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. 1999 May 1;516(Pt 3):757–768. doi: 10.1111/j.1469-7793.1999.0757u.x

Glucocorticoid block of protein kinase C signalling in mouse pituitary corticotroph AtT20 D16:16 cells

Lijun Tian 1, Janet A C Philp 1, Michael J Shipston 1
PMCID: PMC2269291  PMID: 10200423

Abstract

  1. The regulation of large conductance calcium- and voltage-activated potassium (BK) currents by activation of the protein kinase C (PKC) and glucocorticoid signalling pathways was investigated in AtT20 D16:16 clonal mouse anterior pituitary corticotroph cells.

  2. Maximal activation of PKC using the phorbol esters, 4β-phorbol 12-myristate, 13-acetate (PMA), phorbol 12, 13 dibutyrate (PDBu) and 12-deoxyphorbol 13-phenylacetate (dPPA) elicited a rapid, and sustained, inhibition of the outward steady-state voltage- and calcium- dependent potassium current predominantly carried through BK channels.

  3. The effect of PMA was blocked by the PKC inhibitors bisindolylmaleimide I (BIS; 100 nM) and chelerythrine chloride (CHE; 25 μM) and was not mimicked by the inactive phorbol ester analogue 4α-PMA.

  4. PMA had no significant effect on the 1 mM tetraethylammonium (TEA)-insensitive outward current or pharmacologically isolated, high voltage-activated calcium current.

  5. PMA had no significant effect on steady-state outward current in cells pre-treated for 2 h with 1 μM of the glucocorticoid agonist dexamethasone. Dexamethasone had no significant effect on steady-state outward current amplitude or sensitivity to 1 mM TEA and did not block PMA-induced translocation of the phorbol ester-sensitive PKC isoforms, PKCα and PKCε, to membrane fractions.

  6. Taken together these data suggest that in AtT20 D16:16 corticotroph cells BK channels are important targets for PKC action and that glucocorticoids inhibit PKC signalling downstream of PKC activation.

In many neuroendocrine cells of the anterior pituitary gland, activation of the protein kinase C (PKC) intracellular signalling pathway leads to sustained cellular excitability and neurosecretion although the cellular mechanisms and targets for PKC are poorly understood (Ozawa & Sand, 1986; Mason et al. 1988). In anterior pituitary corticotrophs PKC mediates the sustained phase of adrenocorticotrophin (ACTH) secretion stimulated by activation of the phospholipase C pathway by the hypothalamic secretagogue vasopressin (Carvallo & Aguilera, 1989; Oki et al. 1990). Vasopressin elicits a biphasic elevation of intracellular free calcium (Corcuff et al. 1993; Tse & Lee, 1998) and during the sustained phase of calcium influx stimulates PKC translocation and enhances PKC activity at the plasma membrane, an effect that is mimicked by the cell-permeant PKC-activating phorbol esters (Carvallo & Aguilera, 1989).

In AtT20 mouse corticotroph cells phorbol-ester-mediated activation of PKC has been proposed to exert effects both distal and proximal to voltage-dependent calcium influx, which may result from activation of different PKC isoforms (McFerran et al. 1995) to elicit ACTH release (Phillips & Tashjian, 1982; Woods et al. 1992; Clark & Kempainen, 1994; McFerran et al. 1995). Intracellular free calcium measurements in AtT20 cells suggest that PKC-induced calcium influx results, at least in part, from inhibition of TEA-sensitive potassium conductances. This inhibition results in membrane depolarization and subsequent, indirect, enhancement of voltage-gated calcium influx (Reisine & Guild, 1987; Reisine, 1989). However, ionic conductances regulated by PKC activation in corticotrophs have not been identified.

In rat GH4C1 pituitary cells activation of PKC results in inhibition of the TEA-sensitive large conductance calcium- and voltage-activated potassium (BK) channels (Shipston & Armstrong, 1996), which act as immediate negative feedback regulators of voltage-dependent calcium influx in several systems (Robitaille et al. 1993; Yazejian et al. 1997). Furthermore, BK channels are an important target for cellular regulation by two distinct, physiologically relevant, intracellular signalling pathways in AtT20 corticotrophs. Activation of the cAMP-dependent protein kinase pathway results in inhibition of BK channels leading to a robust secretory response. Protein kinase A (PKA)-mediated inhibition of BK channel function is blocked by activation of a protein-synthesis-dependent signalling cascade activated by glucocorticoid hormones. The cross-talk between these two pathways at the level of BK channels is an important determinant of the secretory response in this system (Shipston et al. 1996; Lim et al. 1998; Tian et al. 1998).

As TEA-sensitive BK channels act as immediate negative feedback regulators of voltage-dependent calcium influx in several systems (Robitaille et al. 1993; Yazejian et al. 1997) and PKC-mediated calcium influx is dependent upon inhibition of a TEA-sensitive conductance in AtT20 cells (Reisine & Guild, 1987; Reisine, 1989), we have addressed whether BK channels are an important cellular target for PKC action in this system. Furthermore, as glucocorticoids block PKC-stimulated ACTH release (Phillips & Tashjian, 1982; Woods et al. 1992; Clark & Kempainen, 1994; McFerran et al. 1995) and antagonize PKA-mediated inhibition of BK channels in this system (Shipston et al. 1996; Tian et al. 1998) we have addressed the question as to whether glucocorticoids also block PKC-mediated regulation of BK channels in AtT20 D16:16 corticotroph cells.

METHODS

AtT20 D16:16 cell culture

Clonal mouse anterior pituitary (AtT20 D16:16, passage 18-32) cells were maintained as previously described (Tian et al. 1998), and used 3-7 days post-plating on glass coverslips. Cells were treated with 1 μM of the synthetic glucocorticoid dexamethasone or vehicle (< 0.001 % Me2SO) for 2 h at 37°C in serum-free Dulbecco's modified Eagle's medium (Life Technologies, Paisley, UK), pH 7.4 buffered with 25 mM Hepes and containing 0.25 % bovine serum albumin (DMEM-BSA). Cells were then transferred to the respective bath solution (dexamethasone free) outlined below for electrophysiological recording. Regulation of all currents was performed in parallel on the same passage of cells to avoid potential intra-passage variations in responsiveness.

Electrophysiology

Whole cell currents were recording in the conventional whole cell recording mode of the patch clamp technique. Outward potassium currents were determined in physiological potassium gradients. The bath solution (extracellular) contained (mM): 140 NaCl, 5 KCl, 2 MgCl2, 1 CaCl2, 10 Hepes and 20 glucose; pH 7.4 and containing 0.002 tetrodotoxin. The patch pipette (intracellular) contained (mM): 140 KCl, 2 MgCl2, 10 Hepes, 30 glucose, 1 or 5 BAPTA and 1 ATP; pH 7.3 with intracellular free calcium ([Ca2+]i) buffered to 200 nM. Cells were voltage clamped at -50 mV and depolarized to the respective potentials for 100 ms with leak subtraction applied using a P/4 protocol and series resistance compensation of > 50 %. Steady-state outward current was determined 90 ms into the pulse and was stable for > 30 min under these conditions.

Inward pharmacologically isolated calcium currents were determined in the whole cell recording configuration. The bath (extracellular) solution contained (mM): 120 NaCl, 30 TEA-Cl, 10 Hepes, 10 CaCl2, 2 MgCl2, 10 glucose, 0.1 DIDS and 0.002 tetrodotoxin; pH 7.4. The patch pipette (intracellular) contained (mM): 140 caesium glutamate, 8 NaCl, 10 Hepes, 5 MgCl2, 20 glucose and 1 ATP; pH 7.3. Cells were voltage clamped at -50 mV and calcium currents evoked by 100 ms depolarizations to the respective potential.

Unitary single channel events in the inside out patch configuration were characterized by their large conductance, voltage and calcium sensitivity under physiological potassium gradients. For isolated inside out patches the bath (intracellular solution) contained (mM): 140 KCl, 10 Hepes, 2 MgCl2, 30 glucose, 5 BAPTA and 1 ATP; pH 7.3 with intracellular free calcium buffered to 0.5 μM. The patch pipette (extracellular solution) contained (mM): 140 NaCl, 5 KCl, 5 MgCl2, 0.1 CaCl2 and 20 glucose; pH 7.4. Mean single channel open probability (Po) was determined at 0 mV from at least 20 s of continuous recording for each time point. The total number of channels in the patch was determined by exposing the patch to > 1 μM intracellular free calcium at +80 mV, conditions that maximally activate BK channels in this system.

All data acquisition and voltage protocols were controlled by an Axopatch 200B amplifier and pCLAMP 6 software (Axon Instruments Inc.). All data were sampled at 10 kHz and filtered at 2 kHz. Pipettes were manufactured from Garner no. 7052 glass, Sylgard-coated, with resistances of 1-3 MΩ in physiological saline after fire polishing. Series resistance was < 12 MΩ before series resistance compensation. Cell membrane capacitance was determined from the calibrated compensation controls of the amplifier.

Phorbol esters and inhibitors were applied in bath solution by gravity-driven perfusion at a flow rate of 1-2 ml min−1 or by direct application to the bath. For experiments with purified PKC in isolated inside out patches PKC was applied directly to the bath solution.

Western blotting

Cells were grown in 75 cm2 flasks as described under cell culture to 70-80 % confluency. On the day of the experiment cells were treated with vehicle or 1 μM dexamethasone for 2 h at 37°C in DMEM-BSA and exposed for a further 10 min with or without 100 nM 4β-phorbol 12-myristate, 13-acetate (PMA). Cells were rapidly washed in Hanks’ balanced salt solution (HBSS)/EDTA, pelleted and membrane homogenates prepared by homogenizing ∼107 cells on ice in homogenization buffer (composition (mM): 150 KCl, 5 EGTA and 2 MgCl2; pH 10.6, containing 12 U ml−1 aprotinin, 5 μg ml−1 leupeptin, 6 mM 4-(2-aminoethyl)benzenesulphonyl fluoride (AEBSF) and 4 mM Pepstatin A) followed by two freeze-thaw cycles. After centrifugation at 1000 g for 5 min at 4°C the resultant supernatant was pelleted at 40 000 g to give the crude membrane fraction. Protein samples (10-40 μg) were separated on a 10 % SDS gel and electroblotted to Immobilon polyvinylidenedifluoride (PVDF) membranes. Membranes were blocked for 2 h at room temperature (RT) with PBS containing 0.1 mM EDTA, 0.1 % Triton X-100, pH 7.4 (PBS-TE) and 5 % (w/v) low fat milk (Marvel). Blots were incubated overnight at 4°C with a 1/500 (PKCα) or 1/1000 (PKCε) dilution of the respective anti-PKC isoform antibody (Affiniti Research Products Ltd, Exeter, UK) in PBS-TE containing 1 % (w/v) Marvel. Blots were washed 5 times with PBS-TE and incubated for 45 min at RT with HRP-labelled anti-rabbit IgG (Amersham, 1/5000 final dilution) in PBS- TE containing 5 % (w/v) Marvel. After five washes in PBS-TE blots were incubated with Amersham enhanced chemiluminescence (ECL) reagents according to the manufacturer's protocol and blots exposed to ECL film in the linear response range (Amersham).

Reagents

4β-Phorbol 12-myristate, 13-acetate (PMA), 4α-phorbol 12-myristate, 13-acetate (4α-PMA), phorbol 12, 13 dibutyrate (PDBu), 12-deoxyphorbol 13-phenylacetate (dPPA), chelerythrine chloride (CHE), bisindolylmaleimide I (BIS), okadaic acid, Tyrphostin A25 and dephostatin were from Alexis Corporation (UK) Ltd, Nottingham, UK. Phorbol esters were maintained as stock 10 mM solutions in Me2SO at -20°C. The final concentration of DMSO in the bath was < 0.001 % which had no significant effect on currents. Anti-PKC isoform-specific antibodies were from Affiniti Research Products Ltd. Rat brain PKC was from Sigma or Promega Corporation, Southampton, UK. Recombinant PKCα, PKCε, BAPTA and tetrodotoxin were from Calbiochem-Novabiochem (UK) Ltd, Nottingham, UK. PVDF membranes and reagents for SDS-PAGE and Western blotting were from Bio-Rad Laboratories, Ltd, Hertfordshire, UK. All other reagents were from Sigma or BDH-Merck. Dexamethasone was stored at -20°C at 10 mM in Me2SO.

Statistics

Data are expressed as means ±s.e.m. Statistical significance was determined by Student's t test for paired and unpaired data as appropriate. A P value of less than 0.05 was considered to be significant.

RESULTS

Activation of PKC inhibits outward BK potassium current in AtT20 D16:16 cells

In AtT20 D16:16 cells that were voltage clamped at -50 mV in the whole cell configuration, depolarization for 100 ms elicited large outward, non-inactivating currents in physiological potassium gradients (Fig. 1A). In control cells, depolarized to +40 mV, steady-state outward current density was 245.3 ± 17.7 pA pF−1 (mean ±s.e.m., n = 10). These currents passed predominantly through large conductance calcium- and voltage-activated potassium (BK) channels as the outward steady-state currents were blocked 70.1 ± 7.8 % (n = 7) by 1 mM TEA and 45.9 ± 4.6 % (n = 7) by the specific BK channel blocker iberiotoxin (IbTX; 100 nM) as previously described (Shipston et al. 1996). Under the recording conditions used steady-state outward currents were stable for longer than 30 min.

Figure 1. Phorbol esters inhibit outward potassium currents in AtT20 D16:16 cells.

Figure 1

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A, representative traces of leak-subtracted voltage-activated outward potassium currents at different membrane potentials (-40 to +50 mV) before and 10 min after application of 100 nM PMA. AtT20 cells were voltage clamped at -50 mV in a physiological saline solution in conventional whole-cell recording mode and depolarized for 100 ms to the respective potential. B, current-voltage relationships of the outward steady-state potassium currents (normalized to cell membrane capacitance and expressed as current density (pA pF−1) from control (•) and 10 min after bath application of 100 nM PMA (○). C, time course of the effect of 100 nM PMA (○) or its inactive analogue 4α-PMA (▵) on the steady-state outward currents expressed as a percentage of the pre-treatment control current recorded at time = 0 min at +40 mV. D, summary of effects of 100 nM PDBu (n = 4), 300 nM dPPA (n = 6), 100 nM PMA (n = 10) and 4α-PMA (n = 4) on outward steady-state currents determined 10 min after bath application at +40 mV expressed as percentage inhibition of pre-treatment control current. All data are expressed as means ±s.e.m. for each group. Error bars are within the symbol size unless otherwise indicated.

To examine the regulation of outward currents by PKC we used a range of phorbol ester analogues to directly activate PKC in whole cell recordings. Bath application of 100 nM PMA resulted in a significant inhibition of outward current at all potentials examined (Fig. 1A and B) with maximal inhibition after 5-10 min of application (Fig. 1C). Mean percentage inhibition (determined at +40 mV) 10 min after PMA application was 58.1 ± 1.7 %, a reduction of steady-state current density to 105.2 ± 11.1 pA pF−1, P < 0.01 (Fig. 1B and D). Similar inhibition was observed (Fig. 1D) with 100 nM PDBu (38.1 ± 4.6 % of control, n = 4) and 300 nM dPPA (47.3 ± 5.2 % of control, n = 6: inhibition by 100 nM dPPA was 26.0 ± 3.6 % of control, n = 6). In contrast, the inactive phorbol ester analogue (4α-PMA) had no significant effect over the same time course (1.2 ± 6.6 % of control, n = 4; Fig. 1C and D) suggesting that the effect of phorbol esters is mediated via activation of protein kinase C. Upon washout of phorbol esters a slow and partial reversal of the inhibitory effect on outward current was observed over the time course of experiments (not shown). All subsequent experiments used PMA as the PKC activator.

To verify that the effect of PMA was mediated via activation of PKC, two structurally and functionally distinct PKC inhibitors were used. Bath application of 100 nM BIS (a competitive inhibitor of the ATP binding site of PKC (Toullec et al. 1991)) or 25 μM CHE (a non-competitive inhibitor of PKC (Herbert, 1990)) had no significant effect on steady-state outward current alone (compare Figs 1 and 2). Bath application of 100 nM BIS or 25 μM CHE more than 10 min before application of 100 nM PMA blocked the inhibitory action of PMA: inhibition at +40 mV and 10 min after PMA application was only 10.1 ± 5.7 % of control current for BIS + PMA (n = 5; Fig. 2A-C) and 21.5 ± 6.4 % of control for CHE + PMA (n = 5; Fig. 2C).

Figure 2. The PKC inhibitors bisindolylmaleimide I (BIS) and chelerythrine chloride (CHE) block PMA-mediated inhibition of steady-state outward currents.

Figure 2

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A, representative traces of leak-subtracted voltage-activated outward potassium currents at different membrane potentials (-40 to +50 mV) before and 10 min after application of 100 nM PMA in cells pre-treated with 100 nM BIS. B, current-voltage relationships of the outward steady-state potassium currents (normalized to cell membrane capacitance and expressed as current density (pA pF−1): means ±s.e.m. for each group) from BIS (▪) and 10 min after bath application of 100 nM PMA (□). C, mean inhibition of outward steady-state current determined at +40 mV expressed as a percentage of pre-treatment control current in cells treated as above with 100 nM PMA alone (n = 10), 100 nM PMA + 100 nM BIS (n = 5), or 100 nM PMA + 25 μM CHE (n = 5). Means ±s.e.m. for each group. ** P < 0.01 compared with PMA-treated group.

The effect of PMA was primarily to inhibit the BK component of the outward current as no significant effect of PMA was observed on the 1 mM TEA-insensitive outward current in this system. Mean steady-state TEA-insensitive outward current density was 34.7 ± 3.6 pA pF−1 (n = 4) under the recording conditions used and was reduced to 31.6 ± 2.8 pA pF−1 (n = 4) after 10 min of PMA application, a reduction of only 6.9 ± 9.1 % of control (Fig. 3A and C). Importantly, application of 100 nM IbTX after maximal inhibition of steady-state outward current by PMA resulted in no further significant inhibition of outward current in 3/3 cells confirming IbTX-sensitive BK channels as a major target for PKC action.

Figure 3. PMA has no effect on TEA-insensitive voltage-activated outward current or inward pharmacologically isolated high voltage-activated calcium current.

Figure 3

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A, representative traces (determined at +40 mV) and mean I-V relationships of 1 mM TEA-insensitive steady-state outward current before (•, n = 4) and 10 min after bath application of 100 nM PMA (○, n = 4). I-V relationships are normalized to cell membrane capacitance and expressed as current density (pA pF−1). B, representative traces (determined at +20 mV) and mean I-V relationships of inward voltage-activated calcium current before (▪, n = 6) and 10 min after bath application of 100 nM PMA (□, n = 6). I-V relationships are normalized to cell membrane capacitance and expressed as current density (pA pF−1). C, time course of effect of bath application of 100 nM PMA on TEA-insensitive outward current (○) and inward calcium current (□) determined as described in A and B above and expressed as a percentage of the pre-treatment control value (time 0 min). All data are expressed as means ±s.e.m. with error bars within the symbol size unless otherwise indicated.

As high (> 1 μM) concentrations of phorbol ester or diacylglycerol analogues inhibit dihydropyridine-sensitive voltage-activated calcium influx in AtT20, as well as other neuroendocrine, cells (Lewis & Weight, 1988), we verified that the inhibition of the BK currents was not an indirect result of inhibition of calcium influx. In control cells, peak pharmacologically isolated calcium current density determined at +20 mV was -8.7 ± 0.9 pA pF−1 (n = 6 that were stable for greater than 20 min; Fig. 3B and C). Bath application of 100 nM PMA resulted in no significant inhibition of calcium current at any potential examined over the time course during which PKC-mediated inhibition of BK current was maximal (Fig. 3B and C). Mean current density 10 min after PMA addition determined at +20 mV was -8.3 ± 1.1 pA pF−1 (n = 6), an inhibition of only 6.3 ± 4.5 % of control.

PKC does not directly inhibit BK channels in isolated patches

To examine whether BK channels are direct substrates for PKC-mediated phosphorylation we examined the regulation of single BK channels by PMA, with or without exogenous PKC, in excised inside out patches. PMA (10-100 nM) applied alone or in conjunction with exogenous purified brain PKC (25-100 nM) had no significant effect on single channel mean open probability (Po) of identified BK channels over the time course of PMA action in intact cells (Fig. 4A-C). A similar lack of effect was observed in 3/3 patches using recombinant PKCα or PKCε (not shown). These data strongly suggest that the BK channels themselves are not directly phosphorylated by PKC in this system.

Figure 4. PKC does not inhibit BK channels in isolated inside out patches.

Figure 4

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A, representative consecutive unitary current traces from an excised inside out patch from AtT20 cells containing 4 BK channels before and 10 min after bath application of 100 nM PMA + 25 nM purified rat brain PKC. B, plot of mean single channel open probability (Po), determined from 20 s of continuous recording at each time point, as a function of time. The traces in A are from the 0 and 10 min time points, respectively. C, mean percentage inhibition of Po (with respect to pre-treatment control) in patches after 10 min application of 100 nM PMA (n = 3) alone or 100 nM PMA + 25 nM purified rat brain PKC (n = 3). Patches were depolarized to 0 mV and exposed to 0.5 μM intracellular free calcium and 1 mM ATP using physiological potassium gradients.

In some systems PKC regulation of potassium channel activity is mediated through regulation of tyrosine phosphorylation pathways (Huang et al. 1993; Lev et al. 1995). Indeed, BK channels have been shown to be regulated via tyrosine kinase/phosphatase pathways (Prevarskaya et al. 1995; Holm et al. 1997). However, inhibitors of protein tyrosine kinases (Tyrphostin A25) and tyrosine phosphatases (dephostatin) had no significant effect on outward steady-state potassium current and did not block PMA-induced inhibition of the outward current (Fig. 5A-C). A similar lack of effect was also observed with other inhibitors of tyrosine kinase and phosphatase pathways: genistein and sodium orthovanadate, respectively (not shown).

Figure 5. Inhibitors of protein tyrosine kinases or phosphatases do not block PMA-induced inhibition of outward steady-state potassium current.

Figure 5

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A, mean I-Vrelationships of outward steady-state potassium current before (▪, n = 4) and 10 min after bath application of 100 nM PMA (□, n = 4) in cells pre-treated for > 15 min with 50 μM of the protein tyrosine kinase inhibitor Tyrphostin A25. B, mean I-V relationships of outward steady-state voltage-activated potassium current before (•, n = 4) and 10 min after bath application of 100 nM PMA (○, n = 4) in cells pre-treated for > 15 min with 50 μM of the protein tyrosine phosphatase inhibitor dephostatin. In A and B, I-V relationships are normalized to cell membrane capacitance and expressed as current density (pA pF−1). C, effect of inhibitors alone, and effect of 100 nM PMA in the presence of inhibitors, on steady-state outward potassium current determined at +40 mV and expressed as a percentage of the respective pre-treatment control current amplitude (n = 4 per group). All data are expressed as means ±s.e.m. with error bars within the symbol size unless otherwise indicated.

Glucocorticoids block PKC-mediated inhibition of BK current

As glucocorticoid hormones block PKC-stimulated ACTH release and antagonize PKA-mediated regulation of BK channels we examined whether glucocorticoids also block PKC-mediated inhibition of BK channels in this system. Pre-treatment of intact AtT20 D16:16 cells for 2 h with a maximally effective concentration (1 μM) of the type II glucocorticoid agonist dexamethasone had no significant effect on mean steady-state outward potassium current density (Fig. 6A and B) or sensitivity to TEA (Fig. 6D) as previously described (Shipston et al. 1996). Mean steady-state current density determined at +40 mV was 230.4 ± 20.5 pA pF−1 (n = 7; Fig. 6B; compared with 245.3 ± 17.7 pA pF−1 in control cells); TEA inhibition was 69.1 ± 5.6 % (n = 7; compared with 70.1 ± 7.8 % in control).

Figure 6. Dexamethasone pre-treatment blocks PMA-mediated inhibition of outward steady- state current.

Figure 6

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A, representative traces (determined from -40 to +50 mV) and B, mean I-V relationships of the total outward steady-state potassium current from dexamethasone (DEX) pre-treated (1 μM, 2 h) cells before (♦, n = 7), 10 min after bath application of 100 nM PMA (⋄, n = 7) and after subsequent addition of 1 mM TEA (•, n = 7). I-V relationships are normalized to cell membrane capacitance and expressed as current density (pA pF−1). C, time course of effect of PMA on vehicle-treated control cells (○) and cells pre-treated with dexamethasone (⋄, 1 μM, 2 h) expressed as a percentage of the pre-treatment control current at 0 min. D, summary of effects of 100 nM PMA, 100 nM PMA + 100 nM okadaic acid (OA), 1 mM TEA and 100 nM PMA + 100 μM DRB (transcription inhibitor: 5,6-dichloro-furanosyl-benzimidazole riboside) on outward steady-state potassium currents from control and dexamethasone-treated cells determined at +40 mV and expressed as a percentage of the pre-treatment control current determined at 0 min. Okadaic acid was applied 10 min before and during exposure to PMA; DRB was applied 15 min before and during exposure to DEX. All data are expressed as means ±s.e.m. with error bars within the symbol size unless otherwise indicated. ** P < 0.01 compared with PMA effect in control cells.

In contrast, PMA inhibition of steady-state outward current in dexamethasone-treated cells was significantly blunted (Fig. 6B-D) over the time course during which PMA inhibition of the outward current was maximal in control (vehicle-treated) cells. Mean inhibition determined at +40 mV 10 min after PMA application was only 15.5 ± 5.2 % in dexamethasone-treated cells. To verify that the inhibitory effect of glucocorticoids was mediated via induction of new messenger RNA (Shipston, 1995) cells were exposed to the transcription inhibitor 5,6-dichloro-furanosyl-benzimidazole riboside (DRB) 15 min before and during dexamethasone treatment. In dexamethasone-treated cells pre-exposed to DRB, PMA significantly inhibited outward currents to a similar extent as in control (dexamethasone untreated, Fig. 6D) cells.

As serine/threonine protein phosphatases (PP) mediate the effects of glucocorticoids in this system to regulate PKA-mediated regulation of BK channels (Tian et al. 1998), and PP1 and PP2A have been reported to inhibit recombinant PKCα activity (Ricciarelli & Azzi, 1998), we examined whether blockade of PP1 and PP2A reversed the action of glucocorticoids on PKC signalling. Concentrations of okadaic acid that block PP1 and PP2A in this system (Antaraki et al. 1997; Tian et al. 1998) did not prevent dexamethasone blockade of PKC action (Fig. 6D).

Glucocorticoids do not inhibit PMA-stimulated PKC translocation

In an attempt to delineate the level at which glucocorticoids block PMA-mediated signalling we examined whether PMA-induced translocation of PKC isoforms to membrane fractions was affected in glucocorticoid-treated cells. Previous studies in the AtT20 D16:16 cell line reported expression of the α-, β- and ε- (PMA sensitive) as well as the ζ- (PMA insensitive) PKC isoforms (McFerran et al. 1995). Western blotting for the PMA-sensitive isoforms revealed robust expression of α and ε isoforms (Fig. 7). However, in six separate preparations we were unable to detect the β isoform in cytosol, membrane or whole cell fractions of AtT20 D16:16 cells even with > 40 μg protein per lane. Controls for the β isoform (mouse and rat brain fractions) were positive at < 10 μg per lane (not shown).

Figure 7. PMA-induced translocation of PKCα or PKCε to membrane fractions is not blocked by dexamethasone pre-treatment.

Figure 7

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Representative (of 6 separate cell preparations) Western blots demonstrating PMA-induced translocation of the phorbol ester-sensitive protein kinase C isoforms PKCα and PKCε in AtT20 D16:16 cells. Cells were pre-treated with vehicle (Control) or 1 μM dexamethasone (DEX) for 2 h at 37 °C as described in Methods and exposed to vehicle (-) or 100 nM PMA (+) for a further 10 min. Membrane homogenates (10 μg per lane) were subjected to SDS-PAGE on a 10 % SDS-PAGE gel and electroblotted to PVDF membrane before immunodetection with anti-rabbit PKCα (1/500 dilution) or PKCε (1/1000 dilution) antibodies and subsequent detection by ECL. Rat brain homogenate (Br) was used as an internal standard and molecular mass markers are indicated in kDa.

From vehicle-treated cells, PKCα or PKCε expression in membrane homogenates was low, compared with cytosol fractions (not shown) but detectable (Fig. 7). Bath application of 100 nM PMA to intact cells resulted in translocation of α- and ε-PKC isoforms to the membrane fraction within the time course of PKC-mediated inhibition of BK current activity (Fig. 7) Importantly, glucocorticoid pre-treatment did not prevent PMA-induced translocation of either PKC isoform (Fig. 7) suggesting that the inhibitory action of glucocorticoids is mediated downstream of PMA-induced PKC activation.

DISCUSSION

In this report we demonstrate that activation of PKC inhibits the outward steady-state potassium current that is largely passed through large conductance calcium- and voltage-activated potassium (BK) channels in AtT20 D16:16 mouse pituitary corticotroph cells exposed to physiological potassium gradients. Furthermore, the signalling pathway linking activation of PKC to inhibition of BK channels is blocked by glucocorticoid hormones.

Activation of PKC inhibits BK current in AtT20 D16:16 cells

Activation of PKC plays an important role in the control of excitability and modulation of ACTH secretion, in corticotrophs; however, the molecular targets for PKC action are poorly defined.

Inhibition of BK channels provides the first functional molecular target for PKC action in this system. Previous microfluorimetric analysis of PKC-stimulated calcium influx in AtT20 cells suggested that a major target for PKC action was a TEA-sensitive potassium conductance (Reisine, 1989). As TEA-sensitive BK channels are important regulators of voltage-dependent calcium influx in this (Antoni et al. 1992) and other systems (Robitaille et al. 1993; Yazejian et al. 1997) PKC-mediated inhibition of the TEA-sensitive BK current would provide a molecular mechanism of PKC-induced calcium influx in the absence of significant direct effects of PKC on voltage-dependent calcium currents per se. Importantly, the time course of phorbol ester action reported here mimics the time course of vasopressin- (and phorbol ester-) mediated activation of PKC and stimulation of ACTH secretion that is dependent on voltage-dependent calcium influx in corticotrophs (Carvallo & Aguilera, 1989; Oki et al. 1990). It is likely that co-ordinate regulation of several ion channels, including BK channels reported here, as well as components of the exocytotic machinery (Gillis et al. 1996), determines the secretory response to PKC activation in corticotrophs.

In this study we have used phorbol esters to directly activate PKC in an attempt to dissect the mechanism of PKC action in corticotrophs. The effects of phorbol esters reported here are all mediated through activation of protein kinase C. Firstly, two structurally and mechanistically different inhibitors of PKC significantly attenuated the effects of PMA on outward steady-state potassium current: bisindolylmaleimide I (BIS), a specific competitive inhibitor of ATP binding on PKC based on the broad spectrum kinase inhibitor staurosporine (Toullec et al. 1991), and chelerythrine chloride that blocks PKC activity without affecting ATP or phorbol ester binding (Herbert, 1990). Secondly, the effect of PMA was not mimicked by its inactive 4α-PMA analogue, and finally, the latency (2-5 min) of phorbol action and lack of effect of PMA in isolated patches suggest a phosphorylation cascade rather than a direct effect of phorbol esters on channel gating. Furthermore, the effect of PKC activation is not an indirect result of inhibition of calcium influx through voltage-dependent calcium channels. Indeed, the effects of calcium influx on BK channel behaviour is largely eliminated in these studies using the fast calcium chelating buffer, BAPTA, as the calcium buffer; furthermore, the inhibitory effects of PKC activation are observed at positive membrane potentials when voltage-dependent calcium influx is minimal (not shown).

Although we could demonstrate PMA-induced translocation of PKCα and PKCε isoforms to membrane fractions, application of PMA, active brain PKC extracts or recombinant PKC isoforms to the intracellular face of isolated inside out patches had no significant effect on isolated BK channels. This suggests that PKC is not closely associated with, or involved in regulation of, BK channels in the basal state and that PKC does not directly phosphorylate BK channels in AtT20 cells. However, we cannot exclude the possibility that components of BK channel complexes that mediate PKC action (e.g. putative accessory subunits) may be lost on patch excision. Previous work in a variety of systems has shown that activation of PKC signalling cascades regulates calcium-activated potassium currents (Baraban et al. 1985; Minami et al. 1993; Shipston & Armstrong, 1996; Peers & Carpenter, 1998); however, few studies have directly demonstrated effects of PKC itself on such channels in isolated patches (Reinhart & Levitan, 1995). Furthermore, as PKC can regulate multiple intracellular signalling pathways (Mellor & Parker, 1998) it is likely that phosphorylation cascades downstream of PKC activation may be involved in these cellular responses. In some systems PKC mediates its effects on ion channels through a phosphorylation cascade culminating in regulation of protein tyrosine phosphorylation pathways (Huang et al. 1993; Lev et al. 1995). Indeed, BK channels have been shown to be regulated via tyrosine kinase/phosphatase pathways (Prevarskaya et al. 1995; Holm et al. 1997). However, using a range of tyrosine kinase and phosphatase inhibitors we were unable to block PMA-induced inhibition of BK current in this system, thus the precise pathways linking PKC activation and inhibition of BK currents is presently unknown.

Previous work in AtT20 D16:16 cells suggested, on the basis of putative specific activators (e.g. dPPA) of PKC β1 isoforms, that PKC β1 acts to enhance voltage-dependent calcium influx (McFerran et al. 1995). Although dPPA (at 300 nM) significantly inhibited BK current the effect of dPPA in this report is unlikely to be mediated via PKC β1 as we were unable to detect PKC β isoforms in our AtT20 D16:16 cells. Furthermore, the specificity of dPPA at > 100 nM concentrations for PKC β1 in vivo has been questioned (Kiley et al. 1994) and PKC β1 does not appear to be expressed in the anterior pituitary gland (Garcia-Narvarro et al. 1991).

Cross-talk between PKC and glucocorticoid signalling pathways

Glucocorticoids rapidly (within 2 h) inhibit adrenocorticotrophic hormone (ACTH) release stimulated by activation of the PKA or PKC signalling pathways in corticotrophs (Phillips & Tashjian, 1982; Woods et al. 1992; Clark & Kempainen, 1994; Shipston, 1995). This inhibition is mediated via induction of proteins whose identity and molecular targets are poorly understood (Shipston, 1995). In AtT20 cells glucocorticoids prevent PKA-mediated inhibition of large conductance calcium- and voltage-activated potassium (BK) channels through modulation of okadaic acid-sensitive protein phosphatase 2A-like activity closely associated with the BK channel complex (Shipston et al. 1996; Tian et al. 1998). Although this study demonstrates that glucocorticoids, via induction of de novo mRNA and protein synthesis, block PKC-mediated inhibition of BK currents, the mechanism, and probable site, of blockade of PKC signalling to the BK channel is distinct from that mediating glucocorticoid inhibition of PKA-mediated BK channel regulation. Firstly, we could find no evidence of direct PKC regulation of BK channels in isolated patches; furthermore, the antagonistic effect of glucocorticoids on PKC signalling was not blocked by low concentrations of okadaic acid. Importantly, the effect of glucocorticoids is most likely to be mediated downstream of PKC activation as glucocorticoid pre-treatment did not prevent translocation of PKCα or PKCε to membrane fractions in response to PMA treatment. Increasing evidence suggests that glucocorticoids, via induction of de novo protein synthesis, can rapidly modulate multiple protein phosphorylation/dephosphorylation cascades. For example, in hepatocytes glucocorticoids rapidly induce the p21 cyclin-dependent kinase (CDK) inhibitor that blocks CDK2 phosphorylation pathways (Cha et al. 1998); in epithelial cells and fibroblasts, glucocorticoids upregulate the inducible serine/threonine kinase sgk (Webster et al. 1993); and glucocorticoids block PKC signalling in osteoblasts via a putative tyrosine phosphatase pathway (Hulley et al. 1998).

Whether distinct glucocorticoid-induced proteins regulate the PKC and PKA pathways, respectively, rather than a unique, multifunctional protein remains to be determined. However, as glucocorticoids induce multiple proteins in corticotrophs (Shipston, 1995; Kempainnen & Behrend, 1998), and glucocorticoids modulate other cellular functions in corticotrophs (Booth et al. 1998) and multiple protein phosphorylation signalling cascades in other systems (Webster et al. 1993; Shipston et al. 1996; Cha et al. 1998; Hulley et al. 1998), the former mechanism appears most likely. Interestingly, sustained activation of PKC in AtT20 cells has been shown to downregulate glucocorticoid receptor expression (Sheppard, 1994). Taken together, these data suggest that cross-talk between the PKC and glucocorticoid signalling pathways may play an important role in both the short- and long-term regulation of corticotroph behaviour.

Acknowledgments

We thank Dr A. G. Clark and other members of the Membrane Biology Group for helpful discussions. This work was generously supported by The Wellcome Trust (Ref.: 046787/Z).

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