| Research Abstract |
The Method of intracellular calcium ion assay in which fura 2 and other calcium chelating reagents are used for fluorometry, has been well established. As for an qually important bioactive substance, cyclic AMP assay in living cell has not been successful.
In this study, we tried to assay cyclic AMP in living cells by fluorometry and to observe three dementionally by means of motor-driven focus controller in its image analysis.
cDNA of regulatory subunit of cyclic AMP dependent protein kinase (A kinase) was inserted into escherichiacoli and the protein was prepared.
For fluorescence labelling, tetramethylrhodamine was used for regulatory subunit and fluorescein isothyanate for catalytic subunit. These labelled proteins were microinjected into cells.
When 2 molecules of each of regulatory and catalytic subunit are in tetramer, fluorescence light from catalytic subunit can be the excitation light to regulatory subunit. When these subunit molecules are separated, fluorescence light from catalytic subunit itself is detected. This is the principle of fluorescence assay of cyclic AMP.We used mixed culture of the rat striatum in which D1 and D2 receptors coexisted. As widely known, D1 and D2 receptors act in opposite directions as regards to cyclic AMP level within cells. Activated D1 receptor decrease cyclic AMP, while D2 receptor increase it. For these opposite directions of action to cyclic AMP, it was not always possible to assay cyclic AMP at detectable level.
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