Regulation of cardiac CFTR Cl(-) channel activity by a Mg(2+)-dependent protein phosphatase

Dephosphorylation of the CFTR Cl(-) channel is known to be induced by both okadaic-acid- (OA-) sensitive and -insensitive protein phosphatases (PPs). In the present study, the effects of cytosolic free Mg(2+) on the cardiac CFTR Cl(-) current were examined in relation to the latter PP activity in guinea pig ventricular myocytes. Even when maintaining intracellular Mg-ATP at millimolar concentrations under whole-cell patch-clamp mode, cAMP-activated Cl(-) conductance was reversibly suppressed by cytosolic free Mg(2+), with an IC(50) of around 2.5 mmol/l. In contrast, changes in the cytosolic concentration of free Mg(2+) ([Mg(2+)](i)) had no effect on genistein-activated CFTR Cl(-) currents. The Mg(2+) effect on cAMP-activated CFTR Cl(-) conductance was completely reversed by application of anthracene-9-carboxylic acid (9-AC), which was previously shown to inhibit an OA-insensitive PP in cardiac myocytes. A 9-AC-sensitive fraction of endogenous PP activity in the extract of guinea pig ventricle was found to be activated by free Mg(2+) at millimolar concentrations but to be inactive at micromolar concentrations. The intracellular application of OA failed to activate basal Cl(-) conductance at millimolar [Mg(2+)](i). In the presence of OA, however, basal Cl(-) conductance became activated either by reducing [Mg(2+)](i) to micromolar concentrations or by applying 9-AC. Thus, we conclude that a Mg(2+)-dependent PP sensitive to 9-AC plays a key role in the cAMP-mediated regulation of cardiac CFTR Cl(-) channel at physiological [Mg(2+)](i)under both basal and cAMP-activated conditions. Also, it appears that the genistein-activated conformation of the cardiac CFTR channel is not sensitive to the Mg(2+)-dependent PP.     

Expression of cystic fibrosis transmembrane conductance regulator in human endometrium

BACKGROUND: As a cAMP-regulated Cl- channel, cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in the active secretion of electrolytes and fluid in epithelial cells. Women with CFTR gene mutations are less fertile, generally assumed to be due to cervical factors. However, there is little known about CFTR protein expression in human endometrium and its possible roles in reproduction. METHODS AND RESULTS: CFTR protein and mRNA levels in human endometrium were analysed using immunohistochemical and in situ hybridization methods, respectively. Significant expression of CFTR protein was only seen in the glandular cells from late proliferative to all secretory phases, consistent with western blot analysis. High levels of CFTR mRNA were present only around the ovulatory period. In cultured glandular cells, the production of CFTR protein and mRNA was stimulated by estradiol and inhibited by progesterone. A forskolin-activated Cl- current in endometrial epithelial cells with a linear I-V relationship was detected by the whole-cell patch-clamp technique. CONCLUSIONS: (i) CFTR mRNA and protein were localized in human endometrial epithelial cells and the amounts varied in a cyclic manner; (ii) CFTR expression in cultured glandular cells was up- and downregulated by estradiol and progesterone, respectively; and (iii) CFTR in human endometrium functions as a cAMP-activated Cl- channel.     

Increased expression of apoptosis-linked gene 2 (ALG2) in the rat brain after temporary focal cerebral ischemia

Calcium is an important mediator of programmed cell death induced by transient cerebral ischemia, and calcium-binding proteins have been implicated in calcium-regulated signal transduction. Apoptosis-linked gene 2 is a calcium-binding protein required for cell death induced by different apoptotic stimuli. By Western blot analysis, we found that apoptosis-linked gene 2 protein was expressed in normal brains, and that expression increased in ischemic brains after 20 or 90 min of transient focal cerebral ischemia. Immunocytochemistry showed increased apoptosis-linked gene 2 protein expression in frontal cortex, a region where neurons underwent ischemic stress but still survived, after 20 or 90 min of focal cerebral ischemia. Apoptosis-linked gene 2 protein was also up-regulated in the ischemic border-zone of parietal cortex 24h after 20 min of focal ischemia, and was remarkably over-expressed in the caudate-putamen and parietal cortex, (where cells are destined to die) 24h after 90 min of ischemia. The expression pattern of apoptosis-linked gene 2 protein was similar to that of deoxyribonucleic acid damage detected by Klenow labeling assay.Our results suggest that apoptosis-linked gene 2 may be involved in the regulation of cell death after transient focal cerebral ischemia.     

脑缺血及后适应对树鼩海马内质网应激信号分子PERK及GRP78的影响

目的:探讨脑缺血及后适应对树鼩海马内质网应激信号分子蛋白激酶R样内质网激酶(PERK)及葡萄糖调节蛋白78(GRP78)的影响及后适应的脑保护机制。方法:光化学反应诱导树鼩局部血栓性脑缺血,于脑缺血后3.5 h夹闭、打开缺血侧颈总动脉交替3个循环(每次5 min)以复制缺血后适应模型。HE染色观察缺血侧海马神经元损伤及超微结构变化,RT-PCR检测脑缺血及后适应不同时间海马组织PERK及GRP78 mRNA表达的变化,免疫组织化学法与Western blot法检测PERK及GRP78的蛋白定位及表达变化。结果:海马神经元随脑缺血时间延长而损伤加重,缺血24 h损伤最为严重,后适应可减轻损伤。脑缺血4 h、24 h及72 h PERK的mRNA及蛋白表达较假手术组增高(P0.05),后适应组与相应的缺血组相比,PERK的mRNA及蛋白表达减少,4 h、24 h差异显著(P0.05);脑缺血4 h、24 h及72 h GRP78的mRNA及蛋白表达与假手术组无明显差异,后适应24 h组较缺血24 h组表达增高(P0.05)。结论:树鼩局部血栓性脑缺血可激活缺血侧海马内质网应激反应,引起PERK/e IF2α信号转导通路中相关分子PERK的mRNA及蛋白表达增高。缺血后适应处理通过下调PERK、上调GRP78的表达,减轻内质网应激反应,减少神经元损伤,具有一定的脑保护作用。     

Permanent focal cerebral ischemia activates erythropoietin receptor in the neonatal rat brain

Erythropoietin (Epo) has been shown to act as a neurotrophic and neuroprotective factor via binding to its receptor (EpoR) which is activated in adult brains following hypoxia and ischemia. However, no evidence suggests that cerebral ischemia can activate EpoR in the neonatal brain. In the present study, the changes in EpoR expression were investigated using a modified model of permanent focal cerebral ischemia (FCI) in 7-day-old rat pups. Western blot analysis with an anti-rabbit EpoR antibody revealed a significant increase in the EpoR protein in the ischemic areas, starting from 6 to 12 h after FCI. Moreover, many EpoR-positive cells were detected in the ischemic areas from 12 h after FCI, and the positive cells were identified as neurons and microglia/macrophage but not astrocytes 24 h after FCI. Additionally, double staining with a red in situ apoptosis detection kit and the EpoR antibody indicated that EpoR-positive cells were in apoptotic cell death in the ischemic area. Therefore, these results suggest that EpoR is activated in the ischemic areas of neonatal rats and plays an important role in brain injury during development.     

急性脑缺血损伤大鼠海马神经元谷氨酸转运体的表达

目的：研究急性脑缺血损伤大鼠海马神经元谷氨酸转运体（EAAC1）的表达变化。 方法： 采用EAAC1反义寡核苷酸脑内注射，用插线法建立大鼠局灶性脑缺血模型(MCAO)。运用Western blot法和TTC染色观察缺血区EAAC1表达和梗塞体积；采用RT-PCR 和Western blot法，测定海马EAAC1 mRNA和蛋白在缺血1 h、6 h、24 h的变化。结果： 注射EAAC1反义寡核苷酸组大鼠梗塞体积［(105.67±8.70) mm3］显著小于正义组。缺血1 h大鼠海马EAAC1 mRNA表达（0.963±0.117）与假手术组（0.907±0.113）无明显差异，缺血6h、24h持续高于缺血1 h（分别为1.116±0.104和1.428±0.078）。而海马EAAC1蛋白表达（0.640±0.027）在缺血24 h高于假手术组，缺血1 h和6h EAAC1表达与假手术组比较无显著差异（分别为0.330±0.018、0.330±0.015）。结论： EAAC1可促进缺血脑损伤,在急性脑缺血病理过程中表达增加。     

Molecular studies of CFTR interacting proteins

Transport via the cystic fibrosis transmembrane conductance regulator (CFTR) is activated by its interactions with cytoplasmic cofactors, such as cAMP-activated protein kinases. CFTR activity is also known to couple to other ion channels and transporters. Although the genetic cause of human cystic fibrosis by CFTR mutations has been well established, little is known about the protein machinery that plays a role in linking the CFTR to other regulatory or ion-conducting proteins. Several regions of CFTR proteins are highly conserved among different species. The conserved motifs are thought to determine various aspects of channel and mediate interactions with other regulatory proteins. The C-termini, which are not required for functional expression of the CFTR chloride conductance, are also highly conserved. Several proteins that interact with the conserved C-terminus have now been identified. They contain several distinct protein interaction domains, which may be involved in the assembly of macromolecular CFTR channel complexes in vivo. Molecular understanding of these proteins may provide important insights into CFTR function in cystic fibrosis.     

Protein disulfide isomerase immunoreactivity and protein level changes in neurons and astrocytes in the gerbil hippocampal CA1 region following transient ischemia

We investigated the temporal and spatial alterations of protein disulfide isomerase (PDI) immunoreactivity and protein level in the hippocampus proper after 5 min transient forebrain ischemia in gerbils. PDI immunoreactivity was significantly altered in the hippocampal CA1 region. PDI immunoreactivity in the sham-operated animals was found in non-pyramidal cells. At 30 min after ischemia, PDI immunoreactivity was shown in the pyramidal cells of the stratum pyramidale (SP): the PDI immunoreactivity in the pyramidal cells was increased up to 12 h after ischemia. Thereafter PDI immunoreactivity was decreased, and the PDI immunoreactivity was shown in non-pyramidal cells 2 days after ischemia. Four to 5 days after ischemia, almost pyramidal cells in the CA1 region were lost because the delayed neuronal death occurred. At this time period, PDI immunoreactivity was expressed in some astrocytes as well as some neurons. The results of the Western blot analysis were consistent with the immunohistochemical data. These findings suggest that increase of PDI in pyramidal cells may play a critical role in resistance to ischemic damage at early time after ischemic insult, and that expression of this protein in astrocytes at late time after ischemic insult is partly implicated in the acquisition of tolerance against ischemic stress.     

大鼠肢体缺血再灌注致肺损伤时肺组织中血红素氧合酶的表达

目的:观察大鼠肢体缺血再灌注致肺损伤时肺组织中HO-1表达的变化。方法:采用夹闭大鼠腹主动脉下段造成双下肢缺血和再灌注性肺损伤模型,分别采集假手术组,缺血4h组及缺血4h再灌注4、8、16、24、48h组肺组织标本,以Northern印迹、Western印迹及免疫组化分析,观察HO-1表达的变化。结果:假手术组和单纯缺血组未见HO-1mRNA表达;再灌注4h可见其表达信号,且随着再灌注时间延长表达信号逐渐增强,到16h时达到高峰,之后渐减弱,48h时已消失。蛋白表达水平与mRNA表达一致。免疫组化研究显示,HO-1阳性信号主要出现在气道上皮细胞、血管平滑肌细胞和肺泡巨噬细胞,而假手术组和单纯缺血组均未见阳性信号。结论:假手术及肢体的单纯缺血未诱发肺组织HO-1的表达,而肢体缺血再灌注可诱发其表达,并具有时相变化。     

低氧无血清对乳鼠心肌细胞皮肤桥蛋白表达的影响

 目的 在体外用低氧无血清条件模拟缺血心肌微环境,探讨心肌细胞皮肤桥蛋白(DPT)表达的变化。方法 以低氧无血清处理乳鼠心肌细胞0 、6 、12 和24 h,用实时反转录聚合酶链式反应检测DPT mRNA的表达变化,用Western blot检测DPT的蛋白表达变化。用酶联免疫吸附实验（ELISA）证实低氧无血清处理乳鼠心肌细胞24 h DPT蛋白的表达变化。结果 与0 h组相比,低氧无血清处理心肌细胞6 、12 、24 h DPT的mRNA和蛋白水平均显著升高（p<0.05）。ELISA的方法也检测到低氧无血清处理心肌细胞24 h DPT的表达水平高于正常对照组（p<0.05）。结论 低氧无血清可促进乳鼠心肌细胞DPT的表达,DPT可能在缺血缺氧引起的心室重构中发挥一定的作用。     

Induction of vascular endothelial growth factor receptors and phosphatidylinositol 3'-kinase/Akt signaling by global cerebral ischemia in the rat

Vascular endothelial growth factor is an angiogenic peptide that binds to tyrosine kinase receptors on target cells to activate signal transduction pathways involving phosphatidylinositol 3′-kinase and the serine–threonine protein kinase, Akt. To determine whether this signaling pathway is activated in cerebral ischemia, we examined the expression of vascular endothelial growth factor receptors 1 and 2, and phosphatidylinositol 3′-kinase-activated phospho-Akt, in the cerebral cortex and hippocampus following transient global cerebral ischemia in the rat. Western blot analysis and immunocytochemistry demonstrated induction of vascular endothelial growth factor receptor 1 and 2 expression, and of anti-phosphatidylinositol 3′-kinase-immunoprecipitated phospho-Akt, in vulnerable regions of the cortex and hippocampus after 15 min of global ischemia and 4–72 h of reperfusion.

These findings demonstrate that vascular endothelial growth factor receptors and receptor-coupled signal transduction pathways are induced in ischemic brain in vivo, and could therefore participate in endogenous neuroprotective responses to ischemia.     

Cardiac troponin in ischemic cardiomyocytes: Intracellular decrease before onset of cell death

Aim

Cardiac troponin I (cTnI) and T (cTnT) are the most important biomarkers in the diagnosis of acute myocardial infarction (AMI). Nevertheless, they can be elevated in the absence of AMI. It is unclear if such elevations represent irreversible cardiomyocyte-damage or leakage from viable cardiomyocytes. Our objective is to evaluate whether cTn is released from viable cardiomyocytes in response to ischemia and to identify differences in the release of cTn and its molecular forms.

Methods and results

HL-1 cardiomyocytes (mouse) were subjected to ischemia (modeled by anoxia with glucose deprivation). The total contents and molecular forms of cTn were determined in culture media and cell lysates. Cell viability was assessed from the release of lactate dehydrogenase (LDH). Before the release of LDH, the intracellular cTn content in ischemic cells decreased significantly compared to control (52% for cTnI; 23% for cTnT) and was not matched by a cTn increase in the medium. cTnI decreased more rapidly than cTnT, resulting in an intracellular cTnT/cTnI ratio of 25.5 after 24 h of ischemia. Western blots revealed changes in the relative amounts of fragmented cTnI and cTnT in ischemic cells.

Conclusions

HL-1 cardiomyocytes subjected to simulated ischemia released cTnI and cTnT only in combination with the release of LDH. We find no evidence of cTn release from viable cardiomyocytes, but did observe a significant decrease in cTn content, before the onset of cell death. Intracellular decrease of cTn in viable cardiomyocytes can have important consequences for the interpretation of cTn values in clinical practice.     

氟代柠檬酸抑制缺血后适应对树鼩脑缺血后海马HIF-1α/iNOS信号通路调控的脑损伤机制

目的:观察缺血后适应(PC)对树鼩脑缺血时海马HIF-1α/i NOS信号通路的调控作用,探讨抑制星形胶质细胞(AS)代谢加重脑损伤的机制。方法:光化学法建立血栓性脑缺血动物模型,氟代柠檬酸盐(FC)作为AS代谢抑制剂,于脑缺血后4 h闭/开缺血侧颈总动脉5 min,共3个循环以建立缺血PC模型。67只雄性树鼩随机分为对照组(n=9)、缺血4 h组(n=9)、缺血24 h组(n=9)、缺血PC 4 h组(n=9)、缺血PC 24 h组(n=9)、FC预处理4 h组(n=11)和FC预处理24 h组(n=11)。采用TTC染色观察树鼩脑梗死体积的变化,通过HE染色观察海马神经元病理学改变,用激光多普勒监测缺血区区域性脑血流(r CBF),免疫组化及Western blot检测海马i NOS表达,用分光光度计测定海马NO产量,用ELISA分析海马HIF-1α水平的变化。结果:脑梗死体积随缺血时间延长而增大,以缺血24 h为显著(P 0. 05);脑缺血后4 h和24 h皮层r CBF均下降(P 0. 05);而海马HIF-1α和i NOS表达增强,NO生成显著升高(P 0. 05)。缺血PC可增加皮层r CBF(P 0. 05),显著缩小脑梗死体积(P 0. 05),下调海马i NOS表达并减少NO的生成(P 0. 05)。而FC预处理组r CBF显著低于缺血组(P 0. 05),海马神经元损伤程度较缺血组加重,脑梗死体积随之增大(P 0. 05)。结论:缺血PC通过调控HIF-1α和i NOS表达而减轻缺血性脑损伤,抑制AS功能可削弱缺血PC介导的保护效应而加重脑损伤。     

Mineralocorticoid and glucocorticoid receptor expressions in astrocytes and microglia in the gerbil hippocampal CA1 region after ischemic insult

In the present study, we observed expression and changes of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the gerbil hippocampal CA1 region, but not in the CA2/3 region, after 5 min of transient forebrain ischemia. In blood, corticosterone levels were increased biphasically at 30 min and 12 h after ischemia/reperfusion, and thereafter its levels were decreased. In the sham-operated group, MR and GR immunoreactivities were weakly detected in the CA1 region. By 3 days after ischemia, MR and GR were not significantly altered in the CA1 region: at 12 h after ischemia, GR was expressed in a few neurons in the CA1 region, whereas MR was not expressed in any neurons after ischemic insult. From 4 days after ischemia, MR and GR immunoreactivities were detected in astrocytes and microglia in the CA1 region, and at 7 days after ischemia, MR and GR immunoreactivities peaked in the hippocampal CA1 region. At this time, 55% of astrocytes and 30% of microglia showed MR immunoreactivity, and 20% of astrocytes and 40% of microglia showed GR immunoreactivity. Western blot analyses showed that the pattern of changes in MR and GR protein levels was similar to the immunohistochemical changes observed after transient forebrain ischemia. From 4 days after ischemia, MR and GR protein levels were increased time-dependently after ischemia. In conclusion, enhanced MR and GR expressions in astrocytes and microglia were detected in the hippocampal CA1 region 4-7 days after ischemia/reperfusion. At this time, GR immunoreactivity was abundant in microglia, whereas MR immunoreactivity was prominent in astrocytes. The specific distribution of corticosteroid receptors in the astrocytes and microglia may be associated with the differences of MR and GR functions against ischemic damage.     

小鼠脑缺血时自噬相关基因5的抗损伤作用

 目的: 观察自噬相关基因5(Atg5)在小鼠脑缺血再灌注损伤中的抗损伤作用。方法: 将雄性BALB/c小鼠随机分为假手术(sham)组、缺血再灌注(I/R)组、Atg5 siRNA组和control siRNA组。I/R组采用大脑中动脉阻塞(MCAO)60 min后再灌注24 h。Atg5 siRNA组和control siRNA组将5 μL Atg5 siRNA或scrambled siRNA在MCAO前24 h侧脑室注射。实时荧光定量PCR和Western blot检测Atg5的表达;2,3,5-氯化三苯基四氮唑(TTC)染色法检测抑制Atg5对缺血再灌注损伤后脑梗死面积和水肿率的影响;神经行为学评分法检测抑制Atg5对缺血再灌注损伤后神经症状的影响。结果: MCAO后再灌24 h,缺血半影区Atg5 mRNA和蛋白水平显著增高(P<0.05);Atg5 siRNA明显降低缺血再灌后Atg5 mRNA和蛋白的表达(P<0.05);侧脑室给予Atg5 siRNA能显著增加脑梗死面积和水肿率,并加重神经行为学损伤(P<0.05)。结论: 沉默Atg5加重小鼠脑缺血再灌损伤,提示MCAO后诱导的 Atg5 可减轻小鼠局灶性脑缺血再灌注损伤。     

大鼠脊髓缺血再灌注损伤后caspase-12表达与细胞凋亡

目的:观察大鼠脊髓缺血再灌注损伤过程中细胞凋亡、caspase-12的表达变化规律,以探讨其分子机制。方法:采用自制压迫装置制备脊髓压迫缺血再灌注模型。运用形态学、分子生物学等方法,分别于缺血再灌注后3、7、11、23和47h,观察脊髓缺血再灌注损伤后,脊髓的病理变化和内质网的形态学改变、细胞凋亡及caspase-12的表达变化的规律。结果:脊髓缺血再灌注3h后,出现不同程度的细胞肿胀,神经元退行性变及内质网结构变化;随着再灌注时间的延长,神经元和神经胶质细胞凋亡数明显增加,并伴有caspase-12的表达增强;capspase-12表达与细胞凋亡的时空变化规律相一致。结论:在脊髓缺血再灌注过程中神经细胞凋亡是引起脊髓继发性损伤的主要病理因素,caspase-12可能参与了脊髓缺血再灌注损伤所导致的细胞凋亡。