G_(i/o)蛋白介导的信息转导通路在低氧预处理心肌保护中的作用

实验以低氧 3h后复氧期间心肌细胞的生存率和LDH的释放量为指标 ,观察Gi/o蛋白及其下游成分在低氧预处理 (hypoxicpreconditioning ,HP)心肌保护中的作用。与单纯低氧组相比 ,HP组 ( 2 5min低氧 30min复氧作为HP)细胞生存率增高 ,LDH释放减少 (P <0 0 1)。用NEM预处理 ,能完全模拟HP的心肌细胞保护作用 ;而用PTX阻断Gi/o蛋白 ,或Forskolin和 8 Br cAMP预处理后 ,再给予HP及低氧 3h/复氧 1h ,则细胞生存率降低 ,LDH释放增加 (P <0 0 1) ;U 7312 2预处理后 ,细胞生存率和LDH释放量无差异 (P >0 0 5 )。结果提示 :Gi/o蛋白通过抑制AC ,减少第二信使cAMP的生成介导了HP的心肌保护作用。PLC可能不参与HP的心肌保护作用     

谷氨酰胺对体外培养人小肠上皮细胞缺氧复氧损伤的保护作用

应用原代培养人胎小肠上皮细胞（ＩＥＣ）,观察了谷氨酰胺（ＧＬＮ）对缺氧复氧（Ａ／Ｒ）损伤人ＩＥＣ的影响。结果：缺氧６０ｍｉｎ复氧３０ｍｉｎ后,细胞内乳酸脱氢酶（ＬＤＨ）漏出量显著上升,细胞存活率显著下降。预先应用１～５ｍｍｏｌ／ＬＧＬＮ可使Ａ／Ｒ损伤ＩＥＣ细胞存活率升高和细胞内ＬＤＨ漏出量减少,ＧＬＮ作用的最佳剂量为２ｍｍｏｌ／Ｌ。提示ＧＬＮ对人ＩＥＣ具有直接的保护作用,这可能是其整体保护作用机制之一。     

蛋白激酶C和Na~＋－H~＋交换在ANGⅡ诱发心肌细胞肥大反应中的作用

血管紧张素（ＡＮＧ）Ⅱ在１０－１０－１０－６ｍｏｌ／Ｌ范围内剂量依赖性促进无血清培养新生大鼠心肌细胞蛋白质合成速率。蛋白激酶Ｃ（ＰＫＣ）抑制剂ｓｔａｕｒｏｓｐｏｒｉｎｅ（Ｓｔａｕ２ｎｍｏｌ／Ｌ）对心肌细胞基础状态３Ｈ－Ｌｅｕｃｉｎｅ掺入无明显影响，但Ｓｔａｕ预处理３０ｍｉｎ，则可有效阻断ＡＮＧⅡ（１μｍｏｌ／Ｌ）对细胞蛋白质合成的刺激作用；单纯应用ＰＫＣ激活剂ＰＭＡ（１μｍｏｌ／Ｌ）可使心肌细胞蛋白质合成速率增加，与对照组相比，ＰＭＡ组３Ｈ－Ｌｅｕｃｉｎｅ掺入量增加了４１．０４％。细胞Ｎａ＋－Ｈ＋交换抑制剂Ａｍｉｌｏｒｉｄｅ预处理也能阻断ＡＮＧⅡ刺激３Ｈ－Ｌｅｕｃｉｎｅ掺入细胞蛋白质的作用。以上结果提示ＰＫＣ和Ｎａ＋－Ｈ＋交换的激活，可能是ＡＮＧⅡ诱发的心肌细胞肥大反应的重要胞内信息转导机制。本工作还观察到，阻断细胞Ｎａ＋－Ｈ＋交换后并不影响由ＰＫＣ激活导致的蛋白质合成增加，提示可能存在着ＰＫＣ和Ｎａ＋－Ｈ＋交换彼此相对独立地调节心肌细胞生长的途径。     

低氧对肺动脉内皮细胞PDGF—B链释放及平滑肌细胞生长的影响

应用蛋白ｄｏｔｂｌｏｔ技术检测了低氧内皮细胞条件培养液（ＨＥＣＣＭ）和常氧内皮细胞条件培养液（ＮＥＣＣＭ）内ＰＤＧＦ相对含量,并利用［３Ｈ］－ＴｄＲ掺入法和流式细胞术观察了ＨＥＣＣＭ和ＮＥＣＣＭ及加入特异ＰＤＧＦ抗体对肺动脉平滑肌细胞（ＰＡＳＭＣ）生长的影响。结果表明,ＨＥＣＣＭ中的ＰＤＧＦ含量明显高于ＮＥＣＣＭ;ＨＥＣＣＭ能明显增强ＰＡＳＭＣ内ＤＮＡ合成,促进ＰＡＳＭＣ从Ｇｏ／Ｇ１期进入Ｓ期;当预先加入ＰＤＧＦ－Ｂ链抗体时,则会明显地抑制ＨＥＣＣＭ对ＰＡＳＭＣ的ＤＮＡ合成,阻止ＰＡＳＭＣ从Ｇｏ／Ｇ１期进入Ｓ期。结果提示,低氧时ＰＡＳＭＣ增殖与肺动脉内皮细胞分泌释放ＰＤＧＦ增加有关     

短暂低氧／复氧对培养新生兔心肌细胞丝裂素活化蛋白激酶活性的影响

心肌细胞短暂低氧可诱导对后续长时间低氧所致细胞严重损伤的耐力增强,已在心脏预处理（ＰＣ）模型上得到证实,但ＰＣ发生的细胞内信号传导途径目前尚不清楚。我们在培养的新生兔心肌细胞低氧／复氧模型上,观察丝裂素活化蛋白激酶（ＭＡＰＫ）和核蛋白体Ｓ６激酶（Ｓ６Ｋ）活性改变。结果发现：低氧６０ｍｉｎ后、复氧１５ｍｉｎ,细胞总ＭＡＰＫ和核ＭＡＰＫ活性分别较对照组增加９５％和２３０％（Ｐ〈０．０１）;Ｓ６Ｋ活性在     

大鼠20α羟类固醇脱氢酶在大肠杆菌中的表达

利用谷胱甘肽Ｓ－转移酶（Ｇｌｕｔａｔｈｉｏｎｅ　Ｓ－ｔｒａｎｓｆｅｒａｓｅ,ＧＳＴ）融合基因表达系统,大鼠２０α羟类固醇脱氢酶（２０αＨｙｄｒｏｘｙｓｔｅｒｏｉｄＤｅｈｙｄｒｏｇｅｎａｓｅ,２０αＨＳＤ）在大肠杆菌中得以成功地表达。亲和层析和Ｔｈｒｏｍｂｉｎ消化,可从融合蛋白中回收和纯化重组２０αＨＳＤＳＤＳ－ＰＡＧＥ、Ｗｅｓｔｅｒｎ印迹法和酶活性测定显示,重组２０αＨＳＤ具有天然蛋白质相同分子量、相似的抗原性和酶催化活性,其对ＮＡＤＰ的Ｋ＿ｍ值和Ｖ＿（ｍａｘ）分别为９．５μｍｏｌ／Ｌ、３３４ｎｍｏ１／（ｍｉｎ·ｍｇ）,对底物２０α羟孕酮（２０αＨｙｄｒｏｘｙｐｒｏｇｅｓｔｅｒｏｎｅ,２０αＯＨＰ）的Ｋ＿ｍ值和Ｖ＿（ｍａｘ）分别为５．９μｍｏｌ／Ｌ和３４７ｎｍｏｌ／（ｍｉｎ·ｍｇ）,利用该表达系统大量制备大鼠重组２０αＨＳＤ,为深入研究２０αＨＳＤ的生理活性和功能创造条件。     

神经元缺氧复氧损伤时氧自由基的毒性作用及其机制＊

在原代分离培养Ｗｉｓｔａｒ乳鼠大脑皮质神经元上研究了缺氧复氧损伤（Ｈ／Ｒ）对神经细胞乳酸脱氢酶（ＬＤＨ）,漏出率,死亡率和脂质过氧化物含量的影响,并选用一氧化氮（ＮＯ）合酶抑制剂Ｌ－ＮＧ－硝基－精氨酸（Ｌ－ＮＮＡ）巯基供体Ｎ－乙酰半胱氨酸（ＮＡＣ）和超氧化物歧化酶（Ｃｕ,Ｚｎ－ＳＯＤ）三种自由基清除剂进行预保护等方法来探讨机制。结果表明 Ｈ／Ｒ损伤引起ＬＤＨ漏出率,细胞死亡率和脂过氧化物含量极显著     

神经元缺氧复氧损伤时氧自由基的毒性作用及其机制

在原代分离培养Ｗｉｓｔａｒ乳鼠大脑皮质神经元上研究了缺氧复氧损伤（Ｈ／Ｒ）对神经细胞乳酸脱氢酶（ＬＤＨ）,漏出率,死亡率和脂质过氧化物含量的影响,并选用一氧化氮（ＮＯ）合酶抑制剂Ｌ－ＮＧ－硝基－精氨酸（Ｌ－ＮＮＡ）巯基供体Ｎ－乙酰半胱氨酸（ＮＡＣ）和超氧化物歧化酶（Ｃｕ,Ｚｎ－ＳＯＤ）三种自由基清除剂进行预保护等方法来探讨机制。结果表明 Ｈ／Ｒ损伤引起ＬＤＨ漏出率,细胞死亡率和脂过氧化物含量极显著     

草酸对氧化胁迫下水稻叶片中核酮糖—1,5—二磷酸羧化酶／加氧 …

以杂交稻（汕优６３）为试验材料,在木村Ｂ营养液中培养至三叶期,用草酸５ｍｍｏｌ／Ｌ预处理水稻２ｄ,再处以氧化胁迫（用０．１ｍｍｏｌ／Ｌ浓度的活性氧诱发剂甲基紫精处理）。结果表明：ＭＶ诱发的氧化胁迫下,Ｒｕｂｉｓｃｏ及其它可溶性蛋白快速降解。草酸预处理可明显缓解Ｒｕｂｉｓｃｏ及其它可溶性蛋白的降解,降解速率分别降低１／３和１／２左右,植株经草酸处理后其叶片中几种抗氧化酶如ＡｓＡ－ＰＯＤ、ＳＯＤ、ＣＡ     

高氧预适应对大鼠心肌缺血损伤时抗氧化酶的影响

抗氧化酶具有减轻心肌缺血再灌注损伤的作用,在抗氧化酶中,比较重要的是超氧化物歧化酶（ＳＯＤ）,谷胱甘肽过氧化物酶（ＧｌｕｔａｔｈｉｏｎｅＰｅｒｏｘｉｄａｓｅ,ＧＳＨｐｘ）和过氧化氢酶（ＣＡＴ）。为了解高氧预适应（ＨｙｐｅｒｏｘｉｃＰｒｅｃｏｎｄｉｔｉｏｎｉｎｇ,ＨＯＰ）对大鼠心肌缺血损伤时抗氧化酶的影响,本实验将实验组大鼠放入高压氧舱内,每日吸８０－８５％氧气（１ａｔｍ,１５－２０％为氮气）６ｈ,连续７ｄ。利用Ｌａｎｇｅｎｄｏｒｆ装置做成心肌缺血再灌注模型。实验动物随机分为二个部分。第一部分可逆性心肌缺血（ＨＯＰＡ组与对照Ａ组）：缺血１０ｍｉｎ,再灌注６０ｍｉｎ。观察冠脉回流液中ＳＯＤ活力,检测心肌内抗氧化酶活力（ＳＯＤ,ＧＳＨｐｘ,ＣＡＴ）。第二部分不可逆性心肌缺血（ＨＯＰＢ组与对照Ｂ组）：缺血６０ｍｉｎ,再灌注６０ｍｉｎ。测定冠脉回流液中肌酸磷酸激酶（ＣＰＫ）含量,ＳＯＤ及心肌内抗氧化酶活力。结果表明：对于可逆性心肌缺血：ＳＯＤ,ＧＳＨｐｘ活力升高;对于不可逆性心肌缺血损伤：ＨＯＰ能减少ＣＰＫ释放,ＳＯＤ活力升高。     

缺氧诱导因子-1α在缺氧预处理预防心肌细胞损伤中的作用

本工作旨在研究缺氧预处理(hypoxic preconditioning,HPC)对于心肌细胞外信号调节激酶(extracellular signal-regulated proteinkinases,ERK)活性、缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)表达的影响,及其在缺氧复氧诱导心肌细胞损伤中的作用。通过在培养的SD乳鼠心肌细胞缺氧／复氧(H／R)模型上,观察HPC对于24h后H／R诱导心肌细胞损伤的影响,以台盼蓝排斥实验检测心肌细胞存活率、以TUNEL法检测细胞凋亡、并用荧光素染料Hoechst33258测定心肌细胞凋亡率：制备心肌细胞蛋白提取物,以磷酸化的ERK1／2抗体测定ERK1／2活性,以抗HIF-1α抗体检测HIF-1α的表达,并观察ERKs的上游激酶(MEK1／2)抑制剂PD98059对于HPC诱导的ERKs磷酸化、HIF-1α表达以及心肌细胞保护作用的影响,并分析细胞损伤与ERK1／2活性、HIF-1α表达量之间的相互关系。结果 显示缺氧复氧造成心肌细胞损伤,HPC可以增加心肌细胞H／R后存活率,降低凋亡率,并激活ERKll2,诱导HIF-1α表达：细胞凋亡与ERKs活性、HIF-1α表达量之间存在负相关,即ERKs活化、HIF-1α表达与预防细胞损伤有关：而ERKs活性与HIF-1α表达量之间存在正相关,ERKs的上游激酶MEK抑制剂PD98059可以消除HPC诱导的ERKs磷酸化、HIF-1α表达和心肌细胞保护作用。由此得出的结论是HPC可以提高乳鼠心肌细胞对于H／R的耐受性,其机制涉及ERKs介导的HIF-1α表达。     

急性低氧对体外培养乳鼠心肌细胞肌红蛋白的影响

本实验观察了低氧、复氧时培养的乳鼠心肌细胞肌红蛋白（Ｍｂ）、ｃＡＭＰ、心肌收缩频率的变化以及磷酸二酯酶抑制剂茶碱和抑制肌质网上钙释放的普鲁卡因地低氧下心肌细胞Ｍｂ表达和心肌细胞收缩频率的影响。结果表明,随低氧时间的延长Ｍｂ增加,ｃＡＭＰ和心肌收缩下降,Ｍｂ、ｃＡＭＰ和心肌细胞收缩频率经复氧可以得到恢复。普鲁卡因使低氧时心肌细胞的收缩频率更漫和Ｍｂ的表达减弱;茶碱使低氧下心肌细胞的收缩频率和Ｍｂ的表     

Delayed cytoprotection induced by hypoxic preconditioning in cultured neonatal rat cardiomyocytes: role of GRP78

Hypoxic preconditioning (HPC) has been well demonstrated to have potent protective effects in many cell types; however, the mechanisms responsible for this phenomenon are not fully understood. Recently, glucose-regulated protein 78 (GRP78), an inducible molecular chaperon, was indicated to be associated with ischemic preconditioning. We hypothesized that HPC protects cardiomyocytes against hypoxia by inducing GRP78 in cultured neonatal rat cardiomyocytes. HPC was induced by exposing cardiomyocytes to brief hypoxia (1% O(2), 30 min) followed by reoxygenation. GRP78 was expressed constitutively in cultured cardiomyocytes and its expression was enhanced at 12 h, peaked at 24 h (207.3+/-23.6% of the baseline), and was sustained for up to 72 h after HPC. Twenty-four hours after HPC, the myocytes were subjected to prolonged hypoxia (1% O(2), 12 h). The lactic dehydrogenase (LDH) release and malondialdehyde (MDA) content were reduced, while cell viability and superoxide dismutase (SOD) activity were increased in the preconditioned cells compared with the non-HPC cells. The GRP78 protein level was higher in cells exposed to both HPC and hypoxia than in the cells exposed to HPC alone or hypoxia alone. Heat shock protein 70 (HSP70) was induced in parallel by late HPC. Transfection of GRP78 antisense oligonucleotides blocked GRP78 expression but not HSP70, resulting in attenuated cardioprotection afforded by late HPC. Furthermore, inducing GRP78 by gene transfer protected cardiomyocytes from hypoxic injury. These findings demonstrate that the induction of GRP78 partially mediates the late HPC, suggesting that GRP78 is a novel mechanism responsible for the late cytoprotection of HPC.     

活性氧和线粒体ATP敏感钾通道介导肿瘤坏死因子α对缺氧/复氧心肌的保护作用

在培养的乳鼠心肌细胞上,研究肿瘤坏死因子α(TNF-α)对缺氧/复氧损伤心肌的保护作用的机制。结果发现：(1)用TNF-α(10—500U／ml)预处理,缺氧/复氧后心肌细胞内锰超氧化物歧化酶(Mn-SOD)活性增高、乳酸脱氢酶(LDH)释放量减少(P＜0．05);(2)用抗氧化剂N-乙酰半既氨酸(NAC,1mmol/L)、抗霉素A(antimycin A,50μmol/L)、2-巯基丙酰氨基乙酸(2-MPG,400μmol/L)和铜／锌超氧化物歧化酸(Cu/Zn,SOD)抑制剂二乙基二硫代氨基甲酸盐(DDC,100nmol/L)预处理,可取消TNF-α的抑制缺氧/复氧心肌细胞LDH释放和诱导Mn-SOD活性增高的作用;(3)mitoKATP通道抑制剂5-羟基癸酸(5-HD)预处理可阻断TNF-α对缺氧/复氧心肌细胞的保护作用;选择性mitoKATP通道开放剂diazoxide(50μmol/L)预处理可减少复氧后心肌细胞LDH的释放(P＜0．01),其作用可被5-HD(100μmol/L)和NAC所抑制。上述结果表明,活性氧和线粒体ATP敏感钾通道参与介导TNF-α对缺氧/复氧损伤的心肌保护作用。     

Protective effect of gap junction uncouplers given during hypoxia against reoxygenation injury in isolated rat hearts

It has been shown that cell-to-cell chemical coupling may persist during severe myocardial hypoxia or ischemia. We aimed to analyze the effects of different, chemically unrelated gap junction uncouplers on the progression of ischemic injury in hypoxic myocardium. First, we analyzed the effects of heptanol, 18alpha-glycyrrhetinic acid, and palmitoleic acid on intracellular Ca2+ concentration during simulated hypoxia (2 mM NaCN) in isolated cardiomyocytes. Next, we analyzed their effects on developed and diastolic tension and electrical impedance in 47 isolated rat hearts submitted to 40 min of hypoxia and reoxygenation. All treatments were applied only during the hypoxic period. Cell injury was determined by lactate dehydrogenase (LDH) release. Heptanol, but not 18alpha-glycyrrhetinic acid nor palmitoleic acid, attenuated the increase in cytosolic Ca2+ concentration induced by simulated ischemia in cardiomyocytes and delayed rigor development (rigor onset at 7.31 +/- 0.71 min in controls vs. 14.76 +/- 1.44 in heptanol-treated hearts, P < 0.001) and the onset of the marked changes in electrical impedance (tissue resistivity: 4.02 +/- 0.29 vs. 7.75 +/- 1.84 min, P = 0.016) in hypoxic rat hearts. LDH release from hypoxic hearts was minimal and was not significantly modified by drugs. However, all gap junction uncouplers, given during hypoxia, attenuated LDH release during subsequent reoxygenation. Dose-response analysis showed that increasing heptanol concentration beyond the level associated with maximal effects on cell coupling resulted in further protection against hypoxic injury. In conclusion, gap junction uncoupling during hypoxia has a protective effect on cell death occurring upon subsequent reoxygenation, and heptanol has, in addition, a marked protective effect independent of its uncoupling actions.     

ARC contributes to the inhibitory effect of preconditioning on cardiomyocyte apoptosis

Inhibition of cardiomyocyte apoptosis plays a key role in preconditioning-triggered cardioprotection. However, the molecular mechanism(s) by which preconditioning inhibits apoptosis is not fully understood. Apoptosis repressor with caspase recruitment domain (ARC) possesses the ability to block hypoxia-induced cardiomyocyte apoptosis. We tested whether ARC contributes to the inhibitory effect of preconditioning on cardiomyocyte apoptosis. Cardiomyocytes from 1-day-old male Sprague-Dawley rats were preconditioned by exposing to 10 min of hypoxia, followed by 30 min of reoxygenation. Then, the preconditioned and non-preconditioned cardiomyocytes were exposed to 90 min of hypoxia followed by 120 min of reoxygenation. The results showed that preconditioning inhibited cell death induced by hypoxia and reoxygenation. Hypoxia and reoxygenation could induce a decrease of ARC protein levels. Intriguingly, preconditioning could maintain ARC protein levels. Inhibition of endogenous ARC expression by ARC antisense oligonucleotides reduced the inhibitory effect of preconditioning on apoptosis. Furthermore, preconditioning-induced suppression of the release of mitochondrial cytochrome c to cytosol and caspase-3 activation could be abolished by the inhibition of endogenous ARC expression using ARC antisense oligonucleotides. Conclusion: These data indicate that ARC participates in preconditioning-triggered cardioprotection by interfering with cytochrome c release and caspase-3 activation.     

Hypoxic postconditioning enhances the survival and inhibits apoptosis of cardiomyocytes following reoxygenation: role of peroxynitrite formation

Objectives: Our previous study has shown that slow or “controlled” reperfusion for the ischemic heart reduces cardiomyocyte injury and myocardial infarction, while the mechanisms involved are largely unclear. In this study, we tested the hypothesis that enhancement of survival and prevention of apoptosis in hypoxic/reoxygenated cardiomyocytes by hypoxic postconditioning (HPC) are associated with the reduction in peroxynitrite (ONOO⁻) formation induced by hypoxia/reoxygenation (H/R). Methods: Isolated adult rat cardiomyocytes were exposed to 2 h of hypoxia followed by 3 h of reoxygenation. After 2 h of hypoxia the cardiomyocytes were either abruptly reperfused with pre-oxygenized culture medium or postconditioned by two cycles of 5 min of brief reoxygenation and 5 min of re-hypoxia followed by 160 min of abrupt reoxygenation. Results: H/R resulted in severe injury in cardiomyocytes as evidenced by decreased cell viability, increased LDH leakage in the culture medium, increased apoptotic index (P values all less than 0.01 vs. normoxia control group) and DNA ladder formation, which could be significantly attenuated by HPC treatment applied before the abrupt reoxygenation (P < 0.05 vs. H/R group). In addition, H/R induced a significant increase in ONOO⁻ formation as determined by nitrotyrosine content in cardiomyocytes (P < 0.01 vs. normoxia control). Treatment with the potent ONOO⁻ scavenger uric acid (UA) at reoxygenation significantly decreased ONOO⁻ production and protected myocytes against H/R injury, whereas the same treatment with UA could not further enhance myocyte survival in HPC group (P > 0.05 vs. HPC alone). Statistical analysis showed that cell viability closely correlated inversely with myocyte ONOO⁻ formation (P < 0.01). Conclusion: These data demonstrate that hypoxic postconditioning protects myocytes against apoptosis following reoxygenation and enhances myocytes survival, which is partly attributable to the reduced ONOO⁻ formation following reoxygenation. H.-C. Wang and H.-F. Zhang contributed equally to this study.     

Hypoxia-induced remodelling of PDE4 isoform expression and cAMP handling in human pulmonary artery smooth muscle cells

Human pulmonary artery smooth muscle cells (hPASM cells) express PDE4A10, PDE4A11, PDE4B2, PDE4C and PDE4D5 isoforms. Hypoxia causes a transient up-regulation of PDE4B2 that reaches a maximum after 7 days and sustained up-regulation of PDE4A10/11 and PDE4D5 over 14 days in hypoxia. Seven days in hypoxia increases both intracellular cAMP levels, protein kinase A (PKA) activity and activated, phosphorylated extracellular signal regulated kinase (pERK) but does not alter either PKA isoform expression or total cAMP phosphodiesterase-4 (PDE4) activity or cAMP phosphodiesterase-3 (PDE3) activity. Both the cyclooxygenase inhibitor, indomethacin and the ERK inhibitors, UO126 and PD980589 reverse the hypoxia-induced increase in intracellular cAMP levels back to those seen in normoxic hPASM cells. Challenge of normoxic hPASM cells with prostaglandin E(2) (PGE(2)) elevates cAMP to levels comparable to those seen in hypoxic cells but fails to increase intracellular cAMP levels in hypoxic hPASM cells. The adenylyl cyclase activator, forskolin increases cAMP levels in both normoxic and hypoxic hPASM cells to comparable elevated levels. Challenge of hypoxic hPASM cells with indomethacin attenuates total PDE4 activity whilst challenge with UO126 increases total PDE4 activity. We propose that the hypoxia-induced activation of ERK initiates a phospholipase A(2)/COX-driven autocrine effect whereupon PGE(2) is generated, causing the activation of adenylyl cyclase and increase in intracellular cAMP. Despite the hypoxia-induced increases in the expression of PDE4A10/11, PDE4B2 and PDE4D5 and activation of certain of these long PDE4 isoforms through PKA phosphorylation, we suggest that the failure to see any overall increase in PDE4 activity is due to ERK-mediated phosphorylation and inhibition of particular PDE4 long isoforms. Such hypoxia-induced increase in expression of PDE4 isoforms known to interact with certain signalling scaffold proteins may result in alterations in compartmentalised cAMP signalling. The hypoxia-induced increase in cAMP may represent a compensatory protective mechanism against hypoxia-induced mitogens such as endothelin-1 and serotonin.     

Hypoxic preconditioning up-regulates DJ-1 protein expression in rat heart-derived H9c2 cells through the activation of extracellular-regulated kinase 1/2 pathway

Myocardial preconditioning is a powerful phenomenon that can attenuate ischemia/reperfusion-induced oxidant stress and elicit delayed cardioprotection. Its mechanisms involve activation of intracellular signaling pathways and up-regulation of the protective antioxidant proteins. DJ-1 protein, as a multifunctional intracellular protein, plays an important role in attenuating oxidant stress and promoting cell survival. In the present study, we investigated whether DJ-1 is up-regulated during the late phase of hypoxic preconditioning (HP) and the up-regulation of DJ-1 is mediated by extracellular-regulated kinase 1/2 (ERK1/2) signaling pathway. Rat heart-derived H9c2 cells were exposed to HP. Twenty-four hours later cells were subjected to hypoxia/reoxygenation (H/R) and then cell viability, lactate dehydrogenase (LDH), intracellular reactive oxygen species (ROS), ERK1/2 phosphorylation, and DJ-1 protein were measured appropriately. The results showed that HP efficiently attenuated H/R-induced viability loss and LDH leakage. In addition, HP promoted ERK1/2 activation, up-regulated DJ-1 protein expression, inhibited H/R induced the elevation of ROS. However, when ERK1/2 phosphorylation was specifically inhibited by U0126, the increase in DJ-1 expression occurring during HP was almost completely abolished and, as a result, the delayed cardioprotection induced by HP was abolished, and the inhibitory effect of HP on H/R-induced oxidant stress was also reversed. Furthermore, knocking down DJ-1 by siRNA attenuated the delayed cardioprotection induced by HP. Our data indicate that HP can up-regulate DJ-1 protein expression through the ERK1/2-dependent signaling pathway. Importantly, DJ-1 might be involved in the delayed cardioprotective effect of HP against H/R injury.