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Vasoactive intestinal polypeptide receptor binding sites in the human gastrointestinal tract: Localization by autoradiography
NeuroscienceVol. 31, No. 3, pp. 771-783, 1989 Printed in Great Britain
0306-4522/89$3.00 + 0.00 Pergamon Press plc Q 1989 IBRO
VASOACTIVE INTESTINAL POLYPEPTIDE RECEPTOR BINDING SITES IN THE HUMAN GASTROINTESTINAL TRACT: LOCALIZATION BY AUTORADIOGRAPHY C. R. MANTYH ,* S. R. VICNA,*$$ M. L. WELTON,*~~ E. P. PASSARO JR*[[ and P. W. MANTYH*~$T/ *Center for Ulcer Research and Education, Veterans Administration Medical Center-Wadsworth, Los Angeles, CA 90073, U.S.A. IDepartment of Medicine, ItDepartment of Surgery, and IBrain Research Institute, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90024, U.S.A.
R. P.
ZIMMERMAN,*
T. S. GATES,*
Abstract-Vasoactive intestinal polypeptide (VIP) is a putative neurotransmitter in both the brain and peripheral tissues. To define possible target tissues of VIP we have used quantitative receptor autoradiography to localize and quantify the distribution of [‘251]VIPreceptor binding sites in histologically normal human surgical specimens. While the distribution of VIP binding sites was different for each gastrointestinal segment examined, specific vasoactive intestinal polypeptide binding sites were localized to the mucosa, the muscularis mucosa, the smooth muscle of submucosal arterioles, the circular and longitudinal smooth muscle of the muscularis externa, the myenteric plexus, and lymph nodules. In most segments, the mucosal layer expressed the highest concentration of VIP binding sites, with the duodenal and jejunal mucosa showing the highest density of receptors. These results identify putative VIP target tissues in the human gastrointestinal tract. In correlation with physiological data, VIP binding sites appear to be involved in the regulation of a variety of gastrointestinal functions including mucosal ion transport, gastric secretion, hemodynamic regulation, gastric and intestinal motility, neuronal excitability, and modulation of the immune system.
Vasoactive intestinal polypeptide (VIP) was first described as a potent vasodilator isolated from the porcine intestine2’ but is now known to be a putative peptide neurotransmitter in the central and peripheral nervous systems. Histochemical, pharmacological, and physiological studies have suggested that VIP is involved in regulating several gastrointestinal functions. Immunohistochemical studies have shown that intrinsic VIP immunoreactive fibers are found in the submucous and myenteric plexuses and extend into the lamina propria, the muscularis mucosae, and the external circular muscle in nearly every segment of the rat and guinea-pig gastrointestinal tract.” Pharmacological and physiological studies also indicate VIP has a role in regulating a variety of gastrointestinal functions. VIP has been shown to promote water and electrolyte secretion by intestinal epithelium,3J2.‘3 inhibit gastric acid, pepsinogen,” and gastrin24 secretion, act as a potent vasodilator,2’ and to have varied effects on gastrointestinal motility.2,4.9.”
Demonstration of pharmacologically relevant receptor binding sites is crucial for characterization of VIP action in the gastrointestinal tract since all of its actions at physiological concentrations are thought to be receptor mediated.” Since many neurons are now known to contain multiple neurotransmitters, localization of receptor binding sites for VIP is essential in determining where released VIP will produce a functional response. In the present report, we examined histologically normal surgical specimens obtained from the length of the human gastrointestinal tract to determine whether they express specific [“‘I]VIP binding sites. The objectives of this study are: (1) to demonstrate that intact human surgical specimens can be processed for quantitative receptor autoradiography; (2) to localize possible target tissues for released VIP through identification of cells and tissues expressing specific VIP binding sites in the human GI tract; and (3) to determine the stability of the VIP binding sites in human surgical specimens. EXPERIMENTAL Surgical
~To whom correspondence should be addressed at Research Service 1151), VA Medical Center, 1 Veterans Drive, Minneapolis, MN 55417, U.S.A. $Present address: Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, U.S.A. Abbreviafions: EGTA. ethvleneplvcolbis(aminoethvlether)tetra-acetate; HEPES, -N-2-&droxydthylpiper&ine-h;2-ethansulfonic acid; VIP, vasoactive intestinal polypeptide. 771
PROCEDURES
specimens were obtained within 5 min of removal in order to minimize post-surgical degradation artifacts. Tissue specimens were obtained from the uninvolved margins of extensive resections for carcinoma and determined by a pathologist to be histologically normal. To determine the time course of VIP receptor degradation, samples from a colon specimen were frozen (-70°C) after being kept at room temperature for increasing lengths of time from 0 to 300min. The tissue was then blocked, placed on a brass microtome chuck, frozen on dry ice and processed for
R. P. ZIMMERMAN et al VIP Receptor
VIP Receptor
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Deqradation
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Mc-lumlnol
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Fig 1. Density of surgical removal. (LM), (b) external density of binding of VIP receptors,
180
240
300
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0
60
120
180
Time Elapsed
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300
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[‘2sI]VIP receptor binding sites in human colon vs time stored at room temperature after The stability of the receptor binding sites is shown for (a) external longitudinal muscle circular muscle (CM), (c) basal mucosa (MC) and (d) luminal mucosa (MC). The optical sites at time 0 in each tissue is the maximal binding (100%). Note that the degradation as seen by the decrease in receptor binding site densities with time, is substantial in all tissues but that the time course is different for each region.
quantitative autoradiography as we have previously described.i6 The tissue was serially sectioned (30pm), thawmounted on gelatin coated microscope slides, and stored at -20°C in boxes with desiccant for up to 3 months. The radiolabeled vasoactive intestinal polypeptide used in this study was labeled with I251by the chloramine T method and had specific activity of approximately 2000 Ci/mmol (Amersham). The slide-mounted tissue sections were brought to room temperature and placed in a preincubation medium consisting of 10 mM HEPES, pH 7.4, 25°C for 5 min. The shdemounted sections were then incubated at 25°C for 2 h in a solution of 10mM HEPES, pH 7.4, containing NaCl (130 mM), KC1 (4.7 mM), MnCl, (5 mM), EGTA (1 mM), bovine serum albumin (1%) bacitracin (1 mg/ml), and 100 pM [‘251]VIP. This was done by placing slides on a flat surface and covering the sections with 1 ml of incubation medium. To estimate the non-specific binding, paired serial sections were incubated as described above except that unlabeled VIP was added to the incubation solution to a final concentration of I PM. Following this incubation the slides were rinsed with two washes of incubation solution at 25°C for 15 min each and two washes with dH,O (4°C 5 s each), and then quickly dried in the cold room using a stream of cold air. Sections were left for 3 h to dry in the cold room and then stored in boxes containing desiccant overnight at room temperature. The dried, labeled, slide-mounted tissue sections were then placed in apposition to tritium-sensitive Ultrofilm (LKB) along with commercially available standards (Amersham). After 14 weeks the Ultrofilm was developed in Kodak D-19 developer, fixed and washed. To estimate quantitatively the concentration of radiolabeled VIP binding sites, microdensitometry was performed on the autoradiographic film as previously described.‘8 The exposed film was projected at x 20 on a white horizontal
surface and the densities of areas of the projected image measured with a photocell (Sharp BS-5OOA silicon blue photodiode) connected to a digital voltmeter (Radio Shack). The resolution of the device corresponds to a region about 25 pm in diameter on the projected sections. Previous experiments have established that the Ultrofilm does not respond linearly to linear increases in radioactivity. We therefore constructed a series of standards, exposed these to the Ultrofilm, developed and fixed the film, measured this film densitometrically and used these values with an automatic curve fitting program (Texas Instruments) to obtain a description of the film’s characteristics and to correct for non-linearity. In all cases, specific binding was obtained by subtracting the non-specific binding from the total binding. Non-specific binding was defined as the binding which occurred in the presence of 1PM unlabeled VIP. All results are expressed as a percentage of the maximal binding observed in the gastrointestinal tract which was seen in the luminal region of the mucosa in the duodenum.
RESULTS To assess the stability of VIP receptor binding sites over time, the concentration of specific VIP binding sites was determined on sections of a colon specimen which were frozen on dry ice at various times after removal. As Fig. 1 indicates, the concentration of VIP receptor binding sites decreases with increasing time at room temperature. To minimize degradation artifacts, the surgical specimens used in this study were frozen at - 70°C within 5 min of their removal. The topographical distribution of [‘251]VIP receptor binding sites throughout the human gastro-
VIP receptors in the gastrointestinal tract is shown in Table 1 and Figs 2-g. All receptor binding site concentrations are based on the corrected optical density of the exposed autoradiographic film and are expressed as a percentage of the maximal binding observed (luminal region of duodenal mucosa). The relative concentration of VIP binding sites in each area represents an average value from the specimens which showed detectable specific binding sites. In the following description the relative binding of 50% maximal or more is designated as a high concentration of receptor binding sites, 1550% of maximal binding is described as a moderate concentration of binding sites, and less than 15% maximal binding is called a low concentration of binding sites. In the esophagus, a moderate concentration of VIP binding sites is observed in the mucosa (Table 1). A low concentration of VIP receptor binding sites is found in both the external circular and longitudinal muscle layers. VIP binding sites are also expressed in moderate concentrations by arterioles with a diameter of 0.3-0.5 mm in the serosa of one patient. Three regions of the stomach (cardia, fundus, antrum) were examined for the presence of VIP
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Table 1. Concentrations* of VIP binding sites in the human gastrointestinal tract Tissue
% Max binding site density
Esophagus (3)t Mucosa-luminal (l)$ Circular muscle (3) Longitudinal muscle (2) Arteriole in serosa (1)
29.6 & 2.75 9.Ok2.1 9.2 f 0.7 15.7 k 1.6
Cardia (3) Mucosa-htminal(2) Mucosa-basal(2) Muscularis mucosa (2) Submucosal arteriole (2) Circular muscle (3) Longitudinal muscle (2)
54.8 +_0.8 9.4 + 5.4 19.0 f 6.4 11.7+0.1 53.8 k 23.9 12.5 + 2.7
Fundus (5) Mucosa-htminal(5) Mucosa-basal(4) Muscularis mucosa (5) Submucosal arteriole (2) Circular muscle (4) Longitudinal muscle (2) Lymph nodule (1)
33.2 k 8.8 + 12.6 k 18.0 f 13.0 f 6.9 + 7.8 +
Antrum (6) Mucosa-htminal(6) Mucosa-basal(6)
53.8 + 14.1 12.2 k 2.7
Muscularis mucosa (4) Submucosal arteriole (6) Circular muscle (5)
21.5 f 5.3 19.9 * 3.9 14.4 * 1.0
Duodenum (6) Mucosa-luminal(6) Mucosa-basal(6). Muscularis mucosa (2) Submucosal arteriole (4) Circular muscle (3) Myenteric plexus (2) Longitudinal muscle (3) WC
31,3---H
1100.0f 63.3 f 69.1 + 29.6 + 20.9 f 38.6 f 7.3 f
7.8 4.0 4.0 2.9 2.6 0.8 1.3
18.7 17.6 56.0 15.9 4.8 4.8 2.5
Jejunum (4) Mucosa (4) Submucosal arteriole (2) Circular muscle (4) Myenteric plexus (4) Longitudinal muscle (4)
86.5 + 4.4 10.1 * 5.0 11.1 +2.0 3.0 * 2.0 12.1 f. 2.1
Ileum (4) Mucosa (4) Circular muscle (1) Myenteric plexus (1) Longitudinal muscle (1) Lymph nodule (1)
40.8 f 27.7 k 19.5 * 10.9 * 67.4 +
8.8 2.0 1.4 2.0 2.6
Colon (7) Mucosa-luminal(7) Mucosa-basal(7) Circular muscle (7) Myenteric plexus (7) Longitudinal muscle (7)
54.8 f 17.9 k 10.1 * 17.5 f 9.9 +
9.1 4.8 1.5 5.8 1.6
*The relative concentration of VIP binding sites in each area represents the average optical density of the Ultrofilm obtained from each specimen which showed detectable specific binding sites. For each specimen, fourteen densitometric readings were taken from each tissue layer on the autoradiogram. After subtracting non-specific binding, specific binding site density values from each specimen were averaged together and the standard error of the mean (S.E.M.) was calculated for this group. Nonspecific binding was assessed by incubating sections as above but with addition of 1 PM unlabeled VIP. Nonspecific labeling on the film has an optical density of 0.0, while the luminal mucosa of the duodenum is defined as the highest optical value of 100.0. tNumber of samples examined in parentheses. $Number of samples expressing VIP binding sites in parentheses. §Mean k S.E.M.
receptor binding sites (Table 1). In the cardia VIP binding sites are expressed in high concentrations by the luminal region of the mucosa and by the circular muscle of the muscularis extema (Fig. 2). A moderate concentration of VIP receptor binding sites is observed in the muscularis mucosa. A low concentration of VIP binding sites is found in the basal portion of the mucosa, the smooth muscle cells of the tunica media of a few submucosal arterioles (diameter 0.34.5 mm) but not venules, and the external longitudinal muscle. In the fundus, the luminal aspect of the mucosa expresses a moderate concentration of VIP binding sites (Fig. 3). A lower receptor binding site concentration is associated with the muscularis mucosa, the smooth muscle of a few submucosal arterioles, and the external circular muscle. VIP receptor binding sites are present in low concentrations in the basal region of the mucosa, the external longitudinal muscle, and in one lymph nodule (not shown). The highest concentration of VIP binding sites in the antrum is found in the outermost portion of the mucosa which borders the lumen (Fig. 4). A moderate concentration of VIP receptor binding sites is observed in the muscularis mucosa. and in arterioles
Fig. 2. Distribution of [‘251]VIP binding sites in the cardia. (a) Light-field photomicrograph shows a hematoxylin and eosin (H-E) stained section of cardia from a normal human stomach. (b) Dark-field photomicrograph made from tritium-sensitive film that overlaid section (a) for 10 days showing the total binding. In these and the following dark-field photomicrographs, [‘251]VIP binding sites appear as white silver grains. (c) Contol section showing the non-specific binding that was treated identically to the section shown in (b) except that unlabeled VIP (1 PM) was added to the incubation medium. Specific binding was determined by subtracting the non-specific binding (c) from the total binding (b). Note the high concentrations of specific binding sites in the external circular muscle (CM) and in the luminal region of the mucosa (MC). Moderate VIP binding site concentrations are visible in the muscularis mucosa and low concentrations of VIP binding sites are observed in the basal portion of the mucosa (MC). Scale bar = I mm. 774
Fi g. 3. Localization of [‘*‘I]VIP receptor binding sites in the fundus. (a) Light-field photomicrograph sh.ows a hematoxyhn and eosin (H-E) stained section of normal human antrum. (b) Dark-field Pllotomicrograph of the t~tinm-sensitive film which overlaid section (a) for IO days. (c) Control section sh.owing the non-specific binding. Note the high concentration of specific [‘*‘I]VIP binding sites (white) in the luminal part of the mucosa (MC) and moderate concentration of VIP binding sites in the muscularis mucosa (MM). See Fig. 2 for further explanation. Scale bar = 1.3 mm.
R. P. ZIMMERMAN et al.
Fig. 4. Distribution of [‘251]VIPbinding sites in the antrum. (a) Light-field photomicrograph shows a hematoxylin and eosin (H-E) stained section of human antrum. (b) Dark-field photomicrograph made from tritium-sensitive film that overlaid section (a) for 10 days. Note the high concentrations of specific [1251]VIPbinding sites (white) in the luminal mucosa (MC). Moderate VIP binding site concentrations are seen in the muscularis mucosa (MM). A low concentration of VIP binding sites is present in the basal portion of the mucosa (MC). In this and in the following figures, control sections were essentially blank. See Fig. 2 for further explanation. Scale bar = 0.65 mm.
in the submucosa (not shown). A low concentration of VIP binding sites is found in the basal region of the mucosa and in the circular muscle of the muscularis extema. In the duodenum, the highest concentration of VIP receptor binding sites is observed in the luminal mucosa (Fig. 5). This region was found to contain the highest concentration of VIP binding sites in the entire human gastrointestinal tract (Table 1). High concentrations of VIP receptor binding sites are also associated with the basal mucosa and the muscularis
mucosa. A moderate concentration of binding sites is present in smooth muscle of a few submucosal arterioles, in the external circular muscle, and in the myenteric plexus. The external longitudinal muscle has the lowest concentration of detectable VIP binding sites in the duodenum. The areas with the highest concentration of VIP receptor binding sites in the jejunum are the luminal and basal aspects of the mucosa (Fig. 6). A low concentration of VIP binding sites is found in smooth muscle of submucosal arterioles, the circular and
VIP receptors in the gastrointestinal
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duodenu
Fig. 5. Localization of specific [1251]VIPbinding sites in the duodenum. (a) Light-field photomicrograph shows a hematoxylin and eosin (H-E) stained section of the human duodenum. (b) Dark-field photomicrograph from the tritium-sensitive film that overlaid section (a) for 10 days. Note the high concentrations of receptor binding sites on both the luminal and basal aspect of the mumsa (MC), which has the highest concentration of VIP binding sites in the entire human gastrointestinal tract. The external circular muscle (CM) in this section shows only a low concentration of VIP binding sites. See Fig. 2 for further explanation. Scale bar = 0.65 mm.
longitudinal
muscle layers of the muscularis extema,
and the myenteric
plexus.
In the ileum, submucosal lymph nodules from one specimen show the highest concentration of VIP receptor binding sites in this gastrointestinal segment (Table 1). The ileal mucosa also contains a moderate concentration of VIP binding sites (Fig. 7). Moderate
concentrations external
of VIP binding sites are found in the circular muscle and myenteric plexus. A low
concentration of VIP receptor binding sites is observed on the external longitudinal muscle. In the colon, the luminal region of the mucosa shows a high concentration of VIP binding sites (Fig. 8). The basal portion of the mucosa and the
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Fig. 6. Distribution of [‘251]VIPbinding sites in the jejunum. (a) Light-field photomicrograph shows a hematoxylin and eosin (H-E) stained section of the human jejunum. (b) Dark-field photomicrograph made from tritium-sensitive film that overlaid section (a) for 10 days. Note the high concentrations of receptor binding sites on both the luminal and basal aspect of the mucosa (MC) and moderate concentrations of VIP binding sites in both the circular muscle (CM) and longitudinal muscle (LM) of the muscularis exte’rna. See Fig. 2 for further explanation. Scale bar = 1.5 mm.
myenteric plexus express moderate VIP receptor binding sites. A low VIP binding sites is observed in muscle and the longitudinal muscle externa.
concentrations of concentration of both the circular of the muscularis
DISCUSSION
Variability among human specimens In the present study, we have defined the target tissues for VIP in the normal human gastrointestinal
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Fig. 7. Distribution of specific [1z51]VIPbinding sites in the ileum. (a) Bright-field photomicrograph shows a hematoxylin and eosin (H-E) stained section of the human ileum. (b) Dark-field photomicrograph of the tritium-sensitive film that overlaid section (a) for 10 days. Note the high concentrations of receptor binding sites in the mucosa (MC) and moderate concentrations of VIP binding sites in the external circular muscle (CM). See Fig. 2 for further explanation. Scale bar = 1 mm.
tract bly determining which tissues express highaffinity receptor binding sites for [‘251]VIP.We have demonr itrated that VIP receptor binding sites are relative ly stable after surgical removal and that human surgical specimens can be used for in situ studies to provide quantitative estimates of receptor concenl trations as well as anatomical localization of recepto r binding sites. Although the specimens in the present study were determined to be histologically
normal, the distance between the margin of the resection (which is where the surgical specimens vvere obtained) and the tumor may be related to the variability in the presence of VIP receptor bincting sites observed in certain segments of the gas#trointestinal tract. Thus, when the distance between the surgical specimen and the tumor is greatest (sucl h as in the colon), VIP binding sites in five areas are observed in seven out of the seven specimens ex
Fig X 780
VIP receptors in the gastrointestinal tract
ined. However, when the distance between the specimen and tumor is small (such as in the stomach and esophagus) the variability in the areas expressing VIP binding sites is much greater from specimen to specimen. If the variability in the density of VIP binding sites between specimens obtained from different patients is related to distance from the tumor, this suggests that receptor binding studies may prove useful in detecting biochemical alterations in cancerous tissues which are undetectable using standard histological stains. Vasoactive intestinal polypeptide regulation of electro lyte and water secretion There is a good correlation between the location of VIP receptor binding sites and the areas and cells of the gastrointestinal tract in which VIP has been shown to produce a functional response. The best example is VIP regulation of electrolyte and water secretion in intestinal epithelium.3~‘2~‘3This function is pathologically exaggerated in patients with VIPproducing tumors who have water diarrhea syndrome;” this is the only situation where VIP is known to act as a circulating hormone. Intravenous infusions of VIP cause a dose-dependent decrease in water and sodium absorption and increased chloride secretion in dogI and man.13 This secretory effect of VIP has been found to be coupled to CAMP production.8.23 The present study has shown that the intestinal mucosa contains a high concentration of VIP receptor binding sites and is thus a target tissue for VIP released by neurons innervating this region,7s’4 and suggests that VIP is involved in the regulation of intestinal water and chloride secretion via the VIP receptors on the intestinal epithelium. Vasoactive intestinal pepsinogen secretion
polypeptide
regulation
of
VIP is known to inhibit canine acid and pepsinogen secretion in viva” and these effects are accompanied by an increase in somatostatin-like immunoreactivity. However, VIP has also been shown to stimulate pepsinogen secretion from rat gastric glands” and guinea-pig chief ~~11s~~ in vitro. Whether this inconsistency is due to species differences or to the use of in vivo vs in vitro preparations is unclear. VIP also inhibits canine gastrin secretion in vivo at concentrations which could be reached near VIPergic fiber terminals24 and this inhibition is thought to be mediated by somatostatin.6 VIP-containing fibers have been found to innervate the gastric mucosa’4 and we now demonstrate that there is a high concentration of
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VIP receptor binding sites in the mucosa of the cardia, fundus, and antrum of the stomach, suggesting that VIP may have indirect actions on the mucosal epithelium. It remains, however, for future studies to identify the exact cell types in the gastric mucosa that express VIP receptors. Hemodynamic regulation Since it was first isolated from the porcine intestine, VIP has been noted for its role as a vasodilator. It is known to cause vasodilation of peripheral vascular beds and to increase blood flow to these regions.” The presence of VIP-containing nerve fibers innervating arteries has been contirmed’4 and these fibers release VIP in response to physiological stimuli.22 We have now demonstrated the presence of a moderate concentration of VIP receptor binding sites in the smooth muscle of the tunica media of submucosal arterioles (diameter 0.345 mm) which are located in the cardia, fundus, antrum, duodenum, and jejunum. The localization bf VIP binding sites here identifies the smooth muscle of arterioles as a target tissue for VIP, which therefore may play a role in regulating regional gastrointestinal blood flow. Regulation of gastrointestinal motility There are conflicting reports about the myogenic properties of VIP in the gastrointestinal tract. In the guinea-pig small intestine, low concentrations of VIP have been shown to relax the external circular muscle’ while high concentrations have been found to contract the external longitudinal muscle.” In the guinea-pig colon, VIP has been shown to relax the external circular muscle and contract the external longitudinal muscle,4 whereas in the mouse it has been shown to contract external longitudinal muscle in low concentrations and relax it in high concentrations.’ In addition, VIP immunoreactive fibers have been localized to nerve fibers and cell bodies in the myenteric and submucosal ganglia and around smooth muscle bundles in the external circular and longitudinal muscle layers.’ In the present study, we found VIP receptor binding sites in the myenteric plexus of the duodenum, jejunum, ileum, and colon, suggesting that some VIP myogenic actions may be indirect. We have also shown that both the circular and longitudinal muscle comprising the muscularis extema express VIP receptor binding sites throughout the gastrointestinal tract, indicating that VIP may also have a direct spasmogenic effect on these smooth muscles. The presence of binding sites identifies these tissues as probable sites of VIP action and suggests
Fig. 8. Localization of [‘251]VIPreceptor binding sites in the colon. (a) Bright-field photomicrograph of a hematoxylin and eosin (H-E) stained section of human colon, and (b) dark-field photomicrograph made from tritium-sensitive film that overlaid section (a) for 10 days. (c) Control section showing the non-specific binding. Note the high concentrations of receptor binding sites in the luminal region of the mucosa (MC)and moderate concentrations of VIP binding sites in the basal layer of the mucosa (MC). Low VIP receptor binding site concentrations are observed in the external circular muscle (CM) and longitudinal muscle (LM) of the colon. See Fig. 2 for further explanation. Scale bar = 0.8 mm.
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that VIP may play a direct role in regulating gastric and intestinal motility. The involvement of vasoactive intestinal polypeptide human gastrointestinal disease
in
There is evidence suggesting that VIP may be involved in the pathophysiology of certain diseases such as inflammatory bowel disease. For instance, the VIP content of peripheral nerve fibers. has been reported to increase in inflammatory bowel disease, specifically Crohn’s disease.5 In a study of VIP binding site concentration in colon specimens from patients with inflammatory bowel disease, we found that VIP binding site concentrations were signifi-
cantly lower in the mucosa of specimens from ulcerative colitis patients but not in specimens from Crohn’s patients.16’ Precisely what role VIP and VIP receptors may play in the pathophysiology of ulcerative colitis or Crohn’s disease is not yet clear, but knowledge of the cell types that express VIP receptor binding sites in the normal human gastrointestinal tract provides a basis for comparing the receptor binding site distribution in normal vs diseased states. Acknowledgement-This work was supported by the Southern California Arthritis Foundation, the Alfred Sloan Fellowship, the Smith, Kline and Beckman Fellowship, and NINCDS grant NS23970.
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5 Bishop A. E., Polak J. M., Bryant M. G., Bloom S. R. and Hamilton S. (1980) Abnormalities of vasoactive intestinal polypeptide-containing nerves in Crohn’s disease. Guslroenterology 79, 853-860. 6. Chiba T., Taminato T., Kadowaki S., Abe H., Chihara K., Seino Y., Matsukura S. and Fujita T. (1980) Effects of glucagon, secretin and vasoactive intestinal polypeptide on gastric somatostatin and gastrin release from isolated perfused rat stomach. Gastroenterology 79, 67-71. 7. Costa M., Furness J. B., Buffa R. and Said S. I. (1980) Distribution of enteric nerve cell bodies and axons showing immunoreactivity for VIP in guinea-pig intestine. Neuroscience 5, 587-596. 8. DuPont C., Laburthe M., Broyart J. P., Bataille D. and Rosselin G. (1980) Cyclic AMP production in isolated colonic epithelial crypts: a highly sensitive model for the evaluation of vasoactive intestinal peptide action in human intestine. Eur. J. clin. Invest. 10, 67-76. 9. Fontaine J., Grivegnee A. R. and Robberecht P. (1986) Evidence against VIP as the inhibitory transmitter in non-adrenergic, non-cholinergic nerves supplying the longitudinal muscle of the mouse colon. Br. J. Pharmac. 89, 599-602.
IO. Hiikfelt T., Schultzberg M., Lundberg J. M., Fuxe K., Mutt V., Fahrenkrug J. and Said S. I. (1982) In Vasoactive Intestinal Pepride (ed. Said S. I.), pp.65-90. Raven Press, New York. 11. Jaffer S. S.. Farrar J. T.. Yau W. M. and Makhlouf G. M. (1974) Mode of action and internlav of vasoactive intestinal peptide (ViP), secretin’and octapeptide of cholecystokinin (&TA-CCK) on duodenal and ileal muscle in vitro. Gartroenterology 66, 716. 12. Krejs G. J., Barkley R. M., Read N. W. and Fordtran J. S. (1980) Intestinal secretion induced by vasoactive intestinal peptide: a comparison with cholera toxin in the canine jejunum in vivo. J. c/in. Invest. 61, 1337-1345. 13. Krejs G. J. and Fordtran J. S. (1980) Effect of VIP infusion on water and ion transport in human jejunum. Gaslroenrerology
78, 722-727.
14. Larsson L. I., Fahrenkrug J., Schaffalitzky de Muckadell 0. B., Sundler F., Hakanson R. and Rehfeld J. F. (1976) Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons. Proc. natn. Acad. Sci. U.S.A. 73, 3197-3200.
15. Maghlouf G. M., Zfass A. M., Said S. I. and Schebalin M. (1978) Effects of synthetic vasoactive intestinal polypeptide __ _ (VIP), secretin and their partial sequences on gastric secretion. Proc. Sot. eip. biol. Med. 157, 565-568.16. Mantyh C. R., Vigna S. R., Gates T., Maggio J. E., Welton M. L., Passaro E. P. and Mantyh P. W. (1988) Receptor binding sites for substance P, but not substance K or neuromedin K, are ectopically expressed by arterioles, venules and lymph nodules in Crohn’s disease and ulcerative colitis. Proc. natn. Acad. Sci. U.S.A. 85, 3235-3239. 16a. Mantvh P. W., Catton M., Boehmer C., Gates. T., Mantvh C. R.. Zimmerman R. P.. Welton M. L., Passaro E. P. jr, Maggio J. E. and Vigna S. R. deceptors for sens&y neurotransmitters in human inflammatory diseases: implications for the effector role of sensory neurons. Peprides (in presss). 17. Modlin I. M., Bloom S. R. and Mitchel S. J. (1978) Experimental evidence for vasoactive intestinal polypeptide as the cause of watery diarrhea syndrome. Gastroenteroiogy 75, 1051-1054. 18. Rainbow T. C., Biegon A. and Berck D. J. (1984) Quantitative receptor autoradiography with tritium-labeled ligands: comparison of biochemical and densitometric measurements. J. Neurosci. Merh. 11, 231-241. 19. Raufman J. P., Kasbekar D. K., Jensen R. T. and Gardner J. D. (1983) Potentiation of pepsinogen secretion from dispersed glands from rat stomach. Am. J. Physiol. 245, G525-G530. 20. Raufman J. P., Sutliff V. E., Kasbekar D. K., Jensen R. T. and Gardner J. D. (1984) Pepsinogen secretion from dispersed chief cells from guinea pig stomach. Am. J. Physiol. 247, G95-GlO4. 21. Said S. I. and Mutt V. (1972) Isolation from porcine intestinal wall of a vasoactive octacosapeptide related to secretin and glucagon. Eur. J. Biochem. 28, 199-204.
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22. Schafialitzky de Muckadell 0. B., Fahrenkrug J. and Hoist J. J. (1977) Release of vasoactive intestinal polypeptide (VIP) by electrical stimulation of vagal nerves. Gasrroenterology 72, 373-375. 23. Schwartz C. J., Kimberg D. V., Sheerin H. E., Field M. and Said S. I. (1974) Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa. J. clin. Invest. 54, 536544. 24. Villar H. V., Fender H. R., Rayford P. L., Ramus N. I. and Thompson J. C. (1975) Vasoactive intestinal polypeptide. In Gastrointestinal Hormones (ed. Thompson J. C.), pp. 467-474. University of Texas Press, Austin, TX. (Accepted 3 March 1989)
0306-4522/89$3.00 + 0.00 Pergamon Press plc Q 1989 IBRO
VASOACTIVE INTESTINAL POLYPEPTIDE RECEPTOR BINDING SITES IN THE HUMAN GASTROINTESTINAL TRACT: LOCALIZATION BY AUTORADIOGRAPHY C. R. MANTYH ,* S. R. VICNA,*$$ M. L. WELTON,*~~ E. P. PASSARO JR*[[ and P. W. MANTYH*~$T/ *Center for Ulcer Research and Education, Veterans Administration Medical Center-Wadsworth, Los Angeles, CA 90073, U.S.A. IDepartment of Medicine, ItDepartment of Surgery, and IBrain Research Institute, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90024, U.S.A.
R. P.
ZIMMERMAN,*
T. S. GATES,*
Abstract-Vasoactive intestinal polypeptide (VIP) is a putative neurotransmitter in both the brain and peripheral tissues. To define possible target tissues of VIP we have used quantitative receptor autoradiography to localize and quantify the distribution of [‘251]VIPreceptor binding sites in histologically normal human surgical specimens. While the distribution of VIP binding sites was different for each gastrointestinal segment examined, specific vasoactive intestinal polypeptide binding sites were localized to the mucosa, the muscularis mucosa, the smooth muscle of submucosal arterioles, the circular and longitudinal smooth muscle of the muscularis externa, the myenteric plexus, and lymph nodules. In most segments, the mucosal layer expressed the highest concentration of VIP binding sites, with the duodenal and jejunal mucosa showing the highest density of receptors. These results identify putative VIP target tissues in the human gastrointestinal tract. In correlation with physiological data, VIP binding sites appear to be involved in the regulation of a variety of gastrointestinal functions including mucosal ion transport, gastric secretion, hemodynamic regulation, gastric and intestinal motility, neuronal excitability, and modulation of the immune system.
Vasoactive intestinal polypeptide (VIP) was first described as a potent vasodilator isolated from the porcine intestine2’ but is now known to be a putative peptide neurotransmitter in the central and peripheral nervous systems. Histochemical, pharmacological, and physiological studies have suggested that VIP is involved in regulating several gastrointestinal functions. Immunohistochemical studies have shown that intrinsic VIP immunoreactive fibers are found in the submucous and myenteric plexuses and extend into the lamina propria, the muscularis mucosae, and the external circular muscle in nearly every segment of the rat and guinea-pig gastrointestinal tract.” Pharmacological and physiological studies also indicate VIP has a role in regulating a variety of gastrointestinal functions. VIP has been shown to promote water and electrolyte secretion by intestinal epithelium,3J2.‘3 inhibit gastric acid, pepsinogen,” and gastrin24 secretion, act as a potent vasodilator,2’ and to have varied effects on gastrointestinal motility.2,4.9.”
Demonstration of pharmacologically relevant receptor binding sites is crucial for characterization of VIP action in the gastrointestinal tract since all of its actions at physiological concentrations are thought to be receptor mediated.” Since many neurons are now known to contain multiple neurotransmitters, localization of receptor binding sites for VIP is essential in determining where released VIP will produce a functional response. In the present report, we examined histologically normal surgical specimens obtained from the length of the human gastrointestinal tract to determine whether they express specific [“‘I]VIP binding sites. The objectives of this study are: (1) to demonstrate that intact human surgical specimens can be processed for quantitative receptor autoradiography; (2) to localize possible target tissues for released VIP through identification of cells and tissues expressing specific VIP binding sites in the human GI tract; and (3) to determine the stability of the VIP binding sites in human surgical specimens. EXPERIMENTAL Surgical
~To whom correspondence should be addressed at Research Service 1151), VA Medical Center, 1 Veterans Drive, Minneapolis, MN 55417, U.S.A. $Present address: Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, U.S.A. Abbreviafions: EGTA. ethvleneplvcolbis(aminoethvlether)tetra-acetate; HEPES, -N-2-&droxydthylpiper&ine-h;2-ethansulfonic acid; VIP, vasoactive intestinal polypeptide. 771
PROCEDURES
specimens were obtained within 5 min of removal in order to minimize post-surgical degradation artifacts. Tissue specimens were obtained from the uninvolved margins of extensive resections for carcinoma and determined by a pathologist to be histologically normal. To determine the time course of VIP receptor degradation, samples from a colon specimen were frozen (-70°C) after being kept at room temperature for increasing lengths of time from 0 to 300min. The tissue was then blocked, placed on a brass microtome chuck, frozen on dry ice and processed for
R. P. ZIMMERMAN et al VIP Receptor
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Mc-lumlnol
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Fig 1. Density of surgical removal. (LM), (b) external density of binding of VIP receptors,
180
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60
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[‘2sI]VIP receptor binding sites in human colon vs time stored at room temperature after The stability of the receptor binding sites is shown for (a) external longitudinal muscle circular muscle (CM), (c) basal mucosa (MC) and (d) luminal mucosa (MC). The optical sites at time 0 in each tissue is the maximal binding (100%). Note that the degradation as seen by the decrease in receptor binding site densities with time, is substantial in all tissues but that the time course is different for each region.
quantitative autoradiography as we have previously described.i6 The tissue was serially sectioned (30pm), thawmounted on gelatin coated microscope slides, and stored at -20°C in boxes with desiccant for up to 3 months. The radiolabeled vasoactive intestinal polypeptide used in this study was labeled with I251by the chloramine T method and had specific activity of approximately 2000 Ci/mmol (Amersham). The slide-mounted tissue sections were brought to room temperature and placed in a preincubation medium consisting of 10 mM HEPES, pH 7.4, 25°C for 5 min. The shdemounted sections were then incubated at 25°C for 2 h in a solution of 10mM HEPES, pH 7.4, containing NaCl (130 mM), KC1 (4.7 mM), MnCl, (5 mM), EGTA (1 mM), bovine serum albumin (1%) bacitracin (1 mg/ml), and 100 pM [‘251]VIP. This was done by placing slides on a flat surface and covering the sections with 1 ml of incubation medium. To estimate the non-specific binding, paired serial sections were incubated as described above except that unlabeled VIP was added to the incubation solution to a final concentration of I PM. Following this incubation the slides were rinsed with two washes of incubation solution at 25°C for 15 min each and two washes with dH,O (4°C 5 s each), and then quickly dried in the cold room using a stream of cold air. Sections were left for 3 h to dry in the cold room and then stored in boxes containing desiccant overnight at room temperature. The dried, labeled, slide-mounted tissue sections were then placed in apposition to tritium-sensitive Ultrofilm (LKB) along with commercially available standards (Amersham). After 14 weeks the Ultrofilm was developed in Kodak D-19 developer, fixed and washed. To estimate quantitatively the concentration of radiolabeled VIP binding sites, microdensitometry was performed on the autoradiographic film as previously described.‘8 The exposed film was projected at x 20 on a white horizontal
surface and the densities of areas of the projected image measured with a photocell (Sharp BS-5OOA silicon blue photodiode) connected to a digital voltmeter (Radio Shack). The resolution of the device corresponds to a region about 25 pm in diameter on the projected sections. Previous experiments have established that the Ultrofilm does not respond linearly to linear increases in radioactivity. We therefore constructed a series of standards, exposed these to the Ultrofilm, developed and fixed the film, measured this film densitometrically and used these values with an automatic curve fitting program (Texas Instruments) to obtain a description of the film’s characteristics and to correct for non-linearity. In all cases, specific binding was obtained by subtracting the non-specific binding from the total binding. Non-specific binding was defined as the binding which occurred in the presence of 1PM unlabeled VIP. All results are expressed as a percentage of the maximal binding observed in the gastrointestinal tract which was seen in the luminal region of the mucosa in the duodenum.
RESULTS To assess the stability of VIP receptor binding sites over time, the concentration of specific VIP binding sites was determined on sections of a colon specimen which were frozen on dry ice at various times after removal. As Fig. 1 indicates, the concentration of VIP receptor binding sites decreases with increasing time at room temperature. To minimize degradation artifacts, the surgical specimens used in this study were frozen at - 70°C within 5 min of their removal. The topographical distribution of [‘251]VIP receptor binding sites throughout the human gastro-
VIP receptors in the gastrointestinal tract is shown in Table 1 and Figs 2-g. All receptor binding site concentrations are based on the corrected optical density of the exposed autoradiographic film and are expressed as a percentage of the maximal binding observed (luminal region of duodenal mucosa). The relative concentration of VIP binding sites in each area represents an average value from the specimens which showed detectable specific binding sites. In the following description the relative binding of 50% maximal or more is designated as a high concentration of receptor binding sites, 1550% of maximal binding is described as a moderate concentration of binding sites, and less than 15% maximal binding is called a low concentration of binding sites. In the esophagus, a moderate concentration of VIP binding sites is observed in the mucosa (Table 1). A low concentration of VIP receptor binding sites is found in both the external circular and longitudinal muscle layers. VIP binding sites are also expressed in moderate concentrations by arterioles with a diameter of 0.3-0.5 mm in the serosa of one patient. Three regions of the stomach (cardia, fundus, antrum) were examined for the presence of VIP
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Table 1. Concentrations* of VIP binding sites in the human gastrointestinal tract Tissue
% Max binding site density
Esophagus (3)t Mucosa-luminal (l)$ Circular muscle (3) Longitudinal muscle (2) Arteriole in serosa (1)
29.6 & 2.75 9.Ok2.1 9.2 f 0.7 15.7 k 1.6
Cardia (3) Mucosa-htminal(2) Mucosa-basal(2) Muscularis mucosa (2) Submucosal arteriole (2) Circular muscle (3) Longitudinal muscle (2)
54.8 +_0.8 9.4 + 5.4 19.0 f 6.4 11.7+0.1 53.8 k 23.9 12.5 + 2.7
Fundus (5) Mucosa-htminal(5) Mucosa-basal(4) Muscularis mucosa (5) Submucosal arteriole (2) Circular muscle (4) Longitudinal muscle (2) Lymph nodule (1)
33.2 k 8.8 + 12.6 k 18.0 f 13.0 f 6.9 + 7.8 +
Antrum (6) Mucosa-htminal(6) Mucosa-basal(6)
53.8 + 14.1 12.2 k 2.7
Muscularis mucosa (4) Submucosal arteriole (6) Circular muscle (5)
21.5 f 5.3 19.9 * 3.9 14.4 * 1.0
Duodenum (6) Mucosa-luminal(6) Mucosa-basal(6). Muscularis mucosa (2) Submucosal arteriole (4) Circular muscle (3) Myenteric plexus (2) Longitudinal muscle (3) WC
31,3---H
1100.0f 63.3 f 69.1 + 29.6 + 20.9 f 38.6 f 7.3 f
7.8 4.0 4.0 2.9 2.6 0.8 1.3
18.7 17.6 56.0 15.9 4.8 4.8 2.5
Jejunum (4) Mucosa (4) Submucosal arteriole (2) Circular muscle (4) Myenteric plexus (4) Longitudinal muscle (4)
86.5 + 4.4 10.1 * 5.0 11.1 +2.0 3.0 * 2.0 12.1 f. 2.1
Ileum (4) Mucosa (4) Circular muscle (1) Myenteric plexus (1) Longitudinal muscle (1) Lymph nodule (1)
40.8 f 27.7 k 19.5 * 10.9 * 67.4 +
8.8 2.0 1.4 2.0 2.6
Colon (7) Mucosa-luminal(7) Mucosa-basal(7) Circular muscle (7) Myenteric plexus (7) Longitudinal muscle (7)
54.8 f 17.9 k 10.1 * 17.5 f 9.9 +
9.1 4.8 1.5 5.8 1.6
*The relative concentration of VIP binding sites in each area represents the average optical density of the Ultrofilm obtained from each specimen which showed detectable specific binding sites. For each specimen, fourteen densitometric readings were taken from each tissue layer on the autoradiogram. After subtracting non-specific binding, specific binding site density values from each specimen were averaged together and the standard error of the mean (S.E.M.) was calculated for this group. Nonspecific binding was assessed by incubating sections as above but with addition of 1 PM unlabeled VIP. Nonspecific labeling on the film has an optical density of 0.0, while the luminal mucosa of the duodenum is defined as the highest optical value of 100.0. tNumber of samples examined in parentheses. $Number of samples expressing VIP binding sites in parentheses. §Mean k S.E.M.
receptor binding sites (Table 1). In the cardia VIP binding sites are expressed in high concentrations by the luminal region of the mucosa and by the circular muscle of the muscularis extema (Fig. 2). A moderate concentration of VIP receptor binding sites is observed in the muscularis mucosa. A low concentration of VIP binding sites is found in the basal portion of the mucosa, the smooth muscle cells of the tunica media of a few submucosal arterioles (diameter 0.34.5 mm) but not venules, and the external longitudinal muscle. In the fundus, the luminal aspect of the mucosa expresses a moderate concentration of VIP binding sites (Fig. 3). A lower receptor binding site concentration is associated with the muscularis mucosa, the smooth muscle of a few submucosal arterioles, and the external circular muscle. VIP receptor binding sites are present in low concentrations in the basal region of the mucosa, the external longitudinal muscle, and in one lymph nodule (not shown). The highest concentration of VIP binding sites in the antrum is found in the outermost portion of the mucosa which borders the lumen (Fig. 4). A moderate concentration of VIP receptor binding sites is observed in the muscularis mucosa. and in arterioles
Fig. 2. Distribution of [‘251]VIP binding sites in the cardia. (a) Light-field photomicrograph shows a hematoxylin and eosin (H-E) stained section of cardia from a normal human stomach. (b) Dark-field photomicrograph made from tritium-sensitive film that overlaid section (a) for 10 days showing the total binding. In these and the following dark-field photomicrographs, [‘251]VIP binding sites appear as white silver grains. (c) Contol section showing the non-specific binding that was treated identically to the section shown in (b) except that unlabeled VIP (1 PM) was added to the incubation medium. Specific binding was determined by subtracting the non-specific binding (c) from the total binding (b). Note the high concentrations of specific binding sites in the external circular muscle (CM) and in the luminal region of the mucosa (MC). Moderate VIP binding site concentrations are visible in the muscularis mucosa and low concentrations of VIP binding sites are observed in the basal portion of the mucosa (MC). Scale bar = I mm. 774
Fi g. 3. Localization of [‘*‘I]VIP receptor binding sites in the fundus. (a) Light-field photomicrograph sh.ows a hematoxyhn and eosin (H-E) stained section of normal human antrum. (b) Dark-field Pllotomicrograph of the t~tinm-sensitive film which overlaid section (a) for IO days. (c) Control section sh.owing the non-specific binding. Note the high concentration of specific [‘*‘I]VIP binding sites (white) in the luminal part of the mucosa (MC) and moderate concentration of VIP binding sites in the muscularis mucosa (MM). See Fig. 2 for further explanation. Scale bar = 1.3 mm.
R. P. ZIMMERMAN et al.
Fig. 4. Distribution of [‘251]VIPbinding sites in the antrum. (a) Light-field photomicrograph shows a hematoxylin and eosin (H-E) stained section of human antrum. (b) Dark-field photomicrograph made from tritium-sensitive film that overlaid section (a) for 10 days. Note the high concentrations of specific [1251]VIPbinding sites (white) in the luminal mucosa (MC). Moderate VIP binding site concentrations are seen in the muscularis mucosa (MM). A low concentration of VIP binding sites is present in the basal portion of the mucosa (MC). In this and in the following figures, control sections were essentially blank. See Fig. 2 for further explanation. Scale bar = 0.65 mm.
in the submucosa (not shown). A low concentration of VIP binding sites is found in the basal region of the mucosa and in the circular muscle of the muscularis extema. In the duodenum, the highest concentration of VIP receptor binding sites is observed in the luminal mucosa (Fig. 5). This region was found to contain the highest concentration of VIP binding sites in the entire human gastrointestinal tract (Table 1). High concentrations of VIP receptor binding sites are also associated with the basal mucosa and the muscularis
mucosa. A moderate concentration of binding sites is present in smooth muscle of a few submucosal arterioles, in the external circular muscle, and in the myenteric plexus. The external longitudinal muscle has the lowest concentration of detectable VIP binding sites in the duodenum. The areas with the highest concentration of VIP receptor binding sites in the jejunum are the luminal and basal aspects of the mucosa (Fig. 6). A low concentration of VIP binding sites is found in smooth muscle of submucosal arterioles, the circular and
VIP receptors in the gastrointestinal
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Fig. 5. Localization of specific [1251]VIPbinding sites in the duodenum. (a) Light-field photomicrograph shows a hematoxylin and eosin (H-E) stained section of the human duodenum. (b) Dark-field photomicrograph from the tritium-sensitive film that overlaid section (a) for 10 days. Note the high concentrations of receptor binding sites on both the luminal and basal aspect of the mumsa (MC), which has the highest concentration of VIP binding sites in the entire human gastrointestinal tract. The external circular muscle (CM) in this section shows only a low concentration of VIP binding sites. See Fig. 2 for further explanation. Scale bar = 0.65 mm.
longitudinal
muscle layers of the muscularis extema,
and the myenteric
plexus.
In the ileum, submucosal lymph nodules from one specimen show the highest concentration of VIP receptor binding sites in this gastrointestinal segment (Table 1). The ileal mucosa also contains a moderate concentration of VIP binding sites (Fig. 7). Moderate
concentrations external
of VIP binding sites are found in the circular muscle and myenteric plexus. A low
concentration of VIP receptor binding sites is observed on the external longitudinal muscle. In the colon, the luminal region of the mucosa shows a high concentration of VIP binding sites (Fig. 8). The basal portion of the mucosa and the
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Fig. 6. Distribution of [‘251]VIPbinding sites in the jejunum. (a) Light-field photomicrograph shows a hematoxylin and eosin (H-E) stained section of the human jejunum. (b) Dark-field photomicrograph made from tritium-sensitive film that overlaid section (a) for 10 days. Note the high concentrations of receptor binding sites on both the luminal and basal aspect of the mucosa (MC) and moderate concentrations of VIP binding sites in both the circular muscle (CM) and longitudinal muscle (LM) of the muscularis exte’rna. See Fig. 2 for further explanation. Scale bar = 1.5 mm.
myenteric plexus express moderate VIP receptor binding sites. A low VIP binding sites is observed in muscle and the longitudinal muscle externa.
concentrations of concentration of both the circular of the muscularis
DISCUSSION
Variability among human specimens In the present study, we have defined the target tissues for VIP in the normal human gastrointestinal
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Fig. 7. Distribution of specific [1z51]VIPbinding sites in the ileum. (a) Bright-field photomicrograph shows a hematoxylin and eosin (H-E) stained section of the human ileum. (b) Dark-field photomicrograph of the tritium-sensitive film that overlaid section (a) for 10 days. Note the high concentrations of receptor binding sites in the mucosa (MC) and moderate concentrations of VIP binding sites in the external circular muscle (CM). See Fig. 2 for further explanation. Scale bar = 1 mm.
tract bly determining which tissues express highaffinity receptor binding sites for [‘251]VIP.We have demonr itrated that VIP receptor binding sites are relative ly stable after surgical removal and that human surgical specimens can be used for in situ studies to provide quantitative estimates of receptor concenl trations as well as anatomical localization of recepto r binding sites. Although the specimens in the present study were determined to be histologically
normal, the distance between the margin of the resection (which is where the surgical specimens vvere obtained) and the tumor may be related to the variability in the presence of VIP receptor bincting sites observed in certain segments of the gas#trointestinal tract. Thus, when the distance between the surgical specimen and the tumor is greatest (sucl h as in the colon), VIP binding sites in five areas are observed in seven out of the seven specimens ex
Fig X 780
VIP receptors in the gastrointestinal tract
ined. However, when the distance between the specimen and tumor is small (such as in the stomach and esophagus) the variability in the areas expressing VIP binding sites is much greater from specimen to specimen. If the variability in the density of VIP binding sites between specimens obtained from different patients is related to distance from the tumor, this suggests that receptor binding studies may prove useful in detecting biochemical alterations in cancerous tissues which are undetectable using standard histological stains. Vasoactive intestinal polypeptide regulation of electro lyte and water secretion There is a good correlation between the location of VIP receptor binding sites and the areas and cells of the gastrointestinal tract in which VIP has been shown to produce a functional response. The best example is VIP regulation of electrolyte and water secretion in intestinal epithelium.3~‘2~‘3This function is pathologically exaggerated in patients with VIPproducing tumors who have water diarrhea syndrome;” this is the only situation where VIP is known to act as a circulating hormone. Intravenous infusions of VIP cause a dose-dependent decrease in water and sodium absorption and increased chloride secretion in dogI and man.13 This secretory effect of VIP has been found to be coupled to CAMP production.8.23 The present study has shown that the intestinal mucosa contains a high concentration of VIP receptor binding sites and is thus a target tissue for VIP released by neurons innervating this region,7s’4 and suggests that VIP is involved in the regulation of intestinal water and chloride secretion via the VIP receptors on the intestinal epithelium. Vasoactive intestinal pepsinogen secretion
polypeptide
regulation
of
VIP is known to inhibit canine acid and pepsinogen secretion in viva” and these effects are accompanied by an increase in somatostatin-like immunoreactivity. However, VIP has also been shown to stimulate pepsinogen secretion from rat gastric glands” and guinea-pig chief ~~11s~~ in vitro. Whether this inconsistency is due to species differences or to the use of in vivo vs in vitro preparations is unclear. VIP also inhibits canine gastrin secretion in vivo at concentrations which could be reached near VIPergic fiber terminals24 and this inhibition is thought to be mediated by somatostatin.6 VIP-containing fibers have been found to innervate the gastric mucosa’4 and we now demonstrate that there is a high concentration of
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VIP receptor binding sites in the mucosa of the cardia, fundus, and antrum of the stomach, suggesting that VIP may have indirect actions on the mucosal epithelium. It remains, however, for future studies to identify the exact cell types in the gastric mucosa that express VIP receptors. Hemodynamic regulation Since it was first isolated from the porcine intestine, VIP has been noted for its role as a vasodilator. It is known to cause vasodilation of peripheral vascular beds and to increase blood flow to these regions.” The presence of VIP-containing nerve fibers innervating arteries has been contirmed’4 and these fibers release VIP in response to physiological stimuli.22 We have now demonstrated the presence of a moderate concentration of VIP receptor binding sites in the smooth muscle of the tunica media of submucosal arterioles (diameter 0.345 mm) which are located in the cardia, fundus, antrum, duodenum, and jejunum. The localization bf VIP binding sites here identifies the smooth muscle of arterioles as a target tissue for VIP, which therefore may play a role in regulating regional gastrointestinal blood flow. Regulation of gastrointestinal motility There are conflicting reports about the myogenic properties of VIP in the gastrointestinal tract. In the guinea-pig small intestine, low concentrations of VIP have been shown to relax the external circular muscle’ while high concentrations have been found to contract the external longitudinal muscle.” In the guinea-pig colon, VIP has been shown to relax the external circular muscle and contract the external longitudinal muscle,4 whereas in the mouse it has been shown to contract external longitudinal muscle in low concentrations and relax it in high concentrations.’ In addition, VIP immunoreactive fibers have been localized to nerve fibers and cell bodies in the myenteric and submucosal ganglia and around smooth muscle bundles in the external circular and longitudinal muscle layers.’ In the present study, we found VIP receptor binding sites in the myenteric plexus of the duodenum, jejunum, ileum, and colon, suggesting that some VIP myogenic actions may be indirect. We have also shown that both the circular and longitudinal muscle comprising the muscularis extema express VIP receptor binding sites throughout the gastrointestinal tract, indicating that VIP may also have a direct spasmogenic effect on these smooth muscles. The presence of binding sites identifies these tissues as probable sites of VIP action and suggests
Fig. 8. Localization of [‘251]VIPreceptor binding sites in the colon. (a) Bright-field photomicrograph of a hematoxylin and eosin (H-E) stained section of human colon, and (b) dark-field photomicrograph made from tritium-sensitive film that overlaid section (a) for 10 days. (c) Control section showing the non-specific binding. Note the high concentrations of receptor binding sites in the luminal region of the mucosa (MC)and moderate concentrations of VIP binding sites in the basal layer of the mucosa (MC). Low VIP receptor binding site concentrations are observed in the external circular muscle (CM) and longitudinal muscle (LM) of the colon. See Fig. 2 for further explanation. Scale bar = 0.8 mm.
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that VIP may play a direct role in regulating gastric and intestinal motility. The involvement of vasoactive intestinal polypeptide human gastrointestinal disease
in
There is evidence suggesting that VIP may be involved in the pathophysiology of certain diseases such as inflammatory bowel disease. For instance, the VIP content of peripheral nerve fibers. has been reported to increase in inflammatory bowel disease, specifically Crohn’s disease.5 In a study of VIP binding site concentration in colon specimens from patients with inflammatory bowel disease, we found that VIP binding site concentrations were signifi-
cantly lower in the mucosa of specimens from ulcerative colitis patients but not in specimens from Crohn’s patients.16’ Precisely what role VIP and VIP receptors may play in the pathophysiology of ulcerative colitis or Crohn’s disease is not yet clear, but knowledge of the cell types that express VIP receptor binding sites in the normal human gastrointestinal tract provides a basis for comparing the receptor binding site distribution in normal vs diseased states. Acknowledgement-This work was supported by the Southern California Arthritis Foundation, the Alfred Sloan Fellowship, the Smith, Kline and Beckman Fellowship, and NINCDS grant NS23970.
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