| Co-Investigator(Kenkyū-buntansha) |
ENDO Y. Univ. Occup. Environ. Health. Sch. Med., Asso. Prof., 医学部, 講師 (90194050)
OGAWA K Fukuoka University, Sch. Med., Asso. Prof., 医学部, 助教授 (60078780)
佐川 寿栄子 産業医科大学, 医学部, 助教授 (20035489)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 2003: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Rat adrenal medullnry cells mainly contain the IP3 receptor type 2 (IP3R2). Thus, immunocytochemical studies were done to identify in which organelles the IP3R2 is present. We used two different anti-IP3R2 Abs, the mAb KM1083 and a polyclonal Ab made by Chemicon. Materials immunoreactive to the Chemicon Ab were distributed in the vicinity of the nucleus and at the cell periphery, and most of these immunoreactive materials were bound by BODIPY-IP3, IP3 conjugated with a fluorescent moiety. On the other hand, materials immunoreactive to KM1083 were also present in the vicinity of the nucleus and at the cell periphery, and those near the nucleus were bound by BODIPY IP3. However, the KM1083-immunoreactive materials that were located in a band-like fushion along the cell periphery were devoid of BODIPY-IP3 binding, and were stained with an anti-dopamine-3-hydoxylase Ab, but not with an anti-calnexin Ab. These results indicate that Chemicon Ab-immunoreactive materials were present in the endoplasmic reticulum and KM1083-immunoreactive materials were located not only in the endoplasmic reticulum, but also in secretory granules. The Chemicon Ab recognized IP3R2 of about 250 kDa in immunoblots of adrenal medulla homogenates, whereas KM1083 detected not only the 250 kDa protein, but also an about 200 kDa protein. Intracellular mobilization of Ca^<2+> ions in response to muscarinic receptor stimulation was abolished by a prior exposure to thapsigargin, a SERCA pump inhibitor. Moreover, most of BODIPY thapsigargin binding sites agreed with the distribution of materials immunoreactive to the anti-calnexin Ab. Base on these results, we conclude that the IP3R2 is distributed in the endoplasmic reticulum. KM1083 may recognize not only the IP3R2, but also IP3R2-like proteins in secretory granules.
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