ARTICLE |
Correspondence to:
David M. Hougaard, Dept. of Clinical Biochemistry, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark. E-mail:
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Summary |
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-Aminobutyric acid (GABA) is a neurotransmitter that also occurs in a few non-neuronal cell types, where it may serve as a paracrine modulator. GABA is biosynthesized from glutamate by glutamate decarboxylase (GAD) and from putrescine via diamine oxidase (DAO). GAD is demonstrable in several GABA-positive cell types but is undetectable in the GABA-containing gastrin cells and somatostatin cells of the antropyloric mucosa of the stomach. Using two antisera raised against synthetic peptides corresponding to two different regions of rat DAO, we now demonstrate strong reactivity for DAO in gastrin-positive cells of the rat antropyloric mucosa, whereas somatostatin-positive cells as well as other structures of the antrum are unreactive. Western blotting analysis of antrum and colon demonstrate that both antisera react with a single band of 85 kD, consistent with the predicted molecular weight of DAO. Expression of DAO mRNA in the antrum is demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR). Our results strongly indicate that gastrin cells produce GABA via DAO-catalyzed oxidation of putrescine, and experimental data moreover suggest that the biosynthesis of GABA is regulated by the prandial state. Because GABA modulates release of somatostatin, these results point to a new mechanism of paracrine interaction between gastrin cells and somatostatin cells.
(J Histochem Cytochem 48:839846, 2000)
Key Words: diamine oxidase, DAO, GABA, polyamines, putrescine, gastrin, antrum, immunohistochemistry, paracrine interaction
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Introduction |
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P is a precursor for polyamines, molecules essential for cell growth and function (-aminobutyric aldehyde, which is further converted to
-aminobutyric acid (GABA) by aldehyde dehydrogenase (
GABA is an inhibitory neurotransmitter that also functions as a neuromodulator in the gastroenteric nervous system, where it occurs mainly in nerves in the myenteric plexus and in circular muscle layers of the muscularis externa (
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Materials and Methods |
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Tissue Material
Adult freely fed Wistar rats of either sex were sacrificed with CO2 and intracardially perfused with 12 ml of saline, followed by 30 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4 (PFA), or 0.52.5% glutaraldehyde with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. Specimens from the antrum were postfixed in the respective fixatives overnight, dehydrated in graded ethanol, and embedded in paraffin.
Specimens for Western blotting and RT-PCR were taken from unfixed rats and immediately chilled on ice or frozen in liquid nitrogen, respectively, before further processing.
For quantitative analysis, 18 adult Wistar rats were fasted in wire-bottomed cages with access to drinking water only. After 24 hr, nine rats were sacrificed directly and nine rats were re-fed (in darkness to facilitate eating) with standard rat chow for 3 hr before sacrifice. During sectioning, it was verified that the nine re-fed rats had eaten well and that the stomach of eight of the fasted rats was empty. The stomach of the ninth fasted rat contained hair and other material and was consequently excluded from the study. The remaining 17 rats were processed for immunohistochemistry as described above, using 4% paraformaldehyde as fixative. Paraffin-embedded specimens of antrum were cut longitudinally in sections 5 µm thick and approximately 34 mm long. For each of the 17 rats, four sections were cut from different areas of the curvature major region of the antrum and mounted on a single slide.
Diamine Oxidase Antisera
Two groups of five rabbits each were immunized with synthetic peptides (KJ RoosPetersen; Hørsholm, Denmark) corresponding to the predicted amino acid sequence position 625639 (SERYSSSLYNQNDPW) (DAO625) and 713730 (PQDKGLNRVQRWIPEDRR) (DAO713) of rat colon/lung DAO, which is identical to the amiloride binding protein EMBL accession number P36633 (
Immunocytochemistry
Five-µm paraffin sections of antrum were dewaxed in xylene, hydrated in graded ethanol, and incubated in 1% nonfat milk or 1% BSA in TBS (0.05 M Tris; 0.15 M NaCl, pH 7.4) for 30 min at RT. Sections were then incubated for 20 hr at 4C with one of the DAO antisera diluted 1:5001:2000 in TBS. The DAO antisera were absorbed overnight with 200 µg/ml KLH (anti-DAO625) or 0.25% BSA (anti-DAO713) before use. The site of the antigenantibody reaction was revealed by sequential incubation at RT with biotinylated goat anti-rabbit (DAKO; Glostrup, Denmark) 1:500 in TBS containing 0.25% BSA, 3% rat serum for 1 hr, and alkaline phosphatase-conjugated streptavidin (DAKO) 1:100 in TBS containing 0.25% BSA for 1 hr, followed by development in bromochloroindolyl phosphatenitroblue tetrazolium (BCIPNBT) (Sigma; St Louis, MO) medium containing levamisole. Sections stained for GABA were digested with 00.04% pepsin (Sigma) in 0.2 M HCl for 20 min at RT before incubation with guinea pig anti-GABA antiserum (Affinity; Surrey, UK) followed by Texas Red-conjugated goat anti-guinea pig IgG (Jackson ImmunoResearch Laboratories; West Grove, PA).
For quantitative analysis of DAO-reactive cells in fasted (n = 8) vs re-fed (n = 9) rats, 17 slides with four sections on each (approximately 1216 mm antral mucosa per slide) were stained with anti-DAO625 as described above, and the number of DAO-positive nucleated cells per mm length of mucosa was counted. The MannWhitney U-test was used for statistical comparisons.
Double immunofluorescence stainings were performed with the rabbit DAO antisera, used in combination with guinea pig antiserum to synthetic human gastrin (Milab; Malmö, Sweden) or with monoclonal mouse antibodies to synthetic somatostatin (Novo Nordisk; NovoClone, Bagsværd, Denmark) (
Crossreactivity of secondary antibodies with relevant primary antibodies was ruled out by separate staining experiments. Controls also included preabsorption of primary antisera overnight against 150 µg/ml of synthetic DAO peptides (positions 625639 and 713730), gastrin, or somatostatin (Peninsula Laboratories; Belmont, CA), as well as conventional staining controls as described (
Western Blotting
Homogenized samples of antrum and colon were sonicated in a water bath at 30C for 5 min, boiled for 5 min, and loaded on a 7.5% polyacrylamide gel. The sample buffer was 62.5 mM Tris-HCl, pH 6.8, 0.7 M 2-ß-mercaptoethanol, 10% (v/v) glycerol and 2% (w/v) SDS (Bio-Rad; Hercules, CA). After electrophoresis, the proteins were transferred to a nitrocellulose membrane (Millipore; Bedford, MA) using a semi-dry cell (Pharmacia Biotech; Uppsala, Sweden). The membrane was incubated overnight with 3% nonfat milk in PBS (PBS/milk) at 4C, washed in PBS containing 0.1% Tween-20 (Merck; Darmstadt, Germany) (PBS/Tween), and incubated overnight with the DAO antisera diluted 1:10,000 in PBS/milk at 4C. Detection was achieved by biotinylated goat anti-rabbit (DAKO) 1:1000 in PBS/milk, followed by alkaline phosphatase-conjugated streptavidin (DAKO) 1:200 in PBS/milk, using bromochloroindolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) (Sigma) as substrate. Controls included preabsorption of antisera overnight against 150 µg/ml of the synthetic peptides.
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Total RNA from rat antropyloric mucosa and colon was isolated using TRIzol Reagent (Life Technologies; Grand Island, NY). cDNA was produced employing random hexamers and reverse transcriptase from SuperScript (Life Technologies; cat. no. 18089-011). Primers used in the polymerase chain reaction (PCR) were DAO upper primer, position 16291649 and lower primer, position 21232143, EMBL mRNA sequence accession no. X73911, and ß-actin upper primer, position 23942415, and lower primer, position 30883106, GeneBank DNA sequence accession no. J00691 (primers spanning two introns of total 212 bp).
Amplicons were electrophorized in a 1.5% agarose gel and occasionally transferred to a positively charged nylon membrane (Boehringer Mannheim; Mannheim, Germany) in 0.5 M NaOH, 0.15 M NaCl by vacuum blotting (Vacu-Aid; Hybaid, Teddington, UK). The membrane was heated at 80C for 30 min, exposed to UV radiation for 2 min and prehybridized in hybridization buffer containing 2 x SSC (0.3 M NaCl and 0.03 M sodium citrate), 0.2 mg Ficol (type 400; Sigma), 0.2 mg polyvinylpyrrolidone (MW 25,000; Merck), 0.2 mg BSA, 100 mg dextran sulfate (MW 500,000; Sigma), and 0.1 µg sheared salmon sperm DNA (Sigma) for 2 hr at 60C (
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Results |
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The two antisera, raised against different regions of rat DAO (anti-DAO625 and anti-DAO713), stained endocrine-like cells in the antropyloric mucosa. Anti-DAO625 produced a strong, distinct staining confined to the cytoplasm of several endocrine-like cells (Fig 1A and Fig 1C). Preabsorption of anti-DAO625 against its corresponding synthetic peptide DAO625 abolished the staining (Fig 1B). Anti-DAO713 produced a considerably weaker staining of the endocrine-like cells and a diffuse non-absorbable, and hence unspecific, background staining in many structures (Fig 1D and Fig 1E). Preabsorption of the antisera against unrelated peptides was without effect. No other specific staining was observed in the mucosa or in the submucosa and muscularis externa. Notably, neuronal cells of the submucous plexus and the myenteric plexus present in muscularis externa were unreactive. It was not possible to obtain staining in the antropyloric mucosa with the DAO antisera when glutaraldehyde was included in the fixative, whereas, in contrast, glutaraldehyde was required for a positive staining with the GABA antiserum (not shown).
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The specificity of the DAO-antisera was further corroborated by Western blotting analysis of antrum and colon. Both antisera detected a single band of 85 kD that was not detected by sera preabsorbed with their corresponding peptides (Fig 2). Because the anti-DAO625 produced the strongest specific staining and the lowest background, it was selected for further analysis.
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Examination of antral sections from more than 10 individual rats stained by the double immunofluorescence technique, using anti-DAO625 and guinea pig anti-gastrin, revealed that cells positive for DAO (red) were also positive for gastrin (green) (Fig 3A3F). In general, cells strongly reactive for one of the compounds were also strongly reactive for the other (composite color bright yellow) (Fig 3C). However, some cells stained with different intensities for DAO and gastrin, yielding composite colors ranging from orange to yellow. This variation in staining intensity was most clearly seen at the subcellular level, where compartments that stained orange, yellow, and green could be observed (Fig 3F). Double immunofluorescence staining for DAO and somatostatin, using anti-DAO625 and mouse anti-somatostatin as primary antibodies, revealed no reactivity for DAO in somatostatin-positive cells (Fig 3G3I). Single staining experiments, systematically combining the different primary antisera with each of the fluorescence-labeled secondary antisera documented that all secondary antibodies reacted only with the primary antibody intended.
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The frequency of antropyloric cells containing detectable amounts of DAO depended on the prandial state and varied among individual rats. For each rat, the total length of antral mucosa examined was 1216 mm. In rats sacrificed immediately after 24 hr of fasting, the number of anti-DAO625-reactive cells ranged from 11.2 to 27.6 per mm length antropyloric mucosa, mean value 20.3 cells (Fig 4). After 3 hr of re-feeding, the number of cells detectable with anti-DAO625 was significantly lower (p<0.01), ranging from 3.2 to 18.1 cells per mm mucosa, mean value 10.1 cells.
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The expression of DAO mRNA in the antrum was demonstrated by RT-PCR. Amplicons of the sizes predicted for rat DAO mRNA (515 bp) were produced using cDNA from the antropyloric mucosa and colon as template (Fig 5). No other bands appeared. The identity of the DAO amplicons was verified by Southern blotting. PCR using rat DNA as template produced an amplicon at 1045 bp, indicating that the DAO primers are spanning one or more introns of a total of approximately 530 bp (Fig 5). Possible contamination of the mRNA preparations with genomic DNA was also ruled out by omission of reverse transcriptase during preparation of cDNA, which abolished subsequent production of amplicons, and by control PCR using primers for ß-actin spanning two introns of 212 bp total.
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Discussion |
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Our results demonstrate that DAO is present in antropyloric gastrin cells. Both antisera recognizing DAO stain the cytoplasm of gastrin-producing cells, whereas antropyloric somatostatin cells and neuronal cells of the myenteric and submucous plexi are negative. The localization of DAO in gastrin cells is supported by Western blotting analysis and RT-PCR demonstrating the presence of DAO and DAO mRNA in the rat antrum.
The two antisera produce identical results on Western blots, but anti-DAO625 is preferred for immunohistochemistry because anti-DAO713 produces a disturbing diffuse unspecific background staining of the sections.
Double immunofluorescence staining demonstrates cellular co-localization of gastrin (green) and DAO (red). The resulting composite colors range from orange to bright yellow, indicating that the relationship between the concentration of the two compounds varies, and hence that the cellular level of gastrin and DAO most probably is regulated by separate mechanisms.
Because of different requirements for tissue fixation, it was not possible to stain for DAO and GABA simultaneously. However, it has previously been shown that gastrin-producing cells in the rat antrum contain GABA, predominantly in the extragranular cytoplasmic matrix (
Gastric mucosal growth, differentiation, and function are dependent on gastrin (
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Acknowledgments |
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Supported by the Danish Cancer Society and the Danish MRC.
Received for publication October 25, 1999; accepted February 23, 2000.
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