RFamide-related peptide-3 (RFRP-3), a mammalian ortholog of avian gonadotropin-inhibitory hormone (GnIH), seems to be an important regulator of the hypothalamus-pituitary-gonadal (HPG) reproductive axis. Leptin, a permissive hormonal regulator of fertility, provides energy signal to brain. According to current view, leptin does not act directly on gonadotrophin-releasing hormone (GnRH) neurons. RFRP-3 neurons have been shown to express leptin receptors. The goal of the present study was to examine whether leptin acts through RFRP-3 neurons to modulate activity of the GnRH neurons. For this aim, the effects of leptin on intracellular free  Ca²⁺ levels ([Ca²⁺]_i) in RFRP-3 neurons were investigated by using in vitro calcium imaging system. In the present study, rHypoE-7 cell line was used as a model to explore the effects of leptin on RFRP-3 neurons. rHypoE-7 cells were plated on glass coverslip and loaded with 1μM Fura-2 AM. [Ca²⁺]_i responses were quantified by the changes in 340/380 ratio. Data are analyzed by using unpaired t test, with a 2-tailed P level of <0.05 defining statistical significance. Leptin caused increases in [Ca²⁺]_i in a dose-dependent manner. The mean 340/380 nm ratios were (baseline 100.0±0.0 %): 101.1±0.4 % (0.1µM leptin, n= 15 cells), 113.7±2.9 % (1µM leptin, n=28) and 142.8±4.3 % (10 µM leptin, n=49 ), following application of respective concentrations of leptin. The protein kinase C (PKC) inhibitor chelerythrine chloride significantly attenuated the leptin (10 uM)-induced [Ca²⁺]_i responses (from 100.0±0.0% to 19.2±4.0%, n=30, p<0.001). In  Ca²⁺ free conditions, the stimulatory effect of leptin (10 µM) on [Ca²⁺]_i was weaker (n=18, p<0.01). In conclusion, we demonstrated for the first time that leptin activates intracellular calcium signaling in RFRP-3 neurons through PKC-dependent pathway, and thus leptin may exert its effect on GnRH neurons by means of RFRP-3 cells.

This study was supported by FUBAP Project No. TF.12.88