Secretin Cells in the Gastrointestinal Tract WILLIAM Y. CHEY AND ROBERT ESCOFFERY The Isaac Gordon Center for Digestive Diseases, and the Department of Medicine, Genesee Hospital, University of Rochester School of Medicine and Dentistry Rochester, New York of the antrum and duodenum in both humans and dogs, and throughout the entire length of the canine small intestine. They were not found in the mucosa of the esophagus, fundus of the stomach, or rectum. These cells were either pyramidal in shape or pearshaped and were one-third of the size of gastrin cells. The possible significance of S-cell distribution in the antrum and small intestine is discussed. (Endocrinology 98: 1390, 1976)
ABSTRACT. The morphology and distribution of secretin (S) cells were investigated in the human and the dog. S cells were well-visualized by the indirect immunofluorescence antibody technique, using a highly specific rabbit anti-secretin sera. The fluorescence reaction was not blocked by an excess amount of gastrin, cholecystokinin, glucagon, vasoactive intestinal polypeptide, or motilin, whereas secretin blocked the reaction. S cells were seen in the mucosa
I
T HAS B E E N w e l l e s t a b l i s h e d that t h e
gastrointestinal mucosa contains various endocrine cells (1). Some of these cells are known to produce a well known specific gut hormone, and they have been identified. These cells include G cells for gastrin (2,3), S cells for secretin (4-6), EG cells for enteroglucagon (7), Dj cells for gastric inhibitory polypeptide (8), and cells containing vasoactive intestinal polypeptide (9) and motilin (10). Although the duodenal mucosa has been known to be the major site of the production and release of the oldest hormone, secretin (11), it has been reported that a low secretinlike biological activity is found in the pyloric mucosal extract (12-15), as well as in the more distal parts of the small intestine (1216). Indeed, Bussolati et al. (4) and Polak and her associates (5,6), both using indirect immunofluorescence technique, demonstrated S cells in the duodenum of both dogs (4,5) and man (6). It has not been reported, however, whether S cells are present in any part of the gastrointestinal tract other than the duodenum. We attempted, in the present investigation, to determine whether secretin cells are indeed present in the duodenum and upper jejunum only, or whether they are present elsewhere in the gut as well. We searched for S cells in Received July 21, 1975. Correspondence: W. Y. Chey, M.D., 224 Alexander Street, Rochester, NY 14607.
the esophagus, body of the stomach, pyloric antrum, duodenal bulb, duodenum, and distal small intestine in dogs and in man, using the indirect immunofluorescent technique. Materials and Methods Five fasting mongrel dogs were anesthetized by the intravenous injection of sodium pentobarbital. Through a midline incision of the abdomen, the stomach and the entire small intestine were removed. The stomach was opened along the greater curvature, and 4 mucosal specimens each were obtained from the posterior and the anterior wall of the body, approximately 5 cm from the antralcorpus junction and the antrum, 2-3 cm proximal to the pyloroduodenal (PD) junction. From the small intestine, specimens were obtained from various sites distal to the (PD) junction as follows: 2-3 cm (duodenal bulb), 8 cm (2nd portion of the duodenum), and thereafter at 20 cm intervals throughout the small intestine. Each mucosal specimen studied measured 0.6 x 0.2 cm. In 9 human subjects, serial mucosal biopsy specimens were obtained from the upper gastrointestinal tract. In overnightfasting subjects, an Olympus G.I.F. panendoscope was passed, following the intravenous injection of diazepam in doses ranging from less than 10 mg to 20 mg. Thirty minutes prior to endoscopic procedure, atropine sulfate in a dose of 0.6 mg was given intramuscularly. Two biopsy specimens were obtained from each site, including the lower esophagus 5 cm proximal to the esophagogastric junction, body of the stomach, and the antrum, 2 - 3 cm proximal to the (PD) junction and duodenal bulb, 2 - 3 cm distal
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SECRETIN CELLS IN GASTROINTESTINAL TRACT
to the PD junction, and the duodenum in its second portion. Biopsy specimens were obtained through the scope with an Olympus Model No. Fb-3K biopsy forceps. The size of the specimen was about 2 x 3 mm2. The mucosal specimens obtained from either human subjects or dogs were oriented on nylon mesh and were placed in cold (4 C) 2% 1ethyl-(3-dimethyl-aminopropyl)-carbodiimide in 0.1 M phosphate-buffered saline (PBS), pH 7.2. They were fixed for 24 hours (5). Following fixation, the specimens were washed in PBS for 30 minutes. The washings were repeated three times and fresh PBS was used each time. They were then dehydrated and embedded in paraffin, according to the method described by SainteMarie (17). Using a Leitz rotary microtome, 5 fim sections were cut, and 3 sections each were floated on 6 separate slides with distilled water, and dried at 37 C for lV^ hours. Following removal of the paraffin by xylol and hydration by descending grades of ethanol, immunofluorescence localization of secretin in the tissue was made by an indirect immunofluorescence technique (18). A pure porcine synthetic secretin, prepared by Dr. M. Ondetti and his colleagues (19) was used as the immunogen (20). The synthetic secretin was conjugated with bovine serum albumin by a modification of the method of Goodfriend et al. (21), using l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Six mg of the secretin preparation was dissolved in 0.4 ml of N, N-dimethyl-formamide and added to 0.6 ml of 0.05M potassium phosphate, pH
7.4. Fifty mg of EDC in 0.2 ml of phosphate buffer was added, and the mixture was stirred for 20 hours at 20 C. The resulting suspension was dialyzed for 48 hours at 4 C against 2 liters of 0.15M NaCl-O.OlM potassium phosphate solution at pH 7.4. The dialyzed conjugate was emulsified in an equal volume of complete Freund's adjuvant, and a 0.8 ml aliquot of the emulsion containing 2 mg of secretin was injected into a foot pad of each of the three randomly bred, white, New Zealand rabbits. The immunization was repeated in two and five months following the initial immunization, and two weeks after each immunization the animals were bled by puncture of the ear veins. All three rabbits produced high titers of secretin antisera after a third immunization. The dilution of antisera suitable for radioimmunoassay varied
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from 1:100,000 to 1:300,000. In order to test the specificity of the anti-secretin sera, several hormonal preparations were evaluated for their capacity to inhibit competitively the binding of labeled secretin. Bovine-porcine glucagon, human insulin, porcine cholecystokinin, motilin, vasoactive intestinal polypeptide, and gastric inhibitory polypeptide in doses from 0.1 to 1000 ng did not react in this system (20,22). The rabbit anti-secretin globulins were obtained from the anti-secretin sera by precipitation with an equal volume of saturated ammonium sulfate solution (23), and the precipitate was washed, redissolved, and dialyzed against 0.1M phosphate buffer saline, pH 7.2, for three days to remove the ammonium sulfate. The solution was diluted to a final concentration of 0.3% with buffered saline. Two-tenths to 0.4 ml of the diluent was used as the first incubation layer for one hour, after which the tissue was rinsed in 0.1M phosphate buffer, pH 7.2. Then the tissue was incubated for one hour with a few drops of goat anti-rabbit gamma globulin serum which had been reacted with fluorescein isothiocyanate (Hyland Laboratories, California) and absorbed with acetone-dried human and dog liver powder. After the tissues had been rinsed again, they were examined microscopically as described below. For controls, the following experiments were performed: a) Normal rabbit gamma globulin was used in place of the anti-secretin globulins in the procedure described above, b) The labeled goat antibodies to rabbit gamma globulin were used without prior incubation with the anti-secretin globulins, c) The antibodies to secretin, to which an excess amount, 150 /u-g in 0.25 ml, of either synthetic secretin (Squibb), glucagon (Lilly), vasoactive intestinal polypeptide (VIP), motilin, pure porcine cholecystokinin (produced by Professor V. Mutt) or human synthetic gastrin I had been added, were applied in an attempt to prevent the fluorescence reaction. Only secretin blocked the reaction. In order to study the relative distribution and size of gastrin-producing cells (G cells), compared with the distribution and size of S cells, adjacent biopsy specimens were fixed in methanol-free formaldehyde for 48 hours, processed in the same way as were the biopsy specimens for secretin, and the immunofluorescent localization of the gastrin cells was carried out in a similar fashion, with the same types of controls, using a rabbit anti-gastrin serum to human synthetic
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such units were examined per biopsy at 400 x magnification, and the mean estimation per unit expressed. Each plus represents two positive cells per unit area (Tables 1 and 2).
TABLE 1. Distribution of S cells in the stomach and small intestine of dogs Dog no. Biopsy site
Results
Stomach: body Stomach: antrum Small intestine: 2-3 cm* 8 cm 20 cm
In the dogs, the cells containing specific secretin fluorescence were readily observed in the mucosa of the antrum, duodenum, and jejunum in all five dogs and in the ileum in three of the five. The control slides, on the other hand, showed dull, light gray cytoplasms with dark gray to black nuclei. In the antrum, the S cells were mostly pear-shaped, and their processes were frequently seen to reach the glandular lumen (Fig. 1), and were approximately onethird the size of G cells. These cells were randomly distributed toward the base of the glands (Fig. 1), while G cells were present in the upper portion of the glands (Fig. 2). In the duodenum, the S cells were found in the transitional zone of the mucosal layer (Fig. 3) and in the crypts of the jejunal and ileal mucosa (Fig. 4). The cells were mostly pyramidal in shape. The fluorescence varied from very brilliant to moderate. In human subjects, S cells were observed in both the antrum and the duodenum (Figs. 5 and 6), as we had observed in dogs. The locations and distributions of these cells were also similar to those observed in dogs.
40 cm
60 cm 80 cm 100 cm 120 cm 140 cm 160 cm
0 0 0
0 0 0 0
0
0
•f
* Indicates the distance from the pyloroduodenal junction. \ Each + represents 2 positive cells per unit area.
gastrin I, which was produced in our laboratory using the method described by McGuigan (24). The tissue sections were examined for fluorescence using a Leitz Dialux microscope equipped with a high-pressure mercury lamp, a dark field condenser, 2 mm UG-1 exciting filter, and a K490 secondary filter. One ocular was fitted with a 2 mm grid divided into 100 squares. Photomicrographs were taken using Kodak Tri-X film. Efforts were made to establish a distribution pattern throughout the small intestine by estimating the number of cells demonstrable in a unit area consisting of a segment of mucosa 500 /urn high and 250 /urn wide, which included the base of the villi and the upper portions of the crypts. Ten
TABLE 2. Distribution of S cells in the esophagus, stomach, duodenum, and rectum in man Duodenum Stn 11 J C l l . l l Aorp ann
Case 1 2 3 4 5 6 7 8 9 10 11
f\fl\*/
Clll\_i
2nd
Sex
Esophagus
40 F 72 M 71 M 58 F 67 F 67 M 60 F 58 M 55 F 29 M 28 F
0 0 0 0 0 0
£d\ 1U
Antrum
Bulb
portion
Rectum
0
+
V2 +
++ + + ++
++ ++ + ++ + ++ ++ ++ +++
__ 0
—
0 0
+ ++ ++
+ + + + + + + + +
—
—
—
0 0 0 *
—
Body
y2+
0 0
* Not studied.
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SECRETIN CELLS IN GASTROINTESTINAL TRACT
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FIG. 1. Antral mucosa of a dog showing an S cell in the base of the gland (xl200).
FIG. 2. Antral mucosa of a dog showing G cells in the glands (x650).
The number of these S cells observed in the mucosal specimens of the stomach and small intestine of five dogs are presented in Table 1. S cells were found in the gastric antrum and the small intestine throughout the entire length, including the duodenal bulb. They were seen, however, more frequently in the duodenum and the antrum than in the rest of the small intestine. In three of the five dogs, S cells were found in the distal small intestine as well. In the human subjects (Table 2), again both duodenal and antral mucosa contained S cells. A small number of S cells were seen in the body of the stomach in two human subjects, but they were absent in the mucosa of the esophagus and the rectum.
sera produced in our laboratory (20). There were no cross reactions to those known gut hormones which were available in a synthetic form or in a pure native form. Particularly, the homones which possess structural similarities to secretin, including pancreatic glucagon and vasoactive intestinal polypeptide, did not influence thefluorescencereac-
Discussion The present investigation indicates that S cells are distributed in the antrum and throughout the entire length of the small intestine including the duodenal bulb, although they were seen more frequently in the duodenum, as observed by Polak et al. (5,6) and Bussolati et al. (4). We have shown further in this study that these cells reacted highly specifically to rabbit anti-secretin
FlG. 3. Duodenal mucosa of a dog showing an S cell in the transitional zone (x!400).
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CHEY AND ESCOFFERY
Endo • 1976 Vol 98 • No 6
FIG. 4. Distal ileal mucosa of a dog showing an S cell in the base of the gland (xl350).
FIG. 6. S cells in the transitional zone of human duodenal mucosa (xl540).
tion of our antisera. Secretin and these peptide hormones are considered a secretin family of polypeptides (25). While S cells in the antrum were located in the bottom of the glands, those in the duodenal mucosa were present in the transitional zone, which confirms the observation of Polak et al. (5,6). Furthermore, this observation parallels that by Krawitt et al. (26) who found secretin-like activity pres-
ent primarily in the extract obtained from the transitional zone. These authors also found secretin-like activity in the villi of the canine duodenal mucosal layer but not in the crypts. S cells in the canine jejunum and ileum, on the other hand, were present in the base of the crypts. The reason for the difference in the distribution of these S cells in the glandular units of different regions of the gastrointestinal tract cannot be explained at this time. It was of considerable interest to us that S cells were present in the antrum and small intestine, including the duodenal bulb and distal ileum, in dogs. They were observed also in the human antrum and duodenal bulb in a similar proportion to that observed in dogs. Certainly no S cells were seen in the mucosa of the esophagus, nor have we seen them in the acid-secreting mucosa of the stomach, or in the rectal mucosa in man. Recently we found that the human small intestinal mucosa distal to the duodenum also contained S cells in three subjects studied (unpublished data). In one of them, S cells were found in the distal ileum. The endogenous release of secretin from the upper small intestine in response to the infusion of HC1 solution in the duodenum was demonstrated by observing an
FiG. 5. S cells in human antral mucosa (xl300).
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SECRETIN CELLS IN GASTROINTESTINAL TRACT increase in exocrine pancreatic secretion rich in bicarbonate (11,12,22,27-30). Recently, it has been observed that duodenal infusion of acid resulted in a significant rise in the plasma or serum levels of radioimmunoassayable (RIA) secretin (22,31-33), corroborating observations based on bioassay experiments (11,12,22,27-30). The possible endogenous release of secretin from, and the physiological significance of S cells in the antrum or ileum, await further investigation. Interestingly, in this regard, we have recently observed that S cells are also present in the antrum of rats (unpublished data). The acidification of the antrum of anesthetized rats resulted in a significant increase in exocrine pancreatic secretion, which coincided with a significant increase in plasma RIA secretin levels. The endogenous release of secretin may well begin from the antrum in other animal species as well. Endogenous release of secretin from the antrum by acidic gastric chyme may provide pancreatic and biliary secretion rich in bicarbonate in the duodenum before gastric chyme enters the duodenum. Thus, a prompt neutralization of H + and adequate digestion of foodstuffs must occur in the duodenum. Acknowledgment This work was supported in part by NIH AMDD 16939. We thank Dr. J. C. Brown for his generous supply of motilin and GIP, and Dr. Victor Mutt for the generous supply of pure porcine cholecystokinin and vasoactive intestinal polypeptide.
References 1. Pearse, A. G. E., In Chey, W. Y., and F. P. Brooks (eds.), Endocrinology of the Gut, C. B. Slack, Thorofare, New Jersey, 1974, p. 24. 2. McGuigan, J., Gastroenterology 44: 315, 1968. 3. Creutzfeldt, W., C. Creutzfeldt, and R. Arnold, In Chey, W. Y., and F. P. Brooks (eds.), Endocrinology of the Gut, C. B. Slack, Thorofare, New Jersey, 1974, p. 35. 4. Bussolati, G., C. Capella, E. Solicia, G. Vasallo, and P. Vessadini, Histochemie 26: 281, 1971. 5. Polak, J. M., S. Bloom, I. Couling, and A. G. E. Pearse, Gut 12: 605, 1971. 6. Polak, J. M., I. Couling, S. Bloom, and A. G. E. Pearse, ScandJ Gastroenterol 6: 739, 1971.
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7. Polak, J. M., S. Bloom, I. Couling, and A. G. E. Pearse, Gut 12: 311, 1971. 8. Polak, J. M., S. Bloom, M. Kuzio, J. C. Brown, and A. G. E. Pearse, Gut 14: 284, 1973. 9. Polak, J. M., A. G. E. Pearse, J. C. Garaud, and S. R. Bloom, Gut 15: 720, 1974. 10. Polak, J. M., A. G. E. Pearse, and C. M. Heath, Gut 16: 225, 1975. 11. Bayliss, W. M., and E. H. Starling, / Physiol (Lond) 28: 325, 1902. 12. Weaver, M. M., AmJ Physiol 82: 106, 1927. 13. Drewyer, G. E., and A. C. Ivy, Proc Soc Exp Biol Med 27: 186, 1929. 14. Komarov, S. A., Rev Can Biol 1: 377, 1942. 15. Munch-Peterson, J., G. Ronnow, and B. Uvnas, Ada Physiol Scand 7: 289, 1944. 16. Friedman, M. H. F., and J. E. Thomas, J Lab Clin Med 35: 366, 1950. 17. Sainte-Marie, G.,J Histochem Cytochem 10: 250, 1962. 18. Coons, A. H., E. H. Leduc, and J. M. Connolly, J Exp Med 102: 49, 1955. 19. Ondetti, M. A., W. L. Marayanon, M. von Salta, J. T. Sheehan, E. F. Sabo, and M. Bodansky, / Am Chem Soc 90: 4711, 1968. 20. Boehm, M., Y. Lee, and W. Y. Chey, In Chey, W. Y., and F. P. Brooks (eds.), Endocrinology of the Gut, C. B. Slack, Thorofare, New Jersey, 1974, p. 310. 21. Goodfriend, T. L., L. Levine, and G. D. Gasman, Science 144: 1344, 1964. 22. Chey, W. Y., H. H. Tai, R. Rhodes, K. Y. Lee, and J. Hendricks, In Thompson, J. (ed.), Gastrointestinal Hormones, University of Texas Press, Austin, 1975, p. 269. 23. Nairn, R. C , Fluorescent Protein Tracing, ed. 3, Williams and Wilkins, Baltimore, 1969, p. 303. 24. McGuigan, J., Gastroenterology 54: 1005, 1968. 25. Grossman, M. I., In Chey, W. Y., and F. P. Brooks (eds.), C. B. Slack, Thorofare, New Jersey, 1974, p. 65. 26. Krawitt, E. L., G. R. Zimmerman, and J. A. Clifton, AmJ Physiol 211: 935, 1966. 27. Mellanby, J., and A. S. G. Huggett, / Physiol (Lond) 61: 122, 1926. 28. Thomas, J. E., The External Secretion of the Pancreas, Blackwell, Oxford, 1950. 29. Kanturek, S. J., J. Tasler, and W. Obtulowicz, AmJ Physiol 220: 125, 1971. 30. Meyer, J. H., L. W. Way, and M. I. Grossman, AmJ Physiol 219: 971, 1970. 31. Chey, W. Y., A. Oliai, and M. Boehm, In Chey, W. Y., and F. P. Brooks (eds.), Endocrinology of the Gut, C. B. Slack, Thorofare, New Jersey, 1974, p. 320. 32. Boden, G., N. Essa, O. E. Owen, and F. A. ReichleJ Clin Invest 53: 1185, 1974. 33. Lee, K. Y., H. H. Tai, and W. Y. Chey, Am J Physiol (In press).
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ABSTRACT. The morphology and distribution of secretin (S) cells were investigated in the human and the dog. S cells were well-visualized by the indirect immunofluorescence antibody technique, using a highly specific rabbit anti-secretin sera. The fluorescence reaction was not blocked by an excess amount of gastrin, cholecystokinin, glucagon, vasoactive intestinal polypeptide, or motilin, whereas secretin blocked the reaction. S cells were seen in the mucosa
I
T HAS B E E N w e l l e s t a b l i s h e d that t h e
gastrointestinal mucosa contains various endocrine cells (1). Some of these cells are known to produce a well known specific gut hormone, and they have been identified. These cells include G cells for gastrin (2,3), S cells for secretin (4-6), EG cells for enteroglucagon (7), Dj cells for gastric inhibitory polypeptide (8), and cells containing vasoactive intestinal polypeptide (9) and motilin (10). Although the duodenal mucosa has been known to be the major site of the production and release of the oldest hormone, secretin (11), it has been reported that a low secretinlike biological activity is found in the pyloric mucosal extract (12-15), as well as in the more distal parts of the small intestine (1216). Indeed, Bussolati et al. (4) and Polak and her associates (5,6), both using indirect immunofluorescence technique, demonstrated S cells in the duodenum of both dogs (4,5) and man (6). It has not been reported, however, whether S cells are present in any part of the gastrointestinal tract other than the duodenum. We attempted, in the present investigation, to determine whether secretin cells are indeed present in the duodenum and upper jejunum only, or whether they are present elsewhere in the gut as well. We searched for S cells in Received July 21, 1975. Correspondence: W. Y. Chey, M.D., 224 Alexander Street, Rochester, NY 14607.
the esophagus, body of the stomach, pyloric antrum, duodenal bulb, duodenum, and distal small intestine in dogs and in man, using the indirect immunofluorescent technique. Materials and Methods Five fasting mongrel dogs were anesthetized by the intravenous injection of sodium pentobarbital. Through a midline incision of the abdomen, the stomach and the entire small intestine were removed. The stomach was opened along the greater curvature, and 4 mucosal specimens each were obtained from the posterior and the anterior wall of the body, approximately 5 cm from the antralcorpus junction and the antrum, 2-3 cm proximal to the pyloroduodenal (PD) junction. From the small intestine, specimens were obtained from various sites distal to the (PD) junction as follows: 2-3 cm (duodenal bulb), 8 cm (2nd portion of the duodenum), and thereafter at 20 cm intervals throughout the small intestine. Each mucosal specimen studied measured 0.6 x 0.2 cm. In 9 human subjects, serial mucosal biopsy specimens were obtained from the upper gastrointestinal tract. In overnightfasting subjects, an Olympus G.I.F. panendoscope was passed, following the intravenous injection of diazepam in doses ranging from less than 10 mg to 20 mg. Thirty minutes prior to endoscopic procedure, atropine sulfate in a dose of 0.6 mg was given intramuscularly. Two biopsy specimens were obtained from each site, including the lower esophagus 5 cm proximal to the esophagogastric junction, body of the stomach, and the antrum, 2 - 3 cm proximal to the (PD) junction and duodenal bulb, 2 - 3 cm distal
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SECRETIN CELLS IN GASTROINTESTINAL TRACT
to the PD junction, and the duodenum in its second portion. Biopsy specimens were obtained through the scope with an Olympus Model No. Fb-3K biopsy forceps. The size of the specimen was about 2 x 3 mm2. The mucosal specimens obtained from either human subjects or dogs were oriented on nylon mesh and were placed in cold (4 C) 2% 1ethyl-(3-dimethyl-aminopropyl)-carbodiimide in 0.1 M phosphate-buffered saline (PBS), pH 7.2. They were fixed for 24 hours (5). Following fixation, the specimens were washed in PBS for 30 minutes. The washings were repeated three times and fresh PBS was used each time. They were then dehydrated and embedded in paraffin, according to the method described by SainteMarie (17). Using a Leitz rotary microtome, 5 fim sections were cut, and 3 sections each were floated on 6 separate slides with distilled water, and dried at 37 C for lV^ hours. Following removal of the paraffin by xylol and hydration by descending grades of ethanol, immunofluorescence localization of secretin in the tissue was made by an indirect immunofluorescence technique (18). A pure porcine synthetic secretin, prepared by Dr. M. Ondetti and his colleagues (19) was used as the immunogen (20). The synthetic secretin was conjugated with bovine serum albumin by a modification of the method of Goodfriend et al. (21), using l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Six mg of the secretin preparation was dissolved in 0.4 ml of N, N-dimethyl-formamide and added to 0.6 ml of 0.05M potassium phosphate, pH
7.4. Fifty mg of EDC in 0.2 ml of phosphate buffer was added, and the mixture was stirred for 20 hours at 20 C. The resulting suspension was dialyzed for 48 hours at 4 C against 2 liters of 0.15M NaCl-O.OlM potassium phosphate solution at pH 7.4. The dialyzed conjugate was emulsified in an equal volume of complete Freund's adjuvant, and a 0.8 ml aliquot of the emulsion containing 2 mg of secretin was injected into a foot pad of each of the three randomly bred, white, New Zealand rabbits. The immunization was repeated in two and five months following the initial immunization, and two weeks after each immunization the animals were bled by puncture of the ear veins. All three rabbits produced high titers of secretin antisera after a third immunization. The dilution of antisera suitable for radioimmunoassay varied
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from 1:100,000 to 1:300,000. In order to test the specificity of the anti-secretin sera, several hormonal preparations were evaluated for their capacity to inhibit competitively the binding of labeled secretin. Bovine-porcine glucagon, human insulin, porcine cholecystokinin, motilin, vasoactive intestinal polypeptide, and gastric inhibitory polypeptide in doses from 0.1 to 1000 ng did not react in this system (20,22). The rabbit anti-secretin globulins were obtained from the anti-secretin sera by precipitation with an equal volume of saturated ammonium sulfate solution (23), and the precipitate was washed, redissolved, and dialyzed against 0.1M phosphate buffer saline, pH 7.2, for three days to remove the ammonium sulfate. The solution was diluted to a final concentration of 0.3% with buffered saline. Two-tenths to 0.4 ml of the diluent was used as the first incubation layer for one hour, after which the tissue was rinsed in 0.1M phosphate buffer, pH 7.2. Then the tissue was incubated for one hour with a few drops of goat anti-rabbit gamma globulin serum which had been reacted with fluorescein isothiocyanate (Hyland Laboratories, California) and absorbed with acetone-dried human and dog liver powder. After the tissues had been rinsed again, they were examined microscopically as described below. For controls, the following experiments were performed: a) Normal rabbit gamma globulin was used in place of the anti-secretin globulins in the procedure described above, b) The labeled goat antibodies to rabbit gamma globulin were used without prior incubation with the anti-secretin globulins, c) The antibodies to secretin, to which an excess amount, 150 /u-g in 0.25 ml, of either synthetic secretin (Squibb), glucagon (Lilly), vasoactive intestinal polypeptide (VIP), motilin, pure porcine cholecystokinin (produced by Professor V. Mutt) or human synthetic gastrin I had been added, were applied in an attempt to prevent the fluorescence reaction. Only secretin blocked the reaction. In order to study the relative distribution and size of gastrin-producing cells (G cells), compared with the distribution and size of S cells, adjacent biopsy specimens were fixed in methanol-free formaldehyde for 48 hours, processed in the same way as were the biopsy specimens for secretin, and the immunofluorescent localization of the gastrin cells was carried out in a similar fashion, with the same types of controls, using a rabbit anti-gastrin serum to human synthetic
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CHEY AND ESCOFFERY
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such units were examined per biopsy at 400 x magnification, and the mean estimation per unit expressed. Each plus represents two positive cells per unit area (Tables 1 and 2).
TABLE 1. Distribution of S cells in the stomach and small intestine of dogs Dog no. Biopsy site
Results
Stomach: body Stomach: antrum Small intestine: 2-3 cm* 8 cm 20 cm
In the dogs, the cells containing specific secretin fluorescence were readily observed in the mucosa of the antrum, duodenum, and jejunum in all five dogs and in the ileum in three of the five. The control slides, on the other hand, showed dull, light gray cytoplasms with dark gray to black nuclei. In the antrum, the S cells were mostly pear-shaped, and their processes were frequently seen to reach the glandular lumen (Fig. 1), and were approximately onethird the size of G cells. These cells were randomly distributed toward the base of the glands (Fig. 1), while G cells were present in the upper portion of the glands (Fig. 2). In the duodenum, the S cells were found in the transitional zone of the mucosal layer (Fig. 3) and in the crypts of the jejunal and ileal mucosa (Fig. 4). The cells were mostly pyramidal in shape. The fluorescence varied from very brilliant to moderate. In human subjects, S cells were observed in both the antrum and the duodenum (Figs. 5 and 6), as we had observed in dogs. The locations and distributions of these cells were also similar to those observed in dogs.
40 cm
60 cm 80 cm 100 cm 120 cm 140 cm 160 cm
0 0 0
0 0 0 0
0
0
•f
* Indicates the distance from the pyloroduodenal junction. \ Each + represents 2 positive cells per unit area.
gastrin I, which was produced in our laboratory using the method described by McGuigan (24). The tissue sections were examined for fluorescence using a Leitz Dialux microscope equipped with a high-pressure mercury lamp, a dark field condenser, 2 mm UG-1 exciting filter, and a K490 secondary filter. One ocular was fitted with a 2 mm grid divided into 100 squares. Photomicrographs were taken using Kodak Tri-X film. Efforts were made to establish a distribution pattern throughout the small intestine by estimating the number of cells demonstrable in a unit area consisting of a segment of mucosa 500 /urn high and 250 /urn wide, which included the base of the villi and the upper portions of the crypts. Ten
TABLE 2. Distribution of S cells in the esophagus, stomach, duodenum, and rectum in man Duodenum Stn 11 J C l l . l l Aorp ann
Case 1 2 3 4 5 6 7 8 9 10 11
f\fl\*/
Clll\_i
2nd
Sex
Esophagus
40 F 72 M 71 M 58 F 67 F 67 M 60 F 58 M 55 F 29 M 28 F
0 0 0 0 0 0
£d\ 1U
Antrum
Bulb
portion
Rectum
0
+
V2 +
++ + + ++
++ ++ + ++ + ++ ++ ++ +++
__ 0
—
0 0
+ ++ ++
+ + + + + + + + +
—
—
—
0 0 0 *
—
Body
y2+
0 0
* Not studied.
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SECRETIN CELLS IN GASTROINTESTINAL TRACT
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FIG. 1. Antral mucosa of a dog showing an S cell in the base of the gland (xl200).
FIG. 2. Antral mucosa of a dog showing G cells in the glands (x650).
The number of these S cells observed in the mucosal specimens of the stomach and small intestine of five dogs are presented in Table 1. S cells were found in the gastric antrum and the small intestine throughout the entire length, including the duodenal bulb. They were seen, however, more frequently in the duodenum and the antrum than in the rest of the small intestine. In three of the five dogs, S cells were found in the distal small intestine as well. In the human subjects (Table 2), again both duodenal and antral mucosa contained S cells. A small number of S cells were seen in the body of the stomach in two human subjects, but they were absent in the mucosa of the esophagus and the rectum.
sera produced in our laboratory (20). There were no cross reactions to those known gut hormones which were available in a synthetic form or in a pure native form. Particularly, the homones which possess structural similarities to secretin, including pancreatic glucagon and vasoactive intestinal polypeptide, did not influence thefluorescencereac-
Discussion The present investigation indicates that S cells are distributed in the antrum and throughout the entire length of the small intestine including the duodenal bulb, although they were seen more frequently in the duodenum, as observed by Polak et al. (5,6) and Bussolati et al. (4). We have shown further in this study that these cells reacted highly specifically to rabbit anti-secretin
FlG. 3. Duodenal mucosa of a dog showing an S cell in the transitional zone (x!400).
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1394
CHEY AND ESCOFFERY
Endo • 1976 Vol 98 • No 6
FIG. 4. Distal ileal mucosa of a dog showing an S cell in the base of the gland (xl350).
FIG. 6. S cells in the transitional zone of human duodenal mucosa (xl540).
tion of our antisera. Secretin and these peptide hormones are considered a secretin family of polypeptides (25). While S cells in the antrum were located in the bottom of the glands, those in the duodenal mucosa were present in the transitional zone, which confirms the observation of Polak et al. (5,6). Furthermore, this observation parallels that by Krawitt et al. (26) who found secretin-like activity pres-
ent primarily in the extract obtained from the transitional zone. These authors also found secretin-like activity in the villi of the canine duodenal mucosal layer but not in the crypts. S cells in the canine jejunum and ileum, on the other hand, were present in the base of the crypts. The reason for the difference in the distribution of these S cells in the glandular units of different regions of the gastrointestinal tract cannot be explained at this time. It was of considerable interest to us that S cells were present in the antrum and small intestine, including the duodenal bulb and distal ileum, in dogs. They were observed also in the human antrum and duodenal bulb in a similar proportion to that observed in dogs. Certainly no S cells were seen in the mucosa of the esophagus, nor have we seen them in the acid-secreting mucosa of the stomach, or in the rectal mucosa in man. Recently we found that the human small intestinal mucosa distal to the duodenum also contained S cells in three subjects studied (unpublished data). In one of them, S cells were found in the distal ileum. The endogenous release of secretin from the upper small intestine in response to the infusion of HC1 solution in the duodenum was demonstrated by observing an
FiG. 5. S cells in human antral mucosa (xl300).
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SECRETIN CELLS IN GASTROINTESTINAL TRACT increase in exocrine pancreatic secretion rich in bicarbonate (11,12,22,27-30). Recently, it has been observed that duodenal infusion of acid resulted in a significant rise in the plasma or serum levels of radioimmunoassayable (RIA) secretin (22,31-33), corroborating observations based on bioassay experiments (11,12,22,27-30). The possible endogenous release of secretin from, and the physiological significance of S cells in the antrum or ileum, await further investigation. Interestingly, in this regard, we have recently observed that S cells are also present in the antrum of rats (unpublished data). The acidification of the antrum of anesthetized rats resulted in a significant increase in exocrine pancreatic secretion, which coincided with a significant increase in plasma RIA secretin levels. The endogenous release of secretin may well begin from the antrum in other animal species as well. Endogenous release of secretin from the antrum by acidic gastric chyme may provide pancreatic and biliary secretion rich in bicarbonate in the duodenum before gastric chyme enters the duodenum. Thus, a prompt neutralization of H + and adequate digestion of foodstuffs must occur in the duodenum. Acknowledgment This work was supported in part by NIH AMDD 16939. We thank Dr. J. C. Brown for his generous supply of motilin and GIP, and Dr. Victor Mutt for the generous supply of pure porcine cholecystokinin and vasoactive intestinal polypeptide.
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