Regulation of fundic and antral somatostatin secretion by CCK and gastrin

Yana Zavros, William R. Fleming, Kenneth J. Hardy, and Arthur Shulkes

Department of Surgery, University of Melbourne, Austin and Repatriation Medical Centre, Austin Campus, Melbourne, Victoria 3084, Australia

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CCK and gastrin stimulate somatostatin (SOM) secretion and thus modulate their direct effects on the parietal cell. Although SOM is stored in D cells of the fundus and antrum, the nature of the cell type differs, and it is not known whether both regions respond to the stimulatory effects of CCK and gastrin. The objectives of the present study were to determine the separate effects of CCK and gastrin on fundic and antral SOM secretion and to assess the type of receptor involved, using CCK-A (L-364,718) and CCK-B/gastrin (L-365,260) receptor antagonists. Changes in SOM were measured in plasma collected from cannulas draining blood from the fundus (gastric vein) and antrum (gastroepiploic vein) in anesthetized sheep. Both CCK and gastrin significantly stimulated SOM from the fundus and antrum. Sulfated CCK-8 (CCK-8S) increased SOM secretion from the fundus and antrum through interaction with both type A and B receptors. In contrast to CCK-8S, sulfated gastrin-17 (G-17S) stimulated SOM from the fundus via the type B receptor alone, whereas in the antrum G-17S stimulated SOM secretion independent of the A and B receptors. Histamine mediated, at least in part, the SOM-stimulatory effects; an H2-receptor antagonist blocked CCK-stimulated SOM secretion in both the fundus and antrum and reduced gastrin-stimulated SOM secretion in the fundus. The present study demonstrates regionally distinct regulatory mechanisms for gastric SOM secretion by CCK and gastrin.

sheep; cholecystokinin-A receptor; cholecystokinin-B/gastrin receptor; Helicobacter pylori

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

GASTRIN, secreted by antral G cells, is the principal stimulant of gastric acid secretion (36). The duodenal peptide CCK is structurally related to gastrin and is a major hormonal regulator of gallbladder contraction (11). Both CCK and gastrin stimulate somatostatin (SOM), but the potency and the receptors involved are not identical. CCK appears to be more potent than gastrin (31, 38) and stimulates SOM secretion by binding to both the CCK-A (type A) and CCK-B/gastrin (type B) receptors, whereas gastrin acts through the CCK-B/gastrin receptor alone (38).

Although the stomach appears to be the source of SOM, there are no in vivo data comparing the regulation of antral and fundic SOM by CCK and gastrin. Differences in regulation are likely, as the D cells in the fundus and antrum vary in the response to acidity and food (8). Furthermore, gastrin is present in high concentrations in the antrum, whereas CCK is confined to the duodenum (11, 36). The distribution of the type A and B receptors is not uniform. Ligand binding demonstrates that both type A and type B receptors are present on isolated canine fundic D cells, whereas only type A receptors are detected on isolated antral D cells (3, 19). However, immunohistochemistry in a number of species has shown that the CCK-B/gastrin receptor is present on D cells of the antrum as well as the fundus (7, 32). Immunohistochemistry of canine and human fundic mucosa demonstrated both receptors in the fundus, although the receptors were not localized to the D cell (17, 24). The CCK-B/gastrin receptor present on parietal cells, enterochromaffin-like (ECL) cells, and D cells has an equal affinity for gastrin and CCK, whether sulfated or not (29). The CCK-A receptor has a greater affinity for sulfated CCK than sulfated or nonsulfated gastrin (29).

The role of the CCK-A and CCK-B/gastrin receptors in SOM secretion is often investigated by receptor-specific antagonists. The benzodiazepine derivative L-364,718 (MK-329, devazepide) is highly selective for the CCK-A receptor and antagonizes CCK-regulated gastrointestinal functions including SOM release (15), pancreatic secretion, and gallbladder contraction (12). A related compound, L-365,260, is selective for the CCK-B/gastrin receptor and has been used to study the role of the type B receptor in acid secretion (34).

We and others (8, 25, 38) have suggested that the mechanisms regulating SOM synthesis and secretion differ between the fundus and antrum. The different morphology of the SOM-secreting D cells in these regions supports this proposal. Antral D cells are open cells with apical processes in contact with the lumen and are sensitive to changes in gastric acidity. Fundic D cells are closed cells and are responsive to peptide regulation (8, 26).

Gastrin can stimulate SOM secretion by acting directly on the D cell (19, 31). Histamine may be a mediator of this response, because gastrin stimulates histamine release (22). It is not clear whether the effect of histamine on SOM secretion is a direct action on the D cell (30) or the result of an increase in gastric acid secretion, as studies with H2-receptor antagonists have been contradictory (5, 16, 30). The effect of H2-receptor antagonists on CCK-stimulated SOM secretion has not been reported. There are many more ECL cells in the fundus than in the antrum (6); thus it would be expected that histamine has a more important role in mediating gastrin-stimulated SOM secretion from this region than from the antrum. In addition, ECL cells purified from rat fundus are sensitive to gastrin as well as CCK (21, 22). However, the relative roles of histamine in CCK- and gastrin-stimulated SOM secretion from the fundus and antrum have not been defined.

The objectives of this study were to investigate functional differences in the regulation of fundic and antral SOM secretion. This was achieved by measuring changes in SOM and gastrin secretion in plasma collected from fundic and antral veins of the sheep stomach (abomasum) in response to gastrin and CCK stimulation. The nature of the receptors involved was then determined by repeating the study using the CCK-A and CCK-B/gastrin receptor antagonists. The role of histamine as a mediator of CCK- and gastrin-stimulated SOM secretion was determined by measuring changes in fundic and antral SOM release in response to CCK and gastrin plus the H2-receptor antagonist ranitidine.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

Sulfated forms of CCK-8 (CCK-8S) and G-17 (G-17S) were purchased from Bachem (Torrance, CA). Peptides were dissolved in 0.05 M NH4HCO3-0.5% BSA to a final stock concentration of 50 nmol/ml. Peptide stock concentrations were then diluted in 0.9% saline and 0.1% BSA to obtain the administered peptide dose (150 pmol · kg-1 · h-1). The CCK-A and CCK-B/gastrin receptor antagonists L-364,718 (1 mg/kg) and L-365,260 (3 mg/kg) were supplied by Merck Research Laboratories (Rahway, NJ). Antagonists were dissolved in DMSO-polyethylene glycol-400 (PEG-400) (4.5:0.5, vol/vol) and administered as a 5-ml bolus. The H2-receptor antagonist ranitidine (Glaxo Australia) was diluted in 0.9% saline and 0.1% BSA to obtain the administered dose (bolus 0.5 mg/kg; infusion 1.5 mg · kg-1 · h-1).

Animal Preparation

Merino × Corriedale sheep, ranging in weight from 35 to 45 kg, had food withheld for 24 h before surgery. Anesthesia was induced by intravenous Pentothal sodium (1 g) and maintained throughout the experiment with a 1-2% Fluothane-oxygen mixture (ICI Australia). The abdomen was opened by a midline incision. A cannula (0.76 mm ID, 1.65 mm OD; Dow Corning) was inserted into the gastric vein draining blood from the fundus. The gastroepiploic vein, draining blood from the antrum, was also cannulated. Both the gastric and gastroepiploic veins were ligated on each side of the cannula, allowing drainage of blood from the main branch of the vein and preventing blood from flowing from the fundus to the antrum (Fig. 1). To insert the gastric cannula, a right subcostal incision was made for insertion of the catheter (polyvinyl chloride, 10 mm ID × 15 mm OD) through a purse-string suture into the antral regions of the abomasum. The cannula was secured by tightening the purse string (Fig. 1). Cannulas were also inserted into the jugular veins. One cannula was directed toward the heart (down) for infusion, and the contralateral cannula was directed toward the head (up) for blood sampling (38). After surgery 0.5 h was allowed before the experiment began. All animal experiments were approved by the Austin and Repatriation Medical Center Animal Ethics Committee (95/030).


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Fig. 1.   The equivalent of the stomach in the sheep is the abomasum. The functional division of fundus, antrum, and pylorus, the distribution of gastrin and somatostatin (SOM), and gastric secretions are similar to those in the human. Hormonal regulation of gastric secretion and gastrin-SOM interactions are also similar. Cannulas were inserted into the gastric and gastroepiploic veins draining blood from the fundus and antrum, respectively. A gastric cannula was also inserted into the antral portion of the abomasum.

Experimental Design

Three different experimental protocols were used: CCK and gastrin infusions, CCK and gastrin infusions preceded by administration of either the CCK-A or CCK-B/gastrin receptor antagonists, and CCK and gastrin infusions preceded by administration of the H2-receptor antagonist. A 10-ml blood sample was collected from the jugular vein immediately before anesthesia and after the animal preparation to measure the effect of the anesthetic on basal SOM and gastrin concentrations.

Control experiments. EFFECT OF CCK-8S AND G-17S INFUSION ON SOM SECRETION. The first series of experiments was performed to determine the stimulatory effects of CCK-8S and G-17S alone in the fundus and antrum. After a 60-min basal period, CCK-8S (150 pmol · kg-1 · h-1) was infused for 30 min at 20 ml/h via the jugular vein cannula (down) using a syringe infusion pump (Sage Instruments). A 45-min post-peptide-infusion period was allowed before G-17S (150 pmol · kg-1 · h-1) was infused for a further 30 min, followed by a 30-min postinfusion period. Blood samples were collected simultaneously from the jugular, fundic, and antral veins every 15 min during the basal and postpeptide infusion periods, and every 10 min during the peptide infusions. Gastric secretions were collected every 30 min during the experiment, and pH was measured.

Antagonist experiments. EFFECT OF L-364,718 OR L-365,260 ON CCK-8S- AND G-17S-STIMULATED SOM SECRETION. The second series of experiments was performed to determine the nature of the receptors that mediate SOM secretion from the antrum and fundus in response to gastrin and CCK. These experiments were similar to the control experiments except that the CCK-A (L-364,718) (1 mg/kg) and CCK-B/gastrin (L-365,260) (3 mg/kg) receptor antagonists were administered on separate occasions, 30 min before the infusion of CCK-8S. Blood and gastric juice samples were collected as previously described.

EFFECT OF RANITIDINE ON CCK-8S- AND G-17S-STIMULATED SOM SECRETION. The effect of ranitidine (H2-receptor antagonist) on fundic and antral SOM secretion was studied to determine the role of histamine as a mediator of gastrin- and CCK-stimulated SOM release. After a 60-min basal period ranitidine was administered as a bolus (0.5 mg/kg) followed immediately by an infusion of 1.5 mg · kg-1 · h-1 for 60 min at a rate of 20 ml/h. This dose of ranitidine inhibits ~90% of meal-stimulated gastric acid secretion in dogs (5) and basal and histamine-stimulated gastric acid secretion in sheep. The ranitidine infusion was followed by a 30-min infusion of ranitidine (1.5 mg · kg-1 · h-1) plus CCK-8S (150 pmol · kg-1 · h-1). A 45-min postinfusion period was allowed before ranitidine plus G-17S was infused for a further 30 min, followed by a 30-min postinfusion period. Blood and gastric juice samples were collected as previously described.

Laboratory Methods

Reverse-phase HPLC: predominant SOM molecular form. To determine the proportions of SOM-14 and SOM-28 that were released from the fundus and antrum in response to G-17S and CCK-8S, plasma samples obtained during the infusion of either gastrin or CCK were pooled and concentrated using Sep-Pak C18 cartridges (Millipore). SOM-14 and SOM-28 standards (1 pmol) or Sep-Pak concentrated plasma samples were loaded onto a C18 Bondapak column (Waters) and separated using a 22-min gradient from 28% to 32% acetonitrite and 0.05% trifluoroacetic acid. The flow rate was set at 3 ml/min, and 44 0.5-min fractions were collected. Two hundred microliters of each fraction were dried down under air. Standards and samples were assayed using a buffer standard curve and under normal SOM radioimmunoassay conditions.

Radioimmunoassay. SOM. Plasma SOM was measured in ethanol-extracted plasma as described previously (23). One milliliter of plasma was mixed with 2 ml of absolute ethanol, and the coagulated protein was removed by centrifugation. The ethanol extracts were then divided into 1-ml aliquots, evaporated to dryness, and stored at -20°C until assay. Before assay, extracted plasma was reconstituted in 1 ml assay buffer, and 50-200 µl of reconstituted sample were assayed against a buffer standard curve. Antiserum 8402, which detects SOM-14 and SOM-28 to an equal extent, was used. 125I-Tyr-SOM-14 was prepared using the chloramine T method and purified by reverse-phase HPLC. SOM-14 was used for generating the buffer standard curve. The ID50 was 8 fmol/ml, and the inter- and intra-assay coefficients of variation were less than 5% and 12%, respectively.

GASTRIN. Plasma gastrin was assayed directly, using 50 µl of plasma. The plasma samples were incubated at 4°C in duplicate with the 125I-Met15 human nonsulfated G-17 (G-17NS) label and antiserum 1296. Antiserum 1296 recognizes all COOH-terminal fragments larger than the pentapeptide and measures G-17S and G-17NS identically. G-17NS was used for generating the standard curve. The ID50 was 1 fmol/ml, and inter- and intra-assay coefficients of variation were less than 2% and 11%, respectively.

Statistical Analysis

Results are means ± SE. Statistical comparison between more than two groups was made by one-way analysis of variance followed by the Student-Newman-Keuls method. Data that were not normally distributed were analyzed by the Mann-Whitney test. Statistical comparisons of means between two groups were made using an unpaired t-test. P < 0.05 was considered significantly different. The integrated SOM response was calculated as the area under the plasma SOM concentration time curve with respect to the average of the two basal concentrations preceding the infusion (38).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Control Experiments

Effect of anesthesia on basal SOM and gastrin secretion. A peripheral blood sample was collected before anesthesia and then immediately after completion of the surgery (~90 min after administration of the Fluothane-oxygen mixture) to measure the effect of the anesthetic on basal SOM and gastrin concentrations. Basal plasma SOM concentrations significantly increased from 24 ± 4 to 130 ± 28 pmol/l (P < 0.05). Basal plasma SOM decreased over the subsequent 60-min basal period to a value of 60 ± 8 pmol/l, measured at 0 min (P < 0.05). Basal SOM was highest in the fundic vein (602 ± 97 pmol/l), followed by the antral vein (153 ± 46 pmol/l) and peripheral circulation (60 ± 8 pmol/l).

Peripheral plasma gastrin concentrations significantly decreased from 20 ± 2 pmol/l just before anesthesia to 15 ± 1 pmol/l at the end of surgery (P < 0.05) and then increased over the following 60-min basal period to 37 ± 7 pmol/l (P < 0.05). As expected, basal gastrin concentration was highest in the antral vein (218 ± 83 pmol/l); values in the fundic vein (28 ± 5 pmol/l) and peripheral circulation (37 ± 7 pmol/l) were similar.

Effect of CCK-8S and G-17S infusion on SOM secretion. The first series of experiments was performed to determine the stimulatory effects of CCK and gastrin on fundic and antral SOM secretion before the receptor antagonist study. Both CCK-8S and G-17S increased plasma SOM measured in the peripheral circulation. CCK-8S caused a significant increase in plasma SOM from a basal value of 60 ± 8 pmol/l to a peak response of 82 ± 15 pmol/l (P < 0.05) (Fig. 2A). G-17S also significantly increased plasma SOM from 50 ± 11 to 84 ± 17 pmol/l (P < 0.05) (Fig. 2A).


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Fig. 2.   Effect of infusion (150 pmol · kg-1 · h-1) of sulfated CCK-8 (CCK-8S) and gastrin-17 (G-17S) on plasma SOM concentrations in peripheral (A), antral (B), and fundic (C) plasma. Results are means ± SE (n = 8).

Antral plasma SOM significantly increased in response to CCK-8S, from 153 ± 46 to 267 ± 51 pmol/l (P < 0.05) (Fig. 2B). Antral SOM remained elevated in the postinfusion period, but G-17S then increased plasma SOM from 248 ± 56 to 349 ± 84 pmol/l; this change was not significant (P > 0.05) (Fig. 2B).

Fundic plasma SOM significantly increased in response to CCK-8S, from 602 ± 97 to 930 ± 163 pmol/l (P < 0.05) (Fig. 2C). Fundic SOM secretion also increased in response to G-17S, from 655 ± 219 to 1,282 ± 323 pmol/l (P < 0.05) (Fig. 2C).

The results are also represented as the integrated SOM output (Fig. 3). The integrated SOM output in response to CCK-8S and G-17S in the peripheral circulation (0.47 ± 0.13 and 0.50 ± 0.11 pmol · min-1 · ml-1, respectively), antrum (1.93 ± 0.80 and 1.83 ± 0.78 pmol · min-1 · ml-1, respectively), and fundus (3.76 ± 1.60 and 12.9 ± 3.81 pmol · min-1 · ml-1, respectively) was significantly greater than basal levels (P < 0.05) (Fig. 3). The SOM output in the periphery and antrum in response to CCK-8S was similar to that of G-17S. The response to G-17S in the fundus was significantly greater than the response to CCK-8S (P < 0.05) (Fig. 3).


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Fig. 3.   Peripheral, antral, and fundic integrated SOM outputs in response to CCK-8S (open bars) and G-17S (solid bars). Integrated plasma response (IPR) was calculated as SOM output during the 30-min peptide infusion with respect to basal. Results are means ± SE (n = 8). * P < 0.05 vs. basal; #P < 0.05 vs. CCK-8S.

CCK-8S had no effect on gastrin concentration in plasma from the periphery, antrum, and fundus. The net increase in plasma gastrin during the G-17S infusion was similar in the periphery (from 41 ± 8 to 300 ± 34 pmol/l), antrum (from 172 ± 53 to 465 ± 85 pmol/l), and fundus (from 26 ± 4 to 256 ± 46 pmol/l), with peak responses measured at 105 min (P < 0.05).

CCK-8S caused a significant increase in gastric pH, from a basal value of 3.31 ± 0.25 to 4.01 ± 0.42 (P < 0.05). During the infusion of G-17S the gastric pH did not vary significantly from basal (3.52 ± 0.26).

Antagonist Experiments

Effect of L-364,718 or L-365,260 on CCK-8S- and G-17S-stimulated SOM secretion. The CCK-A (L-364,718) and CCK-B/gastrin (L-365,260) receptor antagonists were used to determine the receptors involved in CCK- and gastrin-stimulated SOM secretion.

CCK-8S-STIMULATED SOM SECRETION. The CCK-A receptor antagonist L-364,718 suppressed both antral and fundic CCK-8S-stimulated SOM secretion. In the presence of L-364,718, antral CCK-8S-stimulated SOM output decreased from 1.93 ± 0.80 to -0.72 ± 0.50 pmol · min-1 · ml-1 (P < 0.05) (Fig. 4). In the fundus the SOM output in response to CCK-8S plus L-364,718 decreased from 3.76 ± 1.60 to -3.70 ± 0.32 pmol · min-1 · ml-1 (P < 0.05) (Fig. 4). The inhibitory effect of L-364,718 on CCK-8S-stimulated antral and fundic SOM output resulted in a decrease in peripheral plasma SOM output from 0.47 ± 0.13 to -0.20 ± 0.22 pmol · min-1 · ml-1 (P < 0.05) (Fig. 4).


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Fig. 4.   Peripheral, antral, and fundic integrated SOM outputs in response to CCK-8S (open bars; n = 8), CCK-8S plus L-364,718 (solid bars; n = 6), and CCK-8S plus L-365,260 (striped bars; n = 3). IPR was calculated as SOM output during the 30-min peptide infusion with respect to basal. Results are means ± SE. * P < 0.05 vs. CCK-8S alone.

Similar to the CCK-A receptor antagonist, the CCK-B/gastrin receptor antagonist L-365,260 significantly suppressed CCK-8S-stimulated SOM secretion in both the antrum (-2.21 ± 1.15 pmol · min-1 · ml-1) and fundus (-3.79 ± 1.18 pmol · min-1 · ml-1) (P < 0.05) (Fig. 4). The decreases were reflected in the peripheral circulation, where SOM output also decreased to -0.39 ± 0.24 pmol · min-1 · ml-1 (P < 0.05) (Fig. 4).

During the infusion of CCK-8S plus either L-364,718 or L-365,260 the pH did not vary significantly from basal (3.80 ± 0.35).

G-17S-STIMULATED SOM SECRETION. L-364,718 and L-365,260 had different effects on G-17S-stimulated SOM secretion in the fundus and antrum. L-364,718 had no effect on either antral, fundic, or peripheral SOM secretion stimulated by G-17S (Fig. 5). The CCK-B/gastrin receptor antagonist L-365,260 significantly suppressed G-17S-stimulated SOM secretion in the fundus, from 12.9 ± 3.81 to 0.87 ± 0.67 pmol ·min-1 · ml-1 (P < 0.05) (Fig. 5) but had no effect on antral secretion (Fig. 5). SOM output in the periphery also decreased from 0.50 ± 0.11 to -0.43 ± 0.34 pmol · min-1 · ml-1 (P < 0.05) (Fig. 5), reflecting the decrease in the fundus.


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Fig. 5.   Peripheral, antral, and fundic integrated SOM outputs in response to G-17S (open bars; n = 8), G-17S plus L-364,718 (solid bars; n = 6), and G-17S plus L-365,260 (striped bars; n = 3). IPR was calculated as SOM output during the 30-min peptide infusion with respect to basal. Results are means ± SE. * P < 0.05 vs. G-17S alone.

The gastric pH did not significantly change from basal during the infusion of G-17S plus L-364,718 (4.03 ± 0.25). However, during the infusion of G-17S plus L-365,260 the pH increased from 4.11 ± 0.18 to 4.67 ± 0.22 (P < 0.05).

Effect of ranitidine on CCK-8S- and G-17S-stimulated SOM secretion. To determine the role of histamine as a mediator in the regulation of SOM secretion, ranitidine was infused with CCK or gastrin. Ranitidine alone decreased SOM concentration in the periphery (from 175 ± 44 to 87 ± 23 pmol/l) and fundus (from 2,645 ± 578 to 1,361 ± 236 pmol/l) (P < 0.05) but had no effect on antral plasma SOM concentrations. Ranitidine blocked the SOM-stimulatory effect of CCK-8S, and this was observed in the antral, fundic, and peripheral circulations (Fig. 6). In contrast, the coadministration of ranitidine plus G-17S did not prevent the release of G-17S-stimulated SOM from the antrum (Fig. 7). In the fundus G-17S-stimulated SOM output was partly suppressed by ranitidine from 12.9 ± 3.81 to 3.63 ± 3.18 pmol · min-1 · ml-1 (Fig. 7). The SOM output in response to G-17S plus ranitidine remained greater than the basal output (P < 0.05) (Fig. 7).