Regulation of the Hypothalamic GnRH Neuron

Child Health And Human Development

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Abstract

Our previous studies in native hypothalamic GnRH neurons and their immortalized counterparts, GT1-7 cells have shown that the autocrine actions of GnRH on its cells of origin cause both inhibitory and stimulatory responses due to the activation of multiple G proteins, and that these actions are essential for episodic GnRH secretion. Also, GnRH neurons were found to express G protein-gated inwardly rectifying potassium (GIRK) channels that are activated by LH/hCG, leading to inhibition of membrane excitability and pulsatile GnRH secretion. The presence of GIRK channels in GnRH neurons is also highly relevant to the mechanism of pulsatile GnRH release, as a consequence of episodic inhibition of neuronal firing and neurosecretion.[unreadable] [unreadable] In more recent studies, the GPR54 receptor and its endogenous ligand, kisspeptin, both of which are essential for activation and regulation of the hypothalamic-pituitary-gonadal axis, were found to be expressed in hypothalamic GnRH neurons and GT1-7 cells. Analysis of RNA extracts from individually identified hypothalamic GnRH neurons with primers for GnRH, KiSS-1, and GPR54 revealed expression of all three gene products. Furthermore, constitutive and GnRH agonist-induced bioluminescence resonance energy transfer BRET(2) between Renilla luciferase (Rluc)-tagged GnRH-R and GPR54 labeled with green fluorescent protein GFP(2), expressed in HEK-293 cells, revealed hetero-oligomerization of the two receptors. Whole-cell patch-clamp recordings from identified GnRH neurons showed that kisspeptin has an initial depolarizing effect on membrane potential, followed by increased action potential firing and an increase in GnRH peak-amplitude and duration. Kisspeptin production and secretion in hypothalamic GnRH neurons and GT1-7 cells was significantly reduced by treatment with GnRH. These findings suggest that kisspeptins can act as paracrine and/or autocrine regulators of the GnRH neuron. The stimulation of GnRH release by kisspeptin, and the opposing effects of GnRH on kisspeptin secretion, indicate that GnRH receptor/GnRH and GPR54/kisspeptin autoregulatory systems are integrated by negative feedback to control the production and secretion of both GnRH and kisspeptin from GnRH neurons. In perifusion studies, treatment of GT1-7 neuronal cells with kisspeptin-10 increased GnRH peak-amplitude and duration. The production and secretion of kisspeptin in cultured hypothalamic neurons and GT1-7 cells was detected by a specific radioimmunoassy, and was significantly reduced by treatment with GnRH. The expression of kisspeptin and GPR54 mRNAs in identified hypothalamic GnRH neurons, as well as kisspeptin secretion, indicate that kisspeptins may act as paracrine and/or autocrine regulators of the GnRH neuron. The stimulation of GnRH release by kisspeptin, and the opposing effects of GnRH on kisspeptin secretion, show that GnRH receptor/GnRH and GPR54/kisspeptin autoregulatory systems are integrated by negative feedback to control the secretion of GnRH and kisspeptin from GnRH neurons.[unreadable] [unreadable] In spontaneously active GnRH neurons, the firing of individual and/or bursts of action potential (AP) is followed by a hyperpolarization which can last from several milliseconds (ms) up to several seconds (s). Such hyperpolarization is mediated by the activation of two families of Ca2+-activated K+ channels. Big conductance (BK) channels contribute to AP repolarization, and small conductance (SK) channels underlie the afterhyperpolarization (AHP) and mediate firing frequency and spike-frequency adaptation. Under whole-cell recording 85% of identified GnRH neurons exhibited spontaneous AP firing. The majority of recorded GnRH neurons (48%) showed irregular AP firing with transition between narrow, high spike amplitude rhythmic firing, and intervals of broader, lower spike amplitude, burst-like AP firing. Narrow high-amplitude APs were followed by fast AHP (fAHP) with a decay constant of 0.9 ms and medium AHP (mAHP) a with decay constant of 27.4 ms. During treatment with GnRH (10 nM) the decay constants of fAHP and mAHP were not significantly changed. However, mAHP was abolished during treatment with GnRH (1 microM), while fAHP remained unchanged and was followed by subthreshold after-depolarization potential (ADP) and significant reduction of the frequency of AP firing. Broader, lower spike amplitude APs during basal recording had a high rate of firing, lacked fAHP and generated mAHP with a decay constant of 39.5 ms. These data provide evidence that the modes of spontaneous action potential firing can determine the nature of the after-hyperpolarizing current in hypothalamic GnRH neurons.[unreadable] [unreadable] An analysis of the role of the first intracellular loop of the mouse GnRH receptor in G protein coupling performed in transfected COS-7 and HEK-293 cells. Previous experimental data, and prediction of coupling by the Hidden Markov model, revealed that the mouse gonadotropin-releasing hormone receptor (mGnRH-R) can couple to Gq11, Gs, and Gi/o. The predicted coupling suggests that residues in the first intracellular (1i) loop between 50 and 63, and in the 3i loop between 228 and 240, are involved in Gq11 coupling. Coupling to Gs is predicted to be in the 1i loop between residues 75 to 85. Amino acids predicted for Gi/o coupling in the 1i loop are residues 57 to 64; 2i loop, residues 137 to 145; and 3i loop, residues 262 to 270. Hidden Markov prediction of coupling suggests that residues in the 1i loop from 50 through 85 are involved in multiple G protein coupling including Gq11, Gs, and Gi/o. Agonist activation of the wild type mGnRH-R expressed in COS-7 cells in a pcDNA 3.1 vector, and in HEK-293 cells using a pGFP2-N vector, caused a monotonic and dose-dependent increase in cAMP production. The predicted promiscuity of the 1i loop of the mGnRH-R in signal transduction was evaluated by mutating selected residues located in its N-terminus. Double Lys/Gln mutations were performed at residues 59 and 62. In contrast to the wild type mGnRH-R, agonist-induced cAMP production by the Lys/Gln mutant showed a biphasic response. cAMP production increased significantly during treatment with 5 pM GnRH and reached its maximal level at 20 pM GnRH. Maximal cAMP production was not sustained by increased GnRH concentrations, and was significantly decreased with a maximal inhibitory effect at 1 microM GnRH. The cAMP profile induced by activation of the mutant mGnRH-R differed significantly from that induced by GnRH activation of the wild type receptor, where maximal cAMP production was obtained with 1 microM GnRH. In summary, mutation of two Lys residues at position 59 and 62 in the 1i loop of the mGnRH-R promotes receptor coupling to Gs at low picomolar GnRH concentrations and increases cAMP production. Conversely, at high nanomolar and micromolar GnRH concentrations the GnRH-R couples to Gi/o and inhibits cAMP production.