Modulation of secretin release by neuropeptides in
secretin-producing cells
Cecilia H.
Chang1,
William Y.
Chey1,
Brian
Erway1,
David H.
Coy2, and
Ta-Min
Chang1
1 The Konar Center for
Digestive and Liver Diseases, Department of Medicine, University of
Rochester School of Medicine and Dentistry, Rochester, New York
14642; and 2 Tulane
University, New Orleans, Louisiana 70112
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ABSTRACT |
Nerve fibers
containing bombesin (BB)/gastrin-releasing polypeptide (GRP), pituitary
adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal
polypeptide (VIP), or galanin are known to innervate the mucosa of the
upper small intestine. Both BB/GRP and PACAP have been
shown to elicit secretin secretion in
vivo. We studied whether the above-mentioned
neuropeptides can act directly on secretin-producing cells, including
the murine neuroendocrine cell line STC-1 and a secretin cell-enriched
preparation isolated from rat upper small intestinal mucosa. Secretin
release from both cell types was stimulated by various agents known to elicit secretin release and by the neuropeptides BB, GRP, and PACAP,
suggesting a comparable response between the two cell preparations. The
effects of neuropeptides were further studied in STC-1 cells. BB, GRP,
and PACAP stimulated secretin release time and concentration dependently. VIP also stimulated secretin release concentration dependently. Stimulation by BB/GRP or PACAP was accompanied by elevation of inositol-1,4,5-trisphosphate
(IP3) or cAMP, respectively. The
stimulatory effect of PACAP on secretin release was synergistically enhanced by BB without any synergistic increase in
IP3 or cAMP production, suggesting
cross talk between different signal transduction pathways downstream of
the production of these two second messengers. The L-type
Ca2+ channel blocker diltiazem (10 µM) and the Ca2+ chelator EGTA
(1 mM) significantly inhibited BB-stimulated secretin release
by 64% and 59%, respectively, and inhibited PACAP-stimulated release
by 75% and 55%, respectively. The protein kinase A-specific inhibitor
Rp-cAMPS (100 µM) also inhibited both BB- and PACAP-stimulated secretin release by 30% and 62%, respectively. Galanin inhibited BB-
and PACAP-stimulated secretin release and production of second messengers in a concentration-dependent and pertussis toxin-sensitive manner. These results suggested that the neuropeptides BB/GRP, PACAP,
VIP, and galanin can modulate secretin release in secretin-producing cells and that STC-1 cells can serve as a useful model for studying the
cellular mechanism of secretin secretion elicited by luminal secretagogues and neuropeptides.
pituitary adenylate cyclase-activating polypeptide; gastrin-releasing polypeptide; galanin; vasoactive intestinal
polypeptide; pertussis toxin; mucosal secretin-producing
cells
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INTRODUCTION |
THE FIRST HORMONE EVER DISCOVERED (4), secretin is a
27-amino acid polypeptide and a member of the
secretin/glucagon/vasoactive intestinal polypeptide (VIP) superfamily
(see Refs. 12, 25 for review). Secretin-producing cells are located
mainly in the mucosa of the upper small intestine in most species (6,
39). In addition, a small amount of secretin-like immunoreactivity (SLI) has been found in the gastric antral mucosa (13) and the brain
(9, 35, 37). The major physiological roles of secretin are stimulation
of exocrine pancreatic secretion of volume and bicarbonate (12, 25),
inhibition of gastric emptying (22, 40), acid secretion, and release of
gastrin (14, 42, 50). Secretin also exerts several other biological
effects, including stimulation of duodenal bicarbonate secretion (21),
inhibition of upper small intestinal motility and lower esophageal
sphincter pressure (12), stimulation of hepatic bile flow (2, 23, 36),
and electrolyte secretion (3, 32, 46).
Secretin is released by intraduodenal administration of acid, digested
products of fat and protein, bile acid, and certain herbal extracts
from the plant kingdom (10, 12, 25). It is now clear that the release
of secretin by duodenal acidification in the rat is mediated via a
secretin-releasing peptide (SRP) that is released into the upper small
intestinal lumen (28). A secretin-releasing factor also exists in
canine pancreatic juice (29). Study of secretin release from mucosal
explants of canine duodenum has led to results indicating that the
release of secretin is controlled by neurohormonal actions (34),
whereas the release and action of SRP are mediated by a neural
mechanism(s) (26). The neurotransmitter(s) or neuropeptide(s) that
affects the action of SRP is not clearly defined at present. However,
it is known that nerve fibers containing gastrin-releasing polypeptide
(GRP)/bombesin (which are peptides of the same family and share
COOH-terminal amino acid sequences; Ref. 17), pituitary adenylate
cyclase-activating polypeptide (PACAP) (45), VIP (16), or galanin (38)
innervate the mucosa of the upper small intestine. In view of this
innervation and the presence of PACAP- and galanin-containing nerve
fibers in the pancreatic islets and their potent stimulation and
inhibition, respectively, of insulin release (38, 49), it is very
likely that some or all of the above-mentioned neuropeptides may be
involved in regulation of secretion from enteric endocrine cells such
as the secretin-producing cells. Indeed, the release of secretin is
stimulated by PACAP in conscious rats (24) and by GRP in pigs
(18) and rats (27).
The cellular mechanism(s) through which the luminal stimulants SRP and
neuropeptide(s) or neurotransmitter(s) elicits the release of secretin
is not clear at present. Using secretin-producing cell-enriched mucosal
cell preparations isolated from the upper small intestine of the dog
(48) and the rat (11) or the mucosal explants of canine duodenum (33),
it has been demonstrated that the release of secretin is regulated
intracellularly by both Ca2+- and
cAMP-dependent mechanisms. Because of their low yield and heterogeneity, it has been difficult to use these preparations to
investigate further intracellular mechanisms of secretin release elicited by each of the luminal stimulants and the neuropeptides.
The purpose of the present study was to establish that the
neuropeptides bombesin/GRP, VIP, and PACAP can act directly on secretin-producing cells to stimulate secretin secretion, using the
murine intestinal neuroendocrine tumor cell line STC-1, which is known
to express and secrete secretin (41, 47), as a model. We also explored
whether, in addition to somatostatin, galanin can act as an inhibitory
regulator of secretin secretion. To ascertain whether or not the
responses of STC-1 cells to the tested neuropeptides resulted from
malignant transformation, we also studied the effects of the
neuropeptides on secretin release from secretin-producing cell-enriched
preparations isolated from rat duodenal mucosa.
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METHODS |
Materials.
Rat secretin, PACAP-27, galanin, and VIP were synthesized by Dr. David
Coy. Porcine GRP was synthesized by Dr. H. Yajima (Kyoto University,
Kyoto, Japan). Bombesin and rat secretin were purchased from Peninsula
Laboratories (Belmont, CA). Forskolin,
4
-12-O-tetradecanoylphorbol 13-acetate (
-TPA), diltiazem, IBMX, and other unspecified
biochemicals were purchased from Sigma Chemical (St. Louis, MO). The Rp
diastereoisomer of cAMPS (Rp-cAMPS) was obtained from Calbiochem (La
Jolla, CA). Camostat
[N,N-dimethylcarbamoylmethyl-p-(p-guanidinobenzoyloxy)phenylacetate methanesulfonate; a synthetic serine protease inhibitor] and
plaunotol [(E,Z,E,)-7-hydroxymethyl-3,11,15-trimethyl-2,6,10,14-hexadecatetraen-1-ol; an antiulcer agent from a herbal extract] were obtained from Ono Pharmaceuticals and Sankyo Pharmaceuticals (both of Tokyo, Japan), respectively. Streptomycin, penicillin, and gentamicin
sulfate were obtained from Flow Laboratories (McLean, VA). All the
tissue culture wares and media were purchased either from GIBCO (Grand Island, NY) or Costar (Cambridge, MA). Radioreceptor assay kit for
inositol-1,4,5-trisphosphate
(IP3) was purchased from
DuPont-NEN (Boston, MA). The RIA kit for cAMP was obtained from
Biomedical Technologies (Stoughton, MA).
All peptides were dissolved and diluted in the incubation medium before
use. Forskolin, plaunotol, and
-TPA were dissolved in DMSO
(Me2SO) and diluted in the
incubation medium immediately before the experiment. The final
concentration of DMSO was kept below 0.02% (vol/vol), and the same
dilution of the solvent alone was added to the control. Camostat and
IBMX were dissolved in diluted NaOH and then diluted in the incubation
medium before each experiment. Sodium oleate solution was prepared by
dissolving oleic acid (Fisher Scientific, Springfield, NJ) in diluted
NaOH and diluted in the incubation medium immediately before use. Stock solutions of camostat, IBMX, and sodium oleate so prepared did not
affect the pH of the medium after being added to the cell culture to
attain the final concentrations of these agents. The concentrations of
nonpeptide secretagogues were selected as the highest nontoxic
concentrations used previously in canine and rat mucosal preparations
(11) and in STC-1 cells (7, 8).
Cell culture.
STC-1 cells were obtained from Dr. Seth Grant (Columbia University, NY)
through Dr. Andrew Leiter (Tufts University, Boston, MA). The cells
were maintained in DMEM containing 15% horse serum, 2.5% fetal bovine
serum, streptomycin (100 µg/ml), penicillin (100 µU/ml), and
gentamicin sulfate (50 µg/ml) in a humidified CO2 incubator at 37°C. Cells
(1 × 106 cell/ml) were
seeded into 12- or 24-well tissue culture plates (Costar) and cultured
for 3-4 days until 80-90% confluency.
Preparation of rat secretin cell-enriched mucosal cells.
Male Sprague-Dawley rats (250-300 g) fed ad libitum were used.
After an overnight fasting with free access to drinking water, a deep
anesthesia was induced in the rat with 50% urethan given intraperitoneally. After a midline laparotomy, a segment of upper small
intestine (15 cm from the pylorus) was excised and flushed with an
incomplete Krebs-Henseleit (KH) buffer (without
Ca2+ and
Mg2+) containing 1 mM
dithiothreitol (DTT), 0.25% BSA, 10 mM HEPES, pH 7.4, and 2.5 mM EDTA
(buffer
A). The intestinal segment was cut
open, and the mucosa was obtained by scraping with a glass slide. The
mucosa was suspended in buffer
A (5 vol of fresh wt) and incubated
for 15 min at room temperature with occasional swirling. The mucosal
suspension was then incubated with 0.008% collagenase (type I; Sigma
Chemical) at 37°C for 10 min under 95%
O2-5%
CO2 with continuous gyration.
Mucosal cells freed of connective tissue were collected by filtering
through a nylon mesh and centrifuged at 500 g for 5 min at room temperature. The
cells were washed three times by resuspension and centrifugation in KH
buffer containing 2 mM
L-glutamine, 10 mM glucose, 5 mM
sodium pyruvate, 1 mM DTT, and 1% BSA plus 20 mM HEPES, pH 7.4 (buffer
B). The final crude cell suspension
was fractionated by centrifugation at 4°C and 875 g for 15 min in a discontinuous
density gradient of 0%, 23%, 34%, and 55% Percoll (Pharmacia
Biotech, Piscataway, NJ) in buffer B. Four cell layers were obtained at
the interfaces of Percoll solutions of 0%/23%
(fraction
1), 23%/34%
(fraction
2), 34%/55% (fraction
3), and as a pellet in 55% Percoll
(fraction
4), respectively. The cells in each
fraction were collected, washed once with
buffer B, and resuspended in the incubation
medium to study secretin release. An aliquot of the cell suspension was
used to determine the cell number. Another aliquot of the cell
suspension was centrifuged to form a pellet and then homogenized in 0.1 N HCl. The homogenate was centrifuged, and the supernatant solution was
lyophilized before reconstitution and determination of secretin content
by a specific RIA.
Studies on secretin release from STC-1 cells.
Monolayer cultures of STC-1 cells in 24-well plates were washed once
with Earle's balanced salt solution (EBSS) containing 10 mM HEPES, pH
7.4, 5 mM sodium pyruvate, 2 mM
L-glutamine, 0.01% soybean
trypsin inhibitor (Sigma Chemical), and 0.2% BSA. The cells were then
incubated in 0.3 ml of the same medium in the presence or absence of
various test agents at 37°C for 60 min or an indicated time period.
The incubation was stopped by cooling the cell culture plates on ice.
The medium was removed for RIA of secretin. The cells were scraped with
a rubber policeman and extracted in 1 ml of 0.1 N HCl and lyophilized
before being reconstituted and assayed for secretin. The amount of SLI
released to the medium was expressed as a percentage of the total cell
content of SLI. The effect of a stimulant was assessed by comparing
secretin release in its presence with the control and was expressed as
the percent increase over the control. All data are presented as means ± SE averaged from the mean of duplicated wells of a specific
number of experiments.
Study on secretin release from secretin-producing cells-enriched
rat mucosal cells.
The release of secretin from rat mucosal cells was studied with
fraction
3, which contained the highest content
of secretin. The cells were suspended in Hanks' balanced salt solution
containing 5 mM sodium pyruvate, 2 mM
L-glutamine, 10 mM HEPES, pH
7.4, and 0.2% BSA plus 0.01% soybean trypsin inhibitor at a cell
concentration of 0.5-1 × 106/ml. An aliquot of 0.5 ml of
the cell suspension was placed in a glass test tube (16 × 100 mm), and a test agent was added. The tube was filled with 95%
O2-5%
CO2, stoppered, and then incubated at 37°C in a gyratory water bath for 1 h. The reaction was stopped by chilling and centrifuged at 4°C, and the supernatant solution was taken and stored at
20°C for secretin RIA. The cell
pellets was extracted with 1 ml of 0.1 N HCl and lyophilized before
secretin assay. Secretin release was expressed as a percentage of the
total cell content.
HPLC.
The molecular form of SLI released in the medium and present in the
STC-1 cells was determined by reverse-phase HPLC on a Varian MCH-10
column (4.6 × 300 mm) after extraction with Sep-Pak C18 cartridges, using the same
chromatographic solvents as described previously (44). However, a
modified gradient (see Fig. 1) was used to resolve porcine and rat
secretins from SLI (murine secretin) of STC-1 cells.
RIA of secretin.
RIA of secretin was carried out as described previously (10), except
for the use of a rabbit anti-secretin serum raised against rat
secretin, 125I-labeled rat
secretin as the tracer, and unlabeled rat secretin as the standard. The
anti-rat secretin serum was raised by the same method as described
previously (10) and used at a final dilution of 1:5 × 106.
125I-labeled rat secretin was
prepared and purified as described previously for
125I-labeled porcine secretin
(10).
Measurement of IP3 production in STC-1
cells.
Monolayer cultures of STC-1 cells were preincubated with a vehicle or
PACAP or galanin in EBSS medium for 15 min at 37°C. Bombesin (10 nM) was then added, and the cells were further incubated for 0-300
s. The reaction was stopped by placing the tissue culture plate on ice,
removing the medium immediately, and then adding 0.5 ml of ice-cold
16.6% TCA. The cell was scraped with a rubber policeman
and transferred in a microcentrifuge tube, vortexed vigorously and
allowed to stand at 4°C for 1 h. After centrifugation at 15,000 g for 1 min at 4°C, 300 µl of
the supernatant solution were extracted with 800 µl of
1,1,2-trichloro-1,2,2-trifluoroethane/tri-n-octylamine (3:1, vol/vol). The IP3 content in
the upper aqueous phase was then determined by the radioreceptor assay
according to the procedure provided by the manufacturer of the assay
kit.
Measurement of intracellular cAMP.
The intracellular cAMP content in STC-1 cells was measured by RIA, as
described previously (7). Briefly, STC-1 cells were first preincubated
with a vehicle or various test agents for 10 min, then PACAP (5 × 10
8 M) was added and the
cells were further incubated for 2 min. Intracellular cAMP was
extracted with 10% TCA. After removing TCA by extraction with ether,
we then assayed the cAMP content by RIA according to the
manufacturer's suggested protocol.
Statistical analysis.
Statistical analyses were performed by using ANOVA for single-factor
experiments with multiple treatments, followed by Dunnett's post hoc
analysis for comparing treatment means with the same control, using a
Systat software program (Systat, Evanston, IL) as described previously
(8). When only two means were compared, Student's paired
t-test was used. A difference of
P < 0.05 between two means was
regarded as significant.
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RESULTS |
SLI in STC-1 cells.
The concentration of SLI in STC-1 cells was 205.9 ± 9.6 fmol/mg
cell protein (n = 18). Reverse-phase
HPLC of the cell extract as shown in Fig. 1
indicated that there was a major peak of SLI stored in STC-1 cells,
which had a retention time of 99 min, that was somewhat different from
those of porcine secretin (93 min) and rat secretin (95 min). The SLI
released from STC-1 cells to the medium on stimulation with 50 mM KCl
also exhibited a major peak at 99 min (Fig. 1). The minor peak eluted
at 105 min could be a degraded product or a precursor molecule but
could not be identified due to the lack of appropriate standards.

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Fig. 1.
Results of reverse-phase HPLC of secretin-like immunoreactivity (SLI)
present in the medium and cell extract of STC-1 cells. Chromatogram of
SLI in the cell extract (solid line) and the medium collected after
stimulation with KCl (dotted line) exhibited a major molecular form
eluted at 99 min, which was different from the retention time of
porcine and rat secretins (pSec and rSec, respectively) (indicated by
arrows).
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SLI in rat secretin-producing cell-enriched cell preparation.
Cell yield and SLI content in the mucosal cell fractions isolated
before and after Percoll density gradient centrifugation are summarized
in Table 1. Secretin-containing cells were
most enriched in the fraction located at the interface between 34% (density = 1.043 g/ml) and 55% (density = 1.056 g/ml) of Percoll (fraction
3) in the Percoll density gradient,
increasing the content of SLI by threefold from 402 ± 48 to 1,341 ± 107 fmol/106
cells. Meanwhile, the viability of the cells as judged by
the trypan blue exclusion method increased from 61.8% to 95%. The total number of cells of fraction
3 from the small intestines of five
rats was 4.1 × 106. The
presence of a considerable amount of SLI in
fractions
1 and
2 suggested that secretin-producing
cells are polydispersed with respect to cell density.
Effect of various agents on SLI release from STC-1 cells.
The results summarized in Table 2 indicate
that the release of SLI from STC-1 cells was stimulated significantly
by dibutyryl-cAMP (data not shown), forskolin, the protein kinase C
(PKC)-activating phorbol ester
-TPA, the
Ca2+ ionophore A-23187, and
depolarization with KCl. The stimulatory effect of
forskolin was enhanced by IBMX (0.5 mM), but the latter alone did not
significantly stimulate the release of SLI. Among the pharmacological
agents studied, KCl was the most potent stimulant. Luminal stimulants
such as camostat, sodium oleate, and plaunotol also significantly
stimulated the release of SLI. Thus the responses of STC-1 cells to
these agents were very similar to those reported previously in
secretin-producing cell-enriched preparations isolated from the mucosae
of canine duodenum (48) and rat upper small intestine (11).
Effects of neuropeptides on SLI release.
To test the hypothesis that neuropeptides participate in regulation of
secretin release, we studied the effect of bombesin/GRP and PACAP-27 on
the release of SLI from both STC-1 cells and the isolated
secretin-producing cell-enriched preparation. Because the elevation of
cellular cAMP level is known to elicit secretin secretion (11, 48), we
used the response of these cells to forskolin (30 µM) plus IBMX (0.5 mM) as a positive control. Thus, if a cell preparation, particularly
the mucosal cells, failed to respond to this mode of stimulation, it
was unlikely to respond to a neuropeptide. As shown in Fig.
2, incubation of STC-1 cells with a
submaximal concentration of GRP (10 nM), bombesin (10 nM), or PACAP (50 nM) at 37°C for 60 min resulted in a significant increase in SLI
release over basal secretion by 116 ± 13%, 105 ± 11%, and 65 ± 14%, respectively. These increases were somewhat smaller than
that of the positive control (238 ± 11%; data not shown)
of forskolin plus IBMX. Similarly, all three peptides stimulated the
release of SLI from rat mucosal cell suspension with significant increases of 43 ± 15% (by GRP), 39 ± 14% (by bombesin), and
43 ± 10% (by PACAP-27) over basal. The effects of these
neuropeptides were comparable to that of the positive control in the
mucosal cell preparation (increased by 69 ± 4%; data not shown).

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Fig. 2.
Effect of bombesin, gastrin-releasing polypeptide (GRP), or pituitary
adenylate cyclase-activating polypeptide-27 (PACAP-27) on secretin
release from STC-1 and mucosal secretin-producing cell-enriched
preparation isolated from rat upper small intestine. Bombesin (10 nM),
GRP (10 nM), and PACAP-27 (50 nM) significantly stimulated secretin
release from both STC-1 cells and rat mucosal cells after 60 min of
incubation. Data (in %stimulation over corresponding controls) are
means ± SE of 4 experiments. Controls were 2.27 ± 0.25% and
3.67 ± 0.66% of the total cellular SLI content for STC-1 and
mucosal cells, respectively.
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Time-dependent effects of neuropeptides on SLI release.
The stimulatory effects of bombesin, GRP, and PACAP-27 on SLI release
from STC-1 cells were time dependent. As shown in Fig. 3, there was a small basal secretion of SLI
(0.74 ± 0.21% of total secretin content,
n = 10) over a period of 120 min at
37°C. In the presence of bombesin (10 nM) or GRP (10 nM), the release of SLI increased rapidly, reaching 1% of the cellular
content within 15 min, and continuously increased at a reduced rate,
reaching 4% at 120 min. In the presence of PACAP-27 (50 nM), there was a smaller but linear increase in the release of SLI over a period of 60 min, reaching 2% of total cellular content. In the subsequent studies,
the cells were incubated with these stimulants for 60 min at 37°C.

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Fig. 3.
Time-dependent effects of GRP, bombesin, and PACAP-27 on secretion of
SLI from STC-1 cells. Bombesin (10 nM), GRP (10 nM), or PACAP-27 (50 nM) continuously stimulated release of SLI from STC-1 cells over the
basal secretion for a 120-min period. Data represent average of 5 experiments.
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Concentration-dependent effects of bombesin, GRP, PACAP-27, and VIP.
The stimulatory effects of these neuropeptides on the release of SLI
from STC-1 cells were also concentration dependent. As shown in Fig.
4, incubation of STC-1 cells with either
GRP or bombesin at 1 nM and higher concentrations resulted in a
significant concentration-dependent increase in SLI secretion that
reached a plateau at 10 nM. In both cases, the
EC50 of bombesin or GRP was ~1
nM. In contrast, higher concentrations of PACAP-27 were required to
stimulate SLI release from STC-1 cells, with a threshold dose of 18 nM,
reaching the same extent of stimulation maximally attained by bombesin
and GRP (100% increase over control) at 180 nM. Because PACAP-27 at
concentrations above 180 nM started to exhibit cross reaction with RIA
of secretin, we did not determine the maximal effect of PACAP-27.
Synthetic VIP appeared to be more potent than PACAP-27, starting to
stimulate secretin release as low as 0.1 nM (52 ± 12% over basal)
and reaching a maximal stimulation at 100 nM (570 ± 78% over
basal) with an EC50 of 10 nM (data
not shown).

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Fig. 4.
Concentration-dependent effect of GRP, bombesin, and PACAP-27 on
secretion of SLI from STC-1 cells. Bombesin and GRP stimulated release
of SLI from STC-1 cells concentration dependently with an
EC50 of 1 nM and a maximal
stimulation attained at 10 nM. PACAP also stimulated release of SLI
concentration dependently but at higher concentrations than the other
peptides. Data represent average of 4 experiments.
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Synergistic effect of PACAP-27 added together with other
secretagogues.
In the presence of IBMX, PACAP-27 and VIP were shown to increase cAMP
production in STC-1 cells (7) and their stimulatory effect was mediated
mainly through the adenylate cyclase signal transduction pathway. Thus
we studied whether PACAP would have a synergistic effect with other
secretagogues that stimulated SLI release via different signal
transduction pathways. As shown in Fig. 5,
PACAP-27 (50 nM) in combination with IBMX (0.5 mM), bombesin (10 nM),
KCl (50 mM), or
-TPA (0.1 µM) produced a significantly greater
stimulation of SLI release from STC-1 cells than the sum of the
individual effects of PACAP and the other secretagogue studied in the
same experiments, thereby suggesting a synergistic effect. A similar synergism between VIP and each of these
agents was also observed (data not shown).

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Fig. 5.
Synergistic effect of PACAP-27 with other secretagogues on SLI release
from STC-1 cells. Incubation of STC-1 cells in the presence of PACAP
(50 nM) together with either IBMX (0.5 mM), KCl (50 mM), bombesin (10 nM), or 4 -12-O-tetradecanoylphorbol 13-acetate ( -TPA; 0.1 µM) resulted in greater stimulation of SLI secretion (hatched bars)
than the sum (solid bars) of the effect of PACAP alone (crosshatched
bars) and that of the other secretagogue alone (open bars) that was
determined in the same experiment, indicating a synergistic effect.
Data are means ± SE of 9 experiments.
* P < 0.05, **
P < 0.01, significant difference
between secretagogue + PACAP and PACAP alone.
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Inhibition by galanin of secretagogue-stimulated SLI secretion.
As shown in Fig. 6, rat galanin alone had
no effect on basal secretion of SLI from STC-1 cells but inhibited
KCl-, bombesin-, and PACAP-stimulated SLI secretion in a
concentration-dependent manner. A small but significant inhibition by
galanin of
-TPA-stimulated secretion was also observed. Pretreatment
of the cells with pertussis toxin (PTx) prevented inhibition by galanin
of SLI secretion stimulated by bombesin (Fig.
7, top),
PACAP (Fig. 7, middle), or KCl (Fig. 7, bottom). In
contrast, PTx alone had no effect on both basal and
secretagogue-stimulated secretions.

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Fig. 6.
Concentration-dependent effect of rat galanin on
secretagogue-stimulated SLI release from STC-1 cells. STC-1 cells were
incubated in the absence (control) or presence of KCl (25 mM), -TPA
(100 nM), bombesin (10 nM), or PACAP (100 nM) with varying doses of
galanin (0 or
10 10-10 7
M) for 1 h, and amount of SLI released was then determined. Galanin
significantly inhibited each secretagogue-stimulated SLI release (at 0 galanin concentration) at concentrations indicated.
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Fig. 7.
Effect of pertussis toxin (PTx) on inhibition by galanin (Gal) of
secretagogue-stimulated SLI release. STC-1 cells were preincubated in
the absence or presence of 100 ng/ml PTx for 10 h and then incubated
with bombesin (10 nM; top), PACAP
(100 nM; middle), or KCl (25 mM;
bottom), either alone or in
combination with galanin (10 nM), for 1 h. The amounts of SLI
released under each set of conditions were determined and compared.
Galanin significantly inhibited secretagogue-stimulated SLI release,
and this effect was reversed by pretreatment of STC-1 cells with PTx.
* P < 0.05, ** P < 0.01 vs. control.
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Effects of galanin and PACAP on bombesin-stimulated
IP3 production.
Bombesin has been shown to elevate intracellular
Ca2+ concentration in STC-1 cells
(35). This effect is most likely due to activation of a
phophatidylinositol phosphate-specific phospholipase C pathway.
Therefore, we determined the effect of bombesin on IP3 production in STC-1 cells. In
addition, we also determined whether or not inhibition by galanin or
potentiation by PACAP on bombesin-stimulated secretin secretion was
mediated by changes in bombesin-stimulated
IP3 production. As shown in Fig.
8, incubation of STC-1 cells with bombesin
(10 nM) resulted in a time-dependent increase of cellular
IP3 content, reaching a maximal
value of 559 ± 120 fmol/mg cell protein at 30 s that was in
equivalent to a 14.8-fold increase over the basal value (27 ± 4 fmol/mg cell protein). The
IP3 level declined subsequently
but remained elevated over basal for 2 min. Preincubation of the cells
with galanin (10 nM) for 15 min significantly reduced the stimulatory
effect of bombesin on IP3
production by 58% (233 ± 13 fmol/mg cell protein at 30 s). Galanin
alone had no significant effect on basal
IP3 production. On the other hand,
preincubation of the cells with PACAP (50 nM) did not affect basal or
bombesin-stimulated IP3 production
(data not shown).

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Fig. 8.
Effect of galanin on bombesin-stimulated inositol-1,4,5-trisphosphate
(IP3) production from STC-1
cells. STC-1 cells were preincubated with or without 10 nM galanin
before incubation in the absence or presence of 5 nM bombesin and
determination of cellular IP3
content as described in METHODS. ,
Basal; , galanin alone; , bombesin alone; , bombesin after
preincubation with galanin. Galanin by itself had no effect on basal
IP3 formation but significantly
inhibited bombesin-stimulated IP3
formation.
|
|
Effect of galanin and bombesin on PACAP-stimulated cAMP production.
We have shown previously that PACAP and VIP stimulated cAMP production
in STC-1 cells (7). Therefore, we explored whether or not the effect of
galanin to inhibit or that of bombesin to enhance PACAP-stimulated
secretin release was mediated via inhibition or enhancement of cAMP
production, respectively. STC-1 cells were preincubated with or without
10 nM galanin or bombesin for 15 min before addition of PACAP (50 nM)
and IBMX and a further incubation of 2 min at 37°C that resulted in
maximal cAMP production (7). As shown in Fig.
9, PACAP stimulated cAMP production by
168% in the presence of IBMX. Preincubation of the cells with galanin resulted in a significant reduction in PACAP-stimulated cAMP production (down to 54% over the control) that corresponded to a 68% inhibition. Galanin alone had no effect on basal cAMP production. As reported previously (7), bombesin did not affect basal or PACAP-stimulated cAMP
production (data not shown).

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Fig. 9.
Effect of galanin on PACAP-stimulated cAMP production in STC-1 cells.
STC-1 cells were preincubated with or without 10 nM galanin before
incubation in the absence or presence of 50 nM PACAP or 0.25 mM IBMX or
both and then cAMP was determined as described in
METHODS. Galanin by itself had no
effect on basal cAMP formation but significantly inhibited
PACAP-stimulated cAMP formation detected in the presence of IBMX.
|
|
Effect of EGTA, diltiazem, and Rp-cAMPS on bombesin- and
PACAP-stimulated secretin release and their synergism.
To determine the involvement of extracellular
Ca2+ and protein kinase A (PKA)
activity on bombesin- and PACAP-stimulated secretin release, the effect
of the Ca2+ chelator EGTA (1 mM),
the L-type voltage-gated Ca2+
channel blocker diltiazem (10 µM), and the PKA-specific inhibitor Rp-cAMPS (100 µM) were studied. As shown in Fig.
10, 5 nM bombesin-stimulated secretin
release was reduced in the presence of EGTA, decreasing from 436 ± 58% to 194 ± 27% over basal with an average inhibition of 59 ± 2% (P < 0.01, n = 4). The inhibited secretin
release, however, remained significantly higher than the basal
secretion (P < 0.01). EGTA also
reduced 50 nM PACAP-stimulated secretin release by 75 ± 13%
(P < 0.01, n = 4), decreasing from 49.0 ± 5%
to 10 ± 6% over basal secretion and thus no longer being
significantly different from basal secretion. As shown in Fig. 10,
diltiazem inhibited bombesin-stimulated secretin release by 64 ± 5% (P < 0.01, n = 11), decreasing from 461 ± 6%
to 172 ± 40% over basal secretion. Again, diltiazem-inhibited
secretion remained significantly greater than basal secretion
(P < 0.05). Diltiazem also inhibited PACAP-stimulated secretin secretion by 55 ± 11%
(P < 0.05, n = 11), reducing it from 52 ± 9%
to 23.4 ± 7% over basal. As shown in Fig.
11, incubation of STC-1 cells with 100 µM Rp-cAMPS resulted in a significant reduction of PACAP-stimulated
secretin release from 69 ± 16% to 26 ± 10% over basal
(P < 0.01, n = 9). Rp-cAMPS also
reduced bombesin-stimulated secretin release by 30 ± 6%
(P < 0.05, n = 9), decreasing from 339 ± 50%
to 239 ± 44% over the basal secretion. Interestingly, none of the
above-mentioned agents prevented potentiation between PACAP and
bombesin, despite their inhibition of the effect of each peptide
individually. Thus, in the presence of EGTA, bombesin plus PACAP
stimulated secretin release by 539 ± 119% over the control (with
EGTA alone) that was significantly greater than the sum of the
individual effects of the two peptides that was 207 ± 37% over the
same control (P < 0.01, n = 3). In the presence of diltiazem,
bombesin plus PACAP increased secretin release by 513 ± 229% over
control (with diltiazem alone), which was significantly greater than
170 ± 52% over the control produced by the sum of the individual
effects of these two peptides with diltiazem.
(P < 0.05, n = 3). In the presence of Rp-cAMPS,
the effect of bombesin plus PACAP was 528 ± 107% over the control
(with Rp-cAMPS alone), which was significantly greater than the sum of
the individual effects of bombesin and PACAP, namely 268 ± 51%
over control (P < 0.05, n = 9).

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Fig. 10.
Effect of EGTA and diltiazem on PACAP- and bombesin-stimulated secretin
release. STC-1 cells were incubated with or without PACAP (50 nM) or
bombesin (5 nM) in the absence or presence of either 1 mM EGTA
(left) or 10 µM diltiazem
(right) to determine secretin
release as described in METHODS. Both
EGTA and diltiazem significantly reduced PACAP- or bombesin-stimulated
SLI release.
|
|

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Fig. 11.
Effect of Rp-cAMPS on PACAP- and bombesin-stimulated secretin release.
STC-1 cells were incubated with PACAP (50 nM) or bombesin (5 nM) in the
absence or presence of 100 µM Rp-cAMPS to determine secretin release
as described in METHODS. Rp-cAMPS
significantly inhibited PACAP- and bombesin-stimulated secretin
release. * P < 0.05 vs. Rp-cAMPS. ** P < 0.01 vs. Rp-cAMPS.
|
|
 |
DISCUSSION |
Study of the cellular mechanisms of secretin secretion from intestinal
endocrine cells has been very limited. The results of previous studies
using secretin-producing cell-enriched mucosal cell preparations
isolated from dog duodenum (48) and rat upper small intestine (11) have
indicated that secretin release from secretin-producing cells is
mediated by Ca2+- and
cAMP-dependent mechanisms and can be elicited by activation of PKC.
However, due to the low yield and heterogeneity of the intestinal
secretin-producing cell preparation, it is difficult to identify the
natural secretagogue that triggers the release of these second
messengers or PKC activation. The discovery of the more homogeneous
secretin-expressing tumor cell lines (41, 47) thus may provide better
models for studying the cellular mechanism of secretin secretion. In
the present study, we have tested the murine intestinal neuroendocrine
tumor cell line STC-1 for use as a model to study secretin release. Our
results have indicated that the release of SLI from STC-1 cells can be
elicited by various pharmacological stimulants, including forskolin,
KCl,
-TPA, and A-23187, and by luminal secretagogues, including
sodium oleate, camostat, and plaunotol. All of these agents have been shown to stimulate the release of secretin either from
secretin-producing cell-enriched mucosal preparations (11, 48) or in
intact animals (10, 12, 25). The observed stimulation of SLI release by forskolin, which activates adenylate cyclase, by A-23187 and KCl, which
increase Ca2+ influx, and by
-TPA, which activates PKC activity, suggested that regulation of
secretin secretion in STC-1 cells, similar to that in the mucosal
secretin-producing cells, is mediated via Ca2+- and cAMP-dependent
mechanisms and through modulation of PKC activity. The responses of
STC-1 cells to sodium oleate, camostat, and plaunotol suggested that
these luminal stimulants can act directly on secretin-producing cells
and it will be possible to use these cells for studying the mechanism
of action by these secretagogues. The observed stimulatory effects of
bombesin, GRP, VIP, and PACAP on SLI secretion suggested that STC-1
cells are also suitable for studying receptor-mediated secretin
release. Indeed, it has been shown that STC-1 cells contain
GRP/bombesin receptors (43). Furthermore, the observation that these
neuropeptides also stimulated secretin release from the mucosal
secretin-producing cell preparation suggested that the stimulation by
these neuropeptides is an intrinsic property of secretin-producing
cells rather than a result of tumorigenic transformation of STC-1
cells. Therefore, it may be concluded that the STC-1 cell line is a
useful model for studying the cellular mechanism of secretin secretion,
despite the fact that this cell line also secrets smaller amounts of
CCK (8), GIP (5), GLP-1 (1), glucagon, and insulin (41).
Previous studies have provided evidence indicating that secretion of
secretin may be subject to neurohormonal regulation. Thus, in cultured
explants of canine duodenal mucosa, the release of SLI was shown to be
stimulated by
-aminobutyric acid that was potentiated by carbamyl
choline but inhibited by somatostatin and Met-enkephalin (34). In rats,
the acid-induced release of secretin is mediated by an SRP (28). It has
been shown that the release and action of SRP are dependent on the
vagal afferent pathways that are independent of cholinergic tone (26).
The noncholinergic neural factor or factors may include a
neuropeptide(s) that acts directly on the secretin-producing cells.
Indeed, it has been shown recently that intravenous infusion of GRP in
pigs (18) and rats (27) and of PACAP (24) in rats resulted in elevation
of plasma secretin concentration. Because both GRP- and
PACAP-containing neurons and nerve fibers exist in the myenteric and
submucous plexus of the upper small intestine (17, 45), it is very
likely that the above-mentioned effects of GRP and PACAP on the release
of secretin may mimic neurocrine actions of these peptides on the
mucosal secretin-producing cells. Similarly, VIP-containing nerve
fibers are known to innervate the intestinal mucosa (16) and may have a
similar effect. To test if these neuropeptides can act directly on
secretin-producing cells, we studied the effects of GRP, bombesin, and
PACAP-27 on secretin secretion from both STC-1 cells and a
secretin-producing cell-enriched mucosal cell preparation isolated from
rat upper small intestine. Our results have demonstrated clearly that
all three peptides can stimulate secretin secretion from the mucosal
cell preparation. This observation suggests that all three peptides can
act directly on mucosal cells. However, due to heterogeneity in the
mucosal cell preparation, it is not certain at present whether these
peptides acted directly on secretin-producing cells or through the
release of SRP and/or another yet unidentified mediator(s). On
the other hand, the observation that all three peptides stimulated
secretion of secretin from STC-1 cells in a concentration- and
time-dependent manner suggests that they definitely can act directly on
secretin-producing cells. We also observed that VIP, which has a high
sequence homology with PACAP and often shares receptors with the latter
peptide, also potently stimulated the release of secretin in STC-1
cells. Therefore, it is very likely that GRP/bombesin and PACAP/VIP
participate in regulation of secretin-producing cell function. In
addition, the release of SLI stimulated by these neuropeptides and
other secretagogues was inhibited by galanin in a
concentration-dependent manner. The effect of galanin appears to be
mediated through a PTx-sensitive G protein-coupled receptor. Moreover,
its inhibitory action appeared to be mediated through inhibition of
bombesin- and PACAP-stimulated production of second messengers. Thus
galanin, which is also abundant in the enteric nervous system (38),
appears to be another candidate neuropeptide involved in the inhibitory regulation of secretin secretion. Therefore, it is likely that the
release of secretin in vivo under various physiological conditions may
be the net effect of the concerted actions of these neuropeptides and
luminal stimulants. This question should be an important and challenging problem for future studies.
Another significant observation of the present study is that there is a
synergistic effect between PACAP and other secretagogues on secretion
of SLI from STC-1 cells. This effect appears to involve a synergistic
effect of different signal cascades. The effect of PACAP on secretin
release was accompanied by production of cAMP, substantially enhanced
by the phosphodiesterase inhibitor IBMX, and inhibited by the
PKA-specific inhibitor Rp-cAMPS, thereby strongly suggesting the
involvement of the cAMP/PKA cascade. In addition, maintenance of
intracellular Ca2+ homeostasis
through influx of extracellular
Ca2+ appears to be as important
because both EGTA and diltiazem inhibited PACAP-stimulated secretin
release. However, these observations should not be regarded as
indicating that PACAP also stimulates secretin secretion via activation
of Ca2+ influx through
Ca2+ channels, including the
L-type Ca2+ channels, although
this could not be ruled out without further study. It should be noted
that several subtypes of adenylate cyclase are known to be either
activated or inhibited by Ca2+ or
Ca2+/calmodulin (15), and that
hormone secretion is a process of regulated exocytosis that is known to
involve an Mg-ATP-dependent priming step before a final step of
Ca2+-dependent secretion (19, 20).
Because we were unable to observe an elevation of intracellular
Ca2+ concentration by PACAP in
STC-1 cells (data not shown), PACAP-stimulated secretin release would
be very likely to be dependent on the extracellular Ca2+ source for this final step of
exocytosis, thereby explaining its inhibition by both EGTA and
diltiazem. In a previous study (7), we demonstrated that PACAP or VIP
was unable to maintain a significantly elevated intracellular cAMP
level in STC-1 cells unless IBMX was present, thereby suggesting a high
level of endogenous phosphodiesterase activity in these cells. It is
possible that a small elevated steady-state level of cAMP stimulated by
PACAP is sufficient to activate a certain subtype(s) of PKA in STC-1 cells to elicit a small but significant stimulation of secretin release. Bombesin has been shown to cause elevation of intracellular Ca2+ concentration that was in
part sensitive to diltiazem (43), and KCl activates voltage-gated
Ca2+ channels (30) in STC-1 cells,
thereby suggesting involvement of a
Ca2+-dependent cascade in their
action on secretin release. Our observation that bombesin stimulated
IP3 production suggests that this
peptide also acts via a receptor coupled to a phosphatidylinositol
phosphate-specific phospholipase C signal cascade that includes a
mobilization of intracellular Ca2+
in response to the production of
IP3 and activation of PKC. In the
present study, we observed that bombesin and PACAP potentiated each
other on secretin release without a synergistic effect on production of
either IP3 or cAMP. Therefore,
their potentiation effect represents synergism between their signal
cascades downstream of the production of these second messengers. We
also observed that synergism between these two peptides was not
affected by EGTA and diltiazem, or by the PKA-specific inhibitor
Rp-cAMPS. These observations appear to suggest that synergism between
these two peptides is not mediated through influx of extracellular
Ca2+ or the activation of PKA
activity, although it cannot be ruled out at present that a small
residual PKA activity is sufficient to potentiate with
bombesin-activated signal molecule(s). The observation of a small but
significant inhibition of bombesin-stimulated secretin release by
Rp-cAMPS appears to indicate that the endogenous PKA activity
participates in interaction with the bombesin-elicited signal cascade
and thus supports this point of view. Nevertheless, the effector(s)
activated by PACAP to involve in potentiation with bombesin remains to
be identified. On the other hand, the observation that
-TPA, a well-known activator of PKC, also potentiates with PACAP to
stimulate secretin release suggests that synergism between bombesin and
PACAP may be in part mediated by PKC. In view of the finding that
bombesin still stimulated secretin release significantly in the
presence of EGTA or diltiazem and elevated intracellular
Ca2+ concentration in the presence
of diltiazem (43), it is very likely that
Ca2+ released from the
intracellular Ca2+ store by
IP3 elevated by bombesin also
participates in the synergistic effect with PACAP. Thus the mechanism
of potentiation between bombesin and PACAP cannot be completely deduced
at present and remains an interesting problem for future study.
Nevertheless, this mode of potentiation appeared to be different from
that recently reported for potentiation between substance P and VIP in
pituitary lactotroph cells (31). In this system, VIP, though
ineffective alone, enhanced IP3
production and translocation of a few subtypes of PKC stimulated by
substance P. Substance P, which had no effect by itself on cAMP
production, also enhanced cAMP formation stimulated by VIP. Thus
synergistic cross talk between
Ca2+ and cAMP signal cascades
appears to be quite diversified and varies among different cell types.
We believe that synergism between neuropeptides with different signal
cascades may be an important characteristic of neuropeptide regulation
of secretin secretion. Thus it is tempting to speculate that
GRP/bombesin and PACAP and possibly VIP may act as coregulators of
secretin secretion under yet to be identified physiological conditions,
whereas under other conditions, these neuropeptides may act
synergistically with luminal stimulants, such as nutrients,
pharmacological agents, and the putative SRP. We believe these are new
and important questions regarding regulation of gut endocrine cells
that merit further study.
 |
ACKNOWLEDGEMENTS |
We thank Frank Roth and David Wagner for technical assistance, Drs.
Andrew Leiter and Seth Grant for providing STC-1 cells, Professor
Haruaki Yajima (retired) (Kyoto University, Kyoto, Japan) for providing
synthetic GRP, and Ono Pharmaceuticals, Inc. and Sankyo
Pharmaceuticals, Inc. (both of Tokyo, Japan) for providing camostat and
plaunotol, respectively.
 |
FOOTNOTES |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-26292 (W. Y. Chey) and DK-18370
(D. H. Coy).
Address for reprint requests: T.-M. Chang, Konar Center for Digestive
and Liver Diseases, Dept. of Medicine, Box 646, Univ. of Rochester
School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY
14642.
Received 3 September 1997; accepted in final form 8 April 1998.
 |
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