he detection of antagonists, CHO-5HT2B
-aequorin cells were incubated with various concentrations of the compounds to be tested. An agonist (α-methyl-5-HT) at a single concentration was then injected on the mixture of cells + antagonist and the emitted light was recorded and integrated for 30 seconds. Non-surmountable antagonists (Methiothepin, Methysergide) as well as surmountable antagonists (Mesulergine, Mianserin, Ketanserin) prevented, in a dosedependent manner the activation of the 5- HT2B
receptor by α-methyl-5-HT (Figure 6B). Similar results were obtained with other antagonists (Ritanserin, rauwolscine, Yohimbine, Spiperone, SB206553 not shown).
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Custom cloning of the G-protein coupled receptor and establishment of a stable cell line, expressing the receptor and apoaequorin.
Selection of the most appropriate calciumcoupling system, if necessary.
Pharmacological characterization of the receptor, development and characterization of the functional assay in a 96-well plate.
A control cell line expressing apoaequorin only is provided (on request) to validate the specificity of the functional response.
Button, D. and Brownstein, M. (1993) Aequorin-expressing mammalian cell lines used to report Ca2+ mobilization. Cell Calcium 14, 663-671.
Milligan, G., Marshall, F., and Rees, S. (1996) Gα16 as a universal G protein adapter: implications for agonist screening strategies. TIPS 17, 235-237.
Mottheakis, L.C. and Ohler, L.D. (2000) Seeing the light: calcium imaging in cells for drug discovery. Drug Discovery Today: HTS supplement 1, (1) 18- 19.
Rink, T.J. (1990) Receptor-mediatPage: All 1 2 3 4 5 6 7 8 Related biology technology :1
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