Naunyn-Schmiedeberg's
Naunyn-Schmiedeberg'sArch. Pharmacol.297, 85- 90 (1977)
Archivesof
Pharmacology 9 by Springer-Verlag1977
The Effect of Clonidine on Gastric Acid Secretion in Rats and Dogs H. M. JENNEWEIN PharmaforschungBiologie,C. H. BoehringerSohn, D-6507 Ingelheim,Federal Republic of Germany
Summary. The effect of clonidine on gastric acid secretion was investigated using rats and dogs. In the stomach lumen perfused rat basal gastric acid secretion was increased by clonidine in the anaesthetized rat but inhibited in the conscious animal. Clonidine also reduced the basal gastric acid secretion in rats with chronic gastric fistula, (EDso 12 l,tg/kg p.o.). In addition, gastric secretion stimulated by insulin hypoglycaemia was inhibited by clonidine in anaesthetized stomach lumen perfused rats and in conscious dogs with gastric fistula. In the rat gastric secretion stimulated by electrical vagus stimulation was inhibited as well. However, clonidine had no effect on the gastric acid secretion stimulated by carbachol in stomach lumen perfused rats and in dogs with denervated fundic pouch. These results suggest that the inhibition of gastric acid secretion by clonidine probably is due to an inhibition of acetylcholine release at the vagus nerve endings. Additional central gastric antisecretory effects can, however, not be excluded by this study.
Key words." Clonidine - Gastric acid secretion - Insulin-hypoglycaemia - Carbachol - Vagus stimulation - Influence of anaesthesia.
INTRODUCTION Since its introduction clonidine (Catapresan | has received wide attention as an effective antihypertensive drug (Pettinger, 1975). Beside this antihypertensive action, which most probably is of central nervous origin (Schmitt and Schmitt, 1969) other effects of clonidine have been described one of which is an inhibition of gastric secretion. This inhibitory effect on gastric secretion was first observed in the pylorus Send offprint requests to the author at the above address
ligated rat (Shay-rat) (Hoefke and Kobinger, 1966; Boissier et al., 1970) and was also seen in man (Ottenjann, 1968; Neuhaus and Humpert, 1968). However, in the pylorus-ligated rat saliva secretion interferes with gastric acid secretion (Levine, 1965; Stockhaus and Wick, 1970) and salivary inhibition is a well known side effect of clonidine in man. This was also confirmed in animal experiments (Rand et al., 1969). Therefore the inhibitory effect of clonidine on gastric secretion in the pylorus ligated rat might be due solely to the absence of saliva acting as an alkaline stimulus for gastric secretion. This is supported by findings using experimental procedures which had no interference by saliva on the gastric secretion (Walz and van Zwieten, 1970; Curwain and Endersby, 1974); in this case clonidine caused an increase in gastric acid secretion. Walz and van Zwieten (1970), using the anaesthetized stomach lumen perfused rat, assumed that clonidine-induced increase of gastric secretion might be caused by the change in reactivity during anaesthesia. However, Curwain and Endersby (1974) performed their experiment in conscious dogs with denervated fundic pouch and observed an enhancement of pentagastrin stimulated gastric secretion after clonidine administration as well. It could be assumed therefore that clonidine would have only a stimulatory effect on gastric acid secretion, whereas saliva secretion is inhibited. Regarding these aspects it seemed of interest to investigate more closely the effect of clonidine on gastric secretion in rats and dogs. METHODS a) Animals. The animals used in this study were rats of the c h b b - T H O M strain and beagle bitches. The dogs were 8 - 12 m o n t h s old and weighed 9 - 11 kg. Both were kept conventionally and were fed with a standard food (Altromin-R, Altromin-H). They were deprived of food 24 h before starting the experiments but had
free accessto water.
86
b) General Procedures and Drugs. In all collected fractions of gastric juice the acid content was estimated by automatic potentiometric titration to pH-7 (TTT-11, Abu-13, PHM-26, Radiometer, Copenhagen). Blood sugar concentration was measured by means of the glucose oxidase assay (Boehringer Mannheim, Kit). The drugs used were clonidine-hydrochloride (Catapresan | C. H. Boehringer, Ingelheim), carbachol-hydrochloride (Lentin | E. Merck, Darmstadt), cimetidine (Smith, Kline and French), ketamine (Ketanest | Parke Davis) and insulin (Insulin Hoechst | Farbwerke Hoechst AG Frankfurt). All the drugs were dissolved or diluted in 0.9 % NaC1. c) Rat Experiments. Gastric fistula rats were prepared and used according to the method of Brodie et al. (1962). Under ether anaesthesia a stainless steel gastric fistula was implanted in the fore stomach of 24 female rats weighing between 180 and 220 g. At least 3 weeks were allowed for the rats to recover from this operation. During the 24 h starvation period, the animals had free access to 5 % glucose solution, which ensured gastric emptying. Gastric juice was collected for 4 h at hourly intervals. Clonidine was given via the gastric fistula 1 h before starting the collection of gastric juice. One week before and 1 week after the clonidine experiment, each rat underwent a NaCl-control. The mean of the gastric secretion values as measured in the 2 control experiments was used as reference in each individual animal and the results were expressed as percent of this control value. Experiments with stomach-lumen-perfusedrats were performed according to Lai's method (Lai, 1964). Male rats weighing between 300 and 350 g whre used. The rats were anaesthetized with urethane 2 g/kg i.m. The stomach lumen was continously perfused with 0.9% NaC1 solution at 37~ in a rate of 1 ml/min and 10min effluent fractions were collected. Gastric secretion was stimulated using 2 gg/kg carbachol i.v., 10 IE/kg insulin i.v. (Hantschmann, 1972), and by electrical vagus nerve stimulation. The stimulation was performed using a Grass stimulator (S-4), a stimulus isolation unit ($IU-5) and a constant current unit (CCU-1). The vagus was stimulated on both sides with platinum wire electrodes delivering square wave pulses of 180 gA, 10 pps, and 5 ms duration over a time period of 10 rain. For the experiments with conscious stomach lumen perfused rats, catheters were implanted in the forestomach and via the pylorus in the antrum under ketamine anaesthesia (150- 200 mg/ kg i.p.). The animals were then immobilized in Bollmann-cages and 2 h were allowed for the animals to recover from the anaesthesia. Then gastric juice was collected as described above and clonidine was given via the tail vein.
Naunyn-Schmiedeberg's Arch. Pharmacol. 297 (1977)
r
.0 25. O 0 09 "O -25 O
< -50o c- -75 o -lO0L~
:3
10 i 0 10(3 360 10'00
Log Dose Clonidine[;ug.kg'] Fig. 1. Dose-effect curve of clonidine in conscious rats with chronically implanted gastric fistula. Clonidine was given via the gastric fistula and 6 animals were used for each dose. For further details see in the text (2 4- SEM)
10- Clonidine
lmg.kg-li.v.
!
c-
~
CIonidine 1 rng. kg -1 i.v. d
-o
d) Dog Experiments. In 6 dogs a Thomas gastric cannula was implanted in the stomach near the greater curvature. Six other dogs were provided with denervated fundic pouches (Heidenhain). Experiments were started at least 3 weeks after the operation. Stimulation of gastric secretion was induced in dogs with gastric fistula using insulin 0.4 IE/kg i.v. and in the dogs with denervated fundic pouch using carbachol 10 !.tg/kg s.c. Gastric secretion was collected in 15 rain fractions.
e) Statistics. The results of the experiments were described by the arithmetic mean (2), the standard error (SEM), and the number of experiments (n). Differences were tested for significance using the Mann-Whitney-U-test, where P < 0.05 was considered as significant.
RESULTS
Rat Experiments. The effect of clonidine on basal gastric secretion was tested in gastric fistula rats and in stomach lumen perfused rats. In gastric fistula rat basal acid secretion was 390 __ 28 I.tEq/4 h (n = 24).
0
1
2
Time [ hours] Fig. 2. The divergent effect of clonidine in 8 anaesthetized and 8 unanaesthetized rats. In both cases gastric secretion was measured using a stomach lumen perfusion technique. Note the greater magnitude of scale for the lower curve. (2 4- SEM)
As shown in Figure 1 clonidine administered via the gastric fistula caused a dose dependent inhibition of basal acid secretion in this animal model. The inhibitory effect of 30 pg/kg is statistically significant (P < 0.01). This is true also for the higher doses. The ED5o as taken from the graph was 12 gg/kg. As shown in Figure 2 in the stomach lumen perfused rat clonidine in a dose of 1 mg/kg i.v. caused a
H. M. Jennewein: Clonidine and Gastric Acid Secretion
87
Control Carbachol 0.9~Nacl Carbachol 20---"2tJg"kg"1i.v. 1 rnl i.v, 21Jg.kg"i.v.
E
nsulin I0 IE' kg-'liv"
0
-=- 10E o
1
~
0Iii ::L !
Carbachol CIonidine C,arbachol 2pg.kg-1i,~ 1mg.kg4iv. 2pg.kg4i,v.
t"
"-o
0
l
,~ 20(i1 GO
o
|
40]/
1-
Clonidine 4 mg.kg -4 i.v.
1~ no~El.,~g.li.v. d h before Insulin
09
.-g_
O
< 10
o
!
E
o
Naunyn-Schmiedeberg'sArch. Pharmacol.297, 85- 90 (1977)
Archivesof
Pharmacology 9 by Springer-Verlag1977
The Effect of Clonidine on Gastric Acid Secretion in Rats and Dogs H. M. JENNEWEIN PharmaforschungBiologie,C. H. BoehringerSohn, D-6507 Ingelheim,Federal Republic of Germany
Summary. The effect of clonidine on gastric acid secretion was investigated using rats and dogs. In the stomach lumen perfused rat basal gastric acid secretion was increased by clonidine in the anaesthetized rat but inhibited in the conscious animal. Clonidine also reduced the basal gastric acid secretion in rats with chronic gastric fistula, (EDso 12 l,tg/kg p.o.). In addition, gastric secretion stimulated by insulin hypoglycaemia was inhibited by clonidine in anaesthetized stomach lumen perfused rats and in conscious dogs with gastric fistula. In the rat gastric secretion stimulated by electrical vagus stimulation was inhibited as well. However, clonidine had no effect on the gastric acid secretion stimulated by carbachol in stomach lumen perfused rats and in dogs with denervated fundic pouch. These results suggest that the inhibition of gastric acid secretion by clonidine probably is due to an inhibition of acetylcholine release at the vagus nerve endings. Additional central gastric antisecretory effects can, however, not be excluded by this study.
Key words." Clonidine - Gastric acid secretion - Insulin-hypoglycaemia - Carbachol - Vagus stimulation - Influence of anaesthesia.
INTRODUCTION Since its introduction clonidine (Catapresan | has received wide attention as an effective antihypertensive drug (Pettinger, 1975). Beside this antihypertensive action, which most probably is of central nervous origin (Schmitt and Schmitt, 1969) other effects of clonidine have been described one of which is an inhibition of gastric secretion. This inhibitory effect on gastric secretion was first observed in the pylorus Send offprint requests to the author at the above address
ligated rat (Shay-rat) (Hoefke and Kobinger, 1966; Boissier et al., 1970) and was also seen in man (Ottenjann, 1968; Neuhaus and Humpert, 1968). However, in the pylorus-ligated rat saliva secretion interferes with gastric acid secretion (Levine, 1965; Stockhaus and Wick, 1970) and salivary inhibition is a well known side effect of clonidine in man. This was also confirmed in animal experiments (Rand et al., 1969). Therefore the inhibitory effect of clonidine on gastric secretion in the pylorus ligated rat might be due solely to the absence of saliva acting as an alkaline stimulus for gastric secretion. This is supported by findings using experimental procedures which had no interference by saliva on the gastric secretion (Walz and van Zwieten, 1970; Curwain and Endersby, 1974); in this case clonidine caused an increase in gastric acid secretion. Walz and van Zwieten (1970), using the anaesthetized stomach lumen perfused rat, assumed that clonidine-induced increase of gastric secretion might be caused by the change in reactivity during anaesthesia. However, Curwain and Endersby (1974) performed their experiment in conscious dogs with denervated fundic pouch and observed an enhancement of pentagastrin stimulated gastric secretion after clonidine administration as well. It could be assumed therefore that clonidine would have only a stimulatory effect on gastric acid secretion, whereas saliva secretion is inhibited. Regarding these aspects it seemed of interest to investigate more closely the effect of clonidine on gastric secretion in rats and dogs. METHODS a) Animals. The animals used in this study were rats of the c h b b - T H O M strain and beagle bitches. The dogs were 8 - 12 m o n t h s old and weighed 9 - 11 kg. Both were kept conventionally and were fed with a standard food (Altromin-R, Altromin-H). They were deprived of food 24 h before starting the experiments but had
free accessto water.
86
b) General Procedures and Drugs. In all collected fractions of gastric juice the acid content was estimated by automatic potentiometric titration to pH-7 (TTT-11, Abu-13, PHM-26, Radiometer, Copenhagen). Blood sugar concentration was measured by means of the glucose oxidase assay (Boehringer Mannheim, Kit). The drugs used were clonidine-hydrochloride (Catapresan | C. H. Boehringer, Ingelheim), carbachol-hydrochloride (Lentin | E. Merck, Darmstadt), cimetidine (Smith, Kline and French), ketamine (Ketanest | Parke Davis) and insulin (Insulin Hoechst | Farbwerke Hoechst AG Frankfurt). All the drugs were dissolved or diluted in 0.9 % NaC1. c) Rat Experiments. Gastric fistula rats were prepared and used according to the method of Brodie et al. (1962). Under ether anaesthesia a stainless steel gastric fistula was implanted in the fore stomach of 24 female rats weighing between 180 and 220 g. At least 3 weeks were allowed for the rats to recover from this operation. During the 24 h starvation period, the animals had free access to 5 % glucose solution, which ensured gastric emptying. Gastric juice was collected for 4 h at hourly intervals. Clonidine was given via the gastric fistula 1 h before starting the collection of gastric juice. One week before and 1 week after the clonidine experiment, each rat underwent a NaCl-control. The mean of the gastric secretion values as measured in the 2 control experiments was used as reference in each individual animal and the results were expressed as percent of this control value. Experiments with stomach-lumen-perfusedrats were performed according to Lai's method (Lai, 1964). Male rats weighing between 300 and 350 g whre used. The rats were anaesthetized with urethane 2 g/kg i.m. The stomach lumen was continously perfused with 0.9% NaC1 solution at 37~ in a rate of 1 ml/min and 10min effluent fractions were collected. Gastric secretion was stimulated using 2 gg/kg carbachol i.v., 10 IE/kg insulin i.v. (Hantschmann, 1972), and by electrical vagus nerve stimulation. The stimulation was performed using a Grass stimulator (S-4), a stimulus isolation unit ($IU-5) and a constant current unit (CCU-1). The vagus was stimulated on both sides with platinum wire electrodes delivering square wave pulses of 180 gA, 10 pps, and 5 ms duration over a time period of 10 rain. For the experiments with conscious stomach lumen perfused rats, catheters were implanted in the forestomach and via the pylorus in the antrum under ketamine anaesthesia (150- 200 mg/ kg i.p.). The animals were then immobilized in Bollmann-cages and 2 h were allowed for the animals to recover from the anaesthesia. Then gastric juice was collected as described above and clonidine was given via the tail vein.
Naunyn-Schmiedeberg's Arch. Pharmacol. 297 (1977)
r
.0 25. O 0 09 "O -25 O
< -50o c- -75 o -lO0L~
:3
10 i 0 10(3 360 10'00
Log Dose Clonidine[;ug.kg'] Fig. 1. Dose-effect curve of clonidine in conscious rats with chronically implanted gastric fistula. Clonidine was given via the gastric fistula and 6 animals were used for each dose. For further details see in the text (2 4- SEM)
10- Clonidine
lmg.kg-li.v.
!
c-
~
CIonidine 1 rng. kg -1 i.v. d
-o
d) Dog Experiments. In 6 dogs a Thomas gastric cannula was implanted in the stomach near the greater curvature. Six other dogs were provided with denervated fundic pouches (Heidenhain). Experiments were started at least 3 weeks after the operation. Stimulation of gastric secretion was induced in dogs with gastric fistula using insulin 0.4 IE/kg i.v. and in the dogs with denervated fundic pouch using carbachol 10 !.tg/kg s.c. Gastric secretion was collected in 15 rain fractions.
e) Statistics. The results of the experiments were described by the arithmetic mean (2), the standard error (SEM), and the number of experiments (n). Differences were tested for significance using the Mann-Whitney-U-test, where P < 0.05 was considered as significant.
RESULTS
Rat Experiments. The effect of clonidine on basal gastric secretion was tested in gastric fistula rats and in stomach lumen perfused rats. In gastric fistula rat basal acid secretion was 390 __ 28 I.tEq/4 h (n = 24).
0
1
2
Time [ hours] Fig. 2. The divergent effect of clonidine in 8 anaesthetized and 8 unanaesthetized rats. In both cases gastric secretion was measured using a stomach lumen perfusion technique. Note the greater magnitude of scale for the lower curve. (2 4- SEM)
As shown in Figure 1 clonidine administered via the gastric fistula caused a dose dependent inhibition of basal acid secretion in this animal model. The inhibitory effect of 30 pg/kg is statistically significant (P < 0.01). This is true also for the higher doses. The ED5o as taken from the graph was 12 gg/kg. As shown in Figure 2 in the stomach lumen perfused rat clonidine in a dose of 1 mg/kg i.v. caused a
H. M. Jennewein: Clonidine and Gastric Acid Secretion
87
Control Carbachol 0.9~Nacl Carbachol 20---"2tJg"kg"1i.v. 1 rnl i.v, 21Jg.kg"i.v.
E
nsulin I0 IE' kg-'liv"
0
-=- 10E o
1
~
0Iii ::L !
Carbachol CIonidine C,arbachol 2pg.kg-1i,~ 1mg.kg4iv. 2pg.kg4i,v.
t"
"-o
0
l
,~ 20(i1 GO
o
|
40]/
1-
Clonidine 4 mg.kg -4 i.v.
1~ no~El.,~g.li.v. d h before Insulin
09
.-g_
O
< 10
o
!
E
o
