ARTICLE |
Correspondence to: Rolf Håkanson, Dept. of Pharmacology, University of Lund, Sölvegatan 10, S-223 62 Lund, Sweden. E-mail: Rolf.Hakanson@farm.lu.se
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Summary |
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The rat stomach is rich in endocrine cells. The acid-producing (oxyntic) mucosa contains ECL cells, A-like cells, and somatostatin (D) cells, and the antrum harbours gastrin (G) cells, enterochromaffin (EC) cells and D cells. Although chromogranin A (CgA) occurs in all these cells, its processing appears to differ from one cell type to another. Eleven antisera generated to different regions of rat CgA, two antisera generated to a human (h) CgA sequences, and one to a bovine (b) CgA sequence, respectively, were employed together with antisera directed towards cell-specific markers such as gastrin (G cells), serotonin (EC cells), histidine decarboxylase (ECL cells) and somatostatin (D cells) to characterize the expression of CgA and CgA-derived peptides in the various endocrine cell populations of the rat stomach. In the oxyntic mucosa, antisera raised against CgA291319 and CGA316321 immunostained D cells exclusively, whereas antisera raised against bCgA8291 and CgA121128 immunostained A-like cells and D cells. Antisera raised against CgA318349 and CgA437448 immunostained ECL cells and A-like cells, but not D cells. In the antrum, antisera against CgA291319 immunostained D cells, and antisera against CgA351356 immunostained G cells. Our observations suggest that each individual endocrine cell type in the rat stomach generates a unique mixture of CgA-derived peptides, probably reflecting cell-specific differences in the post-translational processing of CgA and its peptide products. A panel of antisera that recognize specific domains of CgA may help to identify individual endocrine cell populations. (J Histochem Cytochem 49:918, 2001)
Key Words: chromogranin A, ECL cell, D cell, A-like cell, EC cell, G cell, processing
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Introduction |
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Chromogranins occur in secretory granules of most neuroendocrine cells, together with amines and/or regulatory peptides (
The ECL cells reside within the basal zone of the oxyntic mucosa and are characterized by their expression of histidine decarboxylase (HDC) and histamine (
The objective of this investigation was to use immunohistochemistry to examine the expression and proteolytic processing of CgA in the different endocrine cell populations of the stomach by employing antisera directed to specific domains of CgA and CgA-derived peptides.
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Materials and Methods |
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Antisera Production
All antisera were raised against rat CgA sequences unless otherwise stated (Fig 1). The generation of eleven of the antisera has been described previously (for references see Table 1). One of these antisera (R880) was raised against a bovine (b) rather than a rat CgA sequence (bCgA8291). This sequence differs from the corresponding rat sequence in two positions (F in rCgA, instead of Y, in position 82 and F in rat CgA, instead of L, in position 90). Antiserum (R814) was raised against a human (h) CgA sequence (343356) that differs from the corresponding rat sequence in one position only (R in rCgA instead of K in position 345). An additional three CgA antisera were generated and used here for the first time. These antisera were raised against the C-terminal octapeptide APSKDTVE, representing residues 121128 of rCgA, Y0SGEATDGARPQALPEPMQESK, representing residues 124144 of hCgA (Bachem; La Jolla, CA), and the dodecapeptide KVAHQLQALRRG, representing residues 437448 of rCgA. hCgA124144 exhibits limited sequence homology with the corresponding rCgA sequence except in the sequence 132143. Hence, the antiserum was expected to recognize the corresponding sequence in rCgA, which differs from the human in two positions only (F in rCgA, instead of L, in position 136 and K in rCgA, instead of M, in position 140). The peptides were synthesized by solid-phase methods using Fmoc strategy employing an automated peptide synthesizer (
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Markers for Identification of Individual Endocrine Cell Populations
An antiserum raised in a guinea pig against rat HDC (code no. M9501;
Animals
Male SpragueDawley rats weighing 200250 g were sacrificed by exsanguination from the abdominal aorta under chloral hydrate anesthesia (300 mg/kg, IP). The stomach was opened along the greater curvature, washed in 0.9% saline, and placed, mucosal side up, on a cold glass surface. Tissue specimens from the acid-producing part of the stomach were taken from the ventral side close to the rumen. Specimens from the antrum were collected from the ventral side 12 mm above the pyloric sphincter.
Immunocytochemistry
Tissue specimens were immersion-fixed in 4% paraformaldehyde in 0.1 M PBS, (pH 7.2) for 20 hr at 4C and cryoprotected in 5% (w/v) and 30% sucrose in 0.1 M phosphate buffer containing 0.01% NaN3 before freezing in OCT embedding medium (Miles; Elkhart, IN). Frozen sections (6 µm) were cut using a Leitz cryostat and incubated (18 hr, 4C) with one of the primary antisera (Table 1). Immunoreaction was visualized using rhodamine-conjugated secondary swine anti-rabbit or fluorescein-conjugated rabbit anti-guinea pig IgG antibodies (1 hr, room temperature). Co-localization studies were performed as described previously (
Control studies included liquid-phase preabsorption of each antiserum with its respective antigen (Table 1) and with the antigens used to raise the other antisera to CgA-derived peptides, and also the routine omission of primary antisera and substitution with non-immune rabbit, rat, and guinea pig serum. Truncated and C-terminally extended fragments of the respective antigens were synthesized (as above) and used to further characterize the epitope recognized by the various antisera (Table 2). These fragments included ELSEVLE, ELSEVLEN (of antigen YEDELSEVLE), SKDTVE (of antigen APSKDTVE), QELEK (of antigen Y0KGQELE), LGPPQGLFPG-amide, GPPQGLFPG, PPQGLFPG-amide, PQGLFPG- amide and GPPQGLFP (of antigen GPPQGLFPG-amide), WSKMDQLA, SKMDQLAKE, and ELTAEK (of antigens WSKMDQLAKELTAE (WE-14;
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Results |
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Specificity of Antisera
No immunostaining was observed after omission of the primary antiserum and the inclusion of non-immune rabbit or guinea pig serum. Liquid-phase preabsorption of each antiserum with its respective antigen (1 µg/ml, except for R876 100 µg/ml) overnight at 4C abolished immunostaining (Table 2). Preabsorption with antigens used to raise other CgA antisera did not affect immunostaining. Antisera were further characterized after preabsorption with truncated and/or N-terminally or C-terminally extended peptides to determine epitope recognition sites (Table 2). Antiserum R880, raised against bCgA8291 (YEDELSEVLE), was shown to detect the mid- or C-terminal part of the antigen in that both ELSEVLE and ELSEVLEN abolished immunostaining (10 µg/ml). Immunostaining of antiserum R876 raised against CgA121128 (APSKDTVE, C-terminal end of ß-granin;
Distribution of CgA Immunoreactivity
The pattern of immunostaining observed after application of the various CgA antisera to the different endocrine cell populations of the oxyntic mucosa and antrum are summarized in Table 3. Seven of the 14 antisera immunostained all of the major endocrine cell populations (ECL cells, A-like cells, and D cells) in the oxyntic mucosa, and nine immunostained all endocrine cell populations in the antrum (EC cells, G cells, and D cells). The remaining antisera displayed a selective pattern of immunostaining. In the oxyntic mucosa, the ECL cells, A-like cells, and D cells were immunonegative after incubation with antiserum R635 (raised against CgA351356). However, a discrete population of cells in the oxyntic mucosa, possibly D1/P cells, did exhibit moderate immunostaining. In the antrum, this antiserum immunostained G cells exclusively.
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Oxyntic Mucosa
ECL Cells.
Nine of the antisera immunostained the ECL cells (Table 3), but none of them immunostained the ECL cells exclusively (Fig 2).
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A-like Cells.
Antisera R880 and R876 (raised against bCgA8291 and CgA121128, respectively) failed to stain the ECL cells, whereas D cells and an additional subpopulation of endocrine cells did display staining. Double immunostaining with the somatostatin antiserum revealed that most of these immunopositive cells were distinct from the D cells. The immunopositive cells were larger than D cells, exhibited a distinct spherical shape, and were found throughout the glands of the oxyntic mucosa (Fig 3a and Fig 3b). Their number and morphological features are reminiscent of the A-like cells (
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D Cells. Eleven of the 14 antisera demonstrated the D cells. Seven of these also immunostained the ECL cells, and nine immunostained the A-like cells. Three of the 14 antisera, H98 (raised against CgA318349), R635 (raised against CgA351356), and R881 (raised against CgA437448), failed to stain the D cell population. Double immunostaining with the somatostatin antiserum revealed that both A87A and R633 (raised against CgA291319 and CgA316321, respectively) immunostained D cells exclusively (Fig 3c and Fig 3d).
Antral Mucosa
G Cells.
Antiserum R635 (raised against CgA351356), which failed to stain the ECL cells, A-like, or D cells in the oxyntic mucosa, immunostained the G cells in the antrum. Antiserum A87A (raised against CgA291319) failed to stain the G cell population. All other CgA antisera immunostained the G cells.
EC Cells. None of the antisera immunostained EC cells exclusively. Most antisera immunostained the G, D, and EC cell populations.
D Cells. As in the oxyntic mucosa, A87A (raised against CGA291319) immunostained D cells exclusively. Antisera H98, R635, and R881 (raised against CgA318349, CgA351356 and CgA437448, respectively), failed to stain D cells in the antrum as well as in the oxyntic mucosa.
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Discussion |
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Both light microscopic and electron microscopic techniques have been employed to localize CgA and CgA-derived peptides to gastric endocrine cells (
Oxyntic Mucosa
The ECL cells represent the predominant endocrine cell type of the oxyntic mucosa. In the rat stomach, histamine is found in both mast cells and ECL cells (
The somatostatin-secreting D cells are spindle-shaped cells with long slender projections (
To date, there is no recognized immunohistochemical marker for A-like cells, and their identification is based on indirect criteria. A-like cells are CgA-immunoreactive cells that do not display either HDC or somatostatin immunoreactivity and that exhibit a characteristic spherical shape and occur dispersed throughout the gastric glands in lower numbers than the ECL cells. Eleven of the CgA antisera immunostained this endocrine cell population. However, no single antiserum immunostained these cells exclusively. Two antisera, R880 (raised against CgA8291) and R876 (raised against CgA121128), which were raised against different regions of the N-terminal CgA domain (CgA1128), referred to as ß-granin (
Antrum
The predominant endocrine cell populations of the antral mucosa, the G cells, EC cells, and D cells, are localized in the basal part of the glands. All three endocrine cell populations are of the "open" type. Antiserum R635, specific for the C-terminal end of WE-14, immunostained G cells but not EC cells or D cells. This observation suggests that WE-14 is generated in G cells only.
Antral D cells were immunostained by 11 of the 14 antisera raised against CgA. One antiserum, A87A (raised against CgA291319), immunostained the antral (and oxyntic) D cells but no other endocrine cells. R633 (raised against CgA316321) immunostained antral (and oxyntic) D cells and G cells. Antiserum R36D8 (raised against CgA124144) immunostained EC cells and D cells but not G cells. The fact that the D cells of both the oxyntic mucosa (closed type) and the antrum (open type) displayed identical patterns of CgA immunostaining is notable in view of the fact that they appear to differ from each other both morphologically and functionally (
Eleven of the 14 antisera immunostained the EC cells. The EC cell pattern of CgA immunostaining was identical to that of the A-like cells in the oxyntic mucosa, implying similarities in biosynthesis and processing of CgA.
Conclusions
In this study, 14 well-characterized CgA antisera were tested to generate information on the extent of cell-specific processing in the endocrine cells of the rat stomach. All major endocrine cell types were found to express CgA or CgA-derived peptides. In addition, the application of region-specific antisera raised against a range of CgA-derived peptides has revealed that each endocrine cell type expresses a unique mixture of CgA-derived peptides. This implies that each endocrine cell type has a characteristic spectrum of processing enzymes, e.g., prohormone convertase, carboxypeptidase, and amidating enzymes. It cannot be excluded, however, that additional differences with respect to glycosylation, phosphorylation, sulfation, or the rate and degree of processing will influence the ability of the processed products to react with the antisera. We propose that antisera that recognize specific domains of CgA be used as tools to identify individual endocrine cell populations. This may aid in the identification of cell populations for which no cell-specific markers exist or which, for some reason, are difficult to immunostain with a cell-specific marker (such as D cells and G cells in somatostatin or gastrin knockout animals). The functional significance of the present findings may perhaps be discussed in relation to the prohormones produced by the various endocrine cell types (such as preprogastrin in G cells or prosomatostatin in D cells); the CgA processing pattern may predict how the prohormones are being processed.
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Acknowledgments |
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Supported by grants from the Swedish MRC (04X-1007), the Cancer Foundation (2542-B95-07XCC), the A. Påhlsson Foundation, the Medical Faculty, (University of Lund; Lund, Sweden), and Ipsen Pharmaceuticals, (London, UK).
Received for publication March 16, 2000; accepted July 14, 2000.
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