Role of the cAMP Signaling Pathway in the Regulation of Gonadotropin-Releasing Hormone Secretion in GT1 Cells

Abstract

Development of a line of highly differentiated neuronal cells (GT1 cells) that secrete gonadotropin-releasing hormone (GnRH) has permitted studies on the signaling mechanisms involved in regulating secretion of GnRH. This study focused on the pathways coupling GnRH secretion to elevated levels of cyclic adenosine monophosphate (cAMP) in the GT1 cell line. Two mechanisms were examined: a direct action of increased cAMP by means of cyclic nucleotide-gated (CNG) cation channels and an indirect action through activation of cAMP-dependent protein kinase A (PKA). Patch-clamp experiments were carried out on two lines of GT1 cells in both the on-cell and excised patch configurations. Treatment of perifused GT1 cells with dopamine led to a substantial rise in cAMP levels, which resembled accumulation of intracellular cAMP in GT1 cells exposed to dopamine. GT1 cells were found to express the three CNG subunits present in olfactory neurons: CN2, CNG4.3, and CNG5. The cells possessed functional cAMP-gated cation channels. Activation of PKA did not seem to be required for increased cAMP to stimulate the release of GnRH. Indeed, inhibiting PKA activity was associated with increased basal GnRH secretion. PKA inhibited adenylyl cyclase activity, probably by suppressing the cellular expression of adenylyl cyclase V. Stimulation of GnRH release by increased cAMP seems to result from neuronal depolarization triggered by increased conductance of cations by cAMP-gated cation channels. Activation of PKA may be a negative-feedback means of lowering cAMP levels. The resultant oscillations in cAMP could provide a basis for timing the pulsatile release of GnRH. These findings can be tested in vivo by using transgenic methods to examine the role of cAMP signaling in regulating pulsatile GnRH release in the rat. Proc Natl Acad Sci U S A 2000;97:1861–1866