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Gut, 1988, 29, 762-768 Intramural distribution of regulatory peptides in the sigmoid-recto-anal region of the gut

G-L FERRI, T E ADRIAN, JANET M ALLEN, L SOIMERO, ALESSANDRA CANCELLIERI, JANE C YEATS, MARION BLANK, JULIA M POLAK, AND S R BLOOM From the Department ofAnatomy, 'Tor Vergata' University, Rome, Italy and Departments ofMedicine and Histochemistry, RPMS, Hammersmith Hospital, London

SUMMARY The distribution of regulatory peptides was studied in the separated mucosa, and muscularis externa taken at 10 sampling sites encompassing the whole human (five sites), (two sites), and (three sites). Consistently high concentrations of VIP were measured in the muscle layer at most sites (proximal sigmoid: 286 (16) pmol/g, upper rectum: 269 (17), a moderate decrease being found in the distal smooth (151 (30) pmol/g). Values are expressed as mean (SE). Conversely, substance P concentrations showed an obvious decline in the recto-anal muscle (mid sigmoid: 19 (2 0) pmol/g, distal rectum: 7 1 (1 3), upper anal canal: 1-6 (0 6)). was mainly present in the sigmoid mucosa and submucosa (37 (9 3) and 15 (3-5) pmol/g, respectively) and showed low, but consistent concentrations in the muscle (mid sigmoid: 2-2 (0 7) pmol/g, upper anal canal: 1 5 (0 8)). Starting in the distal sigmoid colon, a distinct peak oftissue NPY was revealed, which was most striking in the muscle (of mid sigmoid: 16 (3-9) pmol/g, upper rectum: 47 (7-8), anal sphincter: 58 (14)). Peptide YY was confined to the mucosa and showed an earlier peak (upper sigmoid: 709 (186) pmol/g, mid-distal sigmoid: 1965 (484)). A clear differential http://gut.bmj.com/ distribution of regulatory peptides was thus shown in the region studied. A possible role is suggested for NPY and VIP containing in the effector control of the human . on September 27, 2021 by guest. Protected copyright.

The whole region of the human gut composed of the by intramural pathways,j which proved to be non- sigmoid colon, rectum and anal canal can probably be adrenergic, non-cholinergic.' considered as a complex regulatory area, devoted to Because little information is available on the the final processing and expulsion of bowel contents, peptide containing innervation of this region of the culminating in .' In fact, although distinct human gut, we have investigated this aspect in detail. sphincteric structures are conspicuously absent at this At each of 10 sampling sites distributed along the level, the rectosigmoid junction itself has been whole region, the main layers of the wall - that is, proposed to act as a regulatory zone.' Below the mucosa, submucosa and muscularis externa, were rectum, a well defined smooth sphincter- that is, the separated and the concentrations of VIP, substance internal anal sphincter, is found at the termination of P, somatostatin, NPY, mammalian bombesin and the alimentary canal.' PYY were measured by radioimmunoassay. In view Within this complex region, intrinsic nerves appear of the immunochemical methods used, results des- to play a crucial role. This is clearly the case for the cribed will refer to peptide like immunoreactivity inhibitory control of the internal anal sphincter, throughout. exerted by the rectoanal reflex and largely mediated Methods Address for correspondence: Dr G-I. Fcrri. D)epartment of Tor Vergata University. via 0. Raimondo. 00173 Roma. Itly. Fresh tissues were obtained from six specimens of Received for publication 4 December 1987. abdominoperineal resection of the sigmoid colon and 762 Gut: first published as 10.1136/gut.29.6.762 on 1 June 1988. Downloaded from

Peptides in hlutantsigmoid-rectliutn-atilzs 763

1 - 30cm - 1 Tatble A ntisera specificity 1 ] 51 F w Spe(ificity Referenice Sigmoid colon nal canal VIP V9 Wholc molcculc (9) Substance P SP 3 C-tcrminal (9) Fig. 1 Sampling placn used in the study. Samples were taken MammaIlian hombesin BN 103 C-tcrminal* (9) at each ofthe equidistant sites 1-5, along the distal30 cm of Somatostatin K 2 Wholc cyclic rcgion (9) colon - that is, virtually the whole sigmoid. One sample each NPY YN7 N-terminal't (m1)) was takent from theproximal and distal halves ofthe rectal PYY Y21 N-tcrminalt (I 1) ampulla (sites 6 and 7, respectively). Three adjacent strips, 7-9 mm wide, were taken across the anal canal (sites 8 to 10), *the antiscrum measurcs fully wholc mniammaliian homhcsin (- the middle one (site 9) at the distal margin ofthe releasing peptide 1-27) and the decapeptidc of mammailiain terminating bomhcsin (gastrin-reicasing pcptilc 18-27); tnot cross-rcacting smooth sphincter. with PYY (<()1)/1%); tnot cross-rcacting with NPY (<()-()1I%). rectum, because of colonic or rectal carcinoma (patients' mean age 67-5 years, range 61-79, three formed as previously described in detail,"'' using the men and three women). Only specimens devoid of antisera listed in the Table. advanced luminal obstruction and/or proximal dilata- tion were investigated, while no was taken for Results the present investigation before sampling for diag- nostic histopathology had been completed. Of the various peptides studied, VIP, substance P, The sampling plan used is summarised in Figure 1. somatostatin and NPY were detected in all three One sample was taken at each of the equidistant sites layers, while bombesin was mainly represented in the 1-5, along the distal 30 cm of colon (thus virtually muscle. Conversely, PYY was virtually confined to covering the whole sigmoid). One sample each was the separated mucosa (99.2 (0-4)% of the total taken from the proximal and distal halves of the content, mean (SE)). The concentration profile of rectal ampulla (sites 6 and 7, respectively), thus over each peptide is shown in Figs 2-4. 2 cm away from any other sample. Three adjacent Vasoactive intestinal polypeptide (Fig. 2, left) was strips, 7-9 mm wide, were taken across the anal canal most abundant in the separated mucosa at all levels, http://gut.bmj.com/ (sites 8 to 10), the middle one (site 9) terminating at lower concentrations being shown in the anal . the distal margin of the smooth sphincter. Thus, the Considerable amounts of the same neuropeptide most distal sample (site 10) did not include a smooth were revealed in the submucosa and muscle layer, muscle layer. As the internal anal sphincter starts as a too, throughout the extension of the internal anal progressive thickening of the muscle layer in the most sphincter. Conversely, substance P (Fig. 2, right) distal rectum, without a distinct proximal border,' appeared to be little represented beyond the rectum,

sites 8 and 9 were considered to include most of it. its muscular concentrations already declining at the on September 27, 2021 by guest. Protected copyright. Tissue samples (n=6, except sites 6 and 7: n=5) distal rectal site. were taken over 5 cm away from the tumour margin, Somatostatin immunoreactivity (Fig. 3, left) was free of mesenteric (or epiploic) fat and other present mainly in the mucosa and, to a lesser extent, structures surrounding the gut wall. was in the submucosa, in which layers it showed a marked checked in parallel samples, in order to exclude decrease in concentration across the rectum. In the neoplastic infiltration in the vicinity of the processed muscle layer, this peptide showed much lower, but tissues. consistent tissue levels down to the proximal anal Samples were separated by microdissection into canal. mucosa (containing and ), Measurable concentrations of NPY (Fig. 3, right) submucosa and muscularis externa, as previously were found in all layers, with the exception of the described in detail.7" From the most distal site mucosa of most of the sigmoid colon (sites 1 to 4). A (number 10), skin and subjacent connective and/or clear increase in NPY concentration was found were obtained in a similar way. For distally, the lower rectum and anal canal showing a peptide extraction, layers were coarsely chopped distinct peak, most evident in the muscle. A similarly with a scalpel, weighed and dropped into preheated striking, but earlier, peak was demonstrated for PYY polypropylene tubes, containing 0-5 mol/l acetic acid concentrations in the mucosa (Fig. 4, upper panel) (approximately 10 ml/g) in a vigorously water between the mid-distal sigmoid and the proximal bath for 15 minutes. After a further 30 minutes at rectum. Mammalian bombesin (Fig. 4, lower panel) room temperature (with slow constant agitation), was largely confined to the sigmoid colon, layers tubes were briefly spun and supernatants were stored other than muscle containing barely detectable frozen (at -70°C). Radioimmunoassays were per- amounts of this peptide. Gut: first published as 10.1136/gut.29.6.762 on 1 June 1988. Downloaded from

764 4Ferri, Adrian, Allen, Soimero, Cancellieri, Yeats, Blank, Polak, and Bloom

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Fig. 2 Concentrations ofvasoactive intestinalpolypeptide (VIP: left) and substance P (Subs. P: right) in the threeseparated layers - that is mucosa (containing epithelium and innervated lamina propria), submucosa and muscularis externa (muscle= circular and longitudinal muscle with intervening ) across the sigmoid-recto-anal region (sigmoid=sigmoid colon, =anal canal; no distinct muscle layer was present at site 10, which was taken distal to the smooth sphincter; means (SE); *=

Discussion as measured in the human gut by various labora- tories7'7-' are probably due to differing character- A distinct differential distribution of regulatory istics of the antisera used, or to the varied tissue peptides was shown along the sigmoid-recto-anal storage and extraction procedures used. region of the human gut. Peptide concentrations A previous study, carried out mainly in the and measured in the sigmoid colon are largely in keeping not including the internal anal sphincter, concluded with previous human studies, which described the that 'a rich VIP supply is characteristic of molecular characterisation of the same peptides and '.2 As far as peptide distribution may be their distribution as far down as the colon.7 I-"' Minor taken to reflect the density of peptide containing differences in somatostatin tissue concentrations, nerves, a comparative abundance of VIP nerves can Gut: first published as 10.1136/gut.29.6.762 on 1 June 1988. Downloaded from

Peptides in human sigmoid-rectum-anus 765 SRIF NPY 50- 100- I, Mucosa pmol/g' 50- 20-

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Fig. 3 Concentrations ofsomatostatin (SRIF: left) and neuropeptide Y (NPY: right) in the three separated layers - that is, mucosa (containing epithelium and innervated laminapropria), submucosa and muscularis externa (muscle=circularand longitudinal muscle with intervening myentericplexus) across the sigmoid-recto-anal region (sigmoid=sigmoid colon, anus=anal canal; no muscle layer was present at site 10, which was taken distal to thesmooth sphincter; means (SE); * =

766 Ferri, Adrian, Allen, Soimero, Cancellieri, Yeats, Blank, Polak, and Bloom

pYy peptide has a powerful vasoconstrictory effect,"7 so 2500 that perivascular NPY-fibres may be involved in gut vasomotor control. Mucosa The distribution of bombesin like peptides, largely confined to the muscle, confirms previous findings obtained in the human , , , and colon.7"47 A study has been reported, in which pmol/g - the human gut wall had been split into preparations of mucosa and 'muscle' (the latter apparently also including submucosa), both of which showed similar 1000- bombesin concentrations at most levels.38 It is relevant to note, however, that human postmorten specimens were used in the latter investigation. A recent study showed an inhibitory effect of somatostatin on the basal tone of human rectal musculature.3" Thus, even the small amounts of 0111 23 5 6 7 8 910 somatostatin shown in the rectal muscularis might 4 have a functional significance. The main localisation 2 Bombesin of this neuroendocrine peptide, however, was shown to be in the endocrine cell containing mucosa, as in other areas of the human gut.7 1837 Finally, the other Muscle endocrine peptide studied - that is, PYY, showed a very clear peak between distal sigmoid and upper rectum. Such finding appears to stress the proposed pmol /g role of PYY in the distal hormonal inhibition of a variety of digestive functions, including gastric emptying, colonic and blood flow.4"3

Most specimens were kindly provided by L Tonelli http://gut.bmj.com/ E and G Biliotti, University of Firenze, Italy. The authors are indebted to G Siracusa, A Riva, and V Eusebi for encouragement and stimulation during the progress of this work, to V Eusebi and A M Mancini for laboratory hospitality. Partly supported 1 23l45 67 8910 by an Italian National Research Council (CNR)

Sigmoid Rectum Anus Research Contract. on September 27, 2021 by guest. Protected copyright. Fig. 4 Concentrations ofpeptide YYin theseparated mucosa (PYY, upperpanel) and mammalian bombesin in References the muscularis externa (muscle=circular and longitudinal muscle with intervening myenteric plexus) across the I Reeve DRE. Anatomy of the sphincters of the alimen- sigmoid-recto-anal region (sigmoid=sigmoid colon, tary canal. In: Thomas PA, Mann CV, eds. Alimentary anus=anal canal; no muscle layer waspresent atsite JO, sphincters and their disorders. London: Macmillan, which was taken distal to the smooth sphincter; means (SE); 1981: 1-26. * =< pmollgfor PYY, <055for mammalian bombesin). 2 Baker WNW, Mann CV. The rectosigmoid junction zone: another sphincter? In: Thomas PA, Mann CV, eds. Alimentary sphincters and their disorders. London: the gut,2j-3' where it was localised to both intrinsic Macmillan, 1981: 20(1-1 1. neurons, which were shown to contain also VIP and 3 Denny-Brown D, Robertson EG. An investigation of PHI like peptides,3' and extrinsic nor-adrenergic the nervous control of defecation. Brain 1935; 58: fibres. As the internal anal sphincter, including that 256-310. of man, is provided with a very rich supply of 4 Burleigh DE, D'Mello A. Neural and pharmacologic noradrenergic nerve the high NPY concen- factors affecting motility of the internal anal sphincter. fibres,33 1983; 84: 409-17. tration shown in this area may reflect the density of 5 Duthie HL. The anal sphincters. In: Thomas PA, Mann extrinsic sympathetic nerves. NPY has been shown to CV, eds. Alimentary sphincters and their disorders. inhibit contraction of smooth muscle,'734 including London: Macmillan, 1981: 213-26. that of the gut," probably through inhibition of 6 Bouvier M, Gonella J. Nervous control of the internal cholinergic neurotransmission." " In addition, this anal sphincter of cat. J Physiol 1981; 310: 457-69. Gut: first published as 10.1136/gut.29.6.762 on 1 June 1988. Downloaded from

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7 Ferri G-L, Adrian TE, Ghatei MA, et al. Tissue 25 Fahrenkrug J, Haglund U, Jodal M, Lundgren 0, Olbe localisation and relative distribution of regulatory pep- L, Schaffalitzky de Muckadell OB. Nervous release of tides in separated layers from the human bowel. Gastro- vasoactive intestinal polypeptide in the gastrointestinal enterology 1983; 84: 777-86. tract of : possible physiological implications. 8 Ferri G-L. Human gut neuroanatomy: methodology for J Physiol 1978; 284: 291-305. a quantitative analysis of nerve elements and neuro- 26 Biancani P, Walsh J, Behar J. Vasoactive intestinal transmitter diversity in the human enteric nervous peptide: a neurotransmitter for relaxation of the rabbit system. Basic Appl Histochem 1988; 32: 117-44. internal anal sphincter. Gastroenterology 1985; 89: 9 Bloom SR, Long RG, eds. Radioimmunoassasy of gut 867-74. regulatory peptides. London: Saunders, 1982. 27 Lundberg JM, Terenius L, Hokfelt T, et al. Neuro- 10 Allen JM, Yeats JC, Adrian TE, Bloom SR. Radio- peptide Y (NPY)-like immunoreactivity in peripheral immunoassay of neuropeptide Y. Regul Pept 1984; 8: noradrenergic neurones and effects of NPY on 61-70. sympathetic function. Acta Physiol Scand 1982; 116: 11 Adrian TE, Ferri G-L, Bacarese-Hamilton AJ, Fuessl 477-90. HS, Polak JM, Bloom SR. Human distribution and 28 Furness JB, Costa M, Emson PC, et al. Distribution, release of a putative new gut , peptide YY. pathways and reactions to treatment of nerves with Gastroenterology 1985; 89: 1070-7. neuropeptide Y- and pancreatic polypeptide-like 12 Holzer P, Bucsics A, Saria A, Lembeck F. A study of the immunoreactivity in the guinea- digestive tract. Cell concentrations of substance P and neurotensin in the Tissue Res 1983; 234: 71-92. gastro-intenstinal tract of various . Neuro- 29 Sundler F, Moghimzadeh E, Hakanson R, Ekelund M, science 1982; 7: 2919-24. Emson P. Nerve fibres in the gut and of the rat 13 Brodin E, Sj6lund K, Hakanson R, Sundler F. Sub- displaying neuropeptide Y immunoreactivity. Intrinsic stance P-containing nerve fibres are numerous in human and extrinsic origin. Cell Tissue Res 1983; 230: 487-93. but not in feline intestinal mucosa. Gastroenterology 30 Ferri G-L, Ali-Rachedi A, Tatemoto K, Bloom SR, 1983; 85: 557-64. Polak JM. Immuno-cytochemical localisation of 14 Price J, Penman E, Wass JAH, Rees LH. Bombesin-like neuropeptide-like immunoreactivity in extrinsic immuno-reactivity in human . noradrenergic and intrinsic gut neurons. Front Horm Regul Pept 1984; 9: 1-10. Res 1984; 12: 81-4. 15 Llewellyn-Smith IJ, Furness JB, Murphy R, O'Brien 31 Lee Y, Shiosaka S, Emson PC, Powell JF, Smith AD, PE, Costa M. Substance P-containing nerves in the Tohyama M. Neuropeptide Y-like immunoreactive human . Gastroenterology 1984; 86: structures in the rat with special reference to 421-35. the noradrenaline neuron system. Gastroenterology http://gut.bmj.com/ 16 Allen JM, Ferri G-L, Hughes J, Polak JM, Bloom SR. 1985; 89: 118-26. Presence, distribution and effects of neuropeptide Y in 32 Ekblad E, Hakanson R, Sundler F. VIP and PHI coexist the mammalian gut. Gut 1985; 26: 547. with an NPY-like peptide in intramural neurones of the 17 Penman E, Wass JAH, Butler MG, et al. Distribution small intestine. Regul Pept 1984; 10: 47-55. and characterisation of immunoreactive somatostatin in 33 Gabella G. Innervation of the gastrointestinal tract. Int human gastro-intestinal tract. Regul Pep 1983; 7: 53-65. Rev Cytol 1979; 59: 129-93. 18 Keast JR, Furness JB, Costa M. Somatostatin in human 34 Allen JM, Adrian TE, Tatemoto K, Polak JM, Hughes

enteric nerves. Cell Tissue Res 1984; 237: 299-308. J, Bloom SR. Two novel peptides, neuropeptide Y on September 27, 2021 by guest. Protected copyright. 19 Baldissera FGA, Holst JJ, Jensen SL, Krarup T. Distri- (NPY) and peptide YY (PYY) inhibit the contraction of bution and molecular forms of peptides containing the electrically stimulated mouse . Neuro- somatostatin immuno-determinants in extracts from the peptides 1982; 3: 71-7. entire gastrointestinal tract of man and pig. Biochim 35 Allen JM, Hughes J, Bloom SR. Presence, distribution Biophys Acta 1985; 838: 132-43. and pharmacological effects of neuropeptide Y in 20 Alumets J, Fahrenkrug J, Hakanson R, Schaffalitzky de mammalian gastrointestinal tract. Dig Dis Sci 1987; 32: Muckadell 0, Sundler F, Uddman R. A rich VIP nerve 506-12. supply is characteristic of sphincters. Nature 1979; 280: 36 Wiley J, Owyang C. Neuropeptide Y inhibits cholinergic 155-6. in the isolated guinea pig colon: mediation 21 McGregor GP, Bishop AE, Blank MA, et al. Compara- through alfa-adrenergic receptors. Proc Natl Acad Sci tive distribution of vasoactive intestinal polypeptide USA 1987; 84: 2047-51. (VIP), substance P and PHI in the enteric sphincters of 37 Ferri G-L, Adrian TE, Soimero L, et al. Regulatory the cat. Experientia 1984; 40: 469-71. peptide distribution in separated layers of the human 22 Ferri G-L, Morreale RA, Soimero L, Biliotti G, jejunum. 1987; 37: 15-21. Dockray GJ. Intra-mural distribution of Met5- 38 Greeley GH, Partin M, Spannagel A, et al. Distribution enkephalin-Arg6-Gly7-Leux in sphincter regions of the of bombesin-like peptides in the alimentary canal of human gut. Neurosci Lett 1987; 74: 304-8. several species. Regul Pept 1986; 16: 169-81. 23 Said SI, ed. Vasoactive intestinal peptide. New York: 39 Lerebours E, Weber J, Chayvialle JA, Galmiche JP, Raven Press, 1982. Colin R, Denis Ph. Somatostatin effect of rectal muscle 24 Kubota M, Szurszewski JH. Effect of VIP, PHI, in human healthy volunteers. Digestion 1987; 36: 42-6. bradykinin and ATP on activity of the canine internal 40 Lundberg JM, Tatemoto K, Terenius L, et al. Localisa- anal sphincter. Gastroenterology 1985; 88: 1458. tion of peptide YY (PYY) in gastrointestinal endocrine Gut: first published as 10.1136/gut.29.6.762 on 1 June 1988. Downloaded from

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cells and effects on intestinal blood flow and motility. 42 Adrian TE, Savage AP, Sagor GR, et al. Effect of Proc Natl Acad Sci USA 1982; 79: 4471-5. peptide YY on gastric, pancreatic, and biliary function 41 Suzuki T, Nakaya M, Itoh Z, Tatemoto K, Mutt V. in . Gastroenterology 1985; 89: 494-9. Inhibition of interdigestive contractile activity in the 43 Pappas TN, Debas HT, Chang AM, Taylor IL. Peptide stomach by peptide YY in Heidenhain pouch dogs. YY release by fatty acids is sufficient to inhibit gastric Gastroenterology 1983; 85: 114-21. emptying in dogs. Gastroenterology 1986; 91: 1386-9. http://gut.bmj.com/ on September 27, 2021 by guest. Protected copyright.