ORIGINAL
RESEARCH
The Expression and Role of HyperpolarizationActivated and Cyclic Nucleotide-Gated Channels in
Endocrine Anterior Pituitary Cells
Karla Kretschmannova, Marek Kucka, Arturo E. Gonzalez-Iglesias,
and Stanko S. Stojilkovic
Section on Cellular Signaling, Program in Developmental Neuroscience, Eunice Kennedy Shriver National
Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland
20892-4510
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 GH3 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. (Molecular Endocrinology 26: 153–164, 2012)
P
ituitary cells fire action potentials (AP) independently
of external stimuli, a phenomenon termed “spontaneous electrical activity.” Each AP is composed of a slow
depolarizing phase, a rapid depolarizing phase or spiking
depolarization, and a rapid or delayed (plateau-bursting
type) repolarizing phase (1). Such rhythmicity fulfills the
need to drive the periodic fluctuations in cytosolic Ca2⫹
concentrations and hormone release, as is well documented for lactotrophs and somatotrophs (2). In general,
intrinsic electrophysiological characteristics of neuronal,
neuroendocrine, and muscle cells reflect the type and den-
sity of numerous voltage- and ligand-gated ion channels
that regulate the flow of ionic currents across the plasma
membrane (3). In that respect, it is not unexpected that
endocrine pituitary cells also possess a rich repertoire of
ion channels, including voltage-gated Na⫹, K⫹, and Ca2⫹
channels, ligand-gated GABA␥-aminobutyric acid-A, and
purinergic P2X receptor channels, as well as a number of
less conventional ionic conductances. Spontaneous electrical activity of these cells is modulated by a large variety
of hypothalamic and intrapituitary agonists and intracellular messenger systems. These include agonists for the G
ISSN Print 0888-8809 ISSN Online 1944-9917
Printed in U.S.A.
Copyright © 2012 by The Endocrine Society
doi: 10.1210/me.2011-1207 Received July 31, 2011. Accepted October 26, 2011.
First Published Online December 1, 2011
Abbreviations: ab, Antibody; AP, action potential; 8-Br-cAMP, 8-bromo-cAMP; 8-Br-cGMP,
8-bromo-cGMP; CNG, cyclic nucleotide-gated; HCN, hyperpolarization-activated and cyclic
nucleotide-gated; Ih, the hyperpolarization-activated cation current; MDL12330A, cis-N-(2phenylcyclopenthyl)azacyclotridec-1-en-2amine HCl; PIP2, phosphatidylinositol 4,5-bisphosphate; s, slope factor.
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HCN Channels in Pituitary Cells
protein-coupled receptors; the Gs-coupled receptors
(CRH, GHRH, vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide) facilitate firing of AP,
the Gi/o-coupled receptors (dopamine and somatostatin) inhibit it, and the Gq/11-coupled receptors (GnRH, TRH, arginine vasopressin) have dual roles, an initial inhibitory and
a sustained stimulatory role (1).
In neurons and cardiac cells, the ionic channels that
generate autonomous pacemaking capabilities are frequently members of the small family of pacemaking
channels. These channels, termed “hyperpolarizationactivated and cyclic nucleotide-gated” (HCN) channels, belong to the superfamily of voltage-gated channels but form a distinct subgroup of channels that are
closely related to voltage-independent cyclic nucleotidegated (CNG) channels (4). Unique voltage dependence of
HCN channels, together with permeability for both K⫹
and Na⫹, explains their pacemaking function in cardiac
tissue (5, 6). HCN and CNG channels also play important
roles in spontaneous and receptor-induced excitability of
other cells by increasing the slope of slow depolarization.
This comes from their regulatory properties. The hyperpolarization-activated cation current, termed “Ih” (h
stands for hyperpolarization), is sensitive to the presence
of cAMP and, to a much weaker extent, cGMP. Cyclic
nucleotides not only accelerate the kinetics of activation
of Ih, but also shift the voltage dependence for activation
toward more depolarized values. On the other hand,
CNG channels expressed in photoreceptors have a strong
preference for cGMP, whereas the olfactory channel is
almost equally sensitive to both ligands (4). The dependence of HCN and CNG channel activation on cyclic
nucleotides provides a rationale for the stimulatory effects of Gs-coupled receptors and nitric oxide-soluble
guanylyl cyclase signaling pathways on the electrical activity in excitable cells. The Gi/o-dependent inhibitory actions on cAMP-mediated regulation of Ih have also been
documented (7). The Gq/11-coupled receptors also influence the gating of HCN and CNG channels by affecting
the phosphatidylinositol 4,5-bisphosphate (PIP2) levels (8).
To date, four mammalian channel subunits, termed
“HCN1– 4,” have been cloned. These subunits, organized
as homo- and heterotetrameric complexes, represent the
molecular correlates of the Ih (9, 10). The pharmacological identification of these channels is based on the sensitivity to several inhibitors, including ZD7288 and extracellular Cs⫹, and insensitivity to extracellular Ba2⫹ (11,
12). In vertebrates, there are six CNG subunits: CNGA1,
CNGA2, CNGA3, CNGA4, CNGB1, and CNGB3.
CNGA1–3 subunits can form homomeric channels in heterologous expression systems, and the other subunits can
coassemble to form functional heteromeric channels (4).
Mol Endocrinol, January 2012, 26(1):153–164
The mRNA transcripts and functional HCN channels
were identified in rat GH3 cells and somatotrophs (13–
15), mouse At-T20 cells (16), and frog melanotrophs
(17), but the expression and role of HCN channels in
electrical activity in other endocrine pituitary cells were
not studied. The mRNA transcripts for CNG channels
were also identified in rat pituitary cells (18), whereas the
dependence of pituitary cell excitability on cAMP vs.
cGMP was not clarified. The influence of the Gq/11 signaling pathway on the activity of these channels was also
not studied.
Here we focus on the expression and role of HCN
channels in electrical activity in cultured somatotrophs,
gonadotrophs, lactotrophs, and thyrotrophs. The biophysical properties of Ih were systematically investigated
in these cell types, including the slow kinetics and voltage
dependence of their activation and the lack of inactivation. We also examined the pharmacological properties of
these channels. Because there is not a common Gs-coupled
receptor among these cells, we used forskolin, an activator of adenylyl cyclase, and cell-permeable 8-Br-cAMP
and 8-Br-cGMP to activate these channels. Similarly, we
down-regulated basal cAMP production and cAMPdependent channel activity by inhibiting adenylyl cyclase.
We also used pharmacological manipulations to alter the
status of phospholipase C activity and levels of phosphoinositides. Single cell recordings were used in electrophysiological studies, and cyclic nucleotides were measured in
mixed pituitary cell populations.
Results
Effects of cyclic nucleotides on electrical activity in
anterior pituitary cells
In secretory anterior pituitary cells, both forskolin and
cAMP stimulate electrical activity. Figure 1, A and E,
shows stimulatory effects of cell permeable 8-Br-cAMP
on electrical activity in thyrotrophs and gonadotrophs,
respectively, and Fig. 1D shows effect of forskolin on
electrical activity in gonadotrophs. In contrast to the depolarizing effect of 8-Br-cAMP, 8-Br-cGMP hyperpolarized the plasma membrane in thyrotrophs (Fig. 1, B and
C), which slowed the firing frequency in spontaneously
firing cells (Fig. 1B). Similar effect of 8-Br-cGMP was also
observed in other pituitary cell types. In addition, an 85fold increase in cGMP production was caused by a
30-min incubation with 10 ␮M 3,3⬘-(hydroxynitrosohydrazino)bis-1-propanamine, a nitric oxide donor
(basal cGMP was at 44 ⫾ 8 fmol/well, n ⫽ 6, 3,3⬘-(hydroxynitrosohydrazino)bis-1-propanamine-stimulated cGMP
was at 3.77 ⫾ 0.1 pmol/well, n ⫽ 6). However, this incu-
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Biophysical characterization of
native HCN channels
Experiments were performed in cultured single pituitary cells 24 h after
dispersion. Somatotrophs were identified by their responses to GHRH. As
recently described, these cells respond
with variable patterns of electrical activity (22). Figure 2 shows that GHRH
induced single spiking in a quiescent
somatotroph (panel A) and increased
the frequency of plateau-bursting type
of electrical activity in a spontaneously
FIG. 1. Expression of HCN cation channels in rat pituitary cells. A–C, Effects of 8-Br-cAMP
active cell (panel B). Both lactotrophs
(A) and 8-Br-cGMP (B and C) on electrical activity in thyrotrophs. Note both the depolarizing
and thyrotrophs express TRH recepeffect of cAMP and the hyperpolarizing effect of 8-Br-cGMP. D and E, Forskolin-(D) and 8-Brtors, but in contrast to lactotrophs,
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
thyrotrophs do not express dopamine
for 24 h. G, Western blot analysis of HCN expression in primary and GH3 immortalized
D2 receptors. Figures 3A and 4A, show
pituitary cells. s, Seconds.
the pattern of electrical activity triggered by TRH in lactotrophs and thybation was still unable to stimulate electrical activity in rotrophs, respectively; the former also responded to the
anterior pituitary cells (data not shown). These results application of bromocriptine, a dopamine D2 agonist,
indicate that HCN channels, but not CNG channels, by hyperpolarization of the plasma membrane (Fig. 3A,
could account for cAMP-stimulated electrical activity in bottom). Note that TRH triggered electrical activity
endocrine pituitary cells, whereas cGMP inhibits electri- with the peak AP amplitude reaching ⫹30 to ⫹40 mV
cal activity through other channels.
in thyrotrophs (Fig. 4A), whereas AP overshooting was
not observed in lactotrophs (Fig. 3A). Gonadotrophs
Expression of HCN channels
were identified by their oscillatory and apamin-sensiTo clarify the expression of HCN channels in endo- tive response to 1 nM GnRH (23, 24) (Fig. 5A). Cells
crine pituitary cells, we performed quantitative RT-PCR
not responding to these agonists were described as
analysis. This analysis confirmed the presence of mRNA
unidentified.
transcripts for HCN1, HCN2, HCN3, and HCN4 in
In these cell types, the hyperpolarization of the plasma
mixed populations of rat anterior pituitary cells cultured
membrane resulted in activation of a slowly developing
for 24 h. The real-time quantitative PCR and the comparinward current in endocrine pituitary cells held at ⫺40
ative Ct method (19) also revealed that HCN2 mRNA
mV and bathed in medium containing 20 mM K⫹. The
transcripts are the most abundantly expressed, followed
current started to appear at voltages approaching ⫺60
by HCN3, HCN4, and HCN1 (Fig. 1F). The presence of
mV, and both the amplitude and the rate of activation of
HCN isoforms was also studied in mixed rat pituitary
cells and GH3 immortalized lactosomatotrophs using this current increased with further hyperpolarization.
Western blot analysis. We were unable to detect any spe- Representative examples of the whole-cell current recific band using HCN1 antibodies. In contrast, double sponse to a hyperpolarizing voltage step to ⫺120 mV
immunoreactive bands for HCN2 and HCN3 were de- from a holding potential of ⫺40 mV are shown in Fig. 2C
tected, consistent with the presence of glycosylated and (somatotrophs), Fig. 3C (lactotrophs), Fig. 4B (thyrononglycosylated forms, whereas HCN4 subunit was ob- trophs), and Fig. 5B (gonadotrophs). Such currents were
served as a single immunoreactive band (Fig. 1G). More- observed in the majority of somatotrophs, thyrotrophs,
over, all immunopositive bands appeared at different and gonadotrophs (Table 1), as well as in unidentified
molecular weights, and their positions were in good agree- pituitary cells (data not shown). In contrast, the majority
ment with earlier data obtained from other tissues ex- of lactotrophs do not express Ih (Fig. 3C, top) or show
pressing HCN channels (20, 21). Finally, rat liver tissue small amplitude current response (middle), whereas only
was used as negative control as no mRNA expression of about 30% of lactotrophs expresses currents with ampliHCN isoforms was detected (results not shown), in ac- tudes comparable to that recorded from somatotrophs
cordance with earlier published data (20, 21).
and gonadotrophs (Fig. 3C, bottom; Table 1).
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HCN Channels in Pituitary Cells
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FIG. 2. Characterization of Ih 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 Ih. 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/Imax
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 Ih current before and 10 min after addition of 100 ␮M 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 Ih current. s, Seconds.
The increase in current in somatotrophs was always fit
best with a single-exponential function, whereas in other cell
types the best approximation for the increase in current was
achieved using single- or double-exponential fits with a time
constant of activation ranging between 0.15 and 5 sec (Fig.
2D). The development of current was fastest in gonadotro-
phs and slowest in somatotrophs. The peak amplitude of
current was comparable in gonadotrophs, lactotrophs, and
somatotrophs and was doubled in thyrotrophs (Table 1).
The currents did not inactivate significantly during the sustained application of a hyperpolarizing pulse (5–7 sec) but
did deactivate rapidly after the end of the pulse.
FIG. 3. Characterization of Ih 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 Ih 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 Ih (top) or show small amplitude current response (middle). Only about 30% of
lactotrophs express Ih with amplitude comparable to other anterior pituitary cells (bottom). E, The activation curve for Ih 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 Ih in an identified lactotroph.
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100 ␮M concentration for 10 min, 62 ⫾ 5% at
10 ␮M concentration for 10 min (n ⫽ 7), and
34 ⫾ 4% when applied at 10 ␮M concentration for 5 min (n ⫽ 6). Effects of 1 mM Cs⫹ on
the Ih amplitude are shown in Figs. 2G, 3B,
and 5E. In all cell types, Cs⫹ (1 mM) inhibited
98 ⫾ 1% of current when applied for 30 sec
(n ⫽ 16). Gonadotrophs expressing Ih (Fig. 5F,
bottom) displayed inward rectifications in response to hyperpolarizing current pulses of
⫺5 pA that were suppressed by 1 mM Cs⫹ (5F,
top) and were absent (5G, top) in cells lacking
Ih (5G, bottom). Similar effects of Cs⫹ were
observed in the three other cell types (data not
shown).
Because of the nonspecific stimulatory efFIG. 4. Characterization of Ih current in rat pituitary thyrotrophs. Panel A,
fect of ZD7288 on electrical activity in pituThyrotrophs 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
itary cells described earlier (27), we could not
lactotrophs (Figs. 3A vs. 4A). Panel B, Representative example of the whole-cell
use this compound in evaluating the role of
current response to a hyperpolarizing voltage step to ⫺120 mV from a holding
HCN channels in spontaneous electrical activpotential of ⫺40 mV. The time course of channel opening was best described by a
double-exponential fit. Panel C, Inhibition of Ih current by 10 ␮M ZD7288. D,
ity. Application of 1 mM Cs⫹ not only inhibits
Stimulation of Ih by 8-Br-cAMP. E, Inhibition of Ih by MDL12230A. Top,
HCN channels effectively, but also silences
Representative trace; bottom, Normalized current- and time-constant (␶) values
spontaneously active inward rectifier K⫹
(mean ⫾ SEM).s, Seconds; C, control.
channels in many cell types, including pituitary cells (28, 29). Consistent with the second
Using the tail current measurements, we also obtained
effect,
we
observed
an increase in the firing frequency of
the activation curve of the Ih current in somatotrophs
(Fig. 2E), lactotrophs (Fig. 3E), and gonadotrophs (Fig. 26 ⫾ 13% in cells not expressing Ih (n ⫽ 13). Figure 5I
⫹
5C). In somatotrophs, a mean V1/2 value of ⫺93.2 ⫾ 3.4 illustrates the stimulatory effect of 1 mM Cs on the firing
mV and a slope value of 9.3 ⫾ 1.1 mV (n ⫽ 7), in lac- frequency in a gonadotroph (n ⫽ 17) in which no Ih curtotrophs a V1/2 value of ⫺94.1 ⫾ 2.7 mV and a slope rent was detected (5I, bottom). Figures 2H and 5H illusvalue of 8.6⫾1.2 mV (n ⫽ 4), and in gonadotrophs a V1/2 trate inhibitory effects of Cs⫹ on the firing frequency of
value of ⫺83.2 ⫾ 3.3 mV and a slope value of 8.3 ⫾ 1.2 Ih-expressing somatotrophs and gonadotrophs, respecmV were recorded. Such kinetic properties of the hyper- tively. Nonetheless, 1 mM Cs⫹ did not abolish spontanepolarization-activated inward currents in anterior pitu- ous firing of AP in any of the cells expressing Ih. The effect
itary cells were similar to the properties of Ih currents of Cs⫹-induced inhibition of HCN channels on the frepresent in neuronal and cardiac cells (11) as well as in quency of AP is probably more pronounced but may be
masked by its concomitant inhibition of classical inward
GH3 pituitary cells (13, 14).
rectifier K⫹ channels, which are also expressed in pituPharmacological characterization of native
itary cells (28, 29). Notwithstanding, these results suggest
HCN channels
that HCN channels contribute to, but are not critical for,
In all cell types, the current development was blocked spontaneous electrical activity in cultured pituitary cells.
by 100 ␮M ZD7288, an organic blocker of Ih (25, 26), and
by 1 mM Cs⫹-containing medium. Consistent with the Regulation of HCN channels
The ability of Cs⫹ to decrease the firing frequency sugliterature, the inhibitory effect of Cs⫹ in our experiments
was instantaneous and reversible, whereas ZD7288 had gested that basal cAMP levels in cultured pituitary cells
to be applied for 10 min to achieve full and irreversible are sufficient to activate these channels. To clarify this
inhibition of this current. Effects of ZD7288 on the cur- hypothesis, we treated pituitary cells with MDL12330A
rent amplitude are shown in somatotrophs (Fig. 2F), lac- (cis-N-(2-phenylcyclopenthyl)azacyclotridec-1-en-2amine
totrophs (Fig. 3D), thyrotrophs (Fig. 4C), and gonadotro- HCl), an inhibitor of the adenylyl cyclase family of enphs (Fig. 5D). The effect of ZD7288 on the current did zymes. Figure 3F, main panel, illustrates the concentranot differ between cell types. In gonadotrophs, ZD7288 tion-dependent effect of MDL12330A on basal cAMP
inhibited 86 ⫾ 6% (n ⫽ 4) of the current when applied at production in cultured pituitary cells. MDL12330A (75
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HCN Channels in Pituitary Cells
Mol Endocrinol, January 2012, 26(1):153–164
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 Ih was
obtained by tail current analysis as described in Fig. 2E. D, Blockade of Ih current by the addition of 100 ␮M ZD7288 to extracellular solution.
ZD7288 was applied to the bath for 10 min to fully develop its blocking effect. E, Inhibition of Ih by 1 mM bath Cs⫹. F and G, Cells expressing Ih (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 Ih (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) Ih. Percentage decrease (H, downward arrow) or increase (I, upward arrow) in frequency is
indicated for each cell group. s, Seconds.
␮M) also had a pronounced effect on Ih in lactotrophs
(Fig. 3F, inset), thyrotrophs (Fig. 4E, top) and other pituitary cell types (data not shown). Figure 4E (bottom) illustrates that the current amplitude was reduced by 53 ⫾
8% (P ⬍ 0.0005, n ⫽ 10), and the current development
was delayed for 222 ⫾ 30% (P ⬍ 0.005, n ⫽ 9) in thyrotrophs treated with MDL12330A. The activation
curve for Ih current was also measured before and after
MDL12330A application. MDL12330A shifted the V1/2
of activation curve to the left (before MDL12330A:
⫺92.5 ⫾ 0.8 mV; after MDL12330A: ⫺105.8 ⫾ 5 .9 mV)
without change in slope factor (s) (s ⫽ 7.8 ⫾ 1.8 mV,
before MDL12330A; s ⫽ 7.7 ⫾ 1.0 mV, after
MDL12330A).
Because there is not a common Gs-coupled receptor
among these cells, we studied effects of forskolin, an activator of adenylyl cyclase, and 8-Br-cAMP, a cell-perme-
able cAMP analog, on the peak amplitude of Ih and the
rate of current development. Ih was measured before
(control) and approximately 5 min after application of
1 ␮M forskolin or 1 mM 8-Br-cAMP. In contrast to
MDL12330A treatment, forskolin slightly but significantly increased the current amplitude (111 ⫾ 3% of
control, P ⬍ 0.01, n ⫽ 7) and facilitated development of
current (␶ ⫽ 66 ⫾ 8% of control, P ⬍ 0.005; n ⫽ 7).
8-Br-cAMP also increased the amplitude of Ih to 112 ⫾
6% of control and decreased the ␶ to 66 ⫾ 9% of control
(n ⫽ 7). In particular, the effect of 8-Br-cAMP on the
amplitude of Ih was rather minor in somatotrophs (3.4%
increase) and much greater in gonadotrophs (14.8%)
and thyrotrophs (36.8%; Fig. 4D), whereas 8-Br-cAMPinduced decrease of ␶ was comparable among these different cell types. These results indicate that basal cAMP
production in pituitary cells in vitro is sufficient to acti-
TABLE 1. Biophysical properties of Ih in anterior pituitary cells
Cells with Ih (%)
Amplitude (pA)
Amplitude (pA/pF)
␶ (s)
Gonadotrophs
73.8
6.4 ⫾ 0.7 (45)
1.1 ⫾ 0.1 (43)
0.9 ⫾ 0.2 (38)
Lactotrophs
33.3
8.5 ⫾ 2.2 (31)
1.5 ⫾ 0.4 (31)
1.2 ⫾ 0.2 (25)
Somatotrophs
92.3
6.6 ⫾ 1.1 (36)
1.7 ⫾ 0.3 (35)
1.7 ⫾ 0.1 (35)
Thyrotrophs
81.3
15.9 ⫾ 3.4 (8)
2.8 ⫾ 0.6 (8)
1.2 ⫾ 0.2 (7)
The Ih was augmented by elevation of K⫹ in extracellular solution to 20 mM before recording. The Ih current was activated by voltage steps from
⫺40 to ⫺120 mV. Data shown are mean ⫾ SEM values and number in parentheses indicate number of cells.
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to the Gq/11 family of G proteins and is coupled to phospholipase C. Because it has been shown that HCN channels are allosterically modulated by phosphoinositides,
we tested the sensitivity of pituitary HCN channels to
phosphoinositides by inhibiting the activity of phosphoinositide 3-kinase, the enzyme responsible for their synthesis. Treatment of pituitary cells with the phosphoinositide 3-kinase inhibitor wortmannin (10 ␮M for ⬎10
min) decreased the Ih current amplitude to 83 ⫾ 5% of
controls (P ⬍ 0.05) and increased the ␶ value to 190 ⫾
23% (P ⬍ 0.01; n ⫽ 8), regardless of pituitary cell type
(Fig. 6, A–C). Furthermore, direct activation of phospholipasec by m-3M3FBS [2,4,6-trimethyl-N-C3-(trifluromethyl)phenylybenzesulfo-nomide] (50 ␮M) strongly attenuated Ih current in pituitary gonadotrophs (Fig. 6E).
Based on these data, we concluded that Ih current is regulated by phosphoinositides in pituitary cells.
Discussion
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 Ih in pituitary cells. Representative traces from
somatotrophs (A) and gonadotrophs (B) and mean values of amplitude
of Ih and ␶ in both cell types (n ⫽ 8). D and E, Inhibition of Ih by 1 nM
GnRH (D) and 50 ␮M m-3M3FBS [2,4,6-trimethyl-N-C3(trifluoromethyl)phenylybenzesulfo-nomide], a phospholipase C
activator (E), in gonadotrophs. s, Seconds; WT, wortmannin.
vate HCN channels and that forskolin can further facilitate gating of these channels. Difference in the sensitivity
of Ih to modulation by cAMP could reflect the cell typespecific expression of different HCN subunits and/or
variations in basal cAMP production among subpopulations of pituitary cells.
In gonadotrophs, we consistently observed attenuation
of Ih current after application of GnRH (1 nM; Fig. 6D)
that could be only slightly recovered by application of
forskolin or 8-Br-cAMP (data not shown). On average
(n ⫽ 16), GnRH decreased the peak amplitude of Ih to
76 ⫾ 6% of control (P ⬍ 0.0005) and increased ␶ to
228 ⫾ 39% (P ⬍ 0.005). The GnRH receptor belongs
It is well established that cyclic nucleotides can modulate
electrical activity through cAMP/cGMP-dependent kinases that phosphorylate several channels, including the
background K⫹ conductance in corticotrophs (30, 31),
voltage-gated K⫹ channels (32), L- and T-type voltagegated Ca2⫹ channels (33–35), tetrodotoxin-sensitive (36)
and -resistant voltage-gated Na⫹ channels in somatotrophs (37), and transient receptor potential type C
channels in all endocrine pituitary cells (38). Cyclic nucleotides could also modulate electrical activity in excitable cells by activation of CNG and HCN channels. Such
a paradoxical role for these channels comes from their
permeability properties (CNG are practically nonselective cation channels, and HCN channels are weakly K⫹selective channels) and unique voltage dependence of
HCN channels. Both second messengers contribute to
regulation of CNG channels, with cAMP being more potent than cGMP in regulation of HCN channels (1).
Pituitary cells express a very sophisticated nitric oxidesynthase-soluble guanylyl cyclase signaling pathway (39 –
41). The mRNA transcripts for CNG channels were also
found in a mixed population of pituitary cells (18).
Should such transcripts be translated to their protein
products, activation of these channels should stimulate
firing of AP. However, here we show that the application
of 8-Br-cGMP caused the reverse effect on the excitability
of pituitary cells for these channels: it hyperpolarized the
plasma membrane and decreased the firing frequency, in
contrast to cAMP, which depolarized the plasma membrane and stimulated firing of AP. The inhibitory effect of
cGMP on electrical activity is consistent with a role of
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protein kinase G in the regulation of BK-type Ca2⫹-controlled K⫹ channels (42). These channels are expressed in
all endocrine pituitary cells, and their activation leads to
inhibition of spontaneous electrical activity (43). Thus,
cGMP plays an opposite role in the control of excitability
of endocrine pituitary cells compared with cAMP, which
is consistent with the inhibitory effect of nitric oxide signaling pathway on hormone release by cultured pituitary
cells (44 – 46). Effects of NO/cGMP in intact pituitary
gland are not only related to the electrical status of endocrine cells, but also may affect pituitary blood flow rates
and oxygen supply by capillaries, as well as oxygen consumption (47, 48).
Previous studies have also described the expression of
the HCN1 subunit and Ih in immortalized AtT-20 pituitary cells (tumor-derived mouse corticotrophs), which
were weakly modulated by forskolin (16). The Ih was also
identified in immortalized rat lactosomatotroph GH3
cells and cultured rat somatotrophs (13, 14) but forskolin-induced changes in the properties of the Ih current
were not observed. Here, we show that transcripts for all
four HCN subunits are expressed in anterior pituitary
cells. The protein expression of HCN2, HCN3, and
HCN4 in pituitary cells and GH3 lactosomatotrophs was
confirmed using Western blot analysis. The apparent molecular weights corresponded to those previously reported for HCN subunits (Refs. 20 and 21 and references
within). HCN2 and HCN3 appeared as doublets, probably due to posttranslational glycosylation (21). Protein
expression of HCN1 could not be demonstrated, consistent with the low level of HCN1-mRNA transcript product and might reflect a cell type-specific expression pattern of HCN subunits in endocrine pituitary cells.
Furthermore, three lines of evidence suggested that
pituitary lactotrophs, somatotrophs, thyrotrophs, and
gonadotrophs, as well as other unidentified endocrine pituitary cells, express functional HCN channels. First, pituitary cells developed a slow-activating inward current
upon hyperpolarization to a value of ⫺60 mV or more
negative. Second, the biophysical properties of this current (activation of this current by hyperpolarizing voltages, time course of activation, lack of inactivation during
sustained recording, rapid deactivation, and activation
curve obtained by tail current analysis) are consistent
with the expression of these channels. Third, pharmacological profile of this current (reversible inhibition by 1
mM Cs⫹ and slow and irreversible inhibition by ZD7288)
also supports the functional expression of HCN channels
in endocrine pituitary cells, as was shown previously in
other neuroendocrine cell types (49).
The majority of gonadotrophs, thyrotrophs, and somatotrophs express HCN channels. On the other hand,
Mol Endocrinol, January 2012, 26(1):153–164
these channels were present in only a fraction of lactotrophs, which is consistent with the functional heterogeneity of these prolactin-producing cells (50, 51). What
is the role of these channels in pituitary cell functions?
Although the current density was relatively low in both
normal and immortalized pituitary cells, such a current
may still profoundly influence the resting membrane potential because pituitary cells are small and the input resistance of these cells is usually more than 5 G⍀. The
contribution of the Ih current to the resting membrane
conductance under normal ion concentrations was confirmed in experiments with hyperpolarizing current injection in gonadotrophs. In these cells, the voltage response
to a hyperpolarizing current pulse showed slowly developing inward rectification, which was blocked by extracellular Cs⫹ and was absent in cells lacking Ih. This could
indicate that Ih currents in pituitary cells may contribute
to set the resting membrane potential in the range where
other channels could account for the slow depolarization.
It is important to stress that our experiments were done in
isolated pituitary cells in vitro, and that in vivo these cells
are organized as a large-scale network (52). The existence
of such network, however, does not argue against the
relevance of voltage-gated Ca2⫹ influx in rapid hormone
release (48). Thus, it is reasonable to speculate that the
operation of HCN channels is also preserved in interconnected cells.
Based on experiments with MDL12330A, an inhibitor
of adenylyl cyclase, we also concluded that the basal cyclic nucleotide production in the majority of pituitary
cells in vitro is high enough to partially or fully activate
the Ih current. A similar effect was also observed using
MDL12330A in GH3 cells and was overcome by the subsequent application of forskolin (14). This also explains
the relatively weak effect of forskolin on Ih activation. We
also frequently observed a decrease in the AP frequency in
spontaneously firing cells with inhibited Ih, indicating
that these channels contribute to the electrical activity in
a manner comparable to that observed in GnRH neurons
(49). In none of our experiments we observed abolition of
spontaneous electrical activity, further indicating the role
of other channels in slow depolarization. Among others,
the background Na⫹ conductance is present in all endocrine pituitary cells (53–55) and contributes to spontaneous electrical activity and accompanied Ca2⫹ transients
(38). Basal cAMP production is down-regulated in vivo
by several hypothalamic and intrapituitary factors, including dopamine and somatostatin (1), which suggest
that facilitation of adenylyl cyclase activity in physiological conditions should lead to activation of HCN channels
and firing of AP.
Mol Endocrinol, January 2012, 26(1):153–164
The activity of these channels is not only determined by
the status of adenylyl cyclase activity, but also affected by
Gq/11-coupled receptors signaling through phospholipase
C pathway. Here we show that pharmacological activation of this enzyme leads to inhibition of Ih in gonadotrophs. Activation of GnRH receptors also causes inhibition
of Ih. Experiments with wortmannin further indicated the
potential role of PIP2 in the regulation of these channels in
gonadotrophs. This is consistent with numerous reports
about the requirement of PIP2 for HCN function (8, 56).
Thus, it is reasonable to suggest that the Gq/11-coupled
receptor-mediated shift from spontaneous electrical activity and Ca2⫹ influx to Ca2⫹ mobilization from intracellular pools is accompanied by a silencing of electrical
activity mediated not only by the activation of apaminsensitive K⫹ channels (23, 24) but also by the inhibition of
HCN channels.
In conclusion, here we show that cAMP and cGMP
exhibit opposite effects on the excitability of isolated endocrine pituitary cells; cGMP inhibits whereas cAMP
stimulates electrical activity. We further show that the
majority of pituitary gonadotrophs, thyrotrophs, somatotrophs, and a fraction of lactotrophs express HCN
channels with biophysical, pharmacological, and immunoreactive properties comparable to those observed in
other excitable cells expressing HCN channels. In cultured cells, basal adenylyl cyclase activity is sufficient to
activate these channels, leading to facilitation of spontaneous firing of AP, and further stimulation of this enzyme
enhances HCN channel activity. In contrast, activation of
phospholipase C inhibits Ih current, presumably by depleting plasma membrane PIP2 levels. In vivo, these channels may contribute to facilitated electrical activity and
enhanced hormone release by activated Gs-coupled receptors and may play an important role in restoration of
electrical activity in lactotrophs and somatotrophs after
removal of their respective inhibitory cues, dopamine and
somatostatin.
Materials and Methods
Chemicals
TRH, GnRH, GHRH, and somatostatin were purchased
from Bachem (Torrance, CA), MDL12330A and 3,3⬘-(hydroxynitrosohydrazino) bis-1-propanamine from Alexis Biochemicals (San Diego, CA), and apomorphine, bromocryptine
mesylate, tetrodotoxin, and ZD7288 were obtained from Tocris
(Ellisville, MO). All other drugs and chemicals were purchased
from Sigma (St. Louis, MO).
mend.endojournals.org
161
Cell cultures
Experiments were performed on anterior pituitary cells from
normal postpubertal female Sprague Dawley rats obtained from
Taconic Farms (Germantown, NY). Euthanasia was performed
by asphyxiation with CO2, and the anterior pituitary glands
were removed after decapitation. Experiments were approved
by the National Institute of Child Health and Human Development (NICHD) Animal Care and Use Committee. Anterior pituitary cells were mechanically dispersed after treatment with
trypsin and cultured as mixed cells or enriched fractions in medium 199 containing Earle’s salts, sodium bicarbonate, 10%
heat-inactivated horse serum, penicillin (100 U/ml), and streptomycin (100 ␮g/ml), as described previously (57).
RT-PCR analysis of HCN isoform expression
Analysis of relative gene expression was done using realtime quantitative PCR and the comparative Ct method (19).
For this purpose the predesigned Taq-Man Gene Expression
Assays with LightCycler TaqMan Master mix (Applied Biosystems, Foster City, CA) and Lightcycler 2.0 Real-time PCR
system (Roche, Indianapolis, IN) were used. To compare the
relative expression levels of individual HCN channels, the
levels were calibrated against HCN2 (set to 100%). Applied
Biosystems predesigned Taq-Man Gene Expression Assays
were used: for HCN1, Rn00584498_m1; HCN2,
Rn01408572_mH; HCN3, Rn00586666_m1; HCN4,
Rn00572232_m1; and GAPDH, Rn01462662_g1.
Western blot analysis of HCN channel isoforms
Primary pituitary cells or GH3 cells in culture were lysed in
radioimmunoprecipitation assay buffer containing 50 mM Tris
HCl (pH 7.4), 150 mM NaCl, 1% Nonider P-40, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate, and supplemented
with protease inhibitors. Samples were separated by Tris-glycine
SDS-PAGE and transferred onto polyvinylidene difluoride
membranes. The membrane was blocked for 1 h at room temperature and then incubated overnight at 4 C with one of the
primary antibodies: anti-HCN2 [Abcam, Inc., Cambridge, MA;
antibody (ab)19346], anti-HCN3 (Abcam, ab109807), or antiHCN4 (Abcam, ab69054), all diluted 1:1000, or anti-␤-tubulin
I antibody (Sigma Aldrich, T7816) diluted 1:10000. All incubations were performed in SuperBlock Blocking Buffer (Thermo
Fisher Scientific. Pittsburgh, PA) supplemented with 0.1%
Tween-20. After incubation with peroxidase-conjugated goat
antirabbit (HCN2 and -4) or donkey antigoat secondary antibody (HCN3) diluted 1:10000 (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA), blots were incubated with SuperSignal West
Pico Chemiluminescent Substrate Kit (Thermo Fisher Scientific), and bands were visualized on FluorChem E Digital imaging System (ProteinSimple, San Jose, CA).
Cyclic nucleotide measurements
Cyclic nucleotide production was monitored using static cultures of anterior pituitary cells. Briefly, cells (1 million per well)
were plated in 24-well plates in medium 199 and incubated
overnight at 37 C under 5% CO2-air and saturated humidity.
The following day, medium was removed, cells were washed
and then stimulated at 37 C under 5% CO2-air and saturated
humidity with 1 mM 3-isobutyl-1-methylxanthine. Cyclic nucleotides were measured in incubation medium by RIA using spe-
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HCN Channels in Pituitary Cells
cific antisera provided by Albert Baukal (NICHD, Bethesda,
MD). 125I-labeled cAMP and 125I-labeled cGMP tracers were
purchased from PerkinElmer Life Sciences (Boston, MA).
Electrophysiological recordings
Membrane voltage potential and whole-cell currents were
measured using amphotericine-perforated patch-clamp technique. All experiments were performed at room temperature.
Cells cultured on 35-mm culture dishes (100,000 per dish) were
washed twice before connecting to a relatively fast gravitydriven microperfusion system (ALA Scientific Instruments,
Westbury, NY) with common outlet. Cells were continuously
perfused with an extracellular solution containing: 150 mM
NaCl, 3 mMKCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, and 10 mM glucose starting from at least 10 min before recording at flow rate
of approximately 2 ml/min. The pH was adjusted to 7.3 with
NaOH. For voltage-clamp measurement of Ih currents, composition of extracellular solution was modified as follows: 113
mMNaCl, 20 mM KCl, and 20 mM tetraethylammonium chloride, 2 mMCaCl2, 1 mM MgCl2, 10 mM 4-(2-hydroxyethyl)
piperazine-1-ethanesulfonic acid, 10 mM glucose, 1 mM 4-aminopyridine, 1 mM BaCl2, 1 mM CdCl2, and 0.001 mM tetrodotoxin.
Patch pipettes were pulled from borosilicate glass (World Precision
Instruments, Sarasota, FL) and heat polished to a tip resistance of
5–7 M⍀. Pipette solution contained: 90 mM K-aspartate, 50 mM
KCl, 3 mM MgCl2, and 10 mM 4-(2-hydroxyethyl)piperazine-1ethanesulfonic acid, with pH adjusted to 7.2 with KOH. Before
measurement, amphotericine B was added to the pipette solution
from a stock solution (20 mg/ml in dimethyl sulfoxide) to obtain a
final concentration of 200 ␮g/ml. Current-clamp and voltageclamp recordings were performed using Axopatch 200B amplifier
(Molecular Devices, Sunnyvale, CA). Recordings started when series resistance dropped below 100 M⍀ for current-clamp or 40 M⍀
for voltage-clamp recordings. Series resistance was compensated
by more than 50%. Membrane potentials were corrected on-line
for liquid junction potential of 9.9 mV. A difference of 1.9 mV
between junction potential of control extracellular solution and
modified 20 mM KCl solution used for Ih current recording was
neglected. In some voltage-clamp experiments, P/4 protocol was
applied on line to subtract leak conductance from current traces.
Drugs dissolved to final concentration in extracellular solution
were delivered to recording chamber by the same perfusion system,
and less than 200 msec was required for exchange solutions around
the patched cells, as described previously (58).
Calculations
The amplitude of Ih was calculated by subtracting the instantaneous current amplitude from the steady-state current at the
end of the test pulse to ⫺120 mV. The density of Ih was calculated by dividing the current amplitude by the membrane capacitance for each given cell. To characterize Ih activation kinetics,
current traces elicited at ⫺120 mV were fitted by a single exponential function or by the sum of two exponentials, and
weighted tau (␶w) was calculated as follows: ␶w ⫽ (␶1*A1⫹
␶2*A2)/(A1⫹A2), where A1 and A2 are the relative amplitudes
of the two exponential components. Activation curves were determined from plots of tail current amplitudes (measured at ⫺40
mV) as a function of test voltage during 7-sec hyperpolarizing
steps. Normalized amplitudes of tail currents I/Imax were then
Mol Endocrinol, January 2012, 26(1):153–164
plotted against test voltage and fitted with the Boltzmann equation: I/Imax ⫽ 1/{1⫹exp[(Vtest ⫺ V1/2)/s]}, where Vtest is the
hyperpolarizing step potential, V1/2 is the half-activation potential, and s is the slope factor. Data values are expressed as
mean ⫾ SEM. Statistical comparisons were made using the onesample Student’s t test. Significance was set at P ⬍ 0.05 or
higher.
Acknowledgments
Address all correspondence and requests for reprints to: Dr.
Stanko Stojilkovic, NICHD; Building 49, Room 6A-36; 49
Convent Drive, Bethesda, Maryland 20892-4510. E-mail:
[email protected] or [email protected].
This work was supported by the Intramural Research Program of the NICHD, NIH.
Disclosure Summary: The authors have nothing to disclose.
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