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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Posttranslational processing of preprosecretin generates several COOH-terminally extended forms of secretin and alpha -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.

                              
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Table 1.   Amino acid analysis of synthetic secretin molecular forms


                              
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Table 2.   Sequence analysis of synthetic secretin molecular forms


                              
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Table 3.   Mass spectral analysis of synthetic secretin molecular forms

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

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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