Formylpeptide Chemoattractant Binding Sites in Ileum

April 14, 2012 

After we developed a method for detecting formylpeptide chemotaxis receptors using autoradiography, we adapted this method to examine these binding sites or receptors in gut tissue.  We exposed freshly harvested segments of guinea pig ileum to iodinated formylhexapeptide, fixed and processed the tissue histologically, and then did autoradiography to localize the radiolabeled peptide in the tissue sections.

Formylpeptide chemoattractants are known to be capable of eliciting contraction of smooth muscle in guinea pig ileum but the cellular target of interaction between peptide and ileum is unknown.  This suggests that receptors specific for formylpeptides may be found on cells in the ileum and may be responsible for ileal smooth muscle contraction.  Formylpeptides may bind directly to ileal smooth muscle and thereby elicit its contraction.  Alternatively, they may act on smooth muscle indirectly by first binding to and stimulating enteric nerves or activating inflammatory cells (macrophages, polymorphonuclear leukocytes, lymphocytes).  Secondary to this, soluble mediators such as neurotransmitters (eg., acetylcholine, substance P) or arachidonate metabolites may be released resulting in smooth muscle contraction.

This study was performed to localize the sites of formylpeptide binding in guinea pig ileum.  Using iodinated formylhexapeptide in conjunction with light microscope autoradiography of sectioned ileum, it appears that formylpeptide binds specifically and in relatively large amounts directly to ileal smooth muscle (muscularis externa and muscularis mucosa).  However, very little receptor-mediated binding to enteric nerve plexuses, lamina propria, infiltrating leukocytes, or ileal epithelium was observed.  

Since formylpeptides are produced in prodigious quantities by bacteria, the release of these peptides in or around the gut may trigger increased smooth muscle contractility.  This may promote both normal gut motility and the augmented motility associated with enteric infections.

 Below is the full text of this paper and a link to the paper in PDF format.

 Formylpeptide Chemoattractant Binding in Ileum

Localization of Formylpeptide Chemoattractant Binding Sites

in Guinea Pig Ileum

by

Robert J.  Walter*

Department of Anatomy

University of Illinois at Chicago

Chicago, IL 60612

Wayne A.  Marasco

Department of Pathology

University of Michigan Medical School

Ann Arbor, MI

Running Head: Formylpeptide Binding in Guinea Pig Ileum

Keywords - formylpeptide, chemoattractant, chemotaxis, smooth muscle, ileum, contractility, receptors, bacterial peptides.

 * To whom reprint requests should be addressed:

Robert J.  Walter, Ph.D.

Department of Anatomy m/c 512

University of Illinois at Chicago

P.O.  Box 6998

Chicago, IL 60680

312/996-9464

ABSTRACT

Formylpeptide chemoattractants are known to be capable of eliciting contraction of smooth muscle in guinea pig ileum but the cellular target of interaction between peptide and ileum is unknown.  To directly ascertain sites for binding and uptake of formylpeptide, we have exposed segments of guinea pig ileum to N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-I¹²⁵-tyrosyl-lysine (10 nM), at either 4ºC or 37ºC.  After this initial incubation, the tissue was rinsed in buffer, fixed, embedded, sectioned (at 3 µm thickness), and autoradiographed.  Developed autoradiographs were examined and silver grains associated with different regions of ileum quantitated.  At both temperatures the smooth muscle layers (outer longitudinal muscle, inner circular muscle, and muscularis mucosa) were heavily labeled with silver grains.  Very few grains were found to be associated with the epithelial cells of the intestinal villi.  At 4ºC, very few grains were observed overlying enteric nerve plexuses, whereas these structures were heavily labeled in ileum exposed to I¹²⁵-hexapeptide at 37ºC.  In control experiments performed at 4ºC where iodinated and excess unlabeled hexapeptide were added simultaneously, labeling was markedly reduced in all regions of ileum. 

            Similarly, in control experiments performed at 37ºC, labeling was also reduced but not to the same extent as that observed at 4ºC.  We conclude from these studies that formylhexapeptide chemoattractants bind specifically and in large amounts to receptors on ileal smooth muscle but not to enteric nerve plexuses.  As demonstrated previously, this binding is receptor-mediated and may directly induce ileal smooth muscle contraction.  The occurrence of such binding sites on ileal smooth muscle may promote both normal gut motility and the augmented motility associated with enteric infections.

Keywords - formylpeptide, chemoattractant, chemotaxis, smooth muscle, ileum, contractility, receptors, bacterial peptides.

INTRODUCTION

            There are several classes of compounds that elicit potent secretory and chemotactic responses from normal polymorphonuclear (PMN) and mononuclear leukocytes.  Of these, the formylmethionylpeptides and the complement cleavage products are by far the most fully characterized.  The formylpeptides are a group of low molecular weight oligopeptides found in bacteria (Snyderman and Pike, 1984a, b) and mitochondria (Carp, 1982).  Purified synthetic analogs of these naturally occurring substances have been synthesized and their leukotactic activities characterized (Snyderman and Pike, 1984a, b; Marasco et al., 1984; Miyake et al., 1983).  C5a, a fragment of the fifth component of complement, is the major source of chemotactic activity found in serum (Snyderman et al., 1971).

In leukocytes, the responses elicited by both classes of chemoattractant compounds are known to be mediated by cell surface receptors specific for each class of attractant.  Furthermore, both types of chemoattractants have been shown to be capable of initiating the contraction of smooth muscle and are potent spasmogenic agents in both guinea pig ileum and lung (Rocha e Silva et al., 1951; Stimler et al., 1981; Regal and Pickering, 1981; Hamel et al., 1984).  The structure-activity relationships (i.e., relative potencies) for a series of related, synthetic N-formylpeptides in initiating ileal smooth muscle contraction are very similar to those seen for leukocyte chemotaxis.  In addition, the competitive formylpeptide receptor antagonist, Boc-Phe-Leu-Phe-Leu-Phe inhibited the ileal contractile response to formylpeptide (Marasco et al., 1982).  This suggests that receptors specific for formylpeptides may be found on cells in the ileum and may be responsible for ileal smooth muscle contraction.  However, formylpeptide binding to these receptors may directly or indirectly elicit smooth muscle contraction.  Formylpeptides may bind directly to ileal smooth muscle and thereby elicit its contraction.  Alternatively, they may act on smooth muscle indirectly by first binding to and stimulating enteric nerves or activating inflammatory cells (macrophages, polymorphonuclear leukocytes, lymphocytes).  Secondary to this, soluble mediators such as neurotransmitters (eg., acetylcholine, substance P) or arachidonate metabolites may be released resulting in smooth muscle contraction.

This study was performed to localize the sites of formylpeptide binding in guinea pig ileum.  Using iodinated formylhexapeptide in conjunction with light microscope autoradiography of sectioned ileum, it appears that formylpeptide binds specifically and in relatively large amounts directly to ileal smooth muscle (muscularis externa and muscularis mucosa).  However, very little receptor-mediated binding to enteric nerve plexuses, lamina propria, infiltrating leukocytes, or ileal epithelium was observed.

METHODS AND MATERIALS

Tissue Isolation

Adult male guinea pigs were anesthetized with ether and 3 cm of terminal ileum isolated and flushed several times with cold Krebs solution (130 mM NaCl, 4.7 mM KCl, 1.18 mM KH₂PO₄, 1.17 mM MgSO₄.7H₂O, 1.6 mM CaCl₂, 14.9 mM NaHCO₃, 5.5 mM dextrose, 2 mg/ml bovine serum albumin).  The isolated ileum was cut into 2 mm long pieces, suspended in Krebs solution and allowed to equilibrate for 60 min at 37ºC in an atmosphere of 5% CO₂ / 95% air with several changes of buffer. 

Hexapeptide Binding Conditions

N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine (Peninsula Laboratories, Belmont, CA) was iodinated by the chloramine-T method using carrier-free I¹²⁵ as described previously (Marasco et al., 1985a: Jesaitis et al., 1983, 1984).  After equilibration in Krebs buffer at 37ºC, segments of ileum were exposed to 10 nM I¹²⁵-hexapeptide at 4ºC or 37ºC for either 3 or 30 min.  Excess (500-fold) unlabeled hexapeptide was added simultaneously with I¹²⁵-hexapeptide to one group of tissue samples so that they might serve as controls for nonspecific hexapeptide binding.  It should be noted that buffer containing formylhexapeptide was in contact with both the serosal and luminal aspects as well as the cut ends of each ileal segment providing a large surface area for entry of this peptide into each segment.

Upon completion of this incubation, ileal segments were rinsed thoroughly in two changes of Krebs solution (4ºC) containing 2 mg/ml bovine serum albumin and a final wash in Krebs solution lacking albumin.  This washing was accomplished in 60 seconds whereupon ileal segments were promptly fixed in 2% glutaraldehyde, 1% paraformaldehyde, 0.1 M cacodylate, pH 7.4 for 18 hours at 4ºC. 

Tissue Preparation and Autoradiography

Fixed ileal segments were rinsed in cacodylate buffer (0.1 M) and dehydrated in graded ethanol series.  Tissue was then embedded in glycol methacrylate (DuPont Instruments, Newtown, CT) and each tissue block trimmed to a depth of approximately 0.5 mm before sectioning was commenced.  Cross-sections of ileum were then cut at 3 µm thickness and sections mounted on acid-cleaned, subbed glass slides.  Slides with adherent sections were then dipped in Kodak NTB-2 emulsion (diluted 1:3 with distilled water), air dried, and stored for 14 days in the dark at 4ºC.  Exposed autoradiographs were developed using Kodak D-19, fixed, stained with 0.1% cresyl violet, and coverglasses mounted using Permount. 

Specimens were examined and photographed using a Nikon Optiphot microscope equipped with an Olympus OM-2n camera.  Bright-field optics were used to identify the specific regions of interest and dark-field optics used to visualize and count silver grains associated with these regions.  Silver grains were counted using an eyepiece reticle at 100X magnification.  The reticle grid divided the field into 27 by 27 µm squares.  Grains within 10 such squares overlying each of the regions of interest were then counted.  The regions examined included: muscularis externa, both outer longitudinal and inner circular layers; submucosa; muscularis mucosa; epithelial cells (villi and crypts); and the lumen.  Three separate experiments were performed, the data from these experiments combined, and the mean number of grains/test area ± S.E.M calculated.

RESULTS

Background and Specificity of Labeling

The emulsion background seen over the lumen was very low (usually less than 1 grain per unit test area = 729 µm²) and only a small percentage (less than 5%) of the total amount of binding seen in the muscle layers (Table 1).  Ileal segments exposed to I¹²⁵-hexapeptide in the presence of excess unlabeled hexapeptide exhibited some non-specific binding of labeled hexapeptide within the ileum (3-4 grains/ 729 µm²).  However, in the muscle layers of the ileum a high degree of specific labeling, 3 to 5 times as many specific as non-specific grains, was evident.

The non-specific binding observed in these experiments could be reduced considerably by extending the time of washing after the initial formylpeptide binding (data not shown) but the total amount of specific labeling was also greatly reduced.  Thus, the background labeling probably results from residual unbound I¹²⁵-hexapeptide not removed during the washing procedure.  However, since hexapeptide binding to the leukocyte formylpeptide receptor is known to be highly reversible (Sklar et al., 1984) longer washings might remove significant amounts of receptor-bound I¹²⁵-hexapeptide.  As a result a tissue background level of 3-4 grains per test area was tolerated in order to maintain maximal specific binding of hexapeptide.

The amount of non-specific labeling seen in ileal segments exposed to I¹²⁵-hexapeptide at 37ºC was much higher than that seen at 4ºC (Table 2).  At low temperatures, labeling is due primarily to specific receptor-mediated binding of formylhexapeptide to the cell surface.  Although not significant at 4ºC, fluid-phase endocytosis becomes a major route of internalization at 37ºC.  Thus, 5 to 15 grains/ 729 µm² were seen over the smooth muscle layers at 37ºC even in the presence of excess unlabeled hexapeptide.  Much of this labeling is probably due to a combination of fluid-phase and receptor-mediated endocytosis of I¹²⁵-hexapeptide.

Localization of Hexapeptide Binding

The amount of specific binding increased markedly (50-100%) in samples of tissue exposed to I¹²⁵-hexapeptide for 30 min at 4ºC as compared to 3 min at 4ºC (Figures 1 and 2).  This reflects a move towards equilibrium binding.  However, a greater increase in binding occurred in deeper layers of the ileum than in the superficial layers (46%, 67%, and 106% in the outer longitudinal, inner circular, and muscularis mucosa, respectively) after 30 minutes.  Thus, the tissue mass or intercellular junctions evidently created a significant barrier to the diffusion and binding of formylpeptide at the early time point.  Nevertheless, labeled hexapeptide had penetrated the deepest layers (i.e., muscularis mucosa and submucosa) within three minutes and had bound extensively in all muscle layers within 30 minutes.  Non-specific binding was not significantly different at these two incubation times. 

I¹²⁵-labeled formylpeptide was localized primarily over the longitudinal and circular smooth muscle layers as well as the muscularis mucosa.  The labeling seen associated with smooth muscle was associated primarily with the cell surface rather than intracellular organelles or cytoplasm (Figure 3 A-D).  Little or no specific binding was found over the submucosa or ileal epithelial cells.  PMN, eosinophils, lymphocytes, and tissue macrophages were seen occasionally in the lamina propria and blood vessels, but very little binding was seen compared to that observed on smooth muscle (Figure 3 E,F).  Furthermore, very little binding to Auerbach`s or Meissner`s plexuses was observed at 4ºC (see Figure 1 at arrows) and very few grains were associated with smooth muscle in blood vessel walls.

The amount of label seen in tissue exposed to I¹²⁵-hexapeptide at 37ºC was increased compared to that seen in parallel incubations at 4ºC (Table 2).  Labeling was increased up to 150% in tissue samples exposed to iodinated hexapeptide for 30 min as compared to 3 min at 37ºC.  Non-specific labeling (Figures 4 and 5) was considerably higher at 37ºC than at 4ºC and comprised a large portion (50-80 % in the muscularis externa) of the total labeling observed at 37ºC.  Specific labeling was seen in ileal smooth muscle layers (muscularis externa and muscularis mucosa) but far less label was associated with the epithelial cell layer.  At 37ºC, large amounts of label were associated with Auerbach`s and Meissner`s nerve plexuses (see Figures 4 and 5).  This labeling was not reduced in the presence of excess unlabeled hexapeptide and was thus considered to be the result of fluid-phase endocytosis of I¹²⁵-hexapeptide.  Samples of terminal guinea pig ileum exposed to hexapeptide at 37ºC also contracted in response to the binding or uptake of formylpeptide as described previously by Marasco et al.  (1982).

DISCUSSION

This study shows that iodinated formylhexapeptide binds primarily to the outer longitudinal and inner circular muscle layers and the muscularis mucosa of the guinea pig ileum.  Binding appears to be specific for smooth muscle in these layers since even neighboring vascular smooth muscle does not bind formylpeptide.  In addition,formylpeptide exhibits little or no specific binding to absorptive or secretory cells found in the intestinal epithelium.  Very little specific binding to infiltrating leukocytes or intravascular leukocytes was detected under the conditions employed for these experiments.  Thus, the possibility that formylpeptides may induce the release of soluble mediators of ileal contraction from leukocytes seems remote.  This relative paucity of binding also suggests that the numbers or binding affinities of formylpeptide receptors present on the smooth muscle cells may be higher than those on leukocytes.  Alternatively, the formylpeptide receptors of infiltrating leukocytes may be blocked or down-regulated due to local, endogenous factors.

Marasco et al. (1982) found formylpeptides to be potent spasmogenic agents for guinea pig ileum.  However the response (eg., contour of dose-response curves, latency period, tachyphylaxis) of ileum to these peptides differed significantly from that of ileum to substance P or histamine.  This suggests that formylpeptides and structurally-related neuropeptides such as substance P may interact with the ileum by way of distinctly different mechanisms.  The occurrence and physiologic activities of substance P as well as tissue distribution of substance P receptors have been characterized in considerable detail (Souquet et al., 1985; Regoli et al., 1984; Costa et al., 1982; Holzer and Petsche, 1983).  Substance P receptors have been demonstrated on ileal smooth muscle as well as enteric nerve plexuses (Costa et al., 1982).  The effects of substance P on smooth muscle contraction are thought to be mediated by endogenous agents such as acetylcholine, prostaglandins, or histamine. 

On the other hand, we have found that iodinated formylpeptide binds with great specificity to smooth muscle but does not bind to enteric nerve plexuses at 4ºC.  At 37ºC, however, formylpeptide is taken up by these plexuses in large amounts.  It is possible that this internalized peptide may elicit subsequent effects on ileal smooth muscle innervated by these nerves but there is no direct evidence that this does occur.  However, the absence of receptor sites specific for formylpeptide on these plexuses (as seen at 4ºC) would argue against such a mechanism.  Substances internalized by bulk fluid pinocytosis are generally targeted for lysosomal destruction (Farquhar, 1983; DeDuve and Wattiaux, 1966) and are therefore not likely to evoke rapid, specific cellular responses of the sort typified by cell surface receptor-mediated phenomena.  The occurrence of very abundant cell surface receptors specific for formylpeptide on cells in the smooth muscle layers indicates that these peptides may exert a direct effect on smooth muscle in these regions.

Normally, resident intestinal flora continually produce and release small quantities of formylpeptides into the gut lumen (Marasco et al., 1984; Miyake et al., 1983).  Magnusson et al.  (1985) have demonstrated that formylpeptides infused intraluminally greatly increase the permeability of ileal epithelia for low molecular weight substances.  Under physiological conditions, this may allow bacterial formylpeptides to penetrate the epithelium more readily and bind directly to ileal smooth muscle.  This would then result in increased smooth muscle contraction and perhaps help to maintain the normal motility of the gut.  The observation that germ-free mice exhibit greatly reduced transit of intraluminal radiotracers lends further credulity to this hypothesis (Abrams and Bishop, 1967).  Under certain pathological conditions involving the gut such as enteric bacterial infections, peristaltic rates and smooth muscle contraction are often dramatically increased.  Under these circumstances, large quantities of bacterial formylpeptides may be elaborated and released into the gut lumen.  These peptides may then penetrate into the muscular layers of the gut and act there to vastly augment the usual contractility of intestinal smooth muscle. 

From this data and other studies, we conclude that synthetic formylpeptides can increase the permeability of the intestinal epithelium, penetrate that epithelium, bind to ileal smooth muscle, and elicit increased smooth muscle contraction.  Since these formylpeptides are synthetic analogs of naturally occurring bacterial peptides, we speculate that intraluminal bacteria may release similar factors that subsequently affect intestinal contractility. 

TABLE I

Silver Grains Associated with Guinea Pig Ileum after Exposure to

I¹²⁵-Hexapeptide at 4ºC

Layer of Ileum                               I¹²⁵-Hexapeptide                      I¹²⁵-Hexapeptide

+ Unlabeled Hexapeptide

                                                3 min                30 min              3 min                30 min

______________________________________________________________________

Muscle

 Outer longitudinal                     10.8 ± 1.6*      15.8 ±1.4**     3.0 ±0.5           3.2 ±0.6

       

 Inner circular                           11.0 ±1.2**     20.1 ±1.3**     3.0 ±0.5           3.5 ±0.8

Submucosa                               4.0 ±1.3           6.7 ±1.5           3.7 ±0.5           4.2 ±0.7

Muscularis mucosa                   8.0 ±1.4**       16.5 ±1.8*       3.7 ±0.6           4.2 ±0.7

Epithelial cells

 (Villi and crypts)                      2.0 ±0.5           2.2 ±0.5           0.7 ±0.3           1.0 ±0.3

Lumen                                      0.7 ±0.3           1.0 ±0.2           0.7 ±0.3           0.8 ±0.3

______________________________________________________________________

2 mm segments of ileum exposed to 10 nM I¹²⁵-hexapeptide in Krebs buffer at 4ºC, rinsed rapidly through 3 changes of Krebs buffer at 4ºC and then fixed in 2% glutaraldehyde with 1% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4 overnight.  To control for nonspecific binding, tissue was exposed to 10 nM I¹²⁵-hexapeptide containing 5 µM unlabeled hexapeptide.

Tissue was dehydrated in ethanol series, embedded in glycol methacrylate, cut into 3 µm thick sections, coated with Kodak NTB-2 emulsion, and exposed in the dark at 4ºC for 14 days.  Autoradiographs were developed in Kodak D-19, stained in cresyl violet, and mounted in Permount.

Silver grains were counted using a reticle at 100X magnification.  Grains within a 27 by 27 µm square were counted overlying each of the regions designated above and 10 squares were counted for each region.  Three separate experiments were performed.  Each entry represents the mean ± SEM for three experiments.

Grain counts for I¹²⁵-hexapeptide compared to I¹²⁵-hexapeptide + excess unlabeled hexapeptide at equivalent times using 2-tailed t-test; * p < 0.03 ; ** p < 0.005

TABLE II

Silver Grains Associated with Guinea Pig Ileum after Exposure to

I¹²⁵-Hexapeptide at 37ºC

Layer of Ileum                              I¹²⁵-Hexapeptide                      I¹²⁵-Hexapeptide +

                                                                                                Unlabeled Hexapeptide

                                                3 min                30 min              3 min                30 min

______________________________________________________________________

Muscle

 Outer longitudinal                     16.2 ± 2.1        23.0 ± 2.8*      5.0 ± 1.1          11.5 ± 2.5

       

 Inner circular                           9.7 ± 1.3          21.2 ± 3.1        8.0 ± 2.8          15.5 ± 3.3

Submucosa                               4.2 ± 1.0          5.0 ± 1.5          6.0 ± 2.2           7.7 ± 2.1

Muscularis mucosa                   8.0 ± 1.5          20.2 ± 3.7        7.2 ± 1.8          10.5 ± 2.6

Epithelial cells

 (Villi and crypts)                      3.0 ± 0.5          5.0 ± 0.3          2.5 ± 0.5          4.0 ± 1.0

Lumen                                      0.7 ± 0.3          0.5 ± 0.2          1.0 ± 0.3          1.1 ± 0.4 ______________________________________________________________________

2 mm segments of ileum exposed to 10 nM I¹²⁵-hexapeptide in Krebs buffer at 37ºC, rinsed rapidly through 3 changes of Krebs buffer at 4ºC and then fixed in 2% glutaraldehyde with 1% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4 overnight.  To control for nonspecific binding, tissue was exposed to 10 nM I¹²⁵-hexapeptide containing 5 µM unlabeled hexapeptide.

Tissue was dehydrated in ethanol series, embedded in glycol methacrylate, cut into 3 µm thick sections, coated with Kodak NTB-2 emulsion, and exposed in the dark at 4ºC for 14 days.  Autoradiographs were developed in Kodak D-19, stained in cresyl violet, and mounted in Permount.

Silver grains were counted using a reticle at 100X magnification.  Grains within a 27 by 27 µm square were counted overlying each of the regions designated above and 10 squares were counted for each region.  Three separate experiments were performed.  Each entry represents the mean

SEM for three experiments.

Grain counts for I¹²⁵-hexapeptide compared to I¹²⁵-hexapeptide + excess unlabeled hexapeptide at equivalent times using 2-tailed t-test; * p < 0.04

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 FIGURES

 

 Figure 1.  Guinea pig ileum exposed to 10 nM I¹²⁵-hexapeptide at 4ºC for 3 min, washed for 1 min in Krebs solution, and then fixed.  Bright-field images of ileal cross-sections (3µm thick) are seen in the left column and corresponding dark-field images in the right.  Many silver grains are seen overlying both layers of the muscularis externa (A,B), both outer longitudinal muscle (olm) and inner circular muscle (icm).  In addition, a number of grains are also seen over the muscularis mucosa (mm).  Auerbach`s plexus (indicated by arrows) is virtually without associated silver grains except at its very periphery.  Very few grains are seen associated with the epithelial cells of the ileum (C,D) and even fewer in the lumen.  Control tissue exposed simultaneously to 10 nM iodinated hexapeptide and 5 µM unlabeled hexapeptide (E,F) shows a great reduction in the number of silver grains.  Magnification bar = 100 µm.

 Figure 2.  Guinea pig ileum exposed to 10 nM I¹²⁵-hexapeptide at 4ºC for 30 min, rinsed in Krebs solution at 4ºC for 1 min, and then fixed.  Three micrometer thick cross-sections of ileum (bright-field, left column; corresponding dark-field, right column) show many silver grains (A,B) associated with the outer longitudinal and inner circular muscle layers (olm and icm, respectively) and also with the muscularis mucosa (mm).  Very few grains are seen in association with the lamina propria or with the epithelial cell layer (C,D).

Under similar conditions, ileal segments exposed to iodinated hexapeptide simultaneously with excess (500-fold) unlabeled hexapeptide show comparatively very few grains overlying the smooth muscle layers (E,F).  An unlabeled Auerbach`s plexus is also seen (arrow).  Magnification bar = 100 µm.

Figure 3.  Ileum initially exposed to I¹²⁵-hexapeptide for 30 min at 4ºC, rinsed, fixed, and cross-sectioned.  At high magnification, cells in both the outer longitudinal (A,B) and inner circular (C, D) muscle layers exhibit silver grains localized on the muscle plasma membrane.  Perivascular tissue macrophage found in the lamina propria (E, F) displays prominent granular cytoplasmic inclusions (dense by bright-field and refractile by dark-field) but no silver grains indicating a lack of I¹²⁵-hexapeptide binding.  Magnification bar = 15 µm.

Figure 4.  Guinea pig ileum exposed to 10 nM I¹²⁵-hexapeptide for 3 min at 37ºC, rinsed in Krebs solution for 1 min at 4ºC, and then fixed.  Cross-sectioned ileum (bright-field, left column; corresponding dark-field, right column) shows a great many silver grains (A,B) overlying the outer longitudinal muscle (olm) as well as the inner circular muscle (icm) layers.  In addition, many grains are seen overlying the Auerbach`s plexus (arrow) beneath the outer longitudinal muscle layer.  Few grains are seen overlying the epithelial cells and very few over the lumen (C,D).

Ileal segments exposed simultaneously to iodinated and excess unlabeled hexapeptide for 3 min at 37ºC display considerable numbers of silver grains associated with the muscularis externa (E,F).  Magnification bar = 100 µm.

 Figure 5.  Segments of guinea pig ileum exposed to I¹²⁵-hexapeptide for 30 min at 37ºC, rinsed in Krebs solution for 1 min at 4ºC, and then fixed.  Cross-sectioned ileum (bright-field, left column; corresponding dark-field, right column) display large numbers of grains over the muscularis externa (outer longitudinal and inner circular layers; olm and icm, respectively), muscularis mucosa (mm), and Auerbach`s plexus (arrow) (A,B).  Increased numbers of grains are also seen associated with the epithelial layer (C,D).  Furthermore, ileal segments exposed simultaneously to labeled and excess unlabeled hexapeptide at 37ºC for 30 min (E,F) also exhibited relatively large numbers of silver grains over the smooth muscle layers of the ileum.  Magnification bar = 100 µm.


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