©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Glucocorticoids Block Protein Kinase A Inhibition of Calcium-activated Potassium Channels (*)

(Received for publication, November 13, 1995; and in revised form, January 19, 1996)

Michael J. Shipston (§) John S. Kelly Ferenc A. Antoni (1)

From the Department of Pharmacology and Medical Research Council Brain Metabolism Unit, University of Edinburgh, 1 George Square, Edinburgh, EH8 7NA, Scotland, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(^1)-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.


MATERIALS AND METHODS

AtT20 D16:16 Cell Culture

Clonal mouse anterior pituitary (AtT20 D16:16, passage 19-30) cells were maintained as described previously (14) except that cells were passaged by brief trypsinization 1 week before replating on glass coverslips. Cells (3-7 days postplating) were treated with dexamethasone or vehicle (0.01% Me(2)SO) for 2 h at 37 °C in serum-free HEPES-buffered Dulbecco's modified Eagle's medium. Cells were then transferred to physiological saline containing (in mM): 140 NaCl, 5 KCl, 25 HEPES, 0.8 MgCl(2), 2 CaCl(2), 30 glucose, and 0.001 tetrodotoxin (TTX), pH 7.4, at room temperature (19-24 °C) for electrophysiological recording. Regulation of currents in control and dexamethasone-treated cells was performed in parallel on the same passage of cells to avoid intrapassage variations.

Electrophysiology

Whole cell currents were recorded under voltage clamp using the amphotericin B (200 µg/ml) perforated patch configuration of the whole cell patch clamp recording technique(21) . Data acquisition and voltage protocols were controlled by an Axopatch 200 amplifier and pCLAMP software (Axon Instruments Inc., Foster City, CA). All traces are leak-subtracted records from cells with compensated series resistance of <15 megaohms. Pipettes were manufactured from Garner 7052 glass with resistances of 1.5-3 megaohms in physiological saline after fire polishing.

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), 2 CaCl(2), 30 glucose, and 0.001 TTX, pH 7.4. The patch pipette contained (in mM): 95 KCH(3)SO(3), 55 KCl, 10 HEPES, 2 MgCl(2), 0.1 CaCl(2), 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(2), 10 HEPES, 1 CaCl(2), 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(o) 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) . (^2)

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(2), 20 HEPES, 10 CaCl(2), and 20 glucose, pH 7.35, with CH(3)SO(3)H. The patch pipette contained (in mM): 95 CsCH(3)SO(3), 35 CsCl, 5 MgCl(2), 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.

ACTH Secretion and Intracellular cAMP Determination

For intracellular cAMP determination and ACTH secretion studies, cells were plated (5 times 10^5cells/well) in 24-well plates and used 4-5 days postplating. Cells were incubated in HEPES-buffered Dulbecco's modified Eagle's medium at 37 °C for 2 h with 1 µM dexamethasone or vehicle (0.01% Me(2)SO). Cells were washed twice and equilibrated for 15 min at room temperature in physiological saline used for outward potassium current determination except that TTX was excluded and 0.1% w/v bovine serum albumin was included to aid ACTH recovery. CRF (100 nM) was then applied for various times as indicated in the legends. IbTx was applied to the cells 15 min before CRF application. For intracellular cAMP determination, medium was aspirated and cells lysed in ice-cold 0.1 N HCl by freeze thawing and cAMP content in the acid extracts determined. ACTH and cAMP were assayed using specific double precipitation radioimmunoassays as described previously(14) .

Reagents

CRF and ACTH were from Bachem (UK) Ltd., Saffron Walden, UK; 8-CPT-cAMP and Rp-cAMPS were from Boehringer Mannheim UK, Lewes, East Sussex, UK; iberiotoxin was from the Peptide Institute, Japan. All other reagents were from Sigma or Aldrich. Dexamethasone was stored at -20 °C at 10 mM in Me(2)SO. Final vehicle concentrations were <0.01% and had no effect on calcium or potassium currents.


RESULTS AND DISCUSSION

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(o) 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(o)) 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.



CRF Inhibition of Potassium Currents Is Mediated by Protein Kinase A

Bath application of a CRF concentration (100 nM) maximally effective with respect to ACTH release in this system (14) inhibited the steady-state outward potassium current (mean ± S.E., 29.6 ± 8.6%, n = 7, p < 0.01, Kruskal-Wallis test, at +30 mV) at all potentials examined (Fig. 1, A and B). The time course of CRF inhibition of potassium currents followed the time course of CRF-stimulated intracellular cAMP accumulation (Fig. 1C). The effect of CRF was significantly reduced by pretreatment of cells with the protein kinase A inhibitor Rp-cAMPS (Fig. 1D)(22) . Steady-state outward potassium current was also significantly inhibited (30.3 ± 2.3%, n = 10, at +30 mV, p < 0.01) after bath application of the cell-permeant protein kinase A activator, 8-CPT-cAMP (0.1 mM).

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) .

The Target of Protein Kinase A Action Is the BK Potassium Channel

Both iberiotoxin (100 nM), a highly specific blocker of BK type channels (27) and TEA (1 mM), a broader spectrum blocker, inhibited the steady-state outward current by 34.0 ± 6.5% (n = 4, Fig. 1D) and 52.9 ± 6.3% (n = 9), respectively. No significant inhibition of the residual outward current by CRF (or 8-CPT-cAMP) occurred in cells pretreated with 100 nM iberiotoxin (mean inhibition, 6.2 ± 5.6%, n = 4, Fig. 1D) or 1 mM TEA (mean inhibition, 5.1 ± 7.2%, n = 4). Similar results were obtained with 100 nM charybdotoxin (n = 2).

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.

Dexamethasone Prevents Inhibition of Potassium Current by Protein Kinase A

Pretreatment with 1 µM dexamethasone for 2 h, which produces maximal inhibition of CRF-stimulated ACTH release (14) , blocked the inhibition of outward steady-state potassium current by 8-CPT-cAMP (Fig. 2, A and D) or CRF (Fig. 2D). This effect of dexamethasone was prevented with actinomycin D (0.1 mM, Fig. 2B) or puromycin (2 µM, n = 2), indicating a requirement for de novo RNA and protein synthesis, a defining feature of early inhibition(8, 14) . In addition an acute (10 min) exposure to dexamethasone did not block CRF inhibition of the steady-state current (not shown).

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% Me(2)SO) 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) .

Effects of Dexamethasone Are Not Secondary to Changes in Calcium Currents

As intracellular calcium ions are potent activators of BK channels and glucocorticoids reportedly (31) enhance calcium entry to stimulate potassium currents in hippocampal neurons, the actions of CRF and dexamethasone on calcium currents were also analyzed.

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.

Iberiotoxin-sensitive Channels Are Pivotal for Early Glucocorticoid Inhibition of CRF-stimulated ACTH Release

Under the same conditions that were used to monitor outward potassium currents, 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 statistically significant (p < 0.05 non-parametric Kruskal-Wallis test) 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, Kruskal-Wallis test) enhanced CRF-stimulated ACTH release to 185 ± 10.5% (n = 5) of basal. AtT20 cells contain multiple voltage-activated potassium conductances(16) , and in this system 1 mM TEA blocks a greater proportion of outward, steady-state, voltage-activated current than 100 nM IbTx (Fig. 2D). The effects of these inhibitors are not additive (not shown), indicating that TEA blocks iberiotoxin-sensitive BK channels as well as other potassium conductances in this system.

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.

Final Comment

Taken together with previous results(8, 12) the present data suggest that concerted cAMP-dependent phosphorylations of L-type calcium channels as well as BK-type potassium channels are required for triggering the ACTH secretory response to CRF. Inhibition of BK channels by cAMP-dependent phosphorylation prevents the calcium-induced feedback hyperpolarization mediated by these channels and thus enhances voltage-activated calcium entry(18) . The critical role of this process in CRF-stimulated ACTH secretion is shown by the observation that dexamethasone prevents protein kinase A inhibition of BK channels and in turn that selective blockage of BK channels abolished the early inhibition of ACTH release.


FOOTNOTES

*
This work was supported by Wellcome Trust Advanced Training Fellowship Grant 038763/Z/93/Z (to M. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Physiology, University of Edinburgh, The Medical School, Teviot Place, Edinburgh, EH8 9AG, Scotland, UK. Tel.: 44 131 650 3253; Fax: 44 131 650 6527; mshipston{at}srv2.med.ed.ac.uk.

(^1)
The abbreviations used are: CRF, corticotropin-releasing factor; ACTH, adrenocorticotropin; Me(2)SO, dimethyl sulfoxide; TTX, tetrodotoxin; IbTx, iberiotoxin; BK, large conductance calcium- and voltage-activated potassium channel; TEA, tetraethylammonium; 8-CPT-cAMP, 8-chlorophenylthio-cAMP; Rp-cAMPS, adenosine 3`,5`-cyclic monophosphothioate-Rp; 4-AP, 4-aminopyridine; pF, picofarads.

(^2)
M. J. Shipston, unpublished data.


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