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. 1998 Aug;43(2):240–247. doi: 10.1136/gut.43.2.240

Distribution and metabolism of intravenously administered trefoil factor 2/porcine spasmolytic polypeptide in the rat

S Poulsen 1, J Thulesen 1, E Nexo 1, L Thim 1
PMCID: PMC1727199  PMID: 10189851

Abstract

Background—Trefoil peptides are secreted by mucus producing cells in the gastrointestinal tract and are supposed to be involved in oligomerisation processes of the mucin glycoproteins in the lumen. Endocrine functions have also been suggested. 
Aims—To target possible binding sites for iodine-125 labelled porcine spasmolytic polypeptide (pSP) in an in vivo rat model. 
Methods—125I-pSP was given by intravenous injection to female Spraque-Dawley rats. The distribution of 125I-pSP was assessed by gamma counting of samples of organs and by autoradiography of paraffin wax embedded sections. The degradation of 125I-pSP was studied by trichloroacetic acid precipitation, and the saturability of binding by administration of excess unlabelled peptide. 
Results—125I-pSP was taken up in the kidneys and the gastrointestinal tract and was excreted almost unmetabolised in the urine. In the stomach, it could be displaced by unlabelled pSP in a dose dependent manner. Autoradiography showed grains in mucous neck cells, parietal cells, the mucus layer, and the pyloric glands of the stomach; in Brunner's glands and the Paneth cells in the small intestine; and in cells in the lower part of the crypts in the colon. 
Conclusions—125I-pSP from the circulatory system is taken up by mucus producing cells in the gastrointestinal tract. The binding can be displaced by non-radioactive pSP, suggesting the presence of a receptor. 



Keywords: trefoil peptides; trefoil factor 2; spasmolytic polypeptide; metabolism; autoradiography; rat

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Figure 1 .

Figure 1

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Uptake of radioactivity six minutes after administration of 125I-pSP, measured as the percentage of total radioactivity given to each rat. Results expressed as mean (SD); n=4. 


Figure 2 .

Figure 2

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Percentage of total radioactivity given to each rat present in blood and the body of the stomach (A) and in the kidney and urine (B) at various times after administration of 125I-pSP. The black part of the bars indicates the fraction which can be precipitated by TCA and the grey part the fraction which cannot be precipitated. Results expressed as mean (SD); n=4. 


Figure 3 .

Figure 3

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(A) Effect of pretreatment with increasing doses of non-iodinated pSP on uptake of radioactivity in the stomach and kidney in rats sacrificed 15 minutes after administration of 125I-pSP. Uptake of 125I-pSP is indicated as the percentage of total radioactivity given to each rat, taken up per g tissue. (B) Effect of pretreatment with the maximum dose of 10 mg non-iodinated pSP on uptake of radioactivity in the intestinal system. Results expressed as mean (SD); n=3. 


Figure 4 .

Figure 4

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Autoradiograph of the kidney 15 minutes after administration of 125I-pSP. Grains are localised to the brush border and the cytoplasm of the cells of the proximal convoluted tubules. Arrows indicate distal tubules. Original magnification ×480. 


Figure 5 .

Figure 5

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Autoradiographs showing uptake of radioactivity in various parts of the gastrointestinal tract. (A) The mucosa of the body of the stomach 15 minutes after administration of 125I-pSP. Grains are localised to the neck part of the gastric glands, mainly to the mucous neck cells. (B) Larger magnification of the neck part showing grains also in the parietal cells. (C) The mucosal surface of the body of the stomach 120 minutes after administration of 125I-pSP. Grains are observed in the lumen of the gastric pits (arrow) and on the luminal surface. (D) The pyloric part of the stomach with grains in the pyloric glands. (E) The duodenum. Grains are localised to Brunner's glands whereas the epithelium of the crypts in the upper part of the picture is negative. (F) The ileum. Grains are localised to the basal part but also in the cytoplasm of the Paneth cells in the bottom of the crypts of Lieberkühn. (G) The colon. Grains are concentrated in the lower part of the crypts. (H) The pancreas. Part of a large excretory duct with grains in small basal buds of the epithelium. Original magnification: A, ×100; B, ×550; D, ×175; C,E,F,G,H, ×440. 


Figure 6 .

Figure 6

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Autoradiographs showing the effect of excess unlabelled pSP on the localisation of radioactivity in the body of the stomach. Dark field microscopy. (A) The mucosa of the body of the stomach 15 minutes after administration of 125I-pSP. Grains are localised to the neck part of the gastric glands. (B) The mucosa 15 minutes after administration of 125I-pSP together with 10 mg unlabelled pSP. Grains are now localised almost exclusively to the luminal surface of the mucosa, whereas the neck part of the glands only appears as a faint transverse band. Original magnification ×70. 


Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Babyatsky M. W., deBeaumont M., Thim L., Podolsky D. K. Oral trefoil peptides protect against ethanol- and indomethacin-induced gastric injury in rats. Gastroenterology. 1996 Feb;110(2):489–497. doi: 10.1053/gast.1996.v110.pm8566596. [DOI] [PubMed] [Google Scholar]
  2. Chinery R., Cox H. M. Immunoprecipitation and characterization of a binding protein specific for the peptide, intestinal trefoil factor. Peptides. 1995;16(4):749–755. doi: 10.1016/0196-9781(95)00045-l. [DOI] [PubMed] [Google Scholar]
  3. Chinery R., Poulsom R., Elia G., Hanby A. M., Wright N. A. Expression and purification of a trefoil peptide motif in a beta-galactosidase fusion protein and its use to search for trefoil-binding sites. Eur J Biochem. 1993 Mar 1;212(2):557–563. doi: 10.1111/j.1432-1033.1993.tb17693.x. [DOI] [PubMed] [Google Scholar]
  4. Dignass A., Lynch-Devaney K., Kindon H., Thim L., Podolsky D. K. Trefoil peptides promote epithelial migration through a transforming growth factor beta-independent pathway. J Clin Invest. 1994 Jul;94(1):376–383. doi: 10.1172/JCI117332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Frandsen E. K., Jørgensen K. H., Thim L. Receptor binding of pancreatic spasmolytic polypeptide (PSP) in rat intestinal mucosal cell membranes inhibits the adenylate cyclase activity. Regul Pept. 1986 Dec 30;16(3-4):291–297. doi: 10.1016/0167-0115(86)90028-5. [DOI] [PubMed] [Google Scholar]
  6. Gajhede M., Petersen T. N., Henriksen A., Petersen J. F., Dauter Z., Wilson K. S., Thim L. Pancreatic spasmolytic polypeptide: first three-dimensional structure of a member of the mammalian trefoil family of peptides. Structure. 1993 Dec 15;1(4):253–262. doi: 10.1016/0969-2126(93)90014-8. [DOI] [PubMed] [Google Scholar]
  7. Hanby A. M., Poulsom R., Singh S., Elia G., Jeffery R. E., Wright N. A. Spasmolytic polypeptide is a major antral peptide: distribution of the trefoil peptides human spasmolytic polypeptide and pS2 in the stomach. Gastroenterology. 1993 Oct;105(4):1110–1116. doi: 10.1016/0016-5085(93)90956-d. [DOI] [PubMed] [Google Scholar]
  8. Hauser F., Poulsom R., Chinery R., Rogers L. A., Hanby A. M., Wright N. A., Hoffmann W. hP1.B, a human P-domain peptide homologous with rat intestinal trefoil factor, is expressed also in the ulcer-associated cell lineage and the uterus. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6961–6965. doi: 10.1073/pnas.90.15.6961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hoffmann W., Hauser F. The P-domain or trefoil motif: a role in renewal and pathology of mucous epithelia? Trends Biochem Sci. 1993 Jul;18(7):239–243. doi: 10.1016/0968-0004(93)90170-r. [DOI] [PubMed] [Google Scholar]
  10. Jeffrey G. P., Oates P. S., Wang T. C., Babyatsky M. W., Brand S. J. Spasmolytic polypeptide: a trefoil peptide secreted by rat gastric mucous cells. Gastroenterology. 1994 Feb;106(2):336–345. doi: 10.1016/0016-5085(94)90590-8. [DOI] [PubMed] [Google Scholar]
  11. Jørgensen K. D., Diamant B., Jørgensen K. H., Thim L. Pancreatic spasmolytic polypeptide (PSP): III. Pharmacology of a new porcine pancreatic polypeptide with spasmolytic and gastric acid secretion inhibitory effects. Regul Pept. 1982 Mar;3(3-4):231–243. doi: 10.1016/0167-0115(82)90128-8. [DOI] [PubMed] [Google Scholar]
  12. Jørgensen K. H., Thim L., Jacobsen H. E. Pancreatic spasmolytic polypeptide (PSP): I. Preparation and initial chemical characterization of a new polypeptide from porcine pancreas. Regul Pept. 1982 Mar;3(3-4):207–219. doi: 10.1016/0167-0115(82)90126-4. [DOI] [PubMed] [Google Scholar]
  13. Jørgensen P. E., Poulsen S. S., Nexø E. Distribution of i.v. administered epidermal growth factor in the rat. Regul Pept. 1988 Nov;23(2):161–169. doi: 10.1016/0167-0115(88)90024-9. [DOI] [PubMed] [Google Scholar]
  14. Lefebvre O., Chenard M. P., Masson R., Linares J., Dierich A., LeMeur M., Wendling C., Tomasetto C., Chambon P., Rio M. C. Gastric mucosa abnormalities and tumorigenesis in mice lacking the pS2 trefoil protein. Science. 1996 Oct 11;274(5285):259–262. doi: 10.1126/science.274.5285.259. [DOI] [PubMed] [Google Scholar]
  15. Lefebvre O., Wolf C., Kédinger M., Chenard M. P., Tomasetto C., Chambon P., Rio M. C. The mouse one P-domain (pS2) and two P-domain (mSP) genes exhibit distinct patterns of expression. J Cell Biol. 1993 Jul;122(1):191–198. doi: 10.1083/jcb.122.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mashimo H., Wu D. C., Podolsky D. K., Fishman M. C. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science. 1996 Oct 11;274(5285):262–265. doi: 10.1126/science.274.5285.262. [DOI] [PubMed] [Google Scholar]
  17. Playford R. J., Marchbank T., Chinery R., Evison R., Pignatelli M., Boulton R. A., Thim L., Hanby A. M. Human spasmolytic polypeptide is a cytoprotective agent that stimulates cell migration. Gastroenterology. 1995 Jan;108(1):108–116. doi: 10.1016/0016-5085(95)90014-4. [DOI] [PubMed] [Google Scholar]
  18. Playford R. J., Marchbank T., Goodlad R. A., Chinery R. A., Poulsom R., Hanby A. M. Transgenic mice that overexpress the human trefoil peptide pS2 have an increased resistance to intestinal damage. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):2137–2142. doi: 10.1073/pnas.93.5.2137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Podolsky D. K., Lynch-Devaney K., Stow J. L., Oates P., Murgue B., DeBeaumont M., Sands B. E., Mahida Y. R. Identification of human intestinal trefoil factor. Goblet cell-specific expression of a peptide targeted for apical secretion. J Biol Chem. 1993 Mar 25;268(9):6694–6702. [PubMed] [Google Scholar]
  20. Rasmussen T. N., Raaberg L., Poulsen S. S., Thim L., Holst J. J. Immunohistochemical localization of pancreatic spasmolytic polypeptide (PSP) in the pig. Histochemistry. 1992 Sep;98(2):113–119. doi: 10.1007/BF00717002. [DOI] [PubMed] [Google Scholar]
  21. Rio M. C., Bellocq J. P., Daniel J. Y., Tomasetto C., Lathe R., Chenard M. P., Batzenschlager A., Chambon P. Breast cancer-associated pS2 protein: synthesis and secretion by normal stomach mucosa. Science. 1988 Aug 5;241(4866):705–708. doi: 10.1126/science.3041593. [DOI] [PubMed] [Google Scholar]
  22. Rio M. C., Chenard M. P., Wolf C., Marcellin L., Tomasetto C., Lathe R., Bellocq J. P., Chambon P. Induction of pS2 and hSP genes as markers of mucosal ulceration of the digestive tract. Gastroenterology. 1991 Feb;100(2):375–379. doi: 10.1016/0016-5085(91)90205-y. [DOI] [PubMed] [Google Scholar]
  23. Suemori S., Lynch-Devaney K., Podolsky D. K. Identification and characterization of rat intestinal trefoil factor: tissue- and cell-specific member of the trefoil protein family. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11017–11021. doi: 10.1073/pnas.88.24.11017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Thim L. A new family of growth factor-like peptides. 'Trefoil' disulphide loop structures as a common feature in breast cancer associated peptide (pS2), pancreatic spasmolytic polypeptide (PSP), and frog skin peptides (spasmolysins). FEBS Lett. 1989 Jun 19;250(1):85–90. doi: 10.1016/0014-5793(89)80690-8. [DOI] [PubMed] [Google Scholar]
  25. Thim L., Jørgensen K. H., Jørgensen K. D. Pancreatic spasmolytic polypeptide (PSP): II. Radioimmunological determination of PSP in porcine tissues, plasma and pancreatic juice. Regul Pept. 1982 Mar;3(3-4):221–230. doi: 10.1016/0167-0115(82)90127-6. [DOI] [PubMed] [Google Scholar]
  26. Thim L., Norris K., Norris F., Nielsen P. F., Bjørn S. E., Christensen M., Petersen J. Purification and characterization of the trefoil peptide human spasmolytic polypeptide (hSP) produced in yeast. FEBS Lett. 1993 Mar 8;318(3):345–352. doi: 10.1016/0014-5793(93)80543-4. [DOI] [PubMed] [Google Scholar]
  27. Thim L. Trefoil peptides: a new family of gastrointestinal molecules. Digestion. 1994;55(6):353–360. doi: 10.1159/000201165. [DOI] [PubMed] [Google Scholar]
  28. Tomasetto C., Rio M. C., Gautier C., Wolf C., Hareuveni M., Chambon P., Lathe R. hSP, the domain-duplicated homolog of pS2 protein, is co-expressed with pS2 in stomach but not in breast carcinoma. EMBO J. 1990 Feb;9(2):407–414. doi: 10.1002/j.1460-2075.1990.tb08125.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Williams R., Stamp G. W., Gilbert C., Pignatelli M., Lalani E. N. pS2 transfection of murine adenocarcinoma cell line 410.4 enhances dispersed growth pattern in a 3-D collagen gel. J Cell Sci. 1996 Jan;109(Pt 1):63–71. doi: 10.1242/jcs.109.1.63. [DOI] [PubMed] [Google Scholar]
  30. Wright N. A., Hoffmann W., Otto W. R., Rio M. C., Thim L. Rolling in the clover: trefoil factor family (TFF)-domain peptides, cell migration and cancer. FEBS Lett. 1997 May 19;408(2):121–123. doi: 10.1016/s0014-5793(97)00424-9. [DOI] [PubMed] [Google Scholar]
  31. Wright N. A., Poulsom R., Stamp G. W., Hall P. A., Jeffery R. E., Longcroft J. M., Rio M. C., Tomasetto C., Chambon P. Epidermal growth factor (EGF/URO) induces expression of regulatory peptides in damaged human gastrointestinal tissues. J Pathol. 1990 Dec;162(4):279–284. doi: 10.1002/path.1711620402. [DOI] [PubMed] [Google Scholar]
  32. Wright N. A., Poulsom R., Stamp G., Van Noorden S., Sarraf C., Elia G., Ahnen D., Jeffery R., Longcroft J., Pike C. Trefoil peptide gene expression in gastrointestinal epithelial cells in inflammatory bowel disease. Gastroenterology. 1993 Jan;104(1):12–20. doi: 10.1016/0016-5085(93)90830-6. [DOI] [PubMed] [Google Scholar]

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