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Endocrinology Vol. 139, No. 10 4404-4415
Copyright © 1998 by The Endocrine Society

ARTICLES

Gastrin-Producing Endocrine Cells: A Novel Source of Histamine in the Rat Stomach

Béla Hunyady, Annamária Zólyomi, Beth J. Hoffman and Éva Mezey

National Institute of Mental Health, National Institute of Health (B.H., B.J.H.), National Institute of Child Health and Disease (A.Z.), Basic Neuroscience Program, National Institute of Neurological Disorders and Stroke (É.M.), NIH, Bethesda, Maryland 20892; First Department of Medicine (B.H.), Medical University of Pécs, H-7643 Pécs, Ifjúság u 13, Hungary

Address all correspondence and requests for reprints to: Éva Mezey, BNP/NINDS/NIH, Building 36 Rm 3A17, 9000 Rockville Pike, Bethesda, Maryland 20892. E-mail: mezey{at}codon.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gastrin and histamine both potently stimulate secretion of acid into the gastric lumen. How these agents interact and how their release is controlled is poorly understood. Therefore, we decided to look for histamine in the antral portion of the rat stomach where the gastrin-producing G cells are located. We used immunocytochemical methods to visualize histamine, histidine decarboxylase (HDC, the enzyme that converts histidine to histamine), and the type 1 vesicular monoamine transporter (VMAT1, the protein responsible for moving histamine into vesicles for storage and release). We were surprised to find that histamine, HDC, and VMAT1 were all present in G cells. Our results suggest that G cells synthesize and secrete gastrin and histamine. Whether histamine acts in concert with gastrin to stimulate acid secretion, or functions as an autocrine inhibitor of gastrin release remains to be seen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GASTRIN and histamine both regulate the endocrine and exocrine functions of the stomach in all studied species, including humans and rats (1, 2, 3, 4). Gastrin is produced in antral G cells; histamine is found in mast cells, ECL cells and nerve fibers in the enteric plexus. Gastrointestinal mast cells are involved in mucosal immune functions responding to antigenic challenges with histamine and serotonin release (5, 6). In addition, mucosal mast cells may also affect exocrine/endocrine functions of the stomach, especially in those species where they outnumber ECL cells (7). ECL cells respond to food intake or vagal nerve stimulation by releasing histamine (8), but the number of these cells is insignificant in the antral stomach. Finally, histaminergic enteric neurons, found in the external muscle layer of the stomach, may be involved in regulating gastric motor function (9), but little is known about their actions.

To date, people who have studied the cellular distribution of histamine in the stomach have focused on the fundus. We wondered where histamine was located in the antrum. In searching for histamine-positive cells in the antral mucosa, we found that gastrin producing G cells also contain histamine, the enzyme that is responsible for its biosynthesis (HDC), and VMAT1. This fourth source of gastric histamine was previously unknown and may be the major source of histamine in the stomach.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue handling
For immunoblotting, four intact adult male Sprague-Dawley rats (Taconic Farms, Germantown, NY; 250–300 g) were killed by decapitation under anesthesia (80 mg/kg pentobarbital sodium ip, Anpro Pharmaceutical, Arcadia, CA). The antral third of each stomach was rapidly removed, frozen on dry ice, and stored at -80 C until used.

For immunohistochemistry, four rats were perfused with 4% paraformaldehyde (Polysciences, Warrington, PA) in PBS (0.0017 M KH2PO4 + 0.005 M Na2HPO4 + 0.15 M NaCl in 1000 ml distilled water, pH 7.4) at 4 C, as described earlier (10). The stomachs and brains were removed, postfixed with the same fixative for 30 min, cryoprotected with 5% (2 h), 10% (4 h), then 20% (overnight, at 4 C) sucrose in PBS, and frozen in 2-methylbutane (Aldrich, Milwaukee, WI). Ten-µm thick sections were cut in a cryostat, then thaw-mounted, air-dried at 37 C onto silanized slides, frozen, and stored at -80 C until used. For studies of histamine and gastrin on adjacent sections 4 µm thin sections were cut.

Unless otherwise noted all the procedures were performed at room temperature (RT, 20 C). NIH Guidelines for Animal Care and Use were followed in all animal procedures.

Immunohistochemistry
Immunofluorescent double labeling (11) was used for cellular co-localization of different monoamines and proteins in the rat stomach. For immunohistochemistry all primary and secondary antibodies were diluted in BSA-diluent (PBS, containing 1% BSA [BSA] and 0.6% Triton X-100). The specification and the dilution of the primary and secondary antibodies are listed in Tables 1Go and 2Go.


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  		Table 1. Characteristics of the antibodies used for immunohistochemistry (1 ) and immunoblotting (2 )

 

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  		Table 2. Affinity purified anti IgG secondary antibodies (F[ab']2 fragments), with minimal cross-reactivity to serum proteins from the listed species1

 
The steps of the double immunolabeling were as follows, with three 5-min washes in PBS between steps: [1] postfixation in 4% formaldehyde fixative for 10 min; 2) blocking of nonspecific staining with BSA-diluent for 30 min; 3) incubation with the primary antiserum, overnight at 4 C; 4) incubation with Cy3- or FITC-conjugated anti-IgG antiserum (F[ab’]2 fragment), affinity purified to minimize cross-reactivity with IgGs other than the primary antibody, 1 h. The steps of the subsequent immunolabeling for the second antigen were analogous to steps 3) and 4), i.e. 5) incubation with a second primary antibody raised in a different species from the hosts of the first primary and secondary antibodies; 6) incubation with a second secondary antibody not cross-reacting with the first primary and secondary antibodies, and conjugated to a different fluorochrome than used in the first labeling. All affinity purified F(ab')2 IgG-fragment secondary antibodies, developed in donkey, were purchased from Jackson ImmonoResearch (Table 2Go).

For double staining with two antibodies from the same host species two different methods were used: either adjacent thin (4 µm) sections were stained with the two antibodies or the tyramide signal amplification (TSA) method was used as previously described (10).

After immunostaining, the sections were washed in distilled water, air dried, and coverslipped using Cytoseal 60 medium (Stephens Scientific, Riverdale, NJ). Immunofluorescent labeling was viewed with a fluorescent microscope (Leitz Dialux 20, Wetzlar, Germany) using fluorescent filters for FITC (appears green) and Cy3 (appears red). Photomicrographs were taken using an Axiphot fluorescent microscope (Zeiss, Germany). Double color images were generated using a double pass fluorescent filter allowing detection of both FITC-green and Cy3-red. Color figures were mounted and printed using a slide scanner (Lacie Silverscanner III, Epson, Japan) and Adobe Photoshop software (Mountain View, CA) with 400-2500 dpi resolution.

Immunohistochemical controls
Four different antibodies to gastrin, two different antibodies to HDC, and three different antibodies to DDC were used in these studies (Table 1Go), all giving consistently identical staining patterns in consecutive sections and/or in double stainings.

As positive controls, the staining properties of the antibodies were verified using brain sections known to contain monoaminergic cells: sections at the level of the mamillary nuclei for histamine and HDC; raphe nuclei and locus ceruleus for serotonin, DDC and VMAT2; substantia nigra for TH, DDC and VMAT2 (12, 13).

The specificities of gastrin and HDC antibodies were also confirmed by immunoabsorption controls and by extended specificity controls recommended by Grube & Weber (14) and Grube (15). In these latter specificity controls primary antibodies were diluted in high sodium, high pH phosphate buffer (NaCl supplemented to 0.5 M, and pH adjusted to 8.6 with 0.1 M NaOH in the regular PBS) containing 1% BSA and 0.6% Triton X-100, and the same buffer was used in the subsequent washes. This method has been shown to be useful in avoiding any possible nonspecific primary antibody binding to gastrin producing G cells. Using this buffer did not effect the immunolabeling of gastrin, histamine or HDC.

For immunoabsorptions, each of the antibodies to gastrin and HDC were incubated with 50 ng/ml of rat gastrin peptide (Sigma Chemical Co., St. Louis, MO; no. G1276) or 100 ng/ml of rat HDC protein that was used for immunization (kind donation of Dr. Lo Persson, Department of Physiology, University of Lund, Sweden) at 4 C, overnight. Anti-HDC and antigastrin sera were also preincubated with the same amounts of noncorresponding peptides (gastrin and HDC, respectively), further confirming the antigen specificity of the absorption control. Similarly, anti-DDC sera were preincubated with the noncorresponding HDC, and the HDC-antisera with the DDC protein in a range of 0.05–100 µg/ml. Subsequently, these preabsorbed/preincubated antisera were used in step (3) of the immunohistochemical protocol in sections run parallel to those incubated with nonabsorbed (regular) primary antibodies.

Further controls included immunostainings with the primary antibodies substituted by nonimmune sera, nonimmune IgGs or BSA diluent in step (3). The appropriate species recognition of the fluorochrome labeled affinity purified F(ab')2 IgG-fragment secondary antibodies was confirmed in our hands by the lack of specific labeling in controls when noncorresponding secondary antibodies were used after the primary antibody. Finally, all the double immunostainings gave identical results when we reversed the order of the primary antibodies.

Western blot analysis of HDC and DDC immunoreactivities in the rat antrum
Fresh frozen antral tissue was weighed (0.2–0.3 g/stomach) and homogenized in five volumes of lysis buffer (PBS containing 0.2% Triton X-100, 5 mM EDTA, 5 mM EGTA, 100 µM AEBSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin and 10 µg/ml aprotinin) using a Tissumizer homogenizer/sonicator (Tekmar, Cincinnati, OH) at 60% of maximal speed for two 20 sec intervals on water-ice. The homogenate was centrifuged at 10,000 x g for 30 min at 4 C to remove cell debris. The supernatant was collected, the protein concentration was determined by the BCA method (Pierce, Rockford, IL) (16) and normalized to 10 mg/ml protein with the lysis buffer. Protein extracts were stored at x20 C until used.

Fifty micrograms of antral protein was subjected to SDS-PAGE and then electroblotted onto Immobilon-P PVDF membrane (Millipore, Bedford, MA) at 25 mV for 2 h in transfer buffer (20% methanol, 25 mM Tris-HCl, 192 mM glycine). Prestained molecular weight markers (Novex, San Diego, CA) were used to estimate the apparent molecular weight of immunoreactive species.

Primary and secondary antibodies were diluted for Western blots as listed in Tables 1Go and 2Go in BSA diluent without Triton X-100. Blots were preblocked in 5% nonfat dry milk in PBS for 1h and incubated overnight at 4 C with rabbit anti-HDC or rabbit anti-DDC antibodies. After three 10-min washes in PBS, the membranes were incubated in 5% nonfat dry milk in PBS for 1 h and again washed three times for 10 min in PBS. Blots were incubated for 1 h with horseradish peroxidase-conjugated antirabbit or antiguinea pig IgGs (depending on the host species of the primary antibody). After several washes in PBS immunoblots were developed using enhanced chemiluminescence (Pierce SuperSignal, Rockford, IL).

Tritiated histamine uptake by the antral mucosa
Histamine uptake was performed as reported by Huszti et al. (17, 18) with some modifications. The antrum was removed and sliced into six equal sections. Following three washes in Krebs ringer buffer or buffer supplemented with 1 mM ascorbate, tissue slices were incubated at 37 C and 0 C with or without uptake inhibitor (thioperamide) for 10 min before addition of 100 nM 3H-histamine for 10 min or 60 min. One section was incubated with 1 µM cold histamine. Uptake was terminated by addition of 10 volumes of ice-cold KRB containing 10 mM cold histamine. Following 3 washes in ice-cold KRB, 3H-histamine was fixed in the tissue slices using 4% paraformaldehyde in PBS for 1 h. After several PBS washes, the slices were soaked in 20% sucrose in PBS overnight and frozen on dry ice in OCT mounting medium. The tissue was then sectioned (12 mikrometer) using a cryostat. Sections were then apposed to Hyperfilm-3H for 3 weeks and developed using conventional Kodak chemicals. The film images were then scanned at high resolution using a flatbad scanner (Lacie Silverscanner III) and the NIH Image 1.61 software. The images were then analyzed and optical density was used for comparison.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Demonstration of histamine and histidine decarboxylase immunoreactivity in antral G cells
In the fundic mucosa, both antibodies to HDC labeled histaminergic epithelial cells with an elongated shape and processes characteristic of ECL cells (Fig. 1AGo). In the antral mucosa, (Fig. 1BGo), where ECL cells are reportedly rare (19, 20, 21), there were also numerous HDC-positive cells. In contrast to fundic histaminergic cells, antral HDC positive cells, located predominantly at the bottom of the pyloric glands, were larger, rather rounded, and appeared not to have processes, suggesting that they may represent a different cell population from ECL cells.



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  		Figure 1. Histidine decarboxylase (HDC) immunoreactive cells in the (A) fundic and (B) antral regions of rat stomach. A, Elongated HDC positive epithelial cells with characteristic bidirectional processes (arrows) correspond to histamine producing enterochromaffin-like (ECL) cells in the fundic mucosa. B, In contrast, most of the antral HDC immunoreactive cells are rounded with no processes (arrowheads), suggesting a different histaminergic cell population here. C and D demonstrate the histological structure of the fundic and antral mucosa, respectively. Boxed areas show approximate sites and orientations for the fields in panels A and B. E and F demonstrate double immunostaining for histidine decarboxylase (HDC) using consecutively the antibodies raised in (E) rabbit and (F) guinea pig. Photomicrograph pair of the same field demonstrating the signal for HDC with (A) the rabbit (labeled by FITC-green) and (B) the guinea pig (labeled by Cy3-red) antibodies. Both antibodies recognize the same cells, some of them are pointed out with arrows. Vertical section. Symbols: mucosal layer (m); lamina propria (lp); muscularis mucosae (mm); submucosa (sm); external muscle layer (em). Vertical sections. Scale bars, 50 µm in (A) and (B), 100 µm in (C–F).

 
To be sure of the specificity of the HDC staining in antral epithelial cells, a panel of immunohistochemical control procedures were applied as described in the Materials and Methods section. First we double labeled with the rabbit and subsequently the guinea pig primary antibodies to HDC, using different fluorochromes (FITC-green and Cy3-red, respectively) to detect them. We found that both HDC antibodies recognized the same cells (Fig. 1Go, E and F). Immunostaining of adjacent sections of the antrum shows that the same cells are positive for gastrin (Fig. 2AGo) and histamine (Fig. 2BGo); using the tyramide amplification procedure on the same sections also demonstrates that the gastrin (Fig. 2CGo) and histamine (Fig. 2DGo) immunopositive cells are identical. Preincubation of either HDC antibody with 100 ng/ml immunizing HDC protein abolished specific staining in both fundic and antral histaminergic cells (Fig. 3AGo). The labeling with HDC antibodies also matched Grube’s extended specificity criteria (14, 15). The staining properties of both antibodies remained unchanged when we diluted them in the high sodium - high pH diluent (0.5 M NaCl, pH 8.6), further confirming that the labeling was specific.



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  		Figure 2. Colocalization of gastrin (A and C) and histamine in the antral mucosa in adjacent 4-µm thin sections (A and B) and in the same section (C and D) using the TSA amplification method and visualizing the primary antibodies with CY3 (red) or FITC (green). Arrows indicate identical cells between A and B or C and D. The images indicate that all gastrin positive cells are also histamine positive. Scale bars, 40 µm in (A) and (B), 100 µm in (C) and (D).

 


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  		Figure 3. Identification of histaminergic epithelial cells in the mucosa of rat stomach. Vertical sections. Scale bar represents 100 µm in all panels. A, Fundic mucosa. Proton pump immunolabeling for parietal cells (FITC-green) and absorption control for the antibody to histidine decarboxylase (HDC). Red + green double exposure photomicrograph. No Cy3-red labeling for HDC can be seen after preincubation of the HDC antibody with 100 ng/ml immunizing HDC protein. If present (without preabsorption), HDC labeling would appear in red. Parietal cells are labeled with FITC-green for topographic orientation in the junction of the antrum and fundus. B–D, Double immunostaining for (B) HDC and (C) histamine in the junction of the antrum and fundus. Photomicrograph triplet of the same area demonstrating the signal for (B) HDC (labeled by FITC-green) and (C) histamine (labeled by Cy3-red). D, Double exposure photomicrograph where overlapping green and red cells appear yellow. All HDC immunoreactive cells are colabeled by the antibody to histamine (arrows). E–G, Double immunostaining for (E) HDC and (F) gastrin in the antral mucosa. Photomicrograph triplet of the same field demonstrating the signal for (E) HDC (labeled by FITC-green) and (F) gastrin (labeled by Cy3-red). G, Double exposure photomicrograph, where overlapping green and red cells appear yellow. Rounded HDC positive antral cells are colabeled by the antibody to gastrin, identifying them as G cells (arrows). A few elongated HDC-containing cells are not immunoreactive for gastrin (arrowheads), they are likely to be scattered ECL cells in the antrum. H, Proton pump immunolabeling for parietal cells (FITC-green) and absorption control for the antibody to gastrin used in panel (F). Red + green double exposure photomicrograph. No Cy3-red labeling for gastrin can be seen after preincubation of the gastrin antibody with 50 ng/ml gastrin peptide. If present (without preabsorption), gastrin labeling would appear in red. Parietal cells are labeled with FITC-green for topographic orientation in the antrum. I and J, Double immunostaining for (I) gastrin (FITC-green) and (J) DDC (Cy3-red) in the antral mucosa. Photomicrograph pair of the same field. While intensely labeled DDC-containing cells did not overlap with the gastrin labeling (arrowheads), a weak but consistent DDC positivity was observed in G cells (arrows). K and L, Double immunostaining for DDC and HDC in the antral mucosa. Photomicrograph pair of the same field demonstrating the signal for (K) DDC (labeled by Cy3-red, arrowheads) and (L) both DDC (Cy3-red) and HDC (FITC-green) using a double pass fluorescent filter. K shows that the weak DDC labeling in HDC-containing G cells (arrows) as well as the general tissue background was reduced when we diluted the primary antibody in a high sodium-high pH diluent. L, In the double exposure photomicrograph DDC (arrowheads) and HDC (arrows) immunoreactivities were observed in distinct mucosal cells. M and N, Double immunostaining for (M) HDC (FITC-green) and (N) VMAT2 (Cy3-red) in the fundic mucosa. Photomicrograph pair of the same field. Fundic HDC-containing cells, considered to be ECL cells, are colabeled for VMAT2 transporter (arrowheads). O–Q, Double immunostaining for (O) HDC and (P) VMAT2 in the antral mucosa. Photomicrograph triplet of the same area demonstrating the signal for (O) HDC (FITC-green) and (P) VMAT2 (Cy3-red). Q shows the double exposure photomicrograph with both HDC and VMAT2 labeling. Rounded HDC positive antral cells, shown to be G cells (arrows), are not colabeled by the antibody to VMAT2. In contrast, elongated HDC-containing cells (arrowheads) that are most likely ECL cells display VMAT2 immunoreactivity. As expected, neuronal plexi are also labeled for VMAT2 (waved arrows). R, Double immunostaining for HDC (FITC-green) and VMAT1 (Cy3-red) in the fundic mucosa. Double exposure photomicrograph using a double pass fluorescent filter for FITC-green and Cy3-red. Overlapping green and red cells would appear yellow, but no VMAT1 labeling is found in fundic HDC-containing cells, considered to be ECL cells (waved arrows). VMAT1 immunoreactivity is observed in an HDC negative fundic cell population (arrowheads), shown to be enterochromaffin cells. S–U, Double immunostaining for (S) HDC and (T) VMAT1 in the antral mucosa. Photomicrograph triplet of the same field demonstrating the signal for (S) HDC (FITC-green) and (T) VMAT1 (Cy3-red). U shows the double exposure photomicrograph, where overlapping green and red cells appear yellow. Rounded HDC-containing cells, shown to be G cells (arrows), are colabeled for VMAT1 transporter. Other VMAT1 positive epithelial cells, negative for HDC (arrowheads), are most likely enterochromaffin (5-HT) cells. Elongated HDC-containing cells (waved arrows), not labeled for VMAT1, might be scattered ECL cells in the antrum.

 
The antigen specificity of the HDC antibodies were also analyzed by immunoblotting. In agreement with previous reports (22, 23, 24), 78 kDa, a 74-kDa and a 53-kDa protein species were detected by both antibodies to HDC (Fig. 4Go). The lower molecular weight species are likely to result from variations in splicing of HDC messenger RNA or proteolytic cleavage of the HDC enzyme, despite the presence of protease inhibitors during the sample preparation. Using the anti-DDC serum, a dominant 60-kDa immunoreactive protein species was detected, with two other fairly weak bands of 68 and 56 kDa. None of the protein species detected by the anti-DDC serum corresponded by apparent molecular weight to those protein species recognized by the anti-HDC serum. Conversely, the anti-HDC serum recognized none of the protein species identified by the anti-DDC antibody. Taken together, immunohistochemical and immunoblotting data suggest that each of these antisera is specific for their respective antigen and do not appear to cross-react with the other to any significant degree.



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  		Figure 4. Immunoblot analysis of the specificity of the antibodies to histidine decarboxylase (HDC) as compared with the immunoreactivity of an antibody to the closely related dopa decarboxylase (DDC). In agreement with previously reported sizes of rat histidine decarboxylase proteins, three immunoreactive protein species were detected by either antibodies to HDC. None of these proteins correspond by apparent molecular weight to those protein species recognized by the anti-DDC serum. These data suggest that each of these antisera are specific for their respective antigen and do not appear to cross-react with each other to any significant degree.

 
After the specificity of HDC labeling had been confirmed in antral epithelial cells, we wanted to demonstrate that these cells indeed contain histamine. With double immunostaining we found that all the cells immunoreactive for HDC were also immunolabeled by the antibody to histamine, including the rounded cells in the antrum (Fig. 3Go, B–D).

Because the localization, the shape, and the histamine content of these cells suggested that they were most likely endocrine cells, we double immunostained for HDC and chromogranin A (CGA), a secretory granule protein known to be present in all monoaminergic endocrine cells and neurons. All HDC immunoreactive epithelial cells were found to contain CGA in the antrum (Fig. 5Go). Some other epithelial cells also showed CGA immunoreactivity.



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  		Figure 5. Double immunostainings for (A) histidine decarboxylase (HDC) and (B) chromogranin A (CGA), a secretory granule protein known to be present in all monoaminergic cells. Photomicrograph pair of the same field demonstrating the signal for (A) HDC (labeled by FITC-green) and (B) the CGA (labeled by Cy3-red) labeling. All HDC-containing antral cells are immunoreactive for CGA (arrows), in addition to other CGA positive endocrine cells (arrowheads). Vertical section. Scale bar represents 100 µm.

 
Based on their shape and localization, we postulated that antral HDC positive cells are different from ECL cells. Thus, we decided to look for HDC in different endocrine cell types of the antral mucosa. We did not find HDC immunoreactivity in either EC cells (Fig. 6Go, A and B) or D cells (Fig. 7Go, A and B), identified by antibodies to 5-HT and somatostatin, respectively. However, an obvious overlap of HDC and gastrin labeling was found, identifying antral histaminergic epithelial cells as G cells (Fig. 3Go, E-G). The overlap of HDC immunoreactivity with G cells was confirmed with all four antibodies to gastrin, and the specificity of the gastrin labeling was also demonstrated by absorption controls (Fig. 3HGo) as well as by Grube’s high sodium-high pH specificity criteria.



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  		Figure 6. Double immunostainings for (A–C) serotonin and HDC, and (D–F) serotonin and DDC in the antral mucosa of rat stomach. A–C and D–F are photomicrograph triplets of the same fields demonstrating the signal for (A) and (D) serotonin (labeled by FITC-green) and for either (B) HDC or (E) DDC (labeled both by Cy3-red). C and F show double exposure photomicrographs combining the signals for serotonin + HDC and serotonin + DDC, respectively. A–C, Serotonin producing enterochromaffin cells (arrowheads) do not contain HDC (arrows). D–F, Serotonin and DDC immunoreactivity overlap in enterochromaffin cells (arrowheads). Surprisingly, serotonin-containing cells in the lamina propria do not seem to react with our antibodies to DDC (waved arrows). Vertical sections. Scale bar represents 100 µm.

 


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  		Figure 7. Double immunostainings for A–C somatostatin and HDC, and D–F somatostatin and DDC in the antral mucosa of rat stomach. A–C and D–F are photomicrograph triplets of the same fields demonstrating the signal for (A) and (D) somatostatin (labeled by FITC-green) and for either (B) HDC or (E) DDC (labeled both by Cy3-red). C and F show double exposure photomicrographs combining the signals for somatostatin + HDC and somatostatin + DDC, respectively. Somatostatin producing D cells (arrowheads) showed neither HDC (arrows in (A–C) nor DDC (arrows in (D–F) immunoreactivities. Vertical sections. Scale bar represents 200 µm in (A–C) and 100 µm in (D–F).

 
Dopa decarboxylase and TH immunoreactivities in the antral mucosa of rats
DDC immunoreactivity was detected in a population of CGA-containing epithelial cells of the antral mucosa using a sheep (Fig. 8Go) or two rabbit (Fig. 9Go) primary antibodies. The labeling for DDC was not abolished by preincubation with HDC protein supporting our results on the lack of cross-reactivity of these antibodies in Western blots (Fig. 4Go). In double stains, DDC positive antral epithelial cells were identified as 5-HT immunoreactive EC cells (Fig. 6Go, C and D). Somatostatin immunoreactive D cells were not DDC positive (Fig. 7Go, C and D).



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  		Figure 8. Double immunostainings for (A) DDC and (B) chromogranin A (CGA, a secretory granule protein known to be present in all monoaminergic cells) in the rat stomach. Photomicrograph pair of the same field demonstrating the signal for (A) DDC (labeled by FITC-green) and (B) CGA (labeled by Cy3-red). All DDC-containing cells are immunoreactive for CGA (arrowheads), in addition to many other CGA positive cells (arrows) in the antrum. Vertical section. Scale bar represents 100 µm.

 


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  		Figure 9. Staining properties of antibodies raised against dopa decarboxylase (DDC) in (A) rabbit and (B) sheep. Photomicrograph pair of the same field demonstrating the signal for DDC with (A) the rabbit (labeled by FITC-green) and (B) the sheep (labeled by Cy3-red) antibody. Both rabbit and sheep antibodies recognized the same cells. In addition to intensely labeled epithelial cells, identified subsequently as enterochromaffin cells (arrowheads), some other epithelial cells showed weak immunoreactivity to DDC with both antibodies (arrows). Vertical section. Scale bar represents 100 µm.

 
In addition, there was weak but consistent DDC staining in some other antral epithelial cells, including G cells (Figs. 3Go, I–J, and 9), with all three antibodies to DDC. This weak labeling was reduced but not abolished by Grube’s high sodium-high pH specificity control, which also decreased the general background of the stainings. DDC and HDC immunoreactivity in intensely labeled cells did not overlap when we diluted the primary antibodies in this high sodium - high pH diluent (Fig. 3Go, K–L).

In accordance with earlier reports on the distribution of TH and dopamine-hydroxylase (DBH) immunoreactivities in the rat stomach (25, 26), we found that TH immunoreactivity was restricted to the enteric neuronal network in the antrum, including sympathetic plexi around blood vessels (Fig. 10Go). We could not detect TH immunoreactivity in DDC-containing antral cells either, suggesting that DDC is involved in the synthesis of serotonin instead of catecholamines in these epithelial cells.



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  		Figure 10. Tyrosine hydroxylase (TH) labeling in the antral mucosa of rat stomach. TH immunoreactivity was restricted to enteric neurons (arrows) including sympathetic fibers around vessels (arrowheads), and it was not detected in epithelial cells of the antral mucosa. Vertical section. Scale bar represents 100 µm.

 
Vesicular monoamine transporters in antral mucosal cells of rats
In accordance with literature data, we detected VMAT2 transporter in ECL cells of the fundic mucosa (Fig. 3Go, M and N). In contrast, VMAT2 was restricted to enteric neurons and rare ECL cells in the antrum, and it was not detectable in antral HDC immunoreactive G cells (Fig. 3Go, O–Q). HDC immunoreactive G cells, however, had VMAT1 immunoreactivity (Fig. 3Go, R and S), which was not detected in ECL cells (Fig. 3Go, T and U). VMAT1 was also detected in DDC/5-HT-containing EC cells in both antral and fundic mucosa (Fig. 11Go).



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  		Figure 11. Double immunostainings for (A and B) DDC and VMAT1, and (C and D) DDC and VMAT2 in the rat stomach. A and B and C and D are photomicrograph pairs of the same fields demonstrating the signal for (A) and (C) DDC (labeled by FITC-green) and for either (B) VMAT1 or (D) VMAT2 (labeled both by Cy3-red). A and B, VMAT1 is present in all DDC-containing cells (arrows), in addition to numerous other VMAT1 positive epithelial cells (arrowheads), some of which are weakly immunoreactive for DDC. C and D, We could not detect VMAT2 immunoreactivity in gastric enterochromaffin (5-HT) cells, labeled for DDC (arrows). Vertical sections. Scale bar represents 100 µm.

 
Monoaminergic properties of mast cells in the rat stomach
All the histamine-containing cells of the stomach have been shown to contain HDC enzyme, including the mast cells located in the connective tissue compartment (Fig. 3Go, B–D). In agreement, we were able to detect HDC in a proportion of immunolabeled mast cells (Fig. 12Go, A and B). Surprisingly, 5-HT-containing cells in the lamina propria and submucosa did not display DDC immunoreactivity with our antibodies (Fig. 6Go, C and D), and we failed to colocalize DDC immunoreactivity to mast cells (Fig. 12Go, C and D). Neither VMAT1 nor VMAT2 transporter was observed in HDC or DDC-containing cells of the lamina propria and submucosa (Figs. 3Go, M–U, and 10).



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  		Figure 12. Double immunostainings for (A and B) mast cells and HDC, and (C and D) mast cells and DDC in the rat stomach. A and B and C and D are photomicrograph pairs of the same fields demonstrating the signal for (A, C) mast cells (labeled by FITC-green) and for either (B) HDC or (D) DDC (labeled both by Cy3-red). A and B, Tangential section shows the submucosa of the antral stomach with a vessel (*). HDC is present at least in a subpopulation of mast cells (arrows). In other mast cells HDC was not detectable (arrowheads). C and D, Vertical section of the antral mucosa. We could not detect DDC immunoreactivity in mast cells (arrowheads) of the lamina propria or submucosa with our antibodies. (arrows) point to DDC positive epithelial cells. Solid white arrows point to small cells in the lamina propria that were nonspecifically labeled by certain batches of secondary antibodies. Scale bar represents 100 µm in all panels.

 
Immunohistochemical characterization of different endocrine/monoaminergic cell types in the antral mucosa of rat stomach is summarized in Table 3Go.


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  		Table 3. Immunohistochemical characterization of different endocrine/monoaminergic cell types in the antral mucosa of rat stomach

 
Results of the additional immunohistochemical controls
In addition to the above described immunohistochemical and Western blot specificity controls of HDC, DDC, and gastrin antibodies, the staining properties of the monoaminergic markers used in these studies matched the known distribution of the respective monoamines in control brain sections (12, 13).

No specific staining was seen when primary or secondary antibodies were omitted or replaced by nonimmune sera. Occasional nonspecific labeling in some small cells in the lamina propria was easily distinguishable from the labeling in epithelial cells. This weak nonspecific labeling persisted even when the primary antibody was absorbed or omitted, and it was typically avoided by using another batch of the same secondary antibody. Changing the order of primary antibodies did not affect the staining pattern in double immunostainings. We also determined in our hands that none of the secondary antibodies showed detectable cross-reactivity with primary antibodies from a noncorresponding host species.

Results of tritiated histamine uptake studies
There is an uptake of tritiated histamine in the antral mucosa (Fig. 13AGo) that is significantly reduced by either the addition of nonlabeled histamine (Fig. 13BGo) or by addition of a specific histamine uptake blocker (Fig. 13CGo).



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  		Figure 13. Uptake of tritiated histamine in the antrum. The left side shows enlarged film images of autoradiography. After the uptake studies were performed, the antrum pieces were fixed in formaldehyde, cryoprotected and 12 µm thin section were cut on a cryostat. These were mounted on glass slides and then opposed to a sensitive x-ray film (Amersham Hyperfilm-3H RPN 12) and developed 4 weeks later. The images were scanned into Adobe Photoshop and analyzed for density gradients using NIH Image software. The result of the image analysis is shown on the right side of the autoradiographic images. The vertical axis shows the relative number of pixels in each of the 10 (1 2 3 4 5 6 7 8 9 10 ) classes going from white to black. The uppermost image shows histamine uptake when 3H-Histamine is added to the incubation. The middle panel shows when an excess of cold histamine is used and the lower panel shows the uptake in the presence of a histamine uptake blocker (Thioperamide). The highest density area in the autoradiographies corresponds to the area where histamine (i.e. gastrin) producing cells are localized in the antral mucosa.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Histamine, a potent stimulant of gastric acid secretion, is stored in enterochromaffin-like (ECL) and mast cells in the fundic part of the rat stomach. However, its source in the antrum was previously unknown. We have found that numerous epithelial cells at the base of pyloric glands contain histamine and histidine decarboxylase (HDC), the enzyme responsible for the synthesis of histamine. ECL cells, known to be the epithelial source of histamine in the fundic mucosa, are reportedly rare in the antrum (21). Furthermore, the majority of antral histaminergic cells did not have the characteristic shape of fundic ECL cells. Thus, we hypothesized that these cells were different from ECL cells, and we attempted to identify them. Immunological markers for different gastrointestinal peptides revealed that the novel antral HDC/histamine-containing endocrine cell population is identical with antral G cells, which also make and release gastrin, another potent secretagogue in the stomach.

In addition to HDC positivity, G cells showed a weak but consistent immunoreactivity to dopa decarboxylase enzyme (DDC). The fact that DDC, but not TH, was present in G cells suggests that they may synthesize small amounts of serotonin, or amines, such as tyramine. Showing this will require further study.

Messenger RNAs of both isoforms of vesicular monoamine transporters (VMAT1 and VMAT2) are reportedly present in the rat stomach (27). VMAT2 has been previously suggested (28, 29, 30) and here confirmed to be present in ECL cells and in enteric neurons. However, cellular localization of VMAT1 in the stomach has not been reported previously. We demonstrate here by double immunostainings that two populations of epithelial cells display VMAT1 immunoreactivity in the antral mucosa. One of these populations have been shown to contain DDC enzyme and 5-HT, thus meeting the definition of EC cells. The other VMAT1 positive cell population has been identified as histaminergic G cells. The presence of the vesicular monoamine transporter in these cells suggests that they can store and release monoamines, -5-HT in the case of EC cells, and histamine in the case of G cells.

Analysis of monoamine storing cells in the connective tissue compartments of the stomach revealed a surprising lack of DDC and VMAT immunoreactivities in mast cells, even though they appear to be rich in HDC. Whether these cells contain immunologically different decarboxylase enzymes and transporters, or the synthesis and storage of monoamines is completely different in these cells compared with epithelial and neuronal monoaminergic cells, remains to be further studied.

Our results indicate the presence of histamine in antral G cells of rat stomach. It may act locally in the antrum as an autocrine stimulator or inhibitor of gastrin secretion; activation of H3 receptors for example, reduces gastric acid secretion, and both histamine and histamine-agonists reportedly reduce histamine secretion by the isolated rat stomach (31). These data, taken together with the newly found histaminergic properties of G cells, suggest that G cell-derived histamine may inhibit gastrin secretion as a negative feedback mechanism. Reduction of gastrin secretion may, in turn, reduce histamine secretion from ECL cells (32, 33, 34, 35). Because histamine H1 receptor agonists and antagonists have no relevant effect on gastric secretion, the receptor subtype potentially involved in this postulated autoregulation of G cells might be either the histamine H2 or H3 receptors. Antral histamine could potentially regulate other local functions, such as mucus, bicarbonate, somatostatin, or serotonin secretion.

Alternatively, G cell-derived histamine may stimulate gastrin secretion and/or contribute to its secretagogue actions in the fundus. Like gastrin, histamine may reach target cells in the fundic part of the stomach, producing acid stimulatory effect on parietal cells. A common cellular origin of antral histamine and gastrin may be the key to their success in reaching their targets in the distant fundic area. Further studies are needed to elucidate the role of G cell-derived histamine in the physiology and/or pathology of the stomach.


   Acknowledgments
 
The authors would like to thank Dr. Lee E. Eiden at the National Institute of Mental Health (Bethesda, MD) for supplying the chromogranin A antibody, Dr. Lo Persson at Department of Physiology, University of Lund (Lund, Sweden) for the donation of HDC protein, and Ricardo Dreyfuss for his help in photomicrography.

Received February 23, 1998.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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