In� ammopharmacology, Vol. 10, No. 4–6, pp. 495–503 (2002)Ó VSP 2002.Also available online - www.vsppub.com

Original study

Autoradiographic and confocal laser microscopicobservation show lafutidine binds toCGRP-immunoreactive nerves as well as gastric parietalcells

MASAHIKO NAKAMURA1;¤, YASUTADA AKIBA 2, HIDENORI MATSUI 3,HIROMASA ISHII 2 and KANJI TSUCHIMOTO4

1 Center for Basic Research, the Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo, Japan2 Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan3 Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan4 Kitasato Institute Hospital, Tokyo, Japan

Received 4 October 2002; accepted 6 November 2002

Abstract—The present study was undertaken to observe the binding sites of lafutidine, a newly in-vented H2 receptor antagonist ((C=¡)-2-(furfurylsul�nyl)-N-[4-[4-(piperidinomethyl)-2-pyridyl]oxy-(Z)-2 butenyl] acetamide), in the Mongolian gerbil and human gastric mucosa using un� xed cryostatsection or incubation with aqueous solution of tritiated lafutidine, followed by in vitro autoradio-graphy or autoradiographyof soluble compounds. The localization of calcitonin gene-related peptideimmunoreactivitywas compared with the lafutidinebinding sites. As a result, lafutidine-speci�c bind-ing sites in the body of the fundic glands were accumulated on the parietal cells, while in the neckand base of the fundic glands, lafutidine was found to bind to the CGRP immunoreactive nerves. Inthe human fundic mucosa, the lafutidine bindings were also observed on the enteric nerves as wellas the parietal cells. In conclusion, autoradiographic studies have shown that lafutidine effector sitescoincided with the CGRP-immunoreactive nerves as well as the parietal cells.

Key words: Mongolian gerbil; human; stomach; lafutidine; cimetidine; autoradiography; entericnerve; CGRP; parietal cell.

1. INTRODUCTION

Lafutidine, (C=¡)-2-(furfurylsul� nyl)-N-[4-[4-(piperidinomethyl)-2-pyridyl]oxy-(Z)-2 butenyl] acetamide, is a newly invented H2 receptor antagonist having charac-teristics of both an acid suppressant and a mucoprotective agent. The cytoprotective

¤To whom correspondence should be addressed. E-mail: nakamura-m@kitasato.or.jp

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effect of lafutidine is thought to be dependent on capsaicin-sensitive nerves and cal-citonin gene-related peptide (CGRP) because systemic treatment of capsaicin andadministration of CGRP receptor antagonist abolish these effects (Kato et al., 2000;Onodera et al., 1995, 1999; Umeda et al., 1999), while the precise morphologicalidenti� cation of the effector sites of lafutidine remains to be elucidated.

Thus, the present study was undertaken to observe the localization of tritiatedlafutidine in the Mongolian gerbil and human gastric mucosa using the combinationof in vitro autoradiography, immunohistochemistry and confocal laser microscopyor autoradiography of soluble compounds by light and electron microscopy.

2. MATERIALS AND METHODS

2.1. Animals

In the animal study, � ve fasted male Mongolian gerbils, weighing 50–100 g, wereused in the following experiments. In addition, human gastric mucosa was obtainedendoscopically after written informed consent from gastric ulcer patients with orwithout Helicobacter pylori (Hp) infection, revealed by the CLO test.

2.2. Procedures for in vitro autoradiography

In vitro autoradiography was performed on cryostat sections of un� xed stomach tis-sues. An aqueous solution of [3H]lafutidine (speci� c activity 389 GBq/mmol, 37MBq/1 ml physiological saline) with and without unlabelled lafutidine or cimeti-dine (100-times higher concentration) was dripped onto the specimens for 30 minand rinsed in physiological saline and � xed with Zamboni’s � xative. Autoradi-ographic emulsion � lms (Konica NR-M2) were applied by the dipping method.After 30-day exposure, the specimens were developed, � xed, and observed bylight microscopy. Some of these sections were then treated with monoclonal an-tibodies against HC,KC-ATPase or calcitonin gene-related peptide (CGRP) (Af� nitiResearch Products, UK) by the indirect immunohistochemical procedures and ob-served with confocal laser microscopy (Leica NT).

2.3. Computer-assisted analysis of autoradiographs

For grain counting, images were digitized under image processing and analysissoftware (Ultimage, Graftek, France). The images were managed in the computerat 512 £ 512-pixel matrices with 256 gray levels. Three hundred images weretransferred for analysis of grains. The area containing silver grains was calculatedusing a gray-level threshold. Student’s t-test was used for statistical analysis.

2.4. Administration of [3H]lafutidine and tissue preparation for autoradiographyof soluble compounds

Gerbils were placed under light anesthesia by intraperitoneal injection of sodiumpentobarbital (20 mg/kg body weight). The abdomen was opened with a lower

Lafutidine binding site in gastric mucosa 497

midlline incision and a polyethylene catheter was inserted into the abdominal aortaup to the opening of the superior mesenteric artery. A dose of 0.5 ml of an aqueoussolution of [3H]lafutidine (speci� c activity 389 GBq/mmol, 37 MBq/100 g b.w.)was infused over 15 min through the intra-aortic catheter, followed by infusion of0.5 ml of physiological saline.

The tissues were excised and cut into small tissue blocks with a razor blade.These blocks were quick-frozen in isopentane cooled to its melting point with liquidnitrogen. The tissue blocks were freeze-dried for 48 h in a special apparatus (OkaScience OTD-1SF) cooled to ¡50±C and evacuated at 9 £ 10¡3 Torr. The freeze-dried tissue blocks were then exposed to osmium vapor, followed by in� ltration withEpon using a dripping unit evacuated by a rotary pump. Then, the tissue blocks wereplaced in a freshly prepared Epon, and Epon was polymerized at 60±C. Semithinand ultrathin sections were cut with an LKB ultramicrotome using ethylene glycolinstead of water (Nagata et al., 1969; Nakamura et al., 1998).

Autoradiographic emulsion (Konica NR-H2 or M2) was diluted (1 : 2 or 1 : 3) withdistilled water at 40±C in a darkroom. Emulsion � lms were made between vinyl-coated iron wire-loops by dipping the wire loops into a solution of autoradiographicemulsion containing dioctyl sodium sulfosuccinate, with the wire-loops left to standat room temperature until the emulsion � lms were almost dry. The emulsion � lmswere then applied to the glass plates or grids on which the sections were mounted.For exposure, the sections were kept in the dark in a refrigerator at 4±C for 4 to8 weeks. After development and � xation, they were observed by light and electronmicroscopy.

3. RESULTS

3.1. Lafutidine binding sites in Mongolian gerbil fundic mucosa

Lafutidine binding sites in the body of the fundic glands were accumulated on theparietal cells (Figs 1 and 2). In the serosal layer the binding sites of lafutidine wereseen on the CGRP-immunoreactive nerve bundles and ganglions (Fig. 3), and in theneck portion of the fundic glands lafutidine was found to bind to the enteric nervoussystem including CGRP-immunoreactive nerve � bers (Fig. 4). The addition of coldligand revealed that the binding of [3H]lafutidine to the parietal cell was blockedby cimetidine, while that to the CGRP-immunoreactive nerves was not inhibited,suggesting the latter is speci� c to lafutidine (Fig. 5).

3.2. Lafutidine binding sites in human fundic mucosa

In the biopsied human fundic mucosa, [3H]lafutidine binding sites were accumu-lated on the parietal cells in the middle and base of the fundic gland (Figs 6and 7) and CGRP-immunoreactive nerve � bers (Fig. 8). By electron microscopy,[3H]lafutidine binding sites were mostly seen on the mesenchymal cells in the lam-ina propria mucosae, coincided with the localization of the enteric nerve � bers

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Figure 1. Confocal laser micrograph (£400/ of [3H]lafutidine-binding sites in control Mongoliangerbil fundic mucosa. In the middle of the fundic glands, HC,KC-ATPase immunoreactivity isrecognized in the parietal cells (P) (a). The silver grains showing the localization of [3H]lafutidinebinding sites are localizedon these cells (b). These cells have strong rhodamine-phalloidinbinding (c).

Figure 2. As Fig. 1, but £1000.

Figure 3. Confocal laser micrograph (£100) of [3H]lafutidine-binding sites in control Mongoliangerbil fundic mucosa. In the nerve bundles and ganglion cells (G) surrounding the arteries in theserosal layer, CGRP immunoreactivity is recognized (left panel). [3H]lafutidinebinding sites (arrows)are seen on these cells (middle panel). These cells show rhodamine–phalloidin binding (right panel).

Lafutidine binding site in gastric mucosa 499

Figure 4. Confocal laser micrograph (£1500) of [3H]lafutidine-binding sites in control Mongoliangerbil fundic mucosa. In the neck portion of the fundic gland, the CGRP-immunoreactive nerve � bersare seen (arrows) (left panel). Silver grains (arrows) are recognized on these nerve � bers (middlepanel). Rhodamine phalloidin bindings are weakly found on the mucous neck cells (right panel).

Figure 5. Effect of cold lafutine and cimetidine on [3H]lafutidinebinding in Mongolian gerbil fundicmucosa. Values are mean § SD (n D 5). The background counting is excluded from each value.¤P < 0:001.

(Fig. 9). By comparison with Hp-negative and positive cases, a similar tendencywas observed in the distribution of lafutidine binding sites on the parietal cells andCGRP-immunoreactive nerve � bers. No signi� cant difference was detected as to the

500 M. Nakamura et al.

Figure 6. Confocal laser and electron micrograph (£1000) of [3H]lafutidine-bindingsites in humanfundic mucosa. In the base of the fundic gland, HC,KC-ATPase immunoreactivity is recognized insome of the parietal cells (P) (a), and the silver grains showing the binding sites of [3H]lafutidine aremostly seen near the basal plasma membrane of the parietal cells (b).

Figure 7. Confocal laser and electron micrograph of [3H]lafutidine-binding sites in human fundicmucosa. In the Epon-embedded 1-¹m sections, the silver grains showing the binding sites (arrows)are seen in the cytoplasmof the parietal cells near the basal plasma membrane. (a) £2000; (b) £6000.

Figure 8. Confocal laser and electron micrograph of [3H]-lafutidine-bindingsites in human fundicmucosa. In the neck portion of the fundic gland, CGRP-immunoreactive nerve � bers are richlydistributed (a). The silver grains are localized near these nerves (b). £1000.

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Figure 9. Confocal laser and electron micrograph of [3H]lafutidine-binding sites in human fundicmucosa. In the Epon-embedded 1-¹m sections, the silver grains are recognized on the mesenchymalcells just adjacent to the fundic glandular cells (arrows) (a). By electron microscopy, the silver grainsare seen on the mesenchymal cells (b). The CGRP-immunoreactive nerve � bers are seen in the laminapropria mucosae just adjacent to the intramucosal smooth muscle cells (c). (a) £800; (b) £12 000;(c) £12000.

Figure 10. Comparison of [3H]lafutidine binding sites in the Hp-positive and negative cases. Valuesare mean § SD (n D 5). The background counting is excluded from each value. ¤P < 0:001.

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localization of the [3H]lafutidine binding sites between the Hp-positive and negativecases (Fig. 10).

4. DISCUSSION

In the present study, we demonstrated that lafutidine binding sites were recognizednot only on the parietal cells but on or near the CGRP-immunoreactive nerve� bers. This result mostly coincides with the pharmacological observations thatlafutidine protects the epithelial cells from noxious agent through the mechanismof capsaicin-sensitive afferent nerves (Onodera et al., 1999). We also observed thatlafutidine bound to the parietal cells in the Mongolian gerbil and human fundicmucosa and this binding was inhibited by cimetidine, which also support the potentantagonism of histamine H2 receptor-mediated effect (Fukushima et al., 2001) fromthe morphological point of view.

In the present study, two kinds of autoradiographic methods were employed.The identi� cation of the lafutidine binding sites on the parietal cell and CGRP-immunoreactive nerves became possible by using in vivo autoradiography usingun� xed cryostat sections (Nakamura et al., 1998), which is a very useful methodto combine autoradiography and immunohistochemistry. The weak point of thismethod is a relatively low resolution, compared with electron microscopic autora-diography. The second method is the autoradiography of soluble compounds (Na-gata et al., 1969). In this method, freeze-drying and direct Epon embedding underlow pressure is used to prevent the isotope-labelled compound from washing awayduring the � xation process, which also enables us to observe the locazation of ra-diolabelled ligand by electron microscopy. The drawback of this method is the lowpreservation of the tissue structure by the freeze-drying. This point must be clari� edin near future by using much lower pressure during freeze-drying.

As to the localization of CGRP-immunoreactive nerves, the abundant distributionwas reported in the human gastric mucosa (Tani et al., 1999), while its alterationduring Hp infection is still unclear. In the present study, the distribution of lafutidinebinding in the Hp-infected and non-infected patients was not signi� cantly different,which suggests little difference in the nerve distribution.

5. CONCLUSION

From these observations, lafutidine effector sites were shown to be localized on theCGRP-immunoreactive nerves as well as parietal cells both in the Mongolian gerbiland human fundic mucosa.

Acknowledgement

The authors express sincere thanks to Adam J. Smolka of Medical University ofSouth Carolina for the monoclonal antibody against HC,KC-ATPase.

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