Am J Physiol Gastrointest Liver Physiol
280: G88–G94, 2001.
Different actions of secretin and Gly-extended
secretin predict secretin receptor subtypes
TRAVIS E. SOLOMON,1,2 GABOR VARGA,3 NING ZENG,1,2 S. VINCENT WU,1,2
JOHN
H. WALSH†,1,2 AND JOSEPH R. REEVE, JR.1,2
1
CURE: Digestive Diseases Research Center, Department of Veterans Affairs Greater Los Angeles
Healthcare System, Los Angeles 90073; 2Digestive Diseases Division, UCLA School of Medicine,
Los Angeles, California 90024; and 3Institute of Experimental Medicine, Hungarian Academy
of Sciences, H-1450 Budapest, Hungary
Received 4 May 2000; accepted in final form 21 August 2000
posttranslational processing; receptor specificity; D cells; somatostatin
in multiple molecular
forms generated by different degrees of posttranslational processing in their cell of synthesis. This differential processing can regulate the expression of biological activity for a peptide if receptor subtypes exist that
select among the processed forms. One example is the
family of PYY and NPY peptides and receptor subtypes: PYY-(1–36) and the more fully processed form
PYY-(3–36) have different spectra of binding to their
receptor subtypes (13). Preliminary evidence (38) sugMANY REGULATORY PEPTIDES EXIST
† Deceased 14 June 2000.
Address for reprint requests and other correspondence: T. E.
Solomon, Research Service (151), Bldg. 115, Rm. 111, VA Greater
Los Angeles Healthcare System, 11301 Wilshire Blvd., Los Angeles,
CA 90073 (E-mail: [email protected]).
G88
gests that two molecular forms of secretin exist in
tissue and plasma: fully processed secretin [secretin(1–27)-amide] and its immediate precursor, secretinGly. We recently reported (26) that COOH-terminally
extended forms of secretin produced during posttranslational processing of preprosecretin were equipotent
with fully processed secretin for stimulating pancreatic
secretion. This was surprising because it suggests that
the ␣-carboxyl amide group at the COOH terminus of
secretin is not a required structural element for efficient binding and activation of the secretin receptor
that stimulates pancreatic fluid secretion. Only one
secretin receptor has been cloned and characterized by
molecular and pharmacological approaches (28), and
this receptor is thought to mediate the stimulatory
effect of secretin on pancreatic fluid and bicarbonate
secretion. However, the relative potencies of COOHterminally extended secretin forms for binding and
activation of this receptor have not been determined.
Secretin has a wide range of actions, including stimulatory effects on pancreatic and biliary secretion, regulatory peptide release, cardiac and neural activity,
and other functions; conversely, secretin has inhibitory
effects on gastric acid secretion and motility (28, 33). In
rats, secretin is a potent inhibitor of gastric acid secretion (25). Several observations indicate that the inhibitory action of secretin on gastric acid secretion is
mediated by local release of somatostatin from oxyntic
gland area D cells (25). Secretin has been reported to
stimulate somatostatin secretion by cultured human
antral D cells (3), suggesting that D cells may be one
target that bears the inhibitory secretin receptor. The
ability of COOH-terminally extended forms of secretin
to inhibit gastric acid secretion has not been previously
examined.
Although the stimulatory and inhibitory effects of
secretin could be exerted through one receptor, there
are precedents for receptor subtypes that differentially
mediate stimulatory and inhibitory actions of other
agonists (34). We examined the relative potencies of
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http://www.ajpgi.org
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Solomon, Travis E., Gabor Varga, Ning Zeng, S. Vincent Wu, John H. Walsh, and Joseph R. Reeve, Jr.
Different actions of secretin and Gly-extended secretin predict secretin receptor subtypes. Am J Physiol Gastrointest
Liver Physiol 280: G88–G94, 2001.—Only one secretin receptor has been cloned and its properties characterized in native
and transfected cells. To test the hypothesis that stimulatory
and inhibitory effects of secretin are mediated by different
secretin receptor subtypes, pancreatic and gastric secretory
responses to secretin and secretin-Gly were determined in
rats. Pancreatic fluid secretion was increased equipotently by
secretin and secretin-Gly, but secretin was markedly more
potent for inhibition of basal and gastrin-induced acid secretion. In Chinese hamster ovary cells stably transfected with
the rat secretin receptor, secretin and secretin-Gly equipotently displaced 125I-labeled secretin (IC50 values 5.3 ⫾ 0.5
and 6.4 ⫾ 0.6 nM, respectively). Secretin, but not secretinGly, caused release of somatostatin from rat gastric mucosal
D cells. Thus the equipotent actions of secretin and secretinGly on pancreatic secretion appear to result from equal
binding and activation of the pancreatic secretin receptor.
Conversely, secretin more potently inhibited gastric acid
secretion in vivo, and only secretin released somatostatin
from D cells in vitro. These results support the existence of a
secretin receptor subtype mediating inhibition of gastric acid
secretion that is distinct from the previously characterized
pancreatic secretin receptor.
SECRETIN RECEPTOR SUBTYPES
secretin and secretin-Gly on pancreatic secretion and
gastric acid secretion to test the hypothesis that stimulatory and inhibitory actions of secretin are mediated
through different secretin receptors. As a further test
of this hypothesis, we compared the relative potencies
of secretin and secretin-Gly for binding to the rat
pancreatic secretin receptor subtype in stably transfected Chinese hamster ovary (CHO) cells and release
of somatostatin from gastric mucosal D cells in vitro.
These studies revealed distinct differences in the patterns of pancreatic and gastric secretory effects and in
vitro actions of secretin and secretin-Gly, suggesting
the existence of a second secretin receptor subtype.
MATERIALS AND METHODS
exit in the midscapular region, filled with saline containing
10 U/ml heparin, and capped. A stainless steel cannula was
inserted into the forestomach and ligated in place with a
purse-string suture. The cannula was brought through the
abdominal wall to the left of the midline and capped, and the
abdominal incision was closed in two layers. After awakening
from anesthesia, the rats were allowed free access to food and
water. Rats were allowed to recover for at least 5 days before
experiments began, and at least 4 days elapsed between
experiments. After each experiment, the rats were returned
to individual cages and given free access to food and water.
All studies reported were performed on animals that showed
normal weight gain and appeared healthy.
Design of pancreatic and gastric secretory studies. After a
60-min stabilization period, pancreatic juice was collected
during sequential 30-min periods. After two basal collections,
canine secretin (n ⫽ 12 rats) or secretin-Gly (n ⫽ 16 rats) was
administered in ascending doses of 1, 3, 10, 30, 100, 300, and
1,000 pmol 䡠 kg⫺1 䡠 h⫺1; each dose was given for 30 min. The
volume of pancreatic juice was determined gravimetrically
using preweighed collection tubes.
In studies of gastric secretion, rats were fasted 24 h before
each study. To determine the effects of secretin and secretinGly on unstimulated (basal) acid secretion, an intravenous
infusion of 0.15 M NaCl containing 1% BSA was given at 4
ml/h, and gastric juice was collected by gravity drainage in
successive 30-min periods. After three control collections,
canine secretin (n ⫽ 25 rats) or secretin-Gly (n ⫽ 23 rats) was
added to the intravenous infusion to deliver 3, 10, 30, 100,
300, or 1,000 pmol 䡠 kg⫺1 䡠 h⫺1, each dose given sequentially
for 30 min. As a control for time-dependent changes in gastric
secretion, other rats (n ⫽ 22) received only saline plus BSA
for the same total number of collections. The volume of each
collection was recorded, and H⫹ was titrated electrometrically.
The effects of secretin forms on gastrin-stimulated secretion were studied in a group of 11 rats. After three control
collections, gastrin-17 was administered intravenously at a
dose of 1.25 nmol 䡠 kg⫺1 䡠 h⫺1 for 3.5 h. This dose was determined to cause maximal stimulation of gastric acid secretion
in other groups of rats tested with a wide range of gastrin-17
doses. After 90 min of gastrin-17, canine secretin or secretinGly was added to deliver doses of 30, 100, 300, and 1,000
pmol 䡠 kg⫺1 䡠 h⫺1, each for 30 min. On a third experimental
day, only gastrin-17 was given for the same total number of
collections. These doses were selected because other studies
in our laboratory indicated that inhibition of acid secretion
was clear-cut at doses ⬎10 pmol 䡠 kg⫺1 䡠 h⫺1 of secretin. Volume and H⫹ were measured as above.
Characterization of secretin receptor binding. Binding experiments were performed with a CHO cell line expressing
the transfected rat secretion receptor (rSecR-1, a gift from
Dr. Laurence J. Miller, Mayo Clinic, Rochester, MN). Cells
were cultured in poly-L-lysine-coated 24-well plates with
DME-F-12 medium containing 10% fetal calf serum. Cells
were grown to a final density of 1–2 ⫻ 106 cells/well. They
were rinsed twice with PBS (150 mM NaCl, 10 mM sodium
and potassium phosphate, pH 7.2), and 1 ml of cell binding
buffer [DME-F-12 medium, 20 mM HEPES, pH 7.4, 0.1%
(wt/vol) bacitracin, 0.2% (wt/vol) BSA] was then added into
each well. Binding assays were started by adding 125I-secretin-(1–27)-amide (rat peptide, 20 pM, ⬃2,000 Ci/mmol) in the
presence of increasing concentrations of unlabeled rat peptides as indicated. The radiolabeled tracer was prepared,
purified, and stored as described previously (26). After 1-h
incubation at 37°C, cells were washed twice with ice-cold
PBS and solubilized in 1 ml of 1% (vol/vol) Triton X-100 in
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Peptides. Synthetic canine and rat secretins with COOHterminal ␣-carboxyl Val-amide [secretin-(1–27)-amide] or
Val-Gly [secretin-(1–27)-Gly] were synthesized by the UCLA
Peptide Synthesis Core Facility under the direction of one of
the authors (J. R. Reeve, Jr.). The peptides were purified by
HPLC and evaluated by amino acid analysis, microsequencing, mass spectroscopy, and analytical reverse-phase HPLC.
The details of these syntheses and purifications have been
reported (26). Secretin peptides were weighed to the nearest
microgram and dissolved in 2% (vol/vol) acetic acid. Aliquots
of these solutions were stored frozen at ⫺70°C; each aliquot
was thawed only once, and any remainder was discarded
after preparation of daily working solutions. Gastrin-17 was
dissolved in 50 mM NH4OH containing 1% (wt/vol) BSA (RIA
grade, Sigma Chemical, St. Louis, MO), divided into aliquots,
and stored in the same manner as for secretin stocks. The
exact peptide concentrations in secretin stock solutions were
determined by quantitative amino acid analysis, and the
same stock solutions were used for all experiments reported.
For administration, stocks were thawed and diluted to appropriate concentrations in 0.15 M NaCl containing 1% BSA.
The diluted solutions were kept on ice until use within a few
hours. Peptide solutions were administered using syringe
pumps mounted with plastic disposable syringes and polyethylene tubing.
Animal models. Male Sprague-Dawley rats (250–320 g)
were purchased from Sasco (Omaha, NE) and housed in an
American Association for Accreditation of Laboratory Animal
Care-approved facility. Rats were kept in the animal facility
for at least 7 days before use and were maintained under
controlled temperature and humidity on a standard rodent
diet and an equal light-dark cycle.
For pancreatic studies, all animals were deprived of food
for 24 h. Anesthesia was induced by administration of urethan (1.25 g/kg, given as divided im and ip injections). A
jugular vein catheter (PE-50) was inserted, and 0.15 M NaCl
containing 1% (wt/vol) BSA was given intravenously at 1.0
ml/h throughout the experiment. A midline celiotomy was
performed, followed by ligation of the pylorus, ligation of the
biliary duct proximal to its investment by pancreatic tissue,
and insertion of a catheter (PE-50) into the distal bile-pancreatic duct for collection of pure pancreatic juice. Core body
temperature was monitored using a rectal thermocouple and
maintained at 35–37°C with a heating pad and heat lamp.
For gastric studies, rats were fasted for 24 h and then
prepared with gastric cannulas and venous catheters using
aseptic surgical techniques similar to those described previously (31). Anesthesia was induced with pentobarbital sodium (35–50 mg/kg ip). A polyethylene catheter (PE-50) was
inserted into the jugular vein, tunneled subcutaneously to
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G90
SECRETIN RECEPTOR SUBTYPES
Fig. 1. Pancreatic volume measured in response to sequential doses
of canine secretin (n ⫽ 12 rats) or secretin-Gly (n ⫽ 16 rats). Values
shown (means ⫾ SE) are increments (⌬) above basal levels, which
were 22 ⫾ 3 and 19 ⫾ 2 ␮l/30 min for secretin and secretin-Gly
groups, respectively. No significant differences were seen between
the 2 groups at any peptide dose.
with secretin and secretin-Gly during basal (Fig. 3) and
gastrin-stimulated (Fig. 4) conditions.
Secretin strongly and significantly inhibited basal
acid output at doses of 100 pmol 䡠 kg⫺1 䡠 h⫺1 and higher
(Fig. 3); inhibition of acid output was the result of
decreases in secretory volume and acid concentration
(data not shown). In contrast, secretin-Gly did not
inhibit basal acid output at any dose up to 1,000
pmol 䡠 kg⫺1 䡠 h⫺1. Acid output actually increased significantly (P ⬍ 0.05 and P ⬍ 0.01) at several doses of
secretin-Gly (Fig. 3); this reflected significant increases
in secretory volume with no change in concentrations
of acid (data not shown).
Secretin caused marked, dose-related inhibition of
gastrin-induced acid output (Fig. 4). Significant inhibi-
RESULTS
Stimulation of pancreatic secretion. Secretin and secretin-Gly caused indistinguishable patterns of pancreatic fluid secretion over the range of doses administered (Fig. 1). Bicarbonate concentration and output
also showed similar increases in response to both secretin forms, and rat secretin and secretin-Gly produced identical results (data not shown).
Inhibition of acid secretion. The patterns of gastric
acid secretion during basal and gastrin-stimulated conditions were stable and reproducible. This is illustrated
in Fig. 2, which shows the time course of basal and
gastrin-induced acid output in control studies. Unstimulated acid secretion did not vary over the 4-h
period of observation. During intravenous infusion of
1.25 nmol 䡠 kg⫺1 䡠 h⫺1 gastrin-17, acid output increased
about fourfold compared with basal output and was
stable for at least 3 h. These data were used for comparison to patterns of acid secretion in rats treated
Fig. 2. Time-dependent control values for acid secretion during iv
saline (n ⫽ 22 rats) or gastrin-17 (G-17; n ⫽ 11 rats) administration.
Acid output did not vary significantly during iv administration of
saline. Gastrin-17 at 1.25 nmol 䡠 kg⫺1 䡠 h⫺1 caused a smooth increase
in acid output to values 3.7- to 3.9-fold higher than preinfusion
values within the first 60 min of administration. No statistically
significant differences occurred in the last 2 h of gastrin-17 administration compared with the plateau level (calculated as the average
of acid output 60 and 90 min after beginning gastrin-17).
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PBS. Radioactivities of bound (cell lysate) and free (medium)
fractions were counted, and these values were used to calculate specific binding, expressed as a percentage of maximal
binding (with tracer alone). Total binding in these experiments was in the range of 4,000–5,000 cpm per well, whereas
nonspecific binding in the presence of 10⫺6 M secretin was
200–300 cpm. Kinetic constants were calculated by nonlinear
regression curve fitting using a one-site model (Prism 3.0,
GraphPad, San Diego, CA).
Somatostatin release from gastric mucosal endocrine cells.
Rat gastric endocrine cells were isolated by a combination of
elutriation and density gradient centrifugation as described
previously (37). The proportion of immunostained D cells in
this preparation ranges from 12 to 30%. Freshly isolated
endocrine cells were rinsed by gentle centrifugation in
growth medium containing DME-F-12 supplemented with 2
mg/ml BSA, 2.5% FCS, 100 ␮M hydrocortisone, 1% penicillinstreptomycin, 5 mg/ml insulin, 5 mg/ml transferrin, and 5
␮g/ml sodium selenite. Aliquots of the washed cell suspension were placed on glass coverslips precoated with Cell-Tak
and incubated at 37°C for 45 min, allowing cell attachment to
the coverslips. Then 0.8 ml of growth medium was added to
the coverslips in six-well plates. Somatostatin release was
determined after 48-h culture by incubating endocrine cells
in Cell-Tak-precoated coverslips in six-well plates. Growth
medium was replaced 3 h before the release experiments.
After cells were incubated with graded concentrations of rat
secretin and secretin-Gly for 2 h, the test medium was harvested, centrifuged, and stored at ⫺20°C for somatostatin
assay. Radioimmunoassay of somatostatin was performed
with anti-somatostatin antiserum (AB 8401) as described
previously (36). In each experiment, somatostatin secretion
was calculated as the average of six wells exposed to each
peptide concentration.
Calculations and statistical analysis. All data are presented as group means ⫾ SE, with n as the number of rats in
each group. Statistical significance of changes in secretory
outputs was assessed both within and across groups. Oneway repeated-measures analysis of variance was followed by
appropriate repeated-measures individual comparisons
within groups. One-way analysis of variance or Student’s
t-test was used for comparisons across groups; appropriate
corrections were made for multiple comparisons. Statistically
significant changes were accepted at P ⬍ 0.05.
SECRETIN RECEPTOR SUBTYPES
tion occurred at 30 pmol 䡠 kg⫺1 䡠 h⫺1 (P ⬍ 0.01), and acid
output was reduced below basal levels at the highest
infused dose of secretin. Secretin-Gly was markedly
less potent for inhibition of gastrin-induced acid out-
Fig. 4. Acid output in response to maximal gastrin-17 with graded
doses of canine secretin or secretin-Gly given on different days to the
same rats (n ⫽ 11) shown in Fig. 2. Data are shown as change from
average plateau values, which were 235 ⫾ 16 and 260 ⫾ 18 ␮mol/30
min (basal values subtracted) for secretin and secretin-Gly groups,
respectively. All 4 doses of secretin significantly inhibited acid secretion. Inhibitory effects of secretin-Gly were less marked and significantly less than those of secretin at 300 and 1,000 pmol 䡠 kg⫺1 䡠 h⫺1.
Statistical symbols as described in Fig. 3.
Fig. 5. Displacement of 125I-secretin from the rat pancreatic secretin
receptor stably transfected into Chinese hamster ovary cells. Cells
were incubated with tracer alone or increasing concentrations of
nonlabeled rat secretin or secretin-Gly at 37°C for 1 h. Data are
expressed as percentage of maximal binding (tracer alone), and
points represent the means of duplicate determinations from 3 experiments. IC50 values for secretin-amide and secretin-Gly were
estimated to be 5.3 ⫾ 0.5 and 6.4 ⫾ 0.6 nM, respectively.
put, being significantly less potent than secretin at
doses of 300 and 1,000 pmol 䡠 kg⫺1 䡠 h⫺1 (Fig. 4). In other
experiments, a dose of 10 nmol 䡠 kg⫺1 䡠 h⫺1 of secretinGly was required for complete inhibition of submaximally stimulated (100 pmol 䡠 kg⫺1 䡠 h⫺1 of gastrin-17)
acid secretion (data not shown). Identical results were
obtained when rat secretin and secretin-Gly were compared under similar conditions.
Secretin receptor binding. As seen in Fig. 5, secretin
and secretin-Gly were equipotent for displacing 125Isecretin from CHO cells bearing the stably transfected
rat secretin receptor. The dissociation constant values
calculated for the two peptides were 5.3 ⫾ 0.5 and
6.4 ⫾ 0.6 nM.
Somatostatin release from gastric mucosal D cells.
Figure 6 demonstrates the effects of secretin and secretin-Gly on somatostatin release from a partially
Fig. 6. Effects of rat secretin and secretin-Gly on somatostatin release by partially purified rat gastric mucosal endocrine cells. Values
shown represent somatostatin increase as percentage of concentration in wells not exposed to peptides. Secretin (n ⫽ 4 separate
preparations) increased somatostatin secretion, whereas secretinGly (n ⫽ 3) was ineffective over the range of concentrations tested.
Each point represents the mean ⫾ SE, and the average of 6 wells for
each condition was determined in each preparation.
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Fig. 3. Effects of canine secretin and secretin-Gly on nonstimulated
gastric acid output. Data are shown as change from average plateau
values, which were 86 ⫾ 7 and 55 ⫾ 6 ␮mol/30 min for secretin (n ⫽
25 rats) and secretin-Gly (n ⫽ 23 rats) groups, respectively. *P ⬍
0.05, ** P ⬍ 0.01 vs. plateau values. Secretin significantly inhibited
basal acid output at doses of 100 pmol 䡠 kg⫺1 䡠 h⫺1 and higher, whereas
secretin-Gly slightly increased acid output. This resulted in marked
differences between the two groups (⫹⫹P ⬍ 0.01).
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SECRETIN RECEPTOR SUBTYPES
purified preparation of rat gastric mucosal endocrine
cells. In contrast to their similar patterns of displacement of radiolabeled secretin from the rat secretin
receptor, there were striking differences in D cell somatostatin release by the two peptides. Secretin increased somatostatin release by nearly threefold at a
concentration of 10⫺7 M, whereas secretin-Gly was
ineffective at concentrations up to 10⫺6 M (Fig. 6).
DISCUSSION
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The results of these studies can be summarized as
follows: secretin and secretin-Gly were equipotent ligands for binding to the transfected rat pancreatic
secretin receptor and for stimulating pancreatic fluid
secretion in vivo, and secretin was significantly more
potent than secretin-Gly for releasing somatostatin
from gastric mucosal D cells in vitro and for inhibiting
basal and gastrin-stimulated acid secretion in vivo.
The most plausible explanation for these findings is the
existence of secretin receptor subtypes. The subtype
that stimulates pancreatic fluid secretion is likely to be
the previously described pancreatic receptor (29),
which we demonstrate here as recognizing secretin and
secretin-Gly equally. A second receptor is preferentially activated by secretin and causes inhibition of
gastric acid secretion. Other explanations for the different actions of secretin and secretin-Gly on gastric
secretion, such as differences in metabolism of the two
peptides or effects on non-secretin receptors, are unlikely.
We were not able to measure plasma levels of secretin-Gly to ensure that similar doses of secretin and
secretin-Gly resulted in similar circulating concentrations. However, several points suggest that differences
in metabolism of secretin and secretin-Gly probably do
not account for the observed effects on pancreatic and
gastric function. Fully processed and COOH-terminally extended secretins have equal or greater potency
for stimulating pancreatic secretion in vivo (26). In
contrast, secretin inhibited but secretin-Gly stimulated basal acid output, and secretin was distinctly
more effective in inhibiting gastrin-induced acid secretion. These observations would require organ-specific
differences in inactivation of the two forms of secretin
if peptide degradation, rather than activation of different secretin receptor subtypes, was responsible for the
two patterns of activity. A more plausible explanation
for these disparate actions of fully processed and
COOH-terminally extended secretin is that two receptors with different patterns of recognition are involved
in stimulation of pancreatic fluid secretion on one hand
and inhibition of gastric acid secretion on the other.
It is also highly unlikely that the actions of fully
processed and COOH-terminally extended secretin
were mediated by nonspecific actions on other receptors, such as those for vasoactive intestinal polypeptide
(VIP) or pituitary adenylate cyclase-activating peptide
(PACAP). A specific secretin receptor has been cloned
from a rat pancreatic cDNA library (14, 29). This secretin receptor is localized to pancreatic duct and aci-
nar cells (30) and almost certainly mediates the direct
stimulatory effects of secretin on bicarbonate secretion
by duct cells and enzyme secretion by acinar cells.
Neither VIP nor PACAP is a potent stimulant of pancreatic secretion in the rat (1, 16). The crucial questions for the hypothetical existence of a different secretin receptor that mediates inhibition of gastric acid
secretion involve the characteristics of secretin-induced inhibition of acid secretion and the localization
(and thus the potential mechanism of action) of such a
receptor. In rats, secretin is a potent inhibitor of gastric
acid secretion under certain conditions (25); VIP does
not inhibit acid secretion in rats (18), and PACAP is a
weak inhibitor that acts indirectly (17, 18).
The mechanisms by which secretin inhibits gastric
acid secretion appear to be indirect via release of somatostatin and prostaglandins. The effects of secretin
on acid secretion are blocked by immunoneutralization
of somatostatin and by inhibition of prostaglandin synthesis (25). Secretin increases gastric venous effluent
levels of somatostatin (5–7, 24, 35) and prostaglandin
E2 (7). These observations indicate that the inhibitory
action of secretin on gastric acid secretion is mediated
by local release of somatostatin by gastric mucosal D
cells and prostaglandin E2 by unknown cells. Secretin
has been reported to stimulate somatostatin secretion
by cultured human antral D cells (3), suggesting that D
cells may be one target that bears the inhibitory secretin receptor. Our finding that secretin— but not secretin-Gly—induced somatostatin release from partially
purified rat mucosal D cells supports this hypothesis.
However, we observed that secretin-Gly was weaker
but not completely ineffective for inhibition of gastric
acid secretion. This suggests that other inhibitory
mechanisms such as prostaglandin generation may be
activated by both molecular forms of secretin.
With Northern blot analysis of mRNA, the single rat
secretin receptor that has been cloned to date (14, 29)
has been localized to several tissues including the
stomach (14). More precise definition of the cell types
bearing this receptor is not currently available. The
existence of secretin receptors on chief cells (23), fundic
or antral D cells (3, 5–7, 24, 35), mucus-secreting cells
(15), and forestomach smooth muscle cells (27) in rats
is suggested by experimental data showing direct actions of secretin on the isolated stomach, mucosa, or
cell populations in vitro. All of these experimental
results were obtained using only fully processed secretin, because COOH-terminally extended secretin forms
have not been available for characterization of their
bioactivities.
Structure-activity relationships for binding and activation of the secretin receptor have been investigated
using the cloned pancreatic receptor subtype. When
this receptor or chimeric receptors composed of portions of the secretin, VIP, and glucagon receptors are
studied, deletion of the NH2-terminal histidyl residue
of secretin reduces binding and activation by 1,000-fold
(32), indicating a critically important role of the NH2terminal structure of secretin for binding and activation. For the pancreatic secretin receptor, the COOH-
SECRETIN RECEPTOR SUBTYPES
tional processing of the secretin receptor has in fact
been shown to alter patterns of secretin binding and
activation (20). It is at least theoretically possible that
this could also result in different agonist selectivity of
the known secretin receptor. It is also theoretically
possible that tissue-specific differences in G protein
coupling could alter receptor selectivity for different
molecular forms of secretin.
Receptor subtypes provide the potential to select
among structurally related agonists that are produced
by different genes (i.e., the CCK-A receptor selects
between CCK and gastrin) or by posttranslational processing of a single gene product [i.e., the Y1 receptor
selects between NPY/PYY-(1–36) and NPY/PYY-(3–
36)]. Our preliminary characterization of the relative
amounts of secretin molecular forms in rat intestine
indicates that secretin-Gly is present in at least 50% of
the amount of secretin (38). The ratio of the two forms
may be regulated by physiological conditions (postnatal development, feeding, acid secretion). These considerations suggest that the existence of secretin receptor
subtypes could be functionally significant for selecting
between secretin and secretin-Gly for actions on pancreatic and gastric secretion, as well as other potential
targets of secretin. The use of secretin and secretin-Gly
as pharmacological probes should facilitate the identification of putative secretin receptor subtypes and
their participation in gastrointestinal and other regulatory events mediated by secretin.
The excellent technical assistance of Louis Bussjaeger and Peter
Chew is gratefully acknowledged.
This research was supported by the CURE Digestive Diseases
Research Center Grant DK-41301 and utilized the Peptide Biochemistry and Molecular Probes, Animal Model, and Antibody Center
Cores. The research was also supported by the Medical Research
Service of the Veterans Health Service.
REFERENCES
1. Alonso RM, Rodriguez AM, Garcia LJ, Lopez MA, and
Calvo JJ. Comparison between the effects of VIP and the novel
peptide PACAP on the exocrine pancreatic secretion of the rat.
Pancreas 9: 123–128, 1994.
2. Bonetto V, Jornvall H, Mutt V, and Sillard R. Two alternative processing pathways for a preprohormone: a bioactive form
of secretin. Proc Natl Acad Sci USA 92: 11985–11989, 1995.
3. Buchan AM, Meloche RM, Kwok YN, and Kofod H. Effect of
cholecystokinin and secretin on somatostatin release from cultured antral cells. Gastroenterology 104: 1414–1419, 1993.
4. Carlquist M and Rokaeus A. Isolation of a proform of porcine
secretin by ion-exchange and reversed-phase high-performance
liquid chromatography. J Chromatogr 296: 143–151, 1984.
5. Chiba T, Taminato T, Kadowaki S, Abe H, Chihara K,
Seino Y, Matsukura S, and Fujita T. Effects of glucagon,
secretin, and vasoactive intestinal polypeptide on gastric somatostatin and gastrin release from isolated perfused rat stomach.
Gastroenterology 79: 67–71, 1980.
6. Chiba T, Taminato T, Kadowaki S, Inoue Y, Mori K, Seino
Y, Abe H, Chihara K, Matsukura S, Fujita T, and Goto Y.
Effects of various gastrointestinal peptides on gastric somatostatin release. Endocrinology 106: 145–149, 1980.
7. Chung I, Li P, Lee K, Chang T, and Chey WY. Dual inhibitory mechanism of secretin action on acid secretion in totally
isolated, vascularly perfused rat stomach. Gastroenterology 107:
1751–1758, 1994.
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terminal region of secretin also appears to be involved
in receptor recognition, although to a lesser degree;
peptides with deletions of the COOH-terminal Valamide or Gly-Leu-Val-amide were only 10- and 50-fold
less potent than intact secretin-(1–27)-amide (12). Recognition of the NH2-terminal histidyl residue of secretin by the pancreatic secretin receptor subtype also
appears to be sensitive to addition of amino acid residues, at least as judged by the distinctly lower in vivo
bioactivity of an alternatively processed secretin form
with an NH2-terminal nonapeptide extension and
COOH-terminal amidation (2). In contrast, COOH-terminal extensions of secretin do not reduce its pancreatic secretory bioactivity in vivo (present results and
Refs. 4, 9, 10, 26) or binding (present results) and
functional activation (19) of the pancreatic secretin
receptor in vitro. The secretin receptor that mediates
inhibition of gastric acid secretion appears to be very
specific for the presence of an ␣-carboxyl amide group.
We show here that addition of a COOH-terminal glycyl
residue markedly reduces the acid inhibitory potency
of secretin, and in unpublished studies we observed
that the free acid form of secretin was also clearly less
potent for this action. The acid-inhibitory effect of
secretin is also affected by the NH2-terminal structure
of the peptide, because secretin-(5–27) is ineffective in
blocking gastrin-induced acid secretion in rats (unpublished observation). Finally, it has been shown that
residues 8–15 of secretin are important for the transfected pancreatic receptor to distinguish secretin from
PACAP (11), suggesting that this region should also be
evaluated for differences between the pancreatic and
acid-inhibitory secretin receptor subtypes.
The data presented here indicate that there are at
least two secretin receptor subtypes with different selectivities for fully processed vs. COOH-terminally extended secretins. In addition, the equipotency of secretin and secretin-Gly for binding to the cloned and
transfected rat pancreatic secretin receptor suggests
that previously described COOH-terminally extended
molecular forms of secretin may be of physiological
importance. There are several possible mechanisms
that could result in the existence of a secretin receptor
subtype with different selectivities for secretin molecular forms. First, there could be two structurally different receptors produced by two different genes. In
general, most families of receptor subtypes appear to
be produced by this mechanism, and members of these
families show a substantial degree of structural homology at the nucleotide and amino acid levels (8). The
presence of multiple bands on Northern blot analysis of
various tissues with a secretin receptor probe is indirect evidence that structurally related receptors might
indeed exist (21). Second, there may be only a single
secretin receptor gene product that undergoes differential mRNA splicing, posttranslational processing, or
G protein coupling to result in tissue-specific differences in agonist selectivity. Different patterns of
mRNA splicing can produce distinct functional differences in receptors, as exemplified by splice variants of
the PACAP receptor (22). The degree of posttransla-
G93
G94
SECRETIN RECEPTOR SUBTYPES
24. Saffouri B, DuVal JW, Arimura A, and Makhlouf GM.
Effects of vasoactive intestinal peptide and secretin on gastrin
and somatostatin secretion in the perfused rat stomach. Gastroenterology 86: 839–842, 1984.
25. Shimizu K, Li P, Lee KY, Chang TM, and Chey WY. The
mechanism of inhibitory action of secretin on gastric acid secretion in conscious rats. J Physiol (Lond) 488: 501–508, 1995.
26. Solomon TE, Walsh JH, Bussjaeger L, Zong Y, Hamilton
JW, Ho FJ, Lee TD, and Reeve JR Jr. COOH-terminally
extended secretins are potent stimulants of pancreatic secretion.
Am J Physiol Gastrointest Liver Physiol 276: G808–G816, 1999.
27. Steiner TS, Mangel AW, McVey DC, and Vigna SR. Secretin
receptors mediating rat forestomach relaxation. Am J Physiol
Gastrointest Liver Physiol 264: G863–G867, 1993.
28. Ulrich CD, Holtmann M, and Miller LJ. Secretin and vasoactive intestinal peptide receptors: members of a unique family
of G protein-coupled receptors. Gastroenterology 114: 382–397,
1998.
29. Ulrich CD, Pinon DI, Hadac EM, Holicky EL, Chang-Miller
A, Gates LK, and Miller LJ. Intrinsic photoaffinity labeling of
native and recombinant rat pancreatic secretin receptors. Gastroenterology 105: 1534–1543, 1993.
30. Ulrich CD, Wood P, Hadac EM, Kopras E, Whitcomb DC,
and Miller LJ. Cellular distribution of secretin receptor expression in rat pancreas. Am J Physiol Gastrointest Liver Physiol
275: G1437–G1444, 1998.
31. Varga G, Campbell DR, Bussjaeger LJ, and Solomon TE.
Role of gastrin and cholecystokinin receptors in regulation of
peptone-stimulated gastric acid secretion in conscious rats. Eur
J Pharmacol 250: 37–42, 1993.
32. Vilardaga JP, Ciccarelli E, Dubeaux C, De Neef P, Bollen
A, and Robberecht P. Properties and regulation of the coupling to adenylate cyclase of secretin receptors stably transfected
in Chinese hamster ovary cells. Mol Pharmacol 45: 1022–1028,
1994.
33. Walsh JH. Gastrointestinal hormones. In: Physiology of the
Gastrointestinal Tract, edited by Johnson LR. New York: Raven,
1994, p. 1–128.
34. Wiley JW, Gross RA, and MacDonald RL. Agonists for neuropeptide Y receptor subtypes NPY-1 and NPY-2 have opposite
actions on rat nodose neuron calcium currents. J Neurophysiol
70: 324–330, 1993.
35. Wolfe MM, Reel GM, and McGuigan JE. Inhibition of gastrin
release by secretin is mediated by somatostatin in cultured rat
antral mucosa. J Clin Invest 72: 1586–1593, 1983.
36. Yamada T, Solomon TE, Petersen H, Levin SR, Lewin K,
Walsh JH, and Grossman MI. Effects of gastrointestinal
polypeptides on hormone content of endocrine pancreas in the
rat. Am J Physiol Gastrointest Liver Physiol 238: G526–G530,
1980.
37. Zeng N, Walsh JH, Kang T, Helander KG, Helander HF,
and Sachs G. Selective ligand-induced intracellular calcium
changes in a population of rat isolated gastric endocrine cells.
Gastroenterology 110: 1835–1846, 1996.
38. Zong Y, Reeve JR Jr, Walsh JH, Gong P, Ho FJ, and
Solomon TE. Secretin-Gly is a major product of preprosecretin
processing in rat small intestine: identification and quantitation
by HPLC and radioimmunoassay (Abstract). Gastroenterology
114: A1197, 1998.
Downloaded from http://ajpgi.physiology.org/ by 10.220.32.247 on July 28, 2017
8. Darlison MG and Richter D. Multiple genes for neuropeptides
and their receptors: co-evolution and physiology. Trends Neurosci 22: 81–88, 1999.
9. Gafvelin G, Carlquist M, and Mutt V. A proform of secretin
with high secretin-like bioactivity. FEBS Lett 184: 347–352,
1985.
10. Gafvelin G, Jornvall H, and Mutt V. Processing of prosecretin: isolation of a secretin precursor from porcine intestine. Proc
Natl Acad Sci USA 87: 6781–6785, 1990.
11. Gourlet P, Vilardaga JP, De Neef P, Vandermeers A, Waelbroeck M, Bollen A, and Robberecht P. Interaction of amino
acid residues at positions 8–15 of secretin with the N-terminal
domain of the secretin receptor. Eur J Biochem 239: 349–355,
1996.
12. Gourlet P, Vilardaga JP, De Neef P, Waelbroeck M, Vandermeers A, and Robberecht P. The C-terminus ends of
secretin and VIP interact with the N-terminal domains of their
receptors. Peptides 17: 825–829, 1996.
13. Grandt D, Teyssen S, Schimiczek M, Reeve JR Jr, Feth F,
Rascher W, Hirche H, Singer MV, Layer P, and Goebell H.
Novel generation of hormone receptor specificity by amino terminal processing of peptide YY. Biochem Biophys Res Commun
186: 1299–1306, 1992.
14. Ishihara T, Nakamura S, Kaziro Y, Takahashi T, Takahashi K, and Nagata S. Molecular cloning and expression of a
cDNA encoding the secretin receptor. EMBO J 10: 1635–1641,
1991.
15. Keates AC and Hanson PJ. Regulation of mucus secretion by
cells isolated from the rat gastric mucosa. J Physiol (Lond) 423:
397–409, 1990.
16. Lee ST, Lee KY, Li P, Coy D, Chang TM, and Chey WY.
Pituitary adenylate cyclase-activating peptide stimulates rat
pancreatic secretion via secretin and cholecystokinin releases.
Gastroenterology 114: 1054–1060, 1998.
17. Li P, Chang T, Coy D, and Chey WY. Inhibition of gastric acid
secretion in rat stomach by PACAP is mediated by secretin,
somatostatin, and PGE2. Am J Physiol Gastrointest Liver
Physiol 278: G121–G127, 2000.
18. Mungan Z, Ozmen V, Ertan A, and Arimura A. Pituitary
adenylate cyclase activating polypeptide-27 (PACAP-27) inhibits
pentagastrin-stimulated gastric acid secretion in conscious rats.
Regul Pept 38: 199–206, 1992.
19. Ng SS, Pang RT, Chow BK, and Cheng CH. Real-time evaluation of human secretin receptor activity using cytosensor microphysiometry. J Cell Biochem 72: 517–527, 1999.
20. Pang RT, Ng SS, Cheng CH, Holtmann MH, Miller LJ, and
Chow BK. Role of N-linked glycosylation on the function and
expression of the human secretin receptor. Endocrinology 140:
5102–5111, 1999.
21. Patel DR, Kong Y, and Sreedharan SP. Molecular cloning
and expression of a human secretin receptor. Mol Pharmacol 47:
467–473, 1995.
22. Pisegna JR and Wank SA. Cloning and characterization of the
signal transduction of four splice variants of the human pituitary adenylate cyclase activating polypeptide receptor. Evidence for dual coupling to adenylate cyclase and phospholipase
C. J Biol Chem 271: 17267–17274, 1996.
23. Raufman JP, Kasbekar DK, Jensen RT, and Gardner JD.
Potentiation of pepsinogen secretion from dispersed glands from
rat stomach. Am J Physiol Gastrointest Liver Physiol 245: G525–
G530, 1983.