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

Abstract

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 ([Ca2+]i) responses were quantified using digital fluorescence imaging system. Kisspeptin-10 (100, 300, and 1000 nM) caused a significant increase in [Ca2+]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 [Ca2+]i levels in kisspeptin cells, but GnIH attenuated the kisspeptin-evoked [Ca2+]i transients (n = 47, p < 0.001). This novel findings of [Ca2+]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.