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.

Fig. 1.

Expression of HCN cation channels in rat pituitary cells. A–C, Effects of 8-Br-cAMP (A) and 8-Br-cGMP (B and C) on electrical activity in thyrotrophs. Note both the depolarizing effect of cAMP and the hyperpolarizing effect of 8-Br-cGMP. D and E, Forskolin-(D) and 8-Br-cAMP-stimulated (E) stimulated electrical activity in pituitary gonadotrophs. F, Quantitative RT-PCR analysis of HCN subunit mRNA transcript expression in anterior pituitary cells cultured for 24 h. G, Western blot analysis of HCN expression in primary and GH₃ immortalized pituitary cells. s, Seconds.

Fig. 2.

Characterization of I_h current in pituitary somatotrophs. A and B, Identification of somatotrophs by GHRH-induced electrical activity in a quiescent cell (A) and modulation of the frequency of AP in a spontaneously active cell (B). C, Representative example of the whole-cell current response to a hyperpolarizing voltage step to −120 mV from a holding potential of −40 mV. D, In all somatotrophs (S), the channel opening was best described by a single-exponential fit. In gonadotrophs (G), lactotrophs (L), and thyrotrophs (T), both single- and double-exponential developments of current were observed. E, Activation curve for I_h. Tail current measurements were used. Cells were held at a holding potential of −40 mV and pulsed in 20-mV increments to test potentials between −60 and −120 mV for 7.5 sec. Normalized amplitudes of tail currents I/I_max were plotted against testing voltage and fitted with the Boltzmann equation (see “Calculations”). Averaged data (means ± sem) from seven cells are shown. F and G, Representative traces of I_h current before and 10 min after addition of 100 μm ZD7288 (F) and before and after application of 1 mm Cs⁺ (G) for 30 sec. H, Reduction in AP frequency by 1 mm Cs⁺ in a spontaneously firing cell expressing I_h current. s, Seconds.

Fig. 3.

Characterization of I_h current in rat pituitary lactotrophs. A, Lactotrophs were identified by their sensitivity to TRH (top) and bromocriptine (bottom). B and D, Whole-cell voltage-clamp recordings of I_h in identified lactotrophs in the presence (gray) and absence (black) of 1 mm Cs⁺ (B) and ZD7288 (D). C, The majority of lactotrophs do not express I_h (top) or show small amplitude current response (middle). Only about 30% of lactotrophs express I_h with amplitude comparable to other anterior pituitary cells (bottom). E, The activation curve for I_h obtained by tail current analysis. F (main panel), Dose-dependent effects of MDL12330A, an adenylyl cyclase inhibitor, on cAMP release in a mixed population of pituitary cells. Inset, Bath application of MDL12330A reduced the amplitude of I_h in an identified lactotroph.

Fig. 4.

Characterization of I_h current in rat pituitary thyrotrophs. Panel A, Thyrotrophs were identified by their response to TRH and their lack of response to bromocriptine. Note the difference in the peak amplitude of AP in thyrotrophs and lactotrophs (Figs. 3A vs. 4A). Panel B, Representative example of the whole-cell current response to a hyperpolarizing voltage step to −120 mV from a holding potential of −40 mV. The time course of channel opening was best described by a double-exponential fit. Panel C, Inhibition of I_h current by 10 μm ZD7288. D, Stimulation of I_h by 8-Br-cAMP. E, Inhibition of I_h by MDL12230A. Top, Representative trace; bottom, Normalized current- and time-constant (τ) values (mean ± sem).s, Seconds; C, control.

Fig. 5.

Properties of HCN channels in pituitary gonadotrophs. A, Cells were identified by their oscillatory response to 1 nm GnRH applied at the end of the recording. B, Representative example of the whole-cell current response to a hyperpolarizing voltage step to −120 mV from a holding potential of −40 mV. The time course of channel opening was best described by a single-exponential fit. C, The activation curve for I_h was obtained by tail current analysis as described in Fig. 2E. D, Blockade of I_h current by the addition of 100 μm ZD7288 to extracellular solution. ZD7288 was applied to the bath for 10 min to fully develop its blocking effect. E, Inhibition of I_h by 1 mm bath Cs⁺. F and G, Cells expressing I_h (F, bottom) displayed inward rectifications in response to hyperpolarizing current pulses of −5 pA that were suppressed by 1 mm Cs⁺ (F, top) and were absent (G, top) in cells lacking I_h (G, bottom). H and I, Difference in the effects of bath Cs⁺ on the frequency of spontaneous firing of AP in gonadotrophs expressing (H) and not expressing (I) I_h. Percentage decrease (H, downward arrow) or increase (I, upward arrow) in frequency is indicated for each cell group. s, Seconds.

Fig. 6.

Regulation of HCN channels by phosphoinositides in gonadotrophs and somatotrophs. A–C, Inhibitory effects of wortmannin, a phosphatidylinositol-3 and phosphatidylinositol-4 kinase inhibitor, on I_h in pituitary cells. Representative traces from somatotrophs (A) and gonadotrophs (B) and mean values of amplitude of I_h and τ in both cell types (n = 8). D and E, Inhibition of I_h by 1 nm GnRH (D) and 50 μm m-3M3FBS [2,4,6-trimethyl-N-C3-(trifluoromethyl)phenylybenzesulfo-nomide], a phospholipase C activator (E), in gonadotrophs. s, Seconds; WT, wortmannin.