OBJECTIVETo investigate the effects of resveratrol (Res) on mitochondrial opening and Ca(2+)-induced Ca(2+) release (CICR) from rat liver cell mitochondria mediated by Ca(2+).

METHODSWistar rat liver cell mitochondria was extracted and Res-induced mitochondrial swelling was assessed spectrophotometrically at 540 nm to examine the permeability transition pore (PTP) opening. The membrane potential changes of Res-treated mitochondria were measured with fluorescence spectrophotometery. Ca(2+) uptake and release by the mitochondria was determined by absorbance change of arsenazo III at 685-675 nm monitored by dual wavelength spectrophotometry.

RESULTSRes promoted Ca(2+)-mediated PTP opening, and this effect was completely inhibited by CsA and lowered by trifluoperazine. CICR accelerated by Res treatment was completely blocked by ruthenium red and partly by trifluoperazine.

CONCLUSIONRes can promote PTP opening by inducing CICR, which may be one of the pathways that Res induces cell apoptosis.

Animals ; Calcium ; metabolism ; Cells, Cultured ; Female ; Hepatocytes ; cytology ; metabolism ; Mitochondria, Liver ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Mitochondrial Swelling ; drug effects ; Rats ; Rats, Wistar ; Stilbenes ; pharmacology

OBJECTIVETo study effects of three pentacyclic triterpenoids, oleanolic acid (OA), ursolic acid (UA) and asiatic acid (AA) on Ca(2+)-induced liver mitochondrial permeability transition (MPT).

METHODEffects of three compounds on liver MPT induced by Ca2+ were assessed by measuring the change in mitochondrial swelling, mitochondrial membrane potential and release of matrix Ca2+ in vitro.

RESULTObvious mitochondrial swelling, loss of mitochondrial membrane potential and release of matrix Ca2+ occurred after the addition of 50 micromol x L(-1) Ca2+. However, preincubation with 50 mg x L(-1) OA, UA or AA significantly blocked the above changes. In addition, it was also found that there are differences in the inhibitions of three compounds on liver MPT induced by Ca2+.

CONCLUSIONThree pentacyclic triterpenoids, OA, UA and AA, have significant mitochondrial protection through blocking on liver MPT and the inhibition on liver MPT of AA is stronger than that of UA and OA.

Animals ; Calcium ; metabolism ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mice ; Mice, Inbred ICR ; Mitochondria, Liver ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; drug effects ; Mitochondrial Swelling ; drug effects ; Pentacyclic Triterpenes ; pharmacology

AIMTo investigate the mechanisms of anti-cancer effect of resveratrol (Res), and the effects of Res in cell apoptosis. The role of Res playing in mitochondrial permeability transition pore (PTP) induction was studied.

METHODSMitochondria was prepared from the liver of Wistar rats. The effects of Res on oxygen consumption of isolated mitochondria from rat liver was measured with Clark-type electrode and resulted in respiration control rate (RCR). Mitochondrial swelling affected by Res was assessed spectrophotometrically, through the changes in absorbance at 540 nm. The PTP opening was learned from the results. Membrane potential of mitochondia was measured through fluorescence spectrophotometry.

RESULTSRes was shown to inhibit the respiration and decrease the RCR of mitochondria. Res can promote the PTP opening mediated by Ca2+. Res was shown to promote the increase of mitochondial membrane potential mediated by Ca2+ and loss of mitochondial membrane potential.

CONCLUSIONRes was shown to inhibit mitochondial respiration and induce PTP opening of mitochondria. These may be one of the pathways that Res showed anti-cancer action and induce cells apoptosis.

Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Calcium ; metabolism ; Female ; Ion Channels ; drug effects ; metabolism ; Membrane Potentials ; drug effects ; Mitochondria, Liver ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; Mitochondrial Swelling ; drug effects ; Rats ; Rats, Wistar ; Stilbenes ; pharmacology

AIMTo clarify whether the activation of mitochondrial ATP sensitive potassium channel and calcium activated potassium channel can influence the permeability transition of normal and ischemic brain mitochondria.

METHODSspectrophotometry was used to determine the effect of the two mitochondrial potassium channel agonists on the swelling of normal and ischemic brain mitochondria respectively.

RESULTSIn normal mitochondria, diazoxide and NS1619 could inhibit the decrease of calcium induced mitochondrial absorbance at 520 nm (A520), which were blocked by atractyloside. When compared with the normal mitochondria, mitochondrial A520 decrease in ischemic brain was even more rapid. Diazoxide and NS1619 could still inhibit the calcium induced mitochondrial A520 decrease, which were blocked by atractyloside.

CONCLUSIONActivation of mitochondrial ATP sensitive potassium channel and calcium activated potassium channel can protect brain mitochondria in vitro probably via influencing the mitochondrial permeability transition.

Animals ; Benzimidazoles ; pharmacology ; Brain ; drug effects ; metabolism ; Brain Ischemia ; metabolism ; Cell Membrane Permeability ; Diazoxide ; pharmacology ; KATP Channels ; metabolism ; Male ; Mitochondria ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; drug effects ; Potassium Channels, Calcium-Activated ; metabolism ; Rats ; Rats, Sprague-Dawley

Opening of mitochondrial permeability transition (MPT) pores leads to mitochondrial injury during oxidative stress. The peripheral benzodiazepine receptor (PBR) located at mitochondrial outer-membrane has been shown to be involved in several mitochondrial functions. In the present study, we used Ro5-4864, a PBR agonist, to test if activation of PBR could prevent MPT pore opening during Ca(2+) overloading. Cardiac mitochondria isolated from Sprague-Dawley rats were treated by 150 mmol/L Ca(2+) to induce MPT. Ro5-4864 (50, 100 and 200 micromol/L) was added into incubation buffer before adding 150 micromol/L Ca(2+). In additional group, atractyloside (ATR, 20 micromol/L), an opener of MPT pores was added 5 min before the addition of 100 micromol/L Ro5-4864. The change of absorbance at 520 nm was monitored with a spectrophotometer at 30 degrees C for 10 min. Western blot was used to detect cytochrome C loss. The mitochondrial membrane potential was monitored with the fluorescence dye JC-1. Ro5-4864 inhibited the decrease of absorbance at 520 nm compared to that in the untreated Ca(2+) group (P<0.01, P<0.05). In the presence of ATR, Ro5-4864 was not able to prevent MPT anymore. Opening of MPT pores by Ca(2+) decreased the content of cytochrome C in mitochondria, but increased cytochrome C content in cytosol. Ro5-4864 preserved cytochrome C content in mitochondria and led to less cytochrome C release to cytosol. ATR treatment reversed the protective effect of Ro5-4864 on cytochrome C content. Opening of MPT pores led to mitochondrial depolarization, whereas Ro5-4864 treatment maintained mitochondrial membrane potential. Thus, prevention of MPT by activation of PBR during calcium overloading maintains mitochondrial cytochrome C content and membrane potential. Activation of PBR during cardiac ischemia and reperfusion may be an alternative way for cardioprotection.
Animals ; Atractyloside ; pharmacology ; Benzodiazepinones ; pharmacology ; Carrier Proteins ; agonists ; metabolism ; physiology ; Female ; Male ; Membrane Potential, Mitochondrial ; physiology ; radiation effects ; Mitochondria, Heart ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA-A ; metabolism ; physiology

OBJECTIVETo explore the intervention effect and the possibly mechanism of the glutamine (Gln) on the opening change of the permeability transition pore (PTP) in the myocardial mitochondrial membrane under the overtraining state.

METHODS30 SD rats were randomly divided into 3 groups (n = 10): control group (CG group), overtraining group (OG group) and supplementary (Gln) + overtraining group group). Spectrophotometry was used to test the openness of the permeability transition pore in the myocardial mitochondrial membrane. Electrochemistry was used to test the malondialdehyde (MDA) and the glutathione (GSH) content and the phospholipase A2 (PLA2) activity.

RESULTSOG group compared with the GOG group, the absorbance (A0) and the absorbance change (Delta A) were decreased significantly (P < 0.05). Rh123 fluorescence (F0) intensity was significantly increased (P < 0.05). Rhodamine123 (Rh123) fluorescence change (delta F) was significantly decreased (P < 0.05). Compared with the GOG, the mitochondrial GSH was significantly decreased (P < 0.05), the PLA2 activity and the content of MDA were significantly increased (P <0.05).

CONCLUSIONOvertraining could lead to opening increase of permeability transition pore in the myocardial mitochondrial membrane, after overtraining, the production of the reactive oxygen species (ROS) and PLA2 activity were increased, GSH content was decreased. But added exogenous Gln had a significant intervention effect for these changes.

Animals ; Glutamine ; pharmacology ; Glutathione ; metabolism ; Male ; Mitochondria, Heart ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; metabolism ; Mitochondrial Membranes ; drug effects ; physiology ; Myocardium ; metabolism ; Permeability ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism

OBJECTIVETo investigate whether activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) and inhibition of mitochondrial permeability transition pore (mitoPTP) were involved in the cardioprotection of ethanol postconditioning in isolated rat heart.

METHODSHearts isolated from male Sprague-Dawley rats were perfused on a langendorff apparatus and subjected to 30 min of regional ischemia (occlusion of left anterior descending artery) followed by 120 min of reperfusion. The ventricular hemodynamic parameters and lactate dehydrogenase (LDH) release during reperfusion were measured. Infarct size was measured by TTC staining method and the expression of ALDH2 at mRNA level of left anterior myocardium was detected by RT-PCR.

RESULTIn contrast to ischemia and reperfusion, ethanol postconditioning improved the recovery of left ventricular developed pressure, maximal rise/fall rate of left ventricular pressure during reperfusion, reduced LDH release and infarct size. The expression of ALDH2 mRNA level was increased. Administration of mitoPTP activator atractyloside attenuated the effect of ethanol postconditioning, LDH release and infarct size were increased, and the recovery of hemodynamic parameters was inhibited. The expression of ALDH2 mRNA was decreased.

CONCLUSIONEthanol postconditioning has cardioprotection effect, which may be associated with upregulating mitochondrial ALDH2 mRNA expression and inhibiting the opening of mitochondrial permeability transition pore.

Aldehyde Dehydrogenase ; drug effects ; genetics ; metabolism ; Aldehyde Dehydrogenase, Mitochondrial ; Animals ; Ethanol ; pharmacology ; In Vitro Techniques ; Ischemic Postconditioning ; L-Lactate Dehydrogenase ; metabolism ; Male ; Mitochondria, Heart ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; drug effects ; metabolism ; Mitochondrial Proteins ; drug effects ; genetics ; metabolism ; Myocardial Reperfusion Injury ; metabolism ; pathology ; prevention & control ; Myocardium ; metabolism ; pathology ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

To investigate the regulatory role of purine nucleotide on uncoupling proteins (UCPs), the activity of UCPs and the expressions of UCP4 and UCP5 in mitochondria of rat brain tissues incubated with GDP were observed in vitro. The cerebral hemispheres of adult male Sprague-Dawley rats were removed and clipped into 8-10 mm3 tissue mass which incubated with 1 mmol/L GDP (GDP group), or only incubation medium (control group), for 30 min in vitro. The mitochondria from incubated tissue mass of rat brain were isolated by centrifugation. The activity of UCPs was detected by the method of [3H]-GTP binding with UCPs specifically. The maximal binding content (Bmax) and the dissociation constant (Kd) were determined from Scatchard plot. The mRNA and protein expressions of UCP4 and UCP5 were measured by RT-PCR and Western blot, respectively. The results showed that Bmax was increased and Kd was decreased in rat brain mitochondria in GDP group compared with that in control group. But the mRNA and protein expressions of UCP4 and UCP5 exhibited no statistically significant changes. It is thus suggested that GDP inhibits the activity of mitochondrial UCPs in rat brain in vitro, but exhibits no effect on the expressions of UCP4 and UCP5.
Animals ; Brain ; drug effects ; metabolism ; Guanosine Diphosphate ; pharmacology ; In Vitro Techniques ; Ion Channels ; metabolism ; Male ; Mitochondria ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Mitochondrial Proteins ; metabolism ; Mitochondrial Uncoupling Proteins ; Nerve Tissue Proteins ; metabolism ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo explore the effect of MnCl(2) on the mitochondrial function of human lung cells, and to study the changes of protein expression level of nuclear respiratory factor-1 (NRF-1) in mitochondrial dysfunction induced by MnCl(2).

METHODSThe effects of MnCl(2) on cell survival rate were assessed by the reductions of tetrazolium dye (MTT) in cultured cell lines 16HBE and A549 cells. All tested16HBE and A549 cells were incubated with different concentrations of MnCl(2). The permeability transition pore (PTP) of mitochondria, mitochondrial membrane potential and the inhibition rate of mitochondrial enzymes as indicators of mitochondrial damage were measured by fluorescent spectrometry and MTT assay, respectively. Apoptosis was determined by flow cytometry. Protein levels of NRF-1 and mtTFA were measured by Western blot assay.

RESULTSMnCl(2) decreased the survival rate of the two cell lines. The IC(50) of 16HBE and A549 cells were 1.91 mmol/L and 1.98 mmol/L, respectively. MnCl(2) caused a concentration-dependent decrease of mitochondrial enzymes and the inhibition rate of mitochondrial enzymes of the two cell lines induced by 1.00 mmol/L MnCl(2) were (52.8 ± 5.4)% and (50.6 ± 2.2)%, respectively. The PTP opening increased in MnCl(2)-treated cells in a dose- and time-dependent manner. Compared with the control group, mitochondrial membrane potential in the two cell lines was decreased by MnCl(2), by (7.9 ± 3.0)%, (26.2 ± 2.2)% and (27.8 ± 4.1)% in the 16HBE cells, and (4.7 ± 1.0)%, (14.9 ± 2.4)% and (27.5 ± 1.2)% in the A549 cells. Increased apoptosis rates of the two cell lines were induced by 1.00 mmol/L MnCl(2), (12.3 ± 1.9)% and (6.0 ± 0.4)%, respectively. The results of Western blot assay revealed that the protein levels of NRF-1 and mtTFA were decreased in manganese-treated cells in a dose-dependent manner, with a significant difference compared with that of the control cells (P < 0.05).

CONCLUSIONMnCl(2) induces mitochondrial dysfunction in 16HBE and A549 cells, and decreases the expression level of nuclear respiratory factor-1 (NRF-1), indicating that NRF-1 may play an important role in mitochondrial dysfunction.

Apoptosis ; drug effects ; Bronchi ; cytology ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cells, Cultured ; Chlorides ; administration & dosage ; toxicity ; DNA-Binding Proteins ; metabolism ; Dose-Response Relationship, Drug ; Epithelial Cells ; cytology ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Manganese Compounds ; administration & dosage ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; Mitochondrial Proteins ; metabolism ; Nuclear Respiratory Factor 1 ; metabolism ; Transcription Factors ; metabolism

AIMTo determine whether the cardioprotection of puerarin (Pue) against ischemia/reperfusion (I/R) is mediated by mitochondrial transmembrane pore or channels.

METHODSMale Sprague-Dawley rats were used for Langendorff isolated heart perfusion. The hearts subjected to global ischemia for 30 min followed by 120 min of reperfusion. Formazan, a product of 2,3,5-triphenyltetrazolium chloride (TTC), which is proportional to myocardial viability, was measured at 490 nm, and the level of lactate dehydrogenase (LDH) in the coronary effluent was measured to evaluate the cardiac injury.

RESULTSThe pretreatment with Pue at 0.24 mmol/L for 5 min before ischemia increased formazan content of myocardium, reduced LDH release, improved the recovery of the left ventricular developed pressure, maximal rise/fall rate of left ventricular pressure, left ventricular end-diastolic pressure and rate pressure product (left ventricular developed pressure multiplied by heart rate) and attenuated the decrease of coronary flow during reperfusion. Administration of atractyloside (20 micromol/L), an opener of mitochondrial permeability transition pore, for 20 min (first 20 min of reperfusion) and 5-hydroxydecanoate (100 micromol/L), the mitochondrial specific K(ATP) blocker, for 20 min before ischemia attenuated the protective effects of Pue.

CONCLUSIONThe findings indicate that in the isolated rat heart, Pue protects myocardium against ischemia/ reperfusion injury via the opening of mitochondrial ATP-sensitive potassium channel and the inhibition of mitochondrial permeability transition pore opening.

Animals ; Decanoic Acids ; metabolism ; Hydroxy Acids ; metabolism ; Isoflavones ; pharmacology ; Male ; Mitochondria, Heart ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; drug effects ; Myocardial Reperfusion Injury ; metabolism ; Rats ; Rats, Sprague-Dawley