TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP-ribose) polymerase
TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP-ribose) polymerase
复制标题
DOI：
10.1038/sj.bjp.0705914
复制
发表时间：
2004-09-01
影响因子：
7.3
通讯作者：
McNulty, S
中科院分区：
文献类型：
--
作者：
Fonfria, E;Marshall, ICB;McNulty, S
TRPM2 （melastatin-like transient receptor potential 2 channel）是一种非选择性阳离子通道，在氧化应激条件下被激活，导致细胞内游离Ca2+浓度（[Ca2+](i)）增加和细胞死亡。我们利用四环素诱导的TRPM2表达细胞系研究了DNA修复酶聚（ADPribose）聚合酶（PARP）在过氧化氢（H2O2）介导的TRPM2激活中的作用在全细胞膜片钳记录中，细胞内腺嘌呤5'-二磷酸核糖（adp -核糖）在四环素诱导的trpm2 -人胚胎肾（HEK293）细胞中触发了内向电流，但在未诱导的细胞中没有。同样，H2O2在负载fluo -4的表达trpm2的HEK293细胞中刺激了[Ca2+](i) （pEC(50) 4.54 +/- 0.02）的增加，但在未诱导的细胞中没有。诱导TRPM2表达增加了H2O2对质膜损伤和线粒体功能障碍的敏感性。这些数据证明了TRPM2在四环素诱导下在TRPM2- hek293细胞中具有功能性表达PARP抑制剂SB750139-B（专利号DE10039610-A1 (Lubisch et al., 2001)）， PJ34 （N-（6-氧- 5,6-二氢菲蒽啶-2-基）-N，N-二甲基乙酰胺）和DPQ（3,4 -二氢-5-[4-（1-哌替啶基）丁氧基]-1(2H)-异喹啉酮）抑制H2O2介导的[Ca2+]增加(i) (pIC(50) vs 100 muM H2O2: 7.64 +/- 0.38；6.68 +/- 0.28；4.78 +/- 0.05)，线粒体功能障碍增加(pIC(50) vs 300 muM H2O2: 7.32 +/- 0.23；6.69 +/- 0.22；分别为5.44 +/- 0.09)和质膜完整性降低(pIC(50) vs 300 muM H2O2: 7.45 +/- 0.27；6.35 +/- 0.18；5.29 +/- 0.12)。在这些实验中，PARP抑制剂（SB750139>PJ34>DPQ）的效价顺序与分离的PARP酶的效价顺序相同在四环素诱导的TRPM2-HEK293细胞中，SB750139-B、PJ34和DPQ对细胞内ADP-核糖引发的内向电流没有影响，提示PARP抑制剂不直接与通道相互作用SB750139-B、PJ34和DPQ抑制内源性表达TRPM2的大鼠胰岛素瘤细胞系（CRI-G1细胞）[Ca2+]的增加(i) (pIC(50) vs 100 muM H2O2: 7.64 +/- 0.38；6.68 +/- 0.28；4.78±0.05)这些数据表明，氧化应激通过激活PARP酶导致重组和内源性表达细胞系统中的TRPM2通道打开。
1 TRPM2 (melastatin-like transient receptor potential 2 channel) is a nonselective cation channel that is activated under conditions of oxidative stress leading to an increase in intracellular free Ca2+ concentration ([Ca2+](i)) and cell death. We investigated the role of the DNA repair enzyme poly( ADPribose) polymerase ( PARP) on hydrogen peroxide (H2O2)-mediated TRPM2 activation using a tetracycline-inducible TRPM2-expressing cell line.2 In whole-cell patch-clamp recordings, intracellular adenine 5'-diphosphoribose (ADP-ribose) triggered an inward current in tetracycline-induced TRPM2-human embryonic kidney (HEK293) cells, but not in uninduced cells. Similarly, H2O2 stimulated an increase in [Ca2+](i) (pEC(50) 4.54 +/- 0.02) in Fluo-4-loaded TRPM2-expressing HEK293 cells, but not in uninduced cells. Induction of TRPM2 expression caused an increase in susceptibility to plasma membrane damage and mitochondrial dysfunction in response to H2O2. These data demonstrate functional expression of TRPM2 following tetracycline induction in TRPM2-HEK293 cells.3 PARP inhibitors SB750139-B ( patent number DE10039610-A1 (Lubisch et al., 2001)), PJ34 (N-(6-oxo- 5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide) and DPQ (3, 4-dihydro-5-[4-(1-piperidinyl) butoxy]-1(2H)-isoquinolinone) inhibited H2O2-mediated increases in [Ca2+](i) (pIC(50) vs 100 muM H2O2: 7.64 +/- 0.38; 6.68 +/- 0.28; 4.78 +/- 0.05, respectively), increases in mitochondrial dysfunction ( pIC(50) vs 300 muM H2O2: 7.32 +/- 0.23; 6.69 +/- 0.22; 5.44 +/- 0.09, respectively) and decreases in plasma membrane integrity (pIC(50) vs 300 muM H2O2: 7.45 +/- 0.27; 6.35 +/- 0.18; 5.29 +/- 0.12, respectively). The order of potency of the PARP inhibitors in these assays (SB750139>PJ34>DPQ) was the same as for inhibition of isolated PARP enzyme.4 SB750139-B, PJ34 and DPQ had no effect on inward currents elicited by intracellular ADP- ribose in tetracycline-induced TRPM2-HEK293 cells, suggesting that PARP inhibitors are not interacting directly with the channel.5 SB750139-B, PJ34 and DPQ inhibited increases in [Ca2+](i) in a rat insulinoma cell line (CRI-G1 cells) endogenously expressing TRPM2 ( pIC(50) vs 100 muM H2O2: 7.64 +/- 0.38; 6.68 +/- 0.28; 4.78 +/- 0.05, respectively).6 These data suggest that oxidative stress causes TRPM2 channel opening in both recombinant and endogenously expressing cell systems via activation of PARP enzymes.