that lack trophic support

In response to different stress signals, the c-Jun NH(2)-terminal kinase (JNK) can trigger cell death. However, JNK also facilitates the survival and cell cycle progression of tumor cells by mechanisms that are poorly defined. Here, we show that schwannoma RN22 cells can survive and proliferate under serum-free conditions although serum withdrawal rapidly induces mitochondrial fission and swelling. Although the morphologic changes observed in the mitochondria did not trigger cytochrome c release, they were accompanied by an increase in the mitochondrial membrane potential (DeltaPsi(M)) and of immunoreactivity for active JNK in these organelles. Pharmacologic inhibition of JNK provoked a further increase of the DeltaPsi(M), an increase in reactive oxygen species (ROS) production, and a sustained decrease in cell viability due to necrosis. This increase in necrosis was prevented by the presence of ROS scavengers. Immunoreactivity for active JNK was also observed in the mitochondria of neuroblastoma 1E-115 and neuroblastoma 2a neuroblastoma cell lines on serum withdrawal, whereas active JNK was barely detected in serum-deprived fibroblasts. Accordingly, the reduction in neural tumor cell viability induced by JNK inhibition was largely attenuated in serum-deprived fibroblasts. These data indicate that local activation of JNK in the mitochondria can protect against necrotic cell death associated with ROS production, facilitating the growth of neural tumor cells subjected to serum deprivation.     

Subcellular heterogeneity of mitochondrial function and dysfunction: Evidence obtained by confocal imaging

Beyond their fundamental role in energy metabolism, mitochondria perform a great variety of other important functions (e.g. in Ca2+ homeostasis, apoptosis, thermogenesis, etc.), thus suggesting their region-specific specializations and intracellular heterogeneity. Although mitochondrial functional heterogeneity has been demonstrated for several cell types, its origin and role under physiological and, in particular, pathophysiological conditions, where the extent of heterogeneity may significantly increase, remain to be elucidated. The present work thus investigated the static and dynamic heterogeneity of mitochondria and mitochondrial function in various cell types in which mitochondria may cope with specific functions including cardiomyocytes, hepatocytes and some cultured carcinoma cells. Modern confocal and two-photon fluorescent microscopy was used for the investigation and direct imaging of region-specific mitochondrial function and heterogeneity. Analysis of the autofluorescence of mitochondrial flavoproteins in hepatocytes and carcinoma cells permitted significant intracellular heterogeneity of mitochondrial redox state to be demonstrated. Comparative homogeneity and clear colocalization of mitochondrial flavoproteins, membrane potential and calcium-sensitive probes were observed in both isolated cardiomyocytes and permeabilized myocardial fibers. After ischemia reperfusion, however, or under conditions of substrate deprivation, significant heterogeneity of all these parameters was detected. Some methodological issues, mechanistic aspects, possible metabolic consequences of mitochondrial functional heterogeneity and its impact under pathological conditions are discussed.     

Cytochalasin B-induced alterations of insulin binding and microfilament organization in cultured fibroblasts

The effect of cytochalasin B (CB) on insulin binding has been investigated in confluent cultures of chick embryo fibroblasts. Time- and dose-dependent increases in binding of [¹²⁵I]insulin was observed after incubation of fibroblasts with CB. At 10 μg/ml, CB caused a 2-fold increase in binding, due to an increase in the number of binding sites from 9.3 × 10³ to 2.0 × 10⁴ per cell. Removal of CB from the growth medium was accompanied by a decrease in [¹²⁵I]insulin binding to control values in 24 h. Increase in the binding of insulin in CB-treated CEF was also accompanied by enhancement of insulin to stimulation of [³H]thymidine incorporation into acid-insoluble material. CB treatment also caused disorganization and disappearance of microfilament bundles and changes in cell shape from flat, with a few blebs and folds on the cell surface, to round with numerous blebs and folds. The data from this study suggest that changes in the number of surface insulin-binding sites may be related to the state of organization of cytoskeletal structures in chick embryo fibroblasts.     

Excessive reactive oxygen species induces apoptosis in fibroblasts: role of mitochondrially accumulated hyaluronic acid binding protein 1 (HABP1/p32/gC1qR)

Constitutively expressed HABP1 in normal murine fibroblast cell line induces growth perturbation, morphological abnormalities along with initiation of apoptosis. Here, we demonstrate that though HABP1 accumulation started in mitochondria from 48 hr of growth, induction of apoptosis with the release of cytochrome c and apoptosome complex formation occurred only after 60 hr. This mitochondrial dysfunction was due to gradual increase in ROS generation in HABP1 overexpressing cells. Along with ROS generation, increased Ca 2+ influx in mitochondria leading to drop in membrane potential was evident. Interestingly, upon expression of HABP1, the respiratory chain complex I was shown to be significantly inhibited. Electronmicrograph confirmed defective mitochondrial ultrastructure. The reduction in oxidant generation and drop in apoptotic cell population accomplished by disruption of HABP1 expression, corroborating the fact that excess ROS generation in HABP1 overexpressing cells leading to apoptosis was due to mitochondrial HABP1 accumulation.     

Cytological effects of platelet-derived growth factor on mitochondrial ultrastructure in fibroblasts

The goal of this study was to evaluate morphofunctional changes in mitochondrial ultrastructure after platelet-derived growth factor application in fibroblasts as an indicator of mitochondrial activation in processes like wound healing. NRK-49F fibroblasts were synchronized, incubated with PDGF (platelet-derived growth factor) and studied by electron microscopy. Volume density (Vv), numerical density (Nv) and surface density (Sv) were measured by stereological analysis. Application of PDGF on NRK-49F caused an increase in mitochondrial volume density by 57% and surface area of cristae per mitochondrion by 65%. The numerical density of the mitochondria was decreased in the PDGF-treated cells by 23%, but at the same time their mean volume was increased. Furthermore, the mitochondria had a complex and highly variable shape both in control and PDGF-treated cells, possibly indicating the existence of a mitochondrial reticulum. The results demonstrated that biochemically active membrane systems in fibroblast mitochondria are enlarged as a direct effect of small doses of platelet-derived growth factor and support the concept that this factor and related peptides serve as mitogens for connective tissue forming cells. Thus, in mitogenic processes like wound healing, the high energy demand of fibroblasts is provided by the increase of the inner surface of mitochondria.     

Functional Significance of the Mitochondrial Membrane Potential

The electrical polarization of the inner mitochondrial membrane largely determines the electrochemical potential of hydrogen ifons, being thereby a significant factor in the energy transformation during oxidation of respiratory substrates and its accumulation in the form of newly synthesized ATP. However, the gradient of the electric potential on the inner mitochondrial membrane (ΔΨm) performs a number of functions not related to energy production. Even under hypoxic conditions, precluding the formation of ATP in mitochondria through oxidative phosphorylation, mitochondria maintain their ΔΨm at the expense of the hydrolysis of cellular ATP, which indicates the exceptional importance of ΔΨm for non-energetic functions of mitochondria. Among these functions, the mitochondrial inward transport of metal cations and proteins carrying a positively charged amino acid sequence and export of anions including nucleic acids possibly providing retrograde signaling, seem very important and essential for maintaining mitochondrial structure and metabolism. ΔΨm is a powerful regulator of mitochondrial generation of reactive oxygen species that perform physiological and pathological functions. And finally, ΔΨm is a critical element in the mechanism of disposal of dysfunctional mitochondria, the so-called quality control machinery of mitochondria. The disturbance of this mechanism leads to increase of heterogeneity in the population of mitochondria in the cell, and the degree of heterogeneity can be considered as an indicator of the pathological cellular phenotype. Correlation between Ψm and cell functions is difficult to identify without adequate quantitative estimates of the magnitude of ΔΨm, which are complicated due to several cellular and mitochondrial processes that affect the experimentally obtained values. Recommendations for assessing the contribution of these processes and avoiding artifacts in the measurements of ΔΨm by standard methods are given.     

Biochemical and stereological analysis of rat liver mitochondria in different thyroid states

The concentrations of the inner mitochondrial membrane markers cardiolipin and cytochrome alpha have been measured in liver homogenates and in purified mitochondria after thyroxine administration to thyroidectomized and normal rats. The biochemical results have been correlated with stereological electron micrographic analyses of hepatocytes in liver sections, and of isolated mitochondrial pellets. There were progressive and parallel increases in homogenate and mitochondrial cardiolipin concentration, and in mitochondrial cytochrome alpha concentration, after administration of 20 microgram of thyroxine on alternate days to thyroidectomized rats, and of 300 microgram on alternate days to normal rats. Electron microscope measurements showed marked differences in the shape of the mitochondria and in the number of cristae in different thyroid states. Hypothyroid mitochondria were shorter and wider than controls, and hyperthyroid mitochondria longer but of similar width. Mitochondrial volume per unit cell volume was virtually unchanged in hypo- and hyperthyroid animals. The most striking changes were a decrease in the area of the inner membrane plus cristae in thyroidectomized rats, and a substantial increase in membrane area after thyroxine administration. The biochemical and electron micrographic results indicate that, in rat liver, thyroid hormone administration leads to a selective increase in the relative amount of mitochondrial inner membranes, with little or no change in the mitochondrial volume per unit cell volume, or in total mitochondrial protein per unit total cell protein.     

丙戊酸钠对抗鱼藤酮诱导的SH-SY5Y细胞损伤的线粒体机制

研究丙戊酸钠（sodiumvalproate,VPA）对抗鱼藤酮（Rotenone）诱导的SH-SY5Y细胞损伤的作用及线粒体机制。以l,10μmol／LVPA预处理SH－SY5Y细胞3h,再加入400nmol／LRotenone作用24h。MTT法检测与相差显微镜观察相结合,分析VPA对抗Rotenone损伤的作用;JC－1染色法与Mito－Tracker染色法分析线粒体膜电位及线粒体数量的变化;Clark氧电极法检测细胞呼吸功能;DCFH-DA探针法检测细胞中Ros的含量;并在离体线粒体上观察VPA对Ca^2＋诱导的线粒体肿胀的影响。结果发现,1,10p．mol／LVPA预处理SH．SY5Y细胞3h可对抗400nmol／LRotenoneI起的细胞损伤,并且可以提高损伤细胞中线粒体的膜电位,增加线粒体的数量,此外,还可以增强损伤细胞的呼吸功能,降低细胞中ROS的含量,但VPA并不能直接作用于离体的线粒体发挥神经保护作用。由此,VPA具有良好的神经保护作用,其机制与增强线粒体功能和数量、从而改善细胞功能有关,这为其应用于帕金森病的预防与治疗提供了实验依据。