Original Paper Digestion 1992;53:179-188
Division of Gastroenterology and Metabolism, Department of Internal Medicine, Philipps University, Marburg, FRG
Keywords Cholecystokinin, B receptor Gastrin receptor antagonists L-365,260 PD 136450 Acid secretion Somatostatin
Effect of Gastrin Receptor Antagonists on Gastric Acid Secretion and Gastrin and Somatostatin Release in the Rat Stomach
Abstract The effects of two recently developed gastrin receptor antago nists, PD 136450 and L-365,260, on pentagastrin-stimulated acid secretion were investigated in rats. PD 136450 at a dose of 6 mg/kg s.c. (9.6 pmol) completely abolished acid secretion induced by pentagastrin. The inhibition of 18 mg/kg PD 136450 s.c. lasted for at least 8 h and was still effective after 14 days of treatment (18 mg/kg s.c. every 8 h). Acute application of L-365,260 at a dose of 3.8 mg/kg, which is equimolar (9.6 pmol) to 6 mg/kg PD 136450 reduced acid responses slightly. However, when L-365,260 was administered intravenously at a dose of 3 mg/kg, this antagonist completely abolished the pentagastrin-stimulated acid secretion. Furthermore, the ef fect of PD 136450 on endogenous gastric somatostatin and gastrin releases was tested in the isolated, vascularly perfused rat stomach. PD 136450 perfused at a concentration of 1 \iM slightly increased somatostatin secretion after stimulation with a high dose of isoproterenol (10~7M). There was no effect of PD 136450 on basal or acetylcholine-stimulated gastrin secretion.
Gastrin is a key stimulus for acid secretion by parietal cells and has also a trophic action on fundic parietal and endocrine cells. Pro found and long-lasting inhibition of acid se cretion by proton pump inhibitors in rats [1-
6] as well as states of complete achlorhydria (pernicious anaemia) in man [7-10] may cause severe hypergastrinaemia and concur rent proliferation of enterochromaffin-like cells. Based upon several studies, it is highly
This study was supported by the Deutsche Forschungsgemeinschaft (Grant Ko 846/2-1) and the Kempkes Foundation (17/90). Marburg.
Dr. R. Eissele Department of Medicine Philipps University Baldingerstrassc D-W-3550 Marburg (FRG)
Received: March 20, 1992 Received in revised form: September 21,1992
© 1992 S. Kargcr AG, Basel 0012-2823/92/ 0534-0179S2.75/0
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:11:41 PM
R. Eissele H. Koop E. Bothe-Sandfort R. Arnold
180
Materials and Methods Animals Female Sprague-Dawlcy rats weighing 200-250 g were used throughout the study. They were housed in cages in temperature- and humidity-controlled rooms. The animals had free access to a commercial labora tory' diet (Altromin 1824: Altromin, Lage. FRG) and water ad libitum. Two days prior to acid secretion studies, the animals were maintained on a liquid diet (Biosorb®, Pfrimmer, Erlangen. FRG) to provide an empty stomach. For studies with the isolated, vascularly perfused stomach, the rats were fasted overnight (15 h). Surgical Procedure Acid Secretion Studies Under anaesthesia with ketamine (Ketanest®: Parke-Davis. Berlin, FRG) and xylazine (Rompun®: Bayer, Leverkusen, FRG), the animals were fitted with intravenous jugular vein catheters. Silastic tubing (Dow Corning, Midland, Mich.. USA). 1 mm in exter nal diameter. was inserted in the right jugular vein, and the catheter was routed subcutaneously to exit at the back of the neck. Acid secretion was determined using a modified in vivo luminal perfusion model [24-26], Briefly, rats were laparatomized by a midline incision. The oesoph agus was ligated, and a polyethylene tube (internal diameter 2 mm) was inserted through a small incision of the rumen. Special care was taken to avoid injury to the nerve and blood supply of the stomach. A distal tube (internal diameter 4 mm) was placed through the pylorus via a duodenotomy, and the pylorus was ligated. The abdominal wall was closed in layers after passing both tubes through the lateral skin. Isolated Vascularly Perfused Stomach Surgical procedure and perfusion technique have been described in detail earlier [27], Briefly, female Spraguc-Dawley rats weighing 220-250 g were anaes thetized with pentobarbital (Bayer). The abdominal aorta was exposed through a midline incision, and the coeliac artery was located. This area of aorta was iso lated in preparation for insertion of the arterial cannu la. The head of the pancreas and the spleen were freed from the greater curvature of the stomach. The duode num with associated pancreas was separated from the stomach at the gastroduodenal junction, care being taken to preserve right gastroepiploic and right gastric
Eisselc/Koop/Bothe-Sandfort/Amold
Gastrin Receptor Antagonists and Acid Secretion
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:11:41 PM
likely that hypergastrinaemia is the major fac tor mediating the trophic action and leading to hvperplasia of enterochromaffin-like cells [11-13], Potent gastrin receptor antagonists to prove the throphic effect of high levels of cir culating gastrin on endocrine and epithelial cells of the stomach were lacking so far. Recently, the benzodiazepine derivative L-365,260 and the dipeptoid PD 136450, re sembling the carboxyl-terminal moiety of gas trin, became available which exert high affin ity for cholccystokinin (CCK) B/gastrin recep tors in brain and stomach [14-20] and antag onize dose dependently the action of gastrin on acid secretion in short-term experiments [16, 18,21], In order to study the trophic role of endog enous gastrin, complete abolition of the gas trin action is required on a long-term basis. Besides effectiveness, a simple method of ad ministration is also important for this pur pose. In the present study varying doses and different routes of administration of the an tagonist L-365,260 and PD 136450 were tested for the ability to completely reverse the stimulation of gastric acid secretion in the rat by pentagastrin. Somatostatin has been shown to act as a key regulator of G cell function [22,23], How ever. there is little information about the influence of gastrin on the secretory activity of the D cell. If somatostatin secretion of D cells is mediated by gastrin receptors, this effect should be altered by gastrin receptor antagonists. Furthermore, in case of feedback regulation of the antral gastrin cells, gastrin receptor antagonists should increase gastrin secretion. In the present study, the influence of gastrin receptor blockade on endogenous gastrin and somatostatin-like immunoreactivity was investigated by the isolated, vascularly perfused rat stomach during perfusion with PD 136450.
Experiments Acid Secretion Studies The gastric lumen was gently rinsed with 100 ml of saline (37 °C) prior to the start of the experiments. Thereafter, the stomach was perfused continuously at a flow rate of 0.5 ml/min with 0.15 M NaCl. The effluent was collected in 10-min aliquots and neutral ized immediately with 0.01 M NaOH. After a basal period of 30 min. maximal stimulation of acid secre tion was achieved by a continuous intravenous infu sion of 32 pg/kg/h pentagastrin (Gastrodiagnost®; Merck. Darmstadt. FRG) [30]. Study I. The water-soluble dipeptoid antagonist PD 136450 (kindly donated by Dr. McKnight. Parke Davies. Cambridge. UK) an a-methyltryptophan de rivative [18], was dissolved in 0.15% NaCl. In all stud ies this antagonist was given subcutaneously: (1) at a dose of 6 mg/kg(9.6 pmol 30 min after and 6 h prior to pentagastrin stimulation; (2) at a dose of 18 rng/kg 6.8, and 12 h prior to pentagastrin stimulation, and (3) in long-term experiments 18 mg/kg PD 136450 was ad ministered at 8-hourly intervals for 14 days. Acid secretion analysis was performed 4 h after the final dose. Study 2. The CCK B/gastrin receptor antagonist L-365,260 (kindly provided by Dr. Evans. Merck Sharp & Dohme. West Point, Pa.. USA), a benzodiaze pine derivative [14], was dissolved in dimethylsuiphoxide (DMSO; Sigma. Dcisenhofen. FRG) for sub cutaneous injections and in DMSO/polyethyleneglycol (PEG; Roth. Karlsruhe. FRG) for intravenous infu
sions and administered at the following dosages: (1) subcutaneously at a dose of 3.8 mg/kg (9.6 pmol) 30 min after pentagastrin stimulation and (2) intrave nously at doses of 1 and 3 mg/kg/h. starting 30 min after pentagastrin stimulation. Control rats receiving the respective vehicle (either DMSO, DMSO/PEG. or saline) alone were investi gated parallel to animals receiving the antagonists. Isolated, Vascularly Perfused Stomach PD 136450 was perfused at a final concentration of I0~6 M which is able to block all gastrin receptors in the stomach [McKnight. unpubl. results]. The gastrin secretion was stimulated by acetylcholine (1O'6 M\ Sig ma. St. Louis, Mo., USA) and the somatostatin secre tion by isoproterenol (10-7 and 1 0 s M\ Sigma). All substances were introduced via side-arm infusions into the perfusate. Sequential perfusion (sec figure 4) con sisted of (1) a 5-min basal period. 5 min perfusion with PD 136450 alone. 10 min simultaneous perfusion with PD 136450 and acetylcholine or isoproterenol. 5 min perfusion with PD 136450 alone, and a 5-min basal period; (2) a 10-min basal period. 10 min PD 136450 and 10 min simultaneous perfusion with PD 136450 and acetylcholine or isoproterenol. Controls were per fused with saline instead of PD 136450.
Evaluation and Statistical Analysis For every' treatment regimen in acid secretion stud ies and every perfusion protocol in the isolated, vascu larly perfused stomach experiments 6-8 rats were used. Mean values ± standard error of the mean were calculated and plotted in the figures for each 10-min period in acid secretion and for l-min periods in hor mone secretion studies. For statistical analysis the total acid output during 1 h (pmol/h). starting 40 min after pentagastrin stimulation, was added up and compared with controls. In the isolated, vascularly perfused stomach the hormone secretion during stimulation was compared in PD 136450 perfused rats and in saline controls. Statistical analysis was performed em ploying the non-parametric Mann-Whitney U test [31].
181
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arteries. Branches of the coeliac arteries supplying the liver and the oesophagus were sectioned. A drainage tube was tied into the pylorus via a duodenotomy. the colon sectioned at the rectum, and small bowel, colon, pancreas, and spleen removed. Arterial perfusion was performed through a cannula placed in the aorta adja cent to the coeliac artery. A second cannula was placed in the portal vein for collecting venous effluent. The perfusate consisted of Krebs bicarbonate buffer con taining 0.2% human serum albumin (Behringwerke, Marburg. FRG), 3% dextran T 70 (Pharmacia. Upp sala, Sweden) and 80 mg/dl glucose and was contin uously gassed with 95% CU 5% CCF to achieve pi 1 7.4. Perfusion was maintained at 3 ml/min, and complete portal vein effluent, following a 20-min period of equilibration, was collected at l-min intervals. Gastrin and somatostatin-like immunorcactivity (SLI) were measured bv well-established radioimmunoassays [28. 29],
Table 1. Influence of the gastrin receptor antagonists PD 136450 and L-365.260 on pentagastrin-stimulaied acid secretion in the rat stomach Antagonist
n
Administration
Dose
Interval“
Acid secretion
L-365.260 L-365.260 Controls
6 6 8
i.v., inf. i.v.. inf. i.v., inf.
1 mg/kg/h 3 mg/kg/h (NaCl)
30 min 30 min 30 min
178± 19 81 ± 10 23.3 ± 2 0
< 0.05
Division of Gastroenterology and Metabolism, Department of Internal Medicine, Philipps University, Marburg, FRG
Keywords Cholecystokinin, B receptor Gastrin receptor antagonists L-365,260 PD 136450 Acid secretion Somatostatin
Effect of Gastrin Receptor Antagonists on Gastric Acid Secretion and Gastrin and Somatostatin Release in the Rat Stomach
Abstract The effects of two recently developed gastrin receptor antago nists, PD 136450 and L-365,260, on pentagastrin-stimulated acid secretion were investigated in rats. PD 136450 at a dose of 6 mg/kg s.c. (9.6 pmol) completely abolished acid secretion induced by pentagastrin. The inhibition of 18 mg/kg PD 136450 s.c. lasted for at least 8 h and was still effective after 14 days of treatment (18 mg/kg s.c. every 8 h). Acute application of L-365,260 at a dose of 3.8 mg/kg, which is equimolar (9.6 pmol) to 6 mg/kg PD 136450 reduced acid responses slightly. However, when L-365,260 was administered intravenously at a dose of 3 mg/kg, this antagonist completely abolished the pentagastrin-stimulated acid secretion. Furthermore, the ef fect of PD 136450 on endogenous gastric somatostatin and gastrin releases was tested in the isolated, vascularly perfused rat stomach. PD 136450 perfused at a concentration of 1 \iM slightly increased somatostatin secretion after stimulation with a high dose of isoproterenol (10~7M). There was no effect of PD 136450 on basal or acetylcholine-stimulated gastrin secretion.
Gastrin is a key stimulus for acid secretion by parietal cells and has also a trophic action on fundic parietal and endocrine cells. Pro found and long-lasting inhibition of acid se cretion by proton pump inhibitors in rats [1-
6] as well as states of complete achlorhydria (pernicious anaemia) in man [7-10] may cause severe hypergastrinaemia and concur rent proliferation of enterochromaffin-like cells. Based upon several studies, it is highly
This study was supported by the Deutsche Forschungsgemeinschaft (Grant Ko 846/2-1) and the Kempkes Foundation (17/90). Marburg.
Dr. R. Eissele Department of Medicine Philipps University Baldingerstrassc D-W-3550 Marburg (FRG)
Received: March 20, 1992 Received in revised form: September 21,1992
© 1992 S. Kargcr AG, Basel 0012-2823/92/ 0534-0179S2.75/0
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:11:41 PM
R. Eissele H. Koop E. Bothe-Sandfort R. Arnold
180
Materials and Methods Animals Female Sprague-Dawlcy rats weighing 200-250 g were used throughout the study. They were housed in cages in temperature- and humidity-controlled rooms. The animals had free access to a commercial labora tory' diet (Altromin 1824: Altromin, Lage. FRG) and water ad libitum. Two days prior to acid secretion studies, the animals were maintained on a liquid diet (Biosorb®, Pfrimmer, Erlangen. FRG) to provide an empty stomach. For studies with the isolated, vascularly perfused stomach, the rats were fasted overnight (15 h). Surgical Procedure Acid Secretion Studies Under anaesthesia with ketamine (Ketanest®: Parke-Davis. Berlin, FRG) and xylazine (Rompun®: Bayer, Leverkusen, FRG), the animals were fitted with intravenous jugular vein catheters. Silastic tubing (Dow Corning, Midland, Mich.. USA). 1 mm in exter nal diameter. was inserted in the right jugular vein, and the catheter was routed subcutaneously to exit at the back of the neck. Acid secretion was determined using a modified in vivo luminal perfusion model [24-26], Briefly, rats were laparatomized by a midline incision. The oesoph agus was ligated, and a polyethylene tube (internal diameter 2 mm) was inserted through a small incision of the rumen. Special care was taken to avoid injury to the nerve and blood supply of the stomach. A distal tube (internal diameter 4 mm) was placed through the pylorus via a duodenotomy, and the pylorus was ligated. The abdominal wall was closed in layers after passing both tubes through the lateral skin. Isolated Vascularly Perfused Stomach Surgical procedure and perfusion technique have been described in detail earlier [27], Briefly, female Spraguc-Dawley rats weighing 220-250 g were anaes thetized with pentobarbital (Bayer). The abdominal aorta was exposed through a midline incision, and the coeliac artery was located. This area of aorta was iso lated in preparation for insertion of the arterial cannu la. The head of the pancreas and the spleen were freed from the greater curvature of the stomach. The duode num with associated pancreas was separated from the stomach at the gastroduodenal junction, care being taken to preserve right gastroepiploic and right gastric
Eisselc/Koop/Bothe-Sandfort/Amold
Gastrin Receptor Antagonists and Acid Secretion
Downloaded by: King's College London 137.73.144.138 - 3/7/2018 8:11:41 PM
likely that hypergastrinaemia is the major fac tor mediating the trophic action and leading to hvperplasia of enterochromaffin-like cells [11-13], Potent gastrin receptor antagonists to prove the throphic effect of high levels of cir culating gastrin on endocrine and epithelial cells of the stomach were lacking so far. Recently, the benzodiazepine derivative L-365,260 and the dipeptoid PD 136450, re sembling the carboxyl-terminal moiety of gas trin, became available which exert high affin ity for cholccystokinin (CCK) B/gastrin recep tors in brain and stomach [14-20] and antag onize dose dependently the action of gastrin on acid secretion in short-term experiments [16, 18,21], In order to study the trophic role of endog enous gastrin, complete abolition of the gas trin action is required on a long-term basis. Besides effectiveness, a simple method of ad ministration is also important for this pur pose. In the present study varying doses and different routes of administration of the an tagonist L-365,260 and PD 136450 were tested for the ability to completely reverse the stimulation of gastric acid secretion in the rat by pentagastrin. Somatostatin has been shown to act as a key regulator of G cell function [22,23], How ever. there is little information about the influence of gastrin on the secretory activity of the D cell. If somatostatin secretion of D cells is mediated by gastrin receptors, this effect should be altered by gastrin receptor antagonists. Furthermore, in case of feedback regulation of the antral gastrin cells, gastrin receptor antagonists should increase gastrin secretion. In the present study, the influence of gastrin receptor blockade on endogenous gastrin and somatostatin-like immunoreactivity was investigated by the isolated, vascularly perfused rat stomach during perfusion with PD 136450.
Experiments Acid Secretion Studies The gastric lumen was gently rinsed with 100 ml of saline (37 °C) prior to the start of the experiments. Thereafter, the stomach was perfused continuously at a flow rate of 0.5 ml/min with 0.15 M NaCl. The effluent was collected in 10-min aliquots and neutral ized immediately with 0.01 M NaOH. After a basal period of 30 min. maximal stimulation of acid secre tion was achieved by a continuous intravenous infu sion of 32 pg/kg/h pentagastrin (Gastrodiagnost®; Merck. Darmstadt. FRG) [30]. Study I. The water-soluble dipeptoid antagonist PD 136450 (kindly donated by Dr. McKnight. Parke Davies. Cambridge. UK) an a-methyltryptophan de rivative [18], was dissolved in 0.15% NaCl. In all stud ies this antagonist was given subcutaneously: (1) at a dose of 6 mg/kg(9.6 pmol 30 min after and 6 h prior to pentagastrin stimulation; (2) at a dose of 18 rng/kg 6.8, and 12 h prior to pentagastrin stimulation, and (3) in long-term experiments 18 mg/kg PD 136450 was ad ministered at 8-hourly intervals for 14 days. Acid secretion analysis was performed 4 h after the final dose. Study 2. The CCK B/gastrin receptor antagonist L-365,260 (kindly provided by Dr. Evans. Merck Sharp & Dohme. West Point, Pa.. USA), a benzodiaze pine derivative [14], was dissolved in dimethylsuiphoxide (DMSO; Sigma. Dcisenhofen. FRG) for sub cutaneous injections and in DMSO/polyethyleneglycol (PEG; Roth. Karlsruhe. FRG) for intravenous infu
sions and administered at the following dosages: (1) subcutaneously at a dose of 3.8 mg/kg (9.6 pmol) 30 min after pentagastrin stimulation and (2) intrave nously at doses of 1 and 3 mg/kg/h. starting 30 min after pentagastrin stimulation. Control rats receiving the respective vehicle (either DMSO, DMSO/PEG. or saline) alone were investi gated parallel to animals receiving the antagonists. Isolated, Vascularly Perfused Stomach PD 136450 was perfused at a final concentration of I0~6 M which is able to block all gastrin receptors in the stomach [McKnight. unpubl. results]. The gastrin secretion was stimulated by acetylcholine (1O'6 M\ Sig ma. St. Louis, Mo., USA) and the somatostatin secre tion by isoproterenol (10-7 and 1 0 s M\ Sigma). All substances were introduced via side-arm infusions into the perfusate. Sequential perfusion (sec figure 4) con sisted of (1) a 5-min basal period. 5 min perfusion with PD 136450 alone. 10 min simultaneous perfusion with PD 136450 and acetylcholine or isoproterenol. 5 min perfusion with PD 136450 alone, and a 5-min basal period; (2) a 10-min basal period. 10 min PD 136450 and 10 min simultaneous perfusion with PD 136450 and acetylcholine or isoproterenol. Controls were per fused with saline instead of PD 136450.
Evaluation and Statistical Analysis For every' treatment regimen in acid secretion stud ies and every perfusion protocol in the isolated, vascu larly perfused stomach experiments 6-8 rats were used. Mean values ± standard error of the mean were calculated and plotted in the figures for each 10-min period in acid secretion and for l-min periods in hor mone secretion studies. For statistical analysis the total acid output during 1 h (pmol/h). starting 40 min after pentagastrin stimulation, was added up and compared with controls. In the isolated, vascularly perfused stomach the hormone secretion during stimulation was compared in PD 136450 perfused rats and in saline controls. Statistical analysis was performed em ploying the non-parametric Mann-Whitney U test [31].
181
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arteries. Branches of the coeliac arteries supplying the liver and the oesophagus were sectioned. A drainage tube was tied into the pylorus via a duodenotomy. the colon sectioned at the rectum, and small bowel, colon, pancreas, and spleen removed. Arterial perfusion was performed through a cannula placed in the aorta adja cent to the coeliac artery. A second cannula was placed in the portal vein for collecting venous effluent. The perfusate consisted of Krebs bicarbonate buffer con taining 0.2% human serum albumin (Behringwerke, Marburg. FRG), 3% dextran T 70 (Pharmacia. Upp sala, Sweden) and 80 mg/dl glucose and was contin uously gassed with 95% CU 5% CCF to achieve pi 1 7.4. Perfusion was maintained at 3 ml/min, and complete portal vein effluent, following a 20-min period of equilibration, was collected at l-min intervals. Gastrin and somatostatin-like immunorcactivity (SLI) were measured bv well-established radioimmunoassays [28. 29],
Table 1. Influence of the gastrin receptor antagonists PD 136450 and L-365.260 on pentagastrin-stimulaied acid secretion in the rat stomach Antagonist
n
Administration
Dose
Interval“
Acid secretion
L-365.260 L-365.260 Controls
6 6 8
i.v., inf. i.v.. inf. i.v., inf.
1 mg/kg/h 3 mg/kg/h (NaCl)
30 min 30 min 30 min
178± 19 81 ± 10 23.3 ± 2 0
< 0.05
