Effects of hypobaric hypoxia on adenine nucleotide pools,adenine nucleotide transporter activity and protein expression in rat liver

AIM： To explore the effect of hypobaric hypoxia on mitochondrial energy metabolism in rat liver. METHODS： Adult male Wistar rats were exposed to a hypobadc chamber simulating 5000 m high altitude for 23 h every day for 0 （H0）, 1 （H1）, 5 （H5）, 15 （H15） and 30 d （H30） respectively. Rats were sacrificed by decapitation and liver was removed. Liver mitochondria were isolated by differential centrifugation program. The size of adenine nucleotide pool （ATP ADP, and AMP） in tissue and mitochondria was separated and measured by high performance liquid chromatography （HPLC）. The adenine nucleotide transporter （ANT） activity was determined by isotopic technique. The ANT total protein level was determined by Western blot. RESULTS： Compared with H0 group, intra-mitochondrial ATP content decreased in all hypoxia groups. However, the H5 group reached the lowest point （70.6%） （P〈 0.01） when compared to the control group. Intra-mitochondrial ADP and AMP level showed similar change in all hypoxia groups and were significantly lower than that in H0 group, In addition, extra-mitochondrial ATP and ADP content decreased significantly in all hypoxia groups. Furthermore, extra-mitochondrial AMP in groups H5, H15 and H30 was significantly lower than that in H0 group, whereas HI group had no marked change compared to the control situation. The activity of ANT in hypoxia groups decreased significantly, which was the lowest in H5 group （55.7%） （P〈0.01） when compared to H0 group. ANT activity in H30 group was higher than in H15 group, but still lower than that in H0 group. ANT protein level in H5, H15, H30 groups, compared with H0 group decreased significantly, which in H5 group was the lowest, being 27.1% of that in H0 group （P〈0.01）. ANT protein level in H30 group was higher than in H15 group, but still lower than in H0 group. CONCLUSION： Hypobaric hypoxia decreases the mitochondrial ATP content in rat liver, while mitochondrial ATP level recovers during long-term hypoxia exposure. The lower level of extra-mitochondrial ATP may be related to the decrease of ANT activity during hypoxia exposure.

Adenine nucleotide translocase 2(Ant2)is required for individualization of spermatogenesis of Drosophila melanogaster

Successful completion of spermatogenesis is crucial for the perpetuation of the species.In Drosophila,spermatid individualization,a process involving changes in mitochondrial structure and function is critical to produce functional mature sperm.Ant2,encoding a mitochondrial adenine nucleotide translocase,is highly expressed in male testes and plays a role in energy metabolism in the mitochondria.However,its molecular function remains unclear.Here,we identified an important role of Ant2 in spermatid individualization.In Ant2 knockdown testes,spermatid individualization complexes composed of F-actin cones exhibited a diffuse distribution,and mature sperms were absent in the seminal vesicle,thus leading to male sterility.The most striking effects in Ant2-knockdown spermatids were decrease in tubulin polyglycylation and disruption of proper mitochondria derivatives function.Excessive apoptotic cells were also observed in Ant2-knockdown testes.To further investigate the phenotype of Ant2 knockdown in testes at the molecular level,complementary transcriptome and proteome analyses were performed.At the mRNA level,868 differentially expressed genes were identified,of which 229 genes were upregulated and 639 were downregulated induced via Ant2 knockdown.iTRAQ-labeling proteome analysis revealed 350 differentially expressed proteins,of which 117 proteins were upregulated and 233 were downregulated.The expression of glutathione transferase(GstD5,GstE5,GstE8,and GstD3),proteins involved in reproduction were significantly regulated at both the mRNA and protein levels.These results indicate that Ant2 is crucial for spermatid maturation by affecting mitochondrial morphogenesis.

Effect of electroacupuncture pretreatment on adenine nucleotides in myocardial tissues of rats with myocardial ischemia-reperfusion injury detected by high performance liquid chromatography(HPLC)

Objective:To observe the effect of electroacupuncture(EA)pretreatment on adenine nucleotides in the myocardial tissues of the myocardial ischemia-reperfusion injury(MIRI)rats,and to explore the mechanism of EA pretreatment on myocardial prevention and protection in MIRI rats.Methods:Forty SPF male Sprague-Dawley(SD)rats were randomly divided into 5 groups:a blank group,a sham operation group,a model group,an EA at Neiguan(PC 6)group and an EA at Hegu(LI 4)group,with 8 rats in each group.Rats in the blank group only received binding to the rat plate,30 min/time,once a day for 7 d;on the 7th day,rats in the sham operation group were subjected to threading for 40 min at the left anterior descending coronary artery without ligation,and then the rats were allowed to stand for 60 min before collection of the specimens;on the 7th day,rats in the model group were subjected to threading at the left anterior descending coronary artery with ligation,for 40 min before the blood flow was restored,and then the rats were allowed to stand for 60 min before collection of the specimens;on the 7th day of pretreatment with EA at Neiguan(PC 6)or Hegu(LI 4)for 30 min per day(once a day for 7 d),rats in the EA at Neiguan(PC 6)group and EA at Hegu(LI 4)group were subjected to modeling and sample collection same as in the model group.The left ventricular myocardium of the lower left anterior descending coronary artery was collected from rats in all 5 groups.Hematoxylin-eosin(HE)staining and transmission electron microscope(TEM)were used to observe the changes in myocardial pathological morphology.The change in the adenine nucleotide level of myocardial tissue was measured by high performance liquid chromatography(HPLC).Results:The HE staining and ultrastructure showed that the myocardial injury was severer in the model group compared with the sham operation group.Compared with the model group,the myocardial injury in the EA at Neiguan(PC 6)and the EA at Hegu(LI 4)groups was mild or hardly any.The adenine nucleotide levels in the sham operation group and the model group were all decreased compared with the blank group(all P<0.05);compared with the sham operation group,the adenine nucleotide level of the model group was also decreased,but the difference was not statistically significant(P>0.05);compared with the model group,the adenine nucleotide level in the EA at Neiguan(PC 6)group was increased(P<0.05),and the adenine nucleotide level in the EA at Hegu(LI 4)group was significantly increased(P<0.01).The adenine nucleotide level in the EA at Hegu(LI 4)group was higher than that in the EA at Neiguan(PC 6)group,but the difference was not statistically significant(P>0.05).Compared with the EA at Neiguan(PC 6)group,the levels of adenosine triphosphate(ATP),adenosine diphosphate(ADP)and adenosine monophosphate(AMP)in the EA at Hegu(LI 4)group were significantly increased(all P<0.01).Conclusion:Both EA at Neiguan(PC 6)and Hegu(LI 4)can alleviate the pathological damage to myocardium in MIRI rats,and increase the adenine nucleotide level in myocardial tissues,and thus protect MIRI rats.EA at Hegu(LI 4)has a better protective effect than Neiguan(PC 6).

A therapeutic anti-CD4 monoclonal antibody inhibits T cell receptor signal transduction in mouse autoimmune cardiomyopathy

Background T cell immune abnormalities in patients with dilated cardiomyopathy （DCM） has been intensively studied over the past 10 years. Our previous study has suggested that immunization of mice with the peptides derived from human adenine nucleotide translocator （ANT） result in the production of autoantibodies against the ANT and histopathological changes similar to those in human DCM. The ANT peptides can induce autoimmune cardiomyopathy like DCM in Balb/c mice. In this study we aimed to focus on the molecular mechanism of T cells in the autoimmune cardiomyopathy mouse model by detecting the expression of the two T cell signaling molecules. Methods The ANT peptides were used to cause autoimmune cardiomyopathy in Balb/c mice. Anti-L3T4 or rat anti-mouse IgG was administered to the mice （n=6 in each group） simultaneously immunized with ANT. ELISA analysis was used to detect autoantibodies against the ANT peptides and the percentages of interferon-y and interleukin-4 producing cells among splenic CD4^＋ lymphocytes was determined by using flow cytometry analysis. The expression of CD45 in spleen T cells was determined by immunohistochemistry and the mRNAs of T cell signaling molecules were detected by real-time PCR. Results Treatment of ANT immunized Balb/c mice with anti-CD4 mAb caused a reduction in the gene expression of P561ck and Zap-70 and a lower level of CD45 expression by spleen T cells. Also, a reverse of the Th1/Th2 ratio that results in the reduced production of antibodies against ANT was found in the anti-CD4 monoclonal antibodies （mAb） group. Whereas irrelevant antibody （rat anti-mouse IgG） did not suppress T cell signaling molecules nor inhibit CD45 expression, and control-antibody mice did not show any significant differences compared with the DCM group. Conclusion The results show that anti-CD4 mAb is a powerful inhibitor of the early initiating events of T cell receptor （TCR） signal transduction in mouse autoimmune dilated cardiomyopathy.

Long-Term Exposure to High Corticosterone Levels Inducing a Decrease of Adenylate Kinase 1 Activity

Corticosterone, a principal glucocorticoid synthesized in the rodent adrenal cortex, can be cumula- tively toxic to hippocampal neurons, the cause of which is not known. The present study determined whether the cytosol adenylate kinase （AK） system long-term exposure to high corticosterone levels. We was involved in the neuronal damage induced by nvestigated the effects of long-term exposure to high corticosterone levels on AK1 activity, AK1 mRNA expression, and energy levels in cultured hippocampal neurons. The results show that long-term exposure to high corticosterone levels induces a reduction of the cultured hippocampal neuron viability, significantly reduces energy levels, and causes a time-dependant re- duction of the AK1 activity. These findings indicate that changes in the AK system might be the mechanism underlying neuronal damage induced by long-term exposure to high corticosterone levels.