- Email: [email protected]
Immuno-Electron Microscopic Study of Differential Localization of Motilin and Serotonin in the Rabbit Duodenal Epithelium
Peptides, Vol. 19, No. 1, pp. 65–73, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00
PII S0196-9781(97)00260-X
Immuno-Electron Microscopic Study of Differential Localization of Motilin and Serotonin in the Rabbit Duodenal Epithelium MINEKO FUJIMIYA,*1 KIYOHITO OKUMIYA,† YIN NAM KWOK,‡ SERGE ST-PIERRE\ AND CHRISTOPHER H. S. McINTOSH‡ * Department of Anatomy, Shiga University of Medical Science, Otsu, Shiga 520-21, Japan † Department of Geriatrics, Kochi Medical School, Nankoku Kochi, 783 Japan ‡ Department of Physiology, University of British Columbia, Health Sciences Mall, Vancouver, BC V6T 1Z3 Canada // INRS-Sante´, Pointe-Claire, QC H9K1G6 Canada Received 17 March 1997; Accepted 13 August 1997 FUJIMIYA, M., K. OKUMIYA, Y. N. KWOK, S. ST-PIERRE AND C. H. S. McINTOSH. Immuno-electron microscopic demonstration of differential localization of motilin and serotonin in the rabbit duodenal epithelium. PEPTIDES 19(1) 65–73, 1998.—A guinea pig antibody against rabbit motilin was generated to study the localization of motilin-containing cells in the rabbit small intestine with special reference to the co-localization of motilin and serotonin. A pre- and post-embedding technique for immuno-electron microscopy was used; duodenal sections were stained with either motilin or serotonin in the pre-embedding DAB-nickel reaction, followed by subsequent staining of ultrathin sections of positive cells with either motilin or serotonin in the post-embedding immunogold reaction. Samples were divided into four groups: 1) pre-motilin, post-motilin, 2) pre-motilin, post-serotonin, 3) pre-serotonin, post-serotonin, and 4) pre-serotonin, post-motilin. Motilin-containing cells in the rabbit duodenum were characterized by round granules (395.3 6 66.1 nm in diameter) with medium electron density, located basally or in the perinuclear cytoplasm. In contrast, serotonin-containing cells were characterized by round to pleomorphic secretory granules (344.5 6 90.5 nm in diameter with electron dense cores and prominent halos. In motilin-containing cells, massive aggregations of immunogold particles reacted to motilin occurred over secretory granules. A few immunogold particles scattered diffusely over the cytoplasm reacted to serotonin; however, this reaction appeared to be background staining because the density was not changed if the section was treated by preabsorption. In serotonin-containing cells, immunogold particles reacted to serotonin were aggregated over the secretory granules and a large number of gold particles were scattered diffusely at the extragranular cytoplasm; however, very few or no immunogold particles were observed within the cells which reacted to motilin. Results of the present study indicate that motilin and serotonin are not co-localized in the epithelial endocrine cells of the rabbit intestine. © 1998 Elsevier Science Inc. Motilin
Serotonin
Immuno-electron microscopy
MOTILIN is a 22 amino acid peptide, synthesized in the small intestine (12,13,19), which has been demonstrated to be a major regulator of the interdigestive migrating motor complex (8,19). It was originally suggested that the biosynthesis of motilin took place in enterochromaffin (EC) cells (17,20). Subsequently a separate cell-type, the M (Mo)-cell,
Immunogold technique
Rabbit
Duodenum
was found to be the major source of motilin in all species examined (3,7). Evidence has been presented for the existence of immunoreactive (IR)-motilin, or a motilin-related peptide, in a subclass of EC cells, the presence of which can only be demonstrated with N-terminal directed antibodies (9). In addition, equivocal results have been obtained con-
1
Requests for reprints should be addressed to Mineko Fujimiya, M.D., Ph.D., Department of Anatomy, Shiga University of Medical Science, Seta, Otsu, Shiga 520-21, Japan, E-mail: [email protected]
65
66
cerning the possible co-localization of motilin and serotonin. These conflicting results may be partially due to the methods used in determining the localization of these substances, since most studies employed the techniques of formaldehyde-induced-fluorescence or argentaffinity to identify serotonin-containing EC cells instead of immunohistochemistry (3,6,10,20), and all used heterologous motilin antisera. Although specific antibody against serotonin has been used in some studies, the distribution of motilinand serotonin-containing cells has only been compared using light microscopy (9). The intestinal actions of motilin and characterization of its receptors have been studied extensively in the rabbit (2,18,21), but information on the cellular localization of IR-motilin in this species are lacking, with the exception of one recent report (22). In that report, distribution of IR-motilin cells was investigated throughout the rabbit gastrointestinal tract; however, possible colocalization of serotonin and motilin in the rabbit intestine was not mentioned. We recently isolated a rabbit motilin cDNA (1), and produced a synthetic peptide based on the predicted sequence (23). Antibodies against this peptide have now been generated, and the objective of the present study was to examine the localization of serotonin and motilin in the epithelial endocrine cells of the rabbit duodenum at the electron microscopic level, with special reference to their possible co-localization. Pre- and post-embedding techniques for immuno-electron microscopy were used; this allowed for the simultaneous reaction (DAB-nickel and immunogold) of different antibodies on a single endocrine cell, thus providing a means to examine the possible colocalization of different antigens. METHOD Production of Motilin Antibody Antisera against rabbit motilin were generated in guinea pigs using synthetic rabbit motilin (INRS-Sante´, Quebec) conjugated to keyhole limpet hemocyanin (1:8 w/w; Calbiochem, La Jolla, CA) using 1-ethyl-3-3-(dimethylaminopropyl) carbodiimide hydrochloride (Pierce, Rockford, IL). The antiserum used in the present study (KC02) was produced after two booster injections. The specificity of this antiserum was checked by using the preabsorption test as described below. The antiserum was also shown not to react with other endocrine peptides such as gastric inhibitory polypeptide, glucagon and secretin as determined by radioimmunoassays. Animals used in the present study were cared for in accordance with the principles of the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care, 1993).
FUJIMIYA ET AL.
Pre- and Post-Embedding Immuno-Electron Microscopic Study for Serotonin and Motilin Two male Japanese white rabbits each weighing 2 kg were anesthetized with pentobarbital (Nembutal®, 50 mg/kg). The animals were perfused systemically via the left ventricle with 2.21 of 0.01 M phosphate buffered saline (PBS) (0.01 M phosphate buffer containing 0.9% NaCl; pH 7.4) at a speed of 300 ml/min and then perfused with 3 l of a cold fixative solution consisting of 0.1 M phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde, 0.5% glutaraldehyde and 0.2% picric acid. The duodenal segment between 5–10 cm distal to the pylorus was then removed, cut open along the mesenterium, stretched on a cork plate, and immersed for 24 h in a post-fixative solution of 0.1 M PB (pH 7.4) containing 4% paraformaldehyde and 0.2% picric acid at 4°C. The duodenum was washed for 4 days with several changes of 0.1 M PB containing 15% sucrose and then dipped in 0.1 M PB containing 10% gelatin at 37°C overnight. The gelatin-embedded specimen was cooled to 4°C, fixed for 3 h with 4% paraformaldehyde in 0.1 M PB, cut into 50-mm thick sections on a vibratome, and the sections were collected in 0.1 M PBS at 4°C. The duodenal sections were treated in the free-floating state throughout the immunohistochemical procedures. They were first pretreated for 10 min with a mixture of 0.01% trypsin (Sigma Chemical Co., St. Louis, MO, USA) and 0.68 mM CaCl2 in 0.05 M Tris-HCl buffer (pH 7.6) at 20°C and washed for 30 min with 0.1 M PBS. Sections were incubated with either motilin antibody (KC02; diluted 1:5000 in 0.1 M PBS) or serotonin antibody (monoclonal antibody generated in a mouse, the detail of immunological property was described in our previous paper (24), diluted 1:10,000 in 0.1 M PBS) for 48 h at 4°C. To inactivate endogenous peroxidase activity, the sections were incubated at room temperature (RT) for 20 min with 0.1% H2O2 in 0.1 M PBS followed by an additional 20 min with 0.1% phenylhydrazine in 0.1 M PBS. After washing for 30 min with 0.1 M PBS, the sections were incubated at RT for 2 h in either biotinylated anti-guinea pig IgG (for motilin staining; Vector Lab. Inc., USA) or biotinylated anti-mouse IgG (for serotonin staining; Vector) diluted 1:1000 in 0.1 M PBS. Sections were then washed and placed in avidin-biotin peroxidase complex (Elite, Vector; 1:2000 in 0.1 M PBS) for 2 h at RT. The immunoreaction was then visualized by reacting with 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01% 3, 3’-diaminobenzidine, 1% ammonium nickel sulfate and 0.0003% H2O2 for 30 min at RT. For light microscopic examination, the stained sections were mounted on gelatin-coated glass slides, dehydrated by graded ethanol, and coverslipped with Entellan® (Merk, Darmstadt, Germany). For the electron microscopic study, serotonin or motilin stained sections were washed for 10 min with 0.1 M PBS, followed by an additional 10 min with distilled water, and dehydrated by a graded series of ethanol as described in our
STUDY FOR MOTILIN AND SEROTONIN
previous paper (16). These sections were then incubated overnight with LR Gold resin (London Resin Co. Ltd., Berkshire, England) at 220°C and incubated for an additional 1 h with LR Gold resin containing 0.1% Benzil (Pelco®, Ted Pella, Inc.) at 220°C. They were then mounted on silicon-coated glass slides and embedded in LR Gold resin containing 0.1% Benzil and covered with silicon-coated cover glasses at 220°C. The embedded specimens were allowed to polymerize for 4 h in an Ultraviolet Cryo Chamber (Pelco®, Ted Pella Inc, CA), where the temperature was kept at 220°C throughout the polymerization period. After removal of the cover glasses, the polymerized sections were examined under light microscopy (3 40) and the intestinal mucosa which contains motilin or serotonin positive cells was dissected out with a razor blade. Ultra-thin sections (60 nm) were cut on an ultramicrotome (Reichert-Jung Ultracut E, Austria) and picked up on nickel grids (thin bar grid, 200-mesh; Nishin EM Co., Tokyo, Japan) supported by collodion. The nickel grids were incubated with 3% normal goat serum (Dako Japan, Tokyo, Japan) dissolved in a reaction buffer (0.1 M PBS containing 0.2% BSA, 0.2% saponin and 0.05% NH4Cl, pH 7.4) for 30 min at RT. The ultrathin sections stained for motilin or serotonin in the pre-embedding procedures were then stained again with either motilin or serotonin in the post-embedding immunohistochemical procedures. The combination was as follows; 1) pre-motilin, post-motilin, 2) pre-motilin, post-serotonin, 3) pre-serotonin, post-serotonin, 4) pre-serotonin, post-motilin. The sections were incubated with antibody against either motilin (1:40) or serotonin (1:300) diluted with the reaction buffer. The sections were washed with 0.1 M PBS and incubated for 1.5 h with either immunogold conjugated goat antiguinea pig IgG (for motilin staining; 10 nm gold, British BioCell International, Cardiff, UK) or immunogold conjugated goat anti-mouse IgG (for 5HT staining; 15 nm gold, British BioCell) 1:40 diluted with the reaction buffer at RT, followed by washings with 0.1 M PBS and then with distilled water. The sections were stained for 10 min at RT with 2% uranyl acetate followed by an additional 5 min with Reynold’s solution. During the above procedures, the nickel grids were floating on drops of incubation medium with the sections on the underside or submerged in the washing media. The stained sections were then examined under electron microscopy (H-7100, Hitachi, Ltd., Japan). In the electron microscopic study, more than 20 motilincontaining cells and more than 30 serotonin-containing cells were observed. One hundred secretory granules located at the basal or the perinuclear region of the cytoplasm were randomly selected from either serotonin- or motilin-containing cells, and the mean diameter of the granules was calculated. In serotonin-containing cells, an additional 50 secretory granules located at the apical cytoplasm were selected, and the mean diameter was measured.
67
Specificity of the Positive Staining The antibody specificity was checked by both radioimmunoassay and immunohistochemical absorption study. As described above, motilin antibody did not crossreact with gastric inhibitory peptide, glucagon and secretin examined by radioimmunoassay. Immunohistochemical absorption study was performed in both pre-embedding and post-embedding procedures, where the primary antibody against motilin or serotonin was substituted with an antibody preabsorbed with the specific antigen. In the pre-embedding staining, the antibodies for motilin (1:5,000) and serotonin (1:10,000) were preabsorbed with 0.01 mM rabbit motilin and 0.1 mM serotonin creatinin sulfate (Merck, Darmstedt, Germany), respectively. In the post-embedding staining, the antibodies for motilin (1:40) and serotonin (1:300) were preabsorbed with 0.2 mM rabbit motilin and 10 mM serotonin creatinin sulfate, respectively. In both steps, the antigen and antibody mixture was incubated for 48 h at 4°C before incubating with the sections. We obtained the negative DAB reactions for motilin and serotonin at light microscopic levels and also in the electron microscopic immunogold reactions as shown in the results. RESULTS Motilin immunoreactive cells were scattered in the mucosal epithelium, and were spindle-like shapes with cytoplasmic processes extending to the intestinal lumen. When observed under electron microscopy, the majority of motilin-containing cells in the rabbit duodenum were similar in morphology. These cells contained round and relatively large granules (395.3 6 66.1 nm in diameter, ranging from 250 to 450 nm, n 5 100) with medium electron density. The granules were packed in the basal and supranuclear region of the cells (Figs. 1A, 1B). Examination under higher magnification revealed that the dense aggregation of immunogold particles was located over the secretory granules situated at both basal and supranuclear cytoplasm (Figs. 1C, 2A). Only a few immunogold particles were detected over the nucleus, other organelles, the extracellular space, or the extragranular matrix of the cytoplasm (Figs. 1C, 2A). To confirm the specificity of the immunogold reaction detected over the secretory granules, ultrathin sections were stained with antibody against motilin which had previously been absorbed with 0.2 mM motilin. Following this treatment, the immunogold reaction over the secretory granules was no longer detectable (Fig. 2D, compared with Fig. 2A). However, the motilin-containing cells were easily identified by the dark DAB-nickel reaction materials precipitated over some of the secretory granules or the cytoplasm (Fig. 2D). To examine the possibility of co-localization of serotonin and motilin in the epithelial cells, duodenal sections previously stained with motilin in the pre-embedding immunohistochemical procedure using the DAB-nickel reaction were additionally stained with serotonin antibody in the
68
FUJIMIYA ET AL.
FIG. 1. Electron micrographs of motilin-containing cells in the rabbit duodenum stained with motilin in both pre- and post-embedding immunohistochemical procedures. Bars 5 1 mm. (A) Round and solid secretory granules were packed in the basal cytoplasm. (B) Secretory granules are located at the supranuclear region in addition to the basal cytoplasm. (C) Higher magnification view of the area enclosed by the rectangle in (A). Immunogold particles are aggregated over the secretory granules; very few of them are located in the extragranular spaces.
STUDY FOR MOTILIN AND SEROTONIN
69
FIG. 2. Electron micrographs of higher magnification view of motilin-containing cells in the rabbit duodenum. All sections are stained with motilin in the pre-embedding immunohistochemical procedure, and stained with motilin (A and D), or serotonin (B and C) in the post-embedding procedure. Motilin-positive cells are easily identified under electron microscopy by dark DAB-nickel reaction products precipitating over secretory granules or cytoplasm (arrow heads). Bars 5 1 mm. (A) Immunogold particles reacted to motilin are densely aggregated over the secretory granules; very few of them located at the extragranular cytoplasm. (B) A small number of immunogold particles reacted to serotonin are scattered diffusely at the cytoplasm. (C) A section stained with serotonin antibody in the postembedding procedure, previously absorbed with 10 mM of serotonin. The density of immunogold particles reacted to serotonin does not change from that seen in (B). (D) A section stained with motilin antibody in the post-embedding procedure, previously absorbed with 0.2 mM motilin. Immunogold particles reacted to motilin seen in (A) have completely disappeared.
70
FUJIMIYA ET AL.
FIG. 3. Electron micrographs of serotonin-containing cells in the rabbit duodenum, stained with serotonin in both pre- and post-embedding immunohistochemical procedures. Bars 5 1 mm. (A) Round or pleomorphic shaped secretory granules are situated in both basal and apical cytoplasm of the cell. (B) Aggregation of secretory granules of various size and electron density is seen at the apical cytoplasm. Immunogold particles are concentrated over the secretory granules, however a considerable number of immunogold particles are scattered diffusely over the extragranular matrix including microvilli (arrow heads).
post-embedding immuno-electron microscopic procedure using the immunogold reaction. The motilin-containing cells were easily identified under electron microscopy as precipitated dark products present over the cytoplasm, resulting from DAB-nickel reaction (Fig. 2B). A small number of serotonin-reactive immunogold particles were scattered throughout the cytoplasm as well as over the secretory granules (Fig. 2B), however the density was quite low when compared to the motilin-immunoreactive particles seen over the secretory granules in Fig. 2A. To examine the specificity of the immunogold reaction for serotonin in the motilincontaining cells, the ultrathin sections were stained with the antibody against serotonin which was previously absorbed with 10 mM of serotonin. No apparent changes was observed in the density of immunogold particles which reacted
to serotonin, even though the sections were treated with antibody previously absorbed with serotonin (Fig. 2C). The ultrastructural characteristics as well as the intracellular localization of immunogold reactions in serotonincontaining cells were completely different from those in motilin-containing cells. Serotonin-containing cells in the rabbit duodenal epithelium contained round or pleomorphic secretory granules with relatively high electron density (Figs. 3A, 3B, 4A). A considerable number of serotonincontaining cells possessed secretory granules at both apical and basal cytoplasm (Figs. 3A, 3B). The size and the electron density of the secretory granules varied considerably within a single cell as shown in Figs. 3A and 3B; the sizes of the secretory granules located in the basal and apical cytoplasm were 344.5 6 90.5 nm (range 200 – 490
STUDY FOR MOTILIN AND SEROTONIN
FIG. 4. Electron micrographs showing higher magnification view of serotonin-containing cells in the rabbit duodenum. All sections are stained with serotonin in the pre-embedding procedure, and stained with serotonin (A, A9 and C) and motilin (B) in the post-embedding procedure. Bars 5 1 mm. (A) Round granules with cores of high electron density and prominent halos are seen at the basal cytoplasm. Immunogold particles are packed diffusely within the cytoplasm, but very few of them are seen outside of the cell. (A9) Higher magnification view of the area enclosed by the rectangle in (A). Immunogold particles reacted to serotonin are concentrated over cores of secretory granules; however, a large number of gold particles are scattered at the extragranular matrix. (B) Very few immunogold particles reacted to motilin are seen. (C) A section reacted with serotonin antibody in the postembedding procedure, which is preabsorbed with 10 mM serotonin. Most of the immunogold reaction observed in (A9) disappeared and a few gold particles are scattered in the cytoplasm.
71
72
nm, n 5 100) and 344.0 6 90.7 nm (range 200 –500 nm, n 5 50), respectively. The pattern of immunogold reaction within the serotonin-containing cells differed from that of motilin-containing cells. Immunogold particles reacted to serotonin were densely aggregated over the cores of the secretory granules, and a large number of immunogold particles were scattered diffusely at the extragranular space of both apical and basal cytoplasm of the cells (Figs. 3A, 3B, 4A9). The specificity of the immunogold reaction was examined in these sections using the preabsorption test. Most of the immunogold reaction observed in Fig. 4A9 disappeared and a few gold particles were scattered in the cytoplasm (Fig. 4C). To examine the co-localization of serotonin and motilin further, additional experiments were performed, in which the ultrathin sections previously stained with serotonin in the pre-embedding procedure were subsequently stained with motilin in the post-embedding procedure. Very few immunogold particles which reacted to motilin were observed throughout the cell body (Fig. 4B). DISCUSSION Previous electron microscopic studies have demonstrated the presence of motilin-containing cells in intestines of human and dogs, but not in rabbits. In the human intestine, motilin-containing cells are characterized by the presence of small (180 nm in diameter), round and solid granules with closely applied limiting membrane (25), while in the dog intestine the granules are of medium size (200 nm in diameter) and pleomorphic (11,25). The present study shows that motilin-containing cells in the rabbit duodenum are characterized by relatively large (395.3 6 66.1 nm in diameter) round granules with closely applied limiting membrane. This finding suggests that the ultrastructural characteristics of the secretory granules appear to be of little value in identifying endocrine cells. In our preliminary study, we examined the distribution of motilin-immunoreactive cells in the duodenum, jejunum, ileum and colon of rabbits. The density of motilin immunoreactive cells was highest in the duodenum, and decreased gradually toward the lower portion of the intestine. This observation was consistent with the data previously reported in the rabbit intestine (22). Within the duodenum, motilin positive cells scattered diffusely in the epithelium and the density was highest in the duodenal bulb. Although the cell number was smaller than that in serotonin containing cells, the distribution pattern in the duodenum was similar in both serotonin and motilin containing cells. To examine the co-localization of serotonin and motilin, the pre- and post-embedding method for immuno-electron microscopy was used, and samples were divided into four groups: 1) pre-motilin, post-motilin, 2) pre-motilin, postserotonin, 3) pre-serotonin, post-serotonin and 4) pre-serotonin, post-motilin. Our previous study has shown that
FUJIMIYA ET AL.
antigenicity was well preserved for post-embedding staining, even though the sections were previously reacted with the same antibody (16). Motilin-containing cells were easily identified under electron microscopy by dark nickel-DAB reaction products precipitated at the cytoplasm. Massive aggregation of immunogold particles reacted to motilin were observed over the secretory granules. Since most of the gold particles disappeared in the sections pre-treated with antibody absorbed with motilin, such reactions are likely to be specific for motilin. When ultrathin sections of motilin-containing cells were stained with serotonin, a few immunogold particles were scattered throughout the cytoplasm; however, this is likely to be non-specific or representing background staining, since the staining remained after the preabsorption test. These results show that motilin containing cells in the rabbit duodenum contain motilin within the secretory granules; however, they do not contain serotonin. The ultrastructural characteristics of serotonin-containing cells varied widely in the rabbit intestine. Some cells contained pleomorphic shaped secretory granules, whereas others contained round granules that were basically electron dense with prominent halos. Most characteristically, in some serotonin-containing cells, a considerable number of secretory granules were located at the apical cytoplasm as well as at the basal cytoplasm of the cells. Such bipolar distribution of secretory granules has also been reported in the rat entero chromaffin (EC) cells (4,5,15). In most serotonin-containing cells, immunogold particles reacted to serotonin were mainly aggregated over the secretory granules; however a considerable number of gold particles were diffusely scattered over the extragranular space of the cytoplasm. These extragranular immunogold reactions were not likely the background staining but specific for serotonin for the following reasons: they disappeared following preabsorption of the antibody with serotonin. Such extragranular localization of 5HT has been reported in EC cells of rats (4,5,14) and assigned to be an intracytoplasmic release of 5HT from secretory granules (5). When ultrathin sections of serotonin-containing cells were stained with motilin, very few or no immunogold particles reacted to motilin were observed, suggesting that serotonin-containing cells in rabbit intestine do not contain motilin. The present study, using immuno-electron microscopy, provides evidence that serotonin and motilin are located in completely separate cells in the rabbit duodenum, i.e., they do not co-localize. This is in disagreement with previous reports indicating that motilin is located in a subtype of EC cell (6,9,17,20). This discrepancy may be partially explained by the differences in techniques used. In the previous studies, heterologous motilin antisera were used, and EC cells were identified by the nature of argentaffinity of
STUDY FOR MOTILIN AND SEROTONIN
73
cells or particular pleomorphic granules at the ultrastructural level rather than serotonin immunoreactivity. In the present study, immuno-electron microscopy was used to define the EC cell localization of serotonin. This reveals that various shapes of secretory granules were present in serotonin-containing cells, and a lack of immunoreactivity for motilin. In conclusion, by using pre- plus post-embedding immuno-electron microscopic procedures the separate cellular localization of serotonin and motilin was demonstrated
in the duodenal epithelium of the rabbit, and the ultrastructure of these cells was examined. ACKNOWLEDGEMENTS These studies were supported by Grant-In-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture, Japan (MF, KO) and British Columbia Health Research Foundation (YNK, CHSM). The authors thank Mr. Takefumi Yamamoto in the Central Research Laboratory of Shiga University of Medical Science for his excellent technical support in the electron microscopic study.
REFERENCES 1. Banfield, D. K.; MacGillivray, R. T. A.; Brown, J. C.; McIntosh, C. H. S. The isolation and characterization of rabbit motilin precursor cDNA. Biochem. Biophys. Acta. 1131:341– 344; 1992. 2. Depoortere, I.; Peeters, T. L.; Vantrappen, G. Solubilization and characterization of motilin receptor complexes from rabbit antral smooth muscle tissue. J. Recept. Res. 13:903–923; 1993. 3. Forssmann, W. G.; Yanaihara, N.; Helmstaedter, V.; Grube, D. Differential demonstration of the motilin-cell and the enterochromaffin-cell. Scand. J. Gastroenterol. 11(suppl. 39): 43– 45; 1976. 4. Fujimiya, M.; Okumiya, K.; Maeda, T. Immuno-electron microscopic demonstration of luminal release of serotonin from enterochromaffin cells of rat embryo. Acta Histochem. Cytochem. 28:555–563; 1995. 5. Fujimiya, M.; Okumiya, K.; Kuwahara A. Immunoelectron microscopic study of the luminal release of serotonin from rat enterochromaffin cells induced by high intraluminal pressure. Histochem. Cell Biol. 8:105–113; 1997. 6. Heitz, P. U.; Kasper, M.; Krey, G.; Polak, J. M.; Pearse, A. G. E. Immunoelectron cytochemical localization of motilin in human duodenal enterochromaffin cells. Gastroenterology 74:713–717; 1978. 7. Helmstaedter, V.; Kreppein, W.; Domschke, W.; Mitznegg, P.; Yanaihara, N.; Wu¨nsch, E.; Forssmann, W. G. Immunohistochemical localization of motilin in endocrine non-enterochromaffin cells of the small intestine of humans and monkey. Gastroenterology 76:897–902; 1979. 8. Itoh, Z. Effect of motilin on gastrointestinal motor activity in the dog. In: Itoh, Z., ed. Motilin. New York: Academic Press; 1990:133–153. 9. Kishimoto, S.; Polak, J. M.; Buchan, A. M. J.; Verhofstad, A. A. J.; Steinbusch, H. W. M.; Yanaihara, N.; Bloom, S. R.; Pearse, A. G. E. Motilin cells investigated by the use of region-specific antisera. Virchow Arch [Cell Pathol] 36:207– 218; 1981. 10. Kobayashi, S.; Iwanaga, T.; Fujita, T.; Yanaihara, N. Do enterochromaffin (EC) cells contain motilin? Arch. Histol. Jap. 43:85–98; 1980. 11. Kobayashi, S.; Uchida, T. Morphological identification of motilin in the gut. In: Itoh, Z., ed. Motilin. New York: Academic Press; 1990:53–72. 12. McIntosh, C. H. S.; Brown, J. C. Purification and chemical structure of porcine and canine motilin and evidence for the existence of motilin in other species. In: Itoh, Z., ed. Motilin. New York: Academic Press; 1990:13–30.
13. McIntosh, C. H. S.; Brown, J. C. Motilin: isolation, secretion, actions and pathophysiology. In: Scarpignato, C.; Bianchi Porro, G., eds. Frontiers of gastrointestinal research clinical investigation of gastric function. Basel: Karger; 1990:307– 352. 14. Nilsson, O.; Ericson, L. E.; Dahlstrom, A.; Ekholm, R.; Steinbusch, H. W. M.; Ahlman, H. Subcellular localization of serotonin immunoreactivity in rat enterochromaffin cells. Histochemistry 82:531–355; 1985. 15. Nilsson, O.; Ahlman, H.; Geffard, M.; Dahlstrom, A.; Ericson, L. E. Bipolarity of duodenal enterochromaffin cells in the rat. Cell Tissue Res. 248:49 –54; 1987. 16. Okumiya, K.; Matsubayashi, K.; Maeda, T.; Fujimiya, M. Change in subcellular localization of gastrin-like immunoreactivity in epithelial cells of rat duodenum induced by carbachol. Peptides 17:225–232; 1996. 17. Pearse, A. G. E.; Polak, J. M.; Bloom, S. R.; Adams, C.; Dryburgh, J. R.; Brown, J. C. Enterochromaffin cells of the mammalian small intestine as the source of motilin. Virchows Arch. B Cell Pathol. 16:111–120; 1974. 18. Peeters, T. L.; Depoortere, I. Motilin receptor: a model for development of prokinetics. Dig. Dis. Sci. 39:76S–78S; 1994. 19. Poitras, P. Motilin. In: Walsh, J. H.; Dockray, G. J., eds. Gut peptides: biochemistry and physiology. New York: Raven Press; 1994:261–304. 20. Polak, J. M.; Pearse, A. G. E.; Heath, C. Complete identification of endocrine cells in the gastrointestinal tract using semithin-thin sections to identify motilin cells in human and animal intestine. Gut 16:225–229; 1975. 21. Sakai, T.; Satoh, M.; Sonobe, K.; Nakajima, M.; Shiba, Y.; Itoh, Z. Autoradiographic study of motilin binding sites in the rabbit gastrointestinal tract. Regul. Pept. 53:249 –257; 1994. 22. Satoh, M.; Sakai, T.; Koyama, H.; Shiba, Y.; Itoh, Z. Immunocytochemical localization of motilin-containing cells in the rabbit gastrointestinal tract. Peptides 16:883– 887; 1995. 23. St. Pierre, S.; Brown, J. C.; McIntosh, C. H. S.; Poitras, P.; Aynsley, S. C. Comparison of synthetic rabbit and natural porcine motilin in a receptor binding assay. Regul. Pept. 40:255; 1992. 24. Tohyama, I.; Kameyama, M.; Kimura, H. Quantitative morphometric analysis of two types of serotonin-immunoreactive nerve fibers differentially responding to p-chlorophenylalinine treatment in the rat brain. Neuroscience 26:971–991; 1988. 25. Usellini, I.; Buchan, A. M. J.; Polak, J. M.; Capella, C.; Cornaggia, M.; Solcia, E. Ultrastructural localization of motilin in endocrine cells of human and dog intestine by the immunogold technique. Histochemistry 81:363–368; 1984.
PII S0196-9781(97)00260-X
Immuno-Electron Microscopic Study of Differential Localization of Motilin and Serotonin in the Rabbit Duodenal Epithelium MINEKO FUJIMIYA,*1 KIYOHITO OKUMIYA,† YIN NAM KWOK,‡ SERGE ST-PIERRE\ AND CHRISTOPHER H. S. McINTOSH‡ * Department of Anatomy, Shiga University of Medical Science, Otsu, Shiga 520-21, Japan † Department of Geriatrics, Kochi Medical School, Nankoku Kochi, 783 Japan ‡ Department of Physiology, University of British Columbia, Health Sciences Mall, Vancouver, BC V6T 1Z3 Canada // INRS-Sante´, Pointe-Claire, QC H9K1G6 Canada Received 17 March 1997; Accepted 13 August 1997 FUJIMIYA, M., K. OKUMIYA, Y. N. KWOK, S. ST-PIERRE AND C. H. S. McINTOSH. Immuno-electron microscopic demonstration of differential localization of motilin and serotonin in the rabbit duodenal epithelium. PEPTIDES 19(1) 65–73, 1998.—A guinea pig antibody against rabbit motilin was generated to study the localization of motilin-containing cells in the rabbit small intestine with special reference to the co-localization of motilin and serotonin. A pre- and post-embedding technique for immuno-electron microscopy was used; duodenal sections were stained with either motilin or serotonin in the pre-embedding DAB-nickel reaction, followed by subsequent staining of ultrathin sections of positive cells with either motilin or serotonin in the post-embedding immunogold reaction. Samples were divided into four groups: 1) pre-motilin, post-motilin, 2) pre-motilin, post-serotonin, 3) pre-serotonin, post-serotonin, and 4) pre-serotonin, post-motilin. Motilin-containing cells in the rabbit duodenum were characterized by round granules (395.3 6 66.1 nm in diameter) with medium electron density, located basally or in the perinuclear cytoplasm. In contrast, serotonin-containing cells were characterized by round to pleomorphic secretory granules (344.5 6 90.5 nm in diameter with electron dense cores and prominent halos. In motilin-containing cells, massive aggregations of immunogold particles reacted to motilin occurred over secretory granules. A few immunogold particles scattered diffusely over the cytoplasm reacted to serotonin; however, this reaction appeared to be background staining because the density was not changed if the section was treated by preabsorption. In serotonin-containing cells, immunogold particles reacted to serotonin were aggregated over the secretory granules and a large number of gold particles were scattered diffusely at the extragranular cytoplasm; however, very few or no immunogold particles were observed within the cells which reacted to motilin. Results of the present study indicate that motilin and serotonin are not co-localized in the epithelial endocrine cells of the rabbit intestine. © 1998 Elsevier Science Inc. Motilin
Serotonin
Immuno-electron microscopy
MOTILIN is a 22 amino acid peptide, synthesized in the small intestine (12,13,19), which has been demonstrated to be a major regulator of the interdigestive migrating motor complex (8,19). It was originally suggested that the biosynthesis of motilin took place in enterochromaffin (EC) cells (17,20). Subsequently a separate cell-type, the M (Mo)-cell,
Immunogold technique
Rabbit
Duodenum
was found to be the major source of motilin in all species examined (3,7). Evidence has been presented for the existence of immunoreactive (IR)-motilin, or a motilin-related peptide, in a subclass of EC cells, the presence of which can only be demonstrated with N-terminal directed antibodies (9). In addition, equivocal results have been obtained con-
1
Requests for reprints should be addressed to Mineko Fujimiya, M.D., Ph.D., Department of Anatomy, Shiga University of Medical Science, Seta, Otsu, Shiga 520-21, Japan, E-mail: [email protected]
65
66
cerning the possible co-localization of motilin and serotonin. These conflicting results may be partially due to the methods used in determining the localization of these substances, since most studies employed the techniques of formaldehyde-induced-fluorescence or argentaffinity to identify serotonin-containing EC cells instead of immunohistochemistry (3,6,10,20), and all used heterologous motilin antisera. Although specific antibody against serotonin has been used in some studies, the distribution of motilinand serotonin-containing cells has only been compared using light microscopy (9). The intestinal actions of motilin and characterization of its receptors have been studied extensively in the rabbit (2,18,21), but information on the cellular localization of IR-motilin in this species are lacking, with the exception of one recent report (22). In that report, distribution of IR-motilin cells was investigated throughout the rabbit gastrointestinal tract; however, possible colocalization of serotonin and motilin in the rabbit intestine was not mentioned. We recently isolated a rabbit motilin cDNA (1), and produced a synthetic peptide based on the predicted sequence (23). Antibodies against this peptide have now been generated, and the objective of the present study was to examine the localization of serotonin and motilin in the epithelial endocrine cells of the rabbit duodenum at the electron microscopic level, with special reference to their possible co-localization. Pre- and post-embedding techniques for immuno-electron microscopy were used; this allowed for the simultaneous reaction (DAB-nickel and immunogold) of different antibodies on a single endocrine cell, thus providing a means to examine the possible colocalization of different antigens. METHOD Production of Motilin Antibody Antisera against rabbit motilin were generated in guinea pigs using synthetic rabbit motilin (INRS-Sante´, Quebec) conjugated to keyhole limpet hemocyanin (1:8 w/w; Calbiochem, La Jolla, CA) using 1-ethyl-3-3-(dimethylaminopropyl) carbodiimide hydrochloride (Pierce, Rockford, IL). The antiserum used in the present study (KC02) was produced after two booster injections. The specificity of this antiserum was checked by using the preabsorption test as described below. The antiserum was also shown not to react with other endocrine peptides such as gastric inhibitory polypeptide, glucagon and secretin as determined by radioimmunoassays. Animals used in the present study were cared for in accordance with the principles of the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care, 1993).
FUJIMIYA ET AL.
Pre- and Post-Embedding Immuno-Electron Microscopic Study for Serotonin and Motilin Two male Japanese white rabbits each weighing 2 kg were anesthetized with pentobarbital (Nembutal®, 50 mg/kg). The animals were perfused systemically via the left ventricle with 2.21 of 0.01 M phosphate buffered saline (PBS) (0.01 M phosphate buffer containing 0.9% NaCl; pH 7.4) at a speed of 300 ml/min and then perfused with 3 l of a cold fixative solution consisting of 0.1 M phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde, 0.5% glutaraldehyde and 0.2% picric acid. The duodenal segment between 5–10 cm distal to the pylorus was then removed, cut open along the mesenterium, stretched on a cork plate, and immersed for 24 h in a post-fixative solution of 0.1 M PB (pH 7.4) containing 4% paraformaldehyde and 0.2% picric acid at 4°C. The duodenum was washed for 4 days with several changes of 0.1 M PB containing 15% sucrose and then dipped in 0.1 M PB containing 10% gelatin at 37°C overnight. The gelatin-embedded specimen was cooled to 4°C, fixed for 3 h with 4% paraformaldehyde in 0.1 M PB, cut into 50-mm thick sections on a vibratome, and the sections were collected in 0.1 M PBS at 4°C. The duodenal sections were treated in the free-floating state throughout the immunohistochemical procedures. They were first pretreated for 10 min with a mixture of 0.01% trypsin (Sigma Chemical Co., St. Louis, MO, USA) and 0.68 mM CaCl2 in 0.05 M Tris-HCl buffer (pH 7.6) at 20°C and washed for 30 min with 0.1 M PBS. Sections were incubated with either motilin antibody (KC02; diluted 1:5000 in 0.1 M PBS) or serotonin antibody (monoclonal antibody generated in a mouse, the detail of immunological property was described in our previous paper (24), diluted 1:10,000 in 0.1 M PBS) for 48 h at 4°C. To inactivate endogenous peroxidase activity, the sections were incubated at room temperature (RT) for 20 min with 0.1% H2O2 in 0.1 M PBS followed by an additional 20 min with 0.1% phenylhydrazine in 0.1 M PBS. After washing for 30 min with 0.1 M PBS, the sections were incubated at RT for 2 h in either biotinylated anti-guinea pig IgG (for motilin staining; Vector Lab. Inc., USA) or biotinylated anti-mouse IgG (for serotonin staining; Vector) diluted 1:1000 in 0.1 M PBS. Sections were then washed and placed in avidin-biotin peroxidase complex (Elite, Vector; 1:2000 in 0.1 M PBS) for 2 h at RT. The immunoreaction was then visualized by reacting with 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01% 3, 3’-diaminobenzidine, 1% ammonium nickel sulfate and 0.0003% H2O2 for 30 min at RT. For light microscopic examination, the stained sections were mounted on gelatin-coated glass slides, dehydrated by graded ethanol, and coverslipped with Entellan® (Merk, Darmstadt, Germany). For the electron microscopic study, serotonin or motilin stained sections were washed for 10 min with 0.1 M PBS, followed by an additional 10 min with distilled water, and dehydrated by a graded series of ethanol as described in our
STUDY FOR MOTILIN AND SEROTONIN
previous paper (16). These sections were then incubated overnight with LR Gold resin (London Resin Co. Ltd., Berkshire, England) at 220°C and incubated for an additional 1 h with LR Gold resin containing 0.1% Benzil (Pelco®, Ted Pella, Inc.) at 220°C. They were then mounted on silicon-coated glass slides and embedded in LR Gold resin containing 0.1% Benzil and covered with silicon-coated cover glasses at 220°C. The embedded specimens were allowed to polymerize for 4 h in an Ultraviolet Cryo Chamber (Pelco®, Ted Pella Inc, CA), where the temperature was kept at 220°C throughout the polymerization period. After removal of the cover glasses, the polymerized sections were examined under light microscopy (3 40) and the intestinal mucosa which contains motilin or serotonin positive cells was dissected out with a razor blade. Ultra-thin sections (60 nm) were cut on an ultramicrotome (Reichert-Jung Ultracut E, Austria) and picked up on nickel grids (thin bar grid, 200-mesh; Nishin EM Co., Tokyo, Japan) supported by collodion. The nickel grids were incubated with 3% normal goat serum (Dako Japan, Tokyo, Japan) dissolved in a reaction buffer (0.1 M PBS containing 0.2% BSA, 0.2% saponin and 0.05% NH4Cl, pH 7.4) for 30 min at RT. The ultrathin sections stained for motilin or serotonin in the pre-embedding procedures were then stained again with either motilin or serotonin in the post-embedding immunohistochemical procedures. The combination was as follows; 1) pre-motilin, post-motilin, 2) pre-motilin, post-serotonin, 3) pre-serotonin, post-serotonin, 4) pre-serotonin, post-motilin. The sections were incubated with antibody against either motilin (1:40) or serotonin (1:300) diluted with the reaction buffer. The sections were washed with 0.1 M PBS and incubated for 1.5 h with either immunogold conjugated goat antiguinea pig IgG (for motilin staining; 10 nm gold, British BioCell International, Cardiff, UK) or immunogold conjugated goat anti-mouse IgG (for 5HT staining; 15 nm gold, British BioCell) 1:40 diluted with the reaction buffer at RT, followed by washings with 0.1 M PBS and then with distilled water. The sections were stained for 10 min at RT with 2% uranyl acetate followed by an additional 5 min with Reynold’s solution. During the above procedures, the nickel grids were floating on drops of incubation medium with the sections on the underside or submerged in the washing media. The stained sections were then examined under electron microscopy (H-7100, Hitachi, Ltd., Japan). In the electron microscopic study, more than 20 motilincontaining cells and more than 30 serotonin-containing cells were observed. One hundred secretory granules located at the basal or the perinuclear region of the cytoplasm were randomly selected from either serotonin- or motilin-containing cells, and the mean diameter of the granules was calculated. In serotonin-containing cells, an additional 50 secretory granules located at the apical cytoplasm were selected, and the mean diameter was measured.
67
Specificity of the Positive Staining The antibody specificity was checked by both radioimmunoassay and immunohistochemical absorption study. As described above, motilin antibody did not crossreact with gastric inhibitory peptide, glucagon and secretin examined by radioimmunoassay. Immunohistochemical absorption study was performed in both pre-embedding and post-embedding procedures, where the primary antibody against motilin or serotonin was substituted with an antibody preabsorbed with the specific antigen. In the pre-embedding staining, the antibodies for motilin (1:5,000) and serotonin (1:10,000) were preabsorbed with 0.01 mM rabbit motilin and 0.1 mM serotonin creatinin sulfate (Merck, Darmstedt, Germany), respectively. In the post-embedding staining, the antibodies for motilin (1:40) and serotonin (1:300) were preabsorbed with 0.2 mM rabbit motilin and 10 mM serotonin creatinin sulfate, respectively. In both steps, the antigen and antibody mixture was incubated for 48 h at 4°C before incubating with the sections. We obtained the negative DAB reactions for motilin and serotonin at light microscopic levels and also in the electron microscopic immunogold reactions as shown in the results. RESULTS Motilin immunoreactive cells were scattered in the mucosal epithelium, and were spindle-like shapes with cytoplasmic processes extending to the intestinal lumen. When observed under electron microscopy, the majority of motilin-containing cells in the rabbit duodenum were similar in morphology. These cells contained round and relatively large granules (395.3 6 66.1 nm in diameter, ranging from 250 to 450 nm, n 5 100) with medium electron density. The granules were packed in the basal and supranuclear region of the cells (Figs. 1A, 1B). Examination under higher magnification revealed that the dense aggregation of immunogold particles was located over the secretory granules situated at both basal and supranuclear cytoplasm (Figs. 1C, 2A). Only a few immunogold particles were detected over the nucleus, other organelles, the extracellular space, or the extragranular matrix of the cytoplasm (Figs. 1C, 2A). To confirm the specificity of the immunogold reaction detected over the secretory granules, ultrathin sections were stained with antibody against motilin which had previously been absorbed with 0.2 mM motilin. Following this treatment, the immunogold reaction over the secretory granules was no longer detectable (Fig. 2D, compared with Fig. 2A). However, the motilin-containing cells were easily identified by the dark DAB-nickel reaction materials precipitated over some of the secretory granules or the cytoplasm (Fig. 2D). To examine the possibility of co-localization of serotonin and motilin in the epithelial cells, duodenal sections previously stained with motilin in the pre-embedding immunohistochemical procedure using the DAB-nickel reaction were additionally stained with serotonin antibody in the
68
FUJIMIYA ET AL.
FIG. 1. Electron micrographs of motilin-containing cells in the rabbit duodenum stained with motilin in both pre- and post-embedding immunohistochemical procedures. Bars 5 1 mm. (A) Round and solid secretory granules were packed in the basal cytoplasm. (B) Secretory granules are located at the supranuclear region in addition to the basal cytoplasm. (C) Higher magnification view of the area enclosed by the rectangle in (A). Immunogold particles are aggregated over the secretory granules; very few of them are located in the extragranular spaces.
STUDY FOR MOTILIN AND SEROTONIN
69
FIG. 2. Electron micrographs of higher magnification view of motilin-containing cells in the rabbit duodenum. All sections are stained with motilin in the pre-embedding immunohistochemical procedure, and stained with motilin (A and D), or serotonin (B and C) in the post-embedding procedure. Motilin-positive cells are easily identified under electron microscopy by dark DAB-nickel reaction products precipitating over secretory granules or cytoplasm (arrow heads). Bars 5 1 mm. (A) Immunogold particles reacted to motilin are densely aggregated over the secretory granules; very few of them located at the extragranular cytoplasm. (B) A small number of immunogold particles reacted to serotonin are scattered diffusely at the cytoplasm. (C) A section stained with serotonin antibody in the postembedding procedure, previously absorbed with 10 mM of serotonin. The density of immunogold particles reacted to serotonin does not change from that seen in (B). (D) A section stained with motilin antibody in the post-embedding procedure, previously absorbed with 0.2 mM motilin. Immunogold particles reacted to motilin seen in (A) have completely disappeared.
70
FUJIMIYA ET AL.
FIG. 3. Electron micrographs of serotonin-containing cells in the rabbit duodenum, stained with serotonin in both pre- and post-embedding immunohistochemical procedures. Bars 5 1 mm. (A) Round or pleomorphic shaped secretory granules are situated in both basal and apical cytoplasm of the cell. (B) Aggregation of secretory granules of various size and electron density is seen at the apical cytoplasm. Immunogold particles are concentrated over the secretory granules, however a considerable number of immunogold particles are scattered diffusely over the extragranular matrix including microvilli (arrow heads).
post-embedding immuno-electron microscopic procedure using the immunogold reaction. The motilin-containing cells were easily identified under electron microscopy as precipitated dark products present over the cytoplasm, resulting from DAB-nickel reaction (Fig. 2B). A small number of serotonin-reactive immunogold particles were scattered throughout the cytoplasm as well as over the secretory granules (Fig. 2B), however the density was quite low when compared to the motilin-immunoreactive particles seen over the secretory granules in Fig. 2A. To examine the specificity of the immunogold reaction for serotonin in the motilincontaining cells, the ultrathin sections were stained with the antibody against serotonin which was previously absorbed with 10 mM of serotonin. No apparent changes was observed in the density of immunogold particles which reacted
to serotonin, even though the sections were treated with antibody previously absorbed with serotonin (Fig. 2C). The ultrastructural characteristics as well as the intracellular localization of immunogold reactions in serotonincontaining cells were completely different from those in motilin-containing cells. Serotonin-containing cells in the rabbit duodenal epithelium contained round or pleomorphic secretory granules with relatively high electron density (Figs. 3A, 3B, 4A). A considerable number of serotonincontaining cells possessed secretory granules at both apical and basal cytoplasm (Figs. 3A, 3B). The size and the electron density of the secretory granules varied considerably within a single cell as shown in Figs. 3A and 3B; the sizes of the secretory granules located in the basal and apical cytoplasm were 344.5 6 90.5 nm (range 200 – 490
STUDY FOR MOTILIN AND SEROTONIN
FIG. 4. Electron micrographs showing higher magnification view of serotonin-containing cells in the rabbit duodenum. All sections are stained with serotonin in the pre-embedding procedure, and stained with serotonin (A, A9 and C) and motilin (B) in the post-embedding procedure. Bars 5 1 mm. (A) Round granules with cores of high electron density and prominent halos are seen at the basal cytoplasm. Immunogold particles are packed diffusely within the cytoplasm, but very few of them are seen outside of the cell. (A9) Higher magnification view of the area enclosed by the rectangle in (A). Immunogold particles reacted to serotonin are concentrated over cores of secretory granules; however, a large number of gold particles are scattered at the extragranular matrix. (B) Very few immunogold particles reacted to motilin are seen. (C) A section reacted with serotonin antibody in the postembedding procedure, which is preabsorbed with 10 mM serotonin. Most of the immunogold reaction observed in (A9) disappeared and a few gold particles are scattered in the cytoplasm.
71
72
nm, n 5 100) and 344.0 6 90.7 nm (range 200 –500 nm, n 5 50), respectively. The pattern of immunogold reaction within the serotonin-containing cells differed from that of motilin-containing cells. Immunogold particles reacted to serotonin were densely aggregated over the cores of the secretory granules, and a large number of immunogold particles were scattered diffusely at the extragranular space of both apical and basal cytoplasm of the cells (Figs. 3A, 3B, 4A9). The specificity of the immunogold reaction was examined in these sections using the preabsorption test. Most of the immunogold reaction observed in Fig. 4A9 disappeared and a few gold particles were scattered in the cytoplasm (Fig. 4C). To examine the co-localization of serotonin and motilin further, additional experiments were performed, in which the ultrathin sections previously stained with serotonin in the pre-embedding procedure were subsequently stained with motilin in the post-embedding procedure. Very few immunogold particles which reacted to motilin were observed throughout the cell body (Fig. 4B). DISCUSSION Previous electron microscopic studies have demonstrated the presence of motilin-containing cells in intestines of human and dogs, but not in rabbits. In the human intestine, motilin-containing cells are characterized by the presence of small (180 nm in diameter), round and solid granules with closely applied limiting membrane (25), while in the dog intestine the granules are of medium size (200 nm in diameter) and pleomorphic (11,25). The present study shows that motilin-containing cells in the rabbit duodenum are characterized by relatively large (395.3 6 66.1 nm in diameter) round granules with closely applied limiting membrane. This finding suggests that the ultrastructural characteristics of the secretory granules appear to be of little value in identifying endocrine cells. In our preliminary study, we examined the distribution of motilin-immunoreactive cells in the duodenum, jejunum, ileum and colon of rabbits. The density of motilin immunoreactive cells was highest in the duodenum, and decreased gradually toward the lower portion of the intestine. This observation was consistent with the data previously reported in the rabbit intestine (22). Within the duodenum, motilin positive cells scattered diffusely in the epithelium and the density was highest in the duodenal bulb. Although the cell number was smaller than that in serotonin containing cells, the distribution pattern in the duodenum was similar in both serotonin and motilin containing cells. To examine the co-localization of serotonin and motilin, the pre- and post-embedding method for immuno-electron microscopy was used, and samples were divided into four groups: 1) pre-motilin, post-motilin, 2) pre-motilin, postserotonin, 3) pre-serotonin, post-serotonin and 4) pre-serotonin, post-motilin. Our previous study has shown that
FUJIMIYA ET AL.
antigenicity was well preserved for post-embedding staining, even though the sections were previously reacted with the same antibody (16). Motilin-containing cells were easily identified under electron microscopy by dark nickel-DAB reaction products precipitated at the cytoplasm. Massive aggregation of immunogold particles reacted to motilin were observed over the secretory granules. Since most of the gold particles disappeared in the sections pre-treated with antibody absorbed with motilin, such reactions are likely to be specific for motilin. When ultrathin sections of motilin-containing cells were stained with serotonin, a few immunogold particles were scattered throughout the cytoplasm; however, this is likely to be non-specific or representing background staining, since the staining remained after the preabsorption test. These results show that motilin containing cells in the rabbit duodenum contain motilin within the secretory granules; however, they do not contain serotonin. The ultrastructural characteristics of serotonin-containing cells varied widely in the rabbit intestine. Some cells contained pleomorphic shaped secretory granules, whereas others contained round granules that were basically electron dense with prominent halos. Most characteristically, in some serotonin-containing cells, a considerable number of secretory granules were located at the apical cytoplasm as well as at the basal cytoplasm of the cells. Such bipolar distribution of secretory granules has also been reported in the rat entero chromaffin (EC) cells (4,5,15). In most serotonin-containing cells, immunogold particles reacted to serotonin were mainly aggregated over the secretory granules; however a considerable number of gold particles were diffusely scattered over the extragranular space of the cytoplasm. These extragranular immunogold reactions were not likely the background staining but specific for serotonin for the following reasons: they disappeared following preabsorption of the antibody with serotonin. Such extragranular localization of 5HT has been reported in EC cells of rats (4,5,14) and assigned to be an intracytoplasmic release of 5HT from secretory granules (5). When ultrathin sections of serotonin-containing cells were stained with motilin, very few or no immunogold particles reacted to motilin were observed, suggesting that serotonin-containing cells in rabbit intestine do not contain motilin. The present study, using immuno-electron microscopy, provides evidence that serotonin and motilin are located in completely separate cells in the rabbit duodenum, i.e., they do not co-localize. This is in disagreement with previous reports indicating that motilin is located in a subtype of EC cell (6,9,17,20). This discrepancy may be partially explained by the differences in techniques used. In the previous studies, heterologous motilin antisera were used, and EC cells were identified by the nature of argentaffinity of
STUDY FOR MOTILIN AND SEROTONIN
73
cells or particular pleomorphic granules at the ultrastructural level rather than serotonin immunoreactivity. In the present study, immuno-electron microscopy was used to define the EC cell localization of serotonin. This reveals that various shapes of secretory granules were present in serotonin-containing cells, and a lack of immunoreactivity for motilin. In conclusion, by using pre- plus post-embedding immuno-electron microscopic procedures the separate cellular localization of serotonin and motilin was demonstrated
in the duodenal epithelium of the rabbit, and the ultrastructure of these cells was examined. ACKNOWLEDGEMENTS These studies were supported by Grant-In-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture, Japan (MF, KO) and British Columbia Health Research Foundation (YNK, CHSM). The authors thank Mr. Takefumi Yamamoto in the Central Research Laboratory of Shiga University of Medical Science for his excellent technical support in the electron microscopic study.
REFERENCES 1. Banfield, D. K.; MacGillivray, R. T. A.; Brown, J. C.; McIntosh, C. H. S. The isolation and characterization of rabbit motilin precursor cDNA. Biochem. Biophys. Acta. 1131:341– 344; 1992. 2. Depoortere, I.; Peeters, T. L.; Vantrappen, G. Solubilization and characterization of motilin receptor complexes from rabbit antral smooth muscle tissue. J. Recept. Res. 13:903–923; 1993. 3. Forssmann, W. G.; Yanaihara, N.; Helmstaedter, V.; Grube, D. Differential demonstration of the motilin-cell and the enterochromaffin-cell. Scand. J. Gastroenterol. 11(suppl. 39): 43– 45; 1976. 4. Fujimiya, M.; Okumiya, K.; Maeda, T. Immuno-electron microscopic demonstration of luminal release of serotonin from enterochromaffin cells of rat embryo. Acta Histochem. Cytochem. 28:555–563; 1995. 5. Fujimiya, M.; Okumiya, K.; Kuwahara A. Immunoelectron microscopic study of the luminal release of serotonin from rat enterochromaffin cells induced by high intraluminal pressure. Histochem. Cell Biol. 8:105–113; 1997. 6. Heitz, P. U.; Kasper, M.; Krey, G.; Polak, J. M.; Pearse, A. G. E. Immunoelectron cytochemical localization of motilin in human duodenal enterochromaffin cells. Gastroenterology 74:713–717; 1978. 7. Helmstaedter, V.; Kreppein, W.; Domschke, W.; Mitznegg, P.; Yanaihara, N.; Wu¨nsch, E.; Forssmann, W. G. Immunohistochemical localization of motilin in endocrine non-enterochromaffin cells of the small intestine of humans and monkey. Gastroenterology 76:897–902; 1979. 8. Itoh, Z. Effect of motilin on gastrointestinal motor activity in the dog. In: Itoh, Z., ed. Motilin. New York: Academic Press; 1990:133–153. 9. Kishimoto, S.; Polak, J. M.; Buchan, A. M. J.; Verhofstad, A. A. J.; Steinbusch, H. W. M.; Yanaihara, N.; Bloom, S. R.; Pearse, A. G. E. Motilin cells investigated by the use of region-specific antisera. Virchow Arch [Cell Pathol] 36:207– 218; 1981. 10. Kobayashi, S.; Iwanaga, T.; Fujita, T.; Yanaihara, N. Do enterochromaffin (EC) cells contain motilin? Arch. Histol. Jap. 43:85–98; 1980. 11. Kobayashi, S.; Uchida, T. Morphological identification of motilin in the gut. In: Itoh, Z., ed. Motilin. New York: Academic Press; 1990:53–72. 12. McIntosh, C. H. S.; Brown, J. C. Purification and chemical structure of porcine and canine motilin and evidence for the existence of motilin in other species. In: Itoh, Z., ed. Motilin. New York: Academic Press; 1990:13–30.
13. McIntosh, C. H. S.; Brown, J. C. Motilin: isolation, secretion, actions and pathophysiology. In: Scarpignato, C.; Bianchi Porro, G., eds. Frontiers of gastrointestinal research clinical investigation of gastric function. Basel: Karger; 1990:307– 352. 14. Nilsson, O.; Ericson, L. E.; Dahlstrom, A.; Ekholm, R.; Steinbusch, H. W. M.; Ahlman, H. Subcellular localization of serotonin immunoreactivity in rat enterochromaffin cells. Histochemistry 82:531–355; 1985. 15. Nilsson, O.; Ahlman, H.; Geffard, M.; Dahlstrom, A.; Ericson, L. E. Bipolarity of duodenal enterochromaffin cells in the rat. Cell Tissue Res. 248:49 –54; 1987. 16. Okumiya, K.; Matsubayashi, K.; Maeda, T.; Fujimiya, M. Change in subcellular localization of gastrin-like immunoreactivity in epithelial cells of rat duodenum induced by carbachol. Peptides 17:225–232; 1996. 17. Pearse, A. G. E.; Polak, J. M.; Bloom, S. R.; Adams, C.; Dryburgh, J. R.; Brown, J. C. Enterochromaffin cells of the mammalian small intestine as the source of motilin. Virchows Arch. B Cell Pathol. 16:111–120; 1974. 18. Peeters, T. L.; Depoortere, I. Motilin receptor: a model for development of prokinetics. Dig. Dis. Sci. 39:76S–78S; 1994. 19. Poitras, P. Motilin. In: Walsh, J. H.; Dockray, G. J., eds. Gut peptides: biochemistry and physiology. New York: Raven Press; 1994:261–304. 20. Polak, J. M.; Pearse, A. G. E.; Heath, C. Complete identification of endocrine cells in the gastrointestinal tract using semithin-thin sections to identify motilin cells in human and animal intestine. Gut 16:225–229; 1975. 21. Sakai, T.; Satoh, M.; Sonobe, K.; Nakajima, M.; Shiba, Y.; Itoh, Z. Autoradiographic study of motilin binding sites in the rabbit gastrointestinal tract. Regul. Pept. 53:249 –257; 1994. 22. Satoh, M.; Sakai, T.; Koyama, H.; Shiba, Y.; Itoh, Z. Immunocytochemical localization of motilin-containing cells in the rabbit gastrointestinal tract. Peptides 16:883– 887; 1995. 23. St. Pierre, S.; Brown, J. C.; McIntosh, C. H. S.; Poitras, P.; Aynsley, S. C. Comparison of synthetic rabbit and natural porcine motilin in a receptor binding assay. Regul. Pept. 40:255; 1992. 24. Tohyama, I.; Kameyama, M.; Kimura, H. Quantitative morphometric analysis of two types of serotonin-immunoreactive nerve fibers differentially responding to p-chlorophenylalinine treatment in the rat brain. Neuroscience 26:971–991; 1988. 25. Usellini, I.; Buchan, A. M. J.; Polak, J. M.; Capella, C.; Cornaggia, M.; Solcia, E. Ultrastructural localization of motilin in endocrine cells of human and dog intestine by the immunogold technique. Histochemistry 81:363–368; 1984.