Signaling Pathways Involved in GnRH Secretion in GT₁ Cells

Abstract

The GT₁ GnRH neuronal cell lines exhibit highly differentiated properties of GnRH neurons. We have used GT_1–1 cells to study the role of the cyclic AMP/ protein kinase A, cyclic GMP/protein kinase G and Ca²⁺/protein kinase C signaling pathways in the regulation of GnRH secretion. Superfusion of GT_1–1 cells with the cyclic AMP analog 8-Br-cyclic AMP (0.5 and 2.5 mM) or the adenylate cyclase activator forskolin (1 and 10 µM) for 100 min increased the amplitude of GnRH secretion 2- to 35-fold. The cyclic GMP analog 8-Br-cyclic GMP (2.5 mM) also stimulated the amplitude of GnRH release from superfused GT_1-1 cells, although to a much lesser extent (1.5- to 3-fold). The amplitude of GnRH pulses was also stimulated (5- to 50-fold) by the protein kinase C activator TPA (1 µM). Increasing intracellular Ca²⁺ with an iono-phore (ionomycin, 1 µM) or by the Ca²⁺ channel activator Bay K 8644 (10 µM) also stimulated GnRH release, while secretion was markedly decreased and spontaneous pulsatility abolished by the L-type Ca²⁺ channel blocker methoxyverapamil (10 µM). These results demonstrate that in GT₁ cells the protein kinase A, protein kinase G and protein kinase C pathways are functionally coupled to regulation of GnRH secretion. Furthermore, pulsatile GnRH secretion is coupled to the entry of extracellular Ca²⁺ via L-type Ca²⁺ channels.

Role of the cAMP signaling pathway in the regulation of gonadotropin-releasing hormone secretion in GT1 cells

“…We previously showed that GT1 cells expressed D1-DA receptors that were positively coupled to AC (8) and that increases in cAMP levels resulted in the secretion of GnRH (13). Since intracellular cAMP is rapidly exported out of a variety of cells (27), we asked whether we could simultaneously follow dynamic changes in GnRH and cAMP.…”

“…Treatment of GT1 cells with DA or NE increased intracellular cAMP levels and stimulated GnRH secretion in a dose-dependent fashion (8,12). The GnRH-releasing effects of DA and NE were mimicked by pharmacologically increasing the intracellular cAMP level with 8-Br-cAMP or forskolin treatment (13). The level of secretion of GnRH was proportional to the activation of AC and increases in cAMP levels.…”

“…The level of secretion of GnRH was proportional to the activation of AC and increases in cAMP levels. These data are all consistent with the hypothesis that the stimulation of AC results in the increased formation of cAMP, activating the signaling cascade responsible for stimulation of GnRH release.Several studies have shown that depolarization followed by the influx of extracellular Ca 2ϩ via voltage-dependent Ca 2ϩ channels is essential for the stimulation of GnRH secretion from GT1 cells (5,13,14). Stimulation of GnRH secretion from GT1 cells by NE was correlated with depolarization of the cells and the influx of extracellular Ca 2ϩ (15).…”

“…Several studies have shown that depolarization followed by the influx of extracellular Ca 2ϩ via voltage-dependent Ca 2ϩ channels is essential for the stimulation of GnRH secretion from GT1 cells (5,13,14). Stimulation of GnRH secretion from GT1 cells by NE was correlated with depolarization of the cells and the influx of extracellular Ca 2ϩ (15).…”

Phosphodiesterase expression targeted to gonadotropin-releasing hormone neurons inhibits luteinizing hormone pulses in transgenic rats

“…The secretion of GnRH in GT1 neurons is stimulated by elevations in cAMP levels (6)(7)(8). The stimulation of GnRH secretion by elevated cAMP levels appears to be regulated via cAMP-gated cation channels expressed by the cells.…”