|Keywords:||Kisspeptin, GPR54, Antagonist, P271, in vitro, single-cell fluorescence microscopy, fura-2-AM|
|Full text PDF:||http://dspace.library.uu.nl:8080/handle/1874/289058|
Kisspeptin is an important regulator of the hypothalamic-pituitary-gonadal axis in mammals. It acts on the GnRH neurons trough the GPR54 receptor initiating GnRH release. The aim of this study is to determine the optimal concentration of kisspeptin-10 to activate the GPR54 receptor in vitro. This data is then used for in vitro testing of the kisspeptin antagonist P271. The GPR54 receptor is a G-protein coupled receptor, which when activated raises the intracellular calcium level. We used a single-cell fluorescence microscopy on Chem-1 cells (expressing the human GPR54 receptor) loaded with the calcium-sensitive fluorescent dye Fura-2. We were unable to create a reproducible concentration-response study, however we found two concentrations of kisspeptin-10 that gave a reproducible peak (10-6M and 10-7M). These concentrations were used to test the antagonist P271. In this study peptide P271 showed agonistic properties and no antagonistic properties. This could be because of the method used in these experiments.