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Immunoreactive somatostatin and vasoactive intestinal peptide in the digestive tract of cats
GASTROENTEROLOGY
79:837-843,198O
Immunoreactive Somatostatin and Vasoactive Intestinal Peptide in the Digestive Tract of Cats JEAN-ALAIN CHAYVIALLE, MASAHIKO MIYATA, PHILLIP L. RAYFORD, and JAMES C. THOMPSON Department
of Surgery,
The University
of Texas Medical
Somatostatin and vasoactive intestinal peptide were measured by radioimmunoassay in acetic acid extracts of tissue samples from the digestive tracts of seven adult Cats. The concentration of immunoreactive somatostatin in antral mucosa (2926 f 978 pmol/g wet weight, mean i SE) was far above the concentrations in the head of the pancreas (442 f 941, distal duodenum (377 i 91), and ileum (355 + 48). Large amounts of immunoreactive vasoactive intestinal peptide were recovered from both mucosal and muscular layers of gastrointestinal tract; the highest values were observed in the muscular layer of the cecum (579 f 82 pmol/g) and the mucosa of the right colon (564 + 244). The mucosal/muscular Iayer ratio of vasoactive intestinal peptide concentrations increased caudally from 0.20 in the esophagus to over 1 in the iieum and colon. Upon gel filtration on G-50 Sephadex, somatostatin in the antrum, duodenum, and pancreas were eluted as a predominant peak in the volume of the tetradecapeptide, but somatostatin from the ileum and cecum was associated with a major faster component. Vasoactive intestinal peptide in the muscular iayer of gastrointestinal tract and in mucosa of antrum and duodenum consisted essentially of a single farm, which coeluted with the purified porcine peptide; a slower component was detected in the mucosa of the
Branch,
Galveston,
Texas
ileum and cecum. These results indicate molecular heterogeneity of both immunoreactive somatostatin and vasoactive intestinal peptide in the digestive tract of Cats, and suggest that the variable ratio of the different molecular farms of each peptide along the gastrointestinal tract may reflect regional specijïcity of biologie effects and metabolism. Somatostatin (STS), a tetradecapeptide isolated from ovine hypothalamus,’ and vasoactive intestinal peptide (VIP), an octacosapeptide purified from porcine intestine,‘- have a wide distribution in the digestive tract of al1 mammals studied. By immunocytochemistry, somatostatin has been detected in some neural elemenfs,3 but is mainly found in endocrine cells of gastrointestinal mucosa and pancreas?’ Vasoactive intestinal peptide has been recognized in nervous sfructures,~’ but the simultaneous location of immunoreactive material in endocrine cells’ is disputed by some investigators.7 In the present study, STS and VIP were comparatively mapped by radioimmunoassay in the digestive tract of Cats, and the immunoreactive components were tested for molecular heterogeneity.
Methods Tissue Sampling
Received August 29, 1979. Accepted May 17.1980. Address requests for reprints to: James C. Thompson, M.D., Department of Surgery. The University of Texas Medical Branch, Galveston, Texas 77550. Dr. Chayvialle is a Visiting Scientist. Parent Institution: Hôpital Edouard-Herriot, Lyon, France. Dr. Miyata is a Visiting Scientist. Parent Institution: Osaka University Medical School, Osaka, Japan. This work was supported by grants from the National Institutes of Health (AM 15241) and the John A. Hartford Foundation, Inc. This work was presented in part at the Annual Meeting of the American Gastroenterological Association, Las Vegas, Nevada, May 1978. 0 1980 by the American Gastroenterological Association 0016-5085/80/110837-07$02.25
Seven adult Cats, weighing 3.5-6 kg, were anesthetized with Nembutal (30 mg/kg intraperitoneally) after an 16-hr fast, and submitted to laparotomy. In the first six animals, tissue specimens were taken at preselected sites of esophagus, fundus, antrum, duodenum, ileum, cecum, right colon, left colon, and upper rectum. For each sample, the mucosa was quickly dissected from the muscular layer, and both parts were frazen on dry ice. Additional specimens were taken from the spleen, gallbladder, and liver. In cat No. 7, samples were taken every centimeter in the fundus, antrum, and duodenum. The mean weight of the specimens from each site varied from 36 to 129 mg for
838
CHAYVIALLE
ET AL.
GASTROENTEROLOGY
gastrointestinal mucosa or muscular layers, and from 102 to 310 mg for the spleen, gallbladder, and liver specimens.
Vol. 79, No. 5, Part 1
mean recovery of 2.5, 5, 10, and 20 ng synthetic cyclic somatostatin added to cat tissue samples before extraction was 83.6% (range: 77%-90%, n = 6 for each dose).
Extraction Samples were successively weighed while they were thawed, boiled in 200~~1 distilled water for 10 min, cut into pieces with a scalpel blade, and homogenized in 2 M acetic acid with a tissue grinder (Kontes size AA, T.M. Kontes, Vineland, N.J.). The homogenate was kept under agitation overnight at 4’, and lyophilized. The dry residue was suspended in 0.1 M ammonium acetate pH 5.5 with 5% horse serum and 1 mg/ml sodium azide, and centrifuged. The supernatant was assayed, at three or more dilutions in duplicates, for STS and VIP on the same day.
Somatostatin
Assay
The method, reported in detail previously,8 has been further characterized. As compared with the synthetic cyclic tetradecapeptide (Serono Pharm Präp GmbH, Freiburg/Breisgau, West Germany), the poteney of neurotensin (Calbiochem, San Diego) is less than 0.0002, while the ID,, of salmon calcitonin (Armour, Phoenix) is 0.4 IU. Lactoperoxydase (Calbiochem) was used for iodination of l-tyrosyl somatostatin (Serono). The tracer was purified on carboxymethylcellulose CM52 (Whatman Ltd., Maidstone, England), using 0.05 M ammonium acetate pH 5.5 as starting buffer and 0.5 M ammonium acetate pH 6.0 for elution. Antiserum 55A fully recognizes 4-Ala somatostatin, while 6-Ala, 8-Als, and 11-Ala somatostatin (gift from Dr. J. Rivier and Prof. R. Guillemin) have relative potencies of 0.002, less than 0.002, and 0.01, respectively. The
Vasoactive
Intestinal
Peptide
Assay
Antiserum 76A was obtained from a New Zealand white rabbit after the second injection of 100 pg highly purified porcine VIP (Prof. V. Mutt) conjugated to 500 pg bovine albumin (Poviet Prod B.V., Amsterdam), with 5 mg lethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (Sigma Chemical Company, St. Louis, Mo.). Porcine secretin (Prof. Mutt), porcine gastric inhibitory polypeptide (Prof. J.C. Brown), bovine pancreatic polypeptide (Dr. R. E. Chance), bovine glucagon and insulin (Novo), synthetic somatostatin (Serono), synthetic human gastrin 1, 1-17 (ICI), salmon calcitonin (Armour) and neurotensin (Calbiochem) did not crossreact at a leve1 of 100 ng/tube, or 0.4 IU/tube for calcitonin. Vasoactive intestinal peptide fragments 1-10 and 18-28 (gift from Prof. M. Bodansky) have relative potenties of less than 0.0001 and of 0.1, respectively. The working final dilution of the antiserum for assay purposes is 1/160,000. Highly purified porcine VIP (1 pg) was labeled with “‘Iodine (0.5 mCi) in 0.4 M ammonium acetate pH 5.0 using lactoperoxydase (Calbiochem, 5 pp) and 5% hydrogen peroxide (5 ~1). The reaction was stopped after 20 min by adding 2 ml starting buffer, and iodinated VIP was purifïed on 1 X 10 cm SP C25 Sephadex column (Pharmacia Fine Chemicals, Piscataway, N.J.) using 0.2 M ammonium acetate pH 5.0 as starting buffer and 0.8 M ammonium acetate pH 5.6 with 5% horse serum for elution. A typical elution profile is shown in Figure 1. The most reactive fractions were diluted in 0.1 M ammonium acetate pH 5.5 and kept frozen. Assays were set up in 0.1 M ammonium acetate buffer pH 5.5 with 5% horse serum and 0.2 mg/ml sodium azide. Tracer VIP (1500-2000 cpm/ tube) and a second antibody (goat antirabbit globulin) were added 24 and 48 hr later. The tubes were centrifuged at 3500 rpm for 30 min at 4’, the supernatant was discarded, and the precipitate was countéd in a well-type gamma spectrometer. The sensitivity of the system, i.e., the minima1 dose giving a significant inhibition of binding when tested as four replicates, was about 5 pg/ml incubate. The within-assay and between-assay variations were less than 10% (coefficient of variation). The mean recovery of 2.5, 5,10, and 20 ng porcine VIP added to cat tissue samples before extraction was 71.5% (range: 62%-83%, n = 6 for each dose).
FRACTION NmfR (1.5 ML)
Figure 1. Elution profile of ?-VIP
on SP C25 Sephadex. Initial
buffer: 0.2 M ammonium acetate pH 5.0. Elution of iodinated peptide: 0.8 M ammonium acetate pH 5.5: 5% aged plasma and Trasylol were added to both solutions. Full line: radioactivity eluted. Free iodine is eluted rapidly with the initial buffer. Two peaks are eluted with the 0.8 M solution. Dotted Ene: percent ra-
dioactivity (about 10,ooO cpm/tube) bound overnight by antiserum at final dilution of 1/8O.o00 (nonspecific binding subtracted). The most immunoreactive fractions are found on the descending slope of the second radioactive peak.
Chromatographic
Studies
Extracts of antrum, duodenum, ileum, cecum, pancreas, and gallbladder were pooled to a volume of 1 ml, incubated for 2 hr in 6 M urea (final concentration) at room temperature, and applied on a 1.5 x 100 cm G-50 superfine Sephadex column (Pharmacia), equilibrated in 0.05 M potassium phosphate-0.1 M sodium chloride pH 5.5 with 5% horse serum and 0.2 mg/ml sodium azide. The column was preloaded with 5 ml 6 M urea immediately before applying the sample. Fractions of 4.0 ml were collected at a flow rate of 8 ml/hr at 4” and assayed for STS and VIP. In some
November
1980
SOMATOSTATIN
experiments, the various cubated at serial dilutions
immunoreactive for comparison
peaks were into the standard
curve. The recovery of both peptides was in the range of 55%-80%. Results are expressed as mean f SE picomoles equivalent synthetic cyclic somatostatin or purified porcine VIP per gram wet weight, or as nanograms per fraction eluted at chromatography.
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Immunoreactive somatostatin was located chiefly in the gastrointestinal mucosa and the pancreas (Figure 2). In mucosa, the concentration in fundus was 77 f 15 pmol/g, rising abruptly to 2926 -r 976 in the antrum and subsequently decreasing to 377 f 91 in the distal duodenum. The concentration from jejunum to rectum varied between 165 f 16 (cecum) and 355 + 46 pmol/g (ileum). Samples taken every centimeter in cat No. 7 showed a sharp gradient of STS concentration across the pylorus (Figure 3). Values in the tail and head of the pancreas were 310 f 49 and 442 f 94 pmol/g, respectively. The STS immunoreactivity detected in al1 these segments, SOMATOSTATIN
PYLZORUS
2 000.
Results
839
AND VIP IN GUT GF CAT
u fs u
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1 cm
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Figure 3. Somatostatin concentration (pmol/g wet weight) in antral and duodenal mucosa. Samples were taken in distal antrum, proximal duodenum, and distal duodenum in cats NOS. 1-6 (dotted Iine, mean f SE) and every centimeter from 2 cm before to 9 cm after the pylorus in cat No. 7 (fuI1 Fine).
when measured in graded doses, gave response curves that were similar to that of the standard (Figure 4). In the gastrointestinal muscular layer, the concentration was in the range of 9 f 4 to 91 f 20 pmol/g, while insignificant amounts were recovered from the spleen, gallbladder, and liver. Immunoreactive VIP was found in both mucosa and muscular layers of the gastrointestinal tract. In mucosa, regional peaks were observed in the proxi-
SAMPLE 4
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1
( 1/x
103)
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z Figure 2. Mean plus or minus one standard error tissue somatostatin concentration (pmol/g wet weight) in gastrointestinal mucosa or parenchymas (right horizontal axis) and gastrointestinal muscular layer (left horizontal axis) in six adult Cats. The peak concentration is observed in mucosa of antrum and proximal duodenum. Substantial amounts of peptide are detected in intestinal mucosa and pancreas.
1
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1 000
(pg/ml)
Figure 4. Inhibition curves generated with graded concentrations of synthetic cyclic somatostatin (standard) and with serial dilutions of various acetic extracts. The immunoreactive component(s) detected in gastrointestinal mucosa and pancreas is not dissimilar from the standard.
VIP 680
GASTROENTEROLOGY
CHAYVIALLE ET AL.
840
CONCENTRATION
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Figure 5. Mean plus or minus one standard error tissue VIP concentration (pmol/g wet weight) in gastrointestinal mucosa and parenchymas (right horizontal axis) and gastrointestinal muscular layer (left horizontal axis) in six adult Cats. Peaks are observed in both mimosa and muscular layer of duodenum and right colon. Substantial amounts of peptide are recovered from the gallbladder wall.
(
Figure
1
mal duodenum (293 + 44 pmol/g) and the right colon (564 + 244, Figure 5). A similar pattern was obtained in the muscular layers, with peaks in the duodenum (415 + 135) and cecum (579 & 82). The mucosa/muscular layer ratio of VIP concentrations increased caudally, with values of 0.20 in the esophagus, 0.45 in the fundus, 0.80 in the antrum and duodenum, 1.16-1.84 in the intestine, and 1.2-2.31 in the colon. The duodenal peak VIP concentration in mucosa and muscular layer was confirmed in cat No. 7 (Figures 6 and 7), in which samples were taken at 1-cm intervals from antrum to duodenum. The concentrations in the tail and head of the pancreas were 15 f 4 and 30 + 4 pmol/g, and 78 + 7 in the gallbladder wall. Insignificant amounts were found in the spleen and liver. Inhibition curves generated by graded doses of VIP in the various tissue segments were similar to that of purified porcine VIP (Figure 8). Chromatography of antral, duodenal, ileal, and cecal mucosal extracts and of pancreatic extracts gave two immunoreactive somatostatin peaks, one coeluting with the cyclic tetradecapeptide, and a faster peak which was the predominant form in the ileum and colon (Figure 9). A third component, eluted slightly after the void volume, was detected in the duodenum, ileum, and cecum, but contributed less than 20% to the total immunoreactivity recovered from the column. Gel filtration of the various gastrointestinal segments gave distinct patterns of VIP immunoreactivity for mucosa and muscular layers (Figure 10). In muscle, VIP was eluted essentially as a single peak in the same position as porcine VIP, similarly to the immunoreactive material detected in the gallbladder. The same component was found in the gastrointesti-
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;
Vol. 79, No. 5, Part
PUNDUS AN*uM-r
DUODENUM
Je l
,t 4
Ce
RC
I
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LC
OJ 6.
Vasoactive intestinal peptide concentration (pmol/g wet weight) in muscular layer of gastrointestinal tract. Samples were taken at intervals from fundus, antrum, duodenum. jujunum (Je), ileum (11) cecum (Ce), right colon (RC), and left colon (LC) in cats NOS. 1-6, and every centimeter in the fundus, antrum, and duodenum in cat No. 7. Peak concentrations occur in proximal duodenum and cecum.
OJ
Figure 7. Vasoactive intestinal peptide concentration (pmol/g) in mucosa of gastrointestinal tract. Diagram is prepared as in Figure 6. Peak concentrations occur in proximal duodenum and right colon.
November 1980
SOMATOSTATIN AND VIP IN GUT OF CAT
STS
na1 mucosa, associated in the ileum and colon with a second peak emerging later than porcine VIP. For both somatostatin and VIP, serial dilutions of pooled fractions corresponding to the various immunoreactive components yielded inhibition curves parallel to that of the standard.
Discussion The present results show that immunoreactive somatostatin in the digestive tract of cats is located predominantly in the mucosa of the gastrointestinal tract, but not in the muscular layer, and in the pancreas. The concentration in antral mucosa was far above that in any other segment, including the fundic mucosa and the pancreas. In this respect, the cat would differ from ratsg”’ or humans,” where values in the fundus or pancreas are of the same order as those in the antrum. Whether this reflects specific features of gastric physiology in the cat, possibly related to dietary habits, is not known. In agreement with other immunocytologic” and radioimmunologic studies,lD”Z immunoreactive somatostatin was detected in intestinal and colonic mucosa. Although the concentration achieved in these segments was only 1O%-15% that in the antrum, it can be suggested that the mass of mucosa from jejunum to rectum represents a large fraction of total digestive somatostatin. In addition to its inhibitory effects on endocrine and exocrine secretions of the stomach and pancreas,13 exogenous somatostatin has been shown to inhibit intestinal absorption,14”5 mo40
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Figure 9. Elution profile of somatostatin-like immunoreactivity in acetic extracts from the antrum, duodenum, ileum, cecum, and pancreas on a 1.5 x 100 cm G-50 superfine Sephadex column. Extracts were preincubated in 6 M urea and applied after 5 ml 6 M urea. Elution buffer: 0.05 M potassium phosphate-0.1 sodium chloride pH 5.5 with 5% horse serum and 0.2 mg/ml sodium azide. Fout milliliter fractions, flow rate: 8 ml/hr. The elution volumes of albumin (Alb), synthetic cyclic somatostatin (STS), and lZ51are shown at the top of the graph.
tility,” and blood flow,14 as wel1 as the release of acetylcholine by the myenteric plexuses.” The immunoreactivity detected in the intestine and colonic mucosa may thus be the basis for important physiologie actions of the peptide in these segments, either in response to intraluminal events, or through the interplay of neurohumoral pathways within the gastrointestinal wall. Several investigators have reported various degrees of molecular heterogeneity of immunoreactive somatostatin in gastrointestinal tract and pancreas in rats and humans,9.“.‘2 but the extra components
GASTROENTEROLOGY
CHAYVIALLE ET AL.
842
VII’
dient of the molecular
125,
the gastrointestinal
+
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30
40
50
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NUMBER
Figure 10. Elution profile of VIP-like immunoreactivity in acetic extracts of mucosa (fu11line) and muscular layer (dotted line) from antrum, duodenum, ileum, cecum, and from gallbladder wall. Same chromatographic conditions as in Figure 9. VIP: elution volume of highly purified porcine VIP.
have not been fully characterized. Vale et a1.18detected a molecular form larger than the tetradecapeptide in tissue extracts from various species only when using antisera directed against the c-terminal moiety of the molecule, which suggested that the large component resulted from extension of the NH,-terminus of the tetradecapeptide. Recently, three forms were found in canine pancreas,lg one coeluting with the 1Camino acid molecule, while the two other forms had apparent molecular weights of about 12,000 and 3500, respectively. The elution volume of the large component detected here in feline extracts of gastrointestinal tract and pancreas, as wel1 as its recognition by a C-terminal directed antiserum, are consistent with its similarity to the components described by the above investigators. The present study demonstrates a consistent gra-
Vol. 79, No. 5, Part 1
forms of somatostatin
along
mucosa, namely an increasing
contribution of the large component to the total immunoreactivity per segment from duodenum to colon. This pattern resembles that of gastrin, since the heptadecapeptide is largely predominant in antrum while big gastrin (G-34) accounts for an increasing percentage of the total immunoreactivity along the The various forms of gastrin duodenojejunum.“” have been shown to have different half-lives and biologic potenties,‘* as wel1 as different patterns of reof the lease and uptake.” Further characterization molecular forms of somatostatin may demonstrate similar differences between smal1 and large components, which would be of interest in view of the short disappearance time and of the diffuse biologie effects of the tetradecapeptide.‘” In contrast to somatostatin, immunoreactive VIP was present in large amounts both in mucosa and muscular layer of the gastrointestinal tract, confirming the observations of Larsson et al.’ These authors stated that the network of VIP-positive nervous fibers, recognized by immunocytology, was predominant at the submucosal and muscular leve1 in the stomach, but became richer in intestinal and colonic mucosa. This is confirmed by the present observation of a caudal increase of mucosal/muscular ratio of VIP concentrations. The reason for such a variation is not known. Exogenous VIP exerts vasodilating and motor effects on the intestine and co1on,23 and stimulates intestinal secretion of water and electrolytes, but neither the physiologic role(s) nor the releasing factors of endogenous VIP have been elucidated so far. The distribution of the peptide along the feline gastrointestinal tract suggests: (a) that either VIP is released by intraluminal factors, or the peptide plays a role in the control of secretory and absorptive functions of intestine and colon, in view of the abundance of mucosal VIP in these segments, and (b) that VIP may be related to the secretory, motor, or vasoactive characteristics of proximal duodenum and right colon. Whether VIP is exclusively located in neural elements of the digestive tract, or simultaneously present in these and in endocrine cells within the mucosa is controversial.6.7 An attractive explanation for this discrepancy was proposed by Dimaline and Dockray’” on the basis that a single molecular form of VIP was found in human colonic muscle, while three different components could be detected in the mucosa, so that antisera with different specificities could recognize either the only known VIP, predominant in muscle, or this and other immunoreactive molecules presumably located in endocrine cells of the mucosa. In the present animals, the immunoreactive VIP detected in gastrointestinal
November
1980
muscle, in gallbladder, and in antral and duodenal mucosa was not clearly heterogeneous on G50 Sephadex, and coeluted with porcine VIP. In contrast, immunoreactive VIP was associated with a slower component in intestinal and cecal mucosa, the elution volume of the latter form being consistent with its similarity to component 111 of Dimaline and Dockray.25 Our results suggest that molecular heterogeneity of VIP in colonic mucosa is not restricted to humans, but is probably a common feature in mammals. Both somatostatin and VIP are broadly distributed in the digestive tract of al1 mammals so far studied, which suggests that the peptides should be involved in genera1 physiologic events such as motility or blood flow. The preferential locations and variations of tissue concentrations of these peptides along the gastrointestinal tract, however, as delineated here in Cats, may provide additional clues for the study of their physiologic role(s) and releasing factors.
References 1. Brazeau
2.
3.
4.
5.
6.
7.
8.
P, Vale W, Burgus R, et al: Hypothalamic peptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179:7779,1973 Said SI, Mutt V: Isolation from porcine intestinal wal1 of a vasoactive octacosapeptide related to secretin and to glucagon. Eur J Biochem 28:199-204: 1972 Hokfelt T, Elfvin LG, Elde R, et al: Occurrence of somatostatin immunoreactivity in some peripheral sympathetic noradrenergic neurons. Proc Nat1 Acad Sci USA 74:3587-359X 1977 Polak JM, Grimelius L, Pearse AGE, et al: Growth-hormone release-inhibiting hormone in gastrointestinal and pancreatic D cells. Lancet 1:1220-1222, 1975 Alumets J, Sundler F, Hakanson R: Distribution, ontogeny and ultrastructure of somatostatin immunoreactive cells in the pancreas and gut. Cel1 Tissue Res 185:465-479, 1977 Bryant MG, Polak JM, Modlin IM, et al: Possible dual role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance. Lancet 1:991-993.1976 Larsson LI, Fahrenkrug J, Schaffalitzky de Muckadell 0, et al: Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons. Proc Nat1 Acad Sci USA 73:31973200, 1976 Chayvialle JA, Descos F, Bernard C, et al: Somatostatin in mu-
SOMATOSTATIN
AND VIP IN GUT OF CAT
843
cosa of stomach and duodenum in gastroduodenal disease. Gastroenterology 75:x3-19, 1978 9. Arimura A, Sato H, Dupont A, et al: Somatostatin: abundance of immunoreactive hormone in rat stomach and pancreas. Science 189:1007-1009, 1975 10. Kronheim S, Berelowitz M, Pimstone BL: A radioimmunoassay for growth hormone release-inhibiting hormone: method and quantitative tissue distribution. Clin Endocrinol 5:619630, 1976 ll.McIntosh C, Arnold R, Bothe E, et al: Gastrointestinal somatostatin: extraction and radioimmunoassay in different species. Gut 19:655-663, 1978 12. Patel YC, Reichlin S: Somatostatin in hypothalamus, extrahypothalamic brain and peripheral tissues of the rat. Endocrinology 102:523-530, 1978 13. Gerich JE, Patton GS: Somatostatin: physiology and clinical applications. Med Clin North Am 62:375-391, 1978 14. Wahren J, Felig P: Influence of somatostatin on carbohydrate disposal and absorption in diabetes mellitus. Lancet 2:12131216, 1976 15. Evensen D, Hanssen KF, Berstad A: The effect on intestinal calcium absorption of somatostatin in man. Stand J Gastroenterol 13:449-451, 1978 16. Johansson C, Effendic S, Wisen 0, et al: Effects of short-time somatostatin infusion on the gastric and intestinal propulsion in humans. Stand J Gastroenterol 13:481-483, 1978 inhibits the release of acetyl17. Guillemin R: Somatostatin choline induced electrically in the myenteric plexus. Endocrinology 99:1653-1654, 1976 18. Vale W, Ling N, Rivier J, et al: Anatomie and phylogenetic distribution of somatostatin. Metabolism 25 (Suppl 1):1491-, 1494, 1976 19. Conlon JM, Zyznar E, Vale W, et al: Multiple forms of somatostatin-like immunoreactivity in canine pancreas. FEBS Lett 94:325-330, 1978 20. Berson SA, Yalow RS: Nature of immunoreactive gastrin extracted from tissues of gastrointestinal tract. Gastroenterology 60:215-222, 1971 21. Walsh JH, Debas HT, Grossman MI: Pure human big gastrin: immunochemical properties, disappearance half-time, and acid stimulating action in dogs. J Clin Invest 54:477-485,1974 22. Thompson JC, Rayford PL, Ramus NI, et al: Patterns of release and uptake of heterogeneous forms of gastrin. In: Gastrointestinal Hormones. Edited by JC Thompson. Austin, University of Texas Press, 1975, p 125-151 23. Makhlouf GM, Said SI: The effect of vasoactive intestinal peptide (VIP) on digestive and hormonal function. In: Gastrointestinal Hormones. Edited by JC Thompson. Austin, University of Texas Press, 1975, p 599-610 24. Barbezat GO, Grossman MI: Intestinal secretion: stimulation by peptides. Science 174:422-424,197l forms of 25. Dimaline R, Dockray GJ: Multiple immunoreactive vasoactive intestinal peptide in human colonic mucosa. Gastroenterology 75:387-392, 1978
79:837-843,198O
Immunoreactive Somatostatin and Vasoactive Intestinal Peptide in the Digestive Tract of Cats JEAN-ALAIN CHAYVIALLE, MASAHIKO MIYATA, PHILLIP L. RAYFORD, and JAMES C. THOMPSON Department
of Surgery,
The University
of Texas Medical
Somatostatin and vasoactive intestinal peptide were measured by radioimmunoassay in acetic acid extracts of tissue samples from the digestive tracts of seven adult Cats. The concentration of immunoreactive somatostatin in antral mucosa (2926 f 978 pmol/g wet weight, mean i SE) was far above the concentrations in the head of the pancreas (442 f 941, distal duodenum (377 i 91), and ileum (355 + 48). Large amounts of immunoreactive vasoactive intestinal peptide were recovered from both mucosal and muscular layers of gastrointestinal tract; the highest values were observed in the muscular layer of the cecum (579 f 82 pmol/g) and the mucosa of the right colon (564 + 244). The mucosal/muscular Iayer ratio of vasoactive intestinal peptide concentrations increased caudally from 0.20 in the esophagus to over 1 in the iieum and colon. Upon gel filtration on G-50 Sephadex, somatostatin in the antrum, duodenum, and pancreas were eluted as a predominant peak in the volume of the tetradecapeptide, but somatostatin from the ileum and cecum was associated with a major faster component. Vasoactive intestinal peptide in the muscular iayer of gastrointestinal tract and in mucosa of antrum and duodenum consisted essentially of a single farm, which coeluted with the purified porcine peptide; a slower component was detected in the mucosa of the
Branch,
Galveston,
Texas
ileum and cecum. These results indicate molecular heterogeneity of both immunoreactive somatostatin and vasoactive intestinal peptide in the digestive tract of Cats, and suggest that the variable ratio of the different molecular farms of each peptide along the gastrointestinal tract may reflect regional specijïcity of biologie effects and metabolism. Somatostatin (STS), a tetradecapeptide isolated from ovine hypothalamus,’ and vasoactive intestinal peptide (VIP), an octacosapeptide purified from porcine intestine,‘- have a wide distribution in the digestive tract of al1 mammals studied. By immunocytochemistry, somatostatin has been detected in some neural elemenfs,3 but is mainly found in endocrine cells of gastrointestinal mucosa and pancreas?’ Vasoactive intestinal peptide has been recognized in nervous sfructures,~’ but the simultaneous location of immunoreactive material in endocrine cells’ is disputed by some investigators.7 In the present study, STS and VIP were comparatively mapped by radioimmunoassay in the digestive tract of Cats, and the immunoreactive components were tested for molecular heterogeneity.
Methods Tissue Sampling
Received August 29, 1979. Accepted May 17.1980. Address requests for reprints to: James C. Thompson, M.D., Department of Surgery. The University of Texas Medical Branch, Galveston, Texas 77550. Dr. Chayvialle is a Visiting Scientist. Parent Institution: Hôpital Edouard-Herriot, Lyon, France. Dr. Miyata is a Visiting Scientist. Parent Institution: Osaka University Medical School, Osaka, Japan. This work was supported by grants from the National Institutes of Health (AM 15241) and the John A. Hartford Foundation, Inc. This work was presented in part at the Annual Meeting of the American Gastroenterological Association, Las Vegas, Nevada, May 1978. 0 1980 by the American Gastroenterological Association 0016-5085/80/110837-07$02.25
Seven adult Cats, weighing 3.5-6 kg, were anesthetized with Nembutal (30 mg/kg intraperitoneally) after an 16-hr fast, and submitted to laparotomy. In the first six animals, tissue specimens were taken at preselected sites of esophagus, fundus, antrum, duodenum, ileum, cecum, right colon, left colon, and upper rectum. For each sample, the mucosa was quickly dissected from the muscular layer, and both parts were frazen on dry ice. Additional specimens were taken from the spleen, gallbladder, and liver. In cat No. 7, samples were taken every centimeter in the fundus, antrum, and duodenum. The mean weight of the specimens from each site varied from 36 to 129 mg for
838
CHAYVIALLE
ET AL.
GASTROENTEROLOGY
gastrointestinal mucosa or muscular layers, and from 102 to 310 mg for the spleen, gallbladder, and liver specimens.
Vol. 79, No. 5, Part 1
mean recovery of 2.5, 5, 10, and 20 ng synthetic cyclic somatostatin added to cat tissue samples before extraction was 83.6% (range: 77%-90%, n = 6 for each dose).
Extraction Samples were successively weighed while they were thawed, boiled in 200~~1 distilled water for 10 min, cut into pieces with a scalpel blade, and homogenized in 2 M acetic acid with a tissue grinder (Kontes size AA, T.M. Kontes, Vineland, N.J.). The homogenate was kept under agitation overnight at 4’, and lyophilized. The dry residue was suspended in 0.1 M ammonium acetate pH 5.5 with 5% horse serum and 1 mg/ml sodium azide, and centrifuged. The supernatant was assayed, at three or more dilutions in duplicates, for STS and VIP on the same day.
Somatostatin
Assay
The method, reported in detail previously,8 has been further characterized. As compared with the synthetic cyclic tetradecapeptide (Serono Pharm Präp GmbH, Freiburg/Breisgau, West Germany), the poteney of neurotensin (Calbiochem, San Diego) is less than 0.0002, while the ID,, of salmon calcitonin (Armour, Phoenix) is 0.4 IU. Lactoperoxydase (Calbiochem) was used for iodination of l-tyrosyl somatostatin (Serono). The tracer was purified on carboxymethylcellulose CM52 (Whatman Ltd., Maidstone, England), using 0.05 M ammonium acetate pH 5.5 as starting buffer and 0.5 M ammonium acetate pH 6.0 for elution. Antiserum 55A fully recognizes 4-Ala somatostatin, while 6-Ala, 8-Als, and 11-Ala somatostatin (gift from Dr. J. Rivier and Prof. R. Guillemin) have relative potencies of 0.002, less than 0.002, and 0.01, respectively. The
Vasoactive
Intestinal
Peptide
Assay
Antiserum 76A was obtained from a New Zealand white rabbit after the second injection of 100 pg highly purified porcine VIP (Prof. V. Mutt) conjugated to 500 pg bovine albumin (Poviet Prod B.V., Amsterdam), with 5 mg lethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (Sigma Chemical Company, St. Louis, Mo.). Porcine secretin (Prof. Mutt), porcine gastric inhibitory polypeptide (Prof. J.C. Brown), bovine pancreatic polypeptide (Dr. R. E. Chance), bovine glucagon and insulin (Novo), synthetic somatostatin (Serono), synthetic human gastrin 1, 1-17 (ICI), salmon calcitonin (Armour) and neurotensin (Calbiochem) did not crossreact at a leve1 of 100 ng/tube, or 0.4 IU/tube for calcitonin. Vasoactive intestinal peptide fragments 1-10 and 18-28 (gift from Prof. M. Bodansky) have relative potenties of less than 0.0001 and of 0.1, respectively. The working final dilution of the antiserum for assay purposes is 1/160,000. Highly purified porcine VIP (1 pg) was labeled with “‘Iodine (0.5 mCi) in 0.4 M ammonium acetate pH 5.0 using lactoperoxydase (Calbiochem, 5 pp) and 5% hydrogen peroxide (5 ~1). The reaction was stopped after 20 min by adding 2 ml starting buffer, and iodinated VIP was purifïed on 1 X 10 cm SP C25 Sephadex column (Pharmacia Fine Chemicals, Piscataway, N.J.) using 0.2 M ammonium acetate pH 5.0 as starting buffer and 0.8 M ammonium acetate pH 5.6 with 5% horse serum for elution. A typical elution profile is shown in Figure 1. The most reactive fractions were diluted in 0.1 M ammonium acetate pH 5.5 and kept frozen. Assays were set up in 0.1 M ammonium acetate buffer pH 5.5 with 5% horse serum and 0.2 mg/ml sodium azide. Tracer VIP (1500-2000 cpm/ tube) and a second antibody (goat antirabbit globulin) were added 24 and 48 hr later. The tubes were centrifuged at 3500 rpm for 30 min at 4’, the supernatant was discarded, and the precipitate was countéd in a well-type gamma spectrometer. The sensitivity of the system, i.e., the minima1 dose giving a significant inhibition of binding when tested as four replicates, was about 5 pg/ml incubate. The within-assay and between-assay variations were less than 10% (coefficient of variation). The mean recovery of 2.5, 5,10, and 20 ng porcine VIP added to cat tissue samples before extraction was 71.5% (range: 62%-83%, n = 6 for each dose).
FRACTION NmfR (1.5 ML)
Figure 1. Elution profile of ?-VIP
on SP C25 Sephadex. Initial
buffer: 0.2 M ammonium acetate pH 5.0. Elution of iodinated peptide: 0.8 M ammonium acetate pH 5.5: 5% aged plasma and Trasylol were added to both solutions. Full line: radioactivity eluted. Free iodine is eluted rapidly with the initial buffer. Two peaks are eluted with the 0.8 M solution. Dotted Ene: percent ra-
dioactivity (about 10,ooO cpm/tube) bound overnight by antiserum at final dilution of 1/8O.o00 (nonspecific binding subtracted). The most immunoreactive fractions are found on the descending slope of the second radioactive peak.
Chromatographic
Studies
Extracts of antrum, duodenum, ileum, cecum, pancreas, and gallbladder were pooled to a volume of 1 ml, incubated for 2 hr in 6 M urea (final concentration) at room temperature, and applied on a 1.5 x 100 cm G-50 superfine Sephadex column (Pharmacia), equilibrated in 0.05 M potassium phosphate-0.1 M sodium chloride pH 5.5 with 5% horse serum and 0.2 mg/ml sodium azide. The column was preloaded with 5 ml 6 M urea immediately before applying the sample. Fractions of 4.0 ml were collected at a flow rate of 8 ml/hr at 4” and assayed for STS and VIP. In some
November
1980
SOMATOSTATIN
experiments, the various cubated at serial dilutions
immunoreactive for comparison
peaks were into the standard
curve. The recovery of both peptides was in the range of 55%-80%. Results are expressed as mean f SE picomoles equivalent synthetic cyclic somatostatin or purified porcine VIP per gram wet weight, or as nanograms per fraction eluted at chromatography.
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Immunoreactive somatostatin was located chiefly in the gastrointestinal mucosa and the pancreas (Figure 2). In mucosa, the concentration in fundus was 77 f 15 pmol/g, rising abruptly to 2926 -r 976 in the antrum and subsequently decreasing to 377 f 91 in the distal duodenum. The concentration from jejunum to rectum varied between 165 f 16 (cecum) and 355 + 46 pmol/g (ileum). Samples taken every centimeter in cat No. 7 showed a sharp gradient of STS concentration across the pylorus (Figure 3). Values in the tail and head of the pancreas were 310 f 49 and 442 f 94 pmol/g, respectively. The STS immunoreactivity detected in al1 these segments, SOMATOSTATIN
PYLZORUS
2 000.
Results
839
AND VIP IN GUT GF CAT
u fs u
\
\
1 cm
\
1 ooo-
Figure 3. Somatostatin concentration (pmol/g wet weight) in antral and duodenal mucosa. Samples were taken in distal antrum, proximal duodenum, and distal duodenum in cats NOS. 1-6 (dotted Iine, mean f SE) and every centimeter from 2 cm before to 9 cm after the pylorus in cat No. 7 (fuI1 Fine).
when measured in graded doses, gave response curves that were similar to that of the standard (Figure 4). In the gastrointestinal muscular layer, the concentration was in the range of 9 f 4 to 91 f 20 pmol/g, while insignificant amounts were recovered from the spleen, gallbladder, and liver. Immunoreactive VIP was found in both mucosa and muscular layers of the gastrointestinal tract. In mucosa, regional peaks were observed in the proxi-
SAMPLE 4
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1
( 1/x
103)
0.5
1
COLON
-SPLEEN
1
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z Figure 2. Mean plus or minus one standard error tissue somatostatin concentration (pmol/g wet weight) in gastrointestinal mucosa or parenchymas (right horizontal axis) and gastrointestinal muscular layer (left horizontal axis) in six adult Cats. The peak concentration is observed in mucosa of antrum and proximal duodenum. Substantial amounts of peptide are detected in intestinal mucosa and pancreas.
1
o
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0
,
15.6
62.5 SOMATOSTATIN
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1 000
(pg/ml)
Figure 4. Inhibition curves generated with graded concentrations of synthetic cyclic somatostatin (standard) and with serial dilutions of various acetic extracts. The immunoreactive component(s) detected in gastrointestinal mucosa and pancreas is not dissimilar from the standard.
VIP 680
GASTROENTEROLOGY
CHAYVIALLE ET AL.
840
CONCENTRATION
40‘0
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(pmoles
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Figure 5. Mean plus or minus one standard error tissue VIP concentration (pmol/g wet weight) in gastrointestinal mucosa and parenchymas (right horizontal axis) and gastrointestinal muscular layer (left horizontal axis) in six adult Cats. Peaks are observed in both mimosa and muscular layer of duodenum and right colon. Substantial amounts of peptide are recovered from the gallbladder wall.
(
Figure
1
mal duodenum (293 + 44 pmol/g) and the right colon (564 + 244, Figure 5). A similar pattern was obtained in the muscular layers, with peaks in the duodenum (415 + 135) and cecum (579 & 82). The mucosa/muscular layer ratio of VIP concentrations increased caudally, with values of 0.20 in the esophagus, 0.45 in the fundus, 0.80 in the antrum and duodenum, 1.16-1.84 in the intestine, and 1.2-2.31 in the colon. The duodenal peak VIP concentration in mucosa and muscular layer was confirmed in cat No. 7 (Figures 6 and 7), in which samples were taken at 1-cm intervals from antrum to duodenum. The concentrations in the tail and head of the pancreas were 15 f 4 and 30 + 4 pmol/g, and 78 + 7 in the gallbladder wall. Insignificant amounts were found in the spleen and liver. Inhibition curves generated by graded doses of VIP in the various tissue segments were similar to that of purified porcine VIP (Figure 8). Chromatography of antral, duodenal, ileal, and cecal mucosal extracts and of pancreatic extracts gave two immunoreactive somatostatin peaks, one coeluting with the cyclic tetradecapeptide, and a faster peak which was the predominant form in the ileum and colon (Figure 9). A third component, eluted slightly after the void volume, was detected in the duodenum, ileum, and cecum, but contributed less than 20% to the total immunoreactivity recovered from the column. Gel filtration of the various gastrointestinal segments gave distinct patterns of VIP immunoreactivity for mucosa and muscular layers (Figure 10). In muscle, VIP was eluted essentially as a single peak in the same position as porcine VIP, similarly to the immunoreactive material detected in the gallbladder. The same component was found in the gastrointesti-
r: / /
;
Vol. 79, No. 5, Part
PUNDUS AN*uM-r
DUODENUM
Je l
,t 4
Ce
RC
I
I
LC
OJ 6.
Vasoactive intestinal peptide concentration (pmol/g wet weight) in muscular layer of gastrointestinal tract. Samples were taken at intervals from fundus, antrum, duodenum. jujunum (Je), ileum (11) cecum (Ce), right colon (RC), and left colon (LC) in cats NOS. 1-6, and every centimeter in the fundus, antrum, and duodenum in cat No. 7. Peak concentrations occur in proximal duodenum and cecum.
OJ
Figure 7. Vasoactive intestinal peptide concentration (pmol/g) in mucosa of gastrointestinal tract. Diagram is prepared as in Figure 6. Peak concentrations occur in proximal duodenum and right colon.
November 1980
SOMATOSTATIN AND VIP IN GUT OF CAT
STS
na1 mucosa, associated in the ileum and colon with a second peak emerging later than porcine VIP. For both somatostatin and VIP, serial dilutions of pooled fractions corresponding to the various immunoreactive components yielded inhibition curves parallel to that of the standard.
Discussion The present results show that immunoreactive somatostatin in the digestive tract of cats is located predominantly in the mucosa of the gastrointestinal tract, but not in the muscular layer, and in the pancreas. The concentration in antral mucosa was far above that in any other segment, including the fundic mucosa and the pancreas. In this respect, the cat would differ from ratsg”’ or humans,” where values in the fundus or pancreas are of the same order as those in the antrum. Whether this reflects specific features of gastric physiology in the cat, possibly related to dietary habits, is not known. In agreement with other immunocytologic” and radioimmunologic studies,lD”Z immunoreactive somatostatin was detected in intestinal and colonic mucosa. Although the concentration achieved in these segments was only 1O%-15% that in the antrum, it can be suggested that the mass of mucosa from jejunum to rectum represents a large fraction of total digestive somatostatin. In addition to its inhibitory effects on endocrine and exocrine secretions of the stomach and pancreas,13 exogenous somatostatin has been shown to inhibit intestinal absorption,14”5 mo40
* 1 \ 12 c
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841
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40
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NUMBER
Figure 9. Elution profile of somatostatin-like immunoreactivity in acetic extracts from the antrum, duodenum, ileum, cecum, and pancreas on a 1.5 x 100 cm G-50 superfine Sephadex column. Extracts were preincubated in 6 M urea and applied after 5 ml 6 M urea. Elution buffer: 0.05 M potassium phosphate-0.1 sodium chloride pH 5.5 with 5% horse serum and 0.2 mg/ml sodium azide. Fout milliliter fractions, flow rate: 8 ml/hr. The elution volumes of albumin (Alb), synthetic cyclic somatostatin (STS), and lZ51are shown at the top of the graph.
tility,” and blood flow,14 as wel1 as the release of acetylcholine by the myenteric plexuses.” The immunoreactivity detected in the intestine and colonic mucosa may thus be the basis for important physiologie actions of the peptide in these segments, either in response to intraluminal events, or through the interplay of neurohumoral pathways within the gastrointestinal wall. Several investigators have reported various degrees of molecular heterogeneity of immunoreactive somatostatin in gastrointestinal tract and pancreas in rats and humans,9.“.‘2 but the extra components
GASTROENTEROLOGY
CHAYVIALLE ET AL.
842
VII’
dient of the molecular
125,
the gastrointestinal
+
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PANCREAS
0. , 10
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30
40
50
, 60
NUMBER
Figure 10. Elution profile of VIP-like immunoreactivity in acetic extracts of mucosa (fu11line) and muscular layer (dotted line) from antrum, duodenum, ileum, cecum, and from gallbladder wall. Same chromatographic conditions as in Figure 9. VIP: elution volume of highly purified porcine VIP.
have not been fully characterized. Vale et a1.18detected a molecular form larger than the tetradecapeptide in tissue extracts from various species only when using antisera directed against the c-terminal moiety of the molecule, which suggested that the large component resulted from extension of the NH,-terminus of the tetradecapeptide. Recently, three forms were found in canine pancreas,lg one coeluting with the 1Camino acid molecule, while the two other forms had apparent molecular weights of about 12,000 and 3500, respectively. The elution volume of the large component detected here in feline extracts of gastrointestinal tract and pancreas, as wel1 as its recognition by a C-terminal directed antiserum, are consistent with its similarity to the components described by the above investigators. The present study demonstrates a consistent gra-
Vol. 79, No. 5, Part 1
forms of somatostatin
along
mucosa, namely an increasing
contribution of the large component to the total immunoreactivity per segment from duodenum to colon. This pattern resembles that of gastrin, since the heptadecapeptide is largely predominant in antrum while big gastrin (G-34) accounts for an increasing percentage of the total immunoreactivity along the The various forms of gastrin duodenojejunum.“” have been shown to have different half-lives and biologic potenties,‘* as wel1 as different patterns of reof the lease and uptake.” Further characterization molecular forms of somatostatin may demonstrate similar differences between smal1 and large components, which would be of interest in view of the short disappearance time and of the diffuse biologie effects of the tetradecapeptide.‘” In contrast to somatostatin, immunoreactive VIP was present in large amounts both in mucosa and muscular layer of the gastrointestinal tract, confirming the observations of Larsson et al.’ These authors stated that the network of VIP-positive nervous fibers, recognized by immunocytology, was predominant at the submucosal and muscular leve1 in the stomach, but became richer in intestinal and colonic mucosa. This is confirmed by the present observation of a caudal increase of mucosal/muscular ratio of VIP concentrations. The reason for such a variation is not known. Exogenous VIP exerts vasodilating and motor effects on the intestine and co1on,23 and stimulates intestinal secretion of water and electrolytes, but neither the physiologic role(s) nor the releasing factors of endogenous VIP have been elucidated so far. The distribution of the peptide along the feline gastrointestinal tract suggests: (a) that either VIP is released by intraluminal factors, or the peptide plays a role in the control of secretory and absorptive functions of intestine and colon, in view of the abundance of mucosal VIP in these segments, and (b) that VIP may be related to the secretory, motor, or vasoactive characteristics of proximal duodenum and right colon. Whether VIP is exclusively located in neural elements of the digestive tract, or simultaneously present in these and in endocrine cells within the mucosa is controversial.6.7 An attractive explanation for this discrepancy was proposed by Dimaline and Dockray’” on the basis that a single molecular form of VIP was found in human colonic muscle, while three different components could be detected in the mucosa, so that antisera with different specificities could recognize either the only known VIP, predominant in muscle, or this and other immunoreactive molecules presumably located in endocrine cells of the mucosa. In the present animals, the immunoreactive VIP detected in gastrointestinal
November
1980
muscle, in gallbladder, and in antral and duodenal mucosa was not clearly heterogeneous on G50 Sephadex, and coeluted with porcine VIP. In contrast, immunoreactive VIP was associated with a slower component in intestinal and cecal mucosa, the elution volume of the latter form being consistent with its similarity to component 111 of Dimaline and Dockray.25 Our results suggest that molecular heterogeneity of VIP in colonic mucosa is not restricted to humans, but is probably a common feature in mammals. Both somatostatin and VIP are broadly distributed in the digestive tract of al1 mammals so far studied, which suggests that the peptides should be involved in genera1 physiologic events such as motility or blood flow. The preferential locations and variations of tissue concentrations of these peptides along the gastrointestinal tract, however, as delineated here in Cats, may provide additional clues for the study of their physiologic role(s) and releasing factors.
References 1. Brazeau
2.
3.
4.
5.
6.
7.
8.
P, Vale W, Burgus R, et al: Hypothalamic peptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179:7779,1973 Said SI, Mutt V: Isolation from porcine intestinal wal1 of a vasoactive octacosapeptide related to secretin and to glucagon. Eur J Biochem 28:199-204: 1972 Hokfelt T, Elfvin LG, Elde R, et al: Occurrence of somatostatin immunoreactivity in some peripheral sympathetic noradrenergic neurons. Proc Nat1 Acad Sci USA 74:3587-359X 1977 Polak JM, Grimelius L, Pearse AGE, et al: Growth-hormone release-inhibiting hormone in gastrointestinal and pancreatic D cells. Lancet 1:1220-1222, 1975 Alumets J, Sundler F, Hakanson R: Distribution, ontogeny and ultrastructure of somatostatin immunoreactive cells in the pancreas and gut. Cel1 Tissue Res 185:465-479, 1977 Bryant MG, Polak JM, Modlin IM, et al: Possible dual role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance. Lancet 1:991-993.1976 Larsson LI, Fahrenkrug J, Schaffalitzky de Muckadell 0, et al: Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons. Proc Nat1 Acad Sci USA 73:31973200, 1976 Chayvialle JA, Descos F, Bernard C, et al: Somatostatin in mu-
SOMATOSTATIN
AND VIP IN GUT OF CAT
843
cosa of stomach and duodenum in gastroduodenal disease. Gastroenterology 75:x3-19, 1978 9. Arimura A, Sato H, Dupont A, et al: Somatostatin: abundance of immunoreactive hormone in rat stomach and pancreas. Science 189:1007-1009, 1975 10. Kronheim S, Berelowitz M, Pimstone BL: A radioimmunoassay for growth hormone release-inhibiting hormone: method and quantitative tissue distribution. Clin Endocrinol 5:619630, 1976 ll.McIntosh C, Arnold R, Bothe E, et al: Gastrointestinal somatostatin: extraction and radioimmunoassay in different species. Gut 19:655-663, 1978 12. Patel YC, Reichlin S: Somatostatin in hypothalamus, extrahypothalamic brain and peripheral tissues of the rat. Endocrinology 102:523-530, 1978 13. Gerich JE, Patton GS: Somatostatin: physiology and clinical applications. Med Clin North Am 62:375-391, 1978 14. Wahren J, Felig P: Influence of somatostatin on carbohydrate disposal and absorption in diabetes mellitus. Lancet 2:12131216, 1976 15. Evensen D, Hanssen KF, Berstad A: The effect on intestinal calcium absorption of somatostatin in man. Stand J Gastroenterol 13:449-451, 1978 16. Johansson C, Effendic S, Wisen 0, et al: Effects of short-time somatostatin infusion on the gastric and intestinal propulsion in humans. Stand J Gastroenterol 13:481-483, 1978 inhibits the release of acetyl17. Guillemin R: Somatostatin choline induced electrically in the myenteric plexus. Endocrinology 99:1653-1654, 1976 18. Vale W, Ling N, Rivier J, et al: Anatomie and phylogenetic distribution of somatostatin. Metabolism 25 (Suppl 1):1491-, 1494, 1976 19. Conlon JM, Zyznar E, Vale W, et al: Multiple forms of somatostatin-like immunoreactivity in canine pancreas. FEBS Lett 94:325-330, 1978 20. Berson SA, Yalow RS: Nature of immunoreactive gastrin extracted from tissues of gastrointestinal tract. Gastroenterology 60:215-222, 1971 21. Walsh JH, Debas HT, Grossman MI: Pure human big gastrin: immunochemical properties, disappearance half-time, and acid stimulating action in dogs. J Clin Invest 54:477-485,1974 22. Thompson JC, Rayford PL, Ramus NI, et al: Patterns of release and uptake of heterogeneous forms of gastrin. In: Gastrointestinal Hormones. Edited by JC Thompson. Austin, University of Texas Press, 1975, p 125-151 23. Makhlouf GM, Said SI: The effect of vasoactive intestinal peptide (VIP) on digestive and hormonal function. In: Gastrointestinal Hormones. Edited by JC Thompson. Austin, University of Texas Press, 1975, p 599-610 24. Barbezat GO, Grossman MI: Intestinal secretion: stimulation by peptides. Science 174:422-424,197l forms of 25. Dimaline R, Dockray GJ: Multiple immunoreactive vasoactive intestinal peptide in human colonic mucosa. Gastroenterology 75:387-392, 1978