Involvement of mtNOS in the Ca2+-induced damages of myocardial mitochondria during the early stage after severe burns

AIM:To study the role of mitochondrial nitric oxide synthase (mtNOS) in the damages of myocardial mitochondria during the early stage after severe burns.METHODS:An experimental model of 30% TBSA full-thickness skin scalding was reproduced in rats. Myocardial mitochondria were isolated from control and burned rats at 1, 3, 6, 12 and 24 h postburn. The mitochondrial respiratory function, content of mitochondrial calcium( [Ca²⁺]_m) and activities of mtNOS and cytochrome c oxidase were determined. RESULTS: (1) Myocardial mitochondrial respiratory control rate(RCR) at 1 h was evidently higher than that of control, but at 3, 6, 12 and 24 h postburn, it was significantly lower than that of the control. The changes in ST₃ is parallel to those of RCR, and ST₄ was evidently increased only at 3 h postburn. (2) [Ca²⁺]_m was higher at all time points postburn and the activity of mtNOS was higher significantly only at 3, 6, 12 and 24 h than that of the control. The activity of cytochrome c oxidase at the 3, 6, 12 and 24 h was low comparing to the control. (3) After severe burns, RCR was negatively correlated with mtNOS activity(r=0.9347, P＜0.05) and mtNOS activity was positive correlated with [Ca²⁺]_m (r=0.8945, P＜0.05). CONCLUSION:The elevation of [Ca²⁺]_m significantly activates mtNOS, which might play an important role in the damages of myocardial mitochondria during the early stage after burn injury.     

Changes of uncoupling proteins activity and the efficiency of oxidative phosphorylation in hypoxic exposed rat brain mitochondria in vitro

AIM: To observe the effect of GDP on uncoupling proteins(UCPs) activity and the efficiency of oxidative phosphorylation in hypoxic exposed rat brain mitochondria.METHODS: Adult SD rats were randomly divided into three groups (control, acute hypoxia and chronic hypoxia groups). The animals were placed into a hypobaric chamber simulated 5 000 m high altitude for 0, 3 and 30 d, respectively. The mitochondria from rat brain were isolated by centrifugation. The activity of UCPs was detected by the method of ［H3］-GTP binding with UCPs specifically. The maximal binding content (Bmax) and the dissociation constant (Kd) were determined by Scatchard plot. The mitochondrial potential was measured by rhodamine 123 method. Oxidative respiratory consumption was measured by Clark electrode. The experiments were conducted under the conditions with or without GDP (1 mmol/L), respectively. RESULTS: For exposed to hypoxia, Bmax and the oxidative consumption of uncoupling respiration were increased. Kd, MMP and RCR were decreased. UCPs activity was inhibited by GDP in three groups. Kd was increased 61.01%, 83.13% and 71.52% and Bmax was decreased 23.18%, 35.20% and 33.38%, respectively. The values in the acute hypoxic group were changed markedly. The sensitivity of UCPs to GDP was elevated significantly by hypoxia. With the reducing of UCPs activity, oxidative consumption of uncoupling respiration was decreased whereas RCR and MMP were increased. The results elucidated increase in the efficiency of oxidative phosphorylation.CONCLUSION: GDP increases the mitochondrial membrane potential and decreases the oxygen consumption of uncoupling respiration in hypoxic exposed rat brain mitochondria by inhibiting UCPs activity. The results suggest that the change in UCPs activity is one of the factors of mitochondrial dysfunction in oxidative phosphorylation induced by hypoxia.     

Changes of the myocardium energy metabolism and adenine nucleotide translocase activity in rats after hypoxic exposure

］AIM: To explore the changes of myocardial energy metabolism and adenine nucleotide translocase (ANT) activity in mitochondria in rats exposed to hypoxia. METHODS: Adult male Wistar rats were exposed to simulated high altitude at 5 000 m for control (0 d), 1 d, 5 d, 15 d, 30 d in hypobaric chamber. Myocardial mitochondria were isolated by centrifugation. Mitochondria respiratory function was measured by Clark oxygen electrode. The size of adenine nucleotides pool (ATP, ADP, AMP) in mitochondria were separated and measured by HPLC. ANT activity was measured by ［3H］-ADP incorporation. RESULTS: Compared to control, mitochondria state Ⅲ respiratory (ST3) and RCR decreased and ST4 increased sharply at 1 d, 5 d and 15 d, ST3 still lower than that in control at 30 d, while RCR level restored. ATP contents and ANT activity decreased at 1 d and 5 d, then restored to control level at 15 d, then decreased again at 30 d. CONCLUSION: The inhibition of mitochondria respiratory function is the main reason that makes ATP contents decrease during hypoxic exposure. ANT activity and ATP content change cooperatively.     

Effect of L-arginine on function and structure of mitochondria in ischemia-reperfusion myocardial cells in rabbits

AIM: To study the effect of L-arginine (L-Arg) on function and structure of mitochondria in ischemia-reperfusion (MRI) myocardial cells. METHODS: Thirty rabbits were randomly divided into three groups (n=10 in each), control group, MIR group and MIR+L-Arg group. The mitochondrial respiratory function, Ca²⁺ concentration ([Ca²⁺]m), malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined. Meanwhile, the contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), total adenylic acid number (TAN) and energy charge (EC) in the myocardial tissue were respectively measured. Moreover, the ultrastructure changes in myocardial mitochondria were observed during MIR. RESULTS: The mitochondrial respiratory control rate (RCR), velocity 3 (V₃), SOD, surface density (Sv) and specific surface (δ) in MIR+L-Arg group were higher than those in MIR group, velocity 4 (V₄), [Ca²⁺]m, MDA, volume density (Vv), horizental diameter (Hd) were lower than those in MIR group. ATP, ADP, TAN and EC levels of myocardial tissue were higher than those in MIR group. There was no significant difference between MIR+L-Arg and control group in V₃, V₄, SOD, MDA, Vv, Sv, δ, Nv, Vd, AMP and TNA. CONCLUSION: It is suggested that L-Arg improves the function and structure of mitochondria in myocardial cells in the reperfusion injury after myocardial ischemia by decreasing oxygen free radical level and Ca²⁺ overload in the mitochondria.     

The relationship between the activity of phospholipase A2 and chronic hypoxic pulmonary hypertension

AIM:To study the relationship between the activity of phospholipase A₂ (PLA₂) and chronic hypoxic pulmonary hypertension. METHODS:29 healthy SD rats were randomly divided into normal control group, chronic hypoxic group and hypoxia plus Polidatin (PD) group. The model of rat chronic hypoxic pulmonary hypertension was made by method of intermittent isobaric hypoxia for 21 days. The mean pulmonary arterial pressure (mPAP) was measured by inserting a microcatheter into the pulmonary artery. RESULTS:After exposing hypoxia for 21 days, the mPAP, R/L+S, the PLA₂ activity, TXB₂, MDA in plasma and lung homogenate increased significantly, while 6-k-PGF_1α, SOD decreased significantly. Pretreatment with PD could relieve the changes mentioned above.CONCLUSION:PLA₂ plays an important inducing role through its metabolic products and the interactional radicals in the formation of chronic hypoxic pulmonary hypertension.     

Effects of urocortin-I preconditioning on myocardial mitochondrial respiratory function and enzyme activity in rats

ATM: To investigate the influence of urocortin-I (Ucn I) preconditioning on the myocardial mitochondrial respiratory function and enzyme activity in the rats with ischemia reperfusion, and to observe the changes of ATP content in the myocardial cells. METHODS: (1) The healthy male Sprague-Dawley rats were randomly divided into 4 groups:normal group (Nor group), ischemia reperfusion group (IR group), Ucn I preconditioning group (Ucn I group), 5-hydroxy acid (5-HD)+Ucn I group. Langendorff perfusion was used to establish the in vitro model of cardiac ischemia reperfusion. At the end of the balance (T₁), before ischemia (T₂) and at the end of the reperfusion (T₃) respectively, the myocardial mitochondria was extracted, the mitochondrial respiratory function and respiratory enzyme activity in each group were determined. (2) The method of MPA isolated heart perfusion was used to isolate myocardial cells of the adult rats. After cultured for 24 h, myocardial cells were divided into 4 groups:Nor group, hypoxia/reoxygenation group (I/R group), Ucn I group, 5-HD+Ucn I group. Hypoxia/reoxygenation model of myocardial cells was established. At the end of reoxygenation, the changes of myocardial ATP content were measured by high performance liquid chromatography.RESULTS: (1) Compared with T₁, T₂ time points, the respiratory function (state 3 respiratory rate, respiratory control rate) and NADH oxidase, succinate oxidase and cytochrome C oxidase activities at T₃ time point were significantly decreased (P<0.05) in all groups except Nor group. At T₃ time point, the myocardial mitochondrial respiratory function and respiratory enzyme activity in Ucn I group were superior to 5-HD+Ucn I group and IR group (P<0.05), but was inferior to Nor group (P<0.05). At T₃ time point, the respiratory function of myocardial mitochondria and respiratory enzyme activities (NADH oxidase, succinate oxidase) in 5-HD+Ucn I group were better than those in IR group (P<0.05), but no statistical difference of the cytochrome C oxidase activity between the 2 groups was observed. The respiratory function and 3 kinds of respiratory enzyme activities at T₁, T₂ time points had no statistical change. (2) At the end of the reoxygenation, the myocardial ATP content in Nor group was higher than that in other groups (P<0.01). The myocardial ATP contents in I/R group and 5-HD+Ucn I group were lower than that in Ucn I group (P<0.05). In additon, 5-HD+Ucn I group was higher ATP content compared with I/R group (P<0.05). CONCLUSION: Ucn I preconditioning attenuates the ischemia/reperfusion induced damages of myocardial mitochondrial respiratory function and respiratory enzyme activity, thus ensuring the myocardial ATP contents under the condition of hypoxia/reoxygenation.     

Effects of acute and chronic hypoxia on rat brain mitochondrial translation activity

AIM: To investigate the effect of simulated high altitude hypoxia on rat brain mitochondrial translation activity. METHODS: Animals were continuously exposed to simulated 4 000 m high altitude in hypobaric chamber for three days and forty days. Mitochondria of rat brain were isolated by homogenizing brain tissue and following centrifuging program. Protein translation activity in isolated mitochondria in vitro was measured with [³H]-Lencine incorporation method. Products labeled with [³⁵S]-methionine in isolated mitochondrial protein synthesis system in vitro were separated on SDS-PAGE and identified by autoradiography. RESULTS: Mitochondrial translation activity in vitro in acute hypoxia exposure were significantly lower than control(P< 0.01). Mitochondrial translation activity in chronic hypoxia exposure were significantly higher than that in acute hypoxia exposure(P< 0.05), but showed no statistical difference between chronic and control. No differences in electrophoretic-autoradiography pattern of -methionine incorporation into translational products was observed among three groups. CONCLUSION: These results indicated that acute hypoxia exposure depressed synthesis of mitochondrial protein encoded by mitochondrial DNA in vitro and the changes in mitochondrial protein synthesis were related with hypoxia exposure time.     

Effects of repeated hypoxic exposures on saturation oxygen,mitochondrial function and ATP levels in the mouse brain

AIM: To study the effects of repeated hypoxic exposures on saturation oxygen and mitochondrial function in the brain. METHODS: A BALB/C mouse was placed in an air-sealed jar and hypoxic environment in the jar was established through consumption of the oxygen by respiration of the mouse. A gasp breath was regarded as the hypoxic tolerant limit of the mouse. The mouse was then transferred to a new jar. The mouse was exposed to hypoxia in this way for 5 times. In each run of hypoxic exposure, the hypoxic tolerance time, local brain saturation oxygen was measured. By the end of each hypoxic exposure, the mitochondrial complex I activity and ATP levels in the brain were also examined. RESULTS: The hypoxic tolerant time of a mouse increased during repeated hypoxic exposures. The saturation oxygen in the brain declined quickly during the first two hypoxic exposures, but recovered to the normal levels after a short fall during the third to fifth hypoxic exposures. The mitochondrial complex I activity decreased during repeated hypoxic exposures. The ATP levels declined by the end of the first hypoxic exposure, but increased by the end of the third and fifth hypoxic exposure. CONCLUSION: Repeated hypoxic exposures lead to increased oxygen saturation and ATP levels but decreased mitochondrial function. The increased ATP levels may be responsible for the increased hypoxic tolerance of the mouse brain.     

Role of K+ channels in attenuation of pulmonary vasoreactivity following chronic hypobaric hypoxia in rats

AIM and METHODS: To determine the role of different K⁺-channels in attenuation of vasoreactivity of intrapulminary artery rings induced by chronic hypoxia. RESULTS: ①Acute hypoxia-induced pulmonary vasoconstriction (HPV) could be significantly attenuated by chronic hypobaric hypoxia for 15 days and for 30 days. ②HPV could be significantly potentiated by ATP-sensitive K⁺-channel (K_ATP) blocker or Ca²⁺-activated K⁺-channel (K_Ca) blocker, and the potentiated scope in chronic hypoxic group was much higher than that observed in control group. ③Delayed rectifier K⁺-channel (K_DR) blocker showed no effect on HPV in both control group and chronic hypoxic group. CONCLUSION: Both K_ATP and K_Ca play an important modulating role in HPV and its potentiation may be a critical mechanism for the attenuated vasoreactivity to acute hypoxia following chronic hypobaric hypoxia.     

Effects of repeated hypoxia on the NPY-like immunoreactivity in the mouse brain

AIM:To observe the effects of repeated hypoxia on the neuropeptide Y(NPY)-like immunoreactivity in the mouse brain. METHODS:Forty male kunming mice were divided into 5 groups: Control, H₁(after the 1^sthypoxic run), H₂(the 2^nd hypoxic run), H₃(the 3^rdhypoxic run)and H₄(the 4^th hypoxic run). The hypoxic groups were subjected to different runs of hypoxia exposure. The NPY-like immunoreactivity in the moue brain was measured by using radioimmunoassary method.RESULTS: The standard tolerance time of the mouse exposed to hypoxia significantly increased following each increase in runs of hypoxia exposure. After the 1^st and 2^nd hypoxic run the NPY-like immunoreactivities in the mice brain significantly increased by 145.5%±3.2% and 147.3%±2.5% compared with the control(P＜0.01), respectively. However, the NPY-like immunoreactivites returned to the control levels after the 3^rd run. CONCLUSION:The repeated exposure to hypoxia can significantly enhance mouse's tolerance to hypoxia by preconditioning, and can induce the increase by only one exposure in NPY-like immunoreactivities of the mouse brain during the early period of formation of hypoxic preconditioning.     

Effect of polidatin on the activity of phospholipase A2and pulmonary structural remodeling in rats with hypoxia-induced pulmonary hypertension

AIM: To study the relationship between the activity of phospholipase A₂ (PLA₂) and pulmonary structural remodeling with the model of chronic isobaric hypoxic pulmonary hypertension. METHODS: 29 healthy Sprague-Dawley rats were randomly divided into normal control group, chronic hypoxic group and hypoxia plus Polidatin (PD) group. By diameter, the arteries were divided into two groups: arteries of group I (30 μm-100 μm) and group II (101 μm-200μm). The mean pulmonary arterial pressure (mPAP) was measured by inserting a microcatheter into the pulmonary artery. The PLA₂ activity was measured with modified microtitrimetic method. The pulmonary tissue and arterioles morphology changes were examined under light microscope. RESULTS: It was found that after 21 days hypoxia, the mean pulmonary arterial pressure (mPAP), the PLA₂ activity in blood and lung homogenate increased significantly. The media thickness of group I arteries increased (P<0.01) while that of group II arteries had no significant changes. The ratio of media area and adventitia of both groups was raised. Under light microscope, it was observed that pulmonary vascular endothelium proliferated, media became thickening and adventitia matrix increased. Pretreatment with PD could attenuate the changes mentioned above. CONCLUSION:PLA₂ plays an important inducing role through promotion of the pulmonary vascular structural remodeling in the formation of chronic hypoxic pulmonary hypertension.     

甜瓜抗蔓枯病基因Gsb-4 的分子标记

 以甜瓜（Cucumis melo L.）抗蔓枯病自交系PI482398（含抗蔓枯病基因Gsb-4）和感病自交系‘白皮脆’以及它们的F₁、BC₁P₁、BC₁P₂、F₂ 群体为材料,苗期进行蔓枯病菌（Didymella bryoniae）接种鉴定,结果表明甜瓜抗蔓枯病基因Gsb-4 为单显性遗传。利用集团分离分析法（bulked segregant analysis,BSA）对89 对SSR 引物进行筛选,引物CMTA170a 在抗性材料中可扩增出约为120 bp 的条带,并与抗性基因Gsb-4 表现出连锁关系。统计了CMTA170a 在118 个F₂ 单株上的多态性,并利用MAPMAKER/Exp version 3.0b 软件进行了计算,其与Gsb-4 的遗传连锁距离为5.14 cM。     

Effect of chronic hypoxia on [Ca2+]iin pulmonary artery endothelial cells and smooth muscle cells under acute hypoxia

AIM AND METHODS: Using Ca²⁺-sensitive fluorescent probe Fura-2,we measured the changes of [Ca²⁺]_iin cultured rat pulmonary artery endothelial cells (PAEC) and porcine pulmonary artery smooth muscle cells (PASMC) from normoxic (NC group) or chronic hypoxic group (CH group) when they were exposed to acute hypoxia. RESULTS: The increase in [Ca²⁺]_iin 6th passage of PASMC caused by acute hypoxia in CH group was significantly lower than that in the same passage of NC group (P<0.05).On the contrary, in PAEC, the acute hypoxia induced increase in _i, which was significantly higher in 5th passage of CH group than that in NC group (P<0.05). CONCLUSION: The decrease of the elevation of [Ca²⁺]_icaused by acute hypoxia in PASMC of CH group indicated that it functioned to lower the constrictive response to hypoxia.The intensive increase in [Ca²⁺]_icaused by acute hypoxia in PAEC of CH group might lead to more relaxing factors derived from PAEC,which results in a decrease in HPV.     

Role of K+ channels in hypoxic pulmonary vasoconstriction in normal and chronic hypoxic rats

AIM:To investigate the role of K⁺ channels in the decreased hypoxic pulmonary vasoconstriction(HPV) in chronic hypoxic rats. METHODS:Blockers of three kinds of K⁺ channels, 4-AP(voltage dependent K⁺ channel blocker), TEA(Ca²⁺ activated K⁺ channel blocker), GLIB(ATP sensitive K⁺ channel blocker) were used in isolated perfused rat lungs to detect the role of K⁺ channels in HPV. RESULTS:In normal rats, 4-AP and TEA, but not GLIB, both elicited a significant increase in pulmonary artery baseline pressure, and also potentiated the acute hypoxic pulmonary vasoconstriction. In chronic hypoxic rats, the HPV is significantly decreased, while 4-AP, TEA, GLIB all elicited a significant but smaller increase in pulmonary artery baseline pressure. Additionally, all these three blockers potentiated the HPV stronger in chronic hypoxic rats than in control rats. CONCLUSION:The opening of Kv, K_Ca, K_ATP might modulate the hypoxic pulmonary vasoconstriction in isolated rat lungs, and the increase in this modulation by potassium channel in chronic hypoxic rats might play a role in its decrease in HPV.     

Changes of expressive proteome of renal tissues in chronic intermittent hypoxic rats

AIM: To obtain the two-dimensional gel electrophoresis maps of renal tissue proteins for identifying the differentially expressed proteins in the chronic intermittent hypoxic rats. METHODS: The rat model of chronic intermittent hypoxia was established. The rats lived in the normoxia environment were used for control. The proteins in the renal tissues underwent two-dimensiona1 gel electrophoresis with immobiline pH gradient isoelectric focusing as the first and vertical SDS-PAGE as the second dimension. Analysis of 2-DE maps was performed to determine the differential expression of proteins between the two groups by the software of ImageMaster 2D Platinum V5.0, in which 4 protein spots expressed differentially were picked up for further identification by MALDI-TOF-MS. RESULTS: Matched and compared with those in control group, 112 protein spots were determined in chronic intermittent hypoxia group. By MALDI-TOF-MS, 4 protein spots with the highest differentially expressive levels were identified as ATP synthase delta subunit mitochondrial precursor, hexokinase, catechol O-methyltransferase and apurinic/apyrimidinic endonudease/redox factor-1. The functions of these identified proteins are involved in cellular energy metabolism, apoptosis, signal transduction, anti-cell injury and hormone metabolism. CONCLUSION: There are obvious differences in expressive proteomes in renal tissue between normoxic and chronic intermittent hypoxic rats. Proteomics can serve as a new approach in the study of obstructive sleep apnea-hypopnea syndrome for discovering new therapeutic targets.     

Moderate hypothermia post hypoxia-ischemia improves the cerebral energy metabolism in the immature rats

AIM: To study the effect of moderate hypothermia on immature rats with hypoxic-ischimic brain damage (HIBD). METHODS: The rats with HIBD were divided into normothermic recovered group (IN) and moderate-hypothermic recovered group (IH). Sham-operated rat pups were normothermic control group (NC) and moderate-hypothermic control group (HC). 0, 2, 6, 24, 48, 72 h after the end of hypoxic-ischemic (HI) insult, the brain was homogenized for measuring glucose and ATP, brain mitochondria was extracted for SDH activity, complex II activity and the capacity of ATP synthesization. RESULTS: In IN group, the brain glucose was significantly lower at 0 h, and recovered as normal at 2 h. The brain ATP and brain-mitochondrial SDH activities were firstly decreased at 2 h, 6 h and then recovered gradually, it was at it's peak value at 72 h. Brain-mitochondrial complex II activity and the capacity of ATP synthesis were recovered at 2 h, but they decreased again at 6 h and came to normal level at 72 h. In moderate-hypothermic group, all the indexes were significantly higher at all the time point than that in IN groups. CONCLUSION: Moderate hypothermia inhibits the decrease in the mitochondrial SDH activity, mitochondrial complex II activity and the capacity of ATP synthesis, increases the brain ATP concentration, improves the energy metabolism, and then protects the brain tissue.     

The effect of L-arginine on protein kinase C of isolated pulmonary arterial rings from rats with hypoxia-induced pulmonary hypertension

AIM: To elucidate whether the mechanism that L-arginine can relieve hypoxia pulmonary hypertension involves inhibition of the activity of protein kinase C(PKC).METHODS: Twenty-one male Wistar rats were randomly divided into NS control, hypoxia and L-arginine(500 mg·kg^-1·d^-1, ip) treatment groups. After two-weeks treatment, mean pulmonary artery pressure (mPAP), mean systematic artery pressure (mSAP) and the ratio of the weight of right ventricle to that of left ventricle plus septum were measured, then two pulmonary arterial rings were isolated to be exposed to PDBu(a specific activator of PKC ) and observed: (1) The maximal response (P₁) to 500 nmol/L PDBu, the time required to achieve a half-maximal response to 500 nmol/L PDBu (t_1/2), the time during which the maximal response to 500 nmol/L PDBu maintained (T) and the isometric responses at different times (2, 4, 8, 12, 20, 40, 60, 80 min). The isometric response was represented as the percentage of the maximal response (P₀) of the same arterial ring to 5μmol/L phenylephrine hydrochloride (P₀%). (2) Dose-response curve in response to PDBu (10-11 000 nmol/L) and the dose producing a half-maximal response in the curve (EC₅₀). RESULTS: mPAP, RV/(LV+S), P₁, T and the isometric responses at 2, 4, 8, 20 min of NS control and L-arginine treatment groups were lower than those of hypoxic group respectively (P＜0.05), while t_1/2 and EC₅₀ were all greater than those of hypoxic group respectively (P＜0.05).CONCLUSION: The activity of PKC was augmented when rats were exposed to two-weeks normobaric hypoxia, which resulted in the increased reactivity of the isolated pulmonary arterial rings. L-arginine can inhibit the activation of PKC, which is likely part of the mechanism by which L-arginine can reduce mPAP and relieve the hypertrophy of right ventricle.     

Hypoxia facilitates rapid pacing-induced calcium transient alternations in ventricular myocytes

AIM:To explore the effect of hypoxia on rapid pacing-induced calcium transient alternations in ventricular myocytes.METHODS:Ventricular myocytes were isolated from the heart of adult SD rats and cultured in serum-free hypoxic fluid to set up an in vitro model of hypoxia-induced cardiac injury. The calcium transient and its alternations were investigated under confocal laser scanning microscope. The mitochondrial function was also examined by WST-8 kit. RESULTS:Under normoxic condition, the ventricular myocytes were claviform. Low frequency of pacing, ranging from 60 to 240 min^-1, induced calcium transient, but not calcium transient alternations, which was elicited by the pacing over a threshold frequency of(288 ±27)min^-1. Exposure of the ventricular myocytes to hypoxia did not obviously affect the morphology of the cells, but reduced the threshold frequency of pacing to(227±26) min^-1(P<0.05). Additionally, exposure of the cells to hypoxia repressed the activity of mitochondrial dehydrogenase from(100.2±8.7)%(control group) to(57.6±7.5)%, which was partially blocked by L-type Ca²⁺ channel inhibitor. CONCLUSION:Hypoxia facilitates calcium transient alternations induced by rapid pacing, and the calcium transient alternations are involved in the hypoxia-injured mitochondria function.     

Effect of rapamycin on apoptosis of mouse astrocytes in vitro

AIM: To observe the effect of rapamycin on the apoptosis of mouse astrocytes in vitro.ME-THODS:The astrocytes from C57BL/6J newborn mouse pups were isolated and primarily cultured. The effect of rapamycin on the viability of astrocytes was assessed by MTT assay. The mean fluorescence intensity of SYTOX^® Green stain in the astrocytes was detected by fluorescence microplate reader in order to analyze the effects of rapamycin on the cell death induced by H₂O₂, ionomycin and/or deferorxamin. DiOC₆(3) staining was used to analyze the mitochondrial membrane potential of the astrocytes induced by H₂O₂. Flow cytometry analysis was used to determine the production of ROS in the astrocytes and mitochondria by staining with H₂DCFDA and MitoSOX^TM Red reagent, respectively.RESULTS: Rapamycin at concentration of 0.5 μmol/L protected the astrocytes against cell death induced by H₂O₂ or deferoxamine plus ionomycin. Rapamycin protected the mitochondrial membrane potential of astrocytes from the injury of H₂O₂. It also reduced the production of ROS in the astrocytes and decreased the level of ROS in the mitochondria.CONCLUSION: Rapamycin reduces the ROS overload in the mitochondria, keeps mitochondrial membrane potential safety and protects the astrocytes against apoptosis in vitro.