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Opposite effects of κ-opioid agonists on gastric emptying of liquids and solids in dogs
GASTROENTEROLOGY
1988;95:927-31
Opposite Effects of K-Opioid Agonists on Gastric Emptying of Liquids and Solids in Dogs M. GUE, J. FIORAMONTI, and L. BUENO
C. HONDE, X. PASCAUD, J. L. JUNIEN,
Department of Pharmacology, Institut National de la Recherche Jouveinal Laboratoires, Fresnes, France
The influence of oral (p.0.) administration of K(U-50488, tifluadom) and EC- (morphine, DAGO) opioid substances on gastric emptying of liquids and solids in a standard canned dog food meal was evaluated using a double-radiolabeled technique in dogs fitted with gastric cannulas. One hour after feeding, 28.6% -+ 3.6% (mean f SD) of the solid phase and 27.1% 2 8.6% of the liquid phase of the meal had been emptied. Both U-50488 and tifluadom given orally (0.01-0.1 mg/kg) significantly increased (p < 0.05) the 1-h emptying of the solid phase of the meal by 23.10/a-49.6%. In contrast, both drugs significantly reduced emptying of liquids. These effects were not reproduced when similar doses were given intravenously. Oral administramglkg) did not tion of morphine or DAGO (0.01-0.1 affect gastric emptying, whereas an inhibited emptying of solids was observed for morphine at a higher dose (1mg/kg p.0.). At a dose of 100 pg/kg i.v. both naloxone and MR 2266 (0.1 mg/kg) abolished the effects of orally administered U-50488 on gastric emptying of solids and liquids. It is concluded that Kbut not Ec_agonists act locally to alter gastric emptying of a standard meal in dogs, having opposite effects on solid and liquid phases. A selective local stimulation of K mucosal or submucosal receptors of the gastroduodenal area may explain such effects.
G
astric emptying depends on the motor activity of two distinct portions of the digestive tract: (a) the lower esophageal sphincter and the proximal stomach (fundus and corpus) and (b) the antropyloroduodenal area (1). It is classically believed that the proximal stomach controls liquid emptying, which may be directly related to the amplitude of the slow sustained contractions and the relaxation following a meal (Z-4). In contrast, gastric emptying of solids
Agronomique,
Toulouse,
and
may result from (a) the rhythmic peristaltic waves of the distal stomach (5--7), (b) the opening of the pylorus (8) affected by digestive hormonal response (1,9), and (c) the size of particles (10,ll). Opiates have long been known to delay gastric emptying of both liquids and solids when systemically administered in dogs, monkeys, and humans (12-14). Although the peripheral and central action of S, p-, and K-agonists on gastric and intestinal motility may be different, their systemic administration usually results in a reduction in gastric emptying; furthermore, these endogenous opiates appear to be involved in the regulation of gastric emptying, as suggested by the tendency of naloxone to increase gastric evacuation of a solid meal in humans (15). Histologic studies performed in dogs have revealed the presence of nerve cells containing enkephalin and dynorphin peptide immunoreactive materials in the submucosal plexus, muscularis mucosae, and villi of the stomach, pylorus, and proximal intestine (16). These observations led us to speculate that opiate-sensitive fibers located at gastric and duodenal levels may influence gastric emptying as well as gastric motor patterns (17). Consequently, the present experiments were conducted to evaluate the influence of orally administered p- and K-agonists on the gastric emptying of the liquid and solid phases of a spontaneously eaten standard (canned food] meal in dogs using a doubleradiolabeling technique (18). Materials
and Methods
Materials were
Six female adult mongrel dogs weighing 12-16 kg used for the experiments. A Thomas cannula was 0 1988 by the American
Gastroenterological 0018-5085/88/$3.50
Association
928
GUE ET AL.
GASTROENTEROLOGY
placed on the greater curvature of the gastric body under halothane (Fluothane, Coopers, Maux, France) anesthesia. The cannula was positioned -10 cm from the pylorus, and was brought through and fixed to the left abdominal wall 5 cm from the last rib and 10 cm from the midline. Animals were allowed 2 wk to recover before testing was begun. The solid phase of the meal consisted of 400 g of canned food (Fiddle, Quaker, Bordeaux, France) containing 21.7% dry matter, 7.7% protein, 4.5% fat, 6.9% carbohydrates, and 2.6% minerals. Sheep liver (20 g) labeled with [57Co]cyanocobalamine was mixed with the canned food (solid phase). Labeled liver was obtained from sheep given an i.v. injection of 10 &i of [57Co]cyanocobalamine (Amersham, Paris, France, sp act 10 &i/pg) and killed 24 h later. One hundred milliliters of tap water (liquid phase) containing 50 mg of polyethylene glycol4OOO (PEG), a nonabsorbable water marker, with 0.5 &i of [14C]PEG (NEN Research Products; sp act, 1 &ilmg) was added to the solid meal before presenting this test meal to the animals. The meal was eaten in <5 min. Total collection of the gastric contents was performed 1 h after the meal. The collected contents were homogenized after determination of the total weight and volume. Two series of five samples (4-5 ml) were rapidly taken; each sample of the first series was weighed and “7Co was counted using a y-counter (MR252; Kontron, Switzerland). The second series was prepared for [14C]PEG determination using a liquid scintillation system (SL 40; Intertechnique, Plaisir, France). The percentages of gastric emptying of liquid and solid phases were calculated from these (18), using the measurements as previously described formula: Esp = [(C,W,
- C,,,W,,,)
x loo]lCLWL,
where ESP designates the emptying of solid phase (expressed as a percentage), CL is counts per minute per gram of marked liver introduced into the meal, WL is the weight of the liver introduced into the meal, Ccct is counts per minute per gram of homogenized gastric contents at time t, and WcCt is the weight of the contents present in the stomach at time t. and
where ELP designates the emptying of liquid phase (expressed as a percentage), [[‘4C]PEGLp& is the initial concentration of the liquid phase of the meal, Vwi is the initial volume of water added, [[r4C]PEGnMt] is the concentration of homogenized meal at time t, and Vu,, is the volume of the homogenized meal at time t.
Experimental
Procedure
First series of experiments. Twenty minutes before the test meal the animals received orally, at s-day intervals and in random fashion, morphine or tifluadom (0.01, 0.1, and 1 mg/kg) and U-50488 or (o-Ala’, N-methyl-Phe4, Gly’-01) enkephalin (DAGO) (0.01 and 0.1 mgikg). Second series of experiments. Ten minutes before
Vol
95, No.
4
feeding U-50488 and morphine were injected intravenously at doses of 0.01 and 0.1 mgikg. Third series of experiments. The oral administration of U-50488 or tifluadom at a dose of 0.1 mgikg was preceded (10 min) by the intravenous injection of naloxone (0.1 mgikg) or MR 2266 (0.1 mgikg). Morphine, DAGO, and U-50488 were purchased from Cooperative Pharmaceutique Francaise (Brive, France), Sigma Chemical Co. (St. Louis, MO.), and Upjohn Diagnostics [Kalamazoo, Mich.), respectively. Tifluadom, naloxone, and MR 2266 were gifts from Sandoz (Basel, Switzerland), Endo Laboratories (Garden City, N.Y.), and Boehringer Ingelheim (Bracknell, U.K.), respectively. All experiments were performed in duplicate on each subject, and gastric emptying of liquids and solids was evaluated 1 h after the ingestion of the meal. Gastric emptying was expressed as the percentage of the meal emptied for both liquids and solids, as previously described (18). Because of the small number of animals (n = 6), statistical analysis of the results was performed using a Wilcoxon matched-pairs signed-rank test and differences were considered significant for p or 0.05. Results were expressed as the mean I SD.
Results Influence of Oral Versus P-Agonists
Administration
of
K-
When measured 1 h after feeding under control (placebo) conditions, the gastric emptying of solids represented 28.6% ? 3.6% (mean + SD, n = 12) of the initial weight of the solid phase; at the same time the volume of liquid emptied was 27.1% ? 8.6% of the initial volume, i.e., 100 ml (Figure 1). The larger standard deviation for emptying rates of liquids compared with that observed for solids may be related to great variations in the total recovery of 14C, as previously reported (18). When given orally at doses of 10 and 100 pgikg, both U-50488 and tifluadom significantly (p < 0.01) increased the percentage of the solid phase emptied after 1 h, whereas a significant (p < 0.02) reduction in the amount of liquid emptied was detected (Figure 1). At a higher oral dose [l mg/kg), tifluadom still enhanced the gastric evacuation of solids but did not affect (p > 0.05) that of liquids. In contrast, neither morphine nor DAGO given orally at similar doses (10 and 100 pgikg] affected gastric emptying of liquids and solids. At a dose of 1 mg/kg, morphine significantly (p < 0.05) reduced emptying of solids by >50%. Comparative Administration
Effects
of Systemic
When injected intravenously 10 min before feeding, U-50488 at doses of 10 and 100 pglkg did not affect the 1-h gastric emptying of solids, whereas
Or,tober
K-AGONISTS
1988
AND GASTRIC
EMPTYLNG
929
1SOLIDS]
TIFLUADOM---
0.01 0.1
,,~O____-__-
X ITigure
1. Comparative
of a standard 0.05. W-test).
slight (28.6%) but significant (p < 0.05) inhibition of liquids was observed at the higher dose (Figure 2). Morphine injected intravenously at doses of 100 ,ug/kg significantly (p < 0.05) inhibited gastric emptying of liquids and solids (Table l), but the lower dose (10 pgikg) was not effective. a
Influence
of Naloxone
and
MR 2266
Naloxone and MR 2266 administered intravenously (0.1 mgikg) 40 min before the meal had no effect per se on the 1-h values of gastric emptying of liquids and solids. When injected 10 min before oral U-50488 (0.1 mgikg) or tifluadom (0.1 mgikg), both naloxone and MR 2266 completely abolished their 50
50
1
40.
B
30
:, 9
l
Table
1. Comparative Influence of Intravenous Administration of U-50488 and Morphine Gastric
Emptying
a Standard
:
1
of Liquid Meal in Dogs” Gastric.
l
20. I
.\’ IO.
IO
0!
0. 0
001 0
Liquids
01
mq/kq
q
0 Sdii
of liquids and solids i SD. n = 12; *p 5
It is believed that parenteral administration of morphine and its derivatives (12,14,15,19), as well (12-14), reduces as enkephalins and their analogues the gastric emptying of both liquids and solids in dogs and humans. The present experiments establish that the slowing effect of morphine is also observed for oral administration, whereas K-agonists administered orally are able to increase gastric emptying of the solid phase of a canned food meal-an effect that is not reproduced by parenteral administration. Furthermore, the K-agonists used herein also inhibit gastric emptying of liquids at systemically inactive
30_
20
P
emptying are mean
Discussion
1
Figurr
(
stimulatory effect on the evacuation of solids as well as their slowing effects on that of liquids (Table 2).
40
g
EMPTYING
INTRAVENOUSI I
p F +
GASTRIC
effects of orally administered U-50488. tifluadom, morphine, and DAGO on gastric meal in dogs (percentage of gastric emptying measured 1 h after feeding. values
0.01 l P
Olmq/kg
2. Comparative influence of oral and intravenous administration of I!-50488 on gastric emptying of liquids and solids of a standard meal in dogs, mean -t SD, n = 12; *p c 0.05, M’-test.
Saline (i.v.) U-50488 0.01 mgikg 0.1 mgikg Morphine 0.01 mgikg 0.1 mgikg
on
und Solid Phases of emptying
(‘36)”
Liquids
Solids
28.3 i- 4.8
27.4 + 6.1
27.5 + 3.5 20.2 -+ 1.6’
29.0 t 2.3 25.3 t 5.1
26.3 JT 2.6 13.2 5 1.3’
27.8 -+ 3.5 16.9 f 4.8’
“Values are mean + SD, n = 12. ” Measured 60 min after the meal. ’ Significantly different (p 5 0.05) from saline values (Wtest).
930
GUE ET AL.
2.
Table
GASTROENTEROLOGY
influence of Naloxone and MR-2266 Gastric
Emptying
of Liquid
and Solid
Against Phases
Effects of Orally Administered of a Standard Meal in Dogs” Gastric emptvinn
u-50488
Tifluadom
on
(%lh Liquid phase
Solid phase Vehicle (placebo)
and
Vol. 95. No. 4
Tifluadom (0.1 mgikg)
U-50488
(0.1 mgikg)
U-50488
Vehicle [placebo]
(0.1 mgikg)
Tifluadom (0.1 mg/kg)
Control
28.6
+ 3.6
39.6
!I 6.0”
42.8
_t 6.8’
27.1
? 6.6
12.6
+ 3.0’.
11.7
+ 3.1’:
Naloxone
30.6
?
5.4
30.8
2 5.1”
33.2
+ 2.4”
35.3
5 7.0
27.8
+- 6.1d
29.5
i
30.8
+ 6.3
29.6
2 5.4”
31.6
t 4.1”
31.9
2 5.6
34.0
?
27.6
+ 5.5”
(0.1
MR-2266 (0.1
3.7”
mgikg i.v.) 7.2”
mgikg i.v.)
’ Values are mean 2 SD, n = 12. ’ Measured control values, respectively (W-test).
60 min after the meal.
doses. These results suggest that local opioid mechanisms are involved in the control of gastric emptying. Opiate receptors of different classes are present in gastric and intestinal walls (20,21), in the myenteric plexus (22,23), and in the gastric smooth muscle (24). This is true particularly in the dog, a species presenting nerve cells containing opiate enkephalin and dynorphin peptide immunoreactive materials in the submucosal plexus, muscularis mucosae, and villi of the stomach, pylorus, and proximal intestine (16). These results support the hypothesis that endogenous or exogenous opioid peptides of different classes may have different effects on the gastric motor activity, and in turn the gastric emptying via local sites of action. In agreement with such a hypothesis, it has been demonstrated that the antral motility was inhibited by p- and S-agonists and stimulated by K-agonists in fed sheep (25). A local action has also been previously suspected for other opiate drugs. For example, it has been shown that loperamide infused into the lumen in very small amounts inhibits colonic motility in humans (26). Furthermore, intraduodenal loperamide stimulates jejunal and colonic contractions in the calf, whereas opposite effects are observed for the same dose given subcutaneously (27). Recently, we have also demonstrated that K-agonists given orally are able to prevent the gastric motor disturbances induced by acoustic stress, whereas they are inactive by the systemic route (17). However, the most striking result is the opposite effects on gastric emptying of liquids and solids induced by U-50488 and tifluadom. Tifluadom and U-50488 are considered to be highly potent and selective K-agonists with minimal p-agonist action in vivo (28,29). Blockade of these effects by both naloxone, a nonselective opiate antagonist, and MR 2266, a selective antagonist of K-receptors (30), strongly suggests that their effects are due to a stimulation of local K-receptors. In agreement with the hypothesis of a local effect of K-agonists, an in
c,d Significantly
(p 5 0.05)
different
from corresponding
vehicle
and
vitro study has shown that K-receptor activation from a mucosal site depresses neurotransmitter release in myenteric plexus (31). The effect of K-agonists on gastric emptying of solids can be related to their stimulatory effects on distal stomach motility, but no evident explanation can be given about their inhibitory effects on gastric emptying of liquids. Parenteral administration of morphine, which delays gastric emptying of a liquid test meal, induces gastric relaxation in the dog (321, as do Leu- and Met-enkephalin in the cat (33). Our observation that K-agonists delay gastric emptying of liquids may be indirect evidence for induction of fundic relaxation. The possible involvement of duodenal motor stimulation by K-agonists (25), acting as a pressure barrier to reduce the gastric evacuation of liquids as for morphine (l5), cannot be easily accepted as enkephalins, which decrease the postprandial spike activity of the duodenum (34), also reduce gastric emptying of liquids (12,141. Furthermore, in dogs, orally administered K-agonists at the doses used herein do not affect the postprandial duodenal motility (unpublished observations), confirming previous data showing that ketazocine and dynorphinel_,, failed to affect gut transit in mice (35). Opioid substances are known to affect gastric acid secretion in dogs (34,363, interacting by this way on gastric emptying. However, K-agonists do not seem to alter gastric secretion in dogs (37). Consequently, the most probable hypothesis to explain the selective inhibitory influence of orally administered U-50488 and tifluadom on gastric emptying of liquids is a potentiation of feeding-induced gastric relaxation, but an inhibitory effect on opening of t’le pylorus cannot be excluded. Finally, the present work establishes that K-opioid agonists given orally affect the gastric emptying of a standard meal in dogs with a discriminative effect on solid and liquid phases. These effects, observed only for oral administration, support the hypothesis of a selective stimulation of mucosal or submucosal K-
October
1988
receptors of the gastroduodenal area affecting both fundic and pyloric motor activity. References RW. The physiology and pathophysiol1. Minami H, McCallum ogy of gastric emptying in humans. Gastroenterology 1984:86: 1592-610. 2. Wilbur BG, Kelly KA. Effect of proximal gastric, complete gastric and truncal vagotomy on canine gastric electric activity, motility and emptying. Ann Surg 1973;178:295-303. Stand J Gastroenterol 1977;12 3. Jahnberg T. Gastric relaxation. (Suppl):l-32. 0, Brandsborg M, Lovgreen NA, et al. Influence of 4. Brandsborg parietal cell vagotomy and selective gastric vagotomy on gastric emptying rate and serum gastrin concentration. Gastroenterology 1977;72:212-4. pressures, 5. Quigley JP, Brody DA. Digestive tract: intraluminal gastrointestinal propulsion, gastric evacuation, pressure-wall tension relationship. In: Glasser 0. ed. Medical physics. Chicago: Year Book Medical Publishers, 1950:280-92. JB, Cohen MB, Schadchehr A, Mandiola 6. Meyer JE, Thomson SA. Sieving of solid food by the canine stomach and sieving after gastric surgery. Gastroenterology 1979;76:804-13. 7. Becker JM, Kelly KA. Antral control of canine gastric emptying of solids. Am J Physiol 1983;245:G334-8. SA, Fraas C. Hart1 A. Cisapride offsets 8 Muller-Lissner dopamine-induced slowing of fasting gastric emptying. Dig Dis Sci 1986;31:807-10. lJT, Code CF. Grossman MI. Effect of gastrin on 9. Strunz electrical activity of antrum and duodenum of dogs. Proc Sot Exp Biol Med 1979;161:25-7. L. Effects of particle 10. ltoh T. Higuchi T, Gardner CK, Caldwell size and food on gastric residence time of non-disintegrating solids in beagle dogs. J Pharm Pharmacol 1986;38:801-6. of polycarbophil: an 11. Russel J. Bass P. Canine gastric emptying indigestible, particulate substance. Gastroenterology 1985;86: 307-12. A, Woussen-Colle MC, De Graef J. Effects of 12. Kostritsky-Pereira morphine, enkephalins and naloxone on postprandial gastric acid secretion, gastric emptying and gastrin release in dogs. Arch lnt Physiol Biochim 1984;92:19-26. PT. Adams N, Arnold J, Dubois A. Effects of 13 Shea-Donohue Met-enkephalin and naloxone on gastric emptying and secretion in rhesus monkeys. Am J Physiol 1983;245:G196-200, SN. Lamki L, Corcoran P. Inhibition of gastric 14. Sullivan emptying by enkephalin analogue. Lancet 1981;ii:86-7. RW. Effects of 15. Mittal RK, Frank EB, Lange RC, McCallum morphine and naloxone on esophageal motility and gastric emptying in man. Dig Dis Sci 1986;31:936-42. HD, Ahmad S, Kostolanska F. 16. Daniel I-E. Fox JET. Allescher Peripheral actions of opiates in canine gastrointestinal tract: at.tions on nerves and muscles. Gastroenterol Clin Biol 1987; 11:35-9. 17. Gue M, Pascaud X, Honde C, Junien JL, Buena L. Peripheral antagonistic action of trimebutine and kappa opioid substances on acoustic stress induced gastric motor inhibition in dogs. Em J Pharmacol 1988:146:57-63. J, Buena L. A simple double radiolabeled 18. Gue M. Fioramonti technique to evaluate gastric emptying of canned food meal in dogs: application to pharmacological tests. Gastroenterol Clin Biol 1988;12:425-30. ML, Donner MW. Morphine in the evaluation of 19. Silbiger gastrointestinal diseases. Radiology 1968;90:1086-90. 20. Costa Ivl, Furness JB, Gibbins IL, Murphy R. Chemical coding and projections of opioid peptide containing neurons in the guinea-pig intestine. Neurosci Lett 1985;S19-55.
K-AGONISTS
AND GASTRIC
EMPTYING
931
M, Kokfelqt T, Nilssen C, et al. Distribution of 21. Schultzberg peptide and catechol-containing neurones in the gastrointes tinal tract of rat and guinea pig: immuno-histochemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin, gastrinicholecystokinin, neurotensin and dopamine P-hydroxylase. Neuroscience 1980;5:689-95. 22. Morita K. North RA. Opiate activation of potassium conductance in myenteric neurons: inhibition by calcium ion. Brain Res 1982;242:145-51. 23. Nishimura E, Buchan AMJ, McIntosh CHS. Autoradiographic localization of mu- and sigma-type opioid receptors in the gastrointestinal tract of the rat and guinea pig. Gastroenterology 1986;91:1084-94. 24. Bitar KN, Makhlouf GM. Specific opiate receptors on isolated mammalian gastric smooth muscle cells. Nature 1983;297:724. 25. Ruckebusch Y. Bardon T, Pairet M. Opioid control of the ruminant stomach motility: functional importance of mu, kappa and sigma receptor. Life Sci 1984;35:1731-8. 26. Altaparmakov I, Wienbeck M. Local inhibition of myoelectrical activity of human colon by loperamide. Dig Dis Sci 1984; 29:232-7. 27. Fioramonti J, Buena L. Effects of loperamide hydrochloride on experimental diarrhea and gastrointestinal myoelectrical activity in calves. Am J Vet Res 1987:48:415-g. 28. Roemer D, Buscher HH, Hill RC, et al. An opioid benzodiazepine. Nature 1982;298:759-60. 29. Wood PL. Kappa agonist analgesics: evidence for mu2 and delta opioid receptor antagonism. Drug Dev Res 1984;4:429. 30. Magnan J, Paterson SJ, Tavani A, Kosterlitz HW. The binding spectrum of narcotic analgesic drugs with different agonist and antagonist properties. Naunyn Schmiedebergs Arch Pharmacol 1982;319:197-205. 31. Cherubini E, North RA. p and K opioids inhibit transmitter release by different mechanisms. Proc Nat1 Acad Sci USA 1985;82:1860-3. 32. Lefebvre RA, Willems JL, Bogaert MG. Gastric relaxation and vomiting by apomorphine, morphine and fentanyl in the conscious dog. Eur J Pharmacol 1981;69:139-45. 33. Edin R, Lunderg L, Terenius A. et al. Evidence for vagal enkephalinergic neural control of the feline pylorus and stomach. Gastroenterology 1980;78:492-7. 34. Konturek SJ, Tasler J, Cieszkowski M, Mikos E, Coy DH, Schally AV. Comparison of methionine-enkephalin and morphine in the stimulation of gastric acid secretion in the dog. Gastroenterology 1980;78:294-300, 35. Porreca F, Galligan JJ, Burks TF. Central opioid receptor involvement in gastrointestinal motility. Trends in Pharmacological Sciences 1986:7:104-7. 36 Soldani G, Del Tacca M, Bernardini MC, Bardon T, Ruckebusch Y. Peripheral opioid receptors mediate gastrointestinal secretory and motor effects of dermorphin N-terminal tetrapeptide (NTT) in the dog. Neuropeptides 1987;10:67-76. 37. Bartolini D, Bernardini C, Del Tacca M, Soldani G. Mu and delta but not kappa opioid agonists mediate gastric secretory effects in the dog (abstr). Br J Pharmacol 1985;86:640.
Received October 15, 1987. Accepted May 13, 1988. Address requests for reprints to: Dr. L. Bueno, Department of Pharmacology, INRA, 180 Chemin de Tournefeuille, 31300 Toulouse, France. This work was supported in part by funds from INRA and Jouveinal Laboratories. The authors thank Mrs. C. Dargelos and C. Delrio for technical assistance and Mrs. C. Santamaria for typing the manuscript.
1988;95:927-31
Opposite Effects of K-Opioid Agonists on Gastric Emptying of Liquids and Solids in Dogs M. GUE, J. FIORAMONTI, and L. BUENO
C. HONDE, X. PASCAUD, J. L. JUNIEN,
Department of Pharmacology, Institut National de la Recherche Jouveinal Laboratoires, Fresnes, France
The influence of oral (p.0.) administration of K(U-50488, tifluadom) and EC- (morphine, DAGO) opioid substances on gastric emptying of liquids and solids in a standard canned dog food meal was evaluated using a double-radiolabeled technique in dogs fitted with gastric cannulas. One hour after feeding, 28.6% -+ 3.6% (mean f SD) of the solid phase and 27.1% 2 8.6% of the liquid phase of the meal had been emptied. Both U-50488 and tifluadom given orally (0.01-0.1 mg/kg) significantly increased (p < 0.05) the 1-h emptying of the solid phase of the meal by 23.10/a-49.6%. In contrast, both drugs significantly reduced emptying of liquids. These effects were not reproduced when similar doses were given intravenously. Oral administramglkg) did not tion of morphine or DAGO (0.01-0.1 affect gastric emptying, whereas an inhibited emptying of solids was observed for morphine at a higher dose (1mg/kg p.0.). At a dose of 100 pg/kg i.v. both naloxone and MR 2266 (0.1 mg/kg) abolished the effects of orally administered U-50488 on gastric emptying of solids and liquids. It is concluded that Kbut not Ec_agonists act locally to alter gastric emptying of a standard meal in dogs, having opposite effects on solid and liquid phases. A selective local stimulation of K mucosal or submucosal receptors of the gastroduodenal area may explain such effects.
G
astric emptying depends on the motor activity of two distinct portions of the digestive tract: (a) the lower esophageal sphincter and the proximal stomach (fundus and corpus) and (b) the antropyloroduodenal area (1). It is classically believed that the proximal stomach controls liquid emptying, which may be directly related to the amplitude of the slow sustained contractions and the relaxation following a meal (Z-4). In contrast, gastric emptying of solids
Agronomique,
Toulouse,
and
may result from (a) the rhythmic peristaltic waves of the distal stomach (5--7), (b) the opening of the pylorus (8) affected by digestive hormonal response (1,9), and (c) the size of particles (10,ll). Opiates have long been known to delay gastric emptying of both liquids and solids when systemically administered in dogs, monkeys, and humans (12-14). Although the peripheral and central action of S, p-, and K-agonists on gastric and intestinal motility may be different, their systemic administration usually results in a reduction in gastric emptying; furthermore, these endogenous opiates appear to be involved in the regulation of gastric emptying, as suggested by the tendency of naloxone to increase gastric evacuation of a solid meal in humans (15). Histologic studies performed in dogs have revealed the presence of nerve cells containing enkephalin and dynorphin peptide immunoreactive materials in the submucosal plexus, muscularis mucosae, and villi of the stomach, pylorus, and proximal intestine (16). These observations led us to speculate that opiate-sensitive fibers located at gastric and duodenal levels may influence gastric emptying as well as gastric motor patterns (17). Consequently, the present experiments were conducted to evaluate the influence of orally administered p- and K-agonists on the gastric emptying of the liquid and solid phases of a spontaneously eaten standard (canned food] meal in dogs using a doubleradiolabeling technique (18). Materials
and Methods
Materials were
Six female adult mongrel dogs weighing 12-16 kg used for the experiments. A Thomas cannula was 0 1988 by the American
Gastroenterological 0018-5085/88/$3.50
Association
928
GUE ET AL.
GASTROENTEROLOGY
placed on the greater curvature of the gastric body under halothane (Fluothane, Coopers, Maux, France) anesthesia. The cannula was positioned -10 cm from the pylorus, and was brought through and fixed to the left abdominal wall 5 cm from the last rib and 10 cm from the midline. Animals were allowed 2 wk to recover before testing was begun. The solid phase of the meal consisted of 400 g of canned food (Fiddle, Quaker, Bordeaux, France) containing 21.7% dry matter, 7.7% protein, 4.5% fat, 6.9% carbohydrates, and 2.6% minerals. Sheep liver (20 g) labeled with [57Co]cyanocobalamine was mixed with the canned food (solid phase). Labeled liver was obtained from sheep given an i.v. injection of 10 &i of [57Co]cyanocobalamine (Amersham, Paris, France, sp act 10 &i/pg) and killed 24 h later. One hundred milliliters of tap water (liquid phase) containing 50 mg of polyethylene glycol4OOO (PEG), a nonabsorbable water marker, with 0.5 &i of [14C]PEG (NEN Research Products; sp act, 1 &ilmg) was added to the solid meal before presenting this test meal to the animals. The meal was eaten in <5 min. Total collection of the gastric contents was performed 1 h after the meal. The collected contents were homogenized after determination of the total weight and volume. Two series of five samples (4-5 ml) were rapidly taken; each sample of the first series was weighed and “7Co was counted using a y-counter (MR252; Kontron, Switzerland). The second series was prepared for [14C]PEG determination using a liquid scintillation system (SL 40; Intertechnique, Plaisir, France). The percentages of gastric emptying of liquid and solid phases were calculated from these (18), using the measurements as previously described formula: Esp = [(C,W,
- C,,,W,,,)
x loo]lCLWL,
where ESP designates the emptying of solid phase (expressed as a percentage), CL is counts per minute per gram of marked liver introduced into the meal, WL is the weight of the liver introduced into the meal, Ccct is counts per minute per gram of homogenized gastric contents at time t, and WcCt is the weight of the contents present in the stomach at time t. and
where ELP designates the emptying of liquid phase (expressed as a percentage), [[‘4C]PEGLp& is the initial concentration of the liquid phase of the meal, Vwi is the initial volume of water added, [[r4C]PEGnMt] is the concentration of homogenized meal at time t, and Vu,, is the volume of the homogenized meal at time t.
Experimental
Procedure
First series of experiments. Twenty minutes before the test meal the animals received orally, at s-day intervals and in random fashion, morphine or tifluadom (0.01, 0.1, and 1 mg/kg) and U-50488 or (o-Ala’, N-methyl-Phe4, Gly’-01) enkephalin (DAGO) (0.01 and 0.1 mgikg). Second series of experiments. Ten minutes before
Vol
95, No.
4
feeding U-50488 and morphine were injected intravenously at doses of 0.01 and 0.1 mgikg. Third series of experiments. The oral administration of U-50488 or tifluadom at a dose of 0.1 mgikg was preceded (10 min) by the intravenous injection of naloxone (0.1 mgikg) or MR 2266 (0.1 mgikg). Morphine, DAGO, and U-50488 were purchased from Cooperative Pharmaceutique Francaise (Brive, France), Sigma Chemical Co. (St. Louis, MO.), and Upjohn Diagnostics [Kalamazoo, Mich.), respectively. Tifluadom, naloxone, and MR 2266 were gifts from Sandoz (Basel, Switzerland), Endo Laboratories (Garden City, N.Y.), and Boehringer Ingelheim (Bracknell, U.K.), respectively. All experiments were performed in duplicate on each subject, and gastric emptying of liquids and solids was evaluated 1 h after the ingestion of the meal. Gastric emptying was expressed as the percentage of the meal emptied for both liquids and solids, as previously described (18). Because of the small number of animals (n = 6), statistical analysis of the results was performed using a Wilcoxon matched-pairs signed-rank test and differences were considered significant for p or 0.05. Results were expressed as the mean I SD.
Results Influence of Oral Versus P-Agonists
Administration
of
K-
When measured 1 h after feeding under control (placebo) conditions, the gastric emptying of solids represented 28.6% ? 3.6% (mean + SD, n = 12) of the initial weight of the solid phase; at the same time the volume of liquid emptied was 27.1% ? 8.6% of the initial volume, i.e., 100 ml (Figure 1). The larger standard deviation for emptying rates of liquids compared with that observed for solids may be related to great variations in the total recovery of 14C, as previously reported (18). When given orally at doses of 10 and 100 pgikg, both U-50488 and tifluadom significantly (p < 0.01) increased the percentage of the solid phase emptied after 1 h, whereas a significant (p < 0.02) reduction in the amount of liquid emptied was detected (Figure 1). At a higher oral dose [l mg/kg), tifluadom still enhanced the gastric evacuation of solids but did not affect (p > 0.05) that of liquids. In contrast, neither morphine nor DAGO given orally at similar doses (10 and 100 pgikg] affected gastric emptying of liquids and solids. At a dose of 1 mg/kg, morphine significantly (p < 0.05) reduced emptying of solids by >50%. Comparative Administration
Effects
of Systemic
When injected intravenously 10 min before feeding, U-50488 at doses of 10 and 100 pglkg did not affect the 1-h gastric emptying of solids, whereas
Or,tober
K-AGONISTS
1988
AND GASTRIC
EMPTYLNG
929
1SOLIDS]
TIFLUADOM---
0.01 0.1
,,~O____-__-
X ITigure
1. Comparative
of a standard 0.05. W-test).
slight (28.6%) but significant (p < 0.05) inhibition of liquids was observed at the higher dose (Figure 2). Morphine injected intravenously at doses of 100 ,ug/kg significantly (p < 0.05) inhibited gastric emptying of liquids and solids (Table l), but the lower dose (10 pgikg) was not effective. a
Influence
of Naloxone
and
MR 2266
Naloxone and MR 2266 administered intravenously (0.1 mgikg) 40 min before the meal had no effect per se on the 1-h values of gastric emptying of liquids and solids. When injected 10 min before oral U-50488 (0.1 mgikg) or tifluadom (0.1 mgikg), both naloxone and MR 2266 completely abolished their 50
50
1
40.
B
30
:, 9
l
Table
1. Comparative Influence of Intravenous Administration of U-50488 and Morphine Gastric
Emptying
a Standard
:
1
of Liquid Meal in Dogs” Gastric.
l
20. I
.\’ IO.
IO
0!
0. 0
001 0
Liquids
01
mq/kq
q
0 Sdii
of liquids and solids i SD. n = 12; *p 5
It is believed that parenteral administration of morphine and its derivatives (12,14,15,19), as well (12-14), reduces as enkephalins and their analogues the gastric emptying of both liquids and solids in dogs and humans. The present experiments establish that the slowing effect of morphine is also observed for oral administration, whereas K-agonists administered orally are able to increase gastric emptying of the solid phase of a canned food meal-an effect that is not reproduced by parenteral administration. Furthermore, the K-agonists used herein also inhibit gastric emptying of liquids at systemically inactive
30_
20
P
emptying are mean
Discussion
1
Figurr
(
stimulatory effect on the evacuation of solids as well as their slowing effects on that of liquids (Table 2).
40
g
EMPTYING
INTRAVENOUSI I
p F +
GASTRIC
effects of orally administered U-50488. tifluadom, morphine, and DAGO on gastric meal in dogs (percentage of gastric emptying measured 1 h after feeding. values
0.01 l P
Olmq/kg
2. Comparative influence of oral and intravenous administration of I!-50488 on gastric emptying of liquids and solids of a standard meal in dogs, mean -t SD, n = 12; *p c 0.05, M’-test.
Saline (i.v.) U-50488 0.01 mgikg 0.1 mgikg Morphine 0.01 mgikg 0.1 mgikg
on
und Solid Phases of emptying
(‘36)”
Liquids
Solids
28.3 i- 4.8
27.4 + 6.1
27.5 + 3.5 20.2 -+ 1.6’
29.0 t 2.3 25.3 t 5.1
26.3 JT 2.6 13.2 5 1.3’
27.8 -+ 3.5 16.9 f 4.8’
“Values are mean + SD, n = 12. ” Measured 60 min after the meal. ’ Significantly different (p 5 0.05) from saline values (Wtest).
930
GUE ET AL.
2.
Table
GASTROENTEROLOGY
influence of Naloxone and MR-2266 Gastric
Emptying
of Liquid
and Solid
Against Phases
Effects of Orally Administered of a Standard Meal in Dogs” Gastric emptvinn
u-50488
Tifluadom
on
(%lh Liquid phase
Solid phase Vehicle (placebo)
and
Vol. 95. No. 4
Tifluadom (0.1 mgikg)
U-50488
(0.1 mgikg)
U-50488
Vehicle [placebo]
(0.1 mgikg)
Tifluadom (0.1 mg/kg)
Control
28.6
+ 3.6
39.6
!I 6.0”
42.8
_t 6.8’
27.1
? 6.6
12.6
+ 3.0’.
11.7
+ 3.1’:
Naloxone
30.6
?
5.4
30.8
2 5.1”
33.2
+ 2.4”
35.3
5 7.0
27.8
+- 6.1d
29.5
i
30.8
+ 6.3
29.6
2 5.4”
31.6
t 4.1”
31.9
2 5.6
34.0
?
27.6
+ 5.5”
(0.1
MR-2266 (0.1
3.7”
mgikg i.v.) 7.2”
mgikg i.v.)
’ Values are mean 2 SD, n = 12. ’ Measured control values, respectively (W-test).
60 min after the meal.
doses. These results suggest that local opioid mechanisms are involved in the control of gastric emptying. Opiate receptors of different classes are present in gastric and intestinal walls (20,21), in the myenteric plexus (22,23), and in the gastric smooth muscle (24). This is true particularly in the dog, a species presenting nerve cells containing opiate enkephalin and dynorphin peptide immunoreactive materials in the submucosal plexus, muscularis mucosae, and villi of the stomach, pylorus, and proximal intestine (16). These results support the hypothesis that endogenous or exogenous opioid peptides of different classes may have different effects on the gastric motor activity, and in turn the gastric emptying via local sites of action. In agreement with such a hypothesis, it has been demonstrated that the antral motility was inhibited by p- and S-agonists and stimulated by K-agonists in fed sheep (25). A local action has also been previously suspected for other opiate drugs. For example, it has been shown that loperamide infused into the lumen in very small amounts inhibits colonic motility in humans (26). Furthermore, intraduodenal loperamide stimulates jejunal and colonic contractions in the calf, whereas opposite effects are observed for the same dose given subcutaneously (27). Recently, we have also demonstrated that K-agonists given orally are able to prevent the gastric motor disturbances induced by acoustic stress, whereas they are inactive by the systemic route (17). However, the most striking result is the opposite effects on gastric emptying of liquids and solids induced by U-50488 and tifluadom. Tifluadom and U-50488 are considered to be highly potent and selective K-agonists with minimal p-agonist action in vivo (28,29). Blockade of these effects by both naloxone, a nonselective opiate antagonist, and MR 2266, a selective antagonist of K-receptors (30), strongly suggests that their effects are due to a stimulation of local K-receptors. In agreement with the hypothesis of a local effect of K-agonists, an in
c,d Significantly
(p 5 0.05)
different
from corresponding
vehicle
and
vitro study has shown that K-receptor activation from a mucosal site depresses neurotransmitter release in myenteric plexus (31). The effect of K-agonists on gastric emptying of solids can be related to their stimulatory effects on distal stomach motility, but no evident explanation can be given about their inhibitory effects on gastric emptying of liquids. Parenteral administration of morphine, which delays gastric emptying of a liquid test meal, induces gastric relaxation in the dog (321, as do Leu- and Met-enkephalin in the cat (33). Our observation that K-agonists delay gastric emptying of liquids may be indirect evidence for induction of fundic relaxation. The possible involvement of duodenal motor stimulation by K-agonists (25), acting as a pressure barrier to reduce the gastric evacuation of liquids as for morphine (l5), cannot be easily accepted as enkephalins, which decrease the postprandial spike activity of the duodenum (34), also reduce gastric emptying of liquids (12,141. Furthermore, in dogs, orally administered K-agonists at the doses used herein do not affect the postprandial duodenal motility (unpublished observations), confirming previous data showing that ketazocine and dynorphinel_,, failed to affect gut transit in mice (35). Opioid substances are known to affect gastric acid secretion in dogs (34,363, interacting by this way on gastric emptying. However, K-agonists do not seem to alter gastric secretion in dogs (37). Consequently, the most probable hypothesis to explain the selective inhibitory influence of orally administered U-50488 and tifluadom on gastric emptying of liquids is a potentiation of feeding-induced gastric relaxation, but an inhibitory effect on opening of t’le pylorus cannot be excluded. Finally, the present work establishes that K-opioid agonists given orally affect the gastric emptying of a standard meal in dogs with a discriminative effect on solid and liquid phases. These effects, observed only for oral administration, support the hypothesis of a selective stimulation of mucosal or submucosal K-
October
1988
receptors of the gastroduodenal area affecting both fundic and pyloric motor activity. References RW. The physiology and pathophysiol1. Minami H, McCallum ogy of gastric emptying in humans. Gastroenterology 1984:86: 1592-610. 2. Wilbur BG, Kelly KA. Effect of proximal gastric, complete gastric and truncal vagotomy on canine gastric electric activity, motility and emptying. Ann Surg 1973;178:295-303. Stand J Gastroenterol 1977;12 3. Jahnberg T. Gastric relaxation. (Suppl):l-32. 0, Brandsborg M, Lovgreen NA, et al. Influence of 4. Brandsborg parietal cell vagotomy and selective gastric vagotomy on gastric emptying rate and serum gastrin concentration. Gastroenterology 1977;72:212-4. pressures, 5. Quigley JP, Brody DA. Digestive tract: intraluminal gastrointestinal propulsion, gastric evacuation, pressure-wall tension relationship. In: Glasser 0. ed. Medical physics. Chicago: Year Book Medical Publishers, 1950:280-92. JB, Cohen MB, Schadchehr A, Mandiola 6. Meyer JE, Thomson SA. Sieving of solid food by the canine stomach and sieving after gastric surgery. Gastroenterology 1979;76:804-13. 7. Becker JM, Kelly KA. Antral control of canine gastric emptying of solids. Am J Physiol 1983;245:G334-8. SA, Fraas C. Hart1 A. Cisapride offsets 8 Muller-Lissner dopamine-induced slowing of fasting gastric emptying. Dig Dis Sci 1986;31:807-10. lJT, Code CF. Grossman MI. Effect of gastrin on 9. Strunz electrical activity of antrum and duodenum of dogs. Proc Sot Exp Biol Med 1979;161:25-7. L. Effects of particle 10. ltoh T. Higuchi T, Gardner CK, Caldwell size and food on gastric residence time of non-disintegrating solids in beagle dogs. J Pharm Pharmacol 1986;38:801-6. of polycarbophil: an 11. Russel J. Bass P. Canine gastric emptying indigestible, particulate substance. Gastroenterology 1985;86: 307-12. A, Woussen-Colle MC, De Graef J. Effects of 12. Kostritsky-Pereira morphine, enkephalins and naloxone on postprandial gastric acid secretion, gastric emptying and gastrin release in dogs. Arch lnt Physiol Biochim 1984;92:19-26. PT. Adams N, Arnold J, Dubois A. Effects of 13 Shea-Donohue Met-enkephalin and naloxone on gastric emptying and secretion in rhesus monkeys. Am J Physiol 1983;245:G196-200, SN. Lamki L, Corcoran P. Inhibition of gastric 14. Sullivan emptying by enkephalin analogue. Lancet 1981;ii:86-7. RW. Effects of 15. Mittal RK, Frank EB, Lange RC, McCallum morphine and naloxone on esophageal motility and gastric emptying in man. Dig Dis Sci 1986;31:936-42. HD, Ahmad S, Kostolanska F. 16. Daniel I-E. Fox JET. Allescher Peripheral actions of opiates in canine gastrointestinal tract: at.tions on nerves and muscles. Gastroenterol Clin Biol 1987; 11:35-9. 17. Gue M, Pascaud X, Honde C, Junien JL, Buena L. Peripheral antagonistic action of trimebutine and kappa opioid substances on acoustic stress induced gastric motor inhibition in dogs. Em J Pharmacol 1988:146:57-63. J, Buena L. A simple double radiolabeled 18. Gue M. Fioramonti technique to evaluate gastric emptying of canned food meal in dogs: application to pharmacological tests. Gastroenterol Clin Biol 1988;12:425-30. ML, Donner MW. Morphine in the evaluation of 19. Silbiger gastrointestinal diseases. Radiology 1968;90:1086-90. 20. Costa Ivl, Furness JB, Gibbins IL, Murphy R. Chemical coding and projections of opioid peptide containing neurons in the guinea-pig intestine. Neurosci Lett 1985;S19-55.
K-AGONISTS
AND GASTRIC
EMPTYING
931
M, Kokfelqt T, Nilssen C, et al. Distribution of 21. Schultzberg peptide and catechol-containing neurones in the gastrointes tinal tract of rat and guinea pig: immuno-histochemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin, gastrinicholecystokinin, neurotensin and dopamine P-hydroxylase. Neuroscience 1980;5:689-95. 22. Morita K. North RA. Opiate activation of potassium conductance in myenteric neurons: inhibition by calcium ion. Brain Res 1982;242:145-51. 23. Nishimura E, Buchan AMJ, McIntosh CHS. Autoradiographic localization of mu- and sigma-type opioid receptors in the gastrointestinal tract of the rat and guinea pig. Gastroenterology 1986;91:1084-94. 24. Bitar KN, Makhlouf GM. Specific opiate receptors on isolated mammalian gastric smooth muscle cells. Nature 1983;297:724. 25. Ruckebusch Y. Bardon T, Pairet M. Opioid control of the ruminant stomach motility: functional importance of mu, kappa and sigma receptor. Life Sci 1984;35:1731-8. 26. Altaparmakov I, Wienbeck M. Local inhibition of myoelectrical activity of human colon by loperamide. Dig Dis Sci 1984; 29:232-7. 27. Fioramonti J, Buena L. Effects of loperamide hydrochloride on experimental diarrhea and gastrointestinal myoelectrical activity in calves. Am J Vet Res 1987:48:415-g. 28. Roemer D, Buscher HH, Hill RC, et al. An opioid benzodiazepine. Nature 1982;298:759-60. 29. Wood PL. Kappa agonist analgesics: evidence for mu2 and delta opioid receptor antagonism. Drug Dev Res 1984;4:429. 30. Magnan J, Paterson SJ, Tavani A, Kosterlitz HW. The binding spectrum of narcotic analgesic drugs with different agonist and antagonist properties. Naunyn Schmiedebergs Arch Pharmacol 1982;319:197-205. 31. Cherubini E, North RA. p and K opioids inhibit transmitter release by different mechanisms. Proc Nat1 Acad Sci USA 1985;82:1860-3. 32. Lefebvre RA, Willems JL, Bogaert MG. Gastric relaxation and vomiting by apomorphine, morphine and fentanyl in the conscious dog. Eur J Pharmacol 1981;69:139-45. 33. Edin R, Lunderg L, Terenius A. et al. Evidence for vagal enkephalinergic neural control of the feline pylorus and stomach. Gastroenterology 1980;78:492-7. 34. Konturek SJ, Tasler J, Cieszkowski M, Mikos E, Coy DH, Schally AV. Comparison of methionine-enkephalin and morphine in the stimulation of gastric acid secretion in the dog. Gastroenterology 1980;78:294-300, 35. Porreca F, Galligan JJ, Burks TF. Central opioid receptor involvement in gastrointestinal motility. Trends in Pharmacological Sciences 1986:7:104-7. 36 Soldani G, Del Tacca M, Bernardini MC, Bardon T, Ruckebusch Y. Peripheral opioid receptors mediate gastrointestinal secretory and motor effects of dermorphin N-terminal tetrapeptide (NTT) in the dog. Neuropeptides 1987;10:67-76. 37. Bartolini D, Bernardini C, Del Tacca M, Soldani G. Mu and delta but not kappa opioid agonists mediate gastric secretory effects in the dog (abstr). Br J Pharmacol 1985;86:640.
Received October 15, 1987. Accepted May 13, 1988. Address requests for reprints to: Dr. L. Bueno, Department of Pharmacology, INRA, 180 Chemin de Tournefeuille, 31300 Toulouse, France. This work was supported in part by funds from INRA and Jouveinal Laboratories. The authors thank Mrs. C. Dargelos and C. Delrio for technical assistance and Mrs. C. Santamaria for typing the manuscript.