The Expression and Role of Hyperpolarization-Activated and Cyclic Nucleotide-Gated Channels in Endocrine Anterior Pituitary Cells

Abstract

Pituitary cells fire action potentials independently of external stimuli, and such spontaneous electrical activity is modulated by a large variety of hypothalamic and intrapituitary agonists. Here, we focused on the potential role of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels in electrical activity of cultured rat anterior pituitary cells. Quantitative RT-PCR analysis showed higher level of expression of mRNA transcripts for HCN2 and HCN3 subunits and lower expression of HCN1 and HCN4 subunits in these cells. Western immunoblot analysis of lysates from normal and GH(3) immortalized pituitary cells showed bands with appropriate molecular weights for HCN2, HCN3, and HCN4. Electrophysiological experiments showed the presence of a slowly developing hyperpolarization-activated inward current, which was blocked by Cs(+) and ZD7288, in gonadotrophs, thyrotrophs, somatotrophs, and a fraction of lactotrophs, as well as in other unidentified pituitary cell types. Stimulation of adenylyl cyclase and addition of 8-Br-cAMP enhanced this current and depolarized the cell membrane, whereas 8-Br-cGMP did not alter the current and hyperpolarized the cell membrane. Both inhibition of basal adenylyl cyclase activity and stimulation of phospholipase C signaling pathway inhibited this current. Inhibition of HCN channels affected the frequency of firing but did not abolish spontaneous electrical activity. These experiments indicate that cAMP and cGMP have opposite effects on the excitability of endocrine pituitary cells, that basal cAMP production in cultured cells is sufficient to integrate the majority of HCN channels in electrical activity, and that depletion of phosphatidylinositol 4,5-bisphosphate caused by activation of phospholipase C silences them.

Loss of Basal and TRH-StimulatedTshbExpression in Dispersed Pituitary Cells

“…Euthanasia was done via asphyxiation with CO 2 , and the anterior pituitary glands were removed after decapitation within 30 minutes. Glands were used for histological preparations and mRNA/protein extraction or cut into approximately 1-mm 3 cubes and used for in vitro incubation or for cell dispersion, the latter done after treatment with trypsin and EDTA as previously described (33). During dispersion of cells, a procedure that requires 2 hours, pituitary fragments were kept on ice, ie, incubations with pituitary fragments and freshly dispersed pituitary cells started at the same time point (about 2.5 h after removal of the glands from animals).…”

Molecular mechanisms of pituitary endocrine cell calcium handling

“…Initially, it was believed that only lactotrophs and GH cells are excitable [9]. It later became obvious that other secretory pituitary cell types also fire APs spontaneously and/or in response to hypothalamic neurohormones: melanotrophs [10], corticotrophs [11, 12], somatotrophs [13], gonadotrophs [14], thyrotrophs [15]. Firing of APs causes transient elevation in [Ca 2+ ] i (intracellular Ca 2+ concentration) as it well documented in gonadotrophs, lactotrophs, somatotrophs [16] and immortalized pituitary cells [12, 17].…”

“…In rat gonadotrophs, the APs are tall and narrow, with amplitudes of more than 60 mV (from initiation to peak), half-widths of less than 50 ms, and spiking frequencies that are typically ~0.7 Hz [20]. Ovine gonadotrophs also fire single APs spontaneously [21], as do rat thyrotrophs [15]. The pattern of activity in lactotrophs and somatotrophs can be similar to that in gonadotrophs, with large and narrow spikes [22, 23].…”

The Molecular Cell Biology of Anterior Pituitary Cells

“…This proof copy is the copyright property of the publisher and is confidential until formal publication. 24 lactotrophs, 25 gonadotrophs and thyrotrophs, 12 and probably corticotrophs. 26 Because they are nonselective cation channels, they are likely to play a role in the initiation of the pacemaker depolarization.…”

“…Secondly, multifunctional cells seem to be more abundant in females than in males, suggesting not only that the female pituitary may be more plastic than the male pituitary, but also that the hormonal changes associated with the female reproductive cycle may promote transdifferentiation. 11 thyrotrophs, 12 and largely in corticotrophs, 13 and is characterized by sharp single APs that are short in duration (less than 100 ms), with spike frequencies of about 0.7 Hz and amplitudes of more than 60 mV. Axonal-type AP spiking is associated with small-amplitude [Ca 21 ] i transients that range from 20 nM to 70 nM and low basal hormonal release in these cells.…”