ARTICLE |
Correspondence to: Kunio Fujiwara, Faculty of Pharmaceutical Sciences, Nagasaki U., Bunkyo-machi 1-14, Nagasaki 852-8131, Japan.
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Summary |
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Histamine (HA), contained in the enterochromaffin-like (ECL) cells of the gastric mucosa in animals, plays an important role in gastric acid secretion, although methods for its exact morphological localization are still lacking. We used a pre-embedding indirect immunoperoxidase approach to define the fine structural localization of HA in rat oxyntic mucosa that was fixed with a glutaraldehyde-based fixative and HA monoclonal antibodies (MAbs AHA-1 and 2). Transmission electron microscopy showed that the peroxidase endproduct not only was concentrated in the cores of cytoplasmic granules but also was distributed to a high degree in the cytoplasm peripheral to the granules of the ECL cells. These results suggest that in ECL cells HA is enzymatically synthesized in the cytoplasm, then is transported and stored in the cores of the granules before its release from the basal lamina. The present HA immunoelectron microscopic method with MAbs would be applicable more generally to the ultrastructural identification of HA-containing cells. (J Histochem Cytochem 47:10311038, 1999)
Key Words: histamine, immunoelectron microscopy, monoclonal antibody, enterochromaffin-like cells
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Introduction |
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HISTAMINE (HA) is a ubiquitous chemical mediator that acts as a regulator of gastric acid secretion, a modulator of inflammatory reactions, and a neurotransmitter (
A large number of argyrophilic endocrine cells, contained in the oxyntic glands of rat stomach, synthesize and store large amounts of HA (
In our recent study of monoclonal antibodies (MAbs AHA-15) against glutaraldehyde-conjugated HA, we identified two MAbs (AHA-1 and 2) that gave the best immunocytochemical results at the light microscopic level when glutaraldehyde was used as a tissue fixative (
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Materials and Methods |
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Antibodies
The MAbs AHA-1 and 2 (IgG1 subisotype) were produced against HA conjugated to bovine serum albumin (BSA) using glutaraldehyde (GA) and sodium borohydride (
Animals
Normal adult male Wistar rats (Otsubo Experimental Animals; Nagasaki, Japan), body weight 200250 g, were used in this study. They were housed in temperature- and light-controlled rooms (21 ± 1C and 12L:12D) and had free access to standard food and tapwater.
Immunocytochemistry (ICC)
Under sodium pentobarbital (Abbott Labs; North Chicago, IL) anesthesia (60 mg/kg), 10 rats were perfused intracardially with PBS at 50 ml/min for 2 min at room temperature (RT) and then with a freshly prepared solution of 2.5% GA in 10 mM phosphate buffer, pH 7.2, for 6 min. Stomachs were quickly excised and immersed overnight at 4C in the same fixative. They were cut into 50-µm-thick sections with a Microslicer (Dosaka EM; Kyoto, Japan) and the sections were treated for 10 min with 1.0% H2O2 to inhibit endogenous peroxidase, 0.2% NaBH4 for 10 min, and incubated at 4C for 48 hr with primary MAb AHA-1 or -2 at concentrations ranging from 10 to 50 ng/ml of 50 mM Tris-HCl buffer, pH 7.4, containing 0.86% NaCl (TBS) [the concentration of which was determined by the conventional sandwich ELISA using chromatographically purified mouse IgG (Zymed; San Francisco, CA) as a standard]. Sections were then incubated with a goat anti-mouse IgG/Fab' labeled with horseradish peroxidase (HRP) (MBL; Nagoya, Japan) 1:200 for 12 hr at 4C. After rinsing with TBS, the HRP was revealed for 510 min with diaminobenzidine and H2O2. The HRP substrate consisted of 10 mg of 3,3'-diaminobenzidine tetrachloride (Sigma; St Louis, MO) dissolved in 20 ml of 50 mM Tris buffer, pH 7.4, supplemented with 20 µl of 30% H2O2 (
Electron Microscopy
The color-developed specimens prepared as above were then fixed with 1.0% osmium tetroxide in 50 mM cacodylate buffer, pH 7.4, for 1 hr at RT and dehydrated in a series of graded ethanol solutions. After immersion in propylene oxide (Nacalai Tesque; Kyoto, Japan) (three times for 10 min each), the samples were immersed in a mixture (1:1) of propylene oxide and Epon 812 resin (Taab; Reading, UK) overnight and embedded in Epon 812 resin in a routine manner. The regions to be studied were cut with a 2-mm diameter punch, mounted on Epon blocks, and sectioned on a horizontal plane into ultrathin sections, which were then immediately observed in a 100 CX Jeol electron microscope.
For quantification, images of electron microscopic photographs (both sample and control specimens) taken under constant conditions were digitized into a computer through a video-camera head (ITC-370M; Olympus-Ikegami, Tokyo, Japan) according to the method of
Control Experiments
In the HA immunocytochemistry study, the specificity of immunostaining was ascertained by incubating control sections with (a) the secondary antiserum alone, (b) type-matched MAb (IgG1) anti-penicillin (3080 ng/ml; Cosmo, Tokyo, Japan), and (c) MAb AHA-1 or -2 preabsorbed with histamineglutaraldehydehuman serum albumin (HA-GA-HSA) conjugate at a concentration of 2 µg/ml.
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Results |
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Immunoelectron microscopic study of HA was carried out employing a pre-embedding method using 50-µm Microslicer sections of the rat gastric fundus and employing MAb AHA-1 or -2 produced against glutaraldehyde-conjugated HA (
HA immunoreactivity was detected by light microscopy mainly in the cells scattered in the basal half of the gastric glands (oxyntic glands) of the gastric fundus and corpus, and in a few cells in the neck portion of the glands (Figure 1A). The HA-immunoreactive cells varied in shape and size and often had one or two processes invading the bases of the adjacent cells. However, no HA-immunoreactive cells were detected in other areas of the stomach, such as pyloric glands, antrum, or other parts of the gastric glands. The mast cells around the blood vessels in the submucosa were very weakly stained by the MAb (Figure 1A), this being in agreement with previous results reported by
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Immunoelectron Microscopy
Among endocrine cells in rat gastric glands, ECL cells and A-like cells were predominantly observed, most of which in the basal half of the mucosa were in contact with the basal lamina of the glands (Figure 2A and Figure 3A). Immunoelectron microscopic examination of the basal parts of the gastric glands showed identical results for MAb AHA-1 and MAb AHA-2: predominant distribution of HA in the ECL cells, which were identified on the basis of their characteristic ultrastructural features; many vesicular granules located in the basal part of the cell, varying in size within the same cell; and a wide electron-lucent halo separating the osmophilic core from the limiting membrane (
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Immunoreactive end deposits were highly concentrated in the cores of various sizes of granules (100500-nm diameter). They were also diffusely distributed throughout the cytoplasm, especially near the surroundings of the ECL cell granules (Figure 3A). In addition, the processes (cytoplasm) characteristic of the ECL cells were positive for HA immunoreactivity (Figure 2B) and were not seen to make contact with the lumen of the gland. However, the nuclei and mitochondria were devoid of immunoreactivity (Figure 2A, Figure 2B, and Figure 3A). The sections stained with the preabsorbed MAb AHA-2 or the anti-penicillin MAb did not show any HA immunoreactivity (Figure 3B). A computer-assisted image analysis of the electron microscopic photographs (Figure 3A and Figure 3B) was carried out by measuring the optical density of the cores of the granules and mitochondria in the ECL cells (Figure 4). The values obtained for the cores of the granules in the sample and control specimens were 184.4 ± 28.3 (SE) and 91 ± 16.4, respectively, with a statistically significant difference (p<0.001), revealing that the cores of the granules were significantly labeled (Figure 4). In contrast, the mitochondria were unlabeled, because no significant difference (p>0.05) was evident between the sample (127.1 ± 8.1) and control (113.7 ± 12.9) (Figure 4). In addition, A-like cells in the gastric glands were analyzed in the same manner, and we found no labeling of the granules in the cells of sample specimens (not shown). None of the other endocrine cells occasionally detected in the specimens, such as D-, P-, and D1-cells, were immunoreactive with both the MAbs (not shown).
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Discussion |
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In the present immunoelectron microscopic study of the subcellular localization of HA in cells of rat gastric glands, using the HA MAbs and GA-based fixation, HA immunoreactivity was demonstrated only in the ECL cells among epithelial endocrine cell types. This agrees with previous results (
In this study, very weak staining or no staining at all was observed in mast cells in the epithelium and around the blood vessels in the submucosa, this being in agreement with the results of
Although HA has been implicated in a variety of biologically important events, such as being the main mediator of allergic reactions and one of the neurotransmitters (
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Acknowledgments |
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We are very grateful to Dr M. Shin for the computer-assisted image analysis, Mr T. Suematsu for technical assistance with electron microscopy, and Prof M. Tiedemann for assistance with editing the manuscript.
Received for publication December 1, 1998; accepted March 23, 1999.
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