• The Korean Journal of Physiology and Pharmacology
• /
• 제16권1호
• /
• pp.71-77
• /
• 2012

Mitochondrial dynamics and distribution is critical for their role in bioenergetics and cell survival. We investigated the consequence of altered fission/fusion on mitochondrial function and motility in INS-1E rat clonal ${\beta}$-cells. Adenoviruses were used to induce doxycycline-dependent expression of wild type (WT-Mfn1) or a dominant negative mitofusin 1 mutant (DN-Mfn1). Mitochondrial morphology and motility were analyzed by monitoring mitochondrially-targeted red fluorescent protein. Adenovirus-driven overexpression of WT-Mfn1 elicited severe aggregation of mitochondria, preventing them from reaching peripheral near plasma membrane areas of the cell. Overexpression of DN-Mfn1 resulted in fragmented mitochondria with widespread cytosolic distribution. WT-Mfn1 overexpression impaired mitochondrial function as glucose- and oligomycin-induced mitochondrial hyperpolarization were markedly reduced. Viability of the INS-1E cells, however, was not affected. Mitochondrial motility was significantly reduced in WT-Mfn1 overexpressing cells. Conversely, fragmented mitochondria in DN-Mfn1 overexpressing cells showed more vigorous movement than mitochondria in control cells. Movement of these mitochondria was also less microtubule-dependent. These results suggest that Mfn1-induced hyperfusion leads to mitochondrial dysfunction and hypomotility, which may explain impaired metabolism-secretion coupling in insulin-releasing cells overexpressing Mfn1.

• The Korean Journal of Physiology and Pharmacology
• /
• 제9권4호
• /
• pp.209-213
• /
• 2005

Interstitial cells of Cajal (ICCs) are the pacemaker cells that generate slow waves in the gastrointestinal (GI) tract. In the present study, we investigated the effect of mitochondrial ATP-sensitive potassium (mitoKATP) channel on pacemaking activity in cultured ICCs from murine small intestine by using whole-cell patch clamp techniques. Under current clamp mode, at 10μM glibenclamide, there was no change in pacemaking activity of ICCs. At $30{\mu}M$ glibenclamide, an inhibitor of the ATP sensitive $K^+$ channels, we could find two examples. If pacemaking activity of ICCs was irregulating, pacemaking activity of ICCs was changed into regulating and if in normal conditions, membrane potential amplitude was increased. At $50{\mu}M$ glibenclamide, the resting membrane potential was depolarized. At 3mM 5-HDA, an inhibitor of the mitoKATP channels, inhibited the pacemaking activity of ICCs. Both the amplitude and the frequency were decreased. At 5 mM 5-HDA, both the amplitude and the frequency were completely abolished. Diazoxide, an opener of the mitoKATP channels, was applied to examine its effect on pacemaking activity of ICCs. At $50{\mu}M$ concentration, the pacemaking activity of ICCs was inhibited. Both the amplitude and the frequency were decreased. At 1 mM concentration, both the amplitude and the frequency were completely abolished and the resting membrane potential was shaked.These results indicate that mitoKATP channel has an important role in pacemaking activity of ICCs.

• 대한한방부인과학회지
• /
• 제19권1호
• /
• pp.1-13
• /
• 2006

Purpose : Polygonum cuspidatum extract is an oriental herb which has been used for uterine diseases. In this study, the effects of Polygonum cuspidatum extract were investigated on inducing growth inhibition and apoptosis of human uterine cervical carcinoma cells. Methods : Viability of Polygonum cuspidatum extract-induced ME-180 cells was measured by MTT assay. Apoptotic cells were visualized by EtBr/AcOr staining under fluorescent microscope. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Cell cycle distribution and changes in mitochondrial membrane potential were observed by flow cytometry. Results : Polygonum cuspidatum extract induced ME-180 cell death in a dose- and time-dependent manner. In the cells treated with Pc, the population of cells at sub-G1 phase significantly increased, and the condensed nuclei, apoptotic bodies and nucleosome-sized DNA were detected. Moreover, reduction in mitochondrial membrane potential was detected. Conclusion : Polygonum cuspidatum extract inhibits the growth and proliferation of ME-180 cells by apoptotic induction and facilitates its activity initiated by depolarization of mitochondria.

• Preventive Nutrition and Food Science
• /
• 제5권4호
• /
• pp.230-233
• /
• 2000

The present work was designed to determine whether change in fluidity of the mitochondrial membrane affects mitochondrial {TEX}$F_{1}${/TEX}{TEX}$F_{0}${/TEX}ATPase characteristics in NIDDM-prone BHE/Cdb rat. Isolated mitochondria fom BHE/Cdb rat fed a 6% coconut oil or corn oil were functionally tested by an analysis of its respiration and the coupling of this process to ATP synthesis in presence of oligomycin, a specific inhibitor of oxidative phosphorylation (OXPHOS), that binds to the {TEX}$F_{1}${/TEX}{TEX}$F_{0}${/TEX}ATPase. Mitochondria from rats fed coconut oil were more responsive to the inhibitory action of oligomycin with respect to state 3 respiration, respiratory control (RC) ratio and ADP:P (P/O) ratio than were mitochondria from rats fed corn oil. In state 3 respiration, mitochondria from rats fed coconut oil consumed less oxygen than did mitochondria from rats fed corn oil. RC ratio was lower in the mitochondria from rats fed coconut oil than was mitochondria from rats fed corn oil. In P/O ratio, the mitochondria from rats fed coconut oil had a lower P/O ratio than did mitochondria from rats fed corn oil. The data showed that the chang influidity of the mitochondrial membrane by dietary fat affected mitochondrial {TEX}$F_{1}${/TEX}{TEX}$F_{0}${/TEX}ATPase characteristics. The present study on diet differences in {TEX}$F_{1}${/TEX}{TEX}$F_{0}${/TEX}ATPase characteristics provides considerable insight into the role diets play in the control of mitochondrial function, expecially OXPHOS in NIDDM with mitochondrial defects.

• The Korean Journal of Physiology and Pharmacology
• /
• 제19권4호
• /
• pp.373-382
• /
• 2015

Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [$Ca^{2+}$]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [$Ca^{2+}$], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [$Ca^{2+}$] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [$Ca^{2+}$], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [$Ca^{2+}$] and TMRE for ${\Psi}_m$ or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [$Ca^{2+}$] concentration was $1.03{\mu}M$. This $1{\mu}M$ cytosolic $Ca^{2+}$ could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [$Ca^{2+}$] increase was limited to ${\sim}30{\mu}M$ in the presence of $1{\mu}M$ cytosolic $Ca^{2+}$. Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

• 대한의생명과학회지
• /
• 제14권4호
• /
• pp.239-242
• /
• 2008

Obesity results from a combination of genetic, environmental, and behavioral factors. Uncoupling proteins (UCP) are members of the larger family of mitochondrial anion carrier proteins (MACP). UCP separates oxidative phosphorylation from ATP synthesis with energy dissipated as heat, also referred to as the mitochondrial proton leak. UCP facilitates the transfer of anions from the inner to the outer mitochondrial membrane and the return transfer of protons from the outer to the inner mitochondrial membrane. Therefore, we investigated the genotype for the G>A polymorphism at the position -866 of UCP2 gene in Koreans and compared genotype of patients with control group. 50 patients (Male 22, Female 28), who previously underwent type 2 diabetcs (T2DM) and 30 controls (Male 14, Female 16) participated in this study. There was a weak significant association between -866 G>A polymorphism in UCP2 gene and T2DM. The present study shows that UCP2 -866 G>A polymorphism may not be associated with the pathogenesis of T2DM as opposed to the previous reports in other countries. Further studies with larger population may be needed for the development of diagnostic methods at genetic level such as DNA chip.

• Biomolecules & Therapeutics
• /
• 제12권2호
• /
• pp.92-100
• /
• 2004

Effect of the depletion or oxidation of GSH on mitomycin c (MMC)-induced mitochondrial damage and cell death was assessed in small cell lung cancer (SCLC) cells. MMC induced cell death and the decrease in the GSH contents in SCLC cells, which were inhibited by z-LEHD.fmk (a cell permeable inhibitor of caspase-9), z-DQMD.fmk (a cell permeable inhibitor of caspase-3) and thiol compound, N-acetylcysteine. MMC caused nuclear damage, release of cytochrome c and activation of caspase-3, which were reduced by N-acetylcysteine. The depletion of GSH due to L-butionine-sulfoximine enhanced the MMC-induced cell death and formation of reactive oxygen species in SCLC cells, whereas the oxidation of GSH due to diamide or $NH_2Cl$ did not affect cytotoxicity of MMC. The results show that MMC may cause cell death in SCLC cells by inducing mitochondrial dysfunction, leading to activation of caspase-9 and -3. The MMC-induced change in the mitochondrial membrane permeability, followed by cell death, in SCLC cells may be significantly enhanced by the depletion of GSH. In contrast, the oxidation of GSH may not affect cytotoxicity of MMC.

• Asian Pacific Journal of Cancer Prevention
• /
• 제15권5호
• /
• pp.2133-2139
• /
• 2014

4-Methylsulfinyl-3-butenyl isothiocyanate (MTBITC) found in the radish (Raphanus sativus L.), is a wellknown anticancer agent. In this study, the mechanisms of the MTBITC induction of cell apoptosis in human A549 lung cancer cells were investigated. Our PI staining results showed that MTBITC treatment significantly increased the apoptotic sub-G1 fraction in a dose-dependent manner. The mechanism of apoptosis induced by MTBITC was investigated by testing the change of mitochondrial membrane potential (${\Delta}{\Psi}m$), the expression of mRNAs of apoptosis-related genes by RT-PCR, and the activities of caspase-3 and -9 by caspase colorimetric assay. MTBITC treatment decreased mitochondrial membrane potential by down-regulating the rate of Bcl-2/Bax and Bcl-xL/Bax, and activation of caspase-3 and -9. Therefore, mitochondrial pathway and Bcl-2 gene family could be involved in the mechanisms of A549 cell apoptosis induced by MTBITC.

• Toxicological Research
• /
• 제23권3호
• /
• pp.215-221
• /
• 2007

Although sanguinarine, a benzophenanthridine alkaloid, possesses anti-cancer properties against several cancer cell lines, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. In order to further explore the critical events leading to apoptosis in sanguinarine-treated MCF-7 human breast carcinoma cells, the following effects of sanguinarine on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration of the mitochondrial membrane potential (MMP), and the expression changes of Bcl-2 family proteins. We show that sanguinarine-induced apoptosis is accompanied by the generation of intracellular ROS and disruption of MMP as well as an increase in pro-apoptotic Bax expression and a decrease of anti-apoptotic Bcl-2 and Bcl-xL expression. The quenching of ROS generation with N-acetyl-L-cysteine, the ROS scavenger, protected the sanguinarine-elicited ROS generation, mitochondrial dysfunction, modulation of Bcl-2 family proteins, and apoptosis. Based on these results, we propose that the cellular ROS generation plays a pivotal role in the initiation of sanguinarine-triggered apoptotic death.

• BMB Reports
• /
• 제50권4호
• /
• pp.214-219
• /
• 2017

Mitochondria play pivotal roles in the ATP production, apoptosis and generation of reactive oxygen species. Although dynamic regulation of mitochondria morphology is a critical step to maintain cellular homeostasis, the regulatory mechanisms are not yet fully elucidated. In this study, we identified miR-200a-3p as a novel regulator of mitochondrial dynamics by targeting mitochondrial fission factor (MFF). We demonstrated that the ectopic expression of miR-200a-3p enhanced mitochondrial elongation, mitochondrial ATP synthesis, mitochondrial membrane potential and oxygen consumption rate. These results indicate that miR-200a-3p positively regulates mitochondrial elongation by downregulating MFF expression.