H2O2-induced Ca2+ influx and its inhibition by N-(p-amylcinnamoyl) anthranilic acid in the beta-cells: involvement of TRPM2 channels

Abstract

Type 2 melastatin-related transient receptor potential channel (TRPM2), a member of the melastatin-related TRP (transient receptor potential) subfamily is a Ca(2+)-permeable channel activated by hydrogen peroxide (H(2)O(2)). We have investigated the role of TRPM2 channels in mediating the H(2)O(2)-induced increase in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in insulin-secreting cells. In fura-2 loaded INS-1E cells, a widely used model of beta-cells, and in human beta-cells, H(2)O(2) increased [Ca(2+)](i), in the presence of 3 mM glucose, by inducing Ca(2+) influx across the plasma membrane. H(2)O(2)-induced Ca(2+) influx was not blocked by nimodipine, a blocker of the L-type voltage-gated Ca(2+) channels nor by 2-aminoethoxydiphenyl borate, a blocker of several TRP channels and store-operated channels, but it was completely blocked by N-(p-amylcinnamoyl)anthranilic acid (ACA), a potent inhibitor of TRPM2. Adenosine diphosphate phosphate ribose, a specific activator of TRPM2 channel and H(2)O(2), induced inward cation currents that were blocked by ACA. Western blot using antibodies directed to the epitopes on the N-terminal and on the C-terminal parts of TRPM2 identified the full length TRPM2 (TRPM2-L), and the C-terminally truncated TRPM2 (TRPM2-S) in human islets. We conclude that functional TRPM2 channels mediate H(2)O(2)-induced Ca(2+) entry in beta-cells, a process potently inhibited by ACA.

Figure 1

H₂O₂ increased [Ca²⁺]_i in INS-1E cells and human β-cells. Cells were perfused with physiological solution containing 3 mM glucose and [Ca²⁺]_i was measured in fura-2 loaded cells by microfluorometry. (A) H₂O₂ (200 μM) increased [Ca²⁺]_i in a reversible manner (n= 16). (B) and (D) [Ca²⁺]_i increase by 500 μM, (n= 24) and 1 mM H₂O₂, (n= 9) did not completely reverse after washout of H₂O₂. (C) baseline [Ca²⁺]_i remained stable when no H₂O₂ was added. (E) the concentration-response curve for H₂O₂-induced [Ca²⁺]_i increase. Data are expressed as percentage of maximal [Ca²⁺]_i increase obtained by 1 mM H₂O₂. Each point represents a mean of 5–24 experiments. (F) and (G) show the effects of H₂O₂ on [Ca²⁺]_i in single human β-cells. H₂O₂ increased [Ca²⁺]_i in 10 out of 12 cells for 200 μM and 4 out of 6 for 500 μM.

Figure 2

H₂O₂-induced [Ca²⁺]_i increase was due to Ca²⁺ entry across the plasma membrane. H₂O₂ (200 μM) was applied to INS-1E cells in the presence of 1.5 mM extracellular Ca²⁺ (A) or in nominally Ca²⁺-free medium (B). Maximal [Ca²⁺]_i change in Ca²⁺ containing medium was 39±2 nM (n= 3) and that in nominally Ca²⁺-free medium was –5±10 nM (n= 3) (C). In (D), cells were first exposed to H₂O₂ (500 μM) in the presence of 1.5 mM extracellular Ca²⁺, and the solution was switched to a nominally Ca²⁺-free medium at the time indicated by the horizontal bar(n= 5) (c.f.Fig. 1B).

Figure 3

Effects of different channel blockers on H₂O₂-induced [Ca²⁺]_i increase in INS-1E cells. (A) and (B) Nimodipine did not inhibit H₂O₂-induced [Ca²⁺]_i response. Cells were pre-incubated with Nimodipine (5 μM) for 10 min. H₂O₂ (500 μM) was applied in the continued presence of nimodipine (the trace is representative of seven independent experiments). (B) Shows control experiments for (A) (the trace is representative of eight independent experiments). (D) and (E) 2-APB (50 μM) did not alter the [Ca²⁺]_i response to H₂O₂ (200 μM). Cells were treated with 2-APB (50 μM) for 10 min. during pre-incubation and H₂O₂ was applied in the continued presence of 2-APB. (E) The control for (A) showing [Ca²⁺]_i response to H₂O₂ in the absence of 2-APB (traces D and E are representatives of three to five independent experiments). (G) ACA (20 μM) did not increase [Ca²⁺]_i in INS-1E cells. (H) ACA (20 μM) completely inhibited H₂O₂-induced [Ca²⁺]_i response. Trace (a) shows [Ca²⁺]_i response to H₂O₂ (200 μM) in the absence of ACA (the trace is representative of three independent experiments). In trace (b), ACA (20 μM) was applied 1 min. before application of H₂O₂ (200 μM) and was continuously present in the perfusion (the trace is representative of three independent experiments). (I) Maximal Ca²⁺ changes by H₂O₂ in controls and in the ACA-treated cells were 23 ± 1 and –9 ± 6 nM, respectively (P < 0.01, n= 6). In (C), (F) and (I), the bars represent mean [Ca²⁺]_i increase obtained by H₂O₂, expressed as the percentage of maximal [Ca²⁺]_i increase obtained by 25 mM KCl in respective experiments.

Figure 4

Whole-cell currents induced by ADP ribose and H₂O₂ in INS-1E cells. The whole-cell configuration was attained at the point indicated with ‘w.c.’. Recordings were performed at RT and the holding potential was –60 mV. Bars indicate times where the standard bath solution was changed to a solution containing either NMDG⁺ or the TRPM2 channel inhibitor ACA. (A) Whole-cell current recorded in the presence of intracellular ADP ribose. The pipette solution contained 0.6 mM ADP ribose and 1 μM Ca²⁺. (B) Whole cell currents recorded without ADP ribose and after application of 1–2 μl 30% H₂O₂ directly into the recording chamber. The estimated final concentration of H₂O₂ in the chamber was ∼10 mM. The pipette solution contained 1 μM free Ca²⁺. (C) Current–voltage relationship of H₂O₂-induced currents as derived from (B), recorded during voltage ramps from –90 to +60 mV of 400 ms duration.

Figure 5

Western blot analysis of TRPM2 proteins in human islets. A total of 90 μg of membrane proteins from human islets were separated in two lanes (A, A1) by 10% SDS-PAGE electrophoresis. In A, the blots were probed with an anti-TRPM2-N antibody (1:300). In A1, the blots were probed with an anti-TRPM2-C antibody (1:300). Standard proteins are shown on the right side. The experiments have been repeated at least three times with similar results.