In vitro studies on secretion of RFRP-3 controlling by leptin in immortalize rHypoE-7 neurons

Hypothalamic RFamide peptides, gonadotropin-inhibitory hormone (GnIH) and orthologous peptides, which were discovered in mammals belonging to the RFamide peptide superfamily (RFRP-3), seem to be important regulators of hypothalamus-pituitary-gonadal (HPG) reproductive axis. Since GnIH is not only involved in reproductive function but also in the regulation of food intake, leptin produced by adipose cells has been suggested to have effects on RFRP-3 neurons. rHypoE-7 cell line was developed as a model to explore the effects of neuroendocrine factors on RFRP-3 neurons. To investigate whether leptin exerts its effects on food intake as well as reproduction by means of RFRP-3 neurons, changes in intracellular free Ca2+ levels ([Ca2+]i) were observed in rHypoE-7 cells treated by leptin. The main aim of the present study is to examine the effects of leptin on RFRP-3 in immortalize rHypoE-7 cells as in vitro. In the present study rHypoE-7 cells relasing RFRP-3 were used. The effects of leptin on RFRP-3 release and calcium signaling in rHypoE-7 cells were investigated. In rHypoE-7 cells loaded with calcium sensitive dye and [Ca+2]i responses were quantified by the changes in 340/380nm ratio using fluorescence imaging system. The ELISA kit was used to determine GnIH concentrations from the media. In conclusion, leptin changes [Ca+2]i level in immortalized RFRP-3 neurons. This indicates that leptin may modulate GnRH activity via RFRP-3 neurones.