大鼠脑缺血诱导的细胞色素c的释放和Bcl-2表达的上调

利用全脑缺血模型,采用免疫印迹和免疫沉淀方法,探讨N-甲基-D-天冬氨酸受体和L-型电压门控钙通道拮抗剂对细胞色素c从线粒体中的释放和Bcl-2的表达变化影响。缺血/复灌后24h,线粒体中细胞色素c明显降低而胞浆中细胞色素c的成分相应增加。Bcl-2的表达呈时间依赖性,其表达在缺血/

Acta Physiologica Sinica, April 25, 2004, 56(2): 147-152ZHANG Chun-Yi et al: Release of Cytochrome c and Bcl-2 Expression in Ischemic Insult

147

Ischemia-induced release of cytochrome c from mitochondria and up-regulation of Bcl-2 expression in rat hippocampus

ZHANG Chun-Yi, SHEN Wan-Hua, ZHANG Guang-Yi*

Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, Xuzhou 221001, China

Abstract: To evaluate the effects of different antagonists on the release of cytochrome c from mitochondria to cytosol and the expressionof Bcl-2 in mitochondria in rat hippocampus after ischemia, we examined Bcl-2 and cytochrome c expression by immunoblotting using 4-vessel occlusion (4-VO) as brain ischemia model. The results showed that after 24 h ischemia/reperfusion (I/R) cytochrome c decreasedmarkedly in mitochondria, which was correspondingly increased in the cytosolic fraction. Bcl-2 expression was time-dependent, reachingits peak level after 6 h I/R. In all those samples, there were no alterations in the subcellular distribution of cytochrome oxidase, amitochondrial respiratory chain protein. The decreases in Bcl-2 and cytochrome c in mitochondria were restored by pretreatment withnon-competitive NMDA receptor antagonist ketamine or L-type voltage-gated Ca2+ channel (L-VGCC) antagonist nifedipine at 20 minprior to ischemia. The results demonstrate that the release of cytochrome c from mitochondria to cytosol and the up-regulation of Bcl-2are possibly mediated by NMDA receptors or L-VGCC following brain ischemia. Cytochrome c release may be injurious while Bcl-2 up-

regulation may be protective to ischemic hippocampus.

Key words

: brain ischemia; cytochrome c; Bcl-2;

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In recent years attention has been paid to the partici-pation of mitochondria in the mechanism of cell deathor apoptosis. Cytochrome c, a water-soluble peripheralmembrane protein of the mitochondria, is known to bean essential component of the mitochondrial respiratorychain[1]. Early in the apoptosis, mitochondria releasescytochrome c from the mitochondrial intermembrane spaceto cytosol and forms a complex with another molecule,apoptotic protease-activating factor-1 (Apaf-1)[2~4], an in-active preform of caspase-9. In the presence of dATP orATP, this complex processes and activates other caspases,in particular, caspase-3,-6,-7[5,6]. The Bcl-2 gene encodes

Received 2003-06-30 Accepted 2003-10-27

This work was supported by the National Nature Science Foundation of China (No. 30070182).

*Corresponding author. Tel: +86-516-5748423; Fax: +86-516-5748423; E-mail: gyzhang@

利用全脑缺血模型,采用免疫印迹和免疫沉淀方法,探讨N-甲基-D-天冬氨酸受体和L-型电压门控钙通道拮抗剂对细胞色素c从线粒体中的释放和Bcl-2的表达变化影响。缺血/复灌后24h,线粒体中细胞色素c明显降低而胞浆中细胞色素c的成分相应增加。Bcl-2的表达呈时间依赖性,其表达在缺血/

148

a 26-kD membrane-associated protein that is located inmultiple subcellular sites, including mitochondria, endoplas-mic reticulum, and nuclear membrane[7]. Overexpressionof Bcl-2, an apoptosis suppressor, blocks cytochrome cefflux in mitochondria induced by a variety of stimuli. Theexpression of Bcl-2 acts to inhibit cytochrome c, therebyblocking caspase-3 activation and apoptotic process, notonly in a cell-free system, but also in intact cells[8,9]. More-over , the Bcl-2 protein renders cells less susceptible toapoptotic stimuli, so the expression of Bcl-2 in neural celllines markedly inhibits cell death induced by L-glutamate,free radicals, Ca2+-ionophores, hypoglycemia or glutathionedepletion[10,11].

Ischemic insults may result in overstimulation ofglutamate receptors and L-type voltage-gated Ca2+ channel(L-VGCC) which then mediate different kinds of down-stream protein expressions[12]. Modification of the NMDAreceptors during hypoxia/ischemia leads to increased in-tracellular calcium and is associated with increased gen-eration of oxygen free radicals[13,14]. Mitochondria, whichundergo harmful Ca2+-loading after NMDA receptoractivation, has an important signaling function in apoptosis[8,9,15]

. Glutamate receptor antagonists, calcium-stabilizingagents and antioxidants have been proven effective in re-ducing ischemic damage to neuron[16]. Ketamine is a volt-age-dependent antagonist of NMDA receptor ion-channelsthat can attenuate the ischemia-induced increase in intrac-ellular calcium influx, while nifedipine can directly inhibitintracellular calcium influx.

Our present studies suggest that global ischemia in-duces the release of cytochrome c and the up-regulationof Bcl-2 protein. Ketamine and nifedipine inhibit the releaseof cytochrome c, but up-regulate Bcl-2 expression in theischemic hippocampus. These results suggest that cyto-chrome c release and Bcl-2 up-regulation may be mediatedby NMDA receptors or L-VGCC and that pharmacologi-cal modulation of cytochrome c release or Bcl-2 expres-sion may become a new strategy to interfere with neuronaldamage.

1 MATERIALS AND METHODS

1.1 Induction of ischemia. Adult male Sprague Dawley(SD) rats (purchased from Sippr-BK Experimental Ani-mal Ltd Co, Shanghai, Grade¢ò, Certificate No D52)weighing 250~300 g were subjected to transient brainischemia by 4-VO[17]. The rats were anesthetized withchloral hydrate and both vertebral arteries were occludedpermanently by electrocoagulation. On the next day, both

Acta Physiologica Sinica, April 25, 2004, 56(2): 147-152

carotid arteries were occluded with aneurysm clips for15 min. The rats that lost their righting reflex within 30s and whose pupils were dilated and unresponsive tolight were selected for the experiments. Electroencepha-logram (EEG) was monitored to ensure isoelectricity.Rectal temperature was continuously monitored and keptat 37~37.5ºC using a heating pad and a temperature-regulated heating lamp. After 15 min occlusion, bloodflow was restored by loosing aneurysm clips. A shamoperation was performed in additional animals using thesame anesthesia and surgical exposure procedures ex-cept that the arteries were not occluded. Ketamine, nife-dipine (Sigma, St. Louis, USA) or dissolvent were ad-ministered to the rats by abdominal injection 20 min be-fore occlusion.

1.2 Brain tissues preparation and drug treatment. At dif-ferent times of reperfusion, animals were killed by de-capitation and hippocampi were removed quickly. Pro-tein extraction of both mitochondrial and cytosolic frac-tion was performed as described with some modifica-tions[2]. Tissues were homogenized in ice cold suspen-sion buffer (pH 7.5) which contained: (in mmol/L)HEPES-KOH 20, sucrose 250, KCl 10, MgCl2 1.5, EDTA1, EGTA 1, dithiothreitol 1, phenylmethylsulfonyl fluo-ride (PMSF) 0.1; (in µg/ml) aprotinin 2, leupeptin 10,pepstatin A 5 and N-acetyl-leu-leu-norleucinal 12.5. Thehomogenates were first centrifuged at 800 g for 10 min,and then at 14000 g for 20 min at 4ºC. Pellets were usedas the mitochondrial fraction, the supernatant was fur-ther centrifuged at 100000 g for 1 h. Protein concentra-tions were determined by the method of Lowry et al.[18].1.3 Western blot. The proteins in mitochondria and cy-tosol were separated by 15% SDS-PAGE and were elec-trotransferred onto nitrocellulose membrane (AmershamPharmacia) by the method of Sambrook et al.[19] andours[20]. Membrane was probed with the indicated anti-bodies at 4ºC overnight. The primary antibodies wereeither a 1:1000 dilution of rabbit cytochrome c and Bcl-2 polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA)or 1 µg/ml cytochrome oxidase subunit (COX ,20E8C12) monoclonal (Molecular Probes, Eugene, OR,USA). Detections were carried out by alkaline phophataseconjugated sheep anti-rabbit IgG or sheep anti-mouseIgG (Sigma, 1:10000) and developed using NBT/BCIPcolor substrate (Promega, Madison, USA). Afterimmunoblot, the bands on the membrane were scannedand analyzed with an image analyzer (LabWorksSoftware, UVP upland, CA).

利用全脑缺血模型,采用免疫印迹和免疫沉淀方法,探讨N-甲基-D-天冬氨酸受体和L-型电压门控钙通道拮抗剂对细胞色素c从线粒体中的释放和Bcl-2的表达变化影响。缺血/复灌后24h,线粒体中细胞色素c明显降低而胞浆中细胞色素c的成分相应增加。Bcl-2的表达呈时间依赖性,其表达在缺血/

ZHANG Chun-Yi et al: Release of Cytochrome c and Bcl-2 Expression in Ischemic Insult

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1.4 Statistical analysis. All data are expressed as mean±SDfrom three independent animals. Statistical analysis ofthe results was carried out by one-way analysis of vari-ance (ANOVA) followed by the Duncan’s new multiplerange method or Newman-Keuls test. The difference wasconsidered to be significant when the P value was lessthan 0.05.

2 RESULTS

2.1 Ischemia-induced release of cytochrome c frommitochondria to cytosol in rat hippocampus after is-chemia/reperfusion

We examined cytochrome c at protein levels in normaland ischemic brains, focusing on the hippocampus wherecells are particularly vulnerable to transient global ischemia.As shown in Fig.1A and B, cytochrome c immunoreactiv-ity was evident as a single 15 kD band, detected by West-

ern blot analysis. No cytochrome c was detected in thecytosolic fraction of the hippocampal region in sham con-trol group. In the ischemia group, a cytochrome c-spe-cific band was observed in the cytosolic fraction from 24h after 15 min ischemia, which was correspondingly de-creased after I/R in the mitochondrial fraction. COX was strongly expressed in the mitochondrial fraction anddid not decrease after I/R (Fig. 1C), and virtually no bandwas seen in the cytosolic fraction in both control and is-chemic hippocampus (Fig. 1D).

2.2 Ketamine and nifedipine effect on ischemia-in-duced release of cytochrome c

To determine the effects of ketamine and nifedipine onthe release of cytochrome c in rat hippocampus after tran-sient global ischemia, ketamine (12.5~50 mg/kg) ornifedipine (10~30 mg/kg) was intraperitoneally injected at20 min prior to ischemia. As shown in Fig.2A and B

, the

Fig. 1. Western blot analysis of mitochondrial cytochrome c and cytochrome oxidase subunit (COX ). A: Western blot analysis ofmitochondrial cytochrome c in rat hippocampus following 24 h ischemia/reperfusion (I/R) after 15 min ischemia. B: Western blot analysisof cytosolic cytochrome c. C: Western blot analysis of mitochondrial COX . D: Western blot analysis of cytosolic COX . From the left,the control and I/R for 24 h in rat hippocampus are shown respectively.

Fig. 2. Antagonists of NMDA receptors and L-VGCC effect on release of cytochrome c. A, B: Western blot analysis of mitochondrialcytochrome c in the hippocampus after 24 h I/R. Different concentrations of ketamine (A) or nifedipine (B) were administered to rats byabdominal injection 20 min before ischemia and hippocampi were removed at 24 h of reperfusion. Dissolvents (0.9% NaCl or DMSO) werealso given in the same way as control. C, D: Western blot analysis of mitochondrial and cytosolic COX . From the left, hippocampus ofthe control, 24 h, vehicle (0.9 % NaCl or DMSO), KT (12.5, 25, 50 mg/kg) or ND (5, 10, 20 mg/kg). E, F: Data are expressed as mean±SDfrom three independent animals (n=3). aP<0.05 vs control, bP<0.05 vs 24 h I/R+vehicle, cP<0.05 vs

KT12.5 or ND5.

利用全脑缺血模型,采用免疫印迹和免疫沉淀方法,探讨N-甲基-D-天冬氨酸受体和L-型电压门控钙通道拮抗剂对细胞色素c从线粒体中的释放和Bcl-2的表达变化影响。缺血/复灌后24h,线粒体中细胞色素c明显降低而胞浆中细胞色素c的成分相应增加。Bcl-2的表达呈时间依赖性,其表达在缺血/

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release of cytochrome c from mitochondria was suppressedin the ketamine or nifedipine-pretreated animals. COX was strongly expressed in the mitochondrial fraction andhad no changes after I/R (Fig. 2C, D).

2.3 Ketamine and nifedipine effect on decreased ex-pression of Bcl-2 after ischemia

As shown in Fig.3A, Bcl-2 expression in hippocampus

Fig. 3. Antagonists of NMDA receptors and L-VGCC effect onBcl-2 expression. A: Western blot analysis of the expression of Bcl-2 in hippocampus following ischemia. From the left, control (=1), 6h (1.53±0.32), 12 h (4.38±0.14), 24 h (0.68±0.234), 72 h (0.43±0.19) are shown respectively. B: Different concentrations of KT wereadministered by abdominal injection 20 min before ischemia andhippocampi were removed at 24 h of reperfusion. Control of 0.9 %NaCl was also given in the same way. As shown from the left, theexpression of Bcl-2 protein after 6 h I/R, 24 h I/R or 0.9 % NaCl, KT(12.5, 25, 50 mg/kg) was analyzed by Western blot. C: Data areexpressed as mean±SD from three independent animals (n=3). aP<0.05 vs I/R 6 h, bP<0.05 vs I/R 24 h+vehicle, cP<0.05 vs KT12.5.D: Western blot analysis of the expression of Bcl-2 protein, asshown from the left, I/R 6 h, I/R 24 h and DMSO, ND (5, 10, 20 mg/kg). E: Data are expressed as mean±SD from three independentanimals (n=3). aP<0.05 vs I/R 6 h, bP<0.05 vs I/R 24 h+vehicle, cP<0.05 vs ND5.

Acta Physiologica Sinica, April 25, 2004, 56(2): 147-152

exhibited a low expression in non-ischemic brain, but itsexpression was induced by cerebral ischemia and its levelincreased at 6 h (4.4-fold) after 15 min of ischemia, thendecreased in a time-dependent manner. At 72 h after is-chemia , Bcl-2 protein expression was barely detectable.For determination of the effects of ketamine and nife-dipine on expression of Bcl-2 in rat hippocampus aftertransient global ischemia, ketamine (12.5~50 mg/kg) andnifedipine (10~30 mg/kg) were injected intraperitoneally20 min before ischemia. In the pretreated animals, Bcl-2was clearly up-regulated in rat hippocampal mitochondria(Fig. 3B, D).

3 DISCUSSION

Attention has been paid to cytochrome c because of arecent cell-free study suggesting its critical role in apoptosis.It is reported that mitochondria are involved in apoptosisby releasing cytochrome c to the cytoplasm where it acti-vates caspase-3, a molecule of the interleukin-1β-convert-ing enzyme (ICE) family[2,6]. On the other hand, the proto-oncogene Bcl-2 plays a key role in regulating programmedcell death in neurons. Bcl-2 is altered in cerebral ischemiaand traumatic brain and acts to inhibit cytochrome c trans-location[8,9], prevents the activation of caspases, inhibitsfree radical formation, regulates calcium sequestration, andblocks the pro-apoptotic actions of other members of theBcl-2 family such as Bax and Bad[21,22]. However, aftercytochrome c is released, Bcl-2 overexpression can notprevent subsequent caspase activation and cell apoptosis,so Bcl-2 anti-apoptotic function acts primarily at the levelof mitochondria[22].

Our studies show that a significant amount of cyto-chrome c in mitochondrial was detected in non-ischemicbrain, which decreased in ischemic brain after 24 h I/R,while the cytosolic fractions from the same samples showeda significant increment. COX , a mitochondrial markerprotein, was not detected in the cytosolic fraction, indi-cating that the detected cytosolic cytochrome c did notresult from mitochondrial damage during preparation ofthe cytosolic fraction. Fifteen minutes of global ischemiainduced a change in Bcl-2 protein expression at hippoc-ampal region from 6 h to 72 h. Our studies also show thatBcl-2 protein is weakly expressed in non-ischemichippocampus, which was induced by cerebral ischemiaand reached its peak level as early as 6 h after I/R. Whenthe neuronal damage was getting more and more severewith time, Bcl-2 expression decreased rapidly, which was

rarely detected at 72 h after I/R. Immunocytochemical

利用全脑缺血模型,采用免疫印迹和免疫沉淀方法,探讨N-甲基-D-天冬氨酸受体和L-型电压门控钙通道拮抗剂对细胞色素c从线粒体中的释放和Bcl-2的表达变化影响。缺血/复灌后24h,线粒体中细胞色素c明显降低而胞浆中细胞色素c的成分相应增加。Bcl-2的表达呈时间依赖性,其表达在缺血/

ZHANG Chun-Yi et al: Release of Cytochrome c and Bcl-2 Expression in Ischemic Insult

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studies have shown that Bcl-2 expression is inducedprimarily in living neurons after ischemia[23,24].

The biochemical and cellular events that lead to ischemicneuronal degeneration are beginning to be understood thatinclude ATP depletion, oxyradical production, and disrup-tion of cellular ion homeostasis[15]. Accordingly, glutamatereceptor antagonists and calcium-stabilizing agents haveproven effective in reducing ischemic damage to neurons[12,16]

, but the downstream mechanism is unclear. Previousstudies have demonstrated that brain hypoxia/ischemiamodifies the recognition and modulatory sites of NMDAreceptor ion-channel complex, resulting in an increase inthe NMDA receptor-mediated calcium influx. An exces-sive and prolonged glutamate release has been found todisrupt neuronal mitochondrial function, leading to activa-tion of apoptotic pathways[25,26]. By immunoprecipitationof cytochrome c from cytosolic fractions, cytochrome cwas detected as early as 3 h following NMDA receptoractivation[27]. Cytochrome c release was found to be asso-ciated with permeability transition pore opening or occur-ring via the voltage-dependent anion channel. Ketamine isa voltage-dependent non-competitive antagonist of theNMDA receptors. When mitochondria are overloaded withCa2+ , that the release of cytochrome c from mitochondriato cytoplasm will increase. The mechanism of the ketamine-dependent inhibition could be due to a reduction in the NMDAreceptor-mediated calcium entry into cells. Nifedipine canalso inhibit intracellular calcium influx directly. Our studysuggested that pretreatment with ketamine or nifedipinecould result in a decrease in ischemia-induced release ofcytochrome c.

Apoptosis or programmed cell death induced by brainischemia is an active process of cell death requiring pro-tein synthesis and release of apoptosis-associated factors.Our studies indicated that ketamine or nifedipine could ef-fectively modulate the expression of Bcl-2 protein afterglobal ischemia. And both of them dramatically elevatedthe Bcl-2 expression at 24 h after reperfusion. The up-regulation of anti-apoptotic Bcl-2 protein in ischemic brainhave been suggested as a protective mechanism producedby injured cells or as a response to stress [28]. It is beneficialfor cell survival to shift the up-regulation of Bcl-2 expres-sion by ketamine or nifedipine after the ischemic insult. Ithas been demonstrated that Bcl-2 participates in the regu-lation of cytoplasmic and intranuclear Ca2+ concentrationfollowing apoptosis. The elevation of Bcl-2 expresion isinduced by phosphorylatoin of CREB (cAMP response el-ement-binding protein), which is regulated by the activa-tion of CaM-K (calcium-dependent kinase). The pretreat-

ment with ketamine or nifedipine could reduce the declinein ATPase and keep the status of active NMDA receptors,which also keep the ion homeostasis. Apoptosis can beinduced by [Ca2+]i in response to various pathological con-ditions and this often depends on the interplay betweenmitochondria and endoplasmic reticulum. Bcl-2 may exertits protective action by interfering with the Ca2+ dynamicsof these two organelles. Moreover, the up-regulation ofBcl-2 acts to inhibit cytochrome c[8,9], which accout foranother protective mechanism of Bcl-2 in vivo.

In conclusion, our results have shown the release ofcytochrome c from mitochondria to cytoplasm and theup-regulation of Bcl-2 in cytoplasm after brain ischemia.NMDA receptors and L-VGCC antagonists suppressed therelease of cytochrome c, whereas they increased Bcl-2expression in the ischemic hippocampus. It suggests thatcytochrome c release or Bcl-2 up-regulation may be medi-ated by NMDA receptors or L-VGCC, and cytochrome crelease may be injurious while Bcl-2 up-regulation may beprotective to ischemic hippocampus.

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利用全脑缺血模型,采用免疫印迹和免疫沉淀方法,探讨N-甲基-D-天冬氨酸受体和L-型电压门控钙通道拮抗剂对细胞色素c从线粒体中的释放和Bcl-2的表达变化影响。缺血/复灌后24h,线粒体中细胞色素c明显降低而胞浆中细胞色素c的成分相应增加。Bcl-2的表达呈时间依赖性,其表达在缺血/

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