| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Our objective of the study was to investigate the mechanisms of Ca^2+ influx as an universal phenomenon in cell biology. To elucidate the intracellular or extracellular Ca^2+ mobilization during Ca^2+ oscillations in the cell system we investigated the spatio-temporal pattern of intracellular Ca^2+ and Ca^2+ influx in mouse eggs using cell-biological techniques that are intacellular Ca^2+ imaging, Mn^2+ quench of fluorescence dye, Fura-2 and microinjection of stimulants or an antibody, which is a functional inhibitor of activation. Ca^2+ oscillations were induced by microinjection of hamster sperm extract (SE) into mouse eggs. We found the following 4 points, l) Competitive Mn^2+/Ca^2+ influx exists at the resting state. A marked Mn^2+/Ga^2+ influx occurs during the first Ca^2+ release upon SE iniection. and persistently facilitated Mn^2+/Ca^2+ influx is observed during steady Ca^2+ oscillations. As intracellular Mn^2+ concentration ([Mn^2+]i) increased progressively, periodic [Mn^2+] rises appear, corresponding to each Ca^2+ transient but taking a slower time course. A numerical simulation and Mn^2+ oscillations calculated from experimental data strongly suggest that repetitive Mn^2+ release develops after Mn2+ entry and uptake into the ER. 2) No significant increase in Mn^2+influx was detected by injection of SE, InsP_3, or Ca^2+, when Ca^2+ release was prevented by pre-injection of an antibody against the InsP_3 receptor, suggesting that none of them itself causes Ca2+ influx. 3) Depletion of the ER using an ER Ca^2+ pump inhibitor, thapsigargin plus repetitive injection of inositol 1, 4, 5-trisphosphate (InsP_3) cause a marked Mn2+ influx, suggesting that mouse eggs have a cpacitative mechanism. 4) Ca^2+ influx is activated during the initial large Ca^2+ release possibly by a capacitative mechanism and kept facilitated during steady Ca^2+ oscillations.
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