Abstract
The orexigenic peptide, ghrelin is known to influence function of GnRH neurons, however, the direct effects of the hormone upon these neurons have not been explored, yet. The present study was undertaken to reveal expression of growth hormone secretagogue receptor (GHS-R) in GnRH neurons and elucidate the mechanisms of ghrelin actions upon them. Ca2+-imaging revealed a ghrelin-triggered increase of the Ca2+-content in GT1-7 neurons kept in a steroid-free medium, which was abolished by GHS-R-antagonist JMV2959 (10µM) suggesting direct action of ghrelin. Estradiol (1nM) eliminated the ghrelin-evoked rise of Ca2+-content, indicating the estradiol dependency of the process. Expression of GHS-R mRNA was then confirmed in GnRH-GFP neurons of transgenic mice by single cell RT-PCR. Firing rate and burst frequency of GnRH-GFP neurons were lower in metestrous than proestrous mice. Ghrelin (40nM-4μM) administration resulted in a decreased firing rate and burst frequency of GnRH neurons in metestrous, but not in proestrous mice. Ghrelin also decreased the firing rate of GnRH neurons in males. The ghrelin-evoked alterations of the firing parameters were prevented by JMV2959, supporting the receptor-specific actions of ghrelin on GnRH neurons. In metestrous mice, ghrelin decreased the frequency of GABAergic mPSCs in GnRH neurons. Effects of ghrelin were abolished by the cannabinoid receptor type-1 (CB1) antagonist AM251 (1µM) and the intracellularly applied DAG-lipase inhibitor THL (10µM), indicating the involvement of retrograde endocannabinoid signaling. These findings demonstrate that ghrelin exerts direct regulatory effects on GnRH neurons via GHS-R, and modulates the firing of GnRH neurons in an ovarian-cycle and endocannabinoid dependent manner.
Highlights
The orexigenic peptide, ghrelin was initially discovered in rat stomach [1], where it is secreted into the circulation under certain physiological conditions such as fasting [2,3,4]
The ghrelin-triggered rise in the intracellular Ca2+-content was abolished by applying 17β-estradiol (E2, 1nM, 24h) in the steroid-free medium indicating an interaction between the ghrelin and E2 signaling mechanisms in GT1-7 neurons (36 cells in three repeated experiments; Figure 1c)
1) growth hormone secretagogue receptor (GHS-R) mediated the effect of ghrelin in Gonadotropin-Releasing Hormone (GnRH) neurons; 2) ghrelin affected GnRH neurons in an estrous-cycle dependent manner; 3) action of ghrelin involved activation of a retrograde endocannabinoid signaling mechanism targeting GABAergic neurotransmission
Summary
The orexigenic peptide, ghrelin was initially discovered in rat stomach [1], where it is secreted into the circulation under certain physiological conditions such as fasting [2,3,4]. Synthesis of ghrelin was later confirmed in other organ systems, too [5]. It serves as endogenous ligand for the growth hormone secretagogue receptor (GHS-R) [1], which belongs to the Gprotein coupled receptor (GPCR) family [6]. The wide-spread expression of ghrelin in various organs shows that it is involved in diverse functions such as growth hormone secretion [10], regulation of food intake and energy balance [2], control of ACTH secretion [11], regulation of insulin and blood glucose levels [12,13], processes of learning and memory, modulation of synaptic plasticity [8,14], and control of reproductive functions [5,15]. Ghrelin production was revealed in the homeostatic centers of the hypothalamus and the pituitary [1,2,16,17,18]
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