Activation of protein kinase C reduces L-type calcium channel activity of GH, pituitary ALLYSON ROBERT

A. HAYMES, YIU WA KWAN, JOSEPH S. KASS, AND PATRICIA M. HINKLE

cells

P. ARENA,

Department of Pharmacology and the Cancer Center, and Department of Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Haymes, Allyson A., Yiu Wa Kwan, Joseph P. Arena, Robert S. Kass, and Patricia M. Hinkle. Activation of protein kinase C reduces L-type calcium channel activity of GH3 pituitary cells. Am. J. Physiol. 262 (CeZlPhysiol. 31): C1211-C1219, 1992.-These studies describe the effect of protein kinase C (PKC) activation on the activity of voltage-sensitive L-type Ca 2+ channels of GH3 pituitary cells. The rate of 45Ca2+ uptake was stimulated >25-fold by depolarization in the presence of BAY K 8644; the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) reduced this response by 70% in a concentration-dependent fashion. Phorbol12,13-dibutyrate (PDBu) inhibited depolarization-induced 45Ca2+ uptake within 1 min and caused a nearly maximal reduction after 1 h; its effects were rapidly reversible. TPA decreased the high K+stimulated increase in intracellular free calcium ion concentration ([Ca”+]i) from 8.5- to 3.2fold by 5 min and to 2.O-fold after 18 h without altering the peak [Ca2+]i response to the peptide hormone TRH. Ca2+ channel current, measured directly using the whole cell configuration of the patch-clamp technique, declined an average of 6.4% over 5 min for control cells and 28.9% when TPA was added to the bathing medium for 5 min. Treatment with 100 nM TPA for 24 h dramatically reduced peak current without shifting the peak of the current-voltage relationship. The mean peak Ca2+ channel current was reduced from 423 to 128 pA, although a few cells seemed completely resistant. To determine whether the effects of phorbol esters were due to the activation of PKC we tested the potency of several drugs to inhibit L-channel activity and to shift the affinity of the epidermal growth factor (EGF) receptor, an established PKC response. The concentrations of TPA, PDBu, 1,2dioctanoylglycerol, and 1 -oleoyl-2-acetylglycerol producing half-maximal inhibition of Ca2+ channel activity and 1251-EGF binding were very close, while 4a-phorbol was inactive. The PKC inhibitor staurosporine strongly inhibited voltage-sensitive Ca2+ channel activity by itself and blunted the ability of TPA to inhibit L-channel activity and 1251-IEF binding, whereas 200 nM K252a was not effective in either assay. Downregulation of PKC with high doses of TPA reduced the ability of phorbol esters to inhibit depolarization-induced 45Ca2+ uptake and rise in [Ca2+]i. Changes in CAMP were not involved in the effects of TPA, since TPA did not lower CAMP, and elevating intracellular CAMP with 3-isobutyl-1-methylxanthine did not reverse TPA effects. The results show that the activity of Ltype Ca2+ channels in GH, cells is inhibited by activation of PKC. intracellular free calcium ion concentration; phorbol esters

A VARIETY of cellular

processes, including contraction, neurite outgrowth, and neurotransmitter and hormone secretion, are dependent on an increase in cytosolic free calcium ion concentration ([Ca’+]i). Rapid changes in [Ca2+]; may be accomplished through release of calcium from intracellular stores or through entry of calcium from the extracellular fluid via calcium channels. Voltage-dependent calcium channels may be divided into three broad categories (14, 26, 29). T-type or tran0363-6143/92

$2.00

Copyright

sient-type channels are activated at a low-threshold membrane voltage, are rapidly inactivated, and have a low unitary conductance (8 pS using barium as charge carrier). L-type or long-lasting-type channels activate at more depolarized potentials, inactivate more slowly, have a higher unitary conductance (25 pS), and are sensitive to the dihydropyridine calcium channel modulators. N- or neuronal-type channels activate at relatively strongly depolarized potentials, as do L-type channels, but are insensitive to dihydropyridines and appear to be found only in neuronal cell types. GH, cells are a clonal line derived from a rat anterior pituitary tumor with the properties of somatomammotrophs (31). These cells secrete both prolactin and growth hormone spontaneously and in response to thyrotropin-releasing hormone (TRH), vasoactive intestinal peptide, 12-0-tetradecanoylphorbol 13-acetate (TPA) and depolarization, which produces a rise in [Ca2+]; due to entry through L-type calcium channels (1, 11). The depolarization-induced increase in hormone secretion from GH, cells can be amplified by inclusion of the dihydropyridine channel agonist BAY K 8644 and completely blocked by dihydropyridine channel antagonists such as nimodipine, as well as by various L-channel blockers (15). Phorbol esters and other activators of protein kinase C, including synthetic lipids and hormones, have been reported by other investigators to modulate calcium conductance through L-type channels. In some cases, protein kinase C activation results in channel activation (3, 6, 10, 17, 21, 30); in others, protein kinase C activation inhibits channel activity (7, 8, 13, 16, 17, 21, 22, 24, 25, 27, 28).

We have investigated the effect of TPA on L-type calcium channels in GH3 cells. Here we demonstrate that TPA reduces depolarization-stimulated increases in radioactive calcium uptake and [Ca2+];, and calcium channel activity measured in electrophysiological experiments. We also provide multiple lines of evidence suggesting that the effect of phorbol esters is due to the activation of protein kinase C. MATERIALS AND METHODS Materials. 45Ca2+ as CaCl, (17 mCi/mg)

was obtained from either New England Nuclear (Boston, MA) or Amersham (Arlington Heights, IL), and carrier-free 125I for protein iodination was from Amersham. Phorbol esters, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) and 1,2-dioctanoylglycerol (di-C,) were purchased from Sigma (St. Louis, MO) and 1-oleoyl-2-acetylglycerol (OAG) from Avanti (Birmingham, AL). Tissue culture media and sera were from GIBCO (Grand Island, NY), and tissue culture plasticware was from Corning (Corning, NY). Epidermal growth factor (EGF,

0 1992 the American

Physiological

Society

Cl211

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receptor grade) was purchased from Collaborative Research (Lexington, MA) and iodinated to a specific activity of 5002,000 Ci/mmol by mild chloramine-T treatment. BAY K 8644 was a gift from Miles Institute of Preclinical Pharmacology (New Haven, CT). Fura- and quin2 free acids and esters were from Molecular Probes (Eugene, OR). Assay kits for the measurement of adenosine 3’,5’-cyclic monophosphate (CAMP) were obtained from Diagnostic Products (Los Angeles, CA). Phorbol esters, di-Cs and OAG, were stored in dimethyl sulfoxide (DMSO) and diluted at least loo-fold into medium or buffer immediately before use. K252a, in chloroform, was evaporated under nitrogen and dissolved in DMSO just before use. K252a and staurosporine were purchased from Calbiochem (San Diego, CA) &ho& GHs cells were grown in monolayer culture in F-10 medium supplemented with 15% horse and 2.5% fetal calf serum as previously described (31); this is referred to as complete F-10 medium. Serum-free F-IO medium contains 126 mM NaCl, 3.8 mM KCl, 0.3 mM CaCl,, and other ions as noted by the manufacturer. As far as we are aware, the effects described in this study are similar or identical for other GH cell lines; we have tested GH3 cells from the American Type Culture Collection, GH,C,, and GH-Y cells. For experiments, GH, cells from a single donor culture were inoculated into 35- or loo-mm dishes and grown for 4-8 days before use. For measurement of 45Ca2+ uptake, 35-mm dishes of GH3 cells were rinsed once with serum-free F-10 medium and then incubated at 37°C in serumfree F-10 medium containing 1-2 &i/ml 45Ca2+. The medium was aspirated and the cell lawn was washed three times with 0.15 M NaCl. Cells were suspended in 0.1 N NaOH or H20, and samples were used for measurement of radioactivity by liquid scintillation counting and for protein determination by the Lowry method (23) using bovine serum albumin as standard. Depolarization-stimulated 45Ca2+ uptake was routinely measured in 5- or lo-min assays as the difference between uptake in medium alone and uptake in medium containing 50 mM KC1 + 1 PM BAY K 8644. 45Ca2+ uptake was typically stimulated 5- to 20-fold by depolarization in the presence of the calcium channel agonist. Data shown are means & range or SE of two to three dishes. Experiments were performed on at least two occasions with similar results. Binding of 1251-EGF was determined by adding 1251-EGF (300,000-800,000 cpm/ml) to serum-free medium containing drugs as shown for either 3 h at 4°C or 1 h at 37°C. Parallel dishes contained a IOO-fold molar excess of unlabeled EGF and served as nonspecific binding controls. Dishes were rinsed three times with 0.15 M NaCl, and cell-associated 125I and protein were measured. Nonspecific binding has been subtracted from all data. To determine the intracellular concentration of CAMP, dishes containing - lo7 cells were rinsed twice with 0.15 M NaCl and treated as described in text. After incubation, dishes were placed on ice, and cells were immediately scraped into 2 ml chilled perchloric acid (3%) and transferred to chilled test tubes. KHCO, (30%) was added dropwise to each tube to bring the final pH to 5.5-6. Cell extracts were spun for 20 min at 2,000 g at 4°C. The concentration of CAMP in the supernatant fluid was assayed using a liquid phase 3H radioimmunoassay kit, with 100 ~1 per sample in duplicate, according to the manufacturer’s instructions. To measure [Ca2+]i, cells in a moderately dense IOO-mm dish were rinsed two times in 5 ml of Hanks’ balanced salt solution plus 10 mM HEPES, pH 7.4 (HBSS), removed from the dish by gentle scraping into 5 ml of HBSS, centrifuged, and incubated for 30 min at 37°C in 2 ml buffer containing 1 PM fura 2-AM or 25 PM quin 2-AM. Cells were diluted to 10 ml with buffer and again centrifuged, and the pellet was resuspended at a density of 2-5 x IO6 cells/ml in HBSS buffer. Cells were placed in a ther-

OF CALCIUM

CHANNELS

mostatted cuvette maintained at 37°C and kept in suspension with a magnetic stirbar. For quin 2 fluorescence, excitation was at 340 nm and emission was at 493 nm, and for fura 2 fluorescence, excitation was at 335 nm and emission at 505 nm. Slit widths were set at IO mm (excitation) and 20 mm (emission). After a stable baseline was attained, cells were depolarized by addition of KC1 (50 mM final concn) or subjected to other treatments as noted in text. Maximum fluorescence (&-& was obtained by lysing cells with digitonin (50 PM final concn) and minimum fluorescence (Fmin) by adding MnCl, (2 mM final concn); for quin 2, Fmin was corrected by the relationship Fmin = Fmin (apparent) + l/6 (Fma, - Fmin) (4). Intracellular calcium concentrations were calculated from the relationship ’ [Ca2+]i = & X (F - Fmin)/(Fma, - F) using 224 and 115 nM as & for the calcium complexes of fura 2 and quin 2, respectively. Calcium channel currents were measured in isolated cells with the whole cell arrangement of the patch-clamp technique. Cells were transferred to a 1.5-ml recording chamber mounted on the stage of an Olympus IMT-1 inverted stage microscope (Lake Success, NY). Cells were bathed in a protein-free saline solution containing (in mM) 60 N-methyl-D-glucamine (NMDG), 10 CsCl, 1 MgC12, 40 BaCl,, 10 HEPES, 10 tetraethylammonium chloride (TEA), 5 glucose, pH 7.3 (with CsOH). Barium chloride was used as the divalent charge carrier to increase signal-to-noise ratio. Sodium channel currents were eliminated by substituting NaCl with NMDG and by holding the membrane potential positive to -40 mV. Electrodes were filled with a solution designed to eliminate potassium channel current and to minimize calcium channel current rundown containing (in mM) 120 CsCl, 2 MgC12, 11 ethylene glycol-bis@aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 1 CaCl,, 10 HEPES, 1 MgATP, 0.1 CAMP, 0.04 GTP, 10 TEA, pH 7.2 (with CsOH). Electrodes (resistance of 3-5 MQ) were fabricated from Gold Seal Accu-fill 90 Micopets (Clay Adams, Parsippany, NJ). Recording was done in the whole cell configuration (12) and calcium channel currents were recorded at room temperature (20-22°C) using a Yale Mark IV patch-clamp amplifier and headstage. Series resistance composition was used in all experiments and was adjusted to give the fastest possible transients without producing ringing. Data were sampled at 650 pus and filtered at 1 kHz with an in-house low-pass filter, digitized, and then stored on a PC-286 computer. Voltage pulses of 300-ms duration were applied at 1 Hz. Linear leak and capacitance currents were subtracted using a protocol consisting of hyperpolarizing pulses, which produced no ionic current. Solutions in the chamber were changed with voltage-controlled values connected to syringe reservoirs. Solutions were drained with suction, and -10 ml of the solution was washed through within 20 s for a solution change. Experiments were performed on four different days. Differences among groups were determined by analysis of variance (ANOVA) or Student’s t test as appropriate. RESULTS

Effect of phorbol esters on calcium uptake. The initial rate of uptake of 45Ca2+ into GH3 cells was stimulated &fold by depolarization with high K+ and X%-fold by depolarization in the presence of the calcium channel agonist BAY K 8644 (Fig. 1). When GH3 cells had been treated with the phorbol ester TPA, the unstimulated uptake of 45Ca2+ was unchanged, but 45Ca2+ uptake in response to depolarization was blunted with a maximal response only one-third that of control cells. The effect of TPA was concentration dependent (Fig. Z), and was observed at all potassium concentrations from 8.8 to 23.8

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PROTEIN

KINASE

C REGULATION

OF CALCIUM

Cl213

CHANNELS

.c 40000 .-v) ? ;30000 m z a 20000 E 3 $ 10000 G i 0 0.0

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.

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Fig. 3. TPA inhibition of depolarization-induced 45Ca2+ uptake. Cells were incubated for 24 h in medium with no additions (0) or with 100 nM TPA (A). 45Ca2+ uptake was measured in serum-free F-10 medium, which contains 3.8 mM K+, and added K+ to give the concentrations shown plus 1 PM BAY K 8644. Values are means t SE of triplicate determinations.

100

r .-cn

+KCI

40000

& BAY

K8644

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Fig. 1. Rate of 4FiCa2+ uptake. Cells were incubated for 18 h in medium with no additions (open symbols) or with 100 nM TPA (solid symbols). 4FjCa2+ uptake was then measured for duplicate points at intervals in serum-free F-10 medium; lines were drawn manually, and errors were within symbol size. 0, No additions; q I, n , 50 mM KCl; A, A, 50 mM KC1 + 1 ,uM BAY K 8644.

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40

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80

100

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MINUTES

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100 nM

1000

Fig. 2. Effect of TPA concentration on depolarization-stimulated 45Ca2+ uptake. Cells were incubated for 18 h with TPA as shown. 45Ca2+ uptake was then measured with no additions (+) or with 50 mM KC1 + 1 PM BAY K 8644 (0). Values are mean t range of duplicate determinations.

mM, though not at the normal medium

concentration

of

3.8 mM (Fig. 3).

The rate of onset and reversal of phorbol ester inhibition of depolarization-induced calcium influx was established using the relatively water soluble phorbol 12,13dibutyrate (PDBu). PDBu was added to cells at intervals before or simultaneously with 45Ca2+ (Fig. 4A). Addition of PDBu inhibited depolarization-induced 45Ca2+ uptake very quickly, causing a 15% reduction in K+-evoked 45Ca2+ flux when added with the 45Ca2+ for a I-min period and a 60% reduction after 60 min. When PDBu was removed and the cells were washed, the K+-induced calcium influx began to increase within 10 min and was nearly restored to control values by 40 min. Longer exposure to PDBu resulted in a more profound and sustained decline in calcium channel activity, which was more slowly reversible when PDBu was removed after 24 h (Fig. 4B). Effect of phorbol esters on [Ca2+/i. To determine whether the decrease in depolarization-induced 45Ca2+ flux resulted in a change in [Ca2+]i, we measured the effects of 5 min or 18 h phorbol ester exposure on [Ca’+]; with intracellularly trapped calcium-sensitive fluorescent dyes. As shown in Fig. 5 and Table 1, TPA did not cause a significant change in the average [Ca2+]i measured in

0

10

20 HOlJ RS

Fig. 4. Reversibility of phorbol ester effect on calcium influx. Cells were incubated with no additions (0) or with 100 nM PDBu (w) for times shown before measurement of rate of 45Ca2+ uptake in the presence of 50 mM KC1 + 1 PM BAY K 8644. At times shown by arrow, medium was replaced on all dishes; PDBu (0) was removed from some cultures (H) and the original incubation conditions retained for all others. In A, 45Ca2+ was added for a 1-min incubation, whereas in B 45Ca2+ uptake was measured in a standard lo-min incubation. Values are expressed as percent of the time 0 control 45Ca2+ uptake (mean t SE of 3 determinations).

resting cells over 5 min. We sometimes observed a slight decline in intracellular calcium after adding TPA, but we never observed an increase. Cells that had been incubated with TPA for 18 h had, on average, a significantly lower resting [Ca”+]; than untreated controls. TPA reduced the increase in [ Ca2+]i after depolarization with high K+ from 8.5- to 3.2-fold after 5 min and to 2.O-fold after 18 h (P < 0.01 vs. control). The ability of phorbol esters to blunt the depolarization-induced increase in [ Ca2+]; was specific, since neither 5 min nor 18 h treatment with TPA

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Cl214

PROTEIN

CONTROL

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CELLS

KCI

OF CALCIUM

CONTROL

CHANNELS

CELLS

CELLS Rx 24h+TPA

I-PA

KCI

KCI

[CL+ Ii, nM

-800 -400 -200

1 min Fig. 5. Effect of TPA on calcium response to depolarization. Cells loaded with quin 2 were incubated in HBSS, and at times shown by arrows 100 nM TPA or 50 mM KC1 was added. A and B: control cells. C: cells treated with 100 nM TPA for 18 h before and during the experiment. Figure shows a typical experiment run in parallel using the same preparation of cells with equal dye loading, as confirmed by the difference between Fmax and Fmine

Table 1. Effect of TPA on [Ca2+J; TPA

Control 5 min

Resting [Ca2+]i, nM Depolarization response ( [ Ca2+] i + K+/basal) TRH response ( [Ca2+]i + TRH/basal) Depolarization after TRH ([Ca’+]; + K+/basal)

18 h

8.5t0.3

(7)

3.2+0.2*(4)

67t6 (12) 2.0t0.2* (4)

2.51tO.l

(5)

2.3tO.l

2.3tO.l

115tll(l4)

12oIk22

6.9k0.8 (5)

(7) (3)

4.2t0.3*(3)

(6)

2.8&0.2* (6)

Resting [Ca2+]i values are given as means t SE of the calculated concentration. Responses to KCl, TRH, and KC1 after TRH were calculated as fold increases for individual points (peak stimulated [Ca2+]Jbasal [Ca”+]J an d are given as means t SE of the fold increases; (n), no. of independent determinations. Either control cells or cells that had been treated with 100 nM TPA for 18 h were loaded with quin2. In some cases, TPA was added for 5 min before the cells were depolarized. [Ca2+]i was determined as described in MATERIALS AND METHODS. In some experiments, once a stable baseline was established, 50 mM KC1 was added and peak [Ca2+]i measured. In others, 100 nM TRH was added and l-2 min later 50 mM KC1 was added. Table shows peak response to each drug, which occurred within 15 s.

changed the peak [Ca2+]i response to the peptide hormone TRH, 2.3- to 2.5fold. TRH acts via the phosphoinositide pathway and causes immediate release of calcium from intracellular stores (1 I). When cells were depolarized several minutes after TRH treatment, the increase in [Ca2+]i was again significantly reduced in TPA-treated cells. We have also found that phorbol ester treatment does not alter the transient increase in [Ca2+]; caused by 1 ,uM A23187, a calcium ionophore (data not shown). Effect of phorbol ester treatment on calcium channel current. To establish whether phorbol ester inhibition of

depolarization-induced 45Ca2+ influx was direct or indirect, we used the whole cell configuration of the patchclamp technique to measure calcium currents (12). Typical current traces and current-voltage relationships are shown in Fig. 6, and data from four separate experiments are summarized in Fig. 7. Calcium channel activity, measured as the peak in the current-voltage curve, declined slightly, an average of 6.4% (n = 17) over 5 min when cells were maintained in buffer alone because of channel rundown. When TPA was added to the bathing medium for 5 min, peak calcium currents declined an average of 28.9% (n = 23) (P < 0.01 vs. control cells at 5 min). Most cells that had been treated with 100 nM TPA overnight had dramatically reduced peak current; a typical cell is depicted in Fig. 6C. The mean peak calcium channel current was reduced from 423 to 128 pA (P < 0.01 vs.

untreated cells) and 18 of 22 cells exposed to TPA displayed a peak calcium current of Cl00 pA (Fig. 7). Despite these highly significant differences, in every experiment one or two cells appeared resistant to the effects of TPA and displayed a current of >300 pA. Mechanism of phorbol ester effects on calcium channels.

To address the question of whether the effects of phorbol esters were due to the activation of protein kinase C, we tested the ability of several drugs to blunt voltage-sensitive calcium channel activity. To estimate the extent to which these drugs activated protein kinase C, we followed another response to protein kinase C activation, the shift in affinity of the EGF receptor, under identical conditions. In numerous cell types, including pituitary GH cells, activation of protein kinase C causes a decrease in the affinity of the high-affinity EGF receptor for 1251-EGF We have previously validated the use of the shift in 12’51_EGF binding as an assay for the activation of protein kinase C in the intact cell (18). Table 2 summarizes the potency of several drugs to block voltage-gated calcium channel activity and to alter the affinity of the EGF receptor. The experiments were performed after a l-h incubation with drug. The concentrations of TPA, PDBu, di-C,, and OAG required to give half-maximal inhibition of calcium channel activity and EGF receptor binding were very close. The drug 4-aphorbol, which does not activate protein kinase C, was inactive (Table 3). Two putative inhibitors of protein

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6

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A

t=o

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1

0

-20

VOLTAGE

-0.5

I

20

40

-80

60

I -60

I -40

I -20

VOLTAGE

(mV)

I 0

1 20

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60

80

(mV)

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Fig. 6. Effect of TPA on calcium channel currents. Calcium channel currents were studied in single-cell voltage-clamp experiments as described in MATERIALS AND METHODS. Recordings were made from untreated control cells before and after incubation for 5 min in buffer alone (A) or in buffer + 100 nM TPA (B) and recorded 5 min later. C: cells that had been incubated overnight in medium containing 100 nM TPA. Representative data are shown. Top traces show a family of leak-subtracted current traces in response to -70, -40, 0, and +30 mV. Currents are in response to 300-ms pulses. Bottom curves show peak current plotted against test voltage. Holding potential was -40 mV. Calibration bars: 0.1 nA, 50 ms.



0

VOLTAGE



20

I

40



60



80

(mV)

kinase C were also tested. K252a did not alter EGF binding or affect phorbol ester inhibition of calcium influx at 200 nM. Staurosporine was tested at concentrations from 10 to 200 nM. Staurosporine strongly inhibited voltagesensitive calcium channel activity by itself at all concentrations adequate to inhibit TPA action, measured as the decrease in 1251-EGF binding Staurosporine at 50 nM did prevent TPA from inhibiting calcium channel activity and from shifting the affinity of the EGF receptor, consistent with the idea that both responses result from protein kinase C activation (Table 3). Effect of protein kinase C depletion. As a further test of the involvement of protein kinase C, we investigated the

effect of conventional downregulation of protein kinase C with high doses of TPA (24 h at 1 PM); we previously showed that this protocol causes disappearance of protein kinase C measured in immunoblots (18). After protein kinase C depletion, cells were challenged with TPA to determine whether it still altered calcium channel activity. As shown in Table 4, after depletion of protein kinase C, phorbol esters did not cause a significant inhibition of depolarization-induced 45Ca2+ uptake. The effect of TPA on the depolarization-stimulated rise in [ Ca2+]i was also monitored, using fura 2, for control cells and cells subjected to protein kinase C downregulation. Resting [Ca2+]i was not affected by pro-

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Cl216

PROTEIN

l?O 100

1 -

KINASE

C REGULATION

X

z800 -e B?

g w E 5

X Js

X X

Y 5 a

activity and EGF binding

-

600

-

400

-

200

-

Expt

1 # X

50 --

2

O-

CONTROL 5 min

Control

+TPA 5 min

TPA 24 h

Fig. 7. Effect of TPA on calcium current. All data from cells recorded on 4 different days, in experiments like that shown in Fig. 6, are compiled. A: change in peak current after 5 min incubation in buffer alone (control) or in buffer + 100 nM TPA. After 5 min, current was reduced an average of 6.4 t 0.8% (n = 17) control vs. 28.9 it 1.4% (n = 23) TPA treated (P < 0.001). B: peak current for control cells and cells treated for 24 h with 100 mM TPA. Mean peak current was 423t27 pA (n = 39) control vs. 128t25 pA (n = 22) TPA treated (P > 0.001).

Table 2. Effect of drugs on calcium uptake and EGF binding Go

PDBu TPA Di-Cs OAG

CI-IANNELS

Table 3. Ability of drugs to inhibit calcium channel

A

+

Drug

OF CALCIUM

Range

l-1,000 O.l-1,000 3-300 100-900

Depolarization-stimulated *Va2+ uptake

nM nM PM /JM

20 0.5 30 200

nM nM /JM PM

‘251-EGF binding

16 2 170 150

nM nM PM pM

Cells were incubated with drugs at various concentrations for 30-60 min in serum-free F-10 medium. Depolarization-stimulated 45Ca2+ uptake and specific 1251-EGF binding were then measured in the continued presence of drug. Table shows concentration range tested and dose giving half-maximal inhibition (I&,). Maximal response for each experiment was obtained with 100 nM PDBu or TPA. PDBu, di-C8, and TPA all gave the same maximal response, whereas OAG was less effective than TPA at the highest dose attainable because of limited solubility. Typically, specific 12”I-EGF binding was reduced 50% by phorbol esters, and the range of 1251-EGF binding was 1,500-4,000 cpm in different experiments.

tein kinase C depletion, but the ability of TPA to block the depolarization-induced increase in [ Ca’+] i was lost (Table 4). Effect of activation of protein kinase C by hormone on calcium channel activity. Protein kinase C is activated

not only by phorbol esters but also indirectly by hormones such as TRH that activate phosphoinositide turnover and cause release of diacylglycerol (11). TRH blunted the depolarization-induced increase in 45Ca2+ (Fig. 8) in much the same way as phorbol esters, although its effect was always lower than that of TPA measured in the same experiment. Possible role of CAMP. It is well established that full activity of L-type calcium channels is dependent on adequate intracellular CAMP (2). To determine whether changes in CAMP levels could be involved in the effects of protein kinase C activators on calcium channel activity, we measured the intracellular CAMP concentration in cultures subjected to TPA treatment identical to that causing inhibition of calcium channel activity. As shown in Table 5, the CAMP concentration in depolarized cells incubated with or without the phosphodiesterase inhibi-

3

Treatment

Control TPA (100 nM) 4-cu-Phorbol (10 PM) None TPA (100 nM) Staurosporine (200 nM) Staurosporine + TPA None TPA (100 nM) K252a (200 nM) K252a + TPA

KCl/BAY -Stimulated 45Ca2+ Uptake, cpm/mg protein

1251-EGF Bound, cpm/dish

206,OOOH3,700 93,400&6,900* 174,000a8,600 254,000~17,500 26,000+1,100* 45,300&2,400 57,000+11,300 295,000+290 65,200+ 1,950” 192,000+12,700 70,100+5,160*

ND ND ND 2,060+100 1,150+110* 2,410+130 2,060klOO 2,060+100 1,150~110* 2,680&90 1,420&60*

Values are means t SE for 3 or 4 dishes. Cells were treated with drugs shown for 30 min and then depolarization-stimulated 45Ca2+ uptake and specific 1251-EGF binding were measured as described in MATERIALS AND METHODS. Unstimulated rates of calcium uptake were not altered by any of the treatments and were 7-14% of maximal stimulated values in different experiments. ND, not determined. *P < 0.01 vs. same treatment without TPA. In the presence of staurosporine, effects of TPA on calcium uptake and 1251-EGF binding were not statistically significant.

tor 3-isobutyl-1-methylxanthine (IBMX) was not decreased by TPA. Furthermore, the ability of phorbol esters to inhibit calcium channel activity was not blocked by the addition of IBMX to increase intracellular CAMP to a maximal level (Table 6). TPA reduced the depolarization-induced increase in 45Ca2+ uptake from lo- to 2.8-fold in control cultures and from 19- to 2.7fold when IBMX was present. DISCUSSION

The evidence that phorbol esters inhibit the activity of L-type voltage-gated calcium channels on GH3 cells is compelling. Phorbol esters decreased depolarizationstimulated 45Ca2+ flux and calcium channel current assessed in electrophysiological experiments. Although GH3 cells express both L- and T-type voltage-sensitive calcium channels, both of these experimental approaches measure primarily the activity of L-type calcium channels (5). Depolarization-stimulated 45Ca2+ uptake is nearly blocked by low concentrations of channel blockers selective for L-type calcium channels, such as the dihydropyridines (15). The calcium current measurements were performed under conditions designed to minimize the contribution of T-channels, and we have previously shown the current measured in this manner is almost completely blocked by nimodipine. The net effect of phorbol ester treatment on [Ca2+]i was to blunt the increase obtained upon depolarization. Taken together, these data establish that calcium current through L-type channels was greatly reduced by treatment with phorbol esters. The effect of phorbol esters on L-type calcium channel activity, when fully developed, was very large, with activity reduced on average to about one-fifth that of control cells. Our results confirm and extend a number of previous studies that characterized phorbol ester effects on GH3 cell calcium homeostasis. Marchetti and Brown (24) previously demonstrated that 4-60 PM OAG reversibly reduces the peak and steady-state calcium currents when added to the bath perfusing GH3 cells held at -90 mV and depolarized to

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PROTEIN

KINASE

C REGULATION

OF CALCIUM

Cl217

CHANNELS

Table 4. Effect of protein kinase C depletion on TPA inhibition of calcium channel activity [Ca’+]i,

Treatment

nM

Resting

Fold

+KCl

Increase

45Ca2+

+KCl

Uptake,

cpm

Resting

Fold

+KCl

Increase

+KCl

Control TPA PKC PKC Values additions

194t19 815t68 4.2 (10) 2,390+240 14,020+1,170 5.9 (3) 229t22 408&50* 1.8*(16) 1,850&370 3,810+10* 2.1*(3) depleted 228k45 529t145 2.5 (4) 2,29Ok380 9,190+220 4.0 (3) depleted + TPA 210t37 5432100 2.6 (6) 2,030t610 8,370t860 4.1 (3) are means t SE; (n), no. of replicates. Before the experiment, GH3 cells were incubated for 24 h in complete F-10 medium with no (control) or with 1 pM TPA to downregulate protein kinase C (PKC depleted). Cells were loaded with furaas described in MATERIALS AND METHODS with or without the inclusion of TPA (100 nM) during the 30-min loading period. A stable baseline fluorescence was obtained and cells were then depolarized by addition of 50 mM KCl. 45Ca2+ uptake was measured on 35-mm GH3 cell dishes subjected to the same downregulation protocol. TPA (100 nM) was added to some dishes 30 min before and during the 5-min incubation with 45Ca2+, and 45Ca2+ uptake was measured in serum-free F-10 medium alone or in medium containing 50 mM KC1 and 1.5 PM BAY K 8644. *P c 0.01 vs. +KCl without TPA. In the PKC-depleted groups, none of the effects of TPA was statistically significant.

Table 5. Effect of TPA on CAMP levels

Control Control+BAY

K

o!



0

I

10



I

‘,V

20

30 [KCI], mM

[cAMPI,

Treatment

TRH Rx+BAY K

,

40



11

50

Fig. 8. Effect of TRH on calcium channel activity. Cells were incubated with or without 100 nM TRH for 18 h and 45Ca2+ uptake measured with or without 1 PM BAY K 8644 as described in the legend to Fig. 3. Values are means t SE of triplicate determinations.

pmol/mg

Table 6. Effect of IBMX +lO mV, and Tornquist

and Tashjian showed (32) that when added simultaneously with high K+, TPA inhibits the rise in [Ca2+]i (32). In our studies, there was considerable heterogeneity between cells even after exposure to a high concentration of phorbol ester overnight. Some cells appeared completely resistant to the effect of phorbol esters even though they displayed normal calcium channel activity; these cells were found at a frequency of about 1 in 10 in electrophysiological studies. Future work will be needed to establish the basis for this heterogeneity. The reported effects of protein kinase C activators on calcium channel activity differ widely depending on the cell type studied. Activators of protein kinase C, or a partially purified protein kinase C subspecies mixture from bovine brain, have been shown to activate voltagesensitive calcium channels in Aplysia neurons (6, 30) and in a vascular smooth muscle cell line (lo), as well as in mouse and human fibroblasts (3). In Aplysia this activation appears to be due to recruitment of a second class of higher unitary conductance calcium channels, rather than an increase in open probability of existing channels. Lacerda et al. (21) have observed an increase followed by a decrease in channel activity in neonatal rat ventricular myocytes with TPA treatment. Izumi et al. (17) describe a rapid increase in [Ca2+]i, via L-channels in pituitary gonadotrophs after TPA application; however, this effect is abolished after 5-30 min TPA or gonadotropin-releasing hormone pretreatment. We saw no evidence of a biphasic effect in GH, cells, and inhibition of calcium channel activity was evident in the first 2 min after phorbol esters were added, the earliest time studied. Further-

protein

None 30t4 5 KCl/BAY K 8644 16t6 8 KCl/BAY K 8644 + IBMX 7Ok7 9 KCl/BAY K 8644 + TPA 29t3 7 KCl/BAY K 8644 + IBMX + TPA 67k4 2 Values are means t SE or range for 5-9 or 2 dishes, respectively. GH, cells were incubated in control medium or in depolarizing medium (50 mM KC1 + 1.5 ,uM BAY K 8644) with or without 100 PM IBMX and/or 100 nM TPA. All treatments were for 30 min at 37°C. CAMP concentration was determined as described in MATERIALS AND METHODS. Within each treatment group, differences t TPA were not statistically significant.

on phorbol ester inhibition-

of calcium channel activity 45Ca2+

Treatment

None TPA IBMX IBMX

+ TPA

Uptake,

cpm/dish

Basal

+KCl/BAY

7,930+ 1,080 3,890+360 5,440+500 3,290&210

76,960&2,870 10,890+980 103,000+6,250 9,010a690

Fold

Stimulation

10.0 2.8 18.9 2.7

Values are means & SE. IBMX (100 PM) and/or TPA (100 nM) were added to dishes of GH3 cells 30 min before and during the 5-min incubation with 45Ca2+ . 45Ca2+ uptake was measured in serum-free F-10 medium alone (basal) or in medium containing 50 mM KC1 and 1.5 PM BAY K 8644.

more, inhibition of calcium channel activity was observed at all doses of phorbol esters tested. In other systems, as in GH3 cells, protein kinase C activators have been found to inhibit calcium channel activity. Inhibition of L-channels occurs in chick sensory neurons (27, 28) and adult dorsal root ganglia (9, 16) and in tissue culture lines, including neuronal PC-12 cells (7, 25), and secretory endocrine cells, including ACTH-secreting AtT-20 cells (22) and RIN m5F insulin-secreting cells (7). It is not known why some cell types respond to phorbol esters with enhanced calcium channel activity and others with profound inhibition of L-channels. In systems in which L-channel activity is blunted by phorbol esters, the response has usually been attributed to the ability of phorbol esters to activate protein kinase C. However, there has recently been considerable disagreement about whether this is the correct mechanism. Protein kinase C has been implicated based on the fact that phorbol esters or synthetic diacylglycerols such as

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OAG are capable of both activating the enzyme and eliciting the effect on calcium channels, but in many cases only a single high drug dose has been tested (8,14,21,22, 29). The argument that protein kinase C is not involved has been based on experiments in which downregulation or putative protein kinase C inhibitors fail to prevent phorbol esters from modulating calcium channels (16). Because of the controversy surrounding this point, we have used a variety of approaches in a systematic manner to test whether protein kinase C mediates the actions of phorbol esters on calcium channels. Our data suggest that the mechanism of the phorbol ester effect involves the activation of protein kinase C. Evidence for this includes structure activity data, the demonstration that a hormone can reproduce the phorbol ester effect, and downregulation studies. In addition to the phorbol esters PDBu and TPA, two lipids known to activate protein kinase C, OAG and di-Cs, also reduced L-channel activity, whereas 4-a-phorbol, which does not activate protein kinase C, did not. The concentrations of drugs required to alter calcium channel activity were close to those required to reduce EGF receptor affinity, an effect attributed to a protein kinase C-catalyzed phosphorylation (18). We also found that TRH, a tripeptide hormone that activates PIP2 hydrolysis and elevates diacylglycerol physiologically, inhibited L-channel activity, as expected from its ability to activate protein kinase C (11). Kramer et al. (20) recently reported that TRH reduces GH3 cell L-channel activity in a perforated patch setup, and they concluded that the effect of TRH was due to the TRHinduced rise in [Ca2+3;, not the activation of protein kinase C, since they found that they could block the TRH effect by chelating intracellular calcium and could not prevent it by protein kinase C downregulation. An alternate possibility to explain the results of Kramer et al. is that calcium buffering reduced [Ca2+]i below the level needed for activation of protein kinase C by phorbol esters; they did not report whether phorbol esters were effective in control, 1,2-bis(2-aminophenyl)ethane-N,N, N’,N’-tetraacetic acid (BAPTA)or EGTA-buffered, or protein kinase C-depleted cells. While we cannot establish whether a calcium spike contributes to the TRH effect on L-channels, we believe that protein kinase C is involved in the response we measure, since it is reduced by protein kinase C depletion (data not shown). Further evidence comes from classical downregulation studies. Cells were subjected to a downregulation protocol similar to that previously shown to deplete GH3 cells of almost all of the p- and t-isoforms of protein kinase C and to decrease the cu-isoform by 50% (19). We have previously shown that it reduces the ability of phorbol esters and TRH to modulate EGF receptor affinity. Protein kinase C depletion greatly reduced the ability of phorbol esters to modulate calcium channel activity, measured as either depolarization-induced increase in 45Ca2+ uptake or [Ca2+]i. Our conclusion differs from that of Tornquist and Tashjian (32), who described the ability of phorbol esters, added together with a depolarizing concentration of potassium, to inhibit calcium uptake and lower the calcium spike. These effects are modified by prior treatment

OF CALCIUM

CHANNELS

with vitamin D. While this previous study used a different experimental paradigm than we have, it is likely that the same phenomenon has been characterized in both cases. Tornquist and Tashjian suggested that protein kinase C may not mediate the phorbol ester effect based on the failure of drugs such as staurosporine and K252a to inhibit, and the failure of OAG and di-C, to mimic, the response. As did Tornquist and Tashjian (32), we found that 200 nM K252a did not prevent TPA from blocking calcium channel activity, but it also failed to prevent TPA from altering 1251-EGF binding; thus the drug probably was not inhibiting protein kinase C in the experiments. Staurosporine itself severely inhibited calcium channel activity at concentrations 250 nM. Although it is difficult to interpret results against this large effect of staurosporine alone, we did find that the effect of TPA was abolished in the presence of staurosporine. The results are consistent with the idea that TPA modulates calcium channel activity via protein kinase C. We found it was necessary to use higher concentrations of OAG and di-Cs than those used in the previous study to inhibit 1251-EGF binding, and at these higher concentrations L-channel activity was likewise inhibited. Activity of L-type calcium channels depends on an adequate intracellular concentration of CAMP in GH3 and other cells. This raises the theoretical possibility that phorbol esters might have decreased channel activity by decreasing the level of CAMP. This seems very improbable for several reasons. First, the patch pipette contained extremely high concentrations of CAMP and ATP, making it unlikely that any phorbol ester-induced changes in CAMP would matter. Second, phorbol esters, with and without depolarization, slightly elevated CAMP; this is the opposite of what would be expected if diminished CAMP were responsible for calcium channel effects. Third, elevating intracellular CAMP in the intact cell with IBMX did not alter the ability of phorbol esters to block L-channel activity. Our attempts to assess the phosphorylation state of the DHP receptor directly have been unsuccessful because of the low number of channels on the cells. The mechanism of the effect of phorbol esters on calcium channels has not been established. Protein kinase C phosphorylation of one of the subunits of the L-type channel is a possibility consistent with the rapid onset of the response, but no direct evidence is available. Because phorbol esters are effective when added to largely dialyzed cells, it seems likely that the substrates for protein kinase are not readily diffusible. Protein kinase C may phosphorylate either the channel or a closely associated membrane protein, as has been suggested for protein kinase A (3, 37), in order to effect its current inhibition. We have shown here that activation of protein kinase C causes a profound inhibition of voltage-sensitive calcium channel activity in pituitary cells. Activation of membrane receptors may affect the activity of ion channels directly, through G proteins, or indirectly, through second messengers such as CAMP, calcium, and diacylglycerol (14). A hormone such as TRH that stimulates polyphosphoinositide hydrolysis both increases intracellular

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calcium and activates wotein kinase C. The Dotential of protein kinase C actiiation to modulate the* activity of L-channels adds to the complexity of the physiological m response to hormones. The authors are grateful to Edward D. Shanshala II for expert technical assistance. This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-19974 (P. M. Hinkle), Cancer Center Core Research Grant CA-11098 (P. M. Hinkle), and National Science Foundation Grant DCB-8822783 (R. S. Kass). Present address of J. P. Arena: Dept. of Biochemical Parasitology, Merck Sharp & Dohme, Rahway, NJ 07065. Address for reprint requests: P. M. Hinkle, Dept. of Pharmacology, Univ. of Rochester Medical School, 601 Elmwood Ave., Rochester, NY 14642. Received 31 October 1991; accepted in final form 19 December 1991. REFERENCES 1. Aizawa, T., and P. M. Hinkle. Differential effects of thyrotropin-releasing hormone, vasoactive intestinal peptide, phorbol ester, and depolarization in GH& rat pituitary cells. EndocrinoZORY 116: 909-919, 1985. Voltage-activated calcium 2. Armstrong, D.‘, and R. Eckert. channels that must be phosphorylated to respond to membrane depolarization. Proc. Natl. Acad. Sci. USA 84: 2518-2522, 1987. 3. Chen, C., and P. Hess. Calcium channels in mouse 3T3 and human fibroblasts (Abstract). Biophys. J. 51: 226a, 1987. 4. Cobbold, P. H., and T. J. Rink. Fluorescence and bioluminescence measurement of cytoplasmic free calcium. Biochem. J. 248: 313-328, 1987. 5. Cohen, C. J., and R. T. McCarthy. Nimodipine block of calcium channels in rat anterior pituitary cells. J. Physiol. Lond. 387: 195-225, 1987. 6. DeReimer, S. A., J. A. Strong, K. A. Albert, P. Greengard, and L. K. Kaczmarek. Enhancement of calcium current in Aplysia neurones by phorbol ester and protein kinase C. Nature Lond. 313: 313-316, 1985. 7. DiVirgilio, G., T. Pozzan, C. Wollheim, L. M. Vicenti, and J. Meldolesi. Tumor promoter phorbol myristate acetate inhibits Ca2+ influx through voltage-gated Ca2+ channels in two secretory cell lines, PC12 and RINm5F. J. Biol. Chem. 261: 32-35, 1986. 8. Doerner, D., T. A. Pitler, and B. E. Alger. Protein kinase C activators block specific calcium and potassium current components in isolated hippocampal neurons. J. Neurosci. 8: 4069-4078, 1988. 9. Ewald, P. A., H. J. G. Matthies, T. M. Perney, M. W. Walker, and R. J. Miller. The effect of down regulation of protein kinase C on the inhibitory modulation of dorsal root ganglion neuron Ca2+ currents by neuropeptide Y. J. Neurosci. 8: 2447-2451, 1988. 10. Fish, R. D., G. Sperti, W. S. Colucci, and D. E. Clapham. Phorbol ester increases the dihydropyridine-sensitive calcium conductance in a vascular smooth muscle cell line. Circ. Res. 62: 1049-1054, 1988. 11. Gershengorn, M. C. Mechanism of the thyrotropin-releasing hormone stimulation of pituitary hormone secretion. Annu. Reu. Physiol. 48: 515-526, 1986. 12. Hamill, 0. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch. 391: 85-100, 1981. 13. Hammond, C., P. Paupardin-Tritsch, A. C. Nairn, P. Greengard, and H. M. Gerschenfeld. Cholecystokinin induces a decrease in Ca2+ current in snail neurons that appears to be mediated by protein kinase C. Nature Lond. 325: 809-811, 1987. 14. Hess, P. Calcium channels in vertebrate cells. Annu. Rev. Neurosci. 13: 337-356, 1990.

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15. Hinkle, P. M., R. N. Day, and J. J. Enyeart. Endocrine effects of calcium channel agonists and antagonists in a pituitary cell system. In: The Calcium Channel: Structure, Function and Implications, edited by M. Morad, W. Nayler, S. Kazda, and M. S&ram. Berlin: Springer-Verlag, 1988, p. 335-346. 16. Hockberger, P., M. Toselli, D. Swandulla, and H. D. Lux. A diacylglycerol analogue reduces neuronal calcium currents independently of protein kinase C activation. Nature Lond. 338: 340342. 1989. 17. Izumi, S., S. Stojilkovic, T. Iida, L. Krsmanovic, R. Omeljaniuk, and K. Catt. Role of voltage-sensitive calcium channels in [Ca2+]i and secretory responses to activators of protein kinase C in pituitary gonadotrophs. Biochem. Biophys. Res. Commun. 170: 359-367, 1990. 18. Kaji, H., J. E. Casnellie, and P. M. Hinkle. Thyrotropin releasing hormone action in pituitary cells: protein kinase C-mediated effects on the epidermal growth factor receptor. J. Biol. Chem. 263: 13588-13593, 1988. 19. Kiley, S., D. Schaap, P. Parker, L. Hsieh, and S. Jaken. Protein kinase C heterogeneity in GH,C, rat pituitary cells: characterization of a Ca2+ -independent phorbol ester receptor. J. BioZ. Chem. 265: 15704-15712, 1990. 20. Kramer, R. H., L. K. Kaczmarek, and E. S. Levitan. Neuropeptide inhibition of voltage-gated calcium channels mediated by mobilization of intracellular calcium. Neuron 6: 557-563, 1991. 21. Lacerda, A. E., D. Rampe, and A. M. Brown. Effects of protein kinase C activators on cardiac Ca2+ channels. Nature Lond. 335: 249-251, 1988. 22. Lewis, D. L., and F. F. Weight. The protein kinase C activator l-oleoyl-2-acetylglycerol inhibits voltage-dependent Ca2+ current in the pituitary cell line AtT-20. Neuroendocrinology 47: 169-175, 1988. 23. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. 24. Marchetti, C., and A. M. Brown. Protein kinase C activator l-oleoyl-2-acetyl-sn-glycerol inhibits two types of calcium currents in GH, cells. Am. J. Physiol. 254 (Cell Physiol. 23): C206C210, 1988. 25. Messing, R. O., C. L. Carpenter, and D. A. Greenberg. Inhibition of calcium influx and calcium channel antagonist binding in the PC 12 neural cell line by phorbol esters and protein kinase C. Biochem. Biophys. Res. Commun. 136: 1049-1056, 1986. 26. Nowycky, M. C., A. P. Fox, and R. W. Tsien. Three types of neuronal calcium channels with different Ca2+ agonist sensitivity. Nature Lond. 316: 440-443, 1985. 27. Rane, S. G., and K. Dunlap. Kinase C activator 1,2-oleoylacetylglycerol attenuates voltage-dependent calcium current in sensory neurones. Proc. Natl. Acad. Sci. USA 83: 184-188, 1986. 28. Rane, S. G., M. P. Walsh, J. R. McDonald, and K. Dunlap. Specific inhibitors of protein kinase C block transmitter-induced modulation of sensory neuron calcium current. Neuron 3: 239245, 1989. 29. Shearman, M. S., K. Sekiguchi, and Y. Nishizuka. Modulation of ion channel activity: a key function of the protein kinase C enzyme family. Pharmacol. Rev. 41: 211-237, 1989. 30. Strong, J. A., A. P. Fox, F. W. Tsien, and L. K. Kaczmarek. Stimulation of protein kinase C recruits covert calcium channels in Aplysia bag cell neurons. Nature Lond. 325: 714-717, 1987. 31. Tashjian, A. H., Jr., Y. Yasumura, L. Levine, G. H. Sato, and M. L. Parker. Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone. Endocrinology 82: 342-352, 1968. 32. Tornquist, K., and A. H. Tashjian, Jr. 12-O-tetradecanoylphorbol-13-acetate decreases influx of extracellular Ca2+ induced by depolarization in GH& cells: effects of pretreatment with 1,25-dihydroxycholecalciferol. Endocrinology 126: 2068-2078, 1990.

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Activation of protein kinase C reduces L-type calcium channel activity of GH3 pituitary cells.

These studies describe the effect of protein kinase C (PKC) activation on the activity of voltage-sensitive L-type Ca2+ channels of GH3 pituitary cell...
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