Abstract
Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H2O2)-induced cytosolic Ca2+ ([Ca2+]i) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca2+]i rise and whole-cell current change in response to H2O2. Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H2O2-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H2O2-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-α-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca2+]i and whole-cell currents to H2O2. TRPM2 overexpression also aggravated the H2O2-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca2+ overload in response to H2O2 and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.
Highlights
Reactive oxygen species (ROS) are key factors in pathophysiology of vascular endothelial cells
In H5V cells, TM2E3 inhibited H2O2-induced Ca2+ influx, which was the second [Ca2+]i rise in response to Ca2+ add-back (Figs. 2E and F)
We found that H2O2 could elicit [Ca2+]i rises, induce whole-cell currents and trigger apoptotic cell death
Summary
Reactive oxygen species (ROS) are key factors in pathophysiology of vascular endothelial cells. Binding of ADP-ribose to TRPM2 opens the channel, allowing Na+ and Ca2+ to enter the cells. ADP-ribose activation of TRPM2 is potentiated by [Ca2+]i, nicotinic acid adenine dinucleotide phosphate and H2O2, which is a major ROS [6,7,8]. In addition to its potentiation effect, H2O2 may directly stimulate TRPM2 activity [9,10]. It has been shown that H2O2induced Ca2+ influx through TRPM2 contributes to ROS-induced cell death in several cell types including neuons, hematopoietic cells and TRPM2-overexpressing HEK293 cells [7,11,12,13]. Besides TRPM2, ROS could activate other Ca2+ influx channels and stimulate intracellular store Ca2+ release, contributing to Ca2+ overload and cell death [14,15,16]
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