Effects of palmitic acid on activity of uncoupling proteins and proton leak in in vitro cerebral mitochondria from the rats exposed to simulated high altitude hypoxia / 生理学报

To reveal the roles of uncoupling proteins (UCPs) in disorder of mitochondrial oxidative phosphorylation induced by free fatty acid during hypoxic exposure, the effects of palmitic acid on activity of UCPs, proton leak and mitochondrial membrane potential in hypoxia-exposed rat brain mitochondria were observed in vitro. Adult Sprague-Dawley (SD) rats were set randomly into control, acute hypoxia and chronic hypoxia groups (n=8 in each group). The acute and chronic hypoxic rats were exposed to simulated 5000 m high altitude in a hypobaric chamber 23 h/d for 3 d and 30 d, respectively. The brain mitochondria were isolated by centrifugation. UCP content and activity were detected by [(3)H]-GTP binding method. The proton leak was measured by TPMP(+) electrode and oxygen electrode. The membrane potential of mitochondria was calculated by detecting the fluorescence from Rodamine 123. Hypoxic exposure resulted in an increase in UCP activity and content as well as proton leak, but a decrease in the membrane potential of rat brain mitochondria. Palmitic acid resulted in further increases in UCP activity and content as well as proton leak, and further decrease in membrane potential of brain mitochondria in vitro from hypoxia-exposed rats, but hypoxic exposure decreased the reactivity of cerebral mitochondria to palmitic acid, especially in the acute hypoxia group. There was a negative correlation between mitochondrial proton leak and K(d) value (representing derivative of UCP activity, P<0.01, r = -0.906), and a positive correlation between proton leak and B(max) (representing the maximal content of UCPs in mitochondrial inner membrane, P<0.01, r = 0.856). Cerebral mitochondrial membrane potential was negatively correlated with proton leak (P<0.01, r = -0.880). It is suggested that hypoxia-induced proton leak enhancement and membrane potential decrease are correlated with the increased activity of UCPs. Hypoxia can also decrease the sensitivity of cerebral mitochondria to palmitic acid, which may be a self-protective mechanism in high altitude environment.

Effects of GDP on the activity and expression of mitochondrial uncoupling proteins in rat brain in vitro / 生理学报

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.

Characteristics of adenine nucleotide translocator in mitochondria of rat cerebral cortex during hypobaric hypoxia exposure / 生理学报

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.

Protein synthesis and characterization in isolated mitochondria from rat brain / 中国应用生理学杂志

<p><b>AIM</b>To establish a system of mitochondrial translation in vitro of rat brain and identify it's production of protein by molecular weight.</p><p><b>METHODS</b>Mitochondria isolated from hemisphere of rat brain by differential centrifugations. The optimization of mitochondrial translation in vitro by 3H-Lencine incorporation was explored. 35S-methionine labeled products of mitochondrial protein synthesis were identified by SDS-PAGE and fluorography.</p><p><b>RESULTS</b>Isolated mitochondria had a highly activity oxidative phosphorylation and respiratory control ratio (RCR) was between 3.5 and 5.5. The activity of 3H-Lencine incorporation in isolated mitochondria in vitro increased with time of incubation in 60 min and maintained a steady level. The maximal activity of 3H-Lencine incorporation per milligram mitochondria protein occured at 1 mg mitochondria/ml of incubation mix.The major auto radiographic bands could be observed at 86, 68, 56, 43, 33, 29, 25 and 18(kD) molecular weight separated on SDS-PAGE.</p><p><b>CONCLUSION</b>The translation system of rat brain mitochondria in vitro is faithful and high activity, can be used to study mtDNA expression and regulation in mammalian brain at the level of translation.</p>

Effects of ATP concentration and hypoxic exposure on RNA and protein synthesis activity in isolated mitochondria from rat brain / 生理学报

To explore the effects of ATP concentration in the medium and hypoxia exposure on mitochondrial DNA expression at transcriptional and translational level, rats were exposed to hypoxia in a hypobaric chamber simulating 4000 m above sea level for 3 d (acute hypoxia) or 40 d (chronic hypoxia). Cerebral cortex mitochondria were isolated from control and hypoxia-exposed rats by centrifugation program. The activities of intramitochondrial RNA and protein synthesis were measured respectively by the methods of incorporation of (3)H-UTP or (3)H-Leucine in a cell-free system in vitro in isolated organelle. The effect of different ATP concentrations in medium on incorporation activity of mitochondria from control rat brains was observed. The results showed that there was a 40% reduction in RNA synthesis and a 60% inhibition in protein synthesis in isolated mitochondria in vitro in acute hypoxia exposure compared to control. But in chronic hypoxic exposure, the inhibition of both RNA synthesis and protein synthesis was alleviated, being 72% and 76% of the normoxic control, respectively. Furthermore, the effect of ATP concentration in medium on mitochondrial RNA and protein synthesis in vitro showed two phases. The mitochondrial RNA and protein synthesis were inhibited when ATP concentration was either above or below 1 mmol/L in the incubation medium. These results indicate that hypoxia exposure affects the expression of mtDNA at both transcription and translation levels. It also suggests that the improvement of mitochondrial semi-automation during chronic hypoxic exposure may be at least one of the cellular mechanisms of body adaptation to hypoxia. The regulation of ATP in mitochondrial RNA and protein synthesis is therefore an economic and effective mode of regulation.

Effects of hypoxic exposure on coordinative expression of cytochrome oxidase subunits I and IV in rat cerebral cortex / 生理学报

This study was intended to evaluate the effects of hypoxic exposure on gene expression and coordination of cytochrome oxidase (COX) subunits I (COX I) and IV (COX IV) encoded by mtDNA and nDNA respectively in rat cerebral cortex. Male Wistar rats were exposed to hypoxia in a hypobaric chamber simulating high altitude at 5000 m for 2, 5, 15 and 30 d. Control rats were fed outside the hypobaric chamber (the height was 300 m above sea level). Rats were sacrificed and mitochondria from cerebral cortex were isolated by differential centrifugation at each time point. COX I and COX IV proteins in isolated rat cerebral cortex mitochondria were detected by Western blot analysis and mRNA in the cerebral cortex by RT-PCR. The ratios of protein and mRNA were used to estimate the coordinative expression of two subunits. The results showed that COX I mRNA increased significantly at 2 and 5 d, and decreased to the control level at 15 and 30 d; COX IV mRNA remarkably increased at 2, 5 and 15 d, and dropped below the control level at 30 d. The mRNA ratio of COX IV to COX I reached a peak at 15 d, but showed no differences between other time points. The Western blot analysis of COX I and COX IV in isolated rat cerebral cortex mitochondria showed no obvious changes during hypoxic exposure. Our findings demonstrate that hypoxia can affect mRNA expression of COX I and COX IV and their coordination, while protein expression of both subunits are stable and coordinative. This study suggests that the expression of COX I and COX IV proteins during hypoxic exposure is coordinately regulated by post-transcriptional mechanisms.