| Mitochondrial oxidative phosphorylation produces the majority of ATP for various cellular energy-requiring processes. Adenine nucleotide translocator(ANT) enables the ADP and ATP exchange at 1:1 ratio across the impermeable mitochondrial membrane. Consequently, the ANT is the key link between energy-producing and energy-consuming processes. Also it is suggested to play a important role in the control of mitochondrial oxidative phosphorylation. It is known that the decreased energy supply during hypoxia is the main factor of hypoxic brain damage. Objective To understand the effect of ANT on oxidative phosphorylation and the change of ANT activity during hypoxia exposure and the possible relative factor, such as the content of ANT,the exchangeable adenine nucleotide pool size(ATP+ADP) and the expression of ANT isoforms (ANT1,ANT2). Methods Adult male Wistar rats were exposed to a hypobaric chamber simulated 5000m high altitude for 23 hours every day for 0,1,5,15,30 days respectively. Rat was sacrificed by decapitation and brain was removed and the cerebral cortex mitochondria was isolated by centrifugation program. Mitochondrial respiratory function was measured by Clark oxygen electrode. Control of ANT on oxygen consumption rate and the content of ANT were estimated using inhibitor titration with carboxyatractyloside(CAT). The ANT activity was detected using the atractyloside(ATR)-inhibitor stop technique. The size of adenylic acid pool (ATP,ADP,AMP) in mitochondria was separated and measured by high performance liquid chromatography(HPLC). The mRNA copies of ANT1,ANT2 in tissues was determined by FQ-RT-PCR. The protein content of ANT in mitochondria was detected by Western blot analysis. Results â’ˆCompared with that in H0 group, mitochondrial state 3 respiration(ST3) decreased in H5,H15 and H30; mitochondrial state 4 respiration(ST4) increased significantly in H1,H5,H15 group, while in H30 fell to the level of H0. Respiratory control rate(RCR) decreased in H1,H5,H15 and H30. â’‰The activity of ANT in hypoxic groups, compared with H0 group , decreased significantly, that in H5 group was 2.75% of that in H0 and reached the lowest point, that in H30 was higher than in H5, but was still lower than in H0. Both Ci and the content of ANT had no significant change among groups. â’ŠAfter hypoxia, the content of ATP in mitochondria declined obviously. At the same time, the exchangeable adenine nucleotide pool(ATP+ADP) size decreased significantly. â’‹Copies of ANT1 and ANT2 increased significantly in H1 and H30 group, compared with H0 group, that in H5 group had no obvious change, copies of ANT1 and ANT2 in H15 increased respectively significantly and obviously. At the same time, the increased ANT2 copies was much more than increased ANT1 copies.Conclusion: â’ˆUnder conditions of hypoxia, mitochondrial respiration function was damaged. Acute hypoxia led to the increasing of ST4 ,suggesting that the increased uneffective oxygen consumption caused mitochondrial dysfuction during hypoxia exposure. â’‰Mitochondrial oxygen consumption rate immediately was lowered with only a few CAT, which is irreversible inhibitor of ANT, suggesting that ANT play a major role in the mitochondrial oxidative phosphorylation. The Ci of ANT on oxygen consumption were seen no change during hypoxia exposures, suggesting that there is to have no influence each other between the decreased mitochondrial respiration and the control of ANT. â’ŠHypoxia exposure slowed significantly the ANT activity without changing the ANT content, suggesting that the oxidative phosphorylation dysfuction was related to the deseased ANT activity during hypoxia.â’‹Our findings demostrate that hypoxia can affect mRNA expression of ANT1 and ANT2. The increased range of ANT2 mRNA was far larger than ANT1 mRNA, suggesting that ANT2 was a more important factor than ANT1 in mitochondrial compensation and mitochondrial adaptation to hypoxia. |
PDF Full Text Request