This project is directed at the elucidation of the molecular and cellular processes involved in the secretion and actions of the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), in pituitary gonadotrophs and hypothalamic neurons. The GnRH receptor is a unique G protein-coupled receptor with unusual structural features, including the lack of a carboxyterminal cytoplasmic region. It signals primarily through Gq/11 and the phosphoinositide/calcium pathway from its second and third intracellular loops, and also activates Gs and the adenylyl cyclase system through its first intracellular loop. The rat and mouse GnRH receptors contain 327 amino acids, whereas human, sheep, and bovine receptors have an additional lysine residue in the second extracellular loop at position 191. The human receptor is less well-expressed in transfected cells, and on activation is internalized much more rapidly than the rodent receptors. Deletion of Lys191 from the human GnRH receptor caused a 4-fold increase in receptor expression in transfected cells, with an increase in binding affinity and responsivity to agonist stimulation, and its internalization rate was reduced to that of the mouse receptor. In contrast to these effects of deletion of Lys191, its replacement by Arg, Glu, Gln, or Ala caused no significant change in receptor expression or function. The mechanism by which a single specific residue in the extracellular region of the human GnRH receptor influences its expression, agonist-induced activation, and internalization is currently under investigation. The episodic profile of gonadotropin release from pituitary gonadotrophs reflects the pulsatile secretory activity of GnRH-producing neurons in the hypothalamus, and is essential for normal reproductive function. Pulsatile release of GnRH is also evident in cultured fetal hypothalamic cells and immortalized GnRH neurons (GT1-7 cells). Both of these cell types express GnRH receptor transcripts as well as high-affinity GnRH binding sites. Furthermore, individual GnRH neurons coexpress GnRH and its receptors as revealed by double immunostaining of hypothalamic cultures. In perifused hypothalamic cells and GT1-7 cells, GnRH receptor agonists increased the amplitude and reduced the frequency of pulsatile GnRH release. In contrast, exposure to GnRH antagonist analogs abolished pulsatile secretion and caused a sustained and progressive rise in GnRH release. These findings indicate that activation of the GnRH receptors expressed in hypothalamic GnRH neurons is required for pulsatile neuropeptide release from the GnRH neuronal network. The effects of GnRH agonist and antagonist analogs on GnRH secretion are consistent with the operation of an ultrashort-loop autocrine feedback mechanism that exerts both positive and negative actions that are necessary for the integrated control of GnRH secretion from the hypothalamus. An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that the more hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Tetrodotoxin-sensitive Na+, T-type Ca2+, and L-type Ca2+ channels were found to be involved in the generation of sharp APs, but of these only the L-type channels contributed to the spike depolarization in cells exhibiting broad APs. In both resting and agonist-stimulated GT1 cells, membrane depolarization limited the participation of Na+ and T-type channels in firing, but facilitated AP-driven Ca2+ influx through L-type and voltage-insensitive pathways. Further analysis revealed that the pattern of firing in GT1 neurons is regulated coordinately by apamin-sensitive SK current and store depletion-activated Ca2+ current. This dual control of pacemaker activity facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process could also provide a general mechanism for the integration of voltage-gated Ca2+ influx into receptor-controlled Ca2+ mobilization.
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