| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥300,000)
Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2006: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
(1) Cell swelling-induced insulin secretion and TRP channels
In isolated rat pancreatic β-cells, hypotonic stimulation elicited an increase in cytosolic Ca^<2+> concentration ([Ca^<2+>]_i) at 2.8 mM glucose. The hypotonically induced [Ca^<2+>]i elevation was significantly suppressed by Gd^<3+>, amiloride, 2-aminoethoxydiphenyl borate (2-APB), and ruthenium red, all cation channel blockers. Whole-cell patch clamp analysis showed that hypotonic stimulation induced membrane depolarization of β-cells and produced outwardly rectifying cation currents; Gd^<3+> inhibited both responses. Hypotonic stimulation also increased insulin secretion from isolated rat islets, and Gd^<3+> significantly suppressed this secretion. Taken together, these results suggest that osmotic cell swelling activates cation channels, possibly TRP channels, in rat pancreatic β-cells, thereby causing membrane depolarization and subsequent activation of voltage-dependent Ca^<2+> channels, thus elevating insulin secretion.
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(2) β-Cell apoptosis and TRP channels
Treatment of pancreatic β-cell line INS-1 with 100 μM H_2O_2 for 18 h significantly increased apoptosis cells. The apoptosis was abolished by BAPTA/AM, an intracellular Ca^<2+> chelator, and by 2-APB, which blocks IP_3 receptors and cation channels, but not by nicardipine. Interestingly, the anti-apoptotic effect of BAPTA/AM or 2-APB was not induced when the drug was applied 30 min after the addition of H_2O_2. H_2O_2 also induced [Ca^<2+>]_i elevation which consisted of two phases; the first transient phase and the second gradually developing one. The [Ca^<2+>]I elevation was significantly inhibited by 2-APB. In addition, PP2, a selective inhibitor of Src family, significantly inhibited the H_2O_2-induced apoptosis. These results suggest that [Ca^<2+>]I elevation induced by IP3-mediated Ca^<2+> release and/or Ca^<2+>entry through cation channels plays a pivotal role in H_2O_2-induced β-cell apoptosis, which is mediated by the activation of Src family, and that the first [Ca^<2+>]_i elevation within 30 min after the addition of H_2O_2 seems to be a obligatory step triggering β-cell apoptosis. Less
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