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Characteristics of adenine nucleotide translocator in mitochondria of rat cerebral cortex during hypobaric hypoxia exposure

Chen Lifen, Liu Junze, Li Bing

Department of Pathophysiology, College of High Altitude Military Medicine, the Third Military Medical University. Chongqing 400038, China

Abstract

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 O_(2) 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.

Key words: Hypoxia;Mitochondria;Adenine nucleotide translocator

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Citing This Article：

Chen Lifen, Liu Junze, Li Bing . Characteristics of adenine nucleotide translocator in mitochondria of rat cerebral cortex during hypobaric hypoxia exposure. Acta Physiol Sin 2006; 58 (1): (in Chinese with English abstract).