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Autoradiographic distribution of vasoactive intestinal polypeptide receptors in rabbit and rat small intestine
Peptides. Vol. 9, pp. 23-30. PergamonJournals Ltd., 1988. Printedin the U.S.A.
0196-9781/88$3.00 + .00
Autoradiographic Distribution of Vasoactive Intestinal Polypeptide Receptors in Rabbit and Rat Small Intestine H A S S A N S A Y A D I , * J O H N W. H A R M O N , * T E R R Y W. M O O D Y t A N D L O U I S Y. K O R M A N * t 1
*Departments o f Medicine and Surgery Veterans Administration Medical Center, Washington, DC 20422 tDepartments of Physiology and Biochemistry George Washington University School of Medicine, Washington, DC R e c e i v e d 8 J u n e 1987 SAYADI, H., J. W. HARMON, T. W. MOODY AND L. Y. KORMAN. Autoradiographic distribution of vasoactive intestinal polypeptide receptors in rabbit and rat small intestine. PEPTIDES 9(1) 23-30, 1988.--Vasoactive intestinal peptide (VIP) is found in the enteric nervous system of all layers of the small intestine. In the gastrointestinal tract, VIP receptors coupled to adenylate cyclase are present on epithelial, smooth muscle and possibly mononuclearcells. This study analyzes the distribution of VIP binding using in vitro autoradiographic techniques. VIP binding was present in high density in the mucosal layer of rabbit duodenum, jejunum and ileum. Low VIP binding was noted over the smooth muscle layers or the lymphoid follicles. Similar results were obtained in rat small intestine. The density of VIP binding was greatest in duodenal mucosa but was present in lower density in jejunal and ileal mucosa. Again, low VIP binding was noted in the smooth muscle layers or lymphoid follicles. Thus, autoradiographic maps of small intestine indicate that VIP receptors are found primarily in the small intestinal mucosa. Vasoactive intestinal peptide (VIP) Lymphoid tissue Mucosa
Autoradiography
Small intestine
VASOACTIVE intestinal peptide (VIP) is a widely distributed neurotransmitter in the gastrointestinal tract and is thought to play a significant role in the regulation of fluid and electrolyte transport in the small intestine [23]. Immunoreactive VIP is found in the mucosal, submucosal, and muscular layers of the entire gastrointestinal tract [6]. In these layers VIP is localized to neurons of the lamina propria, submucosal plexus, and myenteric plexus [5,9]. Numerous studies have demonstrated that VIP acts at several sites in the small intestine. The most thoroughly described actions of VIP are on intestinal secretion and smooth muscle contractility [23]. It is therefore not surprising that several authors have demonstrated VIP receptors on isolated small intestinal epithelial cells and isolated gastric smooth muscle cells [2, 3, 7, 14]. Recently, several groups have suggested that VIP acts as a neuromodulator of immune function by acting on T cells from secondary lymphoid tissue [7, 18, 20, 21]. That VIP should alter the function of several target cells in the small intestine is consistent with the distribution of VIP in all layers of the bowel wall.
Binding
Smooth muscle
However, recent studies of neurotransmitter distribution in the gastrointestinal tract suggest that receptor distribution does not always correlate with the concomitant immunohistochemical localization of neurotransmitters [17]. The diversity of small intestinal preparations used to study stimulus-response coupling may not adequately reflect the site of action of an agent in vivo. In an attempt to provide an alternative view of the role of VIP in small intestinal function we used autoradiographic techniques to examine the in situ distribution of VIP receptors. METHOD
Tissue Preparation Male New Zealand white rabbits were anesthetized with Rompun TM (100 mg/ml) and ketamine tm (100 mg/ml) (2:10) at a dose of 1 ml/kg. The abdomen was opened and segments of duodenum, jejunum and ileum removed, flushed with iced (4°C) 0 . ~ NaC1, then oriented and put on powdered dry ice. Male Sprague Dawley rats weighing 200 to 300 g were
1Requests for reprints should be addressed to Dr. Louis Y. Korman, Department of Medicine (151W), V. A. Medical Center, 50 Irving Street NW, Washington, DC 20422.
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SAYADI, HARMON, MOODY AND KORMAN
A
FIG. 1. A: H&E stained 12 p.m cross-section of rabbit duodenum with the mucosa (Mu), submucosa (S), and circular and longitudinal muscle layers (M) appearing on one side. B: Autoradiograph of an adjacent section demonstrating binding of 'e'~I-VIP.High grain density is present over the mucosal layer and the distribution is uniform from the villus tip to the crypt. Grain density over the muscle layers is much less than over the mucosa. C: Autoradiograph of an adjacent section demonstrating non-specific binding of 'zsI-VIP. Grain density over the muscle layers is similar to that seen in B. Bar indicates 0.5 ram. anesthetized using Chlorapent tm (0.4 mg/100 g). The abdomen was opened and the small intestine removed from the ligament of Treitz to the cecum. Segments of the duodenum, jejunum, and ileum were rapidly excised, flushed with iced (4°C) 0.9% NaC1 and put on powdered dry ice. Segments not used immediately were stored at -70°C and processed within 2 weeks. No differences in binding were noted between specimens used immediately and those stored. Frozen sections 12 microns thick were cut, thaw-mounted on coverslips and air-dried at 20°C.
Autoradiography The distribution of VIP receptors in rabbit and rat small intestine was determined by the incubation of tissue sections with ~25I-VIP(2200 Ci/mmol, New England Nuclear, Boston, MA) [26]. Preliminary studies revealed that specific 'z~I-VIP binding, in the presence of 0.5 /zM VIP (Peninsula Labs, Belmont, CA), represented approximately 2/3 of the total Ie'~I-VIP bound and reached equilibrium at approximately 2 hours. Furthermore, specific '2~I-VIP binding was inhibited by 0.5/xM secretin and 0.5/zM PHI by 68% and 84%, respectively. The concentration of VIP which inhibited onehalf of the specific 'zsI-VIP binding was on the order of 10 nM. Because these data were similar to those seen for brain a similar method for producing the autoradiographs was used [26]. Tissue sections were preincubated for 30 minutes at 20°C in 10 mM HEPES/NaOH (pH 7.4), 4% BSA (bovine serum albumin) and 0.1% bacitracin. The preincubation was
followed by incubation at 20°C in 10 mM HEPES/NaOH (pH 7.4), 130 mM NaC1, 4.7 mM KCI, 5 mM MgC12, 5 mM MnCI2, 1 mM EGTA, 1% BSA, 0.1% bacitracin and 45 pM '251VIP at pH 7.4. Non-specific binding was determined by incubating sequential sections in the same buffer but with 0.5 /~M unlabeled VIP. The incubation was terminated after two and a half hours by 2 successive 5 minute washes at 20°C in the same incubation buffer without ligand. The slide mounted sections were immediately air-dried and placed in an x-ray cassette in tight apposition to an LKB ultrofilm (LKB, Gaithersburg, MD). The cassettes were stored in darkness for one week with even tension applied to the sections to assure proper apposition.
Analysis of Autoradiographic Film and Slides After exposure the film was developed in Kodak D19 developer for 5 minutes and fixed in Kodak rapidfix for 5 minutes. Sequential sections were mounted on slides, fixed and stained with hematoxylin and eosin. Stained slides and their corresponding autoradiograms were studied and compared under a light or phase microscope and photographed. In addition, selected autoradiograms were analyzed by computer based densitometry (LOATS image enhancement system, Baltimore, MD). In brief, the autoradiogram was digitized and grain density assigned relative optical density values. These relative optical density values were then converted to a corresponding color scale.
FACING PAGE FIG. 2. A and B: H&E stained 12 /z cross-section of rabbit jejunum and ileum, respectively. The mucosa, submucosa, circular and longitudinal layers are present in each section. C and D: Autoradiographs of adjacent sections of jejunum and ileum demonstrating high grain density over the mucosa with uniform distribution from the villus tip to the crypt and very low grain density over the submucosal and muscle layers. E and F: Autoradiographs of adjacent sections demonstrating non-specific binding of ~2'~I-VIP.Grain density over the muscle layers is similar to that seen in C and D. Bar indicates 0.5 mm.
VIP RECEPTORS IN SMALL INTESTINE
25
A
B
A
F
j~
26
SAYADI, H A R M O N , MOODY A N D K O R M A N
FIG. 3. A: H&E stained cross-section of rabbit ileum cut through lymphoid follicles of a Peyer's patch (P). These follicles occupy the lamina propria extending from the epithelial layer to the submucosa. B: Autoradiograph of an adjacent section demonstrating the absence of silver grains over the lymphoid follicles and the muscular layers. High grain density can be seen over the epithelial layer of the mucosa. C: Autoradiograph of an adjacent section demonstrating non-specific binding of '2~I-VIP. Grain density over the muscle layers is similar to that seen in B. Bar indicates 0.5 mm. RESULTS The distribution of VIP receptor binding in rabbit small intestine was localized almost exclusively to the mucosal layer. Figure 1A is a cross-section of rabbit duodenum and demonstrates the three characteristic layers of small intestine: mucosa, submucosa and muscularis. The submucosa is the narrow space underlying the muscularis mucosae containing Brnnner's glands as well as vascular and neural elements. The duodenal autoradiograph (Fig. 1B) revealed high grain densities and represented binding of '25I-VIP over the mucosal layer. Binding appeared to be uniformly distributed from the villus tip to the crypt. Compared to the mucsoa very low grain densities were noted over the submucosa and muscular layers. In the muscle layers grain densities were almost indistinguishable from that seen in the presence of 0.5 /zM VIP (Fig. 1C). Image enhancement of the autoradiograph in Fig. 1B using computer-assisted densitometry provided a better differentiation since grain densities are represented by a color opitcal density scale. This image enhancement indicated that binding occurred predominantly in the mucosal layer, and was 3 to 4 times greater for the mucosal than muscular layer. Because the distribution of VIP receptors may vary along the length of the small intestine sections of rabbit jejunum and ileum were examined. Figure 2A and B illustrate crosssections obtained of the jejunum and ileum, respectively. The corresponding autoradiographs (Fig. 2C and D) demonstrated high grain densities over the mucosal layer, replicating the pattern in the duodenum. Grain densities were uniformly distributed along the villi and extended from the crypts to the tip. Grain densities over the submucosa and longitudinal and circular muscle layers were only slightly above non-specific binding (Fig. 2E and F). This was particularly clear in the ileal section, where the muscle band was thicker than in the duodenal or jejunal sections (Fig. 2B and D). VIP receptors are postulated to occur on immune cells of the gastrointestinal tract [18,20]. A cross-section of the rabbit ileum through lymph follicles (Peyer's patch) extending
from the lamina propria into the submucosa was examined (Fig. 3A). Its corresponding autoradiograph (Fig. 3B) demonstrated high grain densities over the mucosa but low grain densities almost identical to those of non-specific binding over the lymph follicles (Fig. 3C). Similar results for rat intestinal lymphoid tissue were obtained (data not shown). To determine whether the pattern of 125I-VIP binding in rabbit was species specific similar studies were done in the rat. Figure 4A is a section of rat duodenum with the mucosa, submucosa and circular and longitudinal muscle layers present. The corresponding autoradiograph (Fig. 4B) demonstrated increased grain densities over the mucosal layer extending from the villus tip to the crypt. In addition, there was a distinct band of increased grain density which appeared to be located at the level of the submucosa very close to the region of the crypt base. In contrast, grain density over the muscle layers was not significantly above tissue non-specific binding (Fig. 4C). In the jejunum and ileum (Fig. 5A and B) increased grain density was noted over the mucosa and again with a distinct dense band in the region of the submucosa bordering the base of the crypts. Examination of the smooth muscle layers did not demonstrate any increase in grain density above the tissue non-specific binding. DISCUSSION The autoradiographic distribution of VIP binding in rabbit and rat small intestinal mucosa is consistent with prior studies indicating that VIP receptors were found on intestinal epithelial cells [7, 8, 15, 25]. In rabbit, a single class of high affinity epithelial binding sites are found with a KD of 23 nM and 78,000 binding sites/cell [7]. In the rat, high and low affinity VIP receptors were identified with dissociation constants and number of binding sites per cell of 1.6 nM and 1.4x 105, and 74 nM and 1 × 106, respectively [15]. Binding to these receptors activates cellular adenylate cyclase, increases intracellular cAMP and results in net intestinal secretion [7, 8, 15]. In addition to functional studies several investigators have characterized the molecular weight and structure of VIP receptors from different cell types [14, 16,
VIP RECEPTORS IN SMALL I N T E S T I N E
27
FIG. 4. A: H&E stained cross-section of rat duodenum. The mucosa, submucosa and circular and longitudinal muscle layers are present. B: Autoradiograph of an adjacent section demonstrating high grain density over the mucosal layer and a distinct band of increased grain density at the level of the submucosa bordering the crypts. C: Autoradiograph of an adjacent section demonstrating non-specific binding of ~'~I-VIP. Grain density over the muscular layers is low and only slightly above background grain density. Bar indicates 0.5 mm.
27]. Although these studies provide significant information on receptor affinity and specificity, intracellular events and cellular response, they do not provide specific information regarding the anatomic distribution of receptors. Receptor autoradiography represents a useful tool for describing the in situ pattern of receptor distribution and has been applied extensively to the brain and to a more limited extent to the gut [17, 22, 26]. The basic assumption underlying autoradiographic mapping is that silver grain localization corresponds to binding sites. However, these binding sites do not necessarily represent functional receptors. The present study demonstrates that in rabbit, VIP binding is uniform from the villus tip to the crypt and from the duodenum to the ileum. Presence of this distribution pattern in duodenum, jejunum and ileum combined with prior functional studies supports the hypothesis that VIP receptors are important regulators of intestinal secretion [7,25]. The level of resolution provided by this technique does not permit localization of VIP receptors to different epithelial cell types. In rat a similar pattern of VIP binding was observed. That is, VIP receptors were found over an area corresponding to the villus tip as well as the crypt. In contrast to the rabbit, in all sections of rat small intestine increased VIP receptor binding was noted in a distinct band at the level of the submucosa adjacent to the crypt. The significance of this localization is uncertain but may represent increased receptor density in the crypt epithelial cells and/or receptors in the submucosal neural plexus. In both these areas networks of VIP containing neurons and their fibers have been demonstrated [9,24]. There is also evidence that VIP acts on
cholinergic neurons in the intestinal neural plexus [13]. The small size of the tissue and the limits of this technique did not permit further characterization of the location of the VIP receptors. Comparing the duodenal, jejunal and ileal autoradiographs from the same experiment indicates differences in the relative abundance of VIP receptors; an observation not seen in the rabbit. The duodenum appears to have more VIP receptors in the mucosa than the jejunum or ileum [23]. These results are similar to those obtained from studies of VIP binding to epithelial cells specifically isolated from these regions indicating that more VIP receptors are found in the duodenum than the jejunum or ileum. This also correlates with regional VIP content which is greatest in the duodenum as compared to the ileum and jejunum ([15], unpublished observations). The combination of increased VIP content and VIP binding capacity in the proximal small intestine may correlate with increased proximal secretion. Confirmation of this hypothesis awaits further study. Several investigators have demonstrated the presence of VIP receptors coupled to adenylate cyclase on immune cells [18,20]. As a result, VIP has been postulated to play a role in regulating the function of some immunocytes in the lamina propria and in distinct lymphoid aggregates of the small intestine [19]. The present study shows no evidence of specific VIP binding to immunocytes present in small intestinal lymph follicles (Fig. 3). This study does not exclude the possibility that some subpopulation of the cells found in this tissue have VIP receptors but that their rarity or a low number of receptors per cell precludes detection by autoradiography. In fact, a prior estimate of VIP binding ca-
FOLLOWING PAGE FIG. 5. A and B: H&E stained 12/xm cross-sections of rat jejunum and ileum, respectively. The mucosa, submucosa and circular and longitudinal muscle layers are present. C and D: Autoradiograph corresponding to the jejunal and ileal sections. High grain density is present over the mucosal layer. Mucosal binding in the jejunum and ileum is more scattered and sparse than in the rat duodenal mucosa. In both specimens there is a distinct band of high grain density at the level of the submucosa bordering the crypts. E and F: Autoradiographs of adjacent sections demonstrating non-specific binding of ~2~I-VIP.Grain density over the muscle layers is similar to that seen in C and D. Bar indicates 0.5 mm.
28
SAYADI, HARMON, MOODY AND KORMAN
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VIP R E C E P T O R S IN S M A L L I N T E S T I N E
29
pacity of lymphocytes from mouse Peyer's patches was 490 sites/cell with a KD of approximately 0.2 nM [20]. Finally, the present study failed to demonstrate the presence of significant numbers of VIP receptors on either circular or longitudinal smooth muscle cells in rabbit or rat small intestine. Although some increase in grain density above background was noted, it is difficult to be certain whether these grains represent specific receptors. Clearly the relative density, as determined by microscopic visualization and by image analysis, was significantly less than for the mucosa and only slightly above both the experimental and tissue non-specific background. In contrast to these autoradiographic studies, substantial data exists supporting the direct action of VIP on smooth muscle and the presence of VIP receptors on isolated smooth muscle cells [3, 10, 11]. These data derive from two types of experiments. First, isolated smooth muscle cells from the guinea pig and human gastric antrum and rat colon were used to demonstrate that VIP inhibited smooth muscle contraction with a half-maximal relaxation dose of approximately 10 nM [2,3]. In addition, VIP augmented IBMX-stimulated cAMP in some of these cells [3]. Second, isolated smooth muscle strips were used to show that VIP antiserum blocked both VIP- and electrically-stimulated smooth muscle relaxation in guinea pig fundic circular muscle and taenia coli [10-12]. Again, the halfmaximal relaxation dose was on the order of 10 nM. The inability to demonstrate VIP receptors on smooth muscle autoradiographically was surprising given these functional data and the immunohistochemical studies indicating that VIP is found in the myenteric plexus of the rat and rabbit. Similar discrepancies between function, immunohistochemical localization and receptor distribution were found for opioid receptors in the rat and guinea pig gastrointestinal tract [17]. In these experiments opioid receptors were found in the circular muscle of the fundus, corpus and antrum but not in the muscle layers of the duodenum and ileum even
though opioids affect the motility of these segments. Several explanations for the current observations are possible. First, VIP receptors are present on small intestinal smooth muscle, but the affinity and/or number of receptors per cell is inadequate to detect binding but sufficient to stimulate a response. Preliminary VIP binding data [1] indicates the presence of a high affinity receptor with inhibition of binding occurring at a concentration of 0.15 nM VIP. Second, it is possible that diffusion of exogenous VIP between layers of smooth muscle cells is too slow to permit adequate binding reaction kinetics. Although VIP receptor binding could not be demonstrated, there is autoradiographic binding of substance P ([4], unpublished observation) to small intestinal smooth muscle. This suggests that the latter explanation is unlikely. Third, high amounts of slowly dissociating, endogenous VIP may mask receptors. Finally, VIP may not act directly on small intestinal smooth muscle thereby explaining either the absence or paucity of VIP receptors in this layer. Previous studies documenting the presence of VIP receptors on smooth muscle were done on isolated stomach and colonic muscle [2, 3, 10-12]. No similar functional or binding studies have been done using isolated small intestinal smooth muscle cells. Given the rabbit and rat epithelial binding [7,15] data this autoradiographic technique detects approximately 150 and 4,500 VIP binding sites per cell in the rabbit and rat, respectively. Although these figures are extrapolations based on previously published data, they do suggest that this technique is capable of detecting small numbers of high affinity receptors. Thus, the absence of VIP binding to smooth muscle suggests a paucity or absence of smooth muscle and T-lymphocyte VIP receptors. ACKNOWLEDGEMENTS The study was supported by funds from the Veterans Administration Medical Research Service. We thank Dr. William Goldberg and Dr. Jerry Bernstein for their support and advice.
REFERENCES 1. Bitar, K. N. and R. T. Jensen. Binding of 125-VIP to isolated gastric smooth muscle cells. Gastroenterology 84: 1107, 1983. 2. Bitar, K. N. and G. M. Makhlouf. Relaxation of isolated gastric smooth muscle cells by vasoactive intestinal peptide. Science 216: 531-533, 1982. 3. Bitar, K. N., B. Saffouri and G. M. Makhlouf. Cholinergic and peptidergic receptors on isolated human antral smooth muscle cells. Gastroenterology 82: 832-837, 1982. 4. Butcher, E., S. H. Buck, W. Lovenberg and T. L. O'Donohue. Characterization and autoradiographic localization of multiple tachykinin binding sites in gastrointestinal tract and bladder. J Pharmacol Exp Ther 236: 819-831, 1986. 5. Costa, M., J. B. Furness, R. Buffa and S. I. Said. Distribution of enteric nerve cell bodies and axons showing immunoreactivity for vasoactive intestinal peptide in guinea-pig intestine. Neuroscience 5: 587-596, 1980. 6. Costa, M., J. B. Furness and I. J. Llewellyn-Smith. Histochemistry of the enteric nervous system. In: Physiology o f the Gastrointestinal Traet, second edition, edited by L. R. Johnson. New York: Raven Press, 1987, pp. 1-40. 7. Dharmsathaphorn, K., V. Harms, D. J. Yamashiro, R. J. Hughes, H. J. Binder and E. M. Wright. Preferential binding of vasoactive intestinal polypeptide to basolateral membrane of rat and rabbit enterocytes. J Clin Invest 71: 21-35, 1983. 8. Donowitz, M. and M. J. Welsh. Regulation of mammalian small intestinal electrolyte secretion. In: Physiology o f the Gastrointestinal Tract. second edition, edited by L. R. Johnson. New York: Raven Press, 1987, pp. 1351-1388.
9. Fehrer, E. and C. Leranth. Light and electron microscopic immunocytochemical localization of vasoactive intestinal polypeptide VIP-like activity in rat small intestine. Neuroscience 10: 97-106, 1983. 10. Grider, J. R., M. B. Cable, K. N. Bitar, S. I. Said and G. M. Makhlouf. Vasoactive intestinal peptide, relaxant neurotransmitter in tenia coli of the guinea pig. Gastroenterology 89: 36-42, 1985. l l. Grider, J. R., M. B. Cable, S. I. Said and G. M. Makhlouf. Vasoactive intestinal peptide as a neural mediator of gastric relaxation. Am J Physiol 248: G73-G78, 1985. 12. Grider, J. R. and G. M. Makhlouf. Colonic peristaltic reflex: identification of vasoactive intestinal peptide as mediator of descending relaxation. Am J Physiol 251: G40-G45, 1986. 13. Kusunoki, M., T. Hsueh, K. Taniyama, and C. Tanaka. Vasoactive intestinal polypeptide provokes acetylcholine release from the myenteric plexus. Am J Physiol 14: G51-G55, 1986. 14. Laburthe, M., B. Breant and C. Rouyer-Fessard. Molecular identification of receptors for vasoactive intestinal peptide in rat intestinal epithelium by covalent cross-linking. Eur .I Biochem 139: 181-187, 1984. 15. Laburthe, M., J. Prieto, B. Amiranoff, C. Dupont, J. Broyart, D. Hui Bon Hoa, Y. Broer and G. Rosselin. VIP receptors in intestinal epithelial cells: distribution throughout the intestinal tract. In: Hormone Receptors in Digestion and Nutrition, edited by G. Rosselin, P. Fromageot and S. Bonfils. Amsterdam: Elsevier/North-Holland Biomedical Press, 1979, pp. 241-254.
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16. Nguyen, T. D., J. A. Williams and G. M. Gray. Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein. Biochemistry 25: 361-368, 1986. 17. Nishimura, E., A. M. J. Buchan and C. H. S. Mclntosh. Autoradiographic localization of mu and delta type opioid receptors in the gastrointestinal tract of the rat and guinea pig. Gastroenterology 91: 1084-1094, 1986. 18. O'Dorisio, M. S., N. S. Hermina, T. M. O'Dorisio and S. P. Balcerzak. Vasoactive intestinal polypeptide modulation of lymphocyte adenylate cyclase. J lmmunol 127:2551-2557, 1981. 19. O'Dorisio, M. S., C. L. Wood and T. M. O'Dorisio. Vasoactive intestinal peptide and neuropeptide modulation of the immune response. J lmmunol 135: 792s-796s, 1985. 20. Ottaway, C. A. and G. R. Greenberg. Interaction of vasoactive intestinal peptide with mouse lymphocytes: specific binding and the modulation of mitogen responses. J lmrnunol 132: 417-423, 1984. 21. Prieto, J. C., M. Laburthe and G. Rosselin. Interaction of vasoactive intestinal peptide with isolated intestinal epithelial cells from rat. 1. Characterization, quantitative aspects and structural requirements of binding sites. Eur J Biochem 96: 22%237, 1979.
SAYADI, HARMON, MOODY AND KORMAN 22. Robinson, P. H., T. H. Moran, M. Goldrich and P. R. McHugh. Development of cholecystokinin binding sites in rat upper gastrointestinal tract. A m J Physiol 252: G52%G534, 1987. 23. Said, S. I. Vasoactive Intestinal Peptide. In: Advances in Peptide Hormone Research Series. New York: Raven Press, 1982. 24. Schultzberg, M., T. Hokfelt, G. Nillsson, L. Terenius, J. F. Rehfeld, M. Brown, R. Elde, M. Goldstein and S. Said. Distribution of peptide- and catecholamine-containing neurons in the gastro-intestinal tract of rat and guinea-pig: immunohistochemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkepbalins, somatostatin, gastrin/cholecystokinin, neurotensin, and dopamine beta-hydroxylase. Neuroscience 5: 68%744, 1980. 25. Schwartz, C. J., D. V. Kimberg, H. E. Sheerin, M. Field and S. I. Said. Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa. J Clin Invest 54: 536-554, 1974. 26. Shaffer, M. M. and T. W. Moody. Autoradiographic visualization of CNS receptors for vasoactive intestinal peptide. Peptides 7: 283-8, 1986. 27. Wood, C. L. and M. S. O'Dorisio. Covalent cross-linking of vasoactive intestinal peptide to its receptors on intact human lymphoblasts. J Biol Chem 260: 1243-1247, 1985.
0196-9781/88$3.00 + .00
Autoradiographic Distribution of Vasoactive Intestinal Polypeptide Receptors in Rabbit and Rat Small Intestine H A S S A N S A Y A D I , * J O H N W. H A R M O N , * T E R R Y W. M O O D Y t A N D L O U I S Y. K O R M A N * t 1
*Departments o f Medicine and Surgery Veterans Administration Medical Center, Washington, DC 20422 tDepartments of Physiology and Biochemistry George Washington University School of Medicine, Washington, DC R e c e i v e d 8 J u n e 1987 SAYADI, H., J. W. HARMON, T. W. MOODY AND L. Y. KORMAN. Autoradiographic distribution of vasoactive intestinal polypeptide receptors in rabbit and rat small intestine. PEPTIDES 9(1) 23-30, 1988.--Vasoactive intestinal peptide (VIP) is found in the enteric nervous system of all layers of the small intestine. In the gastrointestinal tract, VIP receptors coupled to adenylate cyclase are present on epithelial, smooth muscle and possibly mononuclearcells. This study analyzes the distribution of VIP binding using in vitro autoradiographic techniques. VIP binding was present in high density in the mucosal layer of rabbit duodenum, jejunum and ileum. Low VIP binding was noted over the smooth muscle layers or the lymphoid follicles. Similar results were obtained in rat small intestine. The density of VIP binding was greatest in duodenal mucosa but was present in lower density in jejunal and ileal mucosa. Again, low VIP binding was noted in the smooth muscle layers or lymphoid follicles. Thus, autoradiographic maps of small intestine indicate that VIP receptors are found primarily in the small intestinal mucosa. Vasoactive intestinal peptide (VIP) Lymphoid tissue Mucosa
Autoradiography
Small intestine
VASOACTIVE intestinal peptide (VIP) is a widely distributed neurotransmitter in the gastrointestinal tract and is thought to play a significant role in the regulation of fluid and electrolyte transport in the small intestine [23]. Immunoreactive VIP is found in the mucosal, submucosal, and muscular layers of the entire gastrointestinal tract [6]. In these layers VIP is localized to neurons of the lamina propria, submucosal plexus, and myenteric plexus [5,9]. Numerous studies have demonstrated that VIP acts at several sites in the small intestine. The most thoroughly described actions of VIP are on intestinal secretion and smooth muscle contractility [23]. It is therefore not surprising that several authors have demonstrated VIP receptors on isolated small intestinal epithelial cells and isolated gastric smooth muscle cells [2, 3, 7, 14]. Recently, several groups have suggested that VIP acts as a neuromodulator of immune function by acting on T cells from secondary lymphoid tissue [7, 18, 20, 21]. That VIP should alter the function of several target cells in the small intestine is consistent with the distribution of VIP in all layers of the bowel wall.
Binding
Smooth muscle
However, recent studies of neurotransmitter distribution in the gastrointestinal tract suggest that receptor distribution does not always correlate with the concomitant immunohistochemical localization of neurotransmitters [17]. The diversity of small intestinal preparations used to study stimulus-response coupling may not adequately reflect the site of action of an agent in vivo. In an attempt to provide an alternative view of the role of VIP in small intestinal function we used autoradiographic techniques to examine the in situ distribution of VIP receptors. METHOD
Tissue Preparation Male New Zealand white rabbits were anesthetized with Rompun TM (100 mg/ml) and ketamine tm (100 mg/ml) (2:10) at a dose of 1 ml/kg. The abdomen was opened and segments of duodenum, jejunum and ileum removed, flushed with iced (4°C) 0 . ~ NaC1, then oriented and put on powdered dry ice. Male Sprague Dawley rats weighing 200 to 300 g were
1Requests for reprints should be addressed to Dr. Louis Y. Korman, Department of Medicine (151W), V. A. Medical Center, 50 Irving Street NW, Washington, DC 20422.
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SAYADI, HARMON, MOODY AND KORMAN
A
FIG. 1. A: H&E stained 12 p.m cross-section of rabbit duodenum with the mucosa (Mu), submucosa (S), and circular and longitudinal muscle layers (M) appearing on one side. B: Autoradiograph of an adjacent section demonstrating binding of 'e'~I-VIP.High grain density is present over the mucosal layer and the distribution is uniform from the villus tip to the crypt. Grain density over the muscle layers is much less than over the mucosa. C: Autoradiograph of an adjacent section demonstrating non-specific binding of 'zsI-VIP. Grain density over the muscle layers is similar to that seen in B. Bar indicates 0.5 ram. anesthetized using Chlorapent tm (0.4 mg/100 g). The abdomen was opened and the small intestine removed from the ligament of Treitz to the cecum. Segments of the duodenum, jejunum, and ileum were rapidly excised, flushed with iced (4°C) 0.9% NaC1 and put on powdered dry ice. Segments not used immediately were stored at -70°C and processed within 2 weeks. No differences in binding were noted between specimens used immediately and those stored. Frozen sections 12 microns thick were cut, thaw-mounted on coverslips and air-dried at 20°C.
Autoradiography The distribution of VIP receptors in rabbit and rat small intestine was determined by the incubation of tissue sections with ~25I-VIP(2200 Ci/mmol, New England Nuclear, Boston, MA) [26]. Preliminary studies revealed that specific 'z~I-VIP binding, in the presence of 0.5 /zM VIP (Peninsula Labs, Belmont, CA), represented approximately 2/3 of the total Ie'~I-VIP bound and reached equilibrium at approximately 2 hours. Furthermore, specific '2~I-VIP binding was inhibited by 0.5/xM secretin and 0.5/zM PHI by 68% and 84%, respectively. The concentration of VIP which inhibited onehalf of the specific 'zsI-VIP binding was on the order of 10 nM. Because these data were similar to those seen for brain a similar method for producing the autoradiographs was used [26]. Tissue sections were preincubated for 30 minutes at 20°C in 10 mM HEPES/NaOH (pH 7.4), 4% BSA (bovine serum albumin) and 0.1% bacitracin. The preincubation was
followed by incubation at 20°C in 10 mM HEPES/NaOH (pH 7.4), 130 mM NaC1, 4.7 mM KCI, 5 mM MgC12, 5 mM MnCI2, 1 mM EGTA, 1% BSA, 0.1% bacitracin and 45 pM '251VIP at pH 7.4. Non-specific binding was determined by incubating sequential sections in the same buffer but with 0.5 /~M unlabeled VIP. The incubation was terminated after two and a half hours by 2 successive 5 minute washes at 20°C in the same incubation buffer without ligand. The slide mounted sections were immediately air-dried and placed in an x-ray cassette in tight apposition to an LKB ultrofilm (LKB, Gaithersburg, MD). The cassettes were stored in darkness for one week with even tension applied to the sections to assure proper apposition.
Analysis of Autoradiographic Film and Slides After exposure the film was developed in Kodak D19 developer for 5 minutes and fixed in Kodak rapidfix for 5 minutes. Sequential sections were mounted on slides, fixed and stained with hematoxylin and eosin. Stained slides and their corresponding autoradiograms were studied and compared under a light or phase microscope and photographed. In addition, selected autoradiograms were analyzed by computer based densitometry (LOATS image enhancement system, Baltimore, MD). In brief, the autoradiogram was digitized and grain density assigned relative optical density values. These relative optical density values were then converted to a corresponding color scale.
FACING PAGE FIG. 2. A and B: H&E stained 12 /z cross-section of rabbit jejunum and ileum, respectively. The mucosa, submucosa, circular and longitudinal layers are present in each section. C and D: Autoradiographs of adjacent sections of jejunum and ileum demonstrating high grain density over the mucosa with uniform distribution from the villus tip to the crypt and very low grain density over the submucosal and muscle layers. E and F: Autoradiographs of adjacent sections demonstrating non-specific binding of ~2'~I-VIP.Grain density over the muscle layers is similar to that seen in C and D. Bar indicates 0.5 mm.
VIP RECEPTORS IN SMALL INTESTINE
25
A
B
A
F
j~
26
SAYADI, H A R M O N , MOODY A N D K O R M A N
FIG. 3. A: H&E stained cross-section of rabbit ileum cut through lymphoid follicles of a Peyer's patch (P). These follicles occupy the lamina propria extending from the epithelial layer to the submucosa. B: Autoradiograph of an adjacent section demonstrating the absence of silver grains over the lymphoid follicles and the muscular layers. High grain density can be seen over the epithelial layer of the mucosa. C: Autoradiograph of an adjacent section demonstrating non-specific binding of '2~I-VIP. Grain density over the muscle layers is similar to that seen in B. Bar indicates 0.5 mm. RESULTS The distribution of VIP receptor binding in rabbit small intestine was localized almost exclusively to the mucosal layer. Figure 1A is a cross-section of rabbit duodenum and demonstrates the three characteristic layers of small intestine: mucosa, submucosa and muscularis. The submucosa is the narrow space underlying the muscularis mucosae containing Brnnner's glands as well as vascular and neural elements. The duodenal autoradiograph (Fig. 1B) revealed high grain densities and represented binding of '25I-VIP over the mucosal layer. Binding appeared to be uniformly distributed from the villus tip to the crypt. Compared to the mucsoa very low grain densities were noted over the submucosa and muscular layers. In the muscle layers grain densities were almost indistinguishable from that seen in the presence of 0.5 /zM VIP (Fig. 1C). Image enhancement of the autoradiograph in Fig. 1B using computer-assisted densitometry provided a better differentiation since grain densities are represented by a color opitcal density scale. This image enhancement indicated that binding occurred predominantly in the mucosal layer, and was 3 to 4 times greater for the mucosal than muscular layer. Because the distribution of VIP receptors may vary along the length of the small intestine sections of rabbit jejunum and ileum were examined. Figure 2A and B illustrate crosssections obtained of the jejunum and ileum, respectively. The corresponding autoradiographs (Fig. 2C and D) demonstrated high grain densities over the mucosal layer, replicating the pattern in the duodenum. Grain densities were uniformly distributed along the villi and extended from the crypts to the tip. Grain densities over the submucosa and longitudinal and circular muscle layers were only slightly above non-specific binding (Fig. 2E and F). This was particularly clear in the ileal section, where the muscle band was thicker than in the duodenal or jejunal sections (Fig. 2B and D). VIP receptors are postulated to occur on immune cells of the gastrointestinal tract [18,20]. A cross-section of the rabbit ileum through lymph follicles (Peyer's patch) extending
from the lamina propria into the submucosa was examined (Fig. 3A). Its corresponding autoradiograph (Fig. 3B) demonstrated high grain densities over the mucosa but low grain densities almost identical to those of non-specific binding over the lymph follicles (Fig. 3C). Similar results for rat intestinal lymphoid tissue were obtained (data not shown). To determine whether the pattern of 125I-VIP binding in rabbit was species specific similar studies were done in the rat. Figure 4A is a section of rat duodenum with the mucosa, submucosa and circular and longitudinal muscle layers present. The corresponding autoradiograph (Fig. 4B) demonstrated increased grain densities over the mucosal layer extending from the villus tip to the crypt. In addition, there was a distinct band of increased grain density which appeared to be located at the level of the submucosa very close to the region of the crypt base. In contrast, grain density over the muscle layers was not significantly above tissue non-specific binding (Fig. 4C). In the jejunum and ileum (Fig. 5A and B) increased grain density was noted over the mucosa and again with a distinct dense band in the region of the submucosa bordering the base of the crypts. Examination of the smooth muscle layers did not demonstrate any increase in grain density above the tissue non-specific binding. DISCUSSION The autoradiographic distribution of VIP binding in rabbit and rat small intestinal mucosa is consistent with prior studies indicating that VIP receptors were found on intestinal epithelial cells [7, 8, 15, 25]. In rabbit, a single class of high affinity epithelial binding sites are found with a KD of 23 nM and 78,000 binding sites/cell [7]. In the rat, high and low affinity VIP receptors were identified with dissociation constants and number of binding sites per cell of 1.6 nM and 1.4x 105, and 74 nM and 1 × 106, respectively [15]. Binding to these receptors activates cellular adenylate cyclase, increases intracellular cAMP and results in net intestinal secretion [7, 8, 15]. In addition to functional studies several investigators have characterized the molecular weight and structure of VIP receptors from different cell types [14, 16,
VIP RECEPTORS IN SMALL I N T E S T I N E
27
FIG. 4. A: H&E stained cross-section of rat duodenum. The mucosa, submucosa and circular and longitudinal muscle layers are present. B: Autoradiograph of an adjacent section demonstrating high grain density over the mucosal layer and a distinct band of increased grain density at the level of the submucosa bordering the crypts. C: Autoradiograph of an adjacent section demonstrating non-specific binding of ~'~I-VIP. Grain density over the muscular layers is low and only slightly above background grain density. Bar indicates 0.5 mm.
27]. Although these studies provide significant information on receptor affinity and specificity, intracellular events and cellular response, they do not provide specific information regarding the anatomic distribution of receptors. Receptor autoradiography represents a useful tool for describing the in situ pattern of receptor distribution and has been applied extensively to the brain and to a more limited extent to the gut [17, 22, 26]. The basic assumption underlying autoradiographic mapping is that silver grain localization corresponds to binding sites. However, these binding sites do not necessarily represent functional receptors. The present study demonstrates that in rabbit, VIP binding is uniform from the villus tip to the crypt and from the duodenum to the ileum. Presence of this distribution pattern in duodenum, jejunum and ileum combined with prior functional studies supports the hypothesis that VIP receptors are important regulators of intestinal secretion [7,25]. The level of resolution provided by this technique does not permit localization of VIP receptors to different epithelial cell types. In rat a similar pattern of VIP binding was observed. That is, VIP receptors were found over an area corresponding to the villus tip as well as the crypt. In contrast to the rabbit, in all sections of rat small intestine increased VIP receptor binding was noted in a distinct band at the level of the submucosa adjacent to the crypt. The significance of this localization is uncertain but may represent increased receptor density in the crypt epithelial cells and/or receptors in the submucosal neural plexus. In both these areas networks of VIP containing neurons and their fibers have been demonstrated [9,24]. There is also evidence that VIP acts on
cholinergic neurons in the intestinal neural plexus [13]. The small size of the tissue and the limits of this technique did not permit further characterization of the location of the VIP receptors. Comparing the duodenal, jejunal and ileal autoradiographs from the same experiment indicates differences in the relative abundance of VIP receptors; an observation not seen in the rabbit. The duodenum appears to have more VIP receptors in the mucosa than the jejunum or ileum [23]. These results are similar to those obtained from studies of VIP binding to epithelial cells specifically isolated from these regions indicating that more VIP receptors are found in the duodenum than the jejunum or ileum. This also correlates with regional VIP content which is greatest in the duodenum as compared to the ileum and jejunum ([15], unpublished observations). The combination of increased VIP content and VIP binding capacity in the proximal small intestine may correlate with increased proximal secretion. Confirmation of this hypothesis awaits further study. Several investigators have demonstrated the presence of VIP receptors coupled to adenylate cyclase on immune cells [18,20]. As a result, VIP has been postulated to play a role in regulating the function of some immunocytes in the lamina propria and in distinct lymphoid aggregates of the small intestine [19]. The present study shows no evidence of specific VIP binding to immunocytes present in small intestinal lymph follicles (Fig. 3). This study does not exclude the possibility that some subpopulation of the cells found in this tissue have VIP receptors but that their rarity or a low number of receptors per cell precludes detection by autoradiography. In fact, a prior estimate of VIP binding ca-
FOLLOWING PAGE FIG. 5. A and B: H&E stained 12/xm cross-sections of rat jejunum and ileum, respectively. The mucosa, submucosa and circular and longitudinal muscle layers are present. C and D: Autoradiograph corresponding to the jejunal and ileal sections. High grain density is present over the mucosal layer. Mucosal binding in the jejunum and ileum is more scattered and sparse than in the rat duodenal mucosa. In both specimens there is a distinct band of high grain density at the level of the submucosa bordering the crypts. E and F: Autoradiographs of adjacent sections demonstrating non-specific binding of ~2~I-VIP.Grain density over the muscle layers is similar to that seen in C and D. Bar indicates 0.5 mm.
28
SAYADI, HARMON, MOODY AND KORMAN
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• i ~'
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VIP R E C E P T O R S IN S M A L L I N T E S T I N E
29
pacity of lymphocytes from mouse Peyer's patches was 490 sites/cell with a KD of approximately 0.2 nM [20]. Finally, the present study failed to demonstrate the presence of significant numbers of VIP receptors on either circular or longitudinal smooth muscle cells in rabbit or rat small intestine. Although some increase in grain density above background was noted, it is difficult to be certain whether these grains represent specific receptors. Clearly the relative density, as determined by microscopic visualization and by image analysis, was significantly less than for the mucosa and only slightly above both the experimental and tissue non-specific background. In contrast to these autoradiographic studies, substantial data exists supporting the direct action of VIP on smooth muscle and the presence of VIP receptors on isolated smooth muscle cells [3, 10, 11]. These data derive from two types of experiments. First, isolated smooth muscle cells from the guinea pig and human gastric antrum and rat colon were used to demonstrate that VIP inhibited smooth muscle contraction with a half-maximal relaxation dose of approximately 10 nM [2,3]. In addition, VIP augmented IBMX-stimulated cAMP in some of these cells [3]. Second, isolated smooth muscle strips were used to show that VIP antiserum blocked both VIP- and electrically-stimulated smooth muscle relaxation in guinea pig fundic circular muscle and taenia coli [10-12]. Again, the halfmaximal relaxation dose was on the order of 10 nM. The inability to demonstrate VIP receptors on smooth muscle autoradiographically was surprising given these functional data and the immunohistochemical studies indicating that VIP is found in the myenteric plexus of the rat and rabbit. Similar discrepancies between function, immunohistochemical localization and receptor distribution were found for opioid receptors in the rat and guinea pig gastrointestinal tract [17]. In these experiments opioid receptors were found in the circular muscle of the fundus, corpus and antrum but not in the muscle layers of the duodenum and ileum even
though opioids affect the motility of these segments. Several explanations for the current observations are possible. First, VIP receptors are present on small intestinal smooth muscle, but the affinity and/or number of receptors per cell is inadequate to detect binding but sufficient to stimulate a response. Preliminary VIP binding data [1] indicates the presence of a high affinity receptor with inhibition of binding occurring at a concentration of 0.15 nM VIP. Second, it is possible that diffusion of exogenous VIP between layers of smooth muscle cells is too slow to permit adequate binding reaction kinetics. Although VIP receptor binding could not be demonstrated, there is autoradiographic binding of substance P ([4], unpublished observation) to small intestinal smooth muscle. This suggests that the latter explanation is unlikely. Third, high amounts of slowly dissociating, endogenous VIP may mask receptors. Finally, VIP may not act directly on small intestinal smooth muscle thereby explaining either the absence or paucity of VIP receptors in this layer. Previous studies documenting the presence of VIP receptors on smooth muscle were done on isolated stomach and colonic muscle [2, 3, 10-12]. No similar functional or binding studies have been done using isolated small intestinal smooth muscle cells. Given the rabbit and rat epithelial binding [7,15] data this autoradiographic technique detects approximately 150 and 4,500 VIP binding sites per cell in the rabbit and rat, respectively. Although these figures are extrapolations based on previously published data, they do suggest that this technique is capable of detecting small numbers of high affinity receptors. Thus, the absence of VIP binding to smooth muscle suggests a paucity or absence of smooth muscle and T-lymphocyte VIP receptors. ACKNOWLEDGEMENTS The study was supported by funds from the Veterans Administration Medical Research Service. We thank Dr. William Goldberg and Dr. Jerry Bernstein for their support and advice.
REFERENCES 1. Bitar, K. N. and R. T. Jensen. Binding of 125-VIP to isolated gastric smooth muscle cells. Gastroenterology 84: 1107, 1983. 2. Bitar, K. N. and G. M. Makhlouf. Relaxation of isolated gastric smooth muscle cells by vasoactive intestinal peptide. Science 216: 531-533, 1982. 3. Bitar, K. N., B. Saffouri and G. M. Makhlouf. Cholinergic and peptidergic receptors on isolated human antral smooth muscle cells. Gastroenterology 82: 832-837, 1982. 4. Butcher, E., S. H. Buck, W. Lovenberg and T. L. O'Donohue. Characterization and autoradiographic localization of multiple tachykinin binding sites in gastrointestinal tract and bladder. J Pharmacol Exp Ther 236: 819-831, 1986. 5. Costa, M., J. B. Furness, R. Buffa and S. I. Said. Distribution of enteric nerve cell bodies and axons showing immunoreactivity for vasoactive intestinal peptide in guinea-pig intestine. Neuroscience 5: 587-596, 1980. 6. Costa, M., J. B. Furness and I. J. Llewellyn-Smith. Histochemistry of the enteric nervous system. In: Physiology o f the Gastrointestinal Traet, second edition, edited by L. R. Johnson. New York: Raven Press, 1987, pp. 1-40. 7. Dharmsathaphorn, K., V. Harms, D. J. Yamashiro, R. J. Hughes, H. J. Binder and E. M. Wright. Preferential binding of vasoactive intestinal polypeptide to basolateral membrane of rat and rabbit enterocytes. J Clin Invest 71: 21-35, 1983. 8. Donowitz, M. and M. J. Welsh. Regulation of mammalian small intestinal electrolyte secretion. In: Physiology o f the Gastrointestinal Tract. second edition, edited by L. R. Johnson. New York: Raven Press, 1987, pp. 1351-1388.
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16. Nguyen, T. D., J. A. Williams and G. M. Gray. Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein. Biochemistry 25: 361-368, 1986. 17. Nishimura, E., A. M. J. Buchan and C. H. S. Mclntosh. Autoradiographic localization of mu and delta type opioid receptors in the gastrointestinal tract of the rat and guinea pig. Gastroenterology 91: 1084-1094, 1986. 18. O'Dorisio, M. S., N. S. Hermina, T. M. O'Dorisio and S. P. Balcerzak. Vasoactive intestinal polypeptide modulation of lymphocyte adenylate cyclase. J lmmunol 127:2551-2557, 1981. 19. O'Dorisio, M. S., C. L. Wood and T. M. O'Dorisio. Vasoactive intestinal peptide and neuropeptide modulation of the immune response. J lmmunol 135: 792s-796s, 1985. 20. Ottaway, C. A. and G. R. Greenberg. Interaction of vasoactive intestinal peptide with mouse lymphocytes: specific binding and the modulation of mitogen responses. J lmrnunol 132: 417-423, 1984. 21. Prieto, J. C., M. Laburthe and G. Rosselin. Interaction of vasoactive intestinal peptide with isolated intestinal epithelial cells from rat. 1. Characterization, quantitative aspects and structural requirements of binding sites. Eur J Biochem 96: 22%237, 1979.
SAYADI, HARMON, MOODY AND KORMAN 22. Robinson, P. H., T. H. Moran, M. Goldrich and P. R. McHugh. Development of cholecystokinin binding sites in rat upper gastrointestinal tract. A m J Physiol 252: G52%G534, 1987. 23. Said, S. I. Vasoactive Intestinal Peptide. In: Advances in Peptide Hormone Research Series. New York: Raven Press, 1982. 24. Schultzberg, M., T. Hokfelt, G. Nillsson, L. Terenius, J. F. Rehfeld, M. Brown, R. Elde, M. Goldstein and S. Said. Distribution of peptide- and catecholamine-containing neurons in the gastro-intestinal tract of rat and guinea-pig: immunohistochemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkepbalins, somatostatin, gastrin/cholecystokinin, neurotensin, and dopamine beta-hydroxylase. Neuroscience 5: 68%744, 1980. 25. Schwartz, C. J., D. V. Kimberg, H. E. Sheerin, M. Field and S. I. Said. Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa. J Clin Invest 54: 536-554, 1974. 26. Shaffer, M. M. and T. W. Moody. Autoradiographic visualization of CNS receptors for vasoactive intestinal peptide. Peptides 7: 283-8, 1986. 27. Wood, C. L. and M. S. O'Dorisio. Covalent cross-linking of vasoactive intestinal peptide to its receptors on intact human lymphoblasts. J Biol Chem 260: 1243-1247, 1985.