Oral Presentation The International Congress of Neuroendocrinology 2014

Leptin evokes cytosolic calcium responses in immortalized RFamide-related peptide-3 neurons (#110)

Haluk Kelestimur 1 , Tugrul Saatci 1 , Emine Kacar 1 , Mete Ozcan 2 , Bayram Yilmaz 3 , Ahmet Ayar 4
  1. Department of Physiology Faculty of Medicine , Firat University, Elazig, Turkey
  2. Department of Biophysics Faculty of Medicine, Firat University, Elazig, Turkey
  3. Department of Physiology Faculty of Medicine, Yeditepe University, Istanbul, Turkey
  4. Department of Physiology Faculty of Medicine, Karadeniz Technical University, Trabzon, Turkey

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