COOH-terminally extended secretins are potent stimulants of
pancreatic secretion
Travis E.
Solomon1,
John H.
Walsh1,
Louis
Bussjaeger2,
Yumei
Zong1,
James W.
Hamilton2,
F. J.
Ho1,
Terry D.
Lee3, and
Joseph R.
Reeve Jr.1
1 CURE: Digestive
Diseases Research Center, Greater Los Angeles Department of Veterans
Affairs Health Care System, Los Angeles 90073; Digestive Diseases
Division, University of California, Los Angeles, School of
Medicine, Los Angeles, California 90024;
2 Kansas City Department of
Veterans Affairs Medical Center, Kansas City, Missouri 64128;
Department of Biochemistry, Kansas University Medical Center, Kansas
City, Kansas 66160; and 3 Division
of Immunology, Beckman Research Institute, The City of Hope
Research Institute, Duarte, California 91010
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ABSTRACT |
Posttranslational
processing of preprosecretin generates several COOH-terminally extended
forms of secretin and
-carboxyl amidated secretin. We used synthetic
canine secretin analogs with COOH-terminal -amide, -Gly, or
-Gly-Lys-Arg to examine the effects of COOH-terminal extensions of
secretin on bioactivity and detection in RIA. Synthetic products were
purified by reverse-phase and ion-exchange HPLC and characterized by
reverse-phase isocratic HPLC and amino acid, sequence, and mass
spectral analyses. Secretin and secretin-Gly were noted to coelute
during reverse-phase HPLC. In RIA using eight different antisera raised
against amidated secretin, COOH-terminally extended secretins had
little or no cross-reactivity. Bioactivity was assessed by measuring
pancreatic responses in anesthetized rats. Amidated canine and porcine
secretins were equipotent. Secretin-Gly and secretin-Gly-Lys-Arg had
potencies of 81 ± 9% (P > 0.05)
and 176 ± 13% (P < 0.01),
respectively, compared with amidated secretin, and the response to
secretin-Gly-Lys-Arg lasted significantly longer. These data
demonstrate that 1) amidated secretin and secretin-Gly are not separable under some chromatographic conditions, 2) current RIA may not
detect bioactive COOH-terminally extended forms of secretin in tissue
extracts or blood, and 3) the
secretin receptor mediating stimulation of pancreatic secretion recognizes both amidated and COOH-terminally extended secretins.
posttranslational processing; amidation; radioimmunoassay; physiology
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INTRODUCTION |
COOH-TERMINAL AMIDATION occurs during posttranslational
processing of many bioactive peptides, and the resulting structure is
generally thought to be important for binding and activation of
specific receptors for these peptides. This observation was the
theoretical basis for a method of detection and purification of new gut
and brain regulatory peptides (38, 39). One of the
examples used to derive the underlying hypothesis was secretin, because
it was first purified based on its ability to stimulate pancreatic
bicarbonate secretion and found to be a peptide with a COOH-terminal
valine-amide (28). However, apparent exceptions to this requirement for
COOH-terminal amidation for bioactivity of secretin have been reported.
Secretin synthesized by a bacterial expression system incapable of
amidating the COOH terminus of the peptide (35) stimulated pancreatic
secretion in rats. In addition, three COOH-terminally extended forms of
secretin have been isolated from porcine intestinal extracts using an
in vivo bioassay (stimulation of pancreatic secretion in anesthetized cats) to follow the steps of purification (2, 14, 15).
These three COOH-terminally extended peptides correspond to sequential
steps in the posttranslational processing of preprosecretin that
ultimately produce the COOH-terminal amidated form. Figure 1 shows a scheme for porcine preprosecretin
processing. Cleavage of the signal peptide and an
NH2-terminal flanking peptide from preprosecretin would produce an intermediate form consisting of secretin extended by an amidation region (Gly-Lys-Arg-) and the COOH-terminal flanking peptide; this 71-amino acid form has been purified from porcine intestine and shown to be bioactive (15). The
next step in processing probably involves a trypsinlike peptidase specific for pairs of basic residues that cleaves at the COOH side of
the amidation region and yields secretin-Gly-Lys-Arg. A
carboxypeptidase then removes the arginine and lysine residues to give
secretin-Gly. Both of these extended forms have also been purified from
porcine intestine, and the former is reported to be bioactive (2, 14).
In a final step, peptide amide monoamine oxygenase then would produce
secretin with a COOH-terminal valine-amide (8, 9), the molecular form
that was first isolated and sequenced (28). The processing events
described are similar to those seen for other amidated peptides and are
likely to be the steps for processing all mammalian secretins, because
the structures of rat and mouse preprosecretins predicted from cloned
cDNA also have similar amino acid consensus sequences (23, 24). In
addition, an alternative pathway for secretin processing may exist;
isolation of an NH2-terminally
extended, amidated form of secretin (1) suggests that the steps
described above may occur before cleavage of the
NH2-terminal flanking region. The
presence of bioactive COOH-terminally extended forms of secretin in
intestinal extracts raises the possibility that these incompletely
processed peptides might be cosecreted with amidated secretin and
contribute to the physiological actions of secretin.

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Fig. 1.
Two pathways for posttranslational processing of preprosecretin, based
on predicted structure from cDNA of porcine secretin gene product and
structures of peptides purified from porcine intestinal extracts.
Complete sequence of signal peptide is not known but contains at least
20 residues. Signal peptidase is presumed to act on bond between
-Ala-Arg- in positions 10 and 9 upstream from
NH2 terminus of secretin, because
a peptide consisting of amidated secretin with a 9-residue
NH2-terminal flanking sequence has
been purified and chemically characterized. After action of signal
peptidase, prosecretin (80 residues) appears to enter alternative
pathways. In one, NH2-terminal
flanking region is cleaved, leaving a 71-residue peptide with secretin
at its NH2 terminus. Next three
steps occur sequentially and are similar for all peptides with a
COOH-terminal amide. Cleavage after a pair of basic residues yields a
peptide extended beyond its eventual amidation point by -Gly-Lys-Arg.
Carboxypeptidase removes two basic residues in sequence. Remaining
COOH-terminal glycine is then cleaved by monoamine oxidase, producing
amidated peptide and oxylate. In the second pathway, it cannot be
determined whether similar COOH-terminal processing steps occur
completely before eventual cleavage of
NH2-terminal flanking region or
that such cleavage may occur after any step.
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However, there is no information on the relative amounts of
COOH-terminally extended vs. amidated secretin in intestinal mucosa or
blood. Most studies on the molecular forms of secretin in the circulation or intestinal extracts have found a single peptide that
appeared to be identical to amidated secretin on the basis of its
elution pattern in various chromatographic systems (32, 34, 36). In
these studies, secretin was detected by RIA based on antisera raised
against amidated porcine secretin. Most such antisera have been
reported to recognize the COOH-terminal region of secretin
preferentially when used in RIA (5), consistent with the general
observation that COOH-terminal amidation confers highly selective
immunogenic properties on this structure in many peptides. If
COOH-terminal amidation of secretin is required for peptide
cross-reactivity in currently available RIA, it would be impossible to
detect the existence of COOH-terminally extended forms of secretin in
blood or tissue extracts.
None of the studies described above has provided a detailed analysis of
the bioactivity of COOH-terminally extended forms of secretin, nor is
there any information on the cross-reactivity of such forms in
currently available secretin RIA. These molecular forms have not been
previously synthesized or isolated from natural sources in sufficient
quantity to perform such characterizations. An advantage of performing
experiments with synthetic peptides is the lack of contamination by
other bioactive substances, including other molecular forms of the
peptide being evaluated. Thus the goals of the experiments described
here were to synthesize secretin, secretin-Gly, and
secretin-Gly-Lys-Arg and to use these peptides for comparisons of the
bioactivity and immunoreactivity of amidated secretin to its extended forms.
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MATERIALS AND METHODS |
Peptide synthesis.
Peptides were synthesized on a Biosearch 9500 peptide synthesizer
(San Rafael, CA) using 9-fluorenylmethyloxycarbonyl (Fmoc) coupling
strategy. The Fmoc amino acid derivatives Ala, Arg(Mtr), Asp(OtBu),
Glu(OtBu), Gly, Leu, Lys(BOC), Phe, Ser(tBu), Thr(tBu), Val, and
Tyr(OtBu) were purchased from Bachem California (Torrance, CA). The
Fmoc amino acid derivatives His(Bzl) and Gln(Tmob) were purchased from
MilliGen/Biosearch (Milford, MA). Supports for synthesis were
PAL for amidated secretin,
Fmoc-L-Gly-PAC for
secretin-Gly, and
Fmoc-L-Arg(Mtr)-PAC for
secretin-Gly-Lys-Arg and were purchased from MilliGen/Biosearch. The
supports were placed in the reaction vessel, and coupling was
accomplished by the procedure recommended by the manufacturer. The
crude peptide was removed from the resin with 90% trifluoroacetic acid
(TFA) containing 5% thioanisole, 3% ethanedithiol, and 2% anisole.
Peptide purification.
Crude synthetic peptides were dissolved in 0.1% TFA (50 mg/10 ml), and
a small portion (100-300 µg) was injected onto an analytical Vydac C18 column (The Separations
Group, Hesperia, CA; 4.6 mm × 25 cm, 5 µm) equilibrated in
0.1% TFA and eluted with a linear gradient to 50% acetonitrile
containing 0.1% TFA over 100 min. Preparative amounts (200-300
mg) of peptides were injected onto a Dynamax
C8 column (Rainin Instruments,
Emeryville, CA; 2.14 × 25 cm, 12 µm) equilibrated in 0.1% TFA;
the gradient for the preparative purification was based on the position
of the major absorbance peak eluted during the analytical
chromatography. For secretin-Gly-Lys-Arg, an additional preparative
reverse-phase step on a Vydac C18
column (The Separations Group; 2.2 × 25 cm, 10 µm), preparative
strong cation exchanger (Ranin Hydropore SCX 10 mm × 10 cm, 5 µm), and preparative reverse-phase step on a Vydac
C18 column were required to obtain
a peptide of >95% purity.
Purity analysis of synthetic peptides.
Peptides purified on the preparative reverse-phase HPLC were
characterized by analytical reverse-phase HPLC. Analyses were performed
immediately after synthesis and after bioactivity experiments to
determine the stability of peptides during storage at
80°C. The results shown are those after the activity experiments. Secretin, secretin-Gly, and secretin-Gly-Lys-Arg were injected onto an analytical Vydac C18 column equilibrated with
0.1% TFA (buffer A). The peptides were eluted with a linear gradient to 100% buffer
B (70% acetonitrile containing 0.1% TFA) over 70 min
on a Gilson HPLC system (Middleton, WI). The eluent was monitored at
214 nm with a Gilson model 119 variable wavelength detector with a
preparative flow cell (0.2 mm path length, 0.7 µl volume). Data were
collected and plotted with Gilson Unipoint Software.
Amino acid analysis.
Purified peptides (20-50 µg) were hydrolyzed with vaporized HCl
at 105°C for 20 h. The HCl was removed by vacuum, and the amino
acids were dissolved in Beckman dilution buffer. The dissolved amino
acids were chromatographed on a Beckman 9300 amino acid analyzer (Palo
Alto, CA) and quantitated by absorbance after reaction with ninhydrin.
Sequence analysis.
Purified peptides were characterized by microsequence analysis using an
Applied Biosystems 470 gas phase sequencer (Foster City, CA). The
phenylthiohydantoin (PTH) derivatives were analyzed by
reverse-phase HPLC.
Mass spectral analysis.
Purified peptides were dissolved in a few microliters of dimethyl
sulfoxide. A mixture of dithiothreitol-dithioerythreitol (5:1) and
camphor sulfonic acid (6 mM) was used as a sample matrix. Approximately
2 µl of the sample solution were added to 1 µl of sample matrix on
a 1.5 × 6 mm sample stage. Positive ion spectra were obtained
using a JEOL HX-100HF high-resolution double-focusing magnetic sector
mass spectrometer (Peabody, MA) operating at 5-kV accelerating
potential. Sample ionization was accomplished using a 6-keV xenon atom
beam. A JEOL DA5000 data system was used to control instrument
parameters and to collect spectral data.
Radioimmunoassay.
Eight antisera were used to study the properties of synthetic canine
secretin forms in RIA. Antisera were generated in New Zealand White
rabbits immunized with synthetic porcine secretin (E. R. Squibb and
Sons, Princeton, NJ) coupled to bovine serum albumin with carbodiimide;
three of the antisera were generated at the Kansas City Veterans
Affairs Medical Center, whereas the remaining five antisera were
generated at University of California, Los Angeles. Each antiserum was
used for RIA with amidated rat 125I-secretin as a radiolabeled
tracer. Amidated synthetic porcine, canine, and rat secretins gave
similar patterns of cross-reactivities when used as tracers. Secretin
was labeled by dissolving 0.5-1.0 nmol rat secretin in 20 µl of
0.2 M sodium phosphate (pH 7.4), adding 1.0 mCi
125I and 10 µg chloramine-T
(total reaction volume 40 µl), and incubating for 2 min with gentle
shaking. The reaction was stopped by adding 50 µl of 50% (vol/vol)
acetic acid, and the mixture was immediately loaded onto a Sephadex
G10 column (5 ml bed volume)
previously equilibrated with 0.05 M ammonium acetate (pH 6.5)
containing 0.1% (wt/vol) human albumin (acidifying the reaction
mixture as described has the potential to volatilize
125I, requiring the use of a
negative pressure, filtered cabinet). The column was eluted with the
same buffer, and fractions of 400 µl/3 min were collected. The void
volume radioactive peak was loaded onto an analytical Vydac
C18 column and eluted with a
linear gradient of acetonitrile (27.5-35% over 60 min) in 0.1%
TFA at a flow rate of 1 ml/min. Fractions of 1 ml were collected into tubes containing sufficient concentrated bovine albumin (RIA grade) to
yield a final concentration of 0.1%. These procedures resulted in a
single major peak of 125I-secretin
that represented incorporation of ~20% of total added 125I to a specific activity of
2,000 mCi/nmol. Aliquots of this radiolabeled secretin were stored
frozen at
20°C and thawed only once before use.
Assays were conducted using buffer containing 40 mM sodium phosphate
(pH 7.5), 50 mM NaCl, 11 mM EDTA, 3 mM sodium azide, 0.1% (wt/vol)
bovine albumin (RIA grade), and 0.1% (vol/vol) Triton X-100.
Antiserum, radiolabeled secretin (2,000 cpm), and graded concentrations
of peptides were added together to a final volume of 1.0 ml. After
incubation at 4°C for 24 h, free and bound radiolabeled secretin
were separated by dextran-coated charcoal. Both fractions were counted.
Data were analyzed using the software program CRESIA version 3.0 (Creative Research, Los Angeles, CA).
Bioassay.
Male Sprague-Dawley rats were purchased from Sasco (Omaha, NE) and
housed under American Association for Accreditation of Laboratory
Animal Care-approved conditions. Before surgical
preparation, animals were fasted for 24 h with free access to water.
Anesthesia was induced by administration of urethan, 1.25 g/kg, given
as divided intramuscular and intraperitoneal injections. A jugular vein
catheter (PE-50) was inserted and used to administer intravenous injections of peptide solutions; 0.15 M NaCl containing 1% (wt/vol) BSA was also given intravenously throughout the experiment at 1.0 ml/h.
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. Synthetic canine
peptides were weighed to the nearest microgram and dissolved in 2 M
acetic acid. Synthetic porcine secretin was purchased from Peninsula
Laboratories (San Carlos, CA); the entire contents of each vial were
dissolved as above, because the stated amount of peptide per vial is
based on quantitative amino acid analysis performed by the
manufacturer. Peptide solutions were aliquoted and stored at
70°C; an aliquot was thawed only once before use. Doses of
synthetic canine peptides were adjusted for peptide content as
determined by quantitative amino acid analysis described above on
selected aliquots of the same solutions used for bioassay. After a
60-min period of stabilization, pancreatic secretory volumes were
measured every 15 min using a precalibrated length of tubing. Peptides
were injected intravenously over 10 s in a volume of 0.5 ml. Injections
were given every 45 min, a period during which secretory volumes had
returned to basal levels. A two-point parallel line bioassay was
performed by injecting each peptide at low and high doses that produced
similar responses to a standard (either porcine or canine secretin).
Doses of standard and test peptides were randomized according to a
Latin square design to eliminate any carryover effects of previous
secretin doses. A standard and one or two test peptides were given in
each experiment. Results were tabulated, and means ± SE were
calculated. The results shown represent either the absolute secretory
volume or the increment in secretory volume over basal levels (i.e.,
the period before each injection) in microliters per 15 min. The
potency of each test peptide was calculated by expressing responses to
low and high test doses as a percentage of responses to corresponding standard doses in each animal, averaging the low- and high-dose percentages in each rat, and computing the means ± SE for each group, with n equal to the number of
rats. Statistical analysis included corrections for multiple
comparisons and was done on a paired or unpaired basis as appropriate.
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RESULTS |
Peptide synthesis and purification.
The purification of secretin and secretin-Gly required only one
preparative reverse-phase step to yield peptide of >95% purity. Secretin-Gly-Lys-Arg required three reverse-phase steps and one ion-exchange step to obtain peptide of similar purity. The gradient reverse-phase HPLC of each peptide (Fig. 2)
showed a single peak of >95% purity. These patterns indicate that
all peptides were stable during storage as dry powders at
80°C for extended periods of time. Of note was that canine
secretin-amide and secretin-Gly eluted in identical positions under the
conditions described in MATERIALS AND
METHODS. The same pattern of coelution of these two
peptides was seen at acetonitrile gradients as low as 0.25%/min.

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Fig. 2.
Analytical reverse-phase HPLC of purified secretin forms. Secretin
analogs were chromatographed on an analytical Vydac
C18 reverse-phase column eluted
with increasing concentrations of acetonitrile (0-70% in 0.1%
trifluoroacetic acid over 70 min). A:
elution profile of secretin-amide. B:
elution profile of secretin-Gly. C:
elution profile of secretin-Gly-Lys-Arg. Note identical times of
elution of secretin-amide and secretin-Gly.
A214, absorbance at 214 nm; %B,
percent buffer B.
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Chemical characterization of the secretin peptides.
Table 1 shows the amino acid composition of
the three canine secretin forms. All three analyses were consistent
with the structures synthesized. Table 2
shows that the amino acids were in the proper sequence for
the three forms. Table 3 shows the three
forms had the expected mass, based on mass spectral analysis.
Immunological characterization of the secretin peptides.
Figure 3 shows that amidated rat, porcine,
human, and canine secretin were equally cross-reactive with one of the
antisera used for immunological characterization, whereas canine
secretin-Gly or secretin-Gly-Lys-Arg did not inhibit binding of
125I-secretin. Table
4 presents relative cross-reactivities of
the three canine secretin forms in RIA with eight antisera. The
COOH-terminally extended secretins were not well recognized in any of
the assays, with only one antiserum binding secretin-Gly (but not
secretin-Gly-Lys-Arg) at ~20% of its affinity for secretin-amide.

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Fig. 3.
Cross-reactivity of secretin molecular forms with antiserum R400.
Peptides were incubated with antiserum R400 (1:400,000) and
125I-secretin (2,000 cpm) at
4°C for 48 h. Nonspecific binding was 4.3%, and binding in absence
of added peptide (Bo) was 34%.
Neither canine secretin-Gly nor secretin-Gly-Lys-Arg produced any
significant displacement of radiolabeled secretin-amide, whereas
canine, rat, porcine, and human secretin-amide were equipotent
inhibitors of radiolabeled secretin-amide binding.
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Table 4.
Cross-reactivities of canine secretin-amide, secretin-Gly, and
secretin-Gly-Lys-Arg with eight antisera raised against synthetic
porcine secretin-amide
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Bioactivity of the secretin peptides.
The bioassay model was first characterized using synthetic porcine
secretin. Figure 4 shows that incremental
responses to intravenous bolus injections of porcine secretin were
linearly related to the log of dose over a wide range. In a separate
experiment, responses to 3 and 30 pmol/kg porcine secretin were
reproducible when tested by duplicate administration; when calculated
as described in MATERIALS AND METHODS,
a potency estimate of 110 ± 11%
(n = 9 rats) was obtained. Synthetic
canine secretin was similar in potency to synthetic porcine secretin
for stimulation of pancreatic fluid secretion (Fig.
5); its calculated relative potency was 108 ± 8%. In most subsequent assays, canine secretin was therefore used as the comparison standard. On the basis of peak responses, canine
secretin-Gly was slightly less potent, and secretin-Gly-Lys-Arg was
markedly greater in potency when compared with amidated canine secretin
(Fig. 6). The calculated potency of
secretin-Gly was 81 ± 9% vs. canine secretin and 82 ± 8% in a
separate bioassay (n = 8 rats) with
porcine secretin as standard. Although small, this difference in
potency was of borderline significance
(P = 0.0508 and
P = 0.0498) in both assays. The
relative potency of secretin-Gly-Lys-Arg was 176 ± 13%
(P < 0.01) vs. canine secretin. In
addition, the response to secretin-Gly-Lys-Arg lasted substantially longer compared with the other forms (Fig.
7).

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Fig. 4.
Dose-response effects of porcine secretin on pancreatic fluid
secretion. Bolus intravenous injections were made in 5 rats, with order
of doses randomized by a Latin square design. Cumulative pancreatic
volume was measured at 15-min intervals. Values shown are means ± SE of peak increase above basal in 45-min period after each injection.
Basal values ranged from 13.2 ± 2.9 to 15.2 ± 2.8 µl/15 min
and were not significantly different by ANOVA. Responses were linearly
related to log of injected dose
(r2 = 0.9883 by
linear regression analysis over all doses).
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Fig. 5.
Comparison of pancreatic fluid secretion in response to synthetic
amidated porcine and canine secretins. Bolus intravenous injections of
both peptides were given, and cumulative pancreatic volume was measured
at 15-min intervals. Values shown are means ± SE of peak
responses to each dose, after subtracting volume of basal period
immediately preceding each injection. Peak responses occurred in first
or second 15 min postinjection. Basal volumes ranged from 12.4 ± 0.9 to 14.3 ± 1.6 µl/15 min and were not significantly different
by ANOVA. Potency of canine secretin, calculated as described in
MATERIALS AND METHODS, was not
significantly different from that of porcine secretin by paired
Student's t-test;
n = 8 rats, each of which received all
doses of both peptides.
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Fig. 6.
Comparison of pancreatic fluid secretion in response to synthetic
amidated canine secretin, secretin-Gly, and secretin-Gly-Lys-Arg.
Experimental design and data presentation are identical to that
described in Fig. 5. Basal volumes ranged from 13.2 ± 1.6 to 15.5 ± 2.2 µl/15 min and were not significantly different by ANOVA.
Compared with amidated secretin, secretin-Gly had a potency of 81 ± 9% (P < 0.10), and
secretin-Gly-Lys-Arg had a potency of 176 ± 13%
(P < 0.00016). Statistical analysis
consisted of paired Student's t-tests
corrected for two comparisons to amidated secretin group;
n = 12 rats, each of which received
all doses of 3 peptides.
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Fig. 7.
Time course of pancreatic fluid secretion after bolus intravenous
injections of synthetic amidated canine secretin (33 pmol/kg) and
secretin-Gly-Lys-Arg (29 pmol/kg). Values shown are actual volumes of
secretion in basal period and 3 postinjection periods for each peptide.
After basal volume was subtracted from each of 3 postinjection
responses, integrated 45-min response to secretin-Gly-Lys-Arg (50.8 ± 7.1 µl) was found to be significantly greater
(P < 0.002) than that to amidated
secretin (24.0 ± 2.5 µl) by paired Student's
t-test;
n = 12 rats, each of which received
both peptides.
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DISCUSSION |
The main findings from these experiments are that COOH-terminally
extended forms of secretin are fully bioactive for stimulation of
pancreatic secretion but do not cross-react with several
"antisecretin" antisera generated against amidated secretin.
These results suggest that the secretin receptor mediating pancreatic
fluid secretion does not require COOH-terminal amidation of secretin
for binding and activation. In turn, this raises the possibility that
COOH-terminally extended molecular forms of secretin resulting from
posttranslational processing of the secretin gene product could be
involved in the control of pancreatic secretion and other actions of
secretin. The data further indicate that currently available RIA for
secretin would not detect COOH-terminally extended forms of secretin in blood or tissue if they have the same pattern of epitope recognition as
the antisera used in the current study. This leaves unanswered the
question of whether such forms of secretin occur in amounts sufficient
to contribute to the actions of secretin.
The present findings demonstrate that amidation of the COOH terminus of
secretin is not a structural requirement for its ability to stimulate
pancreatic secretion in vivo. The differences in potencies of amidated
vs. -Gly and -Gly-Lys-Arg extended secretins for stimulating pancreatic
secretion may reflect different susceptibilities to degradation in
vivo. Although secretin-Gly was slightly but consistently less potent
than amidated secretin, the practical consequence of this difference
(~20%) is not striking. In other experiments in which both secretin
forms were administered by continuous intravenous infusion, secretin
and secretin-Gly were equipotent for stimulating pancreatic fluid
secretion (33). On the other hand, secretin-Gly-Lys-Arg was almost
twice as potent for stimulating pancreatic fluid secretion and had a
longer duration of effect when similar doses of the two forms were
administered. A longer circulating half-life for secretin-Gly-Lys-Arg
is a plausible explanation for this difference. However, we could not
measure actual plasma levels of the extended forms of secretin because none of the secretin antisera available to us cross-reacted with these
forms in RIA. The possibility that the extended forms are not
inherently bioactive but were converted to amidated secretin, either in
the circulation or during diffusion through interstitial fluid to
pancreatic duct cells, should be examined. However, such processing
would require serial enzyme activity and does not occur with other
COOH-terminally extended peptides (8). It is also difficult to
reconcile such a mechanism with the clearly greater potency of
secretin-Gly-Lys-Arg vs. amidated secretin in vivo. There may also be
different potencies of amidated and extended forms of secretin for
binding and activation of the receptor causing the pancreatic secretory
response, but in vivo studies cannot address this possibility.
It has been proven for many peptides containing an
-carboxyl amide
group that this posttranslational structural modification is of
critical importance for bioactivity. For example, substitution of the
amide group of gastrin by a hydroxyl group or glycine reduces its
potency for stimulation of gastric acid secretion by >1,000-fold (26,
27). However, there are now several examples of an apparent lack of
importance of COOH-terminal amidation for the actions of peptides first
isolated as forms containing this structure. COOH-terminally extended
vasoactive intestinal polypeptide (VIP) (11, 13) and peptide histidine
isoleucinamide (PHI) (25) are bioactive. COOH-terminally extended forms
of VIP (10) and PHI (3) can be the major products of posttranslational
processing of these peptides in some species. Pituitary adenylate
cyclase-activating polypeptide (31) and glucagon-like peptide-1 (30)
are two other examples in which COOH-terminal amidation occurs but is not required for physiological effects. It is easy to understand the
necessity of amidation if all receptors for a certain peptide require
this structure for peptide recognition. It is less apparent why cells
go through the energy-requiring process required to form peptide amides
if receptors do not utilize this structure for recognition of specific
agonists. One possibility is that there are different classes of
receptors, some of which prefer the amidated peptide. For secretin,
this has not been studied because only the amidated peptide has been
available for biological testing. Although the data presented here
suggest that pancreatic fluid secretion is mediated by a receptor that
does not require amidation of secretin, we have preliminary evidence
(16, 33) for more potent inhibition of gastric acid secretion by
amidated vs. COOH-terminally extended secretin.
One secretin receptor has been cloned from a mouse-rat hybrid cell line
(20) and shown to be identical to a receptor cloned from a rat
pancreatic DNA library (41). Human and rabbit secretin receptors with
80% identity to the rat receptor have also been cloned (21, 37). The
rat receptor has been localized to pancreatic duct and acinar cells
(40), indicating that it mediates the effects of secretin on pancreatic
secretion. In cell lines transfected with this receptor or chimeric
receptors composed of portions of the secretin, VIP, and glucagon
receptors, the NH2-terminal structure of secretin is of critical importance for binding and activation; deletion of the
NH2-terminal histidyl residue
reduces binding and activation by 1,000-fold (42). The COOH-terminal region of secretin also appears to be involved in receptor recognition, although to a lesser degree; peptides with deletions of the
COOH-terminal Val-amide or Gly-Leu-Val-amide were only 10- and 50-fold
less potent than intact secretin-(1
27)-amide (18). It is likely that
this secretin receptor mediates the effects of COOH-terminally extended
secretins on pancreatic fluid secretion, although direct studies have
not been done to characterize its recognition of such forms.
The lack of cross-reactivity of secretin-Gly and secretin-Gly-Lys-Arg
in several secretin RIA examined in this study was established by the
use of synthetic peptides. Many groups have developed RIA for secretin
and measured plasma and tissue concentrations and molecular forms of
secretin (5). All such assays have used amidated secretin as both
tracer and standard. With these conditions, we found that eight
different antisera raised against porcine secretin had little or no
ability to recognize COOH-terminally extended secretins, suggesting
that the COOH-terminal Val-amide structure is critically important for
antibody binding. A few secretin RIA have been reported to detect other
regions of secretin (5) and might be expected to recognize
COOH-terminally extended forms. However, this possibility has not been
tested because the appropriate peptides were not available. It has been
a general finding that only one form of secretin is detectable with
these RIA when plasma or intestinal extracts are chromatographed in various systems and that this form coelutes with amidated secretin (32,
34, 36). Three possible explanations for these findings are as follows:
1) the predominant form of stored
and circulating secretin is amidated,
2) the assays used only detect the
amidated form, or 3) the assays
detect extended forms, but these forms are not separated by the
chromatography used. One group has detected another immunoreactive form
of secretin from extracts of rat intestine that did not elute in the
same position as amidated secretin (17). This secretin form was not
characterized chemically or biologically, but it was suggested that it
may represent an extended form of secretin. It is of interest that the
distribution of secretin appears to be much different on the basis of
Northern blot and PCR analysis of secretin mRNA vs. RIA of secretin in
tissue extracts. Secretin mRNA is widely distributed throughout the
small intestine, colon, brain, heart, kidneys, and other organs (23,
29). However, RIA of tissue extracts from these organs has detected
little or no secretin immunoreactivity other than in the small
intestine (4, 23, 29). It is possible that COOH-terminally extended forms of secretin are the major product of mRNA translation in some of
these sites, accounting for lack of detection by amide-specific RIA.
Previous studies on secretin molecular forms in tissue and blood have
also relied on various chromatographic procedures to demonstrate
similar elution patterns of endogenous secretin and purified porcine
secretin as a standard. Typical gel chromatography is unlikely to
resolve small differences in elution resulting from the presence of
glycine instead of amide at the COOH terminus of secretin. Data
reported here (Fig. 2) suggest that even HPLC may not resolve these two
secretin forms under frequently employed conditions.
The marked inhibitory effects of immunoneutralization of endogenous
circulating secretin on pancreatic and other responses (6) would appear
to rule out any significant contribution of COOH-terminally extended
forms of secretin. However, this conclusion is based on the assumption
that the antiserum used for immunoneutralization of amidated secretin
is selective for only this circulating form. In fact, the secretin
antisera used in many reported immunoneutralization studies have been
stated to bind both an internal region of secretin and the
COOH-terminal region (5). Theoretically, this binding pattern would
predict that the actions of all molecular forms of secretin would be
blocked during in vivo immunoneutralization experiments, particularly
with the large doses of antisera used in such experiments. It would be
necessary to test the specificity of such antisera for secretin-amide
vs. COOH-terminally extended secretins in immunoneutralization
experiments before concluding that secretin-amide is the only
circulating form.
Whether quantitatively significant amounts of COOH-terminally extended
forms of secretin are produced during posttranslational processing and
secreted into the circulation cannot be determined until appropriate
methods for detection of such forms become available. Such a
possibility is suggested by the observation that extended forms of
gastrin are present in large amounts in antral mucosa and blood in some
species (7). If this is the case for secretin, it could explain in part
the puzzling finding that the amounts of circulating secretin measured
by RIA after a meal or intestinal perfusion with acid or fatty acids
are not sufficient to account for the degree of pancreatic fluid
secretion measured at the same time (12, 19, 22). Although it has been
assumed that potentiation by CCK, acetylcholine, and other factors is
responsible for the higher than expected pancreatic response to
endogenous secretin, it is possible that bioactive molecular forms of
secretin undetected by available RIA also contribute to this response.
After reexamining the cross-reactivities of the eight antisera reported
above by using 125I-secretin-Gly
as a tracer instead of
125I-secretin, we have recently
established a RIA that detects COOH-terminally extended forms of
secretin (43). Preliminary studies with this assay and HPLC separation
conditions that resolve synthetic secretin and secretin-Gly indicate
the presence of significant amounts of secretin-Gly in rat intestinal
extracts (43). This suggests that some physiological actions of
secretin may indeed be mediated by both amidated secretin and
secretin-Gly.
 |
ACKNOWLEDGEMENTS |
The assistance of Helen Wong in obtaining the secretin antisera is
gratefully acknowledged. Mass spectral analysis was done by Michael T. Davis in the laboratory of T. D. Lee at the Beckman Research Institute
directed by John E. Shively, City of Hope, Duarte, CA. Synthetic
peptides were produced by the UCLA Peptide Synthesis Core Facility
under the direction of J. R. Reeve, Jr.
 |
FOOTNOTES |
This research was supported by National Institute of Diabetes and
Digestive and Kidney Diseases CURE Digestive Diseases Research Center
Grant DK-41301 and used the Peptide Biochemistry and Molecular Probes,
Antibody, and Animal Model Center Cores. The research was also
supported by the Medical Research Service of the Veterans Health Service.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. E. Solomon,
CURE: Digestive Diseases Research Center, Bldg. 115, Rm. 111, West Los
Angeles VAMC, 11301 Wilshire Blvd., Los Angeles, CA 90073 (E-mail:
solomont{at}ucla.edu).
Received 17 February 1998; accepted in final form 22 October 1998.
 |
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