European Journal of Pharmacology, 40 (1976) 233--239 © Eisevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
233
RELATIONSHIP BETWEEN THE PREVENTION OF RAT GASTRIC EROSIONS AND THE INHIBITION OF ACID SECRETION BY PROSTAGLANDINS BRENDAN J.R. WHITTLE Department of Pharmacology, Institute of Basic Medical Sciences, Royal College of Surgeons of England, Lincoln's Inn Fields, London WC2A 3PN, England
Received 4 May 1976, revised MS received 22 July 1976, accepted 27 July 1976
B.J.R. WHITTLE, Relationship between the prevention of rat gastric erosions and the inhibition of acid secretion by prostaglandins, European J. Pharmacol. 40 (1976) 233--239. The formation of gastric mucosal erosions induced by indomethacin in the rat was inhibited in a time- and dose-dependent manner by antisecretory prostaglandins, the methyl analogues of PGE2 being 400 times as active as the parent prostaglandin. PGA2, a methyl analogue of PGF2a and the H2-receptor antagonist metiamide, also inhibited erosion formation. There was a variable relationship between the doses required to inhibit erosions and to inhibit gastric acid secretion. In the anaesthetised rat, the low incidence of erosions with indomethacin was markedly increased by concurrent gastric perfusion with acid saline and taurocholate. This mucosal damage was inhibited by the methyl analogues of PGE2, suggesting protective actions on the mucosa other than inhibition of acid secretion. Gastric acid secretion
Prostaglandins
Bile salt
Indomethacin
Gastric erosions
1. Introduction
2. Materials and methods
Antisecretory prostaglandins of the E series inhibit the formation of gastric erosions and ulcers induced by pyloric-ligation, steroid administration (Robert et al., 1968), stress (Usardi et al., 1974; Kawarada et al., 1975) and treatment with reserpine {Lee et al., 1973) or 5-hydroxytryptamine (Ferguson et al., 1973) in rats. In the present study, the relationship between the inhibition of rat gastric mucosal erosions, induced acutely by the nonsteroid anti-inflammatory agent, indomethacin, and the inhibition of gastric acid secretion by several prostaglandins has been investigated. A preliminary account of this work was presented to the British Pharmacological Society {Whittle, 1975).
2.1. Assessment o f mucosal erosions
Wistar rats (200--250 g) starved for 18 h but allowed water, were killed by cervical dislocation at various time intervals following drug treatment. The stomachs were removed, opened along the lesser curvature and rinsed under a stream of water. They were then pinned flat on a cork board, coded to prevent observer bias and studied with the aid of the binocular microscope (×10). Erosions, which formed only in the glandular mucosa, were counted and each one given a severity rating on a 1--3 scale as previously described (Main and Whittle, 1975a). The total, divided by a factor of 10, was designated the 'erosion index' for that stomach.
234
In further experiments, the gastric lumen of the urethane-anaesthetised rat was perfused {0.1--0.2 ml/min) with acid saline (pH 1--2) using the techniques previously described in detail (Main and Whittle, 1973a), and the erosion index determined 3 h after the start of perfusion.
B.J.R. WHITTLE
c61 3' EROSION INOEX 2
2.2. Statistical analysis Results are expressed as the mean +S.E.M. where (n) is the number of values in the group. The significance of the data was evaluated using the Student's t-test and also the nonparametric Mann--Whitney U-test. p < 0.05 was taken as significant.
2.3. Drugs Indomethacin (Merck, Sharp & Dohme Ltd.) was dissolved in 5% w/v sodium bicarbonate solution (pH 8) to give a final concentration of 10 mg/ml, and immediately injected s.c. Prostaglandins (PGs), stored in methanol (--5 ° C) were made up freshly in saline following evaporation of the methanol when required and injected s.c. Crude sodium taurocholate {Sigma Chemical Co.) dissolved in distilled water, was added to acid saline, filtered {Whatman No. 1 paper), centrifuged (1000 g for 5 rain) and the supernatant diluted to give the desired acid and bile salt concentration. The final bile salt concentration was expressed in terms of the sodium taurocholate content of the crude material. 3. Results
3.1. Formation of gastric erosions by indomethacin Administration of indomethacin (5--30 mg/kg s.c.) gave a dose-dependent increase in the incidence and severity of mucosal erosions over the 6 h period of measurement. A submaximal dose of indomethacin (15 or 20 mg/ kg s.c.) was chosen for subsequent experiments.
2:s
HOURS
Fig. 1. Inhibition of indomethacin (15 mg/kg s.c.)induced gastric mucosal erosions (v, o) by (15S)-15-methyl prostaglandin E2, 2.5 tzg/kg s.c. (o . . . . . . o). Results are shown as the mean ±S.E.M., where (n) is the number of values in each group. Inhibition at 2.5 h was significant (p < 0.001).
3.2. Inhibition of gastric erosions by antisecre tory prostaglandins In an initial series of experiments (15S)15-methyl prostaglandin E~ methyl ester (2.5 pg/kg, s.c.) was administered immediately prior to indomethacin (15 mg/kg s.c.) and erosi6n formation in groups of rats was assessed at various time intervals. As is shown (fig. 1) with this dose of the PGE2 methyl analogue there was a marked inhibition of erosions at 2.5 h (p < 0.001) but this inhibitory activity was much reduced when assessed after 5 h. This presumably reflects the relatively short half-life of the prostaglandin following subcutaneous administration. In subsequent experiments, the potency of prostaglandins as inhibitors of indomethacin-induced erosions was determined 3 h after administration. The dose-dependent reduction of the gastric erosion index by the {15S)-15-methyl analogue of PGE~ is shown in fig. 2; the dose causing 50% reduction of the erosion index (IDs0) was 0.5 pg/kg, s.c. Administration of the antisecretory prostaglandins PGA2, PGE2 and 16,16
233
RELATIONSHIP BETWEEN THE PREVENTION OF RAT GASTRIC EROSIONS AND THE INHIBITION OF ACID SECRETION BY PROSTAGLANDINS BRENDAN J.R. WHITTLE Department of Pharmacology, Institute of Basic Medical Sciences, Royal College of Surgeons of England, Lincoln's Inn Fields, London WC2A 3PN, England
Received 4 May 1976, revised MS received 22 July 1976, accepted 27 July 1976
B.J.R. WHITTLE, Relationship between the prevention of rat gastric erosions and the inhibition of acid secretion by prostaglandins, European J. Pharmacol. 40 (1976) 233--239. The formation of gastric mucosal erosions induced by indomethacin in the rat was inhibited in a time- and dose-dependent manner by antisecretory prostaglandins, the methyl analogues of PGE2 being 400 times as active as the parent prostaglandin. PGA2, a methyl analogue of PGF2a and the H2-receptor antagonist metiamide, also inhibited erosion formation. There was a variable relationship between the doses required to inhibit erosions and to inhibit gastric acid secretion. In the anaesthetised rat, the low incidence of erosions with indomethacin was markedly increased by concurrent gastric perfusion with acid saline and taurocholate. This mucosal damage was inhibited by the methyl analogues of PGE2, suggesting protective actions on the mucosa other than inhibition of acid secretion. Gastric acid secretion
Prostaglandins
Bile salt
Indomethacin
Gastric erosions
1. Introduction
2. Materials and methods
Antisecretory prostaglandins of the E series inhibit the formation of gastric erosions and ulcers induced by pyloric-ligation, steroid administration (Robert et al., 1968), stress (Usardi et al., 1974; Kawarada et al., 1975) and treatment with reserpine {Lee et al., 1973) or 5-hydroxytryptamine (Ferguson et al., 1973) in rats. In the present study, the relationship between the inhibition of rat gastric mucosal erosions, induced acutely by the nonsteroid anti-inflammatory agent, indomethacin, and the inhibition of gastric acid secretion by several prostaglandins has been investigated. A preliminary account of this work was presented to the British Pharmacological Society {Whittle, 1975).
2.1. Assessment o f mucosal erosions
Wistar rats (200--250 g) starved for 18 h but allowed water, were killed by cervical dislocation at various time intervals following drug treatment. The stomachs were removed, opened along the lesser curvature and rinsed under a stream of water. They were then pinned flat on a cork board, coded to prevent observer bias and studied with the aid of the binocular microscope (×10). Erosions, which formed only in the glandular mucosa, were counted and each one given a severity rating on a 1--3 scale as previously described (Main and Whittle, 1975a). The total, divided by a factor of 10, was designated the 'erosion index' for that stomach.
234
In further experiments, the gastric lumen of the urethane-anaesthetised rat was perfused {0.1--0.2 ml/min) with acid saline (pH 1--2) using the techniques previously described in detail (Main and Whittle, 1973a), and the erosion index determined 3 h after the start of perfusion.
B.J.R. WHITTLE
c61 3' EROSION INOEX 2
2.2. Statistical analysis Results are expressed as the mean +S.E.M. where (n) is the number of values in the group. The significance of the data was evaluated using the Student's t-test and also the nonparametric Mann--Whitney U-test. p < 0.05 was taken as significant.
2.3. Drugs Indomethacin (Merck, Sharp & Dohme Ltd.) was dissolved in 5% w/v sodium bicarbonate solution (pH 8) to give a final concentration of 10 mg/ml, and immediately injected s.c. Prostaglandins (PGs), stored in methanol (--5 ° C) were made up freshly in saline following evaporation of the methanol when required and injected s.c. Crude sodium taurocholate {Sigma Chemical Co.) dissolved in distilled water, was added to acid saline, filtered {Whatman No. 1 paper), centrifuged (1000 g for 5 rain) and the supernatant diluted to give the desired acid and bile salt concentration. The final bile salt concentration was expressed in terms of the sodium taurocholate content of the crude material. 3. Results
3.1. Formation of gastric erosions by indomethacin Administration of indomethacin (5--30 mg/kg s.c.) gave a dose-dependent increase in the incidence and severity of mucosal erosions over the 6 h period of measurement. A submaximal dose of indomethacin (15 or 20 mg/ kg s.c.) was chosen for subsequent experiments.
2:s
HOURS
Fig. 1. Inhibition of indomethacin (15 mg/kg s.c.)induced gastric mucosal erosions (v, o) by (15S)-15-methyl prostaglandin E2, 2.5 tzg/kg s.c. (o . . . . . . o). Results are shown as the mean ±S.E.M., where (n) is the number of values in each group. Inhibition at 2.5 h was significant (p < 0.001).
3.2. Inhibition of gastric erosions by antisecre tory prostaglandins In an initial series of experiments (15S)15-methyl prostaglandin E~ methyl ester (2.5 pg/kg, s.c.) was administered immediately prior to indomethacin (15 mg/kg s.c.) and erosi6n formation in groups of rats was assessed at various time intervals. As is shown (fig. 1) with this dose of the PGE2 methyl analogue there was a marked inhibition of erosions at 2.5 h (p < 0.001) but this inhibitory activity was much reduced when assessed after 5 h. This presumably reflects the relatively short half-life of the prostaglandin following subcutaneous administration. In subsequent experiments, the potency of prostaglandins as inhibitors of indomethacin-induced erosions was determined 3 h after administration. The dose-dependent reduction of the gastric erosion index by the {15S)-15-methyl analogue of PGE~ is shown in fig. 2; the dose causing 50% reduction of the erosion index (IDs0) was 0.5 pg/kg, s.c. Administration of the antisecretory prostaglandins PGA2, PGE2 and 16,16
