cAMP modulates the excitability of immortalized hypothalamic (GT1) neuronsvia a cyclic nucleotide-gated channel

GT1 cells are immortalized hypothalamic neurons that show spontaneous burst s of action potentials and oscillations in intracellular calcium concentrat ion [Ca2+](i), as well as pulsatile release of GnRH. We investigated the ro le of cyclic nucleotide gated (CNG) channels in the activity of GT1 neurons using patch clamp and calcium imaging techniques. Excised patches from GT1 cells revealed single channels and macroscopic currents that were activate d by either cAMP or cGMP. CNG channels from GT1 cells showed rapid transiti ons from open to closed states typical of heteromeric CNG channels, were se lective for cations, and had an estimated single channel conductance of 60 picosiemens (pS). Ca2+ inhibited the conductance of macroscopic currents an d caused rectification of currents at increasingly positive and negative po tentials. The membrane permeant cAMP analog Sp-cAMP-monophosphorothioate (S p-cAMPS) increased the frequency of spontaneous Ca2+ oscillations in GT1 ce lls, whereas the Rp-cAMPS isomer had only a slight stimulatory effect on Ca 2+ signaling. Forskolin, norepinephrine, and dopamine, all of which stimula te cAMP production in GT1 cells, each increased the frequency of Ca2+ oscil lations. The effects of Sp-cAMPS or NE on Ca2+ signaling did not appear to be mediated by protein kinase A, since treatment with either H9 or Rp-cAMPS did not inhibit the response. The CNG channel inhibitor L-cis-diltiazem in hibited cAMP-activated channels in GT1 cells. Both L-cis-diltiazem and elev ated extracellular Ca2+ reversibly inhibited the stimulatory effects of cAM P-generating ligands or Sp-cAMP on Ca2+ oscillations. These results indicat e that CNG channels play a primary role in mediating the effects of cAMP on excitability in GT1 cells, and thereby may be important in the modulation of GnRH release.