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cAMP Modulates the Excitability of Immortalized Hypothalamic (GT1) Neurons via a Cyclic Nucleotide-Gated Channel

Department of Neurology (A.C., J.C.) University of California Los Angeles School of Medicine Los Angeles, California 90095
Department of Obstetrics and Gynecology (R.W.) University of California San Francisco School of Medicine San Francisco, California 94143

GT1 cells are immortalized hypothalamic neurons that show spontaneous bursts of action potentials and oscillations in intracellular calcium concentration [Ca²⁺]_i, as well as pulsatile release of GnRH. We investigated the role 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 activated by either cAMP or cGMP. CNG channels from GT1 cells showed rapid transitions from open to closed states typical of heteromeric CNG channels, were selective for cations, and had an estimated single channel conductance of 60 picosiemens (pS). Ca²⁺ inhibited the conductance of macroscopic currents and caused rectification of currents at increasingly positive and negative potentials. The membrane permeant cAMP analog Sp-cAMP-monophosphorothioate (Sp-cAMPS) increased the frequency of spontaneous Ca²⁺ oscillations in GT1 cells, whereas the Rp-cAMPS isomer had only a slight stimulatory effect on Ca²⁺ signaling. Forskolin, norepinephrine, and dopamine, all of which stimulate cAMP production in GT1 cells, each increased the frequency of Ca²⁺ oscillations. 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 inhibited cAMP-activated channels in GT1 cells. Both L-cis-diltiazem and elevated extracellular Ca²⁺ reversibly inhibited the stimulatory effects of cAMP-generating ligands or Sp-cAMP on Ca²⁺ oscillations. These results indicate 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.

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