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

Bari MR, Akbar S, Eweida M, Kühn FJ, Gustafsson AJ, Lückhoff A, Islam MS - J. Cell. Mol. Med. (2009)

Bottom Line: 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)).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.We conclude that functional TRPM2 channels mediate H(2)O(2)-induced Ca(2+) entry in beta-cells, a process potently inhibited by ACA.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Sciences and Education, Karolinska Institutet, Research Centre, Stockholm South Hospital, Stockholm, Sweden.

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.

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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).
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fig02: 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).

Mentions: When Ca2+ was omitted from the extracellular medium, [Ca2+]i response to 200 μM H2O2 was abolished (Fig. 2B and C). In these experiments, cells were exposed to nominally Ca2+-free medium for 1 min. before addition of H2O2. In separate experiments, we established that exposure of cells to nominally Ca2+-free medium for such short period, did not deplete the ER Ca2+ store, because carbachol increased [Ca2+]i by releasing Ca2+ from the ER under such conditions (data not shown). The maximal [Ca2+]i changes in the Ca2+-containing and in the Ca2+-free medium were 39 ± 2 nM and –5 ± 10 nM, respectively (P= 0.01, n= 6). In Fig. 2D, [Ca2+]i was first raised by 500 μM H2O2 in the presence of Ca2+-containing extracellular medium. When [Ca2+]i increased to a plateau, the medium was switched to the nominally Ca2+-free medium. This resulted in the return of [Ca2+]i to the basal level, indicating that the [Ca2+]i increase by H2O2 was due to the entry of Ca2+ across the plasma membrane (Fig. 2D, c.f.Fig. 1B).


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

Bari MR, Akbar S, Eweida M, Kühn FJ, Gustafsson AJ, Lückhoff A, Islam MS - J. Cell. Mol. Med. (2009)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4516483&req=5

fig02: 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).
Mentions: When Ca2+ was omitted from the extracellular medium, [Ca2+]i response to 200 μM H2O2 was abolished (Fig. 2B and C). In these experiments, cells were exposed to nominally Ca2+-free medium for 1 min. before addition of H2O2. In separate experiments, we established that exposure of cells to nominally Ca2+-free medium for such short period, did not deplete the ER Ca2+ store, because carbachol increased [Ca2+]i by releasing Ca2+ from the ER under such conditions (data not shown). The maximal [Ca2+]i changes in the Ca2+-containing and in the Ca2+-free medium were 39 ± 2 nM and –5 ± 10 nM, respectively (P= 0.01, n= 6). In Fig. 2D, [Ca2+]i was first raised by 500 μM H2O2 in the presence of Ca2+-containing extracellular medium. When [Ca2+]i increased to a plateau, the medium was switched to the nominally Ca2+-free medium. This resulted in the return of [Ca2+]i to the basal level, indicating that the [Ca2+]i increase by H2O2 was due to the entry of Ca2+ across the plasma membrane (Fig. 2D, c.f.Fig. 1B).

Bottom Line: 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)).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.We conclude that functional TRPM2 channels mediate H(2)O(2)-induced Ca(2+) entry in beta-cells, a process potently inhibited by ACA.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Sciences and Education, Karolinska Institutet, Research Centre, Stockholm South Hospital, Stockholm, Sweden.

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.

Show MeSH
Related in: MedlinePlus