The purpose of the present study was to explore the effects of hypoxic exposure on mitochondrial adenine nucleotide translocator (ANT) activity and its characteristics. Male Wistar rats were exposed to hypoxia in a hypobaric chamber simulating high altitude at 5 000 m for 1, 5, 15 and 30 d. Control rats were fed outside the hypobaric chamber. Rats were sacrificed by decapitation and mitochondria from the cerebral cortex were isolated by differential centrifugation at each time point. The ANT activity was detected by the atractyloside (ATR)-inhibitor stop technique. Mitochondria was initiated by addition of (3)H-ADP and terminated after 12 s by quick addition of ATR. The radioactivity was measured in a liquid scintillation counter. Nonspecific binding of (3)H-ADP to mitochondria was estimated by incubation of mitochondrial samples with ATR prior to the addition of (3)H-ADP. This blank was substracted from the measured radioactivities. The activity of ANT was expressed as nanomoles (3)H-ADP per minute per milligram protein. The ANT density was determined by titrating the rate of state 3 respiration with increasing concentrations of carboxyatractyloside (CAT). Mitochondria were pre-incubated with CAT in a respiratory medium before ADP addition to initiate state 3 respiration. Plots of O2 consumption versus CAT appeared biphasic with an increasing inhibitory segment followed by a steady respiration, indicating that state 3 respiration was completely inhibited. The density of ANT was determined by the amount of CAT required to completely inhibit state 3 respiration, assuming a 1:1 binding stoichiometry, which was expressed as ANT density per milligram mitochondria protein. (ATP+ADP) in mitochondria was measured by high performance liquid chromatography (HPLC). The results showed that there was an obvious decrease in the ANT activity during hypoxic exposure. The lowest ANT activity was seen in 5 d group. Partial recovery of ANT activity was observed in 15 and 30 d groups, but ANT activity was still lower than that in the control group (P<0.01). Compared with that in normoxic control group, no change of ANT density in mitochondria was observed in hypoxia group (P>0.05). The turnover number of ANT in control, 1, 5, 15, 30 d groups were 16.67, 1.90, 0.40, 1.81, 4.73 pmol ADP/(min.pmol ANT), respectively. However, (ATP+ADP) in mitochondria in 1, 5, 15, 30 d groups were 63.37%, 48.44%, 52.73%, 60.16% of control group respectively. Therefore, the turnover number of energy production and expenditure were reduced. These observations suggest that the change of ANT activity may be one of the mechanisms of cellular oxidative phosphorylation dysfunction during hypoxic exposure.
Altitude ; Animals ; Atractyloside ; pharmacology ; Cerebral Cortex ; metabolism ; physiopathology ; Hypoxia ; metabolism ; physiopathology ; Male ; Mitochondria ; metabolism ; Mitochondrial ADP, ATP Translocases ; metabolism ; Rats ; Rats, Wistar

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L^-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Atractyloside/pharmacology* ; Cyclophilin D ; Male ; Matrix Metalloproteinases ; Mice ; Mitochondrial Membrane Transport Proteins/metabolism* ; Mitochondrial Permeability Transition Pore ; RNA, Messenger ; Rats

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 investigate whether the cardioprotection of mitochondrial Slo channel (mitoSlo(1) channel) is associated with mitochondrial permeability transition in isolated rat hearts subjected to ischemia and reperfusion.

METHODSIsolated perfused rat hearts were subjected to 30 min regional ischemia (occlusion of left anterior descending artery) and 120 min reperfusion. The infarct size, lactate dehydrogenase (LDH) release during reperfusion and ventricular hemodynamic parameters were measured.

RESULTSPretreatment with mitoSlo(1) channel opener, NS1619 10 micromol/L for 10 min reduced the infarct size and LDH release, and improved the recovery of left ventricular developed pressure, left ventricular end-diastolic pressure, maximal rise/fall rate of left ventricular pressure and coronary flow during reperfusion. Administration of atractyloside (20 micromol/L), an opener of mitochondrial permeability transition pore, for 20 min (last 5 min of ischemia and first 15 min of reperfusion) attenuated the reduction of infarct size and LDH release and improvement of left ventricular performance induced by NS1619. In the isolated mitochondria, a significant inhibition of Ca(2+)-induced swelling was observed when mitochondria were incubated with NS1619.

CONCLUSIONMitoSlo(1) channel activation by NS1619 protects the myocardium against ischemia and reperfusion injury by inhibiting mitochondrial permeability transition pore opening.

Animals ; Atractyloside ; pharmacology ; Cardiotonic Agents ; pharmacology ; Heart ; In Vitro Techniques ; Ischemic Preconditioning, Myocardial ; Mitochondria, Heart ; drug effects ; Mitochondrial Membrane Transport Proteins ; Myocardial Reperfusion Injury ; metabolism ; prevention & control ; Permeability ; drug effects ; Potassium Channels ; metabolism ; Potassium Channels, Calcium-Activated ; metabolism ; Rats