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Research
Cell Calcium (1996) 20(1), 1-9
© Pearson Professional Ltd 1996
Recovery from TPA inhibition of
receptor-m ediated Ca2+ m obilization
is paralleled by down-regulation of
protein kinase C-a in CHO cells
expressing the CCK-A receptor
I
R.L.L. Smeets1, K.WI. Garner3, M. Hendriks1, S.E. van Emst-de Vries1,
M.D. Peacock3, W. Hendriks2, J.J.H.H.M. de Pont1, P.H.G.M. Willems1
Departments of ’Biochemistry and 2Cell Biology, University of Nijmegen, The Netherlands
3Divlsion of Biosciences, University of Hertfordshire, Hatfield, Hertfordshire, UK
Digital-imaging microscopy of Fura-2-loaded Chinese hamster ovary cells, stably expressing the
cholecystokinin-A receptor, revealed that both the C-terminal octapeptide of cholecystokinin (CCKa) and its analogue
JMV-180, which acts as an agonist at the high-affinity CCK-A receptor, recruited CHO-CCK-A cells dose-dependently
in terms of receptor-mediated Ca2* mobilization. Agonist-evoked cell recruitment was inhibited by short-term (10 min)
pretreatment with 0.1 |iM 12-O-tetradecanoylphorbol 13-acetate (TPA). In the case of CCKa, inhibition was overcome
with increasing of the hormone concentration. In contrast, increasing of the JMV-180 concentration did not reverse the
inhibitory action of TPA. CHO-CCK-A cells gradually regained their responsiveness to JMV-180 during prolonged TPA
pretreatment. Complete recovery was observed within 1 h following addition of TPA. Western blot analysis using
antibodies directed against the various PKC isotypes revealed that recovery was paralleled by the disappearance of
PKC-a. Surprisingly, short-term (10 min) TPA pretreatment virtually completely inhibited the formation of inositol 1,4,5trisphosphate [lns(1,4,5)P3] in response to CCK8 concentrations at which the effect on cell recruitment was not affected
by short term phorbol ester pretreatment. Together with the finding that JMV-180 does not detectably increase the
cellular lns(1,4,5)P3 content, this suggests a large overproduction of this second messenger by CCKa concentrations
supramaximal in terms of cell recruitment. Again, full responsiveness was observed after long term TPA pretreatment.
The present observations are in agreement with the idea that in CHO-CCK-A cells activation of PKC-a leads to
inhibition of agonist-evoked Ca2+ mobilization through inhibition of receptor-stimulated lns(1,4,5)P3 formation.
Summary
INTRODUCTION
Receptors interacting with the cholecystokinin (CCK)
family of peptides are widely distributed throughout the
Received 12 March 1996
Revised 7 May 1996
Accepted 10 May 1996
gastrointestinal and nervous system [1], CCK receptors
are classified into two subtypes on the basis of their dif­
ference in affinity for sulphated versus nonsulphated
CCK [1]. Thus, the CCK-B receptor displays similar affini­
ties for both forms of the hormone, whereas the CCK-A
receptor has a considerably higher affinity for sulphated
Abbreviations: CCK0, C-terminal octapeptide of cholecystokinin; JMV-180,
Correspondence to: Dr P.H.G.M. Willems, Department of Biochemistry,
University of Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The
Netherlands
Tel. +31 24 361 4589: Fax. +31 24 354 0525
E-mail: [email protected]
Boc-Tyr(S03)-Nle~Gly~Trp-Nle«Asp“2-phenylethylester; TPA, 12-0tetradecanoylphorbol-13-acetate; lns(1,4,5)P3l inositol 1,4,5-trisphosphate;
[Ca2,]t, free cytosolic calcium concentration; PKC-a, protein kinase C-a;
CHO-CCK-A cells, Chinese hamster ovary cells expressing the
cholecystokinin-A receptor.
1
2
RLL Smeetst KM Gamer, M Hendriks et ai
CCK In addition, the CCK-B receptor does not discrimi­
nate between CCK and gastrin, whereas the CCK-A
receptor is highly selective for CCK [1], Finally antago­
nists have been developed, which can effectively discrim­
inate between both subtypes of the CCK receptor [1],
The present study concerns the CCK-A receptor,
which, among others, is present on the pancreatic acinar
cell [2,3]. Hormonal stimulation of these epithelial cells,
which synthesize and store the digestive enzymes,
results in the rapid release of these acinar products by
means of exocytosis. In the case of CCK, triggering of the
secretory event is paralleled by the rapid breakdown of
phosphatidylinositol 4,5-bisphosphate to yield inositol
1,4,5-trisphosphate [Ins(l,4,5)P3] and 1,2-diacylglycerol
(DAG) [4]. Ins(l,4,5)P3 releases Ca2+from the endoplasmic
reticulum, leading to a rapid increase in cytosolic free
Ca2f concentration ([Ca2+]{), and DAG activates protein
kinase C (PKC), Recently, the CCK-A receptor has been
cloned and hydropathy plots of its predicted amino acid
sequence revealed the presence of 7 transmembrane
domains characteristic for members of the G proteincoupled superfamily of receptors [5].
Using freshly isolated pancreatic acinar cells, we [6-9]
and others [10,11] demonstrated that short-term activa­
tion of PKC by 12-Otetradecanoylphorbol 13-acetate
(TPA) results in inhibition of CCK-stimulated enzyme
secretion and Ca2+ mobilization. The inhibitory action of
the phorbol ester appeared to be restricted to the lower
concentrations of CCK, which led to the conclusion that
PKC, directly or indirectly, converts the CCK-A receptor
from a high-affinity state into a low-affinity state [8,9],
This idea is supported by the observation that TPA
causes the complete loss of high-affinity CCK binding
sites [12] and the finding that inhibition by TPA of the
stimulatory action of the CCK analogue JMV-180, which
acts as an agonist only at the high-affinity CCK-A recep­
tor, is not overcome by increasing the agonist concentra­
tion [8,9]. Moreover, TPA has been demonstrated to
induce phosphorylation of the receptor in intact acinar
cells [13-16] and together with the finding that the intra­
cellular domains of the receptor contain 4 potential
phosphorylation sites for PKC [5], this suggests that PKC
might act directly, i.e. without the intermediation of a
receptor kinase, at the level of the CCK-A receptor
The aim of the present study was to investigate
whether signaling through the CCK-A receptor is still
inhibited by TPA when the receptor is expressed in
Chinese hamster ovary (CHO) cells. If so, CHO-CCK-A
cells would provide a suitable mean to assess the role of
the 4 potential phosphorylation sites for PKC by sitedirected mutagenesis. Using a video imaging technique,
we have previously demonstrated that CCK dose-dependently recruits freshly isolated pancreatic acinar cells in
terms of receptor-mediated Ca2+ mobilization [17].
Cel! Cafcium (1996) 20(1), 1-9
Moreover, we have demonstrated that TPA pretreatment
causes a rightward shift of the dose-recruitment curve
[9]. Since this technique, if applicable to CHO-CCK-A
cells, would enable rapid screening of mutated forms of
the CCK-A receptor with respect to inhibition of signal­
ing by TPA, it was investigated whether CCK recruits
CHO-CCK-A cells also in a dose-dependent manner and
whether TPA causes a similar rightward shift of the doserecruitment curve. The level at which PKC interferes
with signaling through the stably expressed CCK-A
receptor was determined by investigating the effect of
TPA pretreatment on the kinetics of the CCK-induced
formation of Ins(l,4,5)P3. Finally, in order to identify the
PKC iso type (s) involved in the inhibitory action of TPA,
CHO-CCK-A cells were treated with the phorbol ester for
prolonged periods of time and the rate of disappearance
of the various PKC isotypes, estimated by means of
immunoblotting, was correlated with the recoveiy from
TPA-induced inhibition of CCK-stimulated Ca2+ mobi­
lization and Ins(l,4,5)P3 formation.
H ie data presented demonstrate that both CCK and
JMV-180 dose-dependently recruit CHO cells stably
expressing the CCK-A receptor and that short-term TPA
pretreatment causes a rightward shift of the dose-recruit­
ment curve for CCK and complete inhibition of JMV-180
evoked cell recruitment. In addition, short-term TPA pre­
treatment was found to effectively inhibit CCK-evoked
Ins(l,4,5)P3 formation. In the continuous presence of
TPA, the cells gradually recovered from these inhibitory
actions of TPA. The latter process was paralleled by the
selective disappearance of PKC-a, suggesting that this
PKC isotype intermediates in the inhibitory action of TPA
on signaling through the CCK-A receptor stably ex­
pressed in CHO cells.
i
MATERIALS AND METHODS
Materials
Fura-2/AM was purchased from Molecular Probes Inc.,
Eugene, OR, USA and i~butyloxycarbonyl-Tyr(S03)-NleGly-Tyr-Nle-Asp-2-phenylethyl ester (JMV-180) from
Research Plus Inc., Bayonne, NJ, USA. CCKg, inositol
1,4,5-trisphosphate, chelerythrine chloride and TPA were
obtained from Sigma Diagnostics, St Louis, MO, USA and
D-myo-[3H]-inositol 1,4,5-trisphosphate (51.4 Ci/mmol)
from Amersham, UK. Isotype-specific PKC antibodies
and tissue culture medium with additives were pur­
chased from Gibco, Paisley, UK and staurosporine from
Boehringer, Mannheim, Germany. I-block Reagent was
obtained from Tropix, Bedford, MA, USA and 4-a-phorbol
12-niyristate 13-acetate from LC Services Corporation,
Woburn, MA, USA. All other chemicals were of reagent
grade.
© Pearson Professional Ltd 1996
Inhibition of CCK-A receptor signaling by protein kinase C
3
Development of a stable CHO-CCK-A cell line
lns(1,4,5)P3 measurements in CHO-CCK-A cells
Full-length cDNA encoding the rat CCK-A receptor [5]
was originally provided by Dr SA. Wank (National
Institutes of Health, Bethesda, MD, USA). However,
because of poor expression, it was decided to use a cDNA
truncated to within three nucleotides of the first in frame
ATG [18]. This truncated cDNA, subcloned into the mam­
malian expression vector pTEJ8, was kindly provided by
Dr CD. Logsdon (University of Michigan, Ann Arbor, MI,
USA). Transcription of the CCK-A receptor is driven by a
ubiquitin promoter. Resistance against G418 is provided
by a neomycin phosphotransferase encoding gene under
the control of the SV40 early promoter. CHO-K1 cells
were cultured in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% FCS in a humidified
atmosphere of 5% C 02 at 37°C. For transfection, cells
were grown to 70% confluency, trypsinized and trans­
ferred to a cuvette (3 x 106 cells/300 \il). The cells were
electroporated (250 V, 960 jllF) in the presence of 20 jig
linearized pTEJ-CCK-A and replated in 100 mm plates (5
\il cell suspension/plate). At 40 h after electroporation,
the plates were washed to remove dead cells and G418
was added at a concentration of 1.2 mg/ml, G418-resistant colonies were selected at 14 days after electropora­
tion. The cells were tested on the presence of functional
CCK-A receptors by digital imaging microscopy.
Cells were plated out in 12-well plates (200,000 cells/well)
24 h previous to hormonal stimulation and Ins(l,4,5)P3
measurement. TPA, at a final concentration of 0.1 jiM,
was added at the indicated times. Immediately before
stimulation, cells were washed twice with a HEPES/Tris
medium (pH 7.4) containing 133 mM NaCl, 4.2 mM KCl,
1.0 mM CaCl2, 1.0 mM MgCl2, 5,8 mM glucose, 0.2 mg/ml
soybean trypsin inhibitor, an amino acid m ixture accord­
ing to Eagle, 1% (w/v) BSA and 10 mM HEPES, adjusted
with Tris to pH 7.4. Each well was stim ulated separately
by the addition of 250 jal HEPES/Tris m edium containing
the indicated concentration of hormone. The reaction
was stopped by addition of 62 |il 50% trichloroacetic acid
at the times indicated. The cells were scraped off the bot­
tom and transferred to a micro test tube (Eppendorf). The
samples were centrifuged for 4 min at 10,000 g
(Eppendorf minifuge) and a 240 jil aliquot of the super­
natant was removed and extracted 3 times w ith 2 ml of
water-saturated diethyl ether. A 150 |al aliquot was then
removed and 4 jrl 50% KHC03 was added to increase the
pH to a value above 7.5. The Ins(l,4,5)P3 content of the
extract was determined by isotope dilution assay as pre­
viously described [8].
Fluorescence measurements in individual
CHO-CCK-A cells
For fluorescence measurements, cells were plated on a
glass cover slip (2 x 104 cells/30 jllI) and allowed to attach
for 30 min. Culture medium was added and th e cells
were grown to subconfluency for another 24 h. Cells
were loaded with 2 |iM Fura-2/AM for 25 min at 37°C. To
remove non-hydrolysed dye, cells were washed 3 times
with a physiological salt solution (PSS) containing 137
mM NaCl, 4.7 mM KCl, 0.56 mM MgCl2, 1.28 mM CaCl2,
1.0 mM Na2H P 04, 2 mM L-glutamine, 5.5 mM D-glucose,
0.1% (w/v) BSA and 10 mM HEPES (pH 7.4). Cover slips
were m ounted in a thermostatic (33°C) perfusion cham­
ber, placed on the stage of an inverted microscope (Nikon
Diaphot). Superfusion with PSS was at a flow rate of 1
ml/min. Routinely, an epifluorescent 40x magnification
oil immersion objective was used to allow simultaneous
monitoring of an average of close to 60 cells. Dynamic
video imaging was carried out as described previously
[17] using the MagiCal hardware and Tardis software pro­
vided by Joyce Loebl (Dukesway, Team Valley, Gateshead,
UK). The fluorescence emission ratio at 492 nm was
monitored as a measure of the cytosolic free Ca2* con­
centration ([Ca2+]j) after excitation at 340 and 380 nm.
© Pearson Professional Ltd 1996
PKC-isotype measurements in CHO-CCK-A cells
Total cell lysates were subjected to SDS-polyacrylamide
gel electrophoresis. Proteins were transferred overnight
to polyvinylidine difluoride membranes (Immobilon P,
Millipore) by Western blotting. Membranes were blocked
for 1 h with PBS (pH 7.4) containing 0.2% (w/v) I-Block
reagent and 0.1% (v/v) Tween-20 and incubated over­
night with PKC-isotype-specific antibodies diluted 1:500
in PBS (pH 7.4) containing 0.1% (w/v) I-Block reagent and
0.2% (v/v) Tween-20. To demonstrate the specificity of
the reaction, control membranes were incubated with
PKC-isotype-specific antibodies in the presence of corre­
sponding PKC-isoform-specific peptides diluted 1:1000.
Membranes were washed with PBS (pH 74) containing
0.3% (v/v) Tween-20 (washing buffer) and incubated for
1 h with goat anti-rabbit IgG antibodies conjugated to
alkaline phosphatase diluted 1:1000 in PBS (pH 7.4) con­
taining 0.1% (w/v) I-Block reagent and 0.2% (v/v) Tween20. Membranes were washed with the above washing
buffer and PBS before staining with 0.1 M diethanolamine, 0.34 mg/ml Nitroblue tetrazonium, 0.18 mg/ml 5bromo-4-chloro-3-indolylphosphate and 1.0 M MgCl2.
Membranes were scanned by an Imaging Densitometer
(Biorad, Munich, Germany) and the optical density values
were calculated with the computer program Molecular
Analyst (Biorad, Munich, Germany).
Cell Calcium (1996) 20(1), 1-9
4
RLL Smeets, KM Garner; M Hendriks et a/
Analysis of the data
Half-maximal CCKg and JMV-180 concentrations for the
recruitment of CHO-CCK-A cells in terms of receptorevoked Ca2+ mobilization and maximal recruitment val­
ues were calculated by means of the nonlinear regression
computer program InPlot (Graphpad Software for Science,
San Diego, CA, USA), Scheffe's F test was used to deter­
mine differences between the mean values.
RESULTS
Effect of short-term TPA pretreatment on agonistevoked calcium mobilization in CHO-CCK-A cells
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Chinese hamster ovary cells, stably expressing the CCKA receptor and grown to subconfluency on glass cover
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Fig. 2 Effect of short-term TPA pretreatment on CCK8-evoked
recruitment of CHO-CCK-A cells. CHO-CCK-A cells were loaded
with Fura-2 and stimulated with the indicated concentrations of
CCKSfor 5 min. TPA-treated cells were preincubated with 0.1 jaM
TPA for 10 min. The data presented are the mean ± SEM of 3
experiments. On average, 60 cells were analysed in each individual
experiment.
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Time (s)
CCK0-evoked increase in free cytosolic Ca2+ concentration in
individual CHO-CCK-A cells. CHO-CCK-A cells were loaded with
Fura-2 and stimulated with (A) 0.01 nM CCKe and (B) 10 nM
CCKe. Changes in [Ca2+], were monitored by digital imaging
microscopy. The ceils were superfused with PSS and stimulated
with CCK8 at the times indicated.
Ceil Calcium (1996) 20(1), 1-9
slips, responded with a rapid increase in [Ca24]. when
stimulated with CCK8 (Fig. 1). At low CCK8 concentra­
tions, the response pattern consisted of repetitive
increases in [Ca2+]j (Fig. 1A). JMV-180, which acts as an
agonist at the high-affinity CCK-A receptor, evoked such
oscillatory changes in [Ca2+]. at all concentrations tested.
In contrast, high CCK8 concentrations induced a sus­
tained increase in [Ca2+]. (Fig. IB). CCK8 recruited CHOCCK-A cells in a dose-dependent m anner in terms of
receptor-mediated Ca2+ mobilization (Fig. 2). Nonlinear
regression analysis revealed that half-maximal recruitm ent was obtained with 0.8 pM CCK8, Pretreatment of
the cells with 0.1 jliM TPA for 10 min resulted in complete
inhibition of cell recruitment by 10 pM CCK8. However,
the inhibitory effect of TPA was overcome with increas­
ing of the CCKg concentration. Thus, at a concentration
of 10 nM, CCK8 evoked an increase in [Ca2+]i in 100% of
the TPA-treated cells. Nonlinear regression analysis
revealed a shift of the EC50 value to 70 pM CCKg in TPA
pretreated cells. Similarly, JMV-180 dose-dependently
increased the num ber of responding CHO-CCK-A cells
(Fig. 3B). The half-maximal stimulatory concentration of
the agonist was calculated to be 0.3 nM. Short-term (10
min) pretreatm ent with TPA (0.1 jiM) completely inhib­
ited cell recruitment by the lower concentrations of JMV© Pearson Professional Ltd 1996
Inhibition of CCK-A receptor signaling by protein kinase C
5
Table 1 Reversal by staurosporine and chelerythrine chloride of the inhibitory action of TPA on JMV-180-evoked recruitment of
CHO-CCK-A cells
Addition
JMV-180 (10 nM)
JMV-180 (10 nM) + TPA (0.1 |iM)
Responding cells
(% of total)
99 ± 0.3
1± 1
JMV-180 (10 nM) + 4-a-phorbol 12-myristate 13-acetate (0,1 ¡llM)
90 ± 10
JMV-180 (10 nM) + TPA (0.1 jxM) + staurosporine (0.1 |j,M)
97 ± 1
JMV-180 (10 nM) + TPA (0.1 |iM) + chelerythrine chloride (2 jiM)
87 ± 8
CHO-CCK-A cells were loaded with Fura-2 and stimulated with 10 nM JMV-180 for 5 min. Phorbol ester-treated cells were incubated with
0.1 jiM TPA in the absence or presence of either 0.1 jiM staurosporine or 2 jiM chelerythrine chloride for 10 min. In case of the inactive
phorbol ester, cells were preincubated with 0.1 ^xM 4-a-phorbol 12-myristate 13-acetate for 10 min. The data presented are the mean ± SEM
of 3 experiments. On average, 60 cells were analysed in each individual experiment.
180. The inhibitory action of 0.1 |uM TPA was partially
overcome with increasing of the JMV-180 concentration.
JMV-180 maximally recruited 60% of TPA (0.1 |xM) pre­
treated cells with a half-maximal stimulatory concentra­
tion of 50 nM. However, increasing of the TPA
concentration to 1 fiM completely blocked the recruit­
ment of cells by JMV-180 in 3 independent experiments.
Table 1 shows that the inhibitory effect of 0.1 |iM TPA on
cell recruitment by 10 nM JMV-180 was not observed
with the inactive phorbol ester, 4-a-phorbol 12-myristate
13-acetate (0.1 ¡nM). Moreover, Table 1 shows th at the
inhibitory action of TPA (0.1 jiM ) was completely reversed
in the presence of either staurosporine (0.1 /±M) or chel­
erythrine chloride (2 jaM), two potent inhibitors of protein
kinase C activity.
Effect of long-term TPA pretreatment on
JMV-180-evoked recruitment of CHO-CCK-A cells
Short-term (10 min) pretreatment of CHO-CCK-A cells
with 0.1 jaM TPA led to a complete loss of responsiveness
to 10 nM JMV-180 in approximately 90% of th e cells (Fig.
3A). However, an increasing number of cells regained
responsiveness to this JMV-180 concentration during
prolongation of phorbol ester pretreatment. Control
recruitment values were obtained within 1 h of TPA pre­
treatment. Surprisingly, the num ber of cells th at regained
responsiveness to JMV-180, at concentrations submaximal in terms of cell recruitment, did not increase to
preinhibitory levels (Fig. 3B). Thus, after 3 h of TPA pre­
treatment, markedly less cells responded to 0.1 nM and 1
nM JMV-180 than in the control situation and even after
24 h of phorbol ester pretreatment control values were
still not reached with these lower concentrations of the
agonist. In contrast, control values were already reached
within 3 h following the onset of TPA pretreatm ent with
a maximally effective JMV-180 concentration of 10 nM.
© Pearson Professional Ltd 1996
Agonist-evoked inositol trisphosphate formation in
CHO-CCK-A cells
In the absence of any stimulus, the Ins(l,4,5)P3 content of
CHO-CCK-A cells am ounted to 6.2 pmol/mg protein (SE
= 1.6, n = 7). Addition of 10 nM CCK8, a concentration
supramaximal in terms of cell recruitment, rapidly
increased the cellular Ins(l,4,5)P3 content to a m axim um
of 42.7 pmol/mg protein (SE = 5,8, n —6, Fig. 4). The max­
imal effect was reached at 10 s following the onset of
stimulation and the cellular Ins(l,4#5)P3 content rem ained
elevated in the continuous presence of the hormone for
at least 5 min. In contrast, 1 juM JMV-180, a concentration
also supramaximal in terms of cell recruitment, did not
significantly increase the cellular Ins(l,4#5)P3 content. The
stimulatory effect of CCK8 was clearly dose-dependent in
that the plateau level readied w ith 1 nM CCKa, a concen­
tration still supramaximal in terms of cell recruitm ent,
am ounted to 12.3 pmol Ins(l,4,5)P3/mg protein (SE = 1.2,
n = 3). Addition of 0.1 nM CCK8, being the lowest maxi­
mally effective CCK8 concentration in term s of cell
recruitment, did not result in a significant increase in cel­
lular Ins(l,4,5,)P3 content (data not shown).
Effect of TPA pretreatment on agonist-evoked inositol
trisphosphate formation in CHO-CCK-A cells
Short-term (10 min) pretreatm ent of CHO-CCK-A cells
with 0.1 jiM TPA virtually completely inhibited the
increase in cellular Ins(l,4,5)P3 content in response to 10
nM CCKg (Fig. 5). Only at 60 s following the onset of stim ­
ulation, a slight, but statistically significant, increase in
cellular Ins(l,4,5)P3 content was observed. It should be
noted, that cell recruitm ent in response to 10 nM CCK8
was not affected by short-term TPA pretreatment. Full
recoveiy from the inhibitory action of TPA was observed
within 3 h of pretreatment with TPA. Similar observations
were reached with 1 nM CCKa (data not shown).
Cell Calcium (1996) 20(1), 1-9
6 RLL Smeets, KM Gamer; M Hendriks et al
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180
240
300
Time of TPA preincubation (m in)
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10 min TPA
3 h TPA
24 h TPA
Fig. 4 Time-dependence of agonist-evoked lns(1,4,5,)P3 formation
in C H O -CCK-A cells, CHO-CCK-A cells were incubated in the
absence (closed circles) and presence of either 10 nM CCKe (open
circles) or 1 |J.M JM V-180 (open squares) for the times indicated.
Ins(1,4,5)P3 was measured by an isotope-dilution assay and
expressed In pmol/mg protein. The data presented are the mean ±
SEM of 3 or 6 (CCKe) experiments. ^Significantly different
(Scheffe’s F test) from control cells.
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Log [J M V -1 8 0 ] (M)
Fig. 3 Time-dependence of the inhibitory effect of TPA
pretreatment on JMV-180-evoked recruitment of CHO-CCK-A cells,
CHO-CCK-A cells were loaded with Fura-2 and stimulated for 5
min with JMV-180. (A) Cells were stimulated with 10 nM JMV-180
at different times following the onset of TPA (0.1 jxM) treatment.
The data presented are from a single experiment. (B) Cells were
stimulated with the indicated concentrations of JMV-180 at different
times following the onset of TPA-treatment. The data presented are
the mean ± SEM of 3 experiments. On average, 60 cells were
analysed in each individual experiment.
Phorbol ester-induced down-regulation of protein
kinase C isotypes in CHO-CCK-A cells
Western blot analysis of total protein obtained from
CHO-CCK-A cells revealed the presence of the protein
kinase C isotypes a, e and The cells did not contain
detectable amounts of the protein kinase C isotypes (3, y
and 8. Treatment of the cells with 0.1 jxM TPA rapidly
Ceil Calcium (1996) 20(1), 1-9
decreased the amount of PKC-a (Fig. 6). A 75% decrease
was observed within 1 h of TPA treatment. Virtually com­
plete disappearance of the signal (5% from the control
mean OD, SD = 3, n = 4) was obtained at 3 h following
the onset of TPA treatment. The amount of PKC-e
remained virtually unchanged (74% from the control
mean OD, SD = 23, n = 3) during the first hour of TPA
treatment but was decreased to approximately 19% (SD =
7, n = 4) of the original value in cells treated with TPA for
24 h. In contrast, the amount of PKC-£ remained virtually
unchanged during 24 h of TPA treatment. After 24 h of
TPA treatment the mean OD was 101% (SD = 10, n — 4).
DISCUSSION
The data presented are in agreement with the idea that
short-term (10 min) activation of the a-subtype of pro­
tein kinase C leads to inhibition of signaling through the
cholecystokinin-A receptor stably expressed in Chinese
hamster ovary cells. In the present study, we made use of
the ability of phorbol esters to down-regulate PKC. The
data presented clearly indicate that the rate of disappearance differed greatly between the 3 PKC isotypes demonstrated in this and other studies [19,20] to be present in
CHO cells. PKC-a was down-regulated by 95% at 3 h following the onset of phorbol ester treatment. In contrast,
PKC-e disappeared at a considerably slower rate, whereas
© Pearson Professional Ltd 1996
Inhibition of CCK-A receptor signaling by protein kinase C 7
PKC-£ was not down-regulated at all. The disappearance
of PKC-a was paralleled by the recovery from the
inhibitory action of TPA on JMV-180-evoked Ca2+ mobi­
lization, suggesting that this PKC isotype is involved in
phorbol ester-induced inhibition of Ca2+
through the CCK-A receptor. Interestingly, complete
recovery was observed only with JMV-180 concentrations
> 10 nM. As a result, the EC50 value for JMV-180-evoked
cell recruitment increased from a control value of 0.3 nM
to values of 1.6 nM and 1.3 nM at 3 h and 24 h of TPA
treatment, respectively. One explanation might be that
low-level PKC-a activation, such as expected to occur
during submaximal stimulation with JMV-180, sensitizes
rather than inhibits cells with respect to receptor-evoked
Ca2+ mobilization. A similar observation was reached in a
recent study with freshly isolated pancreatic acinar cells
[9]. Another explanation might be that prolonged TPA
treatment leads to a loss of functional receptors as a
result of receptor internalization [21],
In principle, inhibition of receptor-evoked Ca2+ mobi­
lization can occur at several levels including the CCK-A
receptor, the G-protein involved in signal transduction,
the phospholipase C involved in phosphatidylinositol
4,5-bisphosphate breakdown, the enzymes involved in
Ins(l,4,5)P3 metabolism, the Ca2+ release channels
involved in Ins(l,4,5,)P3-stimulated Ca2+ release from
internal Ca2+ stores and, finally, the mechanisms involved
in Ca2+ extrusion from the cytosolic compartment.
However, the present observation that CCKg-stimulated
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Time (h)
Fig. 6 Time-dependence of TPA-induced down-regulation of protein
kinase C isotypes in CHO-CCK-A cells. CHO-CCK-A cells were
incubated in the presence of 0.1 jaM TPA for the indicated periods
of time. Total cell lysates were subjected to SDS-polyacrylamide
gei electrophoresis followed by Western blotting. Membranes were
incubated with PKC-isotype-specific antibodies in the absence (-)
and presence (+) of the corresponding PKC-isoform-specific
peptides. PKC-specific bands were quantified densitometrically.
Note the presence of two imm uno reactive bands with anti-PKC-Ç.
The upper one of these two bands is PKC-a. In each experiment,
the mean optical density (OD) of the untreated control is set at
100% to which all other values are related, The data presented are
the mean ± SD of 3 -4 experiments.
20
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0
0
60
120
180
Time (s)
2 40
3 00
Fig. 5 Time-dependence of the inhibitory effect of TPA pretreat­
ment on CCKB-evoked ins(1,4,5,)P3 formation in CHO-CCK-A cells.
Control CHO-CCK-A cells (open circles) and CHO-CCK-A cells
preincubated in the presence of 0.1 jiM TPA for 10 min (closed
circles), 3 h (open squares) or 24 h (closed squares), were stimul­
ated with 10 nM CCKe for the indicated periods of time. Ins(1,4,5)P3
was measured by an isotope-dilution assay and expressed in
pmol/mg protein. The data presented are the mean ± SEM of 3
experiments. ^Significantly different (Scheffe’s F test) from control
cells.
©Pearson Professional Ltd 1996
Ins(l,4,5)P3 formation was significantly inhibited in TPAtreated CHO-CCK-A cells suggests that PKC exerts its
effect upstream to the process of Ins(l,4,5)P3-stimulated
Ca2+ release. It has been reported that phorbol esters can
stimulate the phosphorylation of the CCK-A receptor in
intact rat pancreatic acinar cells [14]. Moreover, recent
elucidation of the primary structure of the CCK-A recep­
tor has revealed the presence of several potential phos­
phorylation sites for PKC [5]. Taken together, most of the
information presently available is in agreement with the
idea that PKC directly phosphorylates the CCK-A recep­
tor, thus inhibiting agonist-induced Ins(l,4,5)P3 formation
Cell Calcium (1996) 20(1), 1-9
8 RLL Smeets, KM Garner, M Hendriks et a!
and subsequent Ca2+ mobilization. It should be noted
that this conclusion is not supported by the finding that,
in rat pancreatic acinar cells, TPA effectively blocked
ongoing Ca2+ oscillations evoked by the G-protein acti­
vating agent mastoparan [22]. Unfortunately however,
the CHO-CCK-A cells used in the present study did not
display a rise in [Ca2+]t in response to mastoparan (data
not shown). Furthermore, the present observation that
PKC inhibits signaling through the CCK-A receptor sta­
bly expressed in CHO cells does not favour the idea that
PKC acts indirectly through a re cep tor-specific kinase.
Evidence in support of such a mechanism has recently
been provided for the p-adrenergic receptor [23].
Comparison of the dose-response curves for the stimu­
latory effects of CCK8 on cell recruitment and Ins(l,4,5)P3
formation revealed that the hormone, at a concentration
of 0.1 nM, already maximally recruited CHO-CCK-A cells
in terms of receptor-mediated Ca2+ mobilization without
detectably increasing the cellular Ins(l,4,5)P3 content.
Significant stimulation of Ins(l,4,5)P3 formation was only
observed with CCKg concentrations > 1 nM that are
supramaximal in terms of cell recruitment. Similar results
have previously been obtained with freshly isolated rab­
bit pancreatic acinar cells [9], In contrast, JMV-180, at
concentrations supramaximal in terms of cell recruit­
ment, did not detectably stimulate the production of
Ins(l,4,5)P3 in CHO-CCK-A cells. Similarly, JMV-180 has
been demonstrated to poorly stimulate Ins(l,4,5)P3 for­
mation in pancreatic acinar cells [24,25], Taken together,
these observations suggest that Ins(l,4,5)P3 is either
overproduced during supramaximal stimulation with
CCKS or not involved in Ca2+ mobilization during submaximal stimulation with CCKa or stimulation with JMV180. Hie latter explanation implies the presence of an
alternative messenger system in both pancreatic acinar
cells and CHO cells. However, recent experiments have
provided substantial evidence that JMV-180 does act
through the intermediation of Ins(l,4,5)P3 to stimulate
the release of Ca2+ from intracellular stores [26-28],
Unlike in pancreatic acinar cells [9], short-term (10 min)
TPA pretreatment dramatically reduced Ins(l;4,5)P3 for­
mation in response to CCK8 concentrations supramaxi­
mal in terms of cell recruitment, Surprisingly, this
reduction did not lead to any decrease in the number of
cells recruited by these supramaximal CCK8 concentra­
tions, indicating that Ins(l,4,5)P3 is produced in sufficient
amounts to promote the release of Ca2+ from internal
stores.
In contrast to freshly isolated pancreatic acinar cells
[8], CHO-CCK-A cells responded with a sustained
increase in Ins(l,4,5)P3, suggesting that either Ins(l,4,5)P3
production continues at a high rate or Ins(l,4,5)P3 metab­
olism occurs at a reduced rate in CHO-CCK-A cells. In
case Ins(l,4,5)P3 production continues at a high rate, this
Ceii Calcium (1996) 20(1), 1-9
might suggest that CHO-CCK-A cells lack a receptor-spe­
cific kinase which rapidly desensitizes the receptor lead­
ing to a drop in Ins(l,4,5)P3 production rate.
PKC-mediated inhibition of signaling through the
CCK-A receptor appeared to be restricted to CCK8 con­
centrations at which the hormone submaximally stimu­
lated the process of cell recruitment in terms of
receptor-mediated Ca2+ mobilization and was completely
overcome with increasing of the CCK8 concentration. In
contrast, inhibition of the stimulatory effect of JMV-180,
which acts as an agonist at the high-affinity CCK-A
receptor, was not overcome with increasing of the ago­
nist concentration. Similar observations, reached with
freshly isolated rabbit pancreatic acinar cells, have led to
the hypothesis that PKC activation results in inhibition
of signaling through the high-affinity CCK-A receptor
[8,9]. Moreover, from the observation that Ins(l,4,5)P3
formation in response to the higher CCK8 concentrations
was not inhibited by TPA, it was concluded that PKC did
not affect signaling through the low-affinity CCK-A
receptor. However, the present study clearly demon­
strates the ability of TPA to significantly inhibit
Ins(l,4,5)P3 formation in response to CCKg acting
through the low-affinity receptor in CHO-CCK-A cells.
This observation suggests that in CHO cells, in contrast
to pancreatic acinar cells, the low-affinity CCK-A recep­
tor is affected in a PKC-dependent manner.
The EC50 value for the stimulatory effect of CCKS on
the recruitment of CHO-CCK-A cells was calculated to
be 0.8 pM. This value is 20-fold lower as compared to the
value of 16.8 pM obtained with rabbit pancreatic acinar
cells [9], Similarly, the ECgo value for the stimulatory
effect of JMV-180 on the recruitment of CHO-CCK-A
cells, calculated to be 0.3 nM, is 30-fold lower as com­
pared to the value of 9 nM obtained with rabbit pancre­
atic acinar cells (R.L.L. Smeets, K.M, Garner, JJ.H.H.M. de
Pont and P.H.G.M. Willems, unpublished observations).
Moreover, JMV-180 maximally recruited 100% of the
CHO-CCK-A cells, whereas in the case of rabbit pancre­
atic acinar cells this value amounted to 70%. These
observations demonstrate that, for hitherto unknown
reasons, CHO-CCK-A cells are more sensitive to stimula­
tion by CCK8 than freshly isolated rabbit pancreatic aci­
nar cells. Thus far, the mechanism underlying the
dose-dependent recruitment of cells in terms of agonistinduced Ca2+ mobilization is unclear. Although it can be
argued that the differences in sensitivity observed within
a population of freshly isolated cells are the result of
receptor damaging during the isolation procedure, this is
not likely to be the case with the CHO-CCK-A cells used
in this study.
In summary, the data presented are in agreement with
the idea that high-level PKC-a activation by TPA inhibits
signaling through the CCK-A receptor in CHO-CCK-A
© Pearson Professional Ltd 1996
Inhibition of CCK-A receptor signaling by protein kinase C
cells at the level of receptor-stimulated Ins(l,4,5)P3 for­
mation. It remains to be elucidated whether PKC-a acts
directly at the receptor protein.
ACKNOWLEDGEMENTS
The research of Dr P.H.G.M. Willems has been made pos­
sible by a fellowship of the Royal Netherlands Academy
of Aits and Sciences.
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