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Stereospecific inhibition of gastrointestinal transit by κ opioid agonists in mice
European Journal of Pharmacology, 155 (1988) 329-331
329
Elsevier EJP 20225
Short communication
Stereospecific inhibition of gastrointestinal transit by x opioid agonists in mice K r i s h n a s w a m i R a m a b a d r a n *, M y l a r r a o Bansinath, H e r m a n T u r n d o r f a n d M a r g a r i t a M. Puig Department of Anesthesiology, School of Medicine, New York University Medical Center, 550 First Avenue, New York, N Y 10016, U.S.A. Received 5 August 1988, accepted 16 August 1988
Systemic administration of highly selective r opiate agonists, U-50488H and U-69593 (3, 10 and 30 mg/kg i.p.) produced significant inhibition of the gastrointestinal transit in mice as assessed by charcoal meal test. In contrast, the (+) stereoisomer of U-50488H, U-53455E did not inhibit the gastrointestinal motility. Furthermore, the r-selective antagonist, Mr 2266 (3 mg/kg) when administered along with the agonists, reversed the effects of the agonists. These results suggest that stereospecific r opiate receptors are involved in inhibition of gut motility in mice. ~¢Opioid agonists; Gastrointestinal transit; U-50488H; U69593; (Stereospecificity, Mouse)
1. Introduction
Analysis of the properties and functions of opiate receptors is complicated by the existence of multiple receptor types and differential distribution and function of these receptor subtypes in different species. The stereospecificity of opiate agonists as well as the blockade by antagonists other than naloxone should be shown in order to confirm the type of opiate receptor for a given pharmacodynamic response (Lesfie, 1987). Although # receptor involvement in the mechanism of opiate-induced inhibition of gastrointestinal transit is well accepted, the implications of receptors remain controversial. U-50488H and U69593 are among the most selective x opioid agonists available to date (Lahti et al., 1985). These compounds were shown not to inhibit gastrointestinal transit in rats and these results were interpreted as evidence to show that ~ opiate receptor subtypes are not involved in the inhibition of gastrointestinal transit (Galligan et al.,
* To whom all correspondence should be addressed.
1984; La Regina et al., 1988; Tavani et al., 1984). However, paucity of x opiate receptors in rats is known. In addition, inhibition of gastrointestinal transit by U-50488H in guinea pigs (CulpepperMorgan et al., 1988) implicate x opiate receptors in gut transit in guinea pigs. Mice have been extensively employed as experimental animals to study the effects of opiates. Species variation in the gastrointestinal effects of opiates is known (Manara and Bianchetti, 1985). In mice, U-50488H has been shown to lack antitransit effect when administered either by intrathecal or i.c.v, routes (Porreca et al., 1984). However, the effects of highly selective r agonists on gastrointestinal transit when administered by i.p. route in mice have not yet been explored. Furthermore, U-53455E, the stereoisomer of r opiate, U-50488H has been available only very recently, and its effect on gastrointestinal motility has not been reported in any of the species. Hence, the current study investigated the role of r receptors in the mechanisms involved in opioid-induced inhibition of intestinal propulsion by studying the pharmacological effects of U-50488H, U-53455E, U-69593 and these drugs in combination with the
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
330
r-selective antagonist, Mr 2266 in charcoal meal test in mice.
TABLE 1
2. Materials and methods
Treatment
Male Swiss Webster mice (25-30 g Taconic Farms, PA) were used after four days acclimation to the housing conditions and after 24 h starvation. During starvation, water was available ad libitum. All the drugs were freshly prepared and injected i.p. in a volume of 10 m l / k g simultaneously with the test meal. Animals in control group received vehicle injections. The animals were given 0.25 ml of a suspension of charcoal meal (10% charcoal in 5% gum acacia) and killed 45 min later. This time point was selected based on the results of peak time for the r agonist U-50488H (Ramabadran, 1983; 1984). The distance travelled by the charcoal meal as well as the total length of the intestine was measured (Bansinath et al., in press). Gastrointestinal transit was expressed as the percentage of the distance travelled by the charcoal relative to the total length of the small intestine. The data are represented as group means + S.E.M. Statistical significance was assessed by the analysis of variance (ANOVA) followed by post-hoc comparison using Student-NewmanKeuls or Student's t-test, as appropriate.
3. Results
The results indicating the stereospecificity of ant±transit effects of the x agonists used are summarized in table 1. Both U-50488H and U-69593 produced a significant inhibition of the gastrointestinal motility, whereas the ( + ) isomer of U50488H, U-53455E did not modify the gastrointestinal transit, indicating the stereospecificity of the K opiate effect on gut. Furthermore the xselective antagonist, Mr 2266 (3 m g / k g i.p.) when administered along with the agonists completely reversed the inhibitory effects of both agonists U-50488H, F(1,51) = 10.5, P < 0.002; U-69593, F(1,52) = 11.7, P < 0.001). The groupwise results indicating the effects of r agonists alone and their effects when administered along with Mr 2266 are
Stereospecific i n h i b i t i o n of i n t e s t i n a l m o t i l i t y b y K o p i a t e agonists in mice. D a t a represent % of charcoal m e a l transit, m e a n s ± S.E.M. n = 8-12 per t r e a t m e n t .
U-50488H U-53455E U-69593
Dose ( m g / k g i.p.)
ANOVA
3
10
30
F
P <
54±4 a 65±4 51 ± 3 a
50+5 a 66±2 44±4 a
47±5 a 69±4 42±2 a
5.6 0.4 11.2
0.004 0.8 0.0001
W h e n c o m p a r e d with vehicle-treated controls P < 0.05.
TABLE 2 Effect of K-selective a n t a g o n i s t , M r 2266 on the i n h i b i t i o n of i n t e s t i n a l m o t i l i t y i n d u c e d by x o p i a t e a g o n i s t s in mice. D a t a represent % of charcoal m e a l transit, m e a n s ± S.E.M. n = 8-12 per treatment. Treatment
Dose ( m g / k g i.p.) 3
10
30
55 + 3 57 + 4
53 + 2 53 + 2
49 + 4 41 ± 3
74+__5 a 63 ± 3 a
49±4 50 ± 3
Agonist alone U-50488H U-69593
With Mr 2266 (3 mg/kg) U-50488H U-69593
70±5 a 82 ± 5 a
W h e n c o m p a r e d with the respective c o n t r o l s (agonist alone) P < 0.05.
a
presented in table 2. The mean % of charcoal meal transit after Mr 2266 (3 m g / k g ) alone, was 69 + 3 and was not different as compared to the percentage of distance travelled (70 + 6) in the vehicle-treated controls.
4. Discussion
These results clearly demonstrate for the first time that stereospecific x receptors are involved in the inhibition of the gastrointestinal transit in mice. The drugs were administered by i.p. route because earlier studies have shown that this route is optimal for the effect of morphine on gut motility (Bianchi et al., 1983). In rats, the /~ agonist, levomethorphan as well as its dextro isomer, dextromethorphan have been shown to inhibit the gastrointestinal motility (Gaginella et al., 1987). In
331
contrast, present results with U-53455E indicate that dextroisomers of x agonists do not inhibit the gastrointestinal motility in mice. Opiates exert their effects on gastrointestinal motility both by central and peripheral mechanisms (Manara and Bianchetti, 1985). Discrepancy between the present results and the earlier report in mice using the x agonist U50488H (Porreca et al., 1984) may be due to differences in experimental details like route of administration, time course and methodology of measurement of transit. Hence, further studies to clarify the sites of action, viz. central and/peripheral for the observed inhibition of the gut transit produced by x-selective agonists in mice are clearly indicated.
Acknowledgements The authors thank Dr. P.F. Von Voigtlander (Upjohn Company, Kalamazoo, USA) and Dr. H. Merz (Boehringer-lngelheim, West Germany) for U-50488H, U-53455E, and U69593 and Mr 2266, respectively.
References Bansinath, M., K. Ramabadran, H. Turndorf and M.M. Puig, Effects of acute and chronic hyperglycemia on morphineinduced inhibition of gastrointestinal transit in mice, Pharmacology (in press). Bianchi, G., P. Ferretti, M. Recchia, M. Rocchetti, A. Tavani and L. Manara, 1983, Morphine tissue levels and reduction of gastrointestinal transit in rats: correlation supports primary action site in the gut, Gastroenterology 85, 852.
Culpepper-Morgan, J., M.J. Kreek, P.R. Holt, D. LaRoche, J. Zhang and L. O'Bryan, 1988, Orally administered x as well as mu opiate agonists delay gastrointestinal transit in the guinea pig, Life Sci. 42, 2073. Gaginella, T.S., R.J. Bertko and J.F. Kachur, 1987, Effect of dextromethorphan and levomethorphan on gastric emptying and intestinal transit in the rat, J. Pharmacol. Exp. Ther. 240, 388. Galligan, J.J., H.I. Mosberg, R. Hurst, V.J. Hruby and T.F. Burks, 1984, Cerebral delta opioid receptors mediate analgesia but not the intestinal motility effects of intracerebroventricularly administered opioids, J. Pharmacol. Exp. Ther. 229, 641. Lahti, R.A., M.M. Mickelson, J.M. Mccall and P.F. Von Voigtlander, 1985, [3H]U-69593 A highly selective ligand for the opioid x receptor, European J. Pharmacol. 109, 281. La Regina, A., P. Pertrillo, M. Sbacchi and A. Tavani, 1988, Interaction of U-69,593 with F-, 8- and r-opioid binding sites and its analgesic and intestinal effects in rat, Life Sci. 42, 293. Leslie, F.M., 1987, Methods used for the study of opioid receptors, Pharmacol. Rev. 39, 197. Manara, L. and A. Bianchetti, 1985, The central and peripheral influences of opioids on gastrointestinal propulsion, Ann. Rev. Pharmacol. Toxicol. 25, 249. Porreca, F., H.I. Mosberg, R. Hurst, V.J. Hruby and T.F. Burks, 1984, Roles of mu, delta and K opioid receptors in spinal and supraspinal mediation of gastrointestinal transit effects and hot-plate analgesia in the mouse, J. Pharmacol. Exp. Ther. 230, 341. Ramabadran, K., 1983, Hyperalgesic effect of a selective kappa agonist, U-50488H in mice, Jap. J. Pharmacol. 33, 1289. Ramabadran, K., 1984, Strain difference in the hyperalgesic effect of a novel kappa receptor agonist, U-50488H in mice, European J Pharmacol. 98, 425. Tavani, A., M.A. Gambino and P. Petrillo, 1984, The opioid kappa-selective compound U-50,488H does not inhibit intestinal propulsion in rats, J. Pharm. Pharmacol. 36, 343.
329
Elsevier EJP 20225
Short communication
Stereospecific inhibition of gastrointestinal transit by x opioid agonists in mice K r i s h n a s w a m i R a m a b a d r a n *, M y l a r r a o Bansinath, H e r m a n T u r n d o r f a n d M a r g a r i t a M. Puig Department of Anesthesiology, School of Medicine, New York University Medical Center, 550 First Avenue, New York, N Y 10016, U.S.A. Received 5 August 1988, accepted 16 August 1988
Systemic administration of highly selective r opiate agonists, U-50488H and U-69593 (3, 10 and 30 mg/kg i.p.) produced significant inhibition of the gastrointestinal transit in mice as assessed by charcoal meal test. In contrast, the (+) stereoisomer of U-50488H, U-53455E did not inhibit the gastrointestinal motility. Furthermore, the r-selective antagonist, Mr 2266 (3 mg/kg) when administered along with the agonists, reversed the effects of the agonists. These results suggest that stereospecific r opiate receptors are involved in inhibition of gut motility in mice. ~¢Opioid agonists; Gastrointestinal transit; U-50488H; U69593; (Stereospecificity, Mouse)
1. Introduction
Analysis of the properties and functions of opiate receptors is complicated by the existence of multiple receptor types and differential distribution and function of these receptor subtypes in different species. The stereospecificity of opiate agonists as well as the blockade by antagonists other than naloxone should be shown in order to confirm the type of opiate receptor for a given pharmacodynamic response (Lesfie, 1987). Although # receptor involvement in the mechanism of opiate-induced inhibition of gastrointestinal transit is well accepted, the implications of receptors remain controversial. U-50488H and U69593 are among the most selective x opioid agonists available to date (Lahti et al., 1985). These compounds were shown not to inhibit gastrointestinal transit in rats and these results were interpreted as evidence to show that ~ opiate receptor subtypes are not involved in the inhibition of gastrointestinal transit (Galligan et al.,
* To whom all correspondence should be addressed.
1984; La Regina et al., 1988; Tavani et al., 1984). However, paucity of x opiate receptors in rats is known. In addition, inhibition of gastrointestinal transit by U-50488H in guinea pigs (CulpepperMorgan et al., 1988) implicate x opiate receptors in gut transit in guinea pigs. Mice have been extensively employed as experimental animals to study the effects of opiates. Species variation in the gastrointestinal effects of opiates is known (Manara and Bianchetti, 1985). In mice, U-50488H has been shown to lack antitransit effect when administered either by intrathecal or i.c.v, routes (Porreca et al., 1984). However, the effects of highly selective r agonists on gastrointestinal transit when administered by i.p. route in mice have not yet been explored. Furthermore, U-53455E, the stereoisomer of r opiate, U-50488H has been available only very recently, and its effect on gastrointestinal motility has not been reported in any of the species. Hence, the current study investigated the role of r receptors in the mechanisms involved in opioid-induced inhibition of intestinal propulsion by studying the pharmacological effects of U-50488H, U-53455E, U-69593 and these drugs in combination with the
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
330
r-selective antagonist, Mr 2266 in charcoal meal test in mice.
TABLE 1
2. Materials and methods
Treatment
Male Swiss Webster mice (25-30 g Taconic Farms, PA) were used after four days acclimation to the housing conditions and after 24 h starvation. During starvation, water was available ad libitum. All the drugs were freshly prepared and injected i.p. in a volume of 10 m l / k g simultaneously with the test meal. Animals in control group received vehicle injections. The animals were given 0.25 ml of a suspension of charcoal meal (10% charcoal in 5% gum acacia) and killed 45 min later. This time point was selected based on the results of peak time for the r agonist U-50488H (Ramabadran, 1983; 1984). The distance travelled by the charcoal meal as well as the total length of the intestine was measured (Bansinath et al., in press). Gastrointestinal transit was expressed as the percentage of the distance travelled by the charcoal relative to the total length of the small intestine. The data are represented as group means + S.E.M. Statistical significance was assessed by the analysis of variance (ANOVA) followed by post-hoc comparison using Student-NewmanKeuls or Student's t-test, as appropriate.
3. Results
The results indicating the stereospecificity of ant±transit effects of the x agonists used are summarized in table 1. Both U-50488H and U-69593 produced a significant inhibition of the gastrointestinal motility, whereas the ( + ) isomer of U50488H, U-53455E did not modify the gastrointestinal transit, indicating the stereospecificity of the K opiate effect on gut. Furthermore the xselective antagonist, Mr 2266 (3 m g / k g i.p.) when administered along with the agonists completely reversed the inhibitory effects of both agonists U-50488H, F(1,51) = 10.5, P < 0.002; U-69593, F(1,52) = 11.7, P < 0.001). The groupwise results indicating the effects of r agonists alone and their effects when administered along with Mr 2266 are
Stereospecific i n h i b i t i o n of i n t e s t i n a l m o t i l i t y b y K o p i a t e agonists in mice. D a t a represent % of charcoal m e a l transit, m e a n s ± S.E.M. n = 8-12 per t r e a t m e n t .
U-50488H U-53455E U-69593
Dose ( m g / k g i.p.)
ANOVA
3
10
30
F
P <
54±4 a 65±4 51 ± 3 a
50+5 a 66±2 44±4 a
47±5 a 69±4 42±2 a
5.6 0.4 11.2
0.004 0.8 0.0001
W h e n c o m p a r e d with vehicle-treated controls P < 0.05.
TABLE 2 Effect of K-selective a n t a g o n i s t , M r 2266 on the i n h i b i t i o n of i n t e s t i n a l m o t i l i t y i n d u c e d by x o p i a t e a g o n i s t s in mice. D a t a represent % of charcoal m e a l transit, m e a n s ± S.E.M. n = 8-12 per treatment. Treatment
Dose ( m g / k g i.p.) 3
10
30
55 + 3 57 + 4
53 + 2 53 + 2
49 + 4 41 ± 3
74+__5 a 63 ± 3 a
49±4 50 ± 3
Agonist alone U-50488H U-69593
With Mr 2266 (3 mg/kg) U-50488H U-69593
70±5 a 82 ± 5 a
W h e n c o m p a r e d with the respective c o n t r o l s (agonist alone) P < 0.05.
a
presented in table 2. The mean % of charcoal meal transit after Mr 2266 (3 m g / k g ) alone, was 69 + 3 and was not different as compared to the percentage of distance travelled (70 + 6) in the vehicle-treated controls.
4. Discussion
These results clearly demonstrate for the first time that stereospecific x receptors are involved in the inhibition of the gastrointestinal transit in mice. The drugs were administered by i.p. route because earlier studies have shown that this route is optimal for the effect of morphine on gut motility (Bianchi et al., 1983). In rats, the /~ agonist, levomethorphan as well as its dextro isomer, dextromethorphan have been shown to inhibit the gastrointestinal motility (Gaginella et al., 1987). In
331
contrast, present results with U-53455E indicate that dextroisomers of x agonists do not inhibit the gastrointestinal motility in mice. Opiates exert their effects on gastrointestinal motility both by central and peripheral mechanisms (Manara and Bianchetti, 1985). Discrepancy between the present results and the earlier report in mice using the x agonist U50488H (Porreca et al., 1984) may be due to differences in experimental details like route of administration, time course and methodology of measurement of transit. Hence, further studies to clarify the sites of action, viz. central and/peripheral for the observed inhibition of the gut transit produced by x-selective agonists in mice are clearly indicated.
Acknowledgements The authors thank Dr. P.F. Von Voigtlander (Upjohn Company, Kalamazoo, USA) and Dr. H. Merz (Boehringer-lngelheim, West Germany) for U-50488H, U-53455E, and U69593 and Mr 2266, respectively.
References Bansinath, M., K. Ramabadran, H. Turndorf and M.M. Puig, Effects of acute and chronic hyperglycemia on morphineinduced inhibition of gastrointestinal transit in mice, Pharmacology (in press). Bianchi, G., P. Ferretti, M. Recchia, M. Rocchetti, A. Tavani and L. Manara, 1983, Morphine tissue levels and reduction of gastrointestinal transit in rats: correlation supports primary action site in the gut, Gastroenterology 85, 852.
Culpepper-Morgan, J., M.J. Kreek, P.R. Holt, D. LaRoche, J. Zhang and L. O'Bryan, 1988, Orally administered x as well as mu opiate agonists delay gastrointestinal transit in the guinea pig, Life Sci. 42, 2073. Gaginella, T.S., R.J. Bertko and J.F. Kachur, 1987, Effect of dextromethorphan and levomethorphan on gastric emptying and intestinal transit in the rat, J. Pharmacol. Exp. Ther. 240, 388. Galligan, J.J., H.I. Mosberg, R. Hurst, V.J. Hruby and T.F. Burks, 1984, Cerebral delta opioid receptors mediate analgesia but not the intestinal motility effects of intracerebroventricularly administered opioids, J. Pharmacol. Exp. Ther. 229, 641. Lahti, R.A., M.M. Mickelson, J.M. Mccall and P.F. Von Voigtlander, 1985, [3H]U-69593 A highly selective ligand for the opioid x receptor, European J. Pharmacol. 109, 281. La Regina, A., P. Pertrillo, M. Sbacchi and A. Tavani, 1988, Interaction of U-69,593 with F-, 8- and r-opioid binding sites and its analgesic and intestinal effects in rat, Life Sci. 42, 293. Leslie, F.M., 1987, Methods used for the study of opioid receptors, Pharmacol. Rev. 39, 197. Manara, L. and A. Bianchetti, 1985, The central and peripheral influences of opioids on gastrointestinal propulsion, Ann. Rev. Pharmacol. Toxicol. 25, 249. Porreca, F., H.I. Mosberg, R. Hurst, V.J. Hruby and T.F. Burks, 1984, Roles of mu, delta and K opioid receptors in spinal and supraspinal mediation of gastrointestinal transit effects and hot-plate analgesia in the mouse, J. Pharmacol. Exp. Ther. 230, 341. Ramabadran, K., 1983, Hyperalgesic effect of a selective kappa agonist, U-50488H in mice, Jap. J. Pharmacol. 33, 1289. Ramabadran, K., 1984, Strain difference in the hyperalgesic effect of a novel kappa receptor agonist, U-50488H in mice, European J Pharmacol. 98, 425. Tavani, A., M.A. Gambino and P. Petrillo, 1984, The opioid kappa-selective compound U-50,488H does not inhibit intestinal propulsion in rats, J. Pharm. Pharmacol. 36, 343.