Effects Of Purine Nucleotide On Activity And Expression Of Mitochondrial UCPs And Its Roles On Energy Synthesis And Oxygen Consumption Of Brain Mitochondria From Rat Exposed To Simulated High Altitude Hypoxia

When electrons are passing through the electron transport chain, protons are pumped across the inner mitochondrial membrane from the matrix to the intermembrane space. This creates an proton gradient (called proton motive ,Δp) which provides energy for ATP syntheses on the F0F1-ATPase. The whole process is called the oxidative phosphorylation. However, the protons return to the matrix not only by the F0F1-ATPase but by others——uncoupling proteins (UCPs). The latter is called mitochondrial proton leak. This leads to decrease of proton gradient, uncoupling of oxidative phosphorylation, decrease of ATP syntheses and efficiency of oxygen utilization. The protons gradient is consumed not only by the ATP-synthase into ATP, but also by UCPs into heat energy. There are increase of uncoupling oxygen consumption and decrease of mitochondria membrane potential (MMP) and ATP synthesis in brain mitochondria during rat exposed to simulated high altitude hypoxia. The UCP4 and UCP5 are expressed predominantly in brain of mammalian. Purine nucleotide (GDP) is the special inhibitor of UCPs. To realize the roles of UCPs on mitochondrial energy produce and efficiency of oxygen utilization in rat brain during hypoxic exposure, we observed the effects of GDP on the activity of UCPs and the expression of UCP4 and UCP5 mRNA and protein and the roles in mitochondria oxygen consumption and energy produce during animal hypoxia exposure.MethodsAdult male SD rats were exposed to a hypobaric chamber simulated 5000m high altitude for 23 hours every day for 0(control),3(acute group),30 days(chronic group) respectively. Rat was sacrificed by decapitation and brain was removed. Brain mitochondria were isolated by centrifugation program. Mitochondria oxidative respiratory function was measured by Clark oxygen electrode. The UCPs'activity and content was detected by [3H]-GTP binding method. Mitochondrial membrane potential was detected by Rhodamine123 method. The content of adenine nucleotide pool (ATP,ADP,AMP) in mitochondria was measured by high performance liquid chromatography(HPLC). The F0F1-ATPase's activity was determined by oligomycin-inhibitor method. The mRNA and protein expression of UCP4 and UCP5 in rat brain was determined by RT-PCR and Western blot analysis respectivly.Results1. The activity, mRNA and protein expression of mitochondrial UCPs from rat brain significantly increased during hypoxia exposure especially in acute hypoxia group, which Kd increased 43.08% and Bmax decreased 1.7-fold respectively. Also UCP4 and UCP5 mRNA expression increased 1.47-fold and 3.69-fold and their protein expression increased2.44-fold and 3.62-fold respectively in acut hypoxia goup. GDP inhibit significantly in UCPs activity in vitro in all groups, especially in acute hypoxia group, but not any changes in UCP4 and UCP5mRNA and protein expression in all groups.2. There were significant decreases in ST3, RCR, P/O and MMP of mitochondria from hypoxia-exposed rat brain, especially in acute hypoxia group, which were lower 16.96%, 38.98%, 23.55%, 8% and 18.04% than control respectively. But ST4 in acute group was significant higher 36.12% than in control. GDP resulted in significant inhibition in mitochondrial oxygen consumption in ST3 and ST4, but increase in RCR, P/O and MMP in vitro in all groups.3. The activity of F0F1-ATPase, ATP content and the ratios of ATP/ADP and ATP/(ATP+ADP+AMP) were decreased in hypoxia-exposed rat brain mitochondria. GDP resulted in significant increases of the above index in vitro in all groups.Conclusion1. Hypobaric hypoxia exposure can result in increase in the activity of UCPs and UCP4 and UCP5mRNA and protein expression in rat brain mitochondria. GDP can inhibit the activity of UCPs but can not change UCP4 and UCP5mRNA and protein expression in vitro.2. Hypobaric hypoxia exposure can induce the increase of uncoupling oxygen consumption and decreases in RCR, OPR and MMP of rat brain mitochondria. GDP can decrease uncoupling respiration and increase RCR, OPR, P/O and MMP of rat brain mitochondria in vitro in all groups, especially in the acute hypoxia group.3. Hypobaric hypoxia exposure can induce the decrease in activity of F0F1-ATPase and drop in content of ATP, the ratios of ATP/ADP and ATP/(ATP+ADP+AMP) in rat brain mitochondria. But GDP can result in increase in activity of brain mitochondria F0F1-ATPase and ATP content in vitro in all groups.Conclusion summary:Hypobaric hypoxia exposure can increase in UCPs activity and content of rat brain mitochondria. This thus results in decreases in mitochondrial MMP, oxidative phosphorylation rate and energy production rate. But there are higher uncoupling respiration and lower efficiency of oxygen consumption. GDP can inhibit the UCPs activity of rat brain mitochondria in vitro, the inhibiting level according to hypoxia-exposed time. Thus GDP can increase MMP and the efficiency of oxidative phosphorylation, especially in hypoxia-exposed rat brain mitochondria.