TRPM2 is a non-selective cation channel that has been implicated in oxidative-stress mediated cell death pathways. This study aimed to address the role of poly(ADP-ribose) polymerase (PARP) in the activation of TRPM2 following exposure to hydrogen peroxide (H₂O₂).

Human embryonic kidney (HEK293) cells expressing tetracycline-inducible FLAG-tagged TRPM2 were seeded into black walled clear-based 96-well plates at 20-25, 000 cells per well and incubated at 37C / 5 % CO₂ overnight. TRPM2 expression was induced by incubating cells overnight with 1 µg/ml tetracycline. Fluorimetric imaging of Fluo-3 AM loaded cells was used to determine intracellular free Ca²⁺ ion concentration ([Ca²⁺ ]_i), changes. Cell death was assessed using a 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay.

Tetracycline-induced cells expressing TRPM2 exhibited a large increase in fluo-3 fluorescence after treatment with H₂O₂ (100 µM – 1 mM) indicative of a rise in [Ca²⁺]_i. This response was 10-fold greater than the corresponding fluorescence increase in non-induced cells. The calcium increase was paralleled by an increase in H₂O₂ induced, TRPM2-mediated cell death at 300 µM H₂O₂. The rises in both [Ca²⁺]_i and cell death in response to H₂O₂ treatment were inhibited by the PARP inhibitors PJ34, SB750139-B and DPQ at concentrations known to inhibit purified PARP enzyme. Complete inhibitiion of the effects of H₂O₂ were achieved by PARP inhibitor concentrations of 10µM._p EC₅₀s of 7.032 ± 0.256 (93 nM), and 7.800 ± 0.478 (16 nM) were obtained for PJ34 and SB750139-B, respectively in experiments monitoring [Ca²⁺]_i. PJ34, SB750139-B, and DPQ exhibited _p EC ₅₀ values of 6.685 ± 0.217 (206 nM), 7.318 ± 0.227 (48 nM), and 5.446 ± 0.081 (3600 nM), respectively in MTT cell death studies. Values are expressed as mean ± standard error, n = 3-5 experiments in triplicate.

These data show that HEK293 cells demonstrate an increased sensitivity to oxidative-stress following induced expression of TRPM2. Furthermore, both oxidative stress-mediated [Ca ⁺²]_i increases and cell death were reduced by pretreatment of cells with PARP inhibitors. We hypothesise that PARP activation is required for oxidative stress-mediated opening of TRPM2 channels. PARP activation has been reported to play a role in a number of pathophysiological conditions. Therefore TRPM2 may be a novel therapeutic target for conditions linked to oxidative stress-mediated cell death.

E. Fonfria and K. Hill are in receipt of EU Framework V Postdoctoral Fellowship.