This project addresses 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 tail-less member of the G protein-coupled receptor (GPCR) family, and signals primarily through the phosphoinositide/calcium pathway and activation of MAP kinases to promote secretory and growth responses. The GnRH receptor is coupled to Gq/11 via its second and third intracellular loops and to Gs via its first loop. The agonist-induced coupling of heptahelical receptors to their G proteins is often dependent on the amino-terminal region of the third intracellular loop. Many GPCRs, including the GnRH receptor, contain an apolar amino acid in this region at a constant distance from conserved Pro and Tyr/Asn residues in the fifth transmembrane domain. In the GnRH and angiotensin AT1 receptors, this residue (Leu237) is an important determinant of receptor expression, signaling, and internalization. Its conservation suggests that an apolar amino acid at this locus is of general importance in the coupling of GPCRs to their cognate G proteins. The pulsatile secretory activity of GnRH-producing neurons in the hypothalamus, and consequently of pituitary gonadotroph cells, is essential for normal reproductive function. This process can be analyzed in vitro, since pulsatile release of GnRH also occurs in cultured fetal hypothalamic cells and immortalized GnRH neurons (GT1-7 cells). Both native and transformed GnRH neurons express GnRH receptors that mediate agonist- and antagonist-induced changes in episodic secretory activity. Such responses reflect the existence and function of autocrine regulation of the GnRH neuron by its neuropeptide product. Pituitary gonadotrophs also release GnRH, as well as LH, and treatment with a GnRH receptor antagonist or GnRH antiserum decreases basal LH release. A minority of gonadotrophs exhibit intracellular Ca2+ oscillations, and contribute to basal LH secretion in cultured pituitary cells. The presence and actions of GnRH in the anterior pituitary gland, the major site of expression of its plasma-membrane receptors, suggest that local regulatory effects of the neuropeptide could supplement the primary hypothalamic mechanism for the control of episodic gonadotropin secretion. In vivo, such tonic GnRH stimulation could provide a mechanism for the maintenance of optimal responsiveness of the gonadotrophs to pulses of GnRH arising in the hypothalamus. Studies on intracellular signaling in immortalized GnRH neurons revealed that cAMP production is elevated by increased extracellular Ca2+ and the Ca2+ channel agonist, BK-8644, and is diminished by low extracellular Ca2+ and treatment with nifedipine. These findings are consistent with the abundant expression of adenylyl cyclase type I (AC I) in GnRH neurons. Potassium-induced depolarization of GT1-7 neurons causes a dose-dependent monotonic increase in [Ca2+]i and elicits a bell-shaped cAMP response. The inhibitory phase of the cAMP response is prevented by pertussis toxin (PTX), consistent with the activation of Gi-related proteins during depolarization. Agonist activation of the endogenous GnRH receptor in GT1-7 neurons also elicits a bell-shaped change in cAMP production. The inhibitory action of high GnRH concentrations is prevented by PTX, indicating coupling of the GnRH receptors to Gi-related proteins at high levels of agonist activation. The stimulation of cAMP production by activation of endogenous luteinizing hormone receptors in GT1-7 cells is enhanced by low (nanomolar) concentrations of GnRH but is abolished by micromolar concentrations of GnRH, again in a PTX-sensitive manner. These findings indicate that GnRH neuronal cAMP production is maintained by Ca2+ entry through voltage-sensitive calcium channels, leading to activation of Ca2+-stimulated type I AC. Furthermore, the Ca2+ influx-dependent activation of AC I acts in conjunction with AC regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production. An interaction between estrogen receptors and intracellular signaling was observed in hypothalamic GnRH neurons and their immortalized counterparts (GT1-7 cells), both of which express estrogen (ERa and ERb) and progesterone receptors. Both cell types were found to exhibit positive immunostaining for plasma membrane ERs, as well as estradiol-induced changes in adenylyl cyclase activity. In GT1-7 cells, short-term treatment (5 min) with estradiol caused dose-dependent inhibition of cAMP production. More prolonged treatment (60 min) with picomolar estradiol concentrations inhibited, while nanomolar concentrations increased, cAMP production. The ER antagonist, ICI 182,780 abolished both inhibitory and stimulatory actions of estradiol on cAMP production. Estradiol-induced inhibition of adenylyl cyclase was also prevented by treatment with pertussis toxin, consistent with coupling of the membrane-bound estradiol receptors to an inhibitory G protein. Inhibitory actions of estradiol on adenylyl cyclase were also evident in membrane fractions, and in cells treated with estrogen-protein conjugates. In perifused GT1-7 cells and hypothalamic neurons, treatment with ovulatory phase estradiol levels increased the GnRH peak interval, shortened peak duration, and increased peak amplitude. These findings have demonstrated that membrane-associated ER expressed in GnRH neurons interact with adenylyl cyclase inhibitory G proteins by a rapid non-genomic mechanism, and modulate intracellular cAMP signaling and neuropeptide secretion.
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