ARTICLE |
Correspondence to: María A. Burrell, Dept. of Cytology and Histology, University of Navarra, 31080 Pamplona, Spain. E-mail: mburrell@unav.es
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Summary |
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The aim of this study was to identify which cell types of the rat gastric epithelium express neuronal nitric oxide synthase (nNOS) because the results of the previous studies have been very divergent regarding this point. By the combination of immunohistochemical (IHC) and in situ hybridization (ISH) techniques, we detected expression of nNOS in chief and mucosecretory cells of the gastric epithelium. Moreover, some gastric endocrine cells were immunoreactive for nNOS, although they could not be distinguished in sections treated with ISH techniques. The strongest signal for all antibodies in IHC techniques was obtained when microwave (MW) heating was performed before the IHC procedure. Our results indicate that in the gastric epithelium a variety of cell types are able to produce NO. The NO produced by the different cell types (chief, mucous, and endocrine) may form a complex network of paracrine communication with an important role in gastric physiology. (J Histochem Cytochem 48:11111119, 2000)
Key Words: nitric oxide synthase, rat, gastric epithelium, chief cells, mucous cells, endocrine cells, immunohistochemistry, in situ hybridization
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Introduction |
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NITRIC OXIDE (NO) is a multifunctional messenger that is involved in a wide range of physiological processes in many systems (
In the gastrointestinal tract, NO may influence muscle tone as well as endocrine and exocrine secretions (
Because the results of the previous studies have been very divergent regarding the localization of nNOS in the gastric mucosa, we tried to ascertain the possible reasons for such dissimilar results. Therefore, the aim of this study was to identify, by the combination of immunohistochemical and in situ hybridization techniques, which cell types of the rat gastric epithelium express nNOS.
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Materials and Methods |
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Adult Wistar rats were sacrificed and pieces from the oxyntic and pyloric regions of the stomach and from the cerebellum were dissected. Part of the material was used for extraction of RNA. Other fragments were used for the histological study, some being fixed in Bouin's fluid or 10% neutral formalin (pH 7.4) for 1224 hr, dehydrated, and embedded in paraffin. Finally, some fragments were fixed in 2% paraformaldehyde/1% glutaraldehyde for 2 hr, washed in PBS 0.1 M, pH 7.4, dehydrated with ethanol, and embedded in Epon 812 for the ultrastructural study.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
RNA was extracted from cerebellum, total gastric wall, or gastric mucosa using Ultraspec (Biotecx Labs; Houston, TX). The RNA (1 µg) was reverse-transcribed with a random hexamer and M-MLV reverse transcriptase (Gibco BRL; Paisley, UK). After an initial denaturation for 5 min at 95C, the resulting complementary DNA (cDNA) was amplified by PCR for 30 cycles of amplification (95C for 1 min, 56C for 1 min, and 68C for 1 min), followed by a 10-min extension at 72C, with a pair of primers (5' CTACAAGGTCCGATTCAACAG 3' sense and 5' CCCACACAGAAGACATCACAG 3' antisense) flanking a 315-bp fragment (28683182) of the rat neural NOS cDNA (
A 315-bp nNOS PCR product was purified by agarose electrophoresis, extracted from the gel, and ligated into a pGEM-T vector (Promega; Madison, WI). The identity of the fragment was confirmed by sequencing (DNA sequencing kit; PE Biosystems, Foster City, CA) in an automated sequencer (310 Genetic Analyzer; PE Biosystems).
Amplification of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed simultaneously on the same samples to assess RNA integrity. The oligonucleotide primer sequences for GAPDH (
Antisera
We used three antisera specific for neuronal NOS. One was a polyclonal antiserum (A1) against the synthetic peptide 520540 of the cloned rat neural NOS, a kind gift from S. Moncada (present address: The Cruciform Project, University College London, London, UK). The amino acid sequence of this peptide was LPLLQANGNDPELFQIPPELC, as described by
Antigen Retrieval Microwave (MW) Heating Technique
Before the immunohistochemical procedure, tissue sections were deparaffinized and rehydrated to water, and endogenous peroxidase was blocked with 3% H2O2 for 15 min. Slides were washed with distilled water for 5 min, placed in a citrate buffer 0.01 M (pH 6.0), and heated in the microwave oven (Balay W-2112, 1150700 W; Madrid, Spain) for 15 min at maximal power and for 15 min at medium power. After rinsing in tapwater, the immunohistochemical procedure was performed as usual. For electron microscopy, thin sections were exposed to MW heating in the same manner but were heated for 50 min at the lowest power.
Immunohistochemistry
Light Microscopy.
Paraffin sections 5 µm thick were mounted on slides coated with Vectabond (SP-1800; Vector Laboratories, Burlingame, CA). Single immunohistochemical staining was performed by the avidinbiotinperoxidase complex method (ABC) (
Electron Microscopy. Thin sections from Epon-embedded material were mounted on nickel grids, MW-treated, and incubated for 1 hr in 1% normal goat serum in gold buffer (Tris-HCl-buffered saline 0.05 M, pH 7.2, 0.15 M ClNa, with 1% bovine serum albumin and 0.05% sodium azide), and then incubated in the polyclonal anti-nNOS, diluted 1:100, overnight at 4C. Subsequent passages, all at room temperature, included rinses in gold buffer, incubation for 1 hr with the gold-labeled secondary antiserum (goat anti-rabbit 20-nm gold conjugate, diluted 1:20; BioCell Research Laboratories, Cardiff, UK), and rinses in gold buffer again. Finally, sections were double stained in uranyl acetate and lead hydroxide.
Probes
The nNOS fragment-containing plasmid was linearized with PstI to create a template for antisense probe production or with SacII to create a template for a sense probe (Boehringer Mannheim; Mannheim, Germany) according to the manufacturer's instructions. Digoxigenin (DIG)-labeled antisense and sense probes were synthesized with T7 or SP6 RNA polymerases, respectively, using a DIG RNA (SP6/T7) labeling kit (Boehringer Mannheim).
In Situ Hybridization
Sections 5 µm thick were mounted on coated slides ProbeON Plus (Fisher Biotech; Pittsburgh, PA), dewaxed, and prepared for hybridization with RNA probes as described by
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Results |
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Expression of nNOS mRNA in Gastric Mucosa
The PCR products amplified using the nNOS-specific primers showed a clear band at the predicted size of 315 bp in control tissue (cerebellum) and in both the total gastric wall and the separate mucosa of the oxyntic and pyloric regions (Fig 1). As a positive control, the rat GADPH was assayed in all the specimens to verify the efficiency of cDNA synthesis from extracted RNA.
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Immunohistochemical Results
The strongest immunostaining signal for all the antisera specific for nNOS used in this study was obtained by MW heating of tissue sections in citrate buffer solution (pH 6.0) before the immunohistochemical procedure. The immunostaining was particularly enhanced in formalin-fixed material, both in control tissues (brain and cerebellum) and in the neural plexi of the digestive organs (Fig 2).
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In the gastric epithelium, immunostaining for nNOS appeared in chief cells (Fig 3 and Fig 4) of the oxyntic region, and in mucosecretory (Fig 3) and endocrine (Fig 5) cells in both the oxyntic and pyloric regions. No immnunostaining was found in parietal cells. This immunolabeling pattern was achieved with the two polyclonal antisera for nNOS used in this study. The antiserum against the synthetic peptide (A1) stained all three cell types, and that against the recombinant rat nNOS (A2) only the chief and mucosecretory cells. The monoclonal antibody (A3) stained neural elements of the plexi but did not give positive results in the gastric mucosa (see Table 1).
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Detection of nNOS mRNA by ISH
ISH studies were consistent with the immunohistochemical results. nNOS mRNA was detected in the chief and mucosecretory cells (Fig 3 and Fig 6) of the gastric glands, both of which were also positive for the nNOS protein. Hybridization was also found in control tissues: brain, cerebellum, and neurons of the gastric plexi (Fig 7A). When the sense probe was used, no stain was detected either in neuronal elements (Fig 7A and Fig 7B) or in the epithelial cells (Fig 7C and Fig 7D). Endocrine cells were the only cell type positive with immunohistochemistry that could not be distinguished in sections treated with ISH techniques. As occurred with IHC, parietal cells were also negative with these techniques (Fig 3E and Fig 3F).
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Description of the NOS-expressing Gastric Cell Types
Chief Cells.
Both immunohistochemical (Fig 3A and Fig 3C) and ISH (Fig 3B and Fig 3D) techniques stained chief cells, which in the rat are preferentially located in the lower region of the oxyntic glands but are absent in the pyloric glands. In these cells the immunolabeling had a granular aspect and was specifically concentrated in the apical region (Fig 3C and Fig 4A), whereas the non-granular staining with ISH was widespread throughout the cell (Fig 3D).
Under the electron microscope, immunostaining for nNOS using the A1 antiserum was also found in chief cells, the signal appearing only in the secretory granules (Fig 4B and Fig 4C).
Mucosecretory Cells. These cells were stained for nNOS both with IHC (Fig 3A and Fig 3E) and ISH (Fig 3B and Fig 3F) techniques. With both techniques, the intensity of the stain was lower than in the chief cells.
Mucous cells in the neck portion of the oxyntic glands were stained with both the A1 and the A2 antiserum (Fig 3E), whereas immunolabeling throughout the pyloric glands was achieved only with the A2 antiserum. The pattern of immunoreactivity of the A2 antiserum was similar to that of the mRNA staining.
Endocrine Cells. Some cells displaying the typical features of gastric endocrine cells were detected with the antiserum A1 in both the oxyntic (Fig 5A) and the pyloric regions (Fig 5B), although they were more easily recognized in the pyloric antrum owing to the absence of chief cells in this region. In the oxyntic region, the stained endocrine cells were more visible when the antiserum was diluted more than the optimal dilution of 1:500, which decreased the staining of chief cells (Fig 5A).
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Discussion |
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This study shows that nNOS mRNA and nNOS protein are present not only in a unique cell type but also in several cell types of the rat gastric epithelium, i.e., the chief, mucous, and endocrine cells. It is important to highlight the coincident pattern of stain we have obtained with immunohistochemical and in situ hybridization techniques, although the difficulty in identifying endocrine cells in ISH-stained sections could be due to their sparse distribution among other more abundant, strongly positive cells: chief cells in the oxyntic glands and mucosecretory cells in the pyloric glands.
The first evidence for the presence of a high content of a calcium-dependent constitutive NOS in the gastric mucosa came from biochemical studies (
Given the striking difference in results, we made an effort to optimize the conditions for the in situ techniques to clarify the differing results about the NO-producing cells in the gastric epithelium. We tested several antisera raised against different regions of nNOS on material processed with different fixatives, with or without MW pretreatment. The use of MW very much enhanced the immunoreactivity in control tissues, thus suggesting that it is an important factor in unmasking this antigen and obtaining a more complete pattern of immunoreactivity (
Our results indicate that in the stomach, as in other systems (
Chief Cells
These are the cell type that shows the most consistent labeling both with immunohistochemical and in situ hybridization techniques. Only in one of the previous studies was NOS activity reported in chief cells (
Mucosecretory Cells
Several previous works reported that mucosecretory cells were the cell type responsible for the generation of NO in the gastric epithelium (
Endocrine Cells
Only in our previous results (
We propose that, in the digestive system, NO could derive not only from different tissues (e.g., neurons, epithelium, muscle), as is proposed by
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Acknowledgments |
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While this manuscript was in revision, Cao et al. (J Histochem Cytochem 48:123131, 2000) reported the use of MW for retrieval of NOS.
Supported by the Spanish Ministry of Education and Science (DGICYT project no. PB93-0711).
We thank Prof S. Moncada (The Cruciform Project, University College London, London, UK) for the antisera against nNOS. We thank I. Ordoqui, B. Irigoyen, T. Echeverría, A. Urbiola, and D. GarcíaRos for technical assistance.
Received for publication October 7, 1999; accepted February 16, 2000.
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