2004 Fiscal Year Final Research Report Summary
STUDY OF THE PHENOTYPES AND FUNCTIONS OF TRP FAMILY MEMBERS EXPRESSED IN NEURONS
| Project/Area Number |
15590059
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | Kyoto University |
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Principal Investigator |
KANEKO Shuji Kyoto University, Graduate School of Pharmaceutical Sciences, Professor, 薬学研究科, 教授 (60177516)
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| Project Period (FY) |
2003 – 2004
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| Keywords | TRPC5 / TRPC4 / TRPM2 / siRNA / Xenopus oocytes / primary culture neuron / cerebral cortex / hydrogen peroxide |
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| Research Abstract |
(1) A brief exposure to hydrogen peroxide (H_2O_2) induces severe deterioration of primary cultured neurons in vitro. We have investigated a possible link between the H_2O_2-induced neuronal death and Ca^<2+>-permeable TRPM2 channels that are supposed to be regulated by ADP-ribose (ADPR) and/or H_2O_2. In most of cultured cerebral cortical neurons from fetal rat, TRPM2 proteins were detected at cell bodies immunocytochemically. Application of H_2O_2 to cultured neurons elicited an increase in intracellular Ca^<2+> concentration ([Ca^<2+>]_i) caused by Ca^<2+> influx and subsequent neuronal death in a similar concentration range. Molecular cloning of rat TRPM2 cDNA revealed several differences in amino acid sequences within Nudix box region as compared with those of human and mouse TRPM2. ADPR-induced current responses, H_2O_2-induced Ca^<2+> influx and H_2O_2-induced cell death were elicited in human embryonic kidney cells heterogeneously expressing rat TRPM2. Treatment of cultured neurons with small interfering RNA (siRNA) against rat TRPM2 caused decrease in immunoreactive TRPM2 content and the H_2O_2-induced Ca^<2+> influx. Moreover, H_2O_2-induced neuronal death was significantly inhibited by the siRNA treatment. These results suggest a critical role of TRPM2 in regulation of neuronal survival as well as the intracellular Ca^<2+> homeostasis.
(2)In cultured cerebral cortical neurons from feral rat, TRPC1,3,4,5 and 6 proteins are detected by immunocytochemistry. In Fura-2 loaded neurons, Ca^<2+> influx was observed after P2Y receptor stimulation with ATP, which was inhibited by La^<3+>, Zn^<2+> and SKF96365. Both TRPC3 immunoreactivity and the receptor-activated Ca^<2+> influx were decreased in the neurons treated with anti-TRPC3 antisense oligodeoxynucleotide. There results suggest that TRPC3 channels are involved in the receptor-operated Ca^<2+> influx in immature cerebral cortical neurons.
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Research Products
(18 results)
