(Received for publication, November 13, 1995; and in revised form, January 19, 1996)
From the
Adrenal corticosteroids have well known and profound effects on neurons and neuroendocrine cells, but the underlying cellular mechanisms are poorly understood. The present study analyzed membrane currents and ACTH release in AtT20 mouse pituitary corticotrope tumor cells. Patch-clamp analysis revealed a significant and selective inhibition of calcium-activated (BK-type) potassium channels upon activation of protein kinase A by corticotropin-releasing factor or 8-chlorophenylthio-cAMP. The synthetic glucocorticoid dexamethasone had no effect on potassium currents evoked by depolarization but prevented the inhibitory effect of protein kinase A activators. The action of dexamethasone had the hallmarks of protein induction, i.e. a lag time and sensitivity to inhibitors of DNA transcription and mRNA translation. In parallel, the specific BK channel blocker iberiotoxin abolished early glucocorticoid inhibition of corticotropin-releasing factor-stimulated ACTH secretion. In summary, the present data show that glucocorticoid-induced proteins render BK-type channels resistant to inhibition by protein kinase A and that this action of the steroid is pivotal for its early inhibitory effect on the secretion of ACTH.
Stressors provoke a cohort of homeostatic defense mechanisms by the central nervous, the immune, and the metabolic control systems of the body(1) . Adrenal corticosteroids released during the stress response terminate many of these adaptive responses through the rapid induction of proteins, the nature and mechanism of which are not understood(1, 2, 3, 4) .
A common motif of corticosteroid action in hippocampal neurons(5, 6) and pituitary somatolactotrope cells (7) is the regulation of potassium channels that are important determinants of cellular excitability. In hippocampal neurons, the target of glucocorticoid action has not been defined. In somatolactotrope cells, glucocorticoid induction of Kv1.5 voltage-regulated potassium channel subunits has been demonstrated (7) .
A physiologically
important early action of glucocorticoids is the suppression of
CRF()-induced ACTH secretion from anterior pituitary
corticotropes (for reviews see (8) and (9) ). CRF
stimulates ACTH release through activation of protein kinase A and the
enhancement of calcium influx through voltage-operated calcium
channels(10, 11, 12) . In turn,
glucocorticoids rapidly (within 2 h) inhibit CRF-stimulated ACTH
secretion through the induction of new
protein(s)(2, 8, 10, 13, 14) ,
while basal secretion remains unchanged. The early inhibition of
stimulated ACTH release in AtT20 corticotropes is antagonized by
membrane depolarization(13, 15) , calcium channel
activator drugs(11, 16) , and potassium channel
blockers(15, 16) , collectively suggesting that the
actions of glucocorticoids may involve potassium channels. However, the
well established glucocorticoid induction of the Kv1.5 channel protein
does not occur in AtT20 corticotropes(17) .
In endocrine cells and neurons large conductance calcium- and voltage-activated potassium (BK) channels have been proposed as important negative feedback regulators of voltage-dependent calcium influx(18) . Such channels have been previously identified in AtT20 corticotrope tumor cells(19, 20) .
This report describes a correlated study of the regulation of BK channels and ACTH release by CRF and glucocorticoids in the mouse anterior pituitary corticotrope cell line AtT20. The data demonstrate that glucocorticoid-induced proteins prevent protein kinase A-dependent inhibition of BK channels and that this is pivotal to early glucocorticoid inhibition of CRF-induced ACTH secretion.
For determination of outward potassium currents cells
were voltage-clamped at -60 mV in physiological saline containing
(in mM): 140 NaCl, 5 KCl, 25 HEPES, 0.8 MgCl, 2
CaCl
, 30 glucose, and 0.001 TTX, pH 7.4. The patch pipette
contained (in mM): 95 KCH
SO
, 55 KCl,
10 HEPES, 2 MgCl
, 0.1 CaCl
, and 200 µg/ml
amphotericin B, pH 7.35. Outward potassium currents were evoked by
100-ms step depolarization (-30 to +50 mV), and the average
steady-state current amplitude between 90 and 100 ms was determined at
each potential. Outward currents were stable for >1 h in this
configuration. Single BK channel events were recorded using the
cell-attached patch configuration. Cells were voltage-clamped at 0 mV
in high potassium saline to eliminate the membrane potential, and the
magnesium to calcium ratio adjusted to limit calcium entry (in
mM: 140 KCl, 5 NaCl, 5 MgCl
, 10 HEPES, 1
CaCl
, 30 glucose, pH 7.4. The patch pipette contained
physiological saline supplemented with 0.001 mM TTX and 100
nM apamin, and single channel events were recorded during
repeated (0.1 Hz) 100-ms depolarizations to +30 mV. Under these
conditions control P
values at +30 mV were
similar (<0.5) to that recorded in isolated patches depolarized to
+30 mV and exposed to <200 nM intracellular free
calcium(20) . (
)
For isolation of high threshold
voltage-activated calcium currents, cells were voltage-clamped at
-40 mV in (mM): 120 N-methyl-D-glucamine, 30 TEA-Cl, 2 MgCl,
20 HEPES, 10 CaCl
, and 20 glucose, pH 7.35, with
CH
SO
H. The patch pipette contained (in
mM): 95 CsCH
SO
, 35 CsCl, 5
MgCl
, 40 HEPES, and 200 µg/ml amphotericin B, pH 7.35,
with CsOH. Calcium currents were evoked by 100-ms depolarization, and
peak calcium current was determined. Under the recording conditions
used currents were stable for >30 min, and inward calcium current
was completely blocked by 200 µM Cd
and
70-80% by 1 µM nifedipine.
In control cells voltage clamped through
amphotericin-perforated patches in physiological saline at -60
mV, a 100-ms depolarization evoked large outward currents that reached
a steady-state level before the end of the pulse. Variations in the
rate of inactivation of the outward currents were observed between
cells (compare Fig. 1A and Fig. 2B)
that were independent of passage number or incubation temperature
(19-24 °C). This slow inactivation was observed in 20%
of both control and glucocorticoid-treated cells. No qualitative
differences in regulation of steady-state current, measured between 90
and 100 ms, reported here were observed between inactivating and
non-inactivating currents.
Figure 1:
CRF inhibits the BK component of the
outward steady-state current via activation of protein kinase A in
intact AtT20 D16:16 cells. A, representative (1 of 7 cells)
leak-subtracted traces of voltage-activated outward current (at
+30 mV) in control cells before and after exposure to 100 nM CRF. AtT20 cells were voltage-clamped through amphotericin
B-perforated patches at -60 mV in physiological saline containing
1 µM TTX. B, the current/voltage relationship
determined from the same cell in A. Average steady-state
outward current was determined between 90 and 100 ms at each potential
as described under ``Materials and Methods.'' C,
time course of 100 nM CRF inhibition of outward steady-state
potassium current (filled circle, measured at +30 mV) and
CRF-stimulated intracellular cAMP accumulation (open circle, n = 3). Inhibition of outward current is expressed as
the percentage inhibition of the outward steady-state current at each
time point with respect to the pre-CRF-treated current amplitude (Io). Intracellular cAMP accumulation was determined under
identical conditions, in the absence of phosphodiesterase inhibitors,
as described under ``Materials and Methods.'' D,
pretreatment of cells with the protein kinase A inhibitor, Rp-cAMPS
(100 µM), or the selective BK inhibitor, IbTx (100
nM), blocks CRF inhibition of outward steady-state current.
Data are expressed as the percent change in the control outward
steady-state current, Io (at +30 mV) by CRF (100
nM) alone, Rp-cAMPS (100 µM) + CRF, IbTx
(100 nM) alone, and IbTx + CRF. Number in parentheses represents number of cells in each group. Means
± S.E. are shown; *, p < 0.05 (non-parametric
Kruskal-Wallis test). E, representative records (1 of 4) from
a cell-attached patch in 140 mM KCl during consecutive (0.1
Hz) 100-ms depolarizations to +30 mV before (control) and 5 min
after CRF application. Open (o) and closed (c) states
of channel are shown. F, representative plot of BK channel
mean open probability versus time from a cell-attached patch
recording as in E. Bath application of 100 nM CRF
reduces average open probability (Po) of the
120-picosiemens BK channel in cell-attached patches. P
was determined during consecutive (0.1 Hz)
100-ms patch depolarization to +30 mV and plotted as a function of
time.
Figure 2:
Dexamethasone prevents protein kinase
A-mediated inhibition of BK currents in intact AtT20 D16:16 cells. A, representative (1 of 8 cells) leak-subtracted traces of
voltage-activated outward current (at +30 mV) before and after
exposure to 8-CPT-cAMP (0.1 mM, 10 min) in AtT20 cells
pretreated for 2 h with the glucocorticoid agonist, dexamethasone (1
µM). B, 8-CPT-cAMP inhibits outward steady-state
current in dexamethasone-treated cells pretreated with the mRNA
transcription inhibitor, actinomycin D (0.1 mM applied 15 min
before and during dexamethasone treatment). A representative trace at
+30 mV from 1 of 3 cells is shown. C, current/voltage
relationship of steady-state outward current density from control (open squares, n = 43) and
dexamethasone-treated (filled squares, n = 29)
cells. Outward steady-state current at each potential was normalized to
membrane capacitance to compensate for variations in cell size and
expressed as mean current density, pA/pF. D, summary of effect
of bath application of CRF (100 nM, 5 min), 8-CPT-cAMP (5 min,
0.1 mM), IbTx (20 min, 100 nM), or TEA (2 min, 1
mM) on outward steady-state potassium current determined at
+30 mV. Data are expressed as the percentage change (% change
Io) in outward steady-state current compared with pretreated
control current amplitude (I) as described in Fig. 1C. Cells were voltage-clamped at -60 mV as
described in Fig. 1and under ``Materials and
Methods.'' The number in parentheses indicates
number of cells per group. Means ± S.E. are
shown.
These data show that the reduction of outward potassium current by CRF is through activation of protein kinase A. This finding corroborates previous evidence that protein kinase A activation in corticotropes results in membrane depolarization and sustained calcium influx through voltage-sensitive L-type calcium channels(19, 23, 24) ; in parallel, inhibition of protein kinase A by various methods also blocks the hormone secretory response to CRF in AtT20 cells(25, 26) .
Bath application of 100 nM CRF significantly (p < 0.05) reduced the average open probability of single large
conductance (120 picosiemens) BK channels in 4/6 patches in
cell-attached patch recordings (Fig. 1, E and F). The time of onset (
2 min) of the effect of CRF was
identical to that seen for the macroscopic current (compare Fig. 1, C and F).
These results clearly show that CRF and cAMP suppress the activity of BK channels in AtT20 cells under conditions when these channels are exposed to elevated intracellular free calcium levels that enhance their open probability(20) . As BK channels are thought to act as immediate negative feedback inhibitors of voltage-dependent calcium influx, inhibition of these channels is a key element for optimal activation of calcium channels involved in hormone secretion (18) .
Previous evidence shows that BK channels may be up- or down-regulated by reversible cAMP-dependent phosphorylation(28) . However, in pituitary cells only protein kinase A-dependent inhibition has been found so far(29, 30) , and the present study is the first example of inhibition of BK channels by a physiologically relevant cAMP-mobilizing hypothalamic peptide, CRF.
In the absence of protein kinase A activation, dexamethasone had no significant effect on mean current density, threshold of activation, or sensitivity to 100 nM IbTx or 1 mM TEA of the steady-state outward current (Fig. 2, C and D), in agreement with a previous study(16) .
Similar to the findings with the macroscopic current, no significant inhibition of single BK channels was observed by CRF in cell-attached patch recordings (0/5 patches) from dexamethasone-treated cells.
These observations show that dexamethasone action is mediated by newly induced proteins and is selective for BK channels inhibited by protein kinase A. Glucocorticoids have also been reported to enhance a 4-aminopyridine (4-AP)-sensitive potassium current in AtT20 cells(16) . In functional secretion assays 4-AP affects ACTH release; however, 4-AP alone does not reverse glucocorticoid inhibition of CRF-stimulated ACTH release(16) . In contrast, blockade of BK channels alone with IbTx (Fig. 3) completely reverses the inhibitory effect of dexamethasone suggesting BK channels are a primary target for glucocorticoid action in AtT20 cells.
Figure 3:
IbTx
prevents early glucocorticoid inhibition of CRF-stimulated ACTH
secretion. A, static incubation assays of CRF-stimulated ACTH
secretion were performed at room temperature (24 °C) under the
conditions used to monitor outward currents as described under
``Materials and Methods.'' CRF (100 nM) was applied
for 30 min. TEA (1 mM) was applied during the CRF exposure,
and IbTx (100 nM) was applied >15 min before and during CRF
exposure. Cells were treated for 2 h with 1 µM dexamethasone or vehicle (0.01% MeSO) as appropriate.
ACTH release for each treatment in dexamethasone-treated cells (open box) is expressed as the percent of the respective
control (absence of dexamethasone, filled box) stimulus
(100%). In these studies CRF (100 nM) stimulated ACTH release
to 157 ± 6% (n = 6) of basal levels. IbTx (100
nM) or TEA (1 mM) alone elicited a small but
significant (p < 0.05) ACTH response (114 ± 2%, n = 3 and 127 ± 7%, n = 5 of
basal, respectively). IbTx had no significant effect on CRF-stimulated
ACTH secretion (155 ± 7.9%, n = 6 of basal
release). TEA (1 mM) significantly (p < 0.05,
non-parametric Kruskal-Wallis test) enhanced CRF-stimulated ACTH
release to 185 ± 10.5%, (n = 5) of basal. Means
± S.E. are shown; *, p < 0.01 compared with control
ACTH release (non-parametric Kruskal-Wallis
test).
The early inhibitory effect of glucocorticoids on CRF-stimulated ACTH release also requires mRNA and protein synthesis(2, 8, 10, 14) , and in this respect the characteristics of the action of dexamethasone on BK channels and ACTH release are identical. The selectivity of the effect of dexamethasone on protein kinase A-inhibited BK channels also helps to explain why early glucocorticoid inhibition suppresses CRF-stimulated ACTH release but does not affect basal output of hormone (2, 8, 13, 14) .
As shown by others previously (12) CRF enhanced peak pharmacologically isolated high voltage-activated calcium currents in control (mean increase at +20 mV was 44 ± 15%, n = 4) and dexamethasone-treated (33 ± 5%, n = 5) cells. Similar enhancement was also seen with 8-CPT-cAMP (n = 2). Dexamethasone pretreatment (2 h, 1 µM) had no significant effect on peak calcium current density (measured at +20 mV: control, 8.9 ± 1.5 pA/pF (n = 8); dexamethasone, 9.2 ± 1.4 pA/pF (n = 7).
On the basis of these data it seems reasonable to suggest that direct modulation of calcium influx through high voltage-activated calcium channels is not responsible for the effects of dexamethasone on BK currents. Moreover, previous work indicates a reduction (11, 32) or no significant change (10) in the average levels of intracellular free calcium after dexamethasone treatment in AtT20 cells, thus excluding increases in intracellular free calcium levels derived from intracellular sources as mediators of the effect of the steroid.
The IC of
dexamethasone to block CRF-induced ACTH release was 7.7 ± 1.9
nM (n = 6). Importantly, IbTx (100
nM) as well as TEA (1 mM) completely blocked the
inhibitory effect of 1 µM dexamethasone on CRF-stimulated
ACTH secretion (Fig. 3). Furthermore, dexamethasone (1
µM) had no significant inhibitory effect on ACTH secretion
stimulated by IbTx or TEA alone.