期刊
BRITISH JOURNAL OF PHARMACOLOGY
卷 143, 期 1, 页码 186-192出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/sj.bjp.0705914
关键词
TRPM2; hydrogen peroxide; PARP; oxidative stress; SB750139-B; PJ34; DPQ; CRI-G1
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.
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