# Cholecystokinin and Somatostatin Negatively Affect Growth of the Somatostatin-RIN-14B Cells

Authors: Karim El-Kouhen; Jean Morisset
Journal: International Journal of Endocrinology
 (2009)

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## Abstract

With the exclusive presence of the pancreatic CCK-2 receptors on the pancreatic delta cells of six different species, this study was undertaken to determine the role of cholecystokinin and gastrin on growth of these somatostatin (SS) cells. For this study, the SS-RIN-14B cells were used in culture and their growth was evaluated by cell counting.Results. To our surprise, we established by Western blot that these RIN cells possess the two CCK receptor subtypes, CCK-1 and CCK-2. Occupation of the CCK-1 receptors by caerulein, a CCK analog, led to inhibition of cell proliferation, an effect prevented by a specific CCK-1 receptor antagonist. Occupation of the CCK-2 receptors by the gastrin agonist pentagastrin had no effect on cell growth. Proliferation was not affected by SS released from these cells but was inhibited by exogenous SS. Conclusions. Growth of the SS-RIN-14B cells can be negatively affected by occupation of their CCK-1 receptors and by exogenous somatostatin.


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## Body

## 1. Introduction

In the pancreas, the cholecystokonin-2 receptor (CCK-2R), formerly
the CCK-B receptor, has been located exclusively on the somatostatin-delta
cells of six different species, including man [1]. To date, nothing is known
about the role of this CCK receptor subtype on the physiology of these delta
cells. It has been reported however that chronic injections of caerulein, a CCK
analog, and secretin caused significant increases in rat pancreatic
somatostatin (SS) content without any effect on total delta cell numbers
[2].  Similar changes in SS contents were
also observed with the same hormones combination in islets cultured for 10 days
with no difference in islets’ total DNA contents [3].It was initially demonstrated that gastrin and CCK-8, at
relatively high doses, caused a quick and transient twofold increase in SS
release by the perfused canine pancreas [4]. When CCK-8 was infused at a dose
that stimulates the secretion of protein in the pancreatic juice of dogs to the
levels normally observed with a meal [5], SS in the pancreatic vein rose
immediately about 2-fold above basal and remained elevated for the 15 minutes
of the hormone infusion [6].  These data
indicate that gastrin and CCK can induce SS secretion from the pancreatic gland
which was later directly confirmed from rat islet cell in culture [7].In order to have an exclusive and direct access to the
pancreatic somatostatin cells and to avoid mixtures of cell types from islet
cell cultures, investigators established and cloned a transplantable rat islet
cell tumor which secretes insulin and somatostatin [8]. Clones of the original
somatostatin cell line were used to study somatostatin secretion (RINT3),
intracellular signalling pathways activated in response to CCK receptor
subtypes occupation (RIN1027-B2) and CCK receptor subtypes characterization and
SS release (RIN-14B) [9–11].This study was undertaken to establish the presence of
the two CCK receptor subtypes on the RIN-14B cells and to determine if one or
both of these types can affect growth of these cells.  To our knowledge, no study has yet been
performed to explore if the gastrointestinal hormones CCK and gastrin, two
specific CCK-1 and CCK-2 receptors agonists, respectively, have any effects on
growth of the pancreatic somatostatin cells.


## 2. Materials and Methods

Products
Somatostatin-14 (SS-14)
was purchased from Bachem Bioscience (King
of Prussia, Pa, USA).  Pentagastrin (PG) was purchased from
Sigma-Aldrich (Oakville, ON, Canada).  Caerulein (Cae) was generously given by Dr. R.
de Castiglione, Farmitalia (Milan,
Italy).  L-364,718 and L-365,260 were gifts from Dr.
V. J. Lotti, Merck Sharp and Dohme Research Laboratories (West Point, Pa).
The RIN-14B cells were obtained from ATCC (Manassas, Va, USA). The COS cells were gifts from
Dr. M. Tremblay (McGill Cancer Centre, Montreal,
Canada). Cell
culture media reagents were obtained from Life Technologies (Burlington, ON, Canada).  The somatostatin (Barbar) antibody, purified
by affinity chromatography, was a gift from Dr. P. Brazeau, Université de
Montréal, Montreal, Canada.  The goat polyclonal anti-CCK-1 receptor
antibody (7509) was raised against the rat internal peptide (KFDASQKKSAKEKR)
and the goat polyclonal anti-CCK-2 receptor antibody (6767) was raised against
the human internal peptides CCK-2 (FDGDSDSDSQSRVRNQ).  These two antibodies and corresponding
peptides were gifts from Dr.  J. Voskuil (Everest Biotech, Oxfordshire, UK).Cell Culture
The rat RIN-14B cell line is
a secondary clone derived from the RIN-m rat islet cell line; these cells
synthesize and secrete somatostatin [8]. Routinely, the cells were grown on
Petri dishes in RPMI 1640 (for RIN-14B) and DMEM (for COS cells) media containing
25 mM glucose, 10% fetal bovine serum (FBS), penicillin (100 UmL−1),
streptomycin (100μg mL−1), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 1.5 g L−1 sodium
bicarbonate.  Cells were kept in culture
at 37°C in a 5% CO2 humidified atmosphere. The culture
media were changed every other day; they were passed weekly by trypsin
(0.25%)-EDTA (2.2 mM)
detachment and subcultured every week at a ratio of 1 : 7.Growth Assay
Cells were plated in 60 mm diameter Petri dishes
at a density of 0.2×106 mL−1 (4 mL/dish). The next
day, cells were transferred to RPMI 1640-0.5% heat inactivated FBS and adapted
overnight to grow in this medium before addition of peptides, antiserum, and
CCK-receptor antagonist.  The medium was
changed daily and cells were supplemented also daily with Cae (10−10 M),
PG (10−5 M), and SS-14 (10−9 M) alone or in combination
with L-364,718 (10−7 M) or Barbar (0.25μg mL−1).  Cell growth was measured after 1, 2, 3, 4,
and 5 days with a cell counter (Coulter counter).SDS-PAGE and Western Blot
The membranes of the RIN-14B and COS cells were prepared as
described in [12].  Briefly, cells were
resuspended in a homogenization buffer (10 mM HEPES, pH 7.5, 250 mM sucrose, 1 mM EGTA, 1 mM EDTA, 0,5 mM PMSF, 20μM leupeptin, 1μM aprotinin, and 2μM pepstatin), homogenized using a Potter homogenizer and sonicated for 10 seconds (40% power).  The membranes were then collected by
centrifugation at 30 000 g for 30 minutes at 4°C
using a Beckman TLS-55 rotor (Palo Alto, Calif, USA) and resuspended at 17 mg mL−1 in the homogenization buffer.  Proteins were determined with the BCA assay of Pierce using bovine serum albumin as standard.
Membrane proteins
(100μg) were separated by electrophoresis on a 10% SDS-polyacrylamide gel
according to [13] and transferred to nitrocellulose membranes
(Sigma-Aldrich).  The membranes were
blocked for 2 hours with NAP-blocker (G Biosciences, St-Louis, Mo, USA) in TBST
(1 : 2 v/v) and incubated overnight at 4°C in NAP-blocker:  TBST (1 : 2 v/v) with either the anti-CCK-1R
7509 (0.1μg mL−1) or the anti-CCK-2R 6767 (0.1μg mL−1)
antibodies. The blots were then washed in TBST and incubated for 1 hour at RT° in NAP-blocker:  TBST (1 : 2 v/v) with
horseradish peroxidase-conjugated antigoat (1 : 10 000).  Blots were washed with TBST and proteins were
detected by chemiluminescence using ECL immunodetection system (Amersham
Biosciences, Baie d’Urfé, QC). Specificity of each CCK receptor
antibody was established by preincubation of each antibody for 2 hours at RT° with its specific peptide antigen:  1μg mL−1 of 7509 and 0.1μg mL−1 of 6767 peptides.Statistical Analysis
Results
were analyzed by the Student’st-test for comparison of independent
samples with a probability value of <.05 which is considered significant.


## 3. Results

### 3.1. Presence of the CCK Receptor Subtypes on the RIN-14B Cells

To establish the presence of both CCK receptor subtypes on the
RIN-14B cells, we used specific antibodies raised against both receptors. With
the specific CCK-1 receptor antibody (7509), we can see that the RIN-14B cells
express the CCK-1 receptor as a 75 kDa protein.
This recognition by the antibody is specific because the band totally
disappeared with preincubation of the antibody with the peptide used to develop
this antibody (see Figure1(a)). Similarly, these cells also express the CCK-2
receptor, also as a 75 kDa protein identified with the antibody 6767;
specificity of the receptor-antibody binding was also revealed with
preincubation of the antibody with the peptide with disappearance of the band (see
Figure 1(b)).Specificity
of the CCK-1 and CCK-2 receptor antibodies established by Western
blotting. Membrane from the RIN-14B and COS cells (100μg) were used for
Western blotting analysis with antibodies 7509 (0.1μg mL−1) and
6767 (0.1μg mL−1). Specificity was established by preincubation of
each primary antibody for 2 hours at RT with each receptor-specific peptide
antigen (1μg mL−1 of 7509 and 0.1μg mL−1 of 6767
peptides).  C:  blue Coomassie gel coloration.
(a)(b)To further strengthen the specificity of both CCK-1 and
CCK-2 receptor antibodies, we established the absence of these two CCK
receptors in COS cells known to be free of
these two receptors [14].  As shown in
Figures 1(a) and 1(b), both antibodies 7509 (anti-CCK-1) and 6767 (anti-CCK-2)
did not recognize the 75 kDa band observed in the RIN-14B cells. This
observation further supports the specificity of the two CCK receptor antibodies
we used in this study to demonstrate their presence in the RIN cells.


### 3.2. Effect of Caerulein on RIN-14B Cell Growth

To determine if the CCK-1 analog caerulein can affect the growth of
the RIN-14B cells, growth assays were performed. As shown in Figure2,
caerulein, used at a concentration which totally occupies the high affinity
CCK-1 receptors and causing maximal amylase release from rat dispersed
pancreatic acini [15], caused significant reductions in RIN-14B cell growth from
day 3 to day 5 of culture. The percentage of growth inhibition at day 5 reached
12%. The observation that this inhibition by caerulein was totally reversed by
the specific CCK-1 receptor antagonist L-364718 indicates that the CCK-1 receptors are
involved in this process of growth inhibition. To eliminate the possibility
that somatostatin released in response to caerulein [11] could be responsible
for growth inhibition by the CCK analog, cells were also incubated with an SS
antibody along with caerulein;  in no
time did we observe
a difference between growth in the caerulein group alone and caerulein plus the
SS antibody (data not shown). As shown in Figure 4, the SS antibody prevented
growth inhibition by SS alone.Figure 2
Growth control of
the RIN-14B cells by the CCK-1 receptor agonist caerulein (Cae) and the
receptor’s specific antagonist, L-364718. Cells were grown for up to 120 hours in the presence of 0.1 nM
caerulein ± 0.1μM L-364718.  Medium and drugs were changed daily and cells
were counted daily for up to 5 days.  In
this experiment, results represent data collected from two separate experiments
with four wells per group; overall they represent 8 different wells in each
group. *Significantly different than control at P<.05.


### 3.3. Effect of Pentagastrin on RIN-14B Cell Growth

To establish if the CCK-2 agonist pentagastrin could affect growth
of these RIN-14B cells, growth assays were also performed.  The results indicate that contrary to what
was observed with occupation of the CCK-1 receptors, occupation of the CCK-2
receptors by 10μM pentagastrin, a gastrin analog, had no effect on RIN-14B cell
growth over the 5-day period examined (see Figure 3). This strongly indicates
that occupation of the CCK-2 receptors did not alter the growth of the RIN-14B
cells.Figure 3
Growth control of
the RIN-14B cells by the CCK-2 receptor agonist pentagastrin (PG). Cells were grown for up to 120 hours in the presence of 10μM PG.
Medium and PG were changed daily and cells were counted daily for up to 5 days.
Results are the means ± SE of 5 wells
per point in each group.Figure 4
Growth control of
the RIN-14B cells by somatostatin and a specific somatostatin antibody.  Cells were grown for up to 120 hours in the presence of 1 nM
somatostatin (SS) ± 0.25μg mL−1 of SS antibody
(Ab anti-SS) or Ab anti-SS alone. Medium and drugs were changed daily and cells
were counted daily for up to 5 days.
Results are the means ± SE of 5
wells per point in each group. *Significantly
different than control at P<.05.


### 3.4. Effect of Somatostatin on RIN-14B Cell Growth

To verify if somatostatin could have an inhibitory effect on growth
of the cells responsible for its synthesis, the RIN-14B cells were incubated
for up to five days in the presence of 1 nM somatostatin-14. As shown in Figure4, somatostatin significantly reduced growth of the RIN-14B cells by 11.3%
after 5 days of culture; this inhibitory effect was already significant after 2
days of culture. Addition of a somatostatin antibody to the culture medium to
neutralize endogenous SS released by the cells had no effect on their growth;
however its addition to exogenous somatostatin reversed its growth inhibition.
These results indicate that endogenous somatostatin released from these cells
did not reach the concentration needed to inhibit their growth but when applied
at the needed concentration, exogenous somatostatin can inhibit their growth.


## 3.1. Presence of the CCK Receptor Subtypes on the RIN-14B Cells

To establish the presence of both CCK receptor subtypes on the
RIN-14B cells, we used specific antibodies raised against both receptors. With
the specific CCK-1 receptor antibody (7509), we can see that the RIN-14B cells
express the CCK-1 receptor as a 75 kDa protein.
This recognition by the antibody is specific because the band totally
disappeared with preincubation of the antibody with the peptide used to develop
this antibody (see Figure1(a)). Similarly, these cells also express the CCK-2
receptor, also as a 75 kDa protein identified with the antibody 6767;
specificity of the receptor-antibody binding was also revealed with
preincubation of the antibody with the peptide with disappearance of the band (see
Figure 1(b)).Specificity
of the CCK-1 and CCK-2 receptor antibodies established by Western
blotting. Membrane from the RIN-14B and COS cells (100μg) were used for
Western blotting analysis with antibodies 7509 (0.1μg mL−1) and
6767 (0.1μg mL−1). Specificity was established by preincubation of
each primary antibody for 2 hours at RT with each receptor-specific peptide
antigen (1μg mL−1 of 7509 and 0.1μg mL−1 of 6767
peptides).  C:  blue Coomassie gel coloration.
(a)(b)To further strengthen the specificity of both CCK-1 and
CCK-2 receptor antibodies, we established the absence of these two CCK
receptors in COS cells known to be free of
these two receptors [14].  As shown in
Figures 1(a) and 1(b), both antibodies 7509 (anti-CCK-1) and 6767 (anti-CCK-2)
did not recognize the 75 kDa band observed in the RIN-14B cells. This
observation further supports the specificity of the two CCK receptor antibodies
we used in this study to demonstrate their presence in the RIN cells.


## 3.2. Effect of Caerulein on RIN-14B Cell Growth

To determine if the CCK-1 analog caerulein can affect the growth of
the RIN-14B cells, growth assays were performed. As shown in Figure2,
caerulein, used at a concentration which totally occupies the high affinity
CCK-1 receptors and causing maximal amylase release from rat dispersed
pancreatic acini [15], caused significant reductions in RIN-14B cell growth from
day 3 to day 5 of culture. The percentage of growth inhibition at day 5 reached
12%. The observation that this inhibition by caerulein was totally reversed by
the specific CCK-1 receptor antagonist L-364718 indicates that the CCK-1 receptors are
involved in this process of growth inhibition. To eliminate the possibility
that somatostatin released in response to caerulein [11] could be responsible
for growth inhibition by the CCK analog, cells were also incubated with an SS
antibody along with caerulein;  in no
time did we observe
a difference between growth in the caerulein group alone and caerulein plus the
SS antibody (data not shown). As shown in Figure 4, the SS antibody prevented
growth inhibition by SS alone.Figure 2
Growth control of
the RIN-14B cells by the CCK-1 receptor agonist caerulein (Cae) and the
receptor’s specific antagonist, L-364718. Cells were grown for up to 120 hours in the presence of 0.1 nM
caerulein ± 0.1μM L-364718.  Medium and drugs were changed daily and cells
were counted daily for up to 5 days.  In
this experiment, results represent data collected from two separate experiments
with four wells per group; overall they represent 8 different wells in each
group. *Significantly different than control at P<.05.


## 3.3. Effect of Pentagastrin on RIN-14B Cell Growth

To establish if the CCK-2 agonist pentagastrin could affect growth
of these RIN-14B cells, growth assays were also performed.  The results indicate that contrary to what
was observed with occupation of the CCK-1 receptors, occupation of the CCK-2
receptors by 10μM pentagastrin, a gastrin analog, had no effect on RIN-14B cell
growth over the 5-day period examined (see Figure 3). This strongly indicates
that occupation of the CCK-2 receptors did not alter the growth of the RIN-14B
cells.Figure 3
Growth control of
the RIN-14B cells by the CCK-2 receptor agonist pentagastrin (PG). Cells were grown for up to 120 hours in the presence of 10μM PG.
Medium and PG were changed daily and cells were counted daily for up to 5 days.
Results are the means ± SE of 5 wells
per point in each group.Figure 4
Growth control of
the RIN-14B cells by somatostatin and a specific somatostatin antibody.  Cells were grown for up to 120 hours in the presence of 1 nM
somatostatin (SS) ± 0.25μg mL−1 of SS antibody
(Ab anti-SS) or Ab anti-SS alone. Medium and drugs were changed daily and cells
were counted daily for up to 5 days.
Results are the means ± SE of 5
wells per point in each group. *Significantly
different than control at P<.05.


## 3.4. Effect of Somatostatin on RIN-14B Cell Growth

To verify if somatostatin could have an inhibitory effect on growth
of the cells responsible for its synthesis, the RIN-14B cells were incubated
for up to five days in the presence of 1 nM somatostatin-14. As shown in Figure4, somatostatin significantly reduced growth of the RIN-14B cells by 11.3%
after 5 days of culture; this inhibitory effect was already significant after 2
days of culture. Addition of a somatostatin antibody to the culture medium to
neutralize endogenous SS released by the cells had no effect on their growth;
however its addition to exogenous somatostatin reversed its growth inhibition.
These results indicate that endogenous somatostatin released from these cells
did not reach the concentration needed to inhibit their growth but when applied
at the needed concentration, exogenous somatostatin can inhibit their growth.


## 4. Discussion

This study reports for the first time that (1) the SS-secreting
RIN-14B cells express the two known and characterized CCK receptor subtypes; (2)
occupation of the CCK-1 receptor subtype leads to inhibition of these cells’
growth, while occupation of the CCK-2 receptor subtype fails to stimulate or
inhibit such growth; (3) somatostatin can also inhibit growth of these RIN-14B
cells probably through an autocrine mechanism.In contrast to substances
which can control somatostatin secretion, nothing is currently known about those
responsible for delta cells’ growth. The observation that the RIN-14B cells express
the two CCK receptor subtypes is not unique to these cells. Indeed, expression
of both receptor subtypes has been documented in normal rat pancreatic acini
with a predominance of the CCK-1 subtype over that of the CCK-2 [16]. These two
receptors were also reported in normal human and rat islets but on different
cell types, the CCK-1 receptors on alpha and beta cells and the CCK-2 receptors
on the delta cells using two different antibodies [17]. Studies performed in
rats demonstrated that in pancreatic malignancies, the CCK-1 receptor is
overexpressed while the CCK-2 receptor is newly expressed [18]. In human
pancreatic tumors however, the distribution of the two CCK receptor subtypes is
still controversial. Indeed, by using the PCR technique, one study reported the
presence of the CCK-2 receptors in all samples of normal pancreas and
pancreatic adenocarcinoma; the CCK-1 receptor expression could not be detected
in normal pancreatic samples but it appeared in all samples of pancreatic
adenocarcinomas [19]. By receptor autoradiography, the CCK-2 receptor was found
occasionally in pancreatic tumors while the CCK-1 receptor was mostly expressed
in these tumors [20]. These data emphasize that the expression of these two CCK
receptor subtypes in many cancer cell types may be an important indicator of
the influence of CCK and gastrin of local or systemic origin on the growth of
these cancers.Indeed, in Elas
CCK-2 receptor transgenic mice, the growth rate of their pancreas was increased
by 40% after birth between 40 and 110 days of age; this expression had a key
role in the development of pre- and neoplastic lesions in their pancreas [21].
While everyone agrees that occupation of the CCK-1 receptors in the pancreas of
rats [22] and other rodents led to growth of the organ, its presence and
stimulation in MiaPaCa-2 and Panc-1 cells led to growth inhibitory responses
[23]. Occupation of the CCK-2 receptor also resulted in surprising opposite
effects when transfected in CHO and Swiss 3T3 cells.  It inhibited proliferation and DNA synthesis
in the CHO-CCK-2 cells while stimulation occurred in the Swiss 3T3-CCK-2 cells;
these opposite effects on growth happened while CCK-8 stimulated the same
common second messenger pathways [24]. Growth inhibition was also observed with
occupation of the transfected CCK-2 receptors in the human pancreatic MiaPaca-2
and Panc-1 cells [23]. Interestingly, coexpression of gastrin and CCK-2
receptors were observed in 5/5 and 7/8 human gastric and colorectal cell lines
and these cells maintain an autocrine growth pathway [25]; the RIN-14B cells
also express gastrin although in small quantities (data not shown).When comparing all
the above results with those obtained with the RIN-14B cells, it seems that
growth inhibition observed in response to caerulein in this study renders these
RIN cells comparable to the transfected CCK-1 MiaPaca-2 and Panc-1 cells [23]. Endogenous
CCK also resulted in growth inhibition of human cholangiocarcinoma; however, no
specific antagonists of either CCK receptor subtypes were used to confirm which
CCK receptor was involved [26]. This study points out that CCK can inhibit
growth not only in cells transfected with both CCK receptor subtypes. Moreover, the inhibition observed in these RIN cells really resulted from occupation of
the CCK-1 receptors because it has been reversed by L-364718, a specific CCK-1 receptor
antagonist.Somatostatin can
inhibit endogenous SS secretion from the delta cells through an auto-feedback
mechanism [27]. Although we have not investigated which somatostatin receptors
are present on the RIN-14B cells, it has been reported that about 70% of the
rat pancreatic delta cells express the SS receptor subtypes 1–4 [28].Since it is
accepted that the antiproliferative effect of somatostatin results from its
action via the endocrine pathway, evidence also exists that somatostatin can
also act via an autocrine/paracrine pathway which has been recently described
in PC-3 and LNCaP cells, two human prostate adenocarcinoma cell lines [29]. We
do not believe that growth of these RIN-14B cells is autoregulated by
somatostatin they release into the medium. This conclusion is based on the
following observations: (a) these cells grew in a 0.5% inactivated FBS medium when
they release into the medium approximately 200 pg mL−1 of
somatostatin per 4 hours [11], and (b) they also grew at their control rates
even in the presence of 0.25μg mL−1 of a specific somatostatin
antibody; an autocrine regulated pathway would have shown these cells grow at a
rate above the controls in the presence of the antibody.  This was previously observed in vivo when the trophic effect of
caerulein on the rat pancreas was significantly enhanced by a simultaneous administration
of a somatostatin antiserum, the same used in this study [30].Although endogenous
somatostatin released by the RIN cells does not seem sufficient to sustain
growth inhibition, these cells are however sensitive to somatostatin as they
exhibited growth inhibition in its presence at a higher concentration. The
magnitude of inhibition is comparable to what was previously described in
AR4-2J cells where at 10 nM, the hormone caused a 25% growth inhibition over a
96-hour period [31]. This inhibitory effect of somatostatin on the RIN cells
proliferation is specific since prevented by the presence of the somatostatin
antibody.In conclusion, our
data indicate for the first time that the somatostatin RIN-14B cells possess
the two CCK receptor subtypes, and that their proliferation can be negatively affected
by occupation of the CCK-1 receptor subtype and exogenous somatostatin.



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