2010 Fiscal Year Final Research Report
Development and application of cAMP imaging in living cell/tissue specimens
| Project/Area Number |
20590195
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
General anatomy (including Histology/Embryology)
|
|---|
| Research Institution | Iwate Medical University |
|---|
Principal Investigator |
SATO Yoichi Iwate Medical University, 医学部, 教授 (40118253)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
SAINO Tomoyuki 岩手医科大学, 医学部, 准教授 (40305991)
AKUTSU Hitomi 岩手医科大学, 医学部, 助教 (30398482)
|
|---|
| Project Period (FY) |
2008 – 2010
|
| Keywords | 細胞・組織 / シグナル伝達 / 生理活性 / cAMP / イメージング |
|---|
| Research Abstract |
We aimed to visualize intracellular cAMP dynamics in living cells/tissues; there were few knowledge on the dynamics, although cAMP has been considered as an important intracellular second messenger. Compared to Ca^<2+> imaging, there was no suitable fluorescent probes for cAMP imaging. In the project, we tried in vivo imaging of cAMP using ratiometric fluorescent probes which are developed for in vitro measurement of cAMP (AKMys and AKMOrn) by Dr. Kudo. These probes are excited by UV laser and sequential fluorescent images were acquired and computed by a laser scanning microscope (Nikon RCM-8000). In the present study, cells loaded AKMLys showed sufficient fluorescent intensity for the measurement. Photobleach of AKMOrn was considerable. Mast cells from rat peritoneal cavity stimulated by Compound 48/80 or ATP showed distinct changes of fluorescent intensity, and computed ratiometric images might represent cAMP changes in the cell. However histamine or cholinergic agonists had no effect on cultured Hela cells, isolated blood vessels, and acinar cells of exocrine glands. 1) Any positive control specimens did not show cAMP changes, 2) mast cell granule matrices can bind various dyes, and 3) high resolution imaging revealed that the exocytosed granules show evident changes of fluorescence. Therefore, at this time, we regret to conclude that the change of fluorescent intensity observed in stimulated mast cells are a kind of artifact. However, in the project, we performed brush-up of the microscopy (Nikon RCM), and consequently many fruitful results on Ca^<2+> imaging have been obtained.
|
