Induction of cell death in rat small intestine by ischemia reperfusion: differential roles of Fas/Fas ligand and Bcl-2/Bax systems depending upon cell types

Although ischemia reperfusion (I/R) induces apoptotic damage of mammalian small intestine, the molecular mechanism is largely unknown. We investigated the appearance of apoptosis at various time-points (0–24 h) of reperfusion after 1-h ischemia and the expression of various apoptosis-related proteins, such as Bcl-2, Bax, Fas, Fas ligand (FasL), activated caspase-3, and cytochrome c, immunohistochemically in rat small intestine. As assessed by TUNEL and electron microscopy, apoptotic cells were increased at 3 h of reperfusion in all intestinal parts (villous epithelium, crypt epithelium, and stroma of intestine). Moreover, the TUNEL-positive cells in the stroma were later identified as T cells. The expression of Fas and FasL as well as activated caspase-3 was markedly increased at 3 h of reperfusion in the stroma. In the villous epithelium, a transient decrease in Bcl-2 expression was found while in the crypt epithelium, Fas expression was induced. Finally, intraperitoneal injection of leupeptin (an SH-protease inhibitor) after I/R resulted in a significant inhibition of the induction of apoptosis in the stroma and crypt epithelium. Our results indicate that the triggering molecules of apoptosis in the I/R rat small intestine may vary depending on cell type and that the use of a broad-spectrum protease inhibitor may reduce intestinal damage.     

Lung injury after ischemia-reperfusion of small intestine in rats involves apoptosis of type II alveolar epithelial cells mediated by TNF-α and activation of Bid pathway

Although ischemia-reperfusion (I/R) of small intestine is known to induce lung cell apoptosis, there is little information on intracellular and extracellular molecular mechanisms. Here, we investigated the mechanisms of apoptosis including the expression of Fas, Fas ligand (FasL), Bid, Bax, Bcl-2, cytochrome c, and activated caspase-3 in the rat lung at various time-points (0–24 h) of reperfusion after 1-h ischemia of small intestine. As assessed by TUNEL, the number of apoptotic epithelial cells, which were subsequently identified as type II alveolar epithelial cells by electron microscopy and immunohistochemical double-staining, increased at 3 h of reperfusion in the lung. However, intravenous injections of anti-TNF-α antibody decreased the number of TUNEL-positive cells, indicating involvement of tumor necrosis factor-α (TNF-α) in the induction of lung cell apoptosis. Western blotting and/or immunohistochemistry revealed a marked up-regulation of Fas, FasL, Bid, Bax, cytochrome c and activated caspase-3 and down-regulation of Bcl-2 in lung epithelial and stromal cells at 3 h of reperfusion. Our results indicate that I/R of small intestine results in apoptosis of rat alveolar type II cells through a series of events including systemic TNF-α, activation of two apoptotic signaling pathways and mitochondrial translocation of Bid.     

Genetic polymorphisms in glutathione S-transferase (GST) superfamily and risk of arsenic-induced urothelial carcinoma in residents of southwestern Taiwan

Background

Myocardial ischemia/reperfusion injury is the major cause of morbidity and mortality for cardiovascular diseases. Dopamine D₂ receptors are expressed in cardiac tissues. However, the roles of dopamine D₂ receptors in myocardial ischemia/reperfusion injury and cardiomyocyte apoptosis are unclear. Here we investigated the effects of both dopamine D₂ receptors agonist (bromocriptine) and antagonist (haloperidol) on apoptosis of cultured neonatal rat ventricular myocytes induced by ischemia/reperfusion injury.

Methods

Myocardial ischemia/reperfusion injury was simulated by incubating primarily cultured neonatal rat cardiomyocytes in ischemic (hypoxic) buffer solution for 2 h. Thereafter, these cells were incubated for 24 h in normal culture medium.

Results

Treatment of the cardiomyocytes with 10 μM bromocriptine significantly decreased lactate dehydrogenase activity, increased superoxide dismutase activity, and decreased malondialdehyde content in the culture medium. Bromocriptine significantly inhibited the release of cytochrome c, accumulation of [Ca²⁺]_i, and apoptosis induced by ischemia/reperfusion injury. Bromocriptine also down-regulated the expression of caspase-3 and -9, Fas and Fas ligand, and up-regulated Bcl-2 expression. In contrast, haloperidol (10 μM) had no significant effects on the apoptosis of cultured cardiomyocytes under the aforementioned conditions.

Conclusions

These data suggest that activation of dopamine D₂ receptors can inhibit apoptosis of cardiomyocytes encountered during ischemia/reperfusion damage through various pathways.     

Role of dopamine D2 receptors in ischemia/reperfusion induced apoptosis of cultured neonatal rat cardiomyocytes

Background

Myocardial ischemia/reperfusion injury is the major cause of morbidity and mortality for cardiovascular diseases. Dopamine D₂ receptors are expressed in cardiac tissues. However, the roles of dopamine D₂ receptors in myocardial ischemia/reperfusion injury and cardiomyocyte apoptosis are unclear. Here we investigated the effects of both dopamine D₂ receptors agonist (bromocriptine) and antagonist (haloperidol) on apoptosis of cultured neonatal rat ventricular myocytes induced by ischemia/reperfusion injury.

Methods

Myocardial ischemia/reperfusion injury was simulated by incubating primarily cultured neonatal rat cardiomyocytes in ischemic (hypoxic) buffer solution for 2 h. Thereafter, these cells were incubated for 24 h in normal culture medium.

Results

Treatment of the cardiomyocytes with 10 μM bromocriptine significantly decreased lactate dehydrogenase activity, increased superoxide dismutase activity, and decreased malondialdehyde content in the culture medium. Bromocriptine significantly inhibited the release of cytochrome c, accumulation of [Ca²⁺]_i, and apoptosis induced by ischemia/reperfusion injury. Bromocriptine also down-regulated the expression of caspase-3 and -9, Fas and Fas ligand, and up-regulated Bcl-2 expression. In contrast, haloperidol (10 μM) had no significant effects on the apoptosis of cultured cardiomyocytes under the aforementioned conditions.

Conclusions

These data suggest that activation of dopamine D₂ receptors can inhibit apoptosis of cardiomyocytes encountered during ischemia/reperfusion damage through various pathways.     

Resveratrol Attenuates Ischemia/Reperfusion Injury in Neonatal Cardiomyocytes and Its Underlying Mechanism

This study was designed to investigate whether Resveratrol (Res) could be a prophylactic factor in the prevention of I/R injury and to shed light on its underlying mechanism. Primary culture of neonatal rat cardiomyocytes were randomly distributed into three groups: the normal group (cultured cardiomyocytes were in normal conditions), the I/R group (cultured cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion), and the Res+I/R group (100 µmol/L Res was administered before cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion). To test the extent of cardiomyocyte injury, several indices were detected including cell viability, LDH activity, Na⁺-K⁺-ATPase and Ca²⁺-ATPase activity. To test apoptotic cell death, caspase-3 activity and the expression of Bcl-2/Bax were detected. To explore the underlying mechanism, several inhibitors, intracellular calcium, SOD activity and MDA content were used to identify some key molecules involved. Res increased cell viability, Na⁺-K⁺-ATPase and Ca²⁺-ATPase activity, Bcl-2 expression, and SOD level. While LDH activity, capase-3 activity, Bax expression, intracellular calcium and MDA content were decreased by Res. And the effect of Res was blocked completely by either L-NAME (an eNOS inhibitor) or MB (a cGMP inhibitor), and partly by either DS (a PKC inhibitor) or Glybenclamide (a K_ATP inhibitor). Our results suggest that Res attenuates I/R injury in cardiomyocytes by preventing cell apoptosis, decreasing LDH release and increasing ATPase activity. NO, cGMP, PKC and K_ATP may play an important role in the protective role of Res. Moreover, Res enhances the capacity of anti-oxygen free radical and alleviates intracellular calcium overload in cardiomyocytes.     

Effects of polyamines on apoptosis induced by simulated ischemia/reperfusion injury in cultured neonatal rat cardiomyocytes

We incubated neonatal Sprague-Dawley rat cardiomyocytes in primary culture in a medium simulating ischemia (consisting of hypoxia plus serum deprivation) for 2 h, then re-incubated them for 24 h in normal culture medium to establish a model of simulated ischemia/reperfusion (I/R) injury. Apoptotic cell death was measured by MTT assay, TUNEL staining and flow cytometry. Morphological alterations were assessed by transmission electron microscopy, the expression of caspases-3 and -9 and Bcl-2 and the release of cytochrome c by Western blotting, and the intracellular free-calcium concentration ([Ca2+]i) by laser confocal scanning microscopy. The results showed that pretreatment with 10 micromol/l spermine or spermidine significantly inhibited apoptosis in the I/R cells, suppressed the expression of caspases-3 and -9 and cytochrome c release, up-regulated Bcl-2 expression and decreased [Ca2+]i. However, pretreatment with 10 micromol/l putrescine had the opposite effects. Evidence for this "double-edged" effect of polyamines on apoptosis in I/R-injured cardiomyocytes is presented for the first time. It may suggest a novel pharmacological target for preventing and treating cardiac ischemia/reperfusion injury.     

Calcium-sensing receptor induces rat neonatal ventricular cardiomyocyte apoptosis

The calcium-sensing receptor (CaSR) exists in many tissues, and its expression has been identified in rat cardiac tissue. However, the physiological importance and pathophysiological involvement of CaSR in homeostatic regulation of cardiac function are unclear. To investigate the relation of CaSR and apoptosis in cardiomyocytes, we examined the role of the CaSR activator gadolinium chloride (GdCl(3)) in rat neonatal ventricular cardiomyocytes. Expression of the CaSR protein was observed by Western blot. The apoptotic ratio of rat neonatal ventricular cardiomyocytes was measured with flow cytometry and immunofluorescence techniques. A laser scan confocal microscope was used to detect the intracellular concentration of calcium ([Ca(2+)](i)) in rat neonatal ventricular cardiomyocytes using the acetoxymethyl ester of fluo-3 (fluo-3/(AM)) as a fluorescent dye. The results showed that GdCl(3) increased the phosphorylation of extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal protein kinases (JNK), and p38. GdCl(3) also activated caspase 9 and increased apoptosis in myocyte by increasing [Ca(2+)](i). In conclusion, these results suggest that CaSR promotes cardiomyocyte apoptosis in rat neonatal ventricular cardiomyocytes through activation of mitogen-activated protein kinases and caspase 9 signaling pathways.     

MicroRNA-15b enhances hypoxia/reoxygenation-induced apoptosis of cardiomyocytes via a mitochondrial apoptotic pathway

Myocardial ischemia reperfusion (I/R) can induce altered expression of microRNAs (miRNAs). The miRNAs—miR-15a, miR-15b and miR-16 have been shown to play a role in apoptosis, although not in cardiac-related models. We investigated the roles of miR-15b in hypoxia/reoxygenation (H/R)-induced apoptosis of cardiomyocytes. Quantitative real time polymerase chain reaction results showed that the expression of miR-15a and miR-15b were up-regulated in Sprague–Dawley rat hearts subjected to I/R. Expression levels of miR-15b increased more than four fold above basal levels. Similar results were obtained for cardiomyocytes exposed to H/R. Recombinant adenoviral vectors were generated to explore the functional role of miR-15b in cultured cardiomyocytes exposed to H/R. Overexpression of miR-15b enhanced cell apoptosis and the loss of mitochondrial membrane potential, as determined by flow cytometric analysis. Conversely, down-regulated expression was cytoprotective. The effects of miR-15b can by mimicked by Bcl-2 short-interfering RNAs. The inhibition of miR-15b increased expression levels of the Bcl-2 protein without affecting Bcl-2 mRNA levels, suppressed the release of mitochondrial cytochrome c to the cytosol and decreased the activities of caspase-3 and 9. It is possible that miR-15b is the upstream regulator of a mitochondrial signaling pathway for H/R induced apoptosis.     

Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes

Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.     

Calcium-sensing receptor activating phosphorylation of PKC�� translocation on mitochondria to induce cardiomyocyte apoptosis during ischemia/reperfusion

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that activates intracellular effectors; for example, it causes inositol phosphate (IP) and 1,2 diacylglycerol (DAG) accumulation, stimulating the release of intracellular calcium and the activation of the protein kinase Cs (PKCs). The activation of CaSR by ischemia/reperfusion (I/R) induces cardiomyocyte apoptosis through the mitochondrial apoptotic pathway; however, the underlying mechanisms remain unclear. In this study, rat hearts were subjected to 30 min of ischemia followed by 2 h of reperfusion in the presence of a CaSR activator, GdCl₃. Our results revealed that, under these conditions, the expression of CaSR was increased, the number of apoptotic cardiomyocytes was significantly increased (as shown by terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay) and the cells with a typical apoptotic morphology were observed using transmission electron microscopy. Our data further showed that mitochondria isolated from hearts that had undergone I/R combined with GdCl₃ exhibited a significant increase in the translocation of phosphorylated PKC?? to the mitochondria, an increase in cytochrome c (cyt c) release from the mitochondria and a marked decrease in mitochondrial potential. The administration of rottlerin, an inhibitor of PKC??, significantly reduced reperfusion-induced apoptosis, phospho-PKC?? translocation to the mitochondria and the release of cyt c from the mitochondria. Thus, the involvement of CaSR in cardiac apoptosis through the mitochondrial pathway during I/R with GdCl₃ is related to phospho-PKC?? translocation to the mitochondria.     

心肌梗死大鼠钙敏感受体表达与心肌细胞凋亡的变化

目的:观察大鼠急性心肌梗死后不同时间心肌钙敏感受体(CaSR)的表达和心肌细胞凋亡的变化情况。方法:健康Wistar大鼠随机分为假手术组(Sham)和心肌梗死(AMI)组,通过结扎左侧冠状动脉前降支的方法,建立大鼠心肌梗死模型,分别在手术后1、2、4周(每组成功存活n=5)检测心脏形态学和血流动力学的改变,检测心肌组织中CaSRmRNA和蛋白的表达,以及Bax、Bcl-2、caspase-3和caspase-9蛋白的表达,检测血清中乳酸脱氢酶(LDH)、肌酸激酶(CK)活性和肌钙蛋白(cTnT)水平,观察心肌细胞凋亡情况。结果:和Sham组相比,随着心肌梗死的发展,AMI组大鼠心肌组织CaSR的mRNA和蛋白的表达、细胞凋亡指数均明显增加(P<0.05),心肌细胞超微结构损伤严重;左心室收缩压(LVSP)、左心室内压最大上升速率(+dp/dtmax)(mmHg/s)和最大下降速率(-dp/dtmax)(mmHg/s)减少,左心室舒张末期压(LVEDP)明显增大(P<0.05);AMI组血清cTnT水平、CK和LDH活性均升高(P<0.05),随着心肌梗死的发展,cTnT水平和CK活性逐渐降低,LDH变化不明显。心肌组织中促凋亡相关蛋白Bax、caspase-3、caspase-9表达增多,抑制凋亡的相关蛋白(或因子)Bcl-2表达减少(P<0.05)。结论:随着AMI的发展,AMI组大鼠心肌组织中CaSR的mRNA和蛋白的表达增多,细胞凋亡数增加,表明CaSR参与了心肌梗死的发展,其机制可能与促进细胞凋亡有关。     

川芎嗪对家兔肺缺血/再灌注损伤时Fas/FasL基因表达的影响

目的:探讨川芎嗪对肺缺血/再灌注损伤(PI/RI)时Fas/FasL基因表达的影响。方法:采用在体兔单肺原位缺血-再灌注模型。实验兔90只,随机均分为假手术对照组(Sham)、肺缺血/再灌注组(I/R)和肺缺血/再灌注加川芎嗪治疗组(LGT)。每组又分为再灌注1h3、h、5 h三个亚组,每个亚组10只,分别于再灌注1 h3、h、5 h三个时点取左肺组织,观察Fas/Fas配体(Fas/FasL)mRNA定位表达、凋亡指数(AI)、肺组织湿、干重比(W/D)、肺损伤组织学定量评价指标(IQA)及光镜、电镜下的组织形态学改变。结果:肺再灌注1 h、3 h、5 h,LGT组Fas/FasLmRNA在肺小动脉内(外)膜、肺小静脉内膜、肺泡上皮及肺支气管上皮弱阳性表达,与I/R组同一时点比较阳性表达明显减弱;AI、W/D和IQA值显著低于I/R组(P<0.01和P<0.05);肺组织形态学异常改变不同程度减轻。结论:川芎嗪可下调肺组织Fas/FasL mRNA的表达而减轻细胞凋亡,对PI/RI发挥积极的防护作用。     

人参皂甙 Rb1对大鼠缺血再灌注心肌细胞Bcl-2、Bax、Bad、Fas基因表达的影响

目的观察人参皂甙Rb1对缺血再灌注心肌细胞Bcl-2、Bax、Bad、Fas基因表达的影响.方法结扎/松解Wistar大鼠左冠状动脉前降支,建立大鼠缺血再灌注动物模型,免疫组化法检测Bcl-2、Bax、Bad、Fas基因的蛋白表达,并利用图象分析系统测量蛋白阳性表达区域平均光密度值,进行定量分析.结果缺血再灌注组及Rb1治疗组Bcl-2、Bax、Bad、Fas基因的表达较假手术组明显增加(P<0.05), Rb1治疗组Bcl-2的表达与缺血再灌注组比较无明显差异(P>0.05),而Bax、Bad、Fas的表达明显下降(P<0.05),人参皂甙Rb1治疗组Bcl-2/Bax、Bcl-2/Bad以及Bcl-2/Fas比值均较假手术组与缺血再灌注组明显增加.结论人参皂甙Rb1治疗可以抑制缺血再灌注心肌细胞中促凋亡基因Bax、Bad、Fas的表达,并使Bcl-2/Bax、Bcl-2/Bad以及Bcl-2/Fas比值增加.     

下肢骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响

目的:探讨在体情况下,骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响。方法:新西兰大白兔36只,随机分成3组(每组随机选取6只进行梗死范围的测定,另外6只进行凋亡测定):①假手术组(Sham组);②缺血/再灌注组(I/R组);③远端后处理组(RPostC组)。在缺血前、后及再灌注60 min、120 min分别抽血测定肌酸激酶(CK),乳酸脱氢酶(LDH)的活性。采用伊文思兰(evans blue)和三苯基氯化四氮唑(TTC)染色方法确定心肌缺血区范围以及心肌坏死区范围。用Tunel法检测兔心肌缺血区细胞凋亡情况,免疫组织化学方法检测心肌缺血区蛋白caspase-3、Bcl-2及Bax的表达。结果:RPostC组心肌坏死程度、再灌注末CK活性较I/R组明显减低。RPostC组缺血区心肌Tunel阳性指数显著低于I/R组(21.79%±1.07%vs35.81%±1.10%,P<0.05)。而RPostC组缺血区心肌细胞caspase-3阳性指数显著低于I/R组(25.03%±1.16%vs39%±2.43%,P<0.05)。与Sham组比较,I/R组及RPostC组Bax蛋白表达指数、Bcl-2蛋白表达指数均升高;但RPostC组的Bax/Bcl-2比值降低,而I/R组的Bax/Bcl-2比值升高。与I/R组相比较,RPostC组Bax蛋白表达指数及Bax/Bcl-2比值显著降低,Bcl-2表达指数显著升高,差异均有统计学意义。结论:远端后处理能够明显的减少缺血/再灌注心肌细胞的坏死和凋亡,其减轻心肌细胞凋亡的机制可能与抑制促凋亡基因caspase-3的活化及Bcl-2表达的上调有关。     

Hypothermia attenuates ischemia/reperfusion-induced endothelial cell apoptosis via alterations in apoptotic pathways and JNK signaling

Hypothermia is the most effective means of protecting the brain, heart and other organs during ischemia/reperfusion (I/R) injury. However, the precise mechanisms for hypothermia to inhibit I/R-induced endothelial cell apoptosis are not fully understood. In the present study, human umbilical endothelial cells (HUVECs) were exposed to ischemia followed by reperfusion under normothermia (37 °C) or hypothermia (33 °C). Our results showed that hypothermia markedly reduced I/R-induced endothelial cell apoptosis, the expression of cleaved caspase-3 and PARP. Moreover, hypothermia markedly reversed I/R-induced activation of Fas/caspase-8, the increase of Bax and decrease of Bcl-2. Furthermore, hypothermia inhibited JNK1/2 activation via MKP-1 induction. Together, these data demonstrate that hypothermia represses I/R-induced endothelial cell apoptosis by inhibiting both extrinsic- and intrinsic-dependent apoptotic pathways and activation of JNK1/2.     

Effect of recombinant erythropoietin on ischemia–reperfusion-induced apoptosis in rat liver

Ischemia–reperfusion (I/R) cannot be avoided in liver transplantation procedures, and apoptosis is a central mechanism of cell death after liver reperfusion. Protective effect of recombinant erythropoietin (rhEPO) on liver apoptosis has not been clearly investigated. This work investigated intraportal (IP) rhEPO-protective effect in a rat model of hepatic I/R-induced apoptosis and its appropriated time and dose of administration. Eight groups were included (n = 10/group): sham-operated, I/R (45 min ischemia and 2 h reperfusion), preconditioned rhEPO I/R (24 h or 30 min before ischemia), and postconditioned rhEPO I/R (before reperfusion) using two different rhEPO doses (1,000 and 5,000 IU/kg). When compared with the sham-operated group, the I/R group showed significant increase of serum levels of aspartate and alanine aminotransferases (AST, ALT), hepatic caspase-9 activity(894.99 ± 176.90 relative fluorescence units (RFU)/mg/min versus 458.48 ± 82.96 RFU/mg/min), and Fas ligand (FasL) expression, histopathological damages, and significant decrease in the antiapoptotic Bcl-xL/apoptotic Bax ratio(0.38 ± 0.21 versus 3.35 ± 0.77) rhEPO-improved ALT and AST but failed to reduce FasL expression in all groups compared with the I/R group. Thirty minutes and 24 h preconditioning with rhEPO (1,000 IU/kg) increased Bcl-xL/Bax ratio and reduced caspase-9 activity, and the same effect was observed when higher dose was given 24 h before ischemia. Preconditioning was more effective than postconditioning in improving caspase-9 activity, and no dose-dependent effect was observed. In conclusion, single IP rhEPO injection 30 min before ischemia has an advantage over rhEPO postconditioning in improving post-hepatic I/R-induced apoptosis with no additional time- and dose-dependent effects which may provide potentially useful guide in liver transplantation procedures.     

Involvement of calcium-sensing receptor in ischemia/reperfusion-induced apoptosis in rat cardiomyocytes

The calcium-sensing receptor (CaR) is a seven-transmembrane G-protein coupled receptor, which activates intracellular effectors, for example, it causes inositol phosphate (IP) accumulation to increase the release of intracellular calcium. Although intracellular calcium overload has been implicated in the cardiac ischemia/reperfusion (I/R)-induced apoptosis, the role of CaR in the induction of apoptosis has not been fully understood. This study tested the hypothesis that CaR is involved in I/R cardiomyocyte apoptosis by increasing [Ca2+]i. The isolated rat hearts were subjected to 40-min ischemia followed by 2 h of reperfusion, meanwhile GdCl3 was added to reperfusion solution. The expression of CaR increased at the exposure to GdCl3 during I/R. By laser confocal microscopy, it was observed that the intracellular calcium was significantly increased and exhibited a Deltapsim, as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) during reperfusion with GdCl3. Furthermore, the number of apoptotic cells was significantly increased as shown by TUNEL assay. Typical apoptotic cells were observed with transmission electron microscopy in I/R with GdCl3 but not in the control group. The expression of cytosolic cytochrome c and activated caspase-9 and caspase-3 was significantly increased whereas the expression of mitochondrial cytochrome c significantly decreased in I/R with GdCl3 in comparison to the control. In conclusion, these results suggest that CaR is involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion through activation of cytochrome c-caspase-3 signaling pathway.