1Department of Clinical Pharmacology, Faculty of Medicine, Tottori University, Tottori, Japan; 2Department of Pharmacology, Institute of Physiological Sciences, University of Lund, Lund, Sweden; and 3Department of Gastroenterology and Hepatology, Kinki University, School of Medicine, Osaka, Japan
Submitted 8 January 2004 ; accepted in final form 17 November 2004
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ABSTRACT |
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enterochromaffin-like cell; ischemia-reperfusion; histidine decarboxylase
Histamine resides in two different cell types in the acid-producing part of the stomach, mast cells and enterochromaffin-like (ECL) cells (2). Mucosal mast cells are relatively few, occurring mainly at the surface of the mucosa and in the submucosa. ECL cells occur predominantly in the basal half of the mucosa. ECL cell histamine is mobilized by gastrin to stimulate acid secretion by activating histamine H2 receptors on the parietal cells (1, 18, 29). In previous studies, administration of acid inhibitory agents (including proton pump inhibitors and H2 receptor blockers) reduced the area of damaged mucosa after ischemia-reperfusion, suggesting that gastric acid contributes to the damage (6, 15). So far, there has been no attempt to study how gastric histamine responds to an insult to the gastric mucosa. The technique of gastric submucosal microdialysis was adopted in our laboratories (8, 14) to make it possible to monitor the mobilization of gastric histamine. It was soon realized that clamping of the celiac artery resulted in the mobilization of massive amounts of histamine from the stomach (present study). To identify the cellular source of the mobilized histamine, we compared wild-type rats with rats devoid of mast cells (19, 20) and examined the effects of -fluoromethylhistidine (
-FMH), an irreversible inhibitor of histidine decarboxylase (HDC) (16), known to eliminate histamine from ECL cells but not from mast cells (1, 2). In addition, we compared the effects of arterial clamping in control rats with those observed in rats pretreated with the histamine H2 receptor antagonist cimetidine or the proton pump inhibitor omeprazole.
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MATERIALS AND METHODS |
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-FMH was purchased from Sigma (St. Louis, MO). The histamine H2 receptor antagonist cimetidine was obtained from Sumitomo Pharmaceutical (Tokyo, Japan). The proton pump inhibitor omeprazole was a kind gift from AstraZeneca (Mölndal, Sweden). All other chemicals were reagent grade and commercially available.
Animals
Male Wistar rats were purchased from SLC (Shizuoka, Japan) or from B&K (Sollentuna, Sweden). They weighed 200260 g at the time of the experiment. The Wistar rats are referred to as wild type (WT). Ws-RC (Ws/Ws) rats are deficient in mast cells (19, 20). -FMH was administered via osmotic minipumps (Alzet 2 MLI; Alza, Palo Alto, CA) implanted subcutaneously in the neck under diethyl ether anesthesia. The studies were approved by the Guidelines for Animal Experimentation in the Faculty of Medicine, Tottori University, and the local Animal Welfare Committee of Lund/Malmö. All rats were fasted for 1824 h (free access to water) before the experiments unless otherwise stated.
Gastric Submucosal Microdialysis
Rats were anesthetized with pentobarbital (50 mg/kg ip) during the experiments. The abdomen was opened by a midline incision, and the microdialysis probe was implanted into the gastric submucosa (8, 14). A flexible microdialysis probe (length 8 mm, outer diameter 0.22 mm, cut-off 50 kDa; Pt-20008-PW; Eicom, Kyoto, Japan) was used. The serosa of the dorsal aspect of the acid-producing part of the stomach was tangentially punctured by a needle (22 gauge), and a tunnel (1215 mm) was made in the submucosa from the greater to the lesser curvature. The probe was gently inserted into the tunnel and kept in place with sutures. The inlet tube was connected to a microinfusion pump, and the outlet was allowed to drain into 0.3-ml polystyrene vials. Immediately after implantation, the microdialysis probes were perfused with degassed saline (1.2 µl/min). Sampling of microdialysate started after 60 min of equilibration. Samples were then collected every 10 min throughout the experiment.
Ischemia-Reperfusion
Gastric mucosal injury was induced by the ischemia-reperfusion technique of Wada et al. (28). The celiac artery was occluded with a small clamp (Sugita standard aneurysm clip, holding force 145 g; Mizuho Ikakogyo, Tokyo, Japan). Reperfusion was initiated 30 min later by removal of the clamp. For comparison, some rats underwent sham operation (surgery but no clamping) or reperfusion (80 min) after a shorter duration (10 min) of ischemia. If not otherwise stated, the rats were killed (exsanguination from the abdominal aorta) after 30 min of arterial clamping and 60 min of reperfusion (removal of the clamp) (Fig. 1). The stomach was removed and opened along the major curvature. The mucosa was exposed, the lesions were photographed, and the injury score was calculated as the total area of erosions with a computer system of imaging analysis (28).
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To examine the role of mast cells in the pathogenesis of the ischemia-reperfusion injury, experiments were performed on mutant rats deficient in mast cells (Ws/Ws rats) (19, 20). Ws/Ws rats and WT rats were compared with respect to changes in the microdialysate histamine concentration and in the area of gastric mucosal lesions (60 min after reperfusion).
Treatment with Antisecretory Drugs
-FMH.
WT rats and Ws/Ws rats received saline or
-FMH (3 mg·kg1·h1) via osmotic minipumps implanted 4 days before the ischemia-reperfusion experiments. This dose of
-FMH is known to abolish histamine formation and to deplete histamine from ECL cells but not from mast cells (1, 2). The
-FMH treatment lowers basal acid secretion and prevents gastrin from stimulating acid secretion (1) as a consequence of impaired ECL cell histamine mobilization (14, 21). Microdialysis was conducted during ischemia-reperfusion, and the total area of mucosal damage was measured 60 min after start of reperfusion.
Histamine H2 receptor antagonist. Cimetidine was dissolved in 0.9% saline and given by subcutaneous injection at doses of 0.1, 1, 3, 10, 30, and 100 mg/kg. The purpose of the experiment was to produce dose-dependent H2 receptor blockade with consequent gastric acid inhibition (5, 7). Ischemia-reperfusion of the celiac artery was performed in rats pretreated with the different doses of cimetidine. The celiac artery was clamped 70 min after the injection of cimetidine. The total area of erosions was measured 60 min after removal of the clamp.
The basal gastric acid output was measured by luminal perfusion of the rat stomach (15). Briefly, a soft catheter was inserted into the esophagus, and another cannula was inserted into the stomach via an incision in the duodenum at a distance of 1 cm from the pylorus. The gastric lumen was perfused with physiological saline solution (NaCl 0.15 mol/l, pH 7.0, 37°C) at a rate of 1 ml/min with a peristaltic pump. Seventy minutes after the subcutaneous injection of different doses of cimetidine (0.1, 1, 3, 10, 30, 100 mg/kg), the perfusate was collected for 30 min and the acid in the perfusate was determined by titration with NaOH (5 mmol/l).
In another experiment, gastric submucosal microdialysis was conducted during ischemia-reperfusion in rats pretreated with a maximally effective dose of cimetidine (100 mg/kg) given subcutaneously 70 min before the ischemia. Controls received vehicle (saline).
Proton pump inhibitor. Omeprazole (a proton pump inhibitor) was dissolved in 0.25% Methocel and given in a dose of 400 µmol/kg once daily by oral gavage for 4 days. This dose is known to induce sustained inhibition of gastric acid secretion (24). The rats were fasted overnight, and omeprazole was given in the morning 2 h before clamping. In one experiment, treatment with omeprazole continued for as long as 9 days after clamping.
Determination of Microdialysate Histamine
Histamine in the microdialysate was measured by enzyme-linked immunosorbent assay with a commercially available kit (Immunotech, Paris, France). The histamine concentration was expressed as picomoles per milliliter. The sensitivity of this method is 0.1 pmol/m1, and the intra-assay variation was 15%.
Determination of Oxyntic Mucosal HDC Activity and Histamine Concentration
Mucosa was scraped off the acid-producing part of the stomach, weighed, and homogenized in ice-cold 0.1 mol/l sodium phosphate buffer, pH 7.4, to a concentration of 100 mg wet wt/ml. Aliquots (80 µl) were incubated with L-[1-14C]histidine (0.02 mCi/ml, specific activity 50 mCi/mmol), 5 x 104 M L-histidine, and 105 M pyridoxal 5'-phosphate in a total volume of 160 µl at 37°C for 1 h under nitrogen (17). HDC activity was expressed as picomoles of 14CO2 per milligram per hour. For determination of histamine, the mucosal homogenates were diluted 1:10 with redistilled water and heated in boiling water for 10 min to release bound histamine. The homogenates were then centrifuged at 6,000 g for 10 min; histamine in the supernatant was measured spectrophotofluorometrically (23).
Statistical Analysis
Results are expressed as means ± SE. Student's t-test was used to evaluate the significance of differences between pairs of data. With multiple comparisons, the statistical significance of the differences was determined by one-way analysis of variance followed by Dunnett's test. P < 0.05 was considered significant.
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RESULTS |
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Ischemia alone (30 min) produced macroscopically visible lesions on the surface of the gastric (oxyntic) mucosa. Ninety minutes of ischemia did not produce more mucosal damage than thirty minutes of ischemia (35.1 ± 10.8 mm2 vs. 48 ± 20 mm2). Reperfusion for 30 or 60 min greatly increased the total area of the erosions (Fig. 2A).
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Effect of Ischemia-Reperfusion on Gastric Mucosal Erosions and Microdialysate Histamine in Mast Cell-Deficient Rats
The gastric mucosa displayed macroscopically visible damage in response to 30 min of arterial clamping and 60 min of reperfusion in both WT rats and Ws/Ws rats (Fig. 3, A and B). There was no statistically significant difference in gastric lesions between the two groups of rats (Fig. 3C). As in the WT rats, the microdialysate histamine concentration in the Ws/Ws rats increased in response to the ischemia. The concentration reached the same level in both strains of rats. (Fig. 3D).
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-FMH.
Pretreatment with
-FMH greatly reduced the area of ischemia-reperfusion-evoked mucosal lesions and virtually abolished the rise in microdialysate histamine in both WT and Ws/Ws rats (Fig. 4).
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The effect of ischemia on gastric mucosal HDC activity was studied in omeprazole-treated rats. HDC is a hallmark feature of rat stomach ECL cells (33). The enzyme is known to be activated by omeprazole treatment (because of the hypergastrinemia; Refs. 17, 33). Ischemia (30 min) of the stomach of omeprazole-treated rats promptly lowered the HDC activity (Fig. 6C). However, the HDC activity started to return to preischemic values after 2 days and was back to normal after 9 days (continued omeprazole treatment).
The histamine concentration was reduced by 50% after ischemia; the concentration was back to normal after 4 days (Fig. 6D).
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DISCUSSION |
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Ischemia Increases Histamine in Submucosal Compartment of Stomach
Microdialysis is a useful technique to monitor the mobilization of histamine from an intracellular to an extracellular compartment. Normally, histamine mobilized from ECL cells will be washed away promptly, only to appear in the portal vein. However, the clamping of the gastric artery will prevent such washout. Hence, the greatly elevated histamine concentration in the gastric submucosa following ischemia probably reflects not only mobilization but also accumulation/entrapment of the amine in the tissue.
Origin of Mobilized Gastric Histamine
To identify the source of the histamine that is mobilized by ischemia of the stomach, we compared mast cell-deficient rats (Ws/Ws rats) and WT rats. First, the area of the gastric mucosal erosions did not differ between Ws/Ws rats and WT rats, suggesting that mast cells do not contribute to the mucosal damage. Second, the rise in microdialysate histamine concentration (and hence in the submucosal histamine concentration; Ref. 8) was the same in the Ws/Ws rats and the WT rats, suggesting that mast cells do not contribute importantly to the histamine response to ischemia. After pretreatment of the two strains of rats with -FMH, which is known to deplete histamine from ECL cells but not from mast cells (1, 2), the ischemia-induced histamine response was virtually abolished (a few percent remaining), in support of the view that the histamine that is mobilized by ischemia comes mainly from the ECL cells.
Mechanism Behind Mobilization of Histamine by Ischemia-Reperfusion
The precise mechanism behind the ischemia-evoked histamine mobilization remains unknown. It is possible but not very likely that the histamine mobilization reflects the death of ECL cells because of damage. This is unlikely because 1) omeprazole-treated rats responded to ischemia-reperfusion with histamine mobilization but not with mucosal damage and 2) clamping of the celiac artery in omeprazole-treated rats reduced the oxyntic mucosal histamine concentration and HDC activity only transiently; the histamine concentration and HDC activity were in fact back to normal 49 days later. Because ECL cells have a long life span (25, 26), the latter findings are in line with the view that although the cells are damaged by 30 min of ischemia, they are able to recover within a matter of days.
Is Mobilized Gastric Histamine Responsible for Gastric Lesions After Ischemia-Reperfusion?
Pretreatment with the histamine H2 receptor antagonist cimetidine or the proton pump inhibitor omeprazole prevented or greatly reduced the gastric mucosal injury in response to ischemia-reperfusion without lowering the amount of histamine mobilized from ECL cells. This is in line with the view that ischemia-evoked mobilization of histamine and development of gastric mucosal injury are independent phenomena. The major cause of the mucosal injury would be ischemia-evoked anoxia in combination with acid secretion. Without acid secretion there is no mucosal damage. The acid output is reduced by 80% after 30 min of ischemia (15). Still, local histamine is probably needed (in small quantities) to stimulate the parietal cells to produce acid. In fact, the elimination of acid by luminal perfusion of the stomach with saline (1 ml/min) protected the gastric mucosa from the ischemia-reperfusion insult, suggesting that luminal acid contributes to the mucosal damage (15). However, it cannot be excluded that it is the energy-demanding process of acid secretion rather than gastric acidity per se that is responsible for the lesions (30). The massive release of histamine in response to ischemia does not in itself cause or aggravate the mucosal damage. In a parallel study, endothelin was administered by submucosal microinfusion, causing local mucosal lesions and local release of spectacular amounts of ECL cell histamine (4), in a manner reminiscent of the events taking place after gastric ischemia. The endothelin-induced mucosal lesions could be prevented by systemic but not by local administration (via the microdialysis probe) of the histamine H2 receptor antagonist ranitidine (4), suggesting that acid per se (or the metabolic demands of acid secretion) is responsible for the damage and that histamine per se is not. Indeed, the failure of local administration of histamine (4) to induce gastric mucosal lesions is in line with this interpretation.
In conclusion, ischemia-reperfusion damages the oxyntic mucosa and causes spectacular histamine release. The lesions do not develop because of the histamine mobilization but because of ischemia plus reperfusion plus gastric acid per se. Histamine comes from ECL cells, and mast cells do not contribute to either the mucosal damage or the histamine output. ECL cell histamine is mobilized by the ischemia, independently of the mucosal damage.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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