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.

We compared the Ca2+ responses to reactive oxygen species (ROS) between mouse endothelial cells derived from large-sized arteries, aortas (aortic ECs), and small-sized arteries, mesenteric arteries (MAECs). Application of hydrogen peroxide (H2O2) caused an increase in cytosolic Ca2+ levels ([Ca2+]i) in both cell types. The [Ca2+]i rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP3) receptors. Removal of Ca2+ from the bath also decreased the [Ca2+]i rises in response to H2O2. In addition, treatment of endothelial cells with H2O2 reduced the [Ca2+]i responses to subsequent challenge of ATP. The decreased [Ca2+]i responses to ATP were resulted from a pre-depletion of intracellular Ca2+ stores by H2O2. Interestingly, we also found that Ca2+ store depletion was more sensitive to H2O2 treatment in endothelial cells of mesenteric arteries than those of aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca2+]i rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H2O2 but not by hydroxyl radical. H2O2 contribution in HX-XO-induced [Ca2+]i rises were more significant in endothelial cells from mesenteric arteries than those from aortas. In summary, H2O2 could induce store Ca2+ release via phospholipase C-IP3 pathway in endothelial cells. Resultant emptying of intracellular Ca2+ stores contributed to the reduced [Ca2+]i responses to subsequent ATP challenge. The [Ca2+]i responses were more sensitive to H2O2 in endothelial cells of small-sized arteries than those of large-sized arteries.

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 (H 2 O 2 )-induced cytosolic Ca 2+ ([Ca 2+ ] 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 [Ca 2+ ] i rise and whole-cell current change in response to H 2 O 2 . Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H 2 O 2 -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 H 2 O 2 -induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-a-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca 2+ ] i and whole-cell currents to H 2 O 2 . TRPM2 overexpression also aggravated the H 2 O 2 -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 Ca 2+ overload in response to H 2 O 2 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.

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