Crosstalk between kisspeptin and gonadotropin-inhibitory hormone in the silence of puberty: preclinical evidence from a calcium signaling study.

Kisspeptin and gonadotropin-inhibitory hormone (GnIH) are among suggested neuroendocrine modulators of reproductive function. Intracellular calcium signaling is a critical component in the regulation of a variety of physiological and pathological processes including neurotransmitter release, and, therefore, can be used as signaling indicator for investigating the involvement of kisspeptin, GnIH, and gonadotropin-releasing hormone (GnRH) release. Hence, this study investigated the effects of kisspeptin and GnIH on calcium signaling using immortalized hypothalamic cells (rHypoE-8) as a model. Kisspeptin neurons were loaded with the ratiometric calcium dye (Fura-2 AM, 1 μmol) and intracellular free calcium ([Ca 2+ ] i ) responses were quantified using digital fluorescence imaging system. Kisspeptin-10 (100, 300, and 1000 nM) caused a significant increase in [Ca 2+ ] i in rHypoE-8 cells ( n  = 58, n  = 64, and n  = 49, respectively, p  < 0.001). The kisspeptin receptor antagonist, P234, inhibited the calcium responses to kisspeptin ( p  < 0.001, n  = 32). GnIH (100 and 1000 nM), alone, did not cause any significant change in the mean basal [Ca 2+ ] i levels in kisspeptin cells, but GnIH attenuated the kisspeptin-evoked [Ca 2+ ] i transients ( n  = 47, p  < 0.001). This novel findings of [Ca 2+ ] i signaling in in vitro setting implicate that kisspeptin and GnIH may exert their effects on hypothalamus-pituitary-gonadal (HPG) axis by modulating kisspeptin neurons. These results also implicate that kisspeptin neurons may have an autocrine regulation.