Naunyn-Schmiedeberg's
Naunyn-Schmiedeberg'sArch. Pharmacol. 303, 87-93 (1978)
Archivesof
Pharmacology 9 by Springer-Verlag1978
Studies on the Mechanism of the Inhibitory Effect of Ethanol on the Gastric Acid Output in the Rat Juhani Puurunen Department of Pharmacology, University of Oulu, SF-90220 Oulu 22, Finland
Summary. The mechanism of the ethanol-induced decrease of the output of gastric acid was studied in the perfused stomach preparation of urethaneanaesthetised rats. Hypotonic (0.8 %) ethanol caused a transient decrease in the spontaneous output of acid. Isotonic (1.7 %) or higher concentrations of ethanol induced a constant inhibition of the gastric acid output. At the concentration of 10 % ethanol strongly inhibited the gastric acid secretion, stimulated by carbachol, histamine or pentagastrin. In the presence of 20% ethanol in the gastric lumen a significant disappearance of added hydrogen ions was observed, whereas 10 ethanol did not cause a loss of acid. This indicates that the decrease of gastric acid output by 10 % or lower concentrations of ethanol is due to the inhibition of the secretory process. Higher concentrations may decrease the gastric acid output also by inducing a backdiffusion of hydrogen ions into the gastric mucosa. Pretreatment of the rats with inhibitors of prostaglandin synthesis, viz. indomethacin, mefenamic acid, meclofenamic acid or phenylbutazone, antagonised the ethanol-induced inhibition of the spontaneous gastric acid secretion. Pretreatment with the phosphodiesterase inhibitors, papaverine (20mg/kg i.v.) and theophylline (60 mg/kg i.v.) increased the secretion of gastric acid. Papaverine antagonised the inhibition of gastric acid output induced by 10% ethanol, but theophylline failed to do so. At the concentration of 10 % ethanol strongly depressed the secretory response to dibutyryl cyclic AMP. The inhibitory effect of ethanol was abolished by a pretreatment with meclofenamic acid. The results suggest that in the gastric mucosa ethanol increases the formation of prostaglandins which exert an inhibitory effect on the gastric acid secretion. Key words: Ethanol - Gastric acid secretion - Backdiffusion - Hyperosmolality - Prostaglandins. Send offprint requests to J. Pnurunen at the above address
Introduction Ethanol has a local inhibitory effect on the gastric acid secretion in the anaesthetised rat (Puurunen and Karppanen, 1975) as well as in the isolated rat stomach (de Saint-Blanquat and Derache, 1966). Davenport (1967) attributed the ethanol-induced decrease of hydrogen ions in the Heidenhain pouch of the dog to a back-diffusion into the gastric mucosa. However, other studies have challanged this view (Shanbour et al., 1973; Kuo et al., 1974, Sernka et al., 1974) and claimed that ethanol inhibits the active secretion of acid by the parietal cells. Sernka and Jackson (1975) suggested that the inhibition of gastric acid secretion by ethanol is due to hyperosmolality of the alcohol solutions. We have previously suggested that the ethanol-induced inhibition of gastric acid secretion in the rat might be partly due to the lowering of cyclic AMP (Puurunen and Karppanen, 1975) and ATP (Puurunen et al., 1977) in the gastric mucosa. Indomethacin, a potent inhibitor of prostaglandin synthesis (Vane, 1971), antagonized the ethanol-induced inhibition of gastric acid secretion in the rat (Karppanen and Puurunen, 1976). This suggested that the inhibition of the gastric acid secretion by ethanol might be in part due to an increased formation of prostaglandins. In the present work the effect of ethanol on the loss of hydrogen ions from the gastric lumen was studied. The effects of hypo- and isotonic ethanol solutions on the gastric acid secretion were investigated in order to test the possibility that the ethanol-induced inhibition of gastric acid secretion is an effect due to hyperosmolality. The ability of ethanol to affect the acid secretion induced by some gastric secretagogues was studied. Furthermore, the possible involvement of prostaglandins was investigated by using non-steroidal antiinflammatory drugs, which inhibit the synthesis of prostaglandins (Vane, 1971; Flower, 1974).
0028-1298/78/0303/0087/$1.40
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Naunyn-Schmiedeberg's Arch. PharmacoI. 303 (1978) 2
M a t e r i a l s and M e t h o d s
B
a) Measurement of Gastric Acid. Male Sprague-Dawley rats weighing 250 - 300 g were fasted for 20 h, but tap water was allowed ad libitum. The rats were anaesthetised with urethane, 1.2 g/kg i.p. The output of gastric acid was determined by the method described previously (Puurunen and Karppanen, 1975). Briefly, 4-ml portions of the perfusion solution (23 ~ C) were added into the stomach as bolus injections every 5 min by means of a polyethylene tube, which was passed through the oesophagus into the ruminal portion of the stomach. The 5-rain samples were collected from a duodenal tube and titrated to pH 6.0 with an automatic potentiometric titrator (TTT 2, Radiometer, Copenhagen). The body temperature of the rats was kept at 35 ~ C by means of a rectal probe and a heating lamp. After the operation the rats were allowed to stabilise for 30-40 min before the measurements of the acid secretion were started. The acid secretion was usually stabilised within 2 0 - 40 rain after the beginning of the measurements of acid. Thereafter the rate of acid output remained constant in each rat, although there were great variations between the individual rats.
b) Perfusion Solutions. Saline, pH 6.0, or 10 ~ (v/v) ethanol, dissolved in saline (pH 6.0), were used in most experiments as the perfusion solutions. Ethanol at the concentrations of 0.8 and 1.7 % (v/v) was dissolved in distilled water (pH 6.0). The loss of acid from the gastric lumen was studied by using 2 mM HC1 in saline and ethanol solutions (in saline) containing the same concentration of hydrogen ions. The 5-rain samples were titrated to the endpoint of saline (pH 6.0). The net flux of acid per each 5-min period was calculated by subtracting the initial amount of hydrogen ions (8 gEq) from that recovered from the stomach.
c) Statistical Analysis. Statistical analysis was performed by using Student's t-test or t-test for paired observations when indicated. The results are expressed as means _+ S.E
d) Drugs Used. Ethanol, 94 % (Oy Alko Ab, Helsinki); indomethacin, phenylbutazone and papaverine (Medipolar Oy, Oulu); mefenamic acid and meclofenamic acid (Parke-Davis & Co, Pontypool); carbachol (Doryl, Merck, Darmstadt); histamine acid phosphate (Koch-Light Laboratories Ltd., Colnbrook Buchs); pentagastrin (Peptavlon, ICI); dibutyryl cyclic AMP (Sigma Chemical Co., St. Louis); theophylline (Aminophyllin, Oy Medica Ab, Helsinki). ][ndomethacin was dissolved in phosphate buffer, pH 8.0, with the aid of NaOH (pH did not exceed 8.0). Eenamates were dissolved in 0.05N NaOH and phenylbutazone in dimethyl sulphoxide (DMSO). Other drugs were dissolved in saline. Control rats received the appropriate solvent.
Results
1. Effect of Ethanol on Loss of Acid from the Gastric Lumen When the stomach was perfused with 2 mM HC1 in saline, there was no net flux of hydrogen ions (Fig. 1 A). The presence of 10 % ethanol in the perfusion solution did not cause loss of acid (Fig. 1 B). However, during the perfusion with 20 % ethanol a disappearance of acid from the gastric lumen was observed (Fig. 1 C).
B. Effect of Hypo- and Isotonic Ethanol on Acid Secretion
Perfusion of the stomach with hypotonic, 0.8%, ethanol decreased initially (i.e., in the first three
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Fig. l. Effect of ethanol on acid loss from the gastric lumen. A Net flux of hydrogen ions during acid saline (2 mM HC1) perfusion. B Effect of 10 % ethanol in acid saline on net flux of acid. C Effect of 20 ~ ethanol in acid saline on net flux of acid. Positive values indicate the secretion of acid into the gastric lumen and negative values loss of acid from the gastric lumen. Each point is the mean _+ S.E. of 4 experiments
samples) the acid secretion in all 6 rats studied. Thereafter, the output of acid increased in one rat to the pre-ethanol level and in another rat above the preethanol level. Except for the initial inhibiton (P < 0.05, paired t-test), the output of acid did not differ from the pre-ethanol values (Fig. 2 A). Isotonic, 1.7 %, ethanol caused a decrease in the acid secretion within 5 rain. Within 20rain the output of acid stabilised to a level which was on the average 53 ~ of the pre-ethanol level (range 3 1 - 8 3 ~ , P < 0.01, paired t-test) (Fig.2B).
C. Effect of Non-Steroidal Anti-Inflammatory Drugs on the Ethanol-Induced Inhibition of Gastric Acid Secretion a) Indomethacin. When the spontaneous output of acid was stabilised, indomethacin in doses of 1,5 or 10 mg/kg was injected as a slow intravenous injection 90rain before the administration of 10% ethanol into the stomach. In the control group the output of acid (1.3 +
J. P u u r u n e n : Ethanol and Gastric Acid Output in the Rat
89
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Naunyn-Schmiedeberg'sArch. Pharmacol. 303, 87-93 (1978)
Archivesof
Pharmacology 9 by Springer-Verlag1978
Studies on the Mechanism of the Inhibitory Effect of Ethanol on the Gastric Acid Output in the Rat Juhani Puurunen Department of Pharmacology, University of Oulu, SF-90220 Oulu 22, Finland
Summary. The mechanism of the ethanol-induced decrease of the output of gastric acid was studied in the perfused stomach preparation of urethaneanaesthetised rats. Hypotonic (0.8 %) ethanol caused a transient decrease in the spontaneous output of acid. Isotonic (1.7 %) or higher concentrations of ethanol induced a constant inhibition of the gastric acid output. At the concentration of 10 % ethanol strongly inhibited the gastric acid secretion, stimulated by carbachol, histamine or pentagastrin. In the presence of 20% ethanol in the gastric lumen a significant disappearance of added hydrogen ions was observed, whereas 10 ethanol did not cause a loss of acid. This indicates that the decrease of gastric acid output by 10 % or lower concentrations of ethanol is due to the inhibition of the secretory process. Higher concentrations may decrease the gastric acid output also by inducing a backdiffusion of hydrogen ions into the gastric mucosa. Pretreatment of the rats with inhibitors of prostaglandin synthesis, viz. indomethacin, mefenamic acid, meclofenamic acid or phenylbutazone, antagonised the ethanol-induced inhibition of the spontaneous gastric acid secretion. Pretreatment with the phosphodiesterase inhibitors, papaverine (20mg/kg i.v.) and theophylline (60 mg/kg i.v.) increased the secretion of gastric acid. Papaverine antagonised the inhibition of gastric acid output induced by 10% ethanol, but theophylline failed to do so. At the concentration of 10 % ethanol strongly depressed the secretory response to dibutyryl cyclic AMP. The inhibitory effect of ethanol was abolished by a pretreatment with meclofenamic acid. The results suggest that in the gastric mucosa ethanol increases the formation of prostaglandins which exert an inhibitory effect on the gastric acid secretion. Key words: Ethanol - Gastric acid secretion - Backdiffusion - Hyperosmolality - Prostaglandins. Send offprint requests to J. Pnurunen at the above address
Introduction Ethanol has a local inhibitory effect on the gastric acid secretion in the anaesthetised rat (Puurunen and Karppanen, 1975) as well as in the isolated rat stomach (de Saint-Blanquat and Derache, 1966). Davenport (1967) attributed the ethanol-induced decrease of hydrogen ions in the Heidenhain pouch of the dog to a back-diffusion into the gastric mucosa. However, other studies have challanged this view (Shanbour et al., 1973; Kuo et al., 1974, Sernka et al., 1974) and claimed that ethanol inhibits the active secretion of acid by the parietal cells. Sernka and Jackson (1975) suggested that the inhibition of gastric acid secretion by ethanol is due to hyperosmolality of the alcohol solutions. We have previously suggested that the ethanol-induced inhibition of gastric acid secretion in the rat might be partly due to the lowering of cyclic AMP (Puurunen and Karppanen, 1975) and ATP (Puurunen et al., 1977) in the gastric mucosa. Indomethacin, a potent inhibitor of prostaglandin synthesis (Vane, 1971), antagonized the ethanol-induced inhibition of gastric acid secretion in the rat (Karppanen and Puurunen, 1976). This suggested that the inhibition of the gastric acid secretion by ethanol might be in part due to an increased formation of prostaglandins. In the present work the effect of ethanol on the loss of hydrogen ions from the gastric lumen was studied. The effects of hypo- and isotonic ethanol solutions on the gastric acid secretion were investigated in order to test the possibility that the ethanol-induced inhibition of gastric acid secretion is an effect due to hyperosmolality. The ability of ethanol to affect the acid secretion induced by some gastric secretagogues was studied. Furthermore, the possible involvement of prostaglandins was investigated by using non-steroidal antiinflammatory drugs, which inhibit the synthesis of prostaglandins (Vane, 1971; Flower, 1974).
0028-1298/78/0303/0087/$1.40
88
Naunyn-Schmiedeberg's Arch. PharmacoI. 303 (1978) 2
M a t e r i a l s and M e t h o d s
B
a) Measurement of Gastric Acid. Male Sprague-Dawley rats weighing 250 - 300 g were fasted for 20 h, but tap water was allowed ad libitum. The rats were anaesthetised with urethane, 1.2 g/kg i.p. The output of gastric acid was determined by the method described previously (Puurunen and Karppanen, 1975). Briefly, 4-ml portions of the perfusion solution (23 ~ C) were added into the stomach as bolus injections every 5 min by means of a polyethylene tube, which was passed through the oesophagus into the ruminal portion of the stomach. The 5-rain samples were collected from a duodenal tube and titrated to pH 6.0 with an automatic potentiometric titrator (TTT 2, Radiometer, Copenhagen). The body temperature of the rats was kept at 35 ~ C by means of a rectal probe and a heating lamp. After the operation the rats were allowed to stabilise for 30-40 min before the measurements of the acid secretion were started. The acid secretion was usually stabilised within 2 0 - 40 rain after the beginning of the measurements of acid. Thereafter the rate of acid output remained constant in each rat, although there were great variations between the individual rats.
b) Perfusion Solutions. Saline, pH 6.0, or 10 ~ (v/v) ethanol, dissolved in saline (pH 6.0), were used in most experiments as the perfusion solutions. Ethanol at the concentrations of 0.8 and 1.7 % (v/v) was dissolved in distilled water (pH 6.0). The loss of acid from the gastric lumen was studied by using 2 mM HC1 in saline and ethanol solutions (in saline) containing the same concentration of hydrogen ions. The 5-rain samples were titrated to the endpoint of saline (pH 6.0). The net flux of acid per each 5-min period was calculated by subtracting the initial amount of hydrogen ions (8 gEq) from that recovered from the stomach.
c) Statistical Analysis. Statistical analysis was performed by using Student's t-test or t-test for paired observations when indicated. The results are expressed as means _+ S.E
d) Drugs Used. Ethanol, 94 % (Oy Alko Ab, Helsinki); indomethacin, phenylbutazone and papaverine (Medipolar Oy, Oulu); mefenamic acid and meclofenamic acid (Parke-Davis & Co, Pontypool); carbachol (Doryl, Merck, Darmstadt); histamine acid phosphate (Koch-Light Laboratories Ltd., Colnbrook Buchs); pentagastrin (Peptavlon, ICI); dibutyryl cyclic AMP (Sigma Chemical Co., St. Louis); theophylline (Aminophyllin, Oy Medica Ab, Helsinki). ][ndomethacin was dissolved in phosphate buffer, pH 8.0, with the aid of NaOH (pH did not exceed 8.0). Eenamates were dissolved in 0.05N NaOH and phenylbutazone in dimethyl sulphoxide (DMSO). Other drugs were dissolved in saline. Control rats received the appropriate solvent.
Results
1. Effect of Ethanol on Loss of Acid from the Gastric Lumen When the stomach was perfused with 2 mM HC1 in saline, there was no net flux of hydrogen ions (Fig. 1 A). The presence of 10 % ethanol in the perfusion solution did not cause loss of acid (Fig. 1 B). However, during the perfusion with 20 % ethanol a disappearance of acid from the gastric lumen was observed (Fig. 1 C).
B. Effect of Hypo- and Isotonic Ethanol on Acid Secretion
Perfusion of the stomach with hypotonic, 0.8%, ethanol decreased initially (i.e., in the first three
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~-2
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~_~.. 2 0
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ETHANOL
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Fig. l. Effect of ethanol on acid loss from the gastric lumen. A Net flux of hydrogen ions during acid saline (2 mM HC1) perfusion. B Effect of 10 % ethanol in acid saline on net flux of acid. C Effect of 20 ~ ethanol in acid saline on net flux of acid. Positive values indicate the secretion of acid into the gastric lumen and negative values loss of acid from the gastric lumen. Each point is the mean _+ S.E. of 4 experiments
samples) the acid secretion in all 6 rats studied. Thereafter, the output of acid increased in one rat to the pre-ethanol level and in another rat above the preethanol level. Except for the initial inhibiton (P < 0.05, paired t-test), the output of acid did not differ from the pre-ethanol values (Fig. 2 A). Isotonic, 1.7 %, ethanol caused a decrease in the acid secretion within 5 rain. Within 20rain the output of acid stabilised to a level which was on the average 53 ~ of the pre-ethanol level (range 3 1 - 8 3 ~ , P < 0.01, paired t-test) (Fig.2B).
C. Effect of Non-Steroidal Anti-Inflammatory Drugs on the Ethanol-Induced Inhibition of Gastric Acid Secretion a) Indomethacin. When the spontaneous output of acid was stabilised, indomethacin in doses of 1,5 or 10 mg/kg was injected as a slow intravenous injection 90rain before the administration of 10% ethanol into the stomach. In the control group the output of acid (1.3 +
J. P u u r u n e n : Ethanol and Gastric Acid Output in the Rat
89
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v~0-1 0
'
ETHANOL 0.8 ~ (V/v)
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