CCK-B receptors produce similar signals but have
opposite growth effects in CHO and Swiss 3T3 cells
KATHARINA DETJEN, DAVID YULE, MIN-JEN TSENG,
JOHN A. WILLIAMS, AND CRAIG D. LOGSDON
Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622
cholecystokinin; cholecystokinin-B receptors; deoxyribonucleic acid synthesis; intracellular calcium; phosphoinositol
hydrolysis; adenosine 38,58-cyclic monophosphate; arachidonic acid release; Chinese hamster ovary cells
tion in the stomach and intestine (16). Gastrin also has
been reported to stimulate cell proliferation in tissue
culture cell models transfected with CCK-B receptors
(15, 26) and a variety of cancer cell lines, including
pancreatic (30), gastric (13), colonic (29), and small
lung tumor (10). In contrast, we recently observed that
activation of exogenously expressed CCK-B receptors
can inhibit the growth of MIA PaCa-2 and PANC-1
human pancreatic cancer cell lines (4). Therefore, it
seems apparent that activation of these receptors can
influence cell growth either positively or negatively
depending on the cell context. However, it is unclear
whether these cell-specific differences in growth responses are due to differences in receptor coupling or to
other mechanisms intrinsic to the different cells.
To develop model systems for investigating mechanisms involved in growth effects of gastrin, we transfected rat CCK-B receptors into Chinese hamster ovary
(CHO) and Swiss 3T3 cells, both of which have been
widely utilized for receptor expression and growth
regulation studies. The effects of CCK-B receptor activation on both growth and second messenger generation
were then determined in these two cell models. We
found that activation of CCK-B receptors inhibited the
growth of CHO cells. In contrast, activation of CCK-B
receptors stimulated the growth of Swiss 3T3 cells. The
inhibitory effect observed in CHO cells did not require
overexpression of receptors, as it occurred in CHO cells
bearing fewer receptors than were present in the Swiss
3T3 cells. In either cell type, CCK-B receptors coupled
to increases in [Ca21]i, phosphoinositide hydrolysis,
arachidonate release, and, to a lesser extent, adenosine
38,58-cyclic monophosphate (cAMP) formation. Thus
cellular context is a major determinant of the biological
effects of CCK-B receptor activation, and differences in
growth responses may occur independently of differences in receptor coupling.
EXPERIMENTAL PROCEDURES
mediate both short- and
long-term actions in a variety of gastrointestinal tissues. Gastrin activates cholecystokinin-B (CCK-B) receptors, which are classic seven-transmembrane-spanning receptors coupled to heterotrimeric G proteins
(33). Gastrin acts in the short term as a secretagogue on
gastric parietal cells (2), and the mechanisms involved
in this secretory effect have been well studied. CCK-B
receptor effects on secretion are believed to be due to
Gq-mediated activation of phosphoinositide-specific
phospholipase C, leading ultimately to increases in
intracellular Ca21 concentration ([Ca21]i ) (2, 3, 34).
Gastrin acts in the long term to stimulate cell proliferaGASTRIN HAS BEEN SHOWN TO
Materials
125I-labeled
cholecystokinin octapeptide (CCK-8), 125IcAMP radioimmunoassay kits, and [3H]arachidonate (100
Ci/mmol) were obtained from DuPont NEN (Boston, MA).
125I-cAMP radioimmunoassay kits were also obtained from
Diagnostic Products (Los Angeles, CA). myo-[3H]inositol (19.1
Ci/mmol), [3H]thymidine, and the ‘‘cell proliferation kit’’ were
obtained from Amersham (Arlington Heights, IL). Fura 2acetoxymethyl ester (fura 2-AM) and fura 2 free acid were
purchased from Molecular Probes (Eugene, OR). Analytical
grade 1-X8 resin (AG 1-X8, 100–200 mesh) and protein assay
reagent were obtained from Bio-Rad (Rockville Center, NY).
Restriction endonucleases were purchased from GIBCO
(Grand Island, NY). Sulfated CCK-8 was purchased from
0363-6143/97 $5.00 Copyright r 1997 the American Physiological Society
C1449
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on March 31, 2017
Detjen, Katharina, David Yule, Min-Jen Tseng, John
A. Williams, and Craig D. Logsdon. CCK-B receptors
produce similar signals but have opposite growth effects in
CHO and Swiss 3T3 cells. Am. J. Physiol. 273 (Cell Physiol.
42): C1449–C1457, 1997.—Rat cholecystokinin-B (CCK-B)
receptors were transfected into Chinese hamster ovary (CHO)K1 (CHO-CCK-B) and Swiss 3T3 (Swiss 3T3-CCK-B) cells,
and the effects of receptor activation on cell proliferation and
intracellular signaling were investigated. CCK octapeptide
(CCK-8) treatment had no effect on cell growth in quiescent
CHO-CCK-B cells but inhibited DNA synthesis, proliferation,
and colony formation when the cells were grown in fetal
bovine serum (FBS). In contrast, CCK-8 stimulated DNA
synthesis in quiescent Swiss 3T3-CCK-B cells and had no
effect when the cells were grown in FBS. These differences in
growth responses were not due to differences in the level of
receptor expression, as similar numbers of receptors were
present in both cell types. To determine whether the different
growth effects were due to differences in receptor coupling to
common second messenger pathways, we investigated the
effects of CCK-8 on several known intracellular signals. In
both cell types, CCK-8 stimulated increases in intracellular
Ca21 concentration and polyphosphoinositide hydrolysis with
similar potencies and efficacies. CCK-8 also stimulated arachidonate release from both cell types, although the potency
was higher in the CHO cells. Adenosine 38,58-cyclic monophosphate generation was observed at high agonist concentrations in both cell types and was much greater in cells with
higher receptor density. In summary, receptor activation had
opposite effects on growth parameters in CHO and Swiss 3T3
cells, but only minor differences were observed in the characteristics of CCK-B receptor coupling to specific second messengers in the two cell types. Thus cellular context is a principal
determinant of the biological effects of CCK-B receptor activation, and differences in biological responses may occur independently of major differences in receptor coupling.
C1450
CELL TYPE DEPENDENCE OF CCK-B RECEPTOR FUNCTION
Bachem (Torrance, CA). Trichloroacetic acid was purchased
from J. T. Baker (Phillipsburg, NJ). Dulbecco’s modified
Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and amphotericin B were obtained from
GIBCO. Tissue culture plasticware (24- and 6-well plates and
10-cm petri dishes) were obtained from Sarstedt (Newton,
NC). CHO-K1 cells were obtained from the American Type
Culture Collection (Rockville, MD), and Swiss 3T3 cells were
obtained from Dr. G. Johnson (National Jewish Center for
Immunology and Respiratory Medicine, Denver, CO). Soybean trypsin inhibitor type I-S, bovine serum albumin fraction V, and all other chemicals were obtained from Sigma
Chemical (St. Louis, MO).
Generation and Characterization of CCK-B
Receptor-Expressing Cell Lines
Analysis of Second Messenger Generation
Measurement of [Ca21]i. Analysis of [Ca21]i changes were
conducted on cells grown to 30–50% confluence on glass
coverslips. Cells were incubated with 5 µM fura 2-AM at 37°C
for 30 min and then washed in HR buffer. Coverslips containing cells were transferred to a closed chamber, mounted on
the stage of a Zeiss Axiovert inverted microscope, and continuously superfused at 1 ml/min with HR buffer at 37°C.
Measurement of emitted fluorescence and calibration of these
signals to yield a measurement of [Ca21]i were performed
using an Attofluor digital imaging system (Rockville, MD),
exactly as described previously (36).
Measurement of [3H]inositol phosphates. For measurement
of total inositol phosphates, cells were cultured in 24-well
dishes at 1 3 105 cells/well for 24 h in the presence of 3 µCi of
myo-[3H]inositol. The cells were then washed twice with HR
buffer containing 10 mM Li1. Incubation with various concentrations of CCK-8 was conducted in the same buffer for 30
min and terminated by addition of an equal volume of 20%
ice-cold trichloroacetic acid (TCA). After centrifugation at
Analysis of Cell Growth Parameters
Measurement of DNA synthesis. DNA synthesis was estimated by measurement of [3H]thymidine incorporation into
TCA-precipitable material. For studies in exponentially growing CHO cells, cells cultured in 5% FBS were treated for 8 h
with the indicated concentrations of CCK-8, and during the
last 1 h of the treatment period [3H]thymidine was added to
each well (final concentration, 1 µCi/ml). In experiments
utilizing quiescent CHO cells, the cells were serum starved
for 24 h and then treated with CCK-8 (10 nM) for 24 h and
exposed to [3H]thymidine for the last 6 h. For studies in Swiss
3T3 cells, cells were made quiescent by incubation in serumfree medium supplemented with 10% conditioned medium
overnight. Cells were then treated with the indicated concentrations of CCK-8 for 24 h and exposed to [3H]thymidine (0.5
µCi/ml) during the final 2 h. After labeling, cells were washed
twice with PBS and then twice with TCA (final concentration,
6%) and removed by dissolving in 0.3 ml of 0.1 N NaOH.
Radioactivity was measured by liquid scintillation counting.
Nuclear labeling. Nuclear labeling was investigated using
an Amersham cell proliferation kit that identifies cells synthesizing DNA by detecting incorporation of 5-bromo-28-deoxyuridine (BrdU). For determination of effects on cells growing in
serum, either CHO or Swiss 3T3 cells expressing CCK-B
receptors were treated with CCK-8 (10 nM) for 8 h, and BrdU
was added during the last 1 h. For determination of effects on
quiescent cells, cells were serum starved for either 24 h
(CHO) or 36 h (Swiss 3T3). Cells were then treated with
CCK-8 (10 nM) for 24 h (CHO) or 40 h (Swiss 3T3), and BrdU
was added for the last 24 h. Subsequently, cells were fixed
with acidic methanol and BrdU-labeled nuclei were detected,
following the manufacturer’s directions.
Analysis of cell numbers. Cells were plated at a density of
20,000 cells/well into 24-well dishes in DMEM with 5% FBS
and allowed to attach overnight. Cells were then treated for
24 h by addition of the indicated concentrations of CCK-8 and
subsequently removed from the dish by gentle trypsin treatment. Cells were counted using a Coulter counter.
Analysis of colony formation in soft agar. Colony formation
was assayed as previously described (5). Cells were plated at
1,000 cells/dish into 35-mm dishes in agar (0.3% in DMEM
supplemented with 5% FBS) and treated with the indicated
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Construction of CCK-B receptor expression vector and development of stable cell lines. A full-length cDNA encoding the
rat CCK-B (33) receptor was kindly provided by S. A. Wank
(National Institutes of Health, Bethesda, MD). The receptor
cDNA was subcloned into the TEJ8 expression vector as
previously described (36). CHO-K1 and Swiss 3T3 cells were
routinely cultured in DMEM supplemented with 5% FBS in a
humidified atmosphere of 5% CO2. For transfection, cells
were grown to 30–40% confluence in 60-mm dishes and
transfected with 3 µg of Pvu I-linearized plasmid DNA using
lipofectin reagent (GIBCO-BRL). G-418-resistant colonies
were selected and screened for CCK-B receptors by binding of
125I-CCK-8. Two or more clones were tested in all assays to
control for clonal variation.
Measurement of CCK-B receptor binding. Ligand binding
was carried out using cells plated in 24-well dishes at 1 3 105
cells/well the day before the binding assay. For binding
assays, 125I-CCK-8 (10 pM) was added to N-2-hydroxyethylpiperazine-N8-2-ethanesulfonic acid (HEPES)-Ringer buffer (HR
buffer) that contained (in mM) 137 NaCl, 4.7 KCl, 0.56 MgCl2,
1 Na2PO4, 1.28 CaCl2, 10 HEPES (pH 7.4), supplemented
with 0.1 mg/ml soybean trypsin inhibitor. Cells were incubated to equilibrium (18 h at 4°C) and then washed twice with
ice-cold phosphate-buffered saline (PBS). The cells were then
scraped and counted in a gamma counter. Nonspecific binding
was determined in the presence of 100 nM CCK-8. Protein
contents were determined on samples after counting. Binding
affinity and capacity were calculated using the LIGAND
analysis program (21).
2,000 g for 20 min, 0.9 ml of each supernatant was washed
twice with water-saturated diethyl ether, neutralized with
100 µl of KHCO3, and diluted with 2.5 ml of water. Analysis of
total [3H]inositol phosphates was carried out by the method
described by Berridge (1).
Measurement of [3H]arachidonic acid release. Measurement of arachidonic acid release was conducted as previously
described (36). Briefly, cells grown to near confluence in
six-well plates were incubated with 1 µCi of [3H]arachidonic
acid for 24 h. The cells were then washed with PBS and
incubated in HR buffer with or without CCK-8 (10 nM) for 30
min. The incubation medium was then removed and counted
in a scintillation counter.
Measurement of cAMP production. Measurement of cAMP
generation was conducted as previously described (36). CHO
and Swiss 3T3 cells grown in six-well dishes were incubated
with indicated concentrations of CCK-8 for 1 h in HR containing 1 mM 3-isobutyl-1-methylxanthine. The incubation was
stopped by addition of 20% ice-cold TCA, and then cells were
scraped from the plate. The TCA was neutralized, and the
cAMP was extracted and assayed by radioimmunoassay using
kits purchased from DuPont NEN (NEK-033) or Diagnostic
Products, following the manufacturers’ recommendations.
CELL TYPE DEPENDENCE OF CCK-B RECEPTOR FUNCTION
C1451
Table 1. Characteristics of CCK-B
receptor-expressing cell lines
CHO-CCK-B cells
Higher density
Lower density
Swiss 3T3-CCK-B cells
Kd , nM
Bmax , fmol/mg protein
0.25 6 0.03
0.20 6 0.06
0.24 6 0.01
852 6 72
329 6 41
462 6 81
concentrations of CCK-8. After 2 wk, the dishes were examined and all colonies .0.5 mm in diameter were counted.
RESULTS
Effects of CCK-B Receptor Activation on Cell Growth
To test whether CCK-B receptors would stimulate
the growth of Swiss 3T3 cells, the cells were transfected
with the rat CCK-B receptor. Swiss 3T3 cells bearing
CCK-B receptors bound ligand with an affinity similar
to what has been reported previously in native cells
(Table 1). When CCK-B receptor-bearing Swiss 3T3
cells growing exponentially in FBS were treated with
CCK-8, there was no effect on DNA synthesis as
measured by [3H]thymidine incorporation (data not
shown). Likewise, no change in nuclear labeling was
observed in the presence of FBS (Fig. 1). In contrast,
stimulation of quiescent Swiss 3T3-CCK-B cells with
CCK-8 (100 nM) led to a fourfold increase in the rate of
DNA synthesis (Fig. 2). This increase in [3H]thymidine
incorporation also corresponded to an increase in
nuclear labeling (Fig 1). The stimulatory effect was
Fig. 2. Effects of CCK-8 on DNA synthesis in Swiss 3T3 cells
expressing CCK-B receptors. Cells were serum starved for 24 h and
then treated with indicated concentrations of CCK-8 for an additional
24 h. Cells were exposed to [3H]thymidine for final 2 h, and
trichloroacetic acid-precipitable incorporation was determined. Data
are means 6 SE for 3 independent determinations, presented as
percentage of control incorporation.
concentration dependent, with significant stimulation
noted at 0.1 nM, half-maximal effects at 2.0 6 0.8 nM
(n 5 4 experiments), and maximal effects at 10 nM
CCK-8 (Fig. 2). Wild-type Swiss 3T3 cells showed no
growth response, either stimulatory or inhibitory, to
CCK-8 stimulation (data not shown).
We previously found that activation of CCK-A receptors could inhibit the growth of CHO cells (5). In the
current study, we examined the effects of activation of
CCK-B receptors in CHO cells. CHO cells expressing
rat CCK-B receptors bound ligand with an affinity
similar to Swiss 3T3 cells (Table 1). In contrast to the
responses observed in the Swiss 3T3 cells, in CHO cells
activation of CCK-B receptors led to a decrease in DNA
Fig. 1. Effects of cholecystokinin octapeptide (CCK-8) on nuclear labeling in Chinese hamster ovary (CHO) and
Swiss 3T3 cells expressing cholecystokinin-B (CCK-B) receptors. Cells either serum starved (2FBS) or grown in
presence of 5% fetal bovine serum (1FBS) were treated with (1CCK) or without (2CCK) CCK-8 (10 nM), and nuclei
undergoing DNA synthesis were detected by 5-bromo-28-deoxyuridine labeling. Shown are representative fields
from 1 of 3 similar experiments.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on March 31, 2017
Values are means 6 SE of 3–6 experiments. Competitive binding
analysis was conducted with cells cultured to near confluence in
24-well dishes. For binding assays, cells were incubated to equilibrium (4 h at 24°C) in presence of 125I-labeled cholecystokinin octapeptide (CCK-8; 10 pM). Cells were then washed, scraped, and counted in
a gamma counter. Nonspecific binding was determined in presence of
100 nM CCK-8. Protein contents were determined from samples after
counting. Binding affinity (Kd ) and capacity (Bmax ) were calculated
using LIGAND analysis program (21). CHO, Chinese hamster ovary;
CCK-B, cholecystokinin-B.
C1452
CELL TYPE DEPENDENCE OF CCK-B RECEPTOR FUNCTION
synthesis (Fig. 3A). This decrease was detectable at 10
pM, inhibition was half-maximal at 0.07 6 0.01 nM
(n 5 4), and a maximal inhibition of 58 6 4% of control
(n 5 4) was observed at 3 nM CCK-8. The observed
inhibition of [3H]thymidine incorporation corresponded
to a reduction in the nuclear labeling of cells cultured in
the presence of serum and CCK-8 (Fig. 1). When
Effects of CCK-B Receptor Activation on Signal
Transduction Pathways
Activation of CCK-B receptors in native cells has
been linked to stimulation of a variety of intracellular
messenger pathways, including an increase in [Ca21]i
(3). This is similar to what has been observed for
CCK-A receptors, for which it is well documented that
this increase involves the release of Ca21 from intracellular pools followed by the influx of extracellular Ca21
(34, 36). For CCK-A receptors, the temporal pattern of
[Ca21]i signaling has been shown to depend on the
concentration of agonist applied, such that at low
Fig. 3. A: effects of CCK-8 on CCK-B receptor-bearing CHO cell DNA
synthesis. CHO cells bearing a higher receptor density (j) or a lower
receptor density (m) were grown in presence of 5% FBS and were
treated with indicated concentrations of CCK-8 for 8 h and exposed
for 1 h to [3H]thymidine, and incorporation into DNA was analyzed.
Results are means 6 SE for 4 experiments, expressed as percentage
of control nontreated cell DNA synthesis. B: effects of CCK-8 on
CCK-B receptor-bearing CHO cell proliferation. Cells were grown in
presence of 5% FBS and of indicated concentrations of CCK-8. After
24 h, cells were counted using a Coulter counter. Results are
expressed as percentage of increase over number of cells plated and
represent 4 separate experiments conducted in quadruplicate. C:
dose dependence of CCK-8-induced inhibition of CHO cell colony
formation in soft agar. Cells were plated at 1,000 cells/dish into
35-mm dishes in agar and treated with indicated concentrations of
CCK-8. After 2 wk, dishes were examined and all colonies .0.5 mm in
diameter were counted. Results are means 6 SE for 4 separate
experiments conducted in triplicate, expressed as a percentage of
colonies formed in control dishes.
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CHO-CCK-B cells were first made quiescent by serum
starvation and then treated with CCK-8, no stimulatory effect was noted at any concentration between 0.01
and 100 nM CCK-8, and at 100 nM the [3H]thymidine
incorporation was 83 6 9% of control (n 5 3). Likewise,
no effect was observed on nuclear labeling under these
conditions (Fig. 1). Effects of CCK-8 treatment on CHO
cell numbers in exponentially growing cell populations
cultured in the presence of 5% FBS were also examined
(Fig. 3B). CCK-8 activation of CCK-B receptors led to
an inhibition of CHO-CCK-B cell proliferation. The
inhibition of cell proliferation was concentration dependent, with half-maximal inhibition occurring with 2.4 6
0.3 nM CCK-8 and a maximal 92 6 8% (n 5 3) inhibition observed with 100 nM CCK-8.
Activation of CCK-A receptors on CHO cells was
previously shown to decrease both anchorage-dependent and anchorage-independent growth, and these
effects were mediated by separate receptor affinity
states (5). To examine the effects of CCK-B receptor
activation on CHO cell anchorage-independent growth,
CHO-CCK-B cells were plated into soft agar in the
presence of various concentrations of CCK-8 and cultured for 2 wk. Analysis of the number of colonies 0.5
mm in diameter or larger indicated a concentrationdependent decrease in colony formation (Fig. 3C).
Significantly lower numbers of colonies were noted in
cultures treated with 0.1 nM CCK-8, half-maximal
inhibition was observed at 1.0 6 0.5 nM (n 5 6), and a
maximal inhibition to 7 6 5% (n 5 6) of control was
observed at 10 nM CCK-8.
CELL TYPE DEPENDENCE OF CCK-B RECEPTOR FUNCTION
tration-dependent manner (Fig 5A). The potency of
stimulation of polyphosphoinositide release was similar for both cell types (Table 1). An increase could be
detected at 300 pM, was half maximal at ,1 nM, and
reached a maximum at 10 nM (Fig. 5A, Table 2). The
maximal effect was slightly larger in CHO cells with a
higher receptor density. However, no difference was
noted between the efficacy of CCK-8 in Swiss 3T3 cells
and CHO cells with a lower receptor density (Fig. 5A).
Arachidonic acid is another potentially important
intracellular messenger. The effects of CCK-B receptor
activation on arachidonate formation in natively expressing cells are unknown. In the current study,
CCK-8 led to a similar increase in arachidonate release
in CHO-CCK-B cells and Swiss 3T3-CCK-B cells that
were prelabeled with [3H]arachidonate (Fig. 5B). The
potency of CCK-8 was greater in the CHO cells bearing
either high or low receptor densities, compared with
the Swiss 3T3 cells.
Activation of CCK-B receptors on gastric parietal
cells has been reported not to increase the intracellular
concentration of cAMP (2). In the current study, CCK-8
activation of CCK-B receptors was able to induce a
large accumulation of cAMP in CHO cells bearing high
numbers of receptors and a small increase in Swiss 3T3
cells (Fig. 5C). However, no significant effect was noted
in CHO cells with a low receptor density.
Fig. 4. Effects of CCK-8 on intracellular Ca21
concentration ([Ca21]i ) signaling in CHO and
Swiss 3T3 cells bearing CCK-B receptors.
Effects of different concentrations (A and C: 50
pM; B and D: 1 nM) of CCK-8 on [Ca21]i in
individual Swiss 3T3-CCK-B cells (A and B)
or CHO-CCK-B cells (C and D). Each trace is a
recording from a representative cell; similar
data were observed in at least 3 experiments,
in each of which .10 cells were imaged.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on March 31, 2017
physiological concentrations of agonists oscillations of
[Ca21]i are seen, whereas at maximal doses a single
large transient peak followed by a much smaller plateau is often observed. In the current study, superfusion of either CHO-CCK-B or Swiss 3T3-CCK-B cells
with CCK-8 at low concentrations (25 pM–0.5 nM)
predominantly resulted in an oscillatory [Ca21]i signal
(Fig. 4). At concentrations above 0.5 nM, a single
transient peak in [Ca21]i followed by a small plateau
was observed. In CHO-CCK-B cells, a maximal concentration of 1 nM increased [Ca21]i from basal values of
78.2 6 9 nM (4 experiments, 178 cells) to a maximal
peak value of 244 6 37 nM (4 experiments, 34 cells). In
Swiss 3T3-CCK-B cells the basal [Ca21]i was 92.1 6 8.2
nM (8 experiments, 120 cells), and stimulation with
CCK-8 increased [Ca21]i to a maximum peak value of
428 6 43 nM (3 experiments, 27 cells). Thus there were
no qualitative differences between the effects of CCK-8
on [Ca21]i in the two cell types.
Activation of CCK-B receptors has previously been
reported to increase polyphosphoinositide hydrolysis in
guinea pig chief cells (22). In the current study, the
effects of activation of CCK-B receptors on formation of
[3H]inositol phosphates were measured in CHO-CCK-B
and Swiss 3T3-CCK-B cells. In both cell types labeled
for 24 h with myo-[3H]inositol, CCK-8 increased the
formation of total [3H]inositol phosphates in a concen-
C1453
C1454
CELL TYPE DEPENDENCE OF CCK-B RECEPTOR FUNCTION
DISCUSSION
In this study we observed that the cloned rat CCK-B
receptor coupled to signal transduction pathways in a
qualitatively similar manner in CHO and Swiss 3T3
cells. Agonist activation of these receptors potently
increased phosphoinositide hydrolysis, [Ca21]i, and
arachidonate release. cAMP formation could also be
detected in both cell types, but it required high concentrations of agonist and was much greater in cells
bearing higher receptor densities. Quantitative responses to agonist in the two cell lines varied somewhat
Fig. 5. Effects of CCK-8 on signaling molecule generation in CHO or
Swiss 3T3 cells bearing CCK-B receptors (CHOhigh and CHOlow, CHO
clones with high and low CCK-B receptor densities, respectively). A:
effects of CCK-8 on total phosphoinositide hydrolysis. Confluent cells
were incubated with myo-[2-3H]inositol for 24 h, after which they
were exposed to indicated concentrations of CCK-8 for 30 min.
Isolation of total [3H]inositol phosphates was performed as detailed
in EXPERIMENTAL PROCEDURES. Data are expressed as multiples of
basal inositol phosphate content and are means 6 SE of 3–5 separate
experiments, with each value measured in triplicate in each experiment. B: effects of CCK-8 on release of arachidonate. Cells were
grown to near confluence in 6-well plates and then incubated with 1
µCi of [3H]arachidonic acid for 24 h. Cells were then washed with
phosphate-buffered saline and incubated in HEPES-Ringer buffer
with indicated concentrations of CCK-8 for 30 min. Incubation
medium was then removed and counted in a scintillation counter.
Data are expressed as multiples of basal arachidonate release and
are means 6 SE of 3–6 separate experiments, with each value
measured in triplicate in each experiment. C: effects of CCK-8 on
cAMP generation. Cells were grown to near confluence in 6-well
plates. Cells were then stimulated with indicated concentrations of
CCK-8 for 30 min. Incubation medium was then removed, cells were
solubilized, and cAMP was extracted and measured as described in
EXPERIMENTAL PROCEDURES. Data are expressed as multiples of basal
value and are means 6 SE of 3–4 separate experiments, with each
value measured in triplicate in each experiment.
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in terms of either efficacy or potency, depending on the
specific pathway examined. However, these differences
were not dramatic. In comparison to these minor
quantitative differences, there were major differences
in the functional responses in the two cell types. CCK-B
receptor activation stimulated the growth of Swiss 3T3
cells and inhibited the growth of CHO cells. These data
illustrate the cell type dependence of receptor signaling
and function.
Exact quantitative comparisons between second messenger production in the two different cell types can be
complicated by a variety of issues, including differences
in receptor numbers. Two CHO cell clones were utilized
in this study. One expressed significantly greater numbers of CCK-B receptors than did the Swiss 3T3 cell
line, and the other expressed approximately equivalent
numbers. Previous studies focused on the influence of
receptor number on coupling to cellular signaling suggest that increased receptor number generally leads to
increased efficacy and, to a lesser extent, increased
potency (8, 32). Due to the phenomenon of ‘‘spare
receptors,’’ which describes the situation when occupation of only a small fraction of receptors is sufficient to
elicit a full biological response, it is often possible to
achieve the same maximal response in cells bearing
multifold different receptor numbers. In the current
study, CCK-8 activation of CHO and Swiss 3T3 cells
resulted in approximately equivalent maximal effects
on [Ca21]i, phosphoinositol hydrolysis, and arachidonate release, despite some variation in receptor number.
This supports the hypothesis that there are significant
spare receptors in terms of coupling to these pathways.
In contrast, the maximal increase in intracellular
concentrations of cAMP was significantly less in Swiss
3T3 cells and CHO cells bearing fewer CCK-B receptors
than in CHO cells bearing a greater number of CCK-B
receptors. This is similar to what has been reported for
b2-adrenergic receptors expressed in CHO cells, in
C1455
CELL TYPE DEPENDENCE OF CCK-B RECEPTOR FUNCTION
Table 2. Coupling of CCK-B receptor activation to second messenger generation in CHO and Swiss 3T3 cells
Phosphoinositide Hydrolysis
CHO-CCK-B cells
Higher density
Lower density
Swiss 3T3-CCK-B cells
Arachidonate Release
cAMP Generation
EC50 ,
nM
Maximum,
multiples of basal value
EC50 , nM
Maximum,
multiples of basal value
EC50 , nM
Maximum,
multiples of basal value
0.9 6 0.2
0.4 6 0.1
0.7 6 0.1
8.9 6 0.4*†
7.8 6 0.3‡
7.0 6 0.4‡
0.3 6 0.2*
0.1 6 0.1*
2.5 6 0.2†‡
2.8 6 0.2
2.0 6 0.2‡
3.0 6 0.1
2.0 6 1.0
ND
ND
18.9 6 3.1*†
1.3 6 0.1‡
2.2 6 0.4‡
Values are means 6 SE for 3–5 experiments. Concentration dependence data for receptor coupling to various second messengers were
analyzed by nonlinear regression, using GRAPHPAD analysis software. EC50 , 50% effective concentration; ND, not determined. Statistically
significant differences (P . 0.05) * vs. Swiss 3T3 cells; † vs. lower receptor density CHO cells; ‡ vs. higher receptor density CHO cells.
In vivo, gastrin stimulates gastric and intestinal
proliferation (16) and CCK stimulates pancreatic proliferation (31). Potentially, these in vivo effects may
involve secondary release of growth regulatory substances, because both CCK and gastrin are known to
increase the secretion of other bioactive molecules.
However, evidence that direct activation of the CCK-A
receptor mediates the effects of CCK on pancreatic
acinar cell growth comes from the observations that
CCK stimulates growth of cultured rat (11) and mouse
(19) acinar cells in vitro. This evidence is compelling, as
these cultures are highly enriched in acinar cells, and
in these species pancreatic acinar cells do not express
CCK-B receptors (33). In vitro, trophic actions of gastrin have been reported in primary cultures of gastrointestinal cells (20). However, these are mixed cultures
consisting of several cell types, so that it is difficult to
rule out the possibility of secondary effects. Direct
trophic effects of gastrin have also been reported in a
variety of pancreatic (30), colonic (29), gastric (13), and
small lung tumor cells (25). Recently, however, it has
become unclear whether the effects of gastrin on the
growth of cancer cell lines are mediated through a
classic CCK-B receptor or involve non-A, non-B CCK
receptors such as those reported in pancreatic acinar
AR42J cells (27) or Swiss 3T3 cells (28). More direct
evidence for trophic actions of the CCK-B receptor
comes from studies using transfected cell lines (15, 26).
Therefore, there is little doubt that both CCK-B and
CCK-A receptors can directly stimulate the growth of
certain cells.
Likewise, both CCK-B and CCK-A receptors have
been shown to inhibit the growth of specific cells.
Activation of endogenous CCK receptors has been
reported to inhibit the growth of lymphocytes (7), a
transplantable rat pancreatic carcinoma (17), and two
human gastrointestinal cancers (12). Activation of exogenously expressed CCK-B or CCK-A receptors inhibited
growth of two pancreatic cancer cell lines (4), and
activation of either CCK-B (current study) or CCK-A (5)
receptors inhibits the growth of CHO cells. Thus the
growth effects of CCK-B and CCK-A receptors do not
appear to differ on the basis of receptor structure but
are determined by the cell context.
The mechanisms involved in the stimulatory or inhibitory growth effects observed in the present study are
unknown. We previously observed inhibition of CHO
cell growth on activation of CCK-A and m3 muscarinic
cholinergic receptors (5, 6). Thus inhibition of CHO cell
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on March 31, 2017
which, over a 200-fold range of receptor expression,
transfectants displayed increasing efficacy of agoniststimulated cAMP accumulation in response to agonist
(8). These results suggest that, in terms of coupling to
Gs and cAMP generation, there are no spare CCK-B
receptors. The potency of CCK-8 stimulation of [Ca21]i
and phosphoinositol hydrolysis was approximately
equivalent for both cell types. However, the potency of
CCK-8 for arachidonate release was much greater in
the CHO cells bearing either high or low receptor
levels, compared with the Swiss 3T3 cells. Thus both
the receptor expression level and the cellular context
were important determinants of receptor coupling to
specific signaling pathways.
Previously, we reported that CCK-A receptors expressed in CHO cells also coupled to increased phosphoinositide hydrolysis, [Ca21]i, arachidonate release, and
cAMP (36). Comparing the two studies, we found no
major differences between the coupling of CCK-A and
CCK-B receptors in CHO cells. CCK-B receptors have
also been shown to increase [Ca21]i and polyphosphoinositide hydrolysis in natively receptor-expressing gastric cells (2, 3). In contrast, in guinea pig pancreatic
acini, which endogenously possess both CCK-A and
CCK-B receptors, only occupation of the CCK-A receptor has been reported to stimulate activation of phospholipase C (35). The current data suggest that the observed lack of coupling to phospholipase C may be due
to the relatively small number of CCK-B receptors
present in the guinea pig pancreatic cells.
Important cell-specific differences have previously
been noted in the influence of CCK-B receptor activation on cellular cAMP levels. Activation of CCK-B
receptors found on guinea pig pancreatic acinar cells
(35) or rabbit parietal cells (2) does not lead to formation of cAMP. Activation of CCK-B receptors has been
reported to inhibit adenylate cyclase activity in membranes prepared from the pancreatic AR42J cell line
(24). In other reports, CCK-B receptor activation has
been shown to increase cAMP in certain tumor cells
(14). In the present study, the ability of the CCK-B
receptor to increase cAMP generation in two different
cell models supports the hypothesis that the CCK-B
receptor, like the CCK-A receptor, is capable of coupling
to increased cAMP. However, cAMP generation in the
current study was highly dependent on receptor expression levels. Thus it is likely that the level of receptor
expression in native cells is too low for generation of a
significant increase in cellular cAMP concentration.
C1456
CELL TYPE DEPENDENCE OF CCK-B RECEPTOR FUNCTION
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-35912, DK-41225, DK34933 (Michigan Gastrointestinal Peptide Center), and DK-20572
(Michigan Diabetes Research and Training Center).
Address for reprint requests: C. D. Logsdon, Box 0622, Dept. of
Physiology, The University of Michigan, 1150 W. Medical Center Dr.,
Ann Arbor, MI 48109-0622.
Received 11 July 1996; accepted in final form 30 June 1997.
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