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Autoradiographic demonstration of gastrin-releasing peptide-binding sites in the rat gastric mucosa
GASTROENTEROLOGY 1988;94:968-76
Autoradiographic Demonstration of Gas&n-Releasing Peptide-Binding Sites in the Rat Gastric Mucosa MASAHIKO YASUFUMI HIROKAZU
NAKAMURA, MASAYA ODA, KOTARO KANEKO, AKAIWA, NOBUHIRO TSUKADA, and MASAHARU TSUCHIYA KOMATSU,
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan
The location of [1251]iodotyrosyl gastrin-releasing peptide-binding sites in the rat fundic mucosa was studied. Peptide specificity was demonstrated by competitive binding studies using the addition of a large amount of cold gastrin-releasing peptide or substance P. Autoradiography of the stomach tissue was carried out by freeze-drying, embedding in Epon, wet-sectioning with ethylene glycol, and drymounting the emulsion film by the wire-loop method to prevent loss of the labeled substance. Specific binding sites of gastrin-releasing peptide were found on D cells, surface mucus cells, and parietal cells, whereas few binding sites were seen on the chief or mucus neck cells.
G
astrin-releasing peptide (GRP) was first isolated from nonantral gastric tissue as a 27-amino acid peptide (1). In the gastrointestinal tract, GRP is thought to be located in nerve fibers and cell bodies of the myenteric and submucosal nerve plexus and to act as a neuromodulator in regulating the secretion of various kinds of peptides from the neuroendocrine cells (2). In the stomach, GRP is thought to release gastrin from the antral G cells and to stimulate acid secretion from the parietal cells (3). Recent histochemical observations, however, have shown that most of the GRP-containing fibers are located in the fundic region, not in the antral region, suggesting the direct action of GRP on the fundic glandular cells (4).
Therefore, this study was designed to clarify the binding sites of [3-1251]iodotyrosyl GRP in the rat fundic mucosa by light and electrop micrdsqopic autoradiography, combined with the immunohistochemical technique.
Materials
and Methods
Administration of [3251]Iodotyrosyl GastrinReleasing Peptide Male Wistar rats weighing 200-250 g were used in the following experiments. The rats were divided into control and cold GRP-treated groups. In the cold GRPtreated group, lo-“, 10-13, lo-“, and lo-’ mol GRP/kg body wt were mixed with radiolabeled GRP and administered through an intraaortic catheter in the following manner. Under light anesthesia by intraperitoneal injection of sodium pentobarbital (20 mg/kg body wt), the abdomen was opened by a lower middle incision and a polyethylene catheter was inserted into the abdominal aorta up to the opening of the celiac artery. A dose of 0.7 ml of an aqueous solution of [3-‘251]iodotyrosyl synthetic porcine GRP (50 &i/kg body wt; 2000 Cilmmol; 2.5 X lo-l3 mol/kg body wt; Amersham International plc, Amersham, U.K.) was infused for 10 min through the aortic catheter using an infusion pump at a constant rate of 2.1 ml/h, followed by infusion of physiologic saline for 5 min to wash out the unbound ligand. As the control, the same amount of lZ51-substance P (Amersham), one of the gastrointestinal peptides structurally related to GRP, was infused and the distribution of the binding sites of ‘251-substance P was compared with that of lz51-GRP. Tissue Preparation for Light and Microscopic Autoradiography
Electron
Immediately after the infusion, the fundic portion of the stomach was taken out and cut into small tissue blacks (2 x 2 x 2 mm3) with a razor blade. These blocks were quickly frozen in isopentane cooled to its melting point with liquid nitrogen. The stomach tissue blocks were freeze-dried for 48 h in an apparatus (Oka Science OTDAbbreviation
used in this paper:
GRP, gastrin-releasing
peptide. 0 1988
by the American Gastroenterological 0016~5Q85/88l$3.50
Association
GRP-BINDING SITES ON RAT GASTRIC MUCOSA
April 1988
1SF) cooled to -50°C and evacuated at 9 X lo-” torr. The freeze-dried tissue blocks were then exposed to osmium vapor, followed by infiltration in Epon by a dripping unit evacuated by rotary pump (5). The tissue blocks were removed and embedded in freshly prepared Epon and polymerized at 60°C. Semithin (1 pm thick) or ultrathin (1000-2000 A thick) sections were cut with an LKB ultramicrotome using ethylene glycol instead of water. Autoradiographic emulsion (Sakura NR-H2 or M2) was diluted (1:2 or 1:3) with distilled water at 40°C in the darkroom. According to the previously described drymounting procedure (6), an emulsion film was made between vinyl-coated iron-wire loops by dipping the wire loops into a solution of dioctyl sodium sulfosuccinate, with the wire loops being left to stand at normal temperature until the emulsion film was almost dry. The emulsion film was then applied to the glass plates or grids on which sections were mounted. The sections were kept in the dark in a refrigerator at 4°C for 4-8 wk for exposure. After development and fixation, they were examined under light and electron microscopes. Some of the stomach tissue sections were processed for electron microscopy, and the parietal cell morphology was quantified according to Black’s classification (7) to clarify the functional status of the parietal cells. Oxyntic cells were classified into one of four categories by the structure of the intracellular canaliculi and the tubulovesicles, i.e., into resting, stimulated, partially stimulated, and returning states. The percentages of the cells in the four conditions were compared in the GRP-infused and control rats. Analysis
of Autoradiographs
The silver grains on the chief, parietal, mucus neck, surface mucus, and D cells were counted in the cold GRP-treated and control groups. The background counting rate was determined by counting the grains in the stomach tissue obtained from the rat infused with saline alone and treated in the same fashion described above. The number of grains on the cells was then estimated by subtracting the background count. D cells were identified by an indirect immunohistochemical technique using the antisomatostatin antibody. The sections used for light microscopic autoradiography were then processed for the immunocytochemical demonstration of somatostatin using rabbit antiserum to human somatostatin (Milab) at a dilution of 1:lOOO. The site of the antigen-antibody reaction was revealed with the indirect
Table
1. Analysis
of Morphology
IO-l3
969
10-'
10-l'
UNLABELED PEPTIDE (M) Figure
1. Inhibition of [1251]iodotyrosyl GRP binding by unlabeled GRP or substance P. The rats were treated with 2.5 x lOeTa molikg body wt [‘251]iodotyrosyl GRP mixed with varying concentrations of unlabeled GRP or substance P by intraaortic infusion, and the radioactivity remaining in the gastric mucosa was counted using a y-counter.
immunohistochemical method (8). The preparations were incubated with diluted primary antisera for 24 h at 4°C in a humid chamber, then washed in phosphate-buffered saline and incubated for 1 h with a horseradish peroxidase-conjugated second antibody (goat immunoglobulins to rabbit immunoglobulins) at a dilution of 1:40. After being washed in phosphate-buffered saline, the sections were treated with dimethylaminoazobenzene solution for 5 min (9). For control sections, we used antiserum that had been inactivated by the addition of an excess amount of antigen.
Assay
of Binding
Releasing
Peptide
of [“51]Iodotyrosyl to the Gastric
Gastrin-
Mucosa
Soon after the continuous intraaortic infusion of cold GRP or substance P mixed with radiolabeled GRP, a portion of the stomach tissue was homogenized with a glass homogenizer. The accumulation of [‘251]iodotyrosyl GRP on the fundic mucosa was estimated with a y-counter (ARC-300), and the radioactivity in the gastric mucosa was
of Control and Gastrin-Releasing
Peptide-Stimulated
Rat Gastric Parietal
Cells
Morphologic appearance I% of totall Condition
n (n’)
Resting
50 (51 50 (5)
Stimulated n, number of calculated 0.03 vs. resting condition
Stimulated 0 64"
parietal cells; n’, number of rats used. Morphologic by binomial distribution.
Partially stimulated 12 24 appearance
Returning
Resting
16 8
72 40 was judged according
to Black’s
criteria.
ap <
970
Figure
NAKAMURA
ET AL.
GASTROENTEROLOGY
2. Electron micrographs of the typical parietal cells of the GRP-infused and control rats. a. In the GRP-infused parietal cells indicate large numbers of greatly expanded intracellular canaliculi (ICI. Magnification, x 7000. rats, most of the cells contain abundant tubulovesicles (TV). Magnification, x 10,000.
estimated by determining the protein method of Lowry et al. (10).
content
using
the
Results Assay of [‘Z5J]IodotyrosyJ Gastrin-Releasing Peptide Binding to Gastric Mucosa As is clear in Figure 1,unlabeled GRP in the range of lo-l5to lo-'mol/kg body wt competitively inhibited the binding of [‘251]iodotyrosyl GRP to the fundic mucosa in a continuous dose-dependent fashFigure
ion, and substance P had almost soever on the binding of GRP.
Vol. 94, No. 4
rats, most of the b. In the control
no influence
what-
Alteration of Parietal Cell Ultrastructure After Gastrin-Releasing Peptide Infusion The distribution profile of parietal cells from the GRP-infused stomach was markedly different from that of the control stomach (Table 1). The cells of the former were mostly classified as stimulated, whereas the latter indicated the large majority of cells as resting (Figure 2). These morphologic data >
3. Light microscopic autoradiographs of the gastric mucosa of the rat infused with [‘Z51]iodotyrosyl GRP or ‘Z51-substance P. a-d. Tip portion of the gastric mucosa. The grains (arrowheads) are mainly recognized on the surface epithelial cells [a, b). On the other hand, through the addition of an excess amount of cold GRP (c), the binding of GRP on the epithelial cells is only slightly recognized, with only a little found on the capillaries (arrowheads]. In the rats receiving substance P, few grains are seen on the epithelial cells. a, c, and d were unstained, and b was counterstained with toluidine blue. e-h. Middle and basal portions of the gastric mucosa. Most of the grains (arrowheads) are found on the parietal cells, and not on the chief cells having toluidine blue-positive granules (d, e). whereas few grains are found on the parietal cells through the addition of an excess amount of cold GRP cfl. In the substance P-treated rats, no grains were found on these epithelial cells. e. g. and h were unstained, and f was counterstained with toluidine blue. Magnification, X 1200.
April 1988
GRP-BINDING SITES ON RAT GASTRIC MUCOSA
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GASTROENTEROLOGY Vol.
NAKAMURA ET AL.
94, No.
Figure 4. Electron microscopic autoradiographs of the surface mucus cell of the tip portion of the rat gastric mucosa infused with [‘251]iodotyrosyl GRP. a. The silver grains (arrowheads], corresponding to the GRP-binding sites, are found near the basolateral membrane of the cells. Magnification, x 9000. b. An electron micrograph of the surface mucus cells fixed with mixed solution of 4% formaldehyde and 1% glutaraldehyde. No silver grains are observed on these cells. Magnification, x7000.
confirm that GRP administration stimulates hydrochloric acid secretion from the parietal cell. Distribution of [‘251]Iodotyrosyi Gastrin-Releasing Peptide-Binding the Gastric Mucosa
Sites in
Silver grains corresponding to the localization of ‘251-GRP were seen mainly in the tip and middle portions of the gastric mucosa, whereas few grains were recognized by the addition of an excess amount of cold GRP, showing the localization of these silver grains specific for GRP (Figure 3). Few grains were found after the administration of ‘9-substance P, which also suggested the specificity of localization of silver grains after 9-GRP administration. Under higher magnification, most of the cells having silver grains as a result of lz51-GRP administration were found to be surface mucus cells in the tip portion of the gastric mucosa (Figure 41, and the two types of cells in the middle portion of the gastric
mucosa, i.e., parietal cells and basally granulated cells (Figure 5). The immunohistochemical method, using the antisomatostatin antibody, showed the latter cells to be D cells (Figure 6). Effects of Administration of Cold GastrinReleasing Peptide and Substance P on the [‘251]Iodotyrosyl Gastrin-Releasing Peptide Binding Studied by Light Microscopic Autoradiography After the mixed administration of lo-" M cold GRP, significantly fewer grains were seen on the parietal, surface mucus, or D cells compared with the group treated by [9]iodotyrosyl GRP alone. On the other hand, the same amount of substance P demonstrated no significant effect on GRP binding (Table 2). These results, together with the data shown in Figure 1, suggest that the localization of radioactivity found after [‘251]iodotyrosyl GRP administration is specific for GRP-binding sites.
April 1988
Figure
GRP-BINDING
SITES ON RAT GASTRIC
MUCOSA
973
5. Electron microscopic autoradiographs of the parietal and basally granulated cells in the rat infused with [‘2sI]iodotyrosyl GRF’. o. The GRP-binding sites on the parietal cells (arrowheads) are mainly found in the cytoplasm of the parietal cells. Magnification: ~15,000. c. The GRP-binding sites (arrowheads) on the basally granulated cells are found near the plasma membrane. Magnification: ~12,000. a, c. Freeze-dried specimens. b, d. Electron micrographs of the parietal (b) and D cell (d) fixed with a mixed solution of 4% glutaraldehyde and 1% formaldehyde. No grains are found on these cells. Magnification: b, x12,000; d, x10,000. Uranyl acetate and lead citrate stains. respectively.
974
NAKAMIJRA ET AL
GASTROENTEROLOGY Vol. 94,No. 4
Figure 6. Light microscopic autoradiographs of the GRP-binding sites on the basal portion of the gastric mucosa. o. Some grains (arrowheads) are recognized on the small cells just adjacent to the parietal cells. b, c. By the application of the indirect immunohistochemical technique using antisomatostatin antibody, these cells (arrowheads) are found to be positive to somatostatin, corresponding to D cells. c. Counterstained with toluidine blue. Magnification, x 1200.CV, collecting venule.
Discussion Somatostatin-containing D cells are located in the upper portion of the digestive system, mainly from the fundic region of the stomach to the duodenum (11)and pancreas (12)) and are thought to act as a neuromodulator and to regulate the secretory status of the endocrine or exocrine cells in terms of paracrine. According to a recent pharmacologic study (3), GRP suppresses the secretion of somatostatin in the antral region of the stomach, and then, directly or indirectly, increases gastrin secretion. These phenomena result in augumented secretion of hydrochloric acid from the parietal cells.
In a histochemical study (4), GRP-like immunoreactive fibers were found to be more numerous in the oxyntic region than in the antral region. It is postulated from these results that GRP released from the nerve fibers reaches the antrum through the circulation and stimulates the G cell. It is more reasonable, however, to think that GRP plays a more significant role in the fundic region (13). D cells in the fundic region are thought to directly inhibit acid secretion from the parietal cells. In the present investigation, the GRP-binding sites in the fundus were found to be abundant on the D cells, suggesting the direct regulation by GRP of somatostatin secretion in the oxyntic gland.
April
1988
GRP-BINDING
SITES ON RAT GASTRIC
MUCOSA
975
Table 2. Localization of [‘251JIodotyrosyl Gas&in-Releasing Peptide-Binding Sites in Rat Gastric Mucosa Number
[ “‘I]Iodotyrosyl GRP + 10~” M cold GRP
(‘2”I]Iodotyrosyl GRP alone Surface mucus cells Mucus neck cells Parietal cells Chief cells D cells
5.4 + 1.6 + 9.2 ? 1.6 t 13 ?
of grains/cell
1.2 0.42 2.3 1.0 3.2
(“‘I]Iodotyrosyl GRP + 16” cold substance
0.80 t 0.40" 0.80 It 0.40 1.0 f 0.42" 0.80 + 0.40 1.2 t 0.700
4.8 ? 1.0 ? 8.8 k 1.0 k 14 k
M P
2.1" 0.63 1.8': 0.63 3.8"
Values are means 2 SD (n = 5)." p < 0.001 vs. [I"Ijiodotyrosyl GRP alone. b 0.001 < p < 0.01 vs. [‘““Iliodotyrosyl GRP + lo- I’ M cold GRP. ’ p < 0.001 vs. [ “‘I]iodotyrosyl GRP + lo-” M cold GRP. Specific binding sites of GRP are found on the surface mucus, parietal. and D cells.
The parietal cells were also found to have specific binding sites for GRP. In a pharmacologic study with the infusion of GRP (l4), gastrin was found to increase immediately after the infusion, with a IS-min lag period of acid secretion from the parietal cells. Complete removal of gastrin-producing antral G cells by antrectomy totally eliminates the stimulatory effect of bombesin on gastric acid secretion (15). These data suggest that GRP action on the parietal cell is mediated by gastrin. Therefore, the present data may indicate the direct action of GRP on the parietal cell in some fashion other than the direct stimulation of acid secretion, whereas another report suggests the direct action of GRP on acid secretion from the parietal cell (16). The binding sites on the parietal cells were dispersed in the cytoplasm and not on the cell membrane. Although this may suggest the internalization of GRP, it may result from the movement of the metabolites of GRP into the cytoplasm, in view of the rapid turnover of GRP induced by the membranebound peptidase (17). With respect to the mucus secretion, few mediators have been reported. In our recent study, few binding sites of quinuclidinyl benzilate (18), pirenzepine (6), or gastrin (19) have been seen on the mucus-secreting cells. In this study, specific GRPbinding sites were found on the surface mucus cells, suggesting that the cholinergic stimulation of the mucus secretion may be mediated by GRP. In conclusion, direct binding of GRP to the parietal and D cells was confirmed by light and electron microscopic autoradiography. References 1. McDonald
TJ, Jornvall H, Nilsson G, et al. Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochem Biophys Res Commun 1979;90:227-33. 2. Dockray GJ, Vaillant C, Walsh JH. The neuronal origin of bombesin-like immunoreactivity in the rat gastrointestinal tract. Neuroscience 1979;4:1561-8.
3. Nishi S. Seino Y, Takemura J, et al. Vagal regulation of GRP. gastric somatostatin, and gastric secretion in vitro. Am J Physiol 1985;248:E425-31. 4. Yanaihara N. Yanaihara C, Mochizuki T, Iwahara K, Fujita T, Iwanaga T. Immunoreactive GRP. Peptides 1981;2(Suppl 2): 185-91. 5. Nakamura
M, Oda M, Yonei Y. et al. Muscarinic
receptors
in rat gastric
mucosa-a
using a potent muscarinic tochemistry 1985;83:479-87. 6. Nagata
T, Nawa
antagonist,
T, Yokota
acetylcholine
radioautographic
S. A new
technique
microscopic dry-mounting radioautography pounds. Histochemie 1969;18:241-9.
isolated
piglet
on ultrastructure gastric
mucosa.
His-
for electron
of soluble
7. Black JA. Forte TM, and Forte JG. Inhibition and the effects
study
3H-pirenzepine.
com-
of HCl secretion
and electrical
resistance
Gastroenterology
in
1981:81:509-
19. 6. Nakane
PK, Pierce
tion and Histochem 9. Graham
GB. Enzyme-labelled
application Cytochem RC, Karnovsky
injected
horseradish
mouse
kidney:
nique.
J Histochem
10. Lowry
HO,
MJ. The early peroxidase
with
Preparaantigens.
J
of adsorption
of
tubules
by a new
of
tech-
1966:14:291-302.
NJ, Farr
the
stages
cytochemistry
Cytochem
Rosebrough
of
in the proximal
ultrastructural
measurement
antibodies.
for the localization 1966;14:929-31.
Folin
AL,
phenol
Randall
RJ. Protein
reagent.
J Biol
Chem
1951;193:265-75. 11. Kobayashi human
S. Fujita duodenal
Arch Histol
T, Sasagawa mucosa.
T. The
An
endocrine
electron
cells
microscope
of
study.
Jpn 1970;31:477-94.
12. Fujita T. The identification of the argyrophil cells of pancreatic islets with D cells. Arch Histol Jpn 1964;25:189-97. 13. Iwanaga
T. Gastrin-releasing
peptide
immunoreactivity
in the neurons
and lung.
Res 1983:4:93-104.
14. Knigge
Biomed U, Holst
peptide:
JJ, Knuhtsen
pharmacokinetics
pancreatic
hormones
S, et al. and
and gastric
effects
human
dependent
gastric
mechanisms.
function
gastro-entero-
in normal
men.
AA. Bombesin
by gastrin-dependent
Peptides
of the gut
Gastrin-releasing on
secretion
Clin Endocrinol Metab 1984;59:310-5. 15. Walsh JH, Maxwell V, Ferrari J. Varner ulates
(GRP)/bombesin-like
and paraneurons
1981;2(Suppl
J
stimand
in-
2):193-8.
16. Knigge U, Knutson S, Holst JJ, Christiansen PM. The effect of the peptides TRH and GRP on gastric acid secretion in normal men. Stand J Gastroenterol Suppl 1983;18:1834. R, Orloff MS, Reeve JR, Turner AJ, 17. Bunnett NW, Kobayashi Walsh JH. Catabolism of gastrin releasing peptide and sub-
976
NAKAMLJRA ET AL.
stance P by gastric membrane-bound peptidases. Peptides 1985;6:277-83. 18. Nakamura M, Oda M, Yonei Y, et al. Demonstration of the localization of muscarinic acetylcholine receptors in the rat gastric mucosa-light and electron microscopic autoradiographic studies using 3H-quinuclidinyl benzilate. Acta Histothem Cytochem 1984;17:297-309. 19. Nakamura M, Oda M, Kaneko K, et al. Radioautographic demonstration of gastrin binding sites in the rat gastric mucosa. Peptides 1987;8:391-8.
GASTROENTEROLOGY Vol. 94, No. 4
Received October 28, 1986. Accepted November 30, 1987. Address requests for reprints to: Masaharu Tsuchiya, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan(No. 56107005). The authors thank Tatsushi Fujiwara and Dr. Koji Kami for their encouragement and valuable suggestions.
Autoradiographic Demonstration of Gas&n-Releasing Peptide-Binding Sites in the Rat Gastric Mucosa MASAHIKO YASUFUMI HIROKAZU
NAKAMURA, MASAYA ODA, KOTARO KANEKO, AKAIWA, NOBUHIRO TSUKADA, and MASAHARU TSUCHIYA KOMATSU,
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan
The location of [1251]iodotyrosyl gastrin-releasing peptide-binding sites in the rat fundic mucosa was studied. Peptide specificity was demonstrated by competitive binding studies using the addition of a large amount of cold gastrin-releasing peptide or substance P. Autoradiography of the stomach tissue was carried out by freeze-drying, embedding in Epon, wet-sectioning with ethylene glycol, and drymounting the emulsion film by the wire-loop method to prevent loss of the labeled substance. Specific binding sites of gastrin-releasing peptide were found on D cells, surface mucus cells, and parietal cells, whereas few binding sites were seen on the chief or mucus neck cells.
G
astrin-releasing peptide (GRP) was first isolated from nonantral gastric tissue as a 27-amino acid peptide (1). In the gastrointestinal tract, GRP is thought to be located in nerve fibers and cell bodies of the myenteric and submucosal nerve plexus and to act as a neuromodulator in regulating the secretion of various kinds of peptides from the neuroendocrine cells (2). In the stomach, GRP is thought to release gastrin from the antral G cells and to stimulate acid secretion from the parietal cells (3). Recent histochemical observations, however, have shown that most of the GRP-containing fibers are located in the fundic region, not in the antral region, suggesting the direct action of GRP on the fundic glandular cells (4).
Therefore, this study was designed to clarify the binding sites of [3-1251]iodotyrosyl GRP in the rat fundic mucosa by light and electrop micrdsqopic autoradiography, combined with the immunohistochemical technique.
Materials
and Methods
Administration of [3251]Iodotyrosyl GastrinReleasing Peptide Male Wistar rats weighing 200-250 g were used in the following experiments. The rats were divided into control and cold GRP-treated groups. In the cold GRPtreated group, lo-“, 10-13, lo-“, and lo-’ mol GRP/kg body wt were mixed with radiolabeled GRP and administered through an intraaortic catheter in the following manner. Under light anesthesia by intraperitoneal injection of sodium pentobarbital (20 mg/kg body wt), the abdomen was opened by a lower middle incision and a polyethylene catheter was inserted into the abdominal aorta up to the opening of the celiac artery. A dose of 0.7 ml of an aqueous solution of [3-‘251]iodotyrosyl synthetic porcine GRP (50 &i/kg body wt; 2000 Cilmmol; 2.5 X lo-l3 mol/kg body wt; Amersham International plc, Amersham, U.K.) was infused for 10 min through the aortic catheter using an infusion pump at a constant rate of 2.1 ml/h, followed by infusion of physiologic saline for 5 min to wash out the unbound ligand. As the control, the same amount of lZ51-substance P (Amersham), one of the gastrointestinal peptides structurally related to GRP, was infused and the distribution of the binding sites of ‘251-substance P was compared with that of lz51-GRP. Tissue Preparation for Light and Microscopic Autoradiography
Electron
Immediately after the infusion, the fundic portion of the stomach was taken out and cut into small tissue blacks (2 x 2 x 2 mm3) with a razor blade. These blocks were quickly frozen in isopentane cooled to its melting point with liquid nitrogen. The stomach tissue blocks were freeze-dried for 48 h in an apparatus (Oka Science OTDAbbreviation
used in this paper:
GRP, gastrin-releasing
peptide. 0 1988
by the American Gastroenterological 0016~5Q85/88l$3.50
Association
GRP-BINDING SITES ON RAT GASTRIC MUCOSA
April 1988
1SF) cooled to -50°C and evacuated at 9 X lo-” torr. The freeze-dried tissue blocks were then exposed to osmium vapor, followed by infiltration in Epon by a dripping unit evacuated by rotary pump (5). The tissue blocks were removed and embedded in freshly prepared Epon and polymerized at 60°C. Semithin (1 pm thick) or ultrathin (1000-2000 A thick) sections were cut with an LKB ultramicrotome using ethylene glycol instead of water. Autoradiographic emulsion (Sakura NR-H2 or M2) was diluted (1:2 or 1:3) with distilled water at 40°C in the darkroom. According to the previously described drymounting procedure (6), an emulsion film was made between vinyl-coated iron-wire loops by dipping the wire loops into a solution of dioctyl sodium sulfosuccinate, with the wire loops being left to stand at normal temperature until the emulsion film was almost dry. The emulsion film was then applied to the glass plates or grids on which sections were mounted. The sections were kept in the dark in a refrigerator at 4°C for 4-8 wk for exposure. After development and fixation, they were examined under light and electron microscopes. Some of the stomach tissue sections were processed for electron microscopy, and the parietal cell morphology was quantified according to Black’s classification (7) to clarify the functional status of the parietal cells. Oxyntic cells were classified into one of four categories by the structure of the intracellular canaliculi and the tubulovesicles, i.e., into resting, stimulated, partially stimulated, and returning states. The percentages of the cells in the four conditions were compared in the GRP-infused and control rats. Analysis
of Autoradiographs
The silver grains on the chief, parietal, mucus neck, surface mucus, and D cells were counted in the cold GRP-treated and control groups. The background counting rate was determined by counting the grains in the stomach tissue obtained from the rat infused with saline alone and treated in the same fashion described above. The number of grains on the cells was then estimated by subtracting the background count. D cells were identified by an indirect immunohistochemical technique using the antisomatostatin antibody. The sections used for light microscopic autoradiography were then processed for the immunocytochemical demonstration of somatostatin using rabbit antiserum to human somatostatin (Milab) at a dilution of 1:lOOO. The site of the antigen-antibody reaction was revealed with the indirect
Table
1. Analysis
of Morphology
IO-l3
969
10-'
10-l'
UNLABELED PEPTIDE (M) Figure
1. Inhibition of [1251]iodotyrosyl GRP binding by unlabeled GRP or substance P. The rats were treated with 2.5 x lOeTa molikg body wt [‘251]iodotyrosyl GRP mixed with varying concentrations of unlabeled GRP or substance P by intraaortic infusion, and the radioactivity remaining in the gastric mucosa was counted using a y-counter.
immunohistochemical method (8). The preparations were incubated with diluted primary antisera for 24 h at 4°C in a humid chamber, then washed in phosphate-buffered saline and incubated for 1 h with a horseradish peroxidase-conjugated second antibody (goat immunoglobulins to rabbit immunoglobulins) at a dilution of 1:40. After being washed in phosphate-buffered saline, the sections were treated with dimethylaminoazobenzene solution for 5 min (9). For control sections, we used antiserum that had been inactivated by the addition of an excess amount of antigen.
Assay
of Binding
Releasing
Peptide
of [“51]Iodotyrosyl to the Gastric
Gastrin-
Mucosa
Soon after the continuous intraaortic infusion of cold GRP or substance P mixed with radiolabeled GRP, a portion of the stomach tissue was homogenized with a glass homogenizer. The accumulation of [‘251]iodotyrosyl GRP on the fundic mucosa was estimated with a y-counter (ARC-300), and the radioactivity in the gastric mucosa was
of Control and Gastrin-Releasing
Peptide-Stimulated
Rat Gastric Parietal
Cells
Morphologic appearance I% of totall Condition
n (n’)
Resting
50 (51 50 (5)
Stimulated n, number of calculated 0.03 vs. resting condition
Stimulated 0 64"
parietal cells; n’, number of rats used. Morphologic by binomial distribution.
Partially stimulated 12 24 appearance
Returning
Resting
16 8
72 40 was judged according
to Black’s
criteria.
ap <
970
Figure
NAKAMURA
ET AL.
GASTROENTEROLOGY
2. Electron micrographs of the typical parietal cells of the GRP-infused and control rats. a. In the GRP-infused parietal cells indicate large numbers of greatly expanded intracellular canaliculi (ICI. Magnification, x 7000. rats, most of the cells contain abundant tubulovesicles (TV). Magnification, x 10,000.
estimated by determining the protein method of Lowry et al. (10).
content
using
the
Results Assay of [‘Z5J]IodotyrosyJ Gastrin-Releasing Peptide Binding to Gastric Mucosa As is clear in Figure 1,unlabeled GRP in the range of lo-l5to lo-'mol/kg body wt competitively inhibited the binding of [‘251]iodotyrosyl GRP to the fundic mucosa in a continuous dose-dependent fashFigure
ion, and substance P had almost soever on the binding of GRP.
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rats, most of the b. In the control
no influence
what-
Alteration of Parietal Cell Ultrastructure After Gastrin-Releasing Peptide Infusion The distribution profile of parietal cells from the GRP-infused stomach was markedly different from that of the control stomach (Table 1). The cells of the former were mostly classified as stimulated, whereas the latter indicated the large majority of cells as resting (Figure 2). These morphologic data >
3. Light microscopic autoradiographs of the gastric mucosa of the rat infused with [‘Z51]iodotyrosyl GRP or ‘Z51-substance P. a-d. Tip portion of the gastric mucosa. The grains (arrowheads) are mainly recognized on the surface epithelial cells [a, b). On the other hand, through the addition of an excess amount of cold GRP (c), the binding of GRP on the epithelial cells is only slightly recognized, with only a little found on the capillaries (arrowheads]. In the rats receiving substance P, few grains are seen on the epithelial cells. a, c, and d were unstained, and b was counterstained with toluidine blue. e-h. Middle and basal portions of the gastric mucosa. Most of the grains (arrowheads) are found on the parietal cells, and not on the chief cells having toluidine blue-positive granules (d, e). whereas few grains are found on the parietal cells through the addition of an excess amount of cold GRP cfl. In the substance P-treated rats, no grains were found on these epithelial cells. e. g. and h were unstained, and f was counterstained with toluidine blue. Magnification, X 1200.
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Figure 4. Electron microscopic autoradiographs of the surface mucus cell of the tip portion of the rat gastric mucosa infused with [‘251]iodotyrosyl GRP. a. The silver grains (arrowheads], corresponding to the GRP-binding sites, are found near the basolateral membrane of the cells. Magnification, x 9000. b. An electron micrograph of the surface mucus cells fixed with mixed solution of 4% formaldehyde and 1% glutaraldehyde. No silver grains are observed on these cells. Magnification, x7000.
confirm that GRP administration stimulates hydrochloric acid secretion from the parietal cell. Distribution of [‘251]Iodotyrosyi Gastrin-Releasing Peptide-Binding the Gastric Mucosa
Sites in
Silver grains corresponding to the localization of ‘251-GRP were seen mainly in the tip and middle portions of the gastric mucosa, whereas few grains were recognized by the addition of an excess amount of cold GRP, showing the localization of these silver grains specific for GRP (Figure 3). Few grains were found after the administration of ‘9-substance P, which also suggested the specificity of localization of silver grains after 9-GRP administration. Under higher magnification, most of the cells having silver grains as a result of lz51-GRP administration were found to be surface mucus cells in the tip portion of the gastric mucosa (Figure 41, and the two types of cells in the middle portion of the gastric
mucosa, i.e., parietal cells and basally granulated cells (Figure 5). The immunohistochemical method, using the antisomatostatin antibody, showed the latter cells to be D cells (Figure 6). Effects of Administration of Cold GastrinReleasing Peptide and Substance P on the [‘251]Iodotyrosyl Gastrin-Releasing Peptide Binding Studied by Light Microscopic Autoradiography After the mixed administration of lo-" M cold GRP, significantly fewer grains were seen on the parietal, surface mucus, or D cells compared with the group treated by [9]iodotyrosyl GRP alone. On the other hand, the same amount of substance P demonstrated no significant effect on GRP binding (Table 2). These results, together with the data shown in Figure 1, suggest that the localization of radioactivity found after [‘251]iodotyrosyl GRP administration is specific for GRP-binding sites.
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5. Electron microscopic autoradiographs of the parietal and basally granulated cells in the rat infused with [‘2sI]iodotyrosyl GRF’. o. The GRP-binding sites on the parietal cells (arrowheads) are mainly found in the cytoplasm of the parietal cells. Magnification: ~15,000. c. The GRP-binding sites (arrowheads) on the basally granulated cells are found near the plasma membrane. Magnification: ~12,000. a, c. Freeze-dried specimens. b, d. Electron micrographs of the parietal (b) and D cell (d) fixed with a mixed solution of 4% glutaraldehyde and 1% formaldehyde. No grains are found on these cells. Magnification: b, x12,000; d, x10,000. Uranyl acetate and lead citrate stains. respectively.
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Figure 6. Light microscopic autoradiographs of the GRP-binding sites on the basal portion of the gastric mucosa. o. Some grains (arrowheads) are recognized on the small cells just adjacent to the parietal cells. b, c. By the application of the indirect immunohistochemical technique using antisomatostatin antibody, these cells (arrowheads) are found to be positive to somatostatin, corresponding to D cells. c. Counterstained with toluidine blue. Magnification, x 1200.CV, collecting venule.
Discussion Somatostatin-containing D cells are located in the upper portion of the digestive system, mainly from the fundic region of the stomach to the duodenum (11)and pancreas (12)) and are thought to act as a neuromodulator and to regulate the secretory status of the endocrine or exocrine cells in terms of paracrine. According to a recent pharmacologic study (3), GRP suppresses the secretion of somatostatin in the antral region of the stomach, and then, directly or indirectly, increases gastrin secretion. These phenomena result in augumented secretion of hydrochloric acid from the parietal cells.
In a histochemical study (4), GRP-like immunoreactive fibers were found to be more numerous in the oxyntic region than in the antral region. It is postulated from these results that GRP released from the nerve fibers reaches the antrum through the circulation and stimulates the G cell. It is more reasonable, however, to think that GRP plays a more significant role in the fundic region (13). D cells in the fundic region are thought to directly inhibit acid secretion from the parietal cells. In the present investigation, the GRP-binding sites in the fundus were found to be abundant on the D cells, suggesting the direct regulation by GRP of somatostatin secretion in the oxyntic gland.
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Table 2. Localization of [‘251JIodotyrosyl Gas&in-Releasing Peptide-Binding Sites in Rat Gastric Mucosa Number
[ “‘I]Iodotyrosyl GRP + 10~” M cold GRP
(‘2”I]Iodotyrosyl GRP alone Surface mucus cells Mucus neck cells Parietal cells Chief cells D cells
5.4 + 1.6 + 9.2 ? 1.6 t 13 ?
of grains/cell
1.2 0.42 2.3 1.0 3.2
(“‘I]Iodotyrosyl GRP + 16” cold substance
0.80 t 0.40" 0.80 It 0.40 1.0 f 0.42" 0.80 + 0.40 1.2 t 0.700
4.8 ? 1.0 ? 8.8 k 1.0 k 14 k
M P
2.1" 0.63 1.8': 0.63 3.8"
Values are means 2 SD (n = 5)." p < 0.001 vs. [I"Ijiodotyrosyl GRP alone. b 0.001 < p < 0.01 vs. [‘““Iliodotyrosyl GRP + lo- I’ M cold GRP. ’ p < 0.001 vs. [ “‘I]iodotyrosyl GRP + lo-” M cold GRP. Specific binding sites of GRP are found on the surface mucus, parietal. and D cells.
The parietal cells were also found to have specific binding sites for GRP. In a pharmacologic study with the infusion of GRP (l4), gastrin was found to increase immediately after the infusion, with a IS-min lag period of acid secretion from the parietal cells. Complete removal of gastrin-producing antral G cells by antrectomy totally eliminates the stimulatory effect of bombesin on gastric acid secretion (15). These data suggest that GRP action on the parietal cell is mediated by gastrin. Therefore, the present data may indicate the direct action of GRP on the parietal cell in some fashion other than the direct stimulation of acid secretion, whereas another report suggests the direct action of GRP on acid secretion from the parietal cell (16). The binding sites on the parietal cells were dispersed in the cytoplasm and not on the cell membrane. Although this may suggest the internalization of GRP, it may result from the movement of the metabolites of GRP into the cytoplasm, in view of the rapid turnover of GRP induced by the membranebound peptidase (17). With respect to the mucus secretion, few mediators have been reported. In our recent study, few binding sites of quinuclidinyl benzilate (18), pirenzepine (6), or gastrin (19) have been seen on the mucus-secreting cells. In this study, specific GRPbinding sites were found on the surface mucus cells, suggesting that the cholinergic stimulation of the mucus secretion may be mediated by GRP. In conclusion, direct binding of GRP to the parietal and D cells was confirmed by light and electron microscopic autoradiography. References 1. McDonald
TJ, Jornvall H, Nilsson G, et al. Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochem Biophys Res Commun 1979;90:227-33. 2. Dockray GJ, Vaillant C, Walsh JH. The neuronal origin of bombesin-like immunoreactivity in the rat gastrointestinal tract. Neuroscience 1979;4:1561-8.
3. Nishi S. Seino Y, Takemura J, et al. Vagal regulation of GRP. gastric somatostatin, and gastric secretion in vitro. Am J Physiol 1985;248:E425-31. 4. Yanaihara N. Yanaihara C, Mochizuki T, Iwahara K, Fujita T, Iwanaga T. Immunoreactive GRP. Peptides 1981;2(Suppl 2): 185-91. 5. Nakamura
M, Oda M, Yonei Y. et al. Muscarinic
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16. Knigge U, Knutson S, Holst JJ, Christiansen PM. The effect of the peptides TRH and GRP on gastric acid secretion in normal men. Stand J Gastroenterol Suppl 1983;18:1834. R, Orloff MS, Reeve JR, Turner AJ, 17. Bunnett NW, Kobayashi Walsh JH. Catabolism of gastrin releasing peptide and sub-
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stance P by gastric membrane-bound peptidases. Peptides 1985;6:277-83. 18. Nakamura M, Oda M, Yonei Y, et al. Demonstration of the localization of muscarinic acetylcholine receptors in the rat gastric mucosa-light and electron microscopic autoradiographic studies using 3H-quinuclidinyl benzilate. Acta Histothem Cytochem 1984;17:297-309. 19. Nakamura M, Oda M, Kaneko K, et al. Radioautographic demonstration of gastrin binding sites in the rat gastric mucosa. Peptides 1987;8:391-8.
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Received October 28, 1986. Accepted November 30, 1987. Address requests for reprints to: Masaharu Tsuchiya, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan(No. 56107005). The authors thank Tatsushi Fujiwara and Dr. Koji Kami for their encouragement and valuable suggestions.