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Synergistic opioid inhibition of colonic Cl− secretion by κ-opioid receptor agonism plus μ-opioid receptor antagonism
European Journal of Pharmacology, 230 (1993) 235-237 © 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
235
EJP 21167 Short communication
Synergistic opioid inhibition of colonic C1- secretion by K-opioid receptor agonism plus/x-opioid receptor antagonism Wolfgang Kromer Department of Pharmacology, Byk Gulden Pharmaceuticals, Byk-Gulden-Strasse 2, D-7750 Konstanz, Germany Received 22 July 1992, revised MS received 6 November 1992, accepted 10 November 1992
On the basis of the novel concept of a dual prosecretory/antisecretory intestinal opioid system, a voltage-clamp experiment was performed with guinea-pig colonic mucosa, and net anion (CI-) secretion was measured after stimulation with prostaglandin E 1 (PGE 0 plus theophylline. Synergism between K-opioid receptor agonism by U69593 and ~-opioid receptor antagonism by CTOP-NH 2 (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) was observed. Opioids; Receptor subtypes; C1 secretion (intestinal)
1. Introduction
It has recently been shown that submucosal K-opioid receptors inhibit and /x-opioid receptors enhance prostaglandin E x (PGE~) plus theophylline-stimulated net chloride secretion in guinea-pig colonic mucosa in vitro (Kromer, 1991). T h e / x - o p i o i d receptor antagonist C T O P - N H 2 (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-ThrNH2; Gulya et al., 1986) also impairs net water secretion (enteropooling) in the rat jejunum in vivo. The antisecretory effect of C T O P - N H 2 was prevented by prior administration of high concentrations of naloxone, both in vivo and in vitro (Kromer and Beubler, in preparation). These data suggested a dual prosecret o r y / a n t i s e c r e t o r y role of intestinal opioid systems and prompted the present experiments to test for an overadditive, antisecretory effect of concomitant activation of K-opioid receptors and blockade of/x-opioid receptors.
2. Materials and methods
2.1. Experimental system and conditions Mucosal preparations with adherent submucosal plexus, freed of circular and longitudinal muscle layers, were obtained from male guinea-pigs (350-450 g body
Correspondence to: W. Kromer, Byk Gulden, Byk-Gulden-Strasse 2, D-7750 Konstanz, Germany. Tel. 49-7531/84-2628.
weight) and mounted in Ussing-type chambers (see Kromer, 1991). Short circuit current (Isc) was measured as an indicator of net anion secretion in the presence of 100 /xmol/1 amiloride after stimulation with 1 / x m o l / l P G E 1 plus 100 /xmol/1 theophylline. Amiloride was used to block Na + fluxes. U n d e r these conditions, the attribution of changes in I sc to changes in C I - secretion is based on control experiments performed in the absence of C I - and has been discussed elsewhere (Kromer, 1991). Experimental conditions were as described by Kromer (1991), except for the composition of the buffer, which was (mmol/l): NaC1 113.6, KC1 5.4, CaC12 1.2, MgCI 2 1.2, N a z H P O 4 2.4, N a H 2 P O 4 0.6, N a H C O 3 21, glucose 10, p H 7.4, 37°C, and gassed with 95% O 2 plus 5% CO 2. 2.2. Experimental protocol After 60 min of equilibration, drugs were administered in a single experiment in the following order: amiloride, followed 7.5 rain later by C T O P - N H 2 , followed 2.5 min later by the K-opioid receptor agonist U69593 (Lahti et al., 1985), followed 10 min later by theophylline, and immediately thereafter by P G E 1. Amiloride was applied to the mucosal side and PGE1 to the serosal side. The other compounds were applied to either side. Isc recordings were made every 2.5 min. The difference between the mean value of four recordings after PGE~ and the basal value immediately before stimulation was taken as AIsc (see fig. 1). 1 /xA = 0.037/xEq × h - ~ × cm 2. A concentration-inhibition curve was made for the K-opioid receptor agonist U69593 in the absence or
236 presence of the /z-opioid receptor antagonist CTOPN H 2, 100 nmol/l. C T O P - N H 2 alone was tested in parallel. In addition, U69593- and CTOP-NH2-free control experiments were performed. For each concentration of the K-opioid receptor agonist, an intraindividual comparison was made, with random allocation of the different experimental conditions to adjacent colonic segments of each animal.
2.3. Calculation and statistical analysis The existence of synergism between U69593 and C T O P - N H 2 was demonstrated according to the method of P6ch and Holzmann (1980), by expressing the C T O P - N H 2 concentration in terms of the equipotent concentration of U69593. This transformed value was then added to the concentration of U69593 used in the combination experiment with CTOP-NH2, and the expected effect of this additive concentration was read from the concentration-inhibition curve of U69593 and compared to the combined effect actually observed. The statistical significance of synergistic effects was tested with the one-sample t-test, one-sided. This test rejects the Ho hypothesis which claims correspondence between the empirical distribution and the theoretical mean value for additive synergism. All of the other comparisons were assessed with Student's t-test for paired samples, two-sided.
2.4. Drugs Amiloride hydrochloride, PGEt, U69593 (5a,7a, 8/3)( - )-N-methyl-N-(7-[ 1-pyrrolidinyl]- 1-oxaspiro[4,5]dec-8-yl)benzeneacetamide (all Sigma, FRG), CTOPNH2 (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NHz; Peninsula, USA) and theophylline (Byk Gulden, F R G ) were used. Aqueous solutions were prepared except for P G E 1, for which an ethanolic stock solution was made and further diluted with water.
4. Discussion The present work clearly demonstrates that ~-opioid receptors constitute a prosecretory counterpart to the antisecretory function of K-opioid receptors in guineapig colonic mucosa with adherent submucosal plexus. It should be noted, however, that Nakayama et al. (1990) reported the presynaptic inhibition of acetylcholine release via K-opioid receptors, but the inhibition of both acetylcholine and noradrenaline release via /z-opioid receptors in the guinea-pig ileum in vitro. While the inhibitory modulation of noradrenaline release by p.-opioid receptors under conditions that cause stimulation of sympathetic nerve endings in the mucosa may well explain the antisecretory action of the #opioid receptor antagonist C T O P - N H 2 in the present study, the inhibition of acetylcholine release via #opioid receptors may relate to an additional antisecretory component of /z-opioid receptor activation possibly missed under the present experimental conditions. The method of P6ch and Holzmann (1980), used to demonstrate overadditive effects, i.e., synergism, required a C T O P - N H 2 concentration that caused a significant but nevertheless small effect on its own. Lower (10 n m o l / l ) or higher (1 p.mol/l) concentrations of C T O P - N H 2 had either no significant or too pronounced an inhibitory effect on their own. Therefore, synergism could not be demonstrated between these C T O P - N H 2 concentrations and U69593 (not shown). The maximum effect achieved at 1 /~mol/l U69593 (fig. 1) may represent the total antisecretory efficacy of the intestinal opioid system under the experimental conditions used. In conclusion, this is the first time that a synergistic antisecretory effect of K-opioid receptor agonism and
'E 4 .u
3. Results
[]
[]
~7
o-2 2
IH v
C T O P - N H 2 shifted the concentration-inhibition curve of U69593 in a non-parallel fashion to the left, without significantly affecting the maximum effect (fig. 1). The response to the two compounds was more pronounced than theoretically expected on the basis of individual effects (middle curve). Absolute values for lsc observed in controls (Co.; column) were 3.85 + 0.42, corresponding to Alsc values (differences to individual, basal values) of 6.29 _+ 1.73 (mean + S.E.M.; /zEq × h -1 × cm-2). The AIsc value for C T O P - N H 2 alone (100 n m o l / 1 ) was 4.23 _+ 1.95, corresponding to an equieffective concentration of U69593 of 22 nmol/l.
[]
.
10r
r
T i ilrriT9
i
i r irlu,8
r
i'l
1111117
i
i , ,lilt,6
Co.
- log (mol/I)
Fig. 1. Change in PGE 1 plus theophylline-stimulated lsc (ordinate) elicited by either U69593 alone (v) or in combination with CTOPNH2, 100 nmol/l (ll). The abscissa refers to the concentration of U69593 (-log mol/l). © represents the effect of 100 nmol/l CTOPNH 2 alone. The middle curve (D) indicates the theoretical curve for additive effects, the column represents controls (Co.) without U69593 or CTOP-NH2. n = 7 (U69593, 0.1 nmol/l) or 9 (U69593, 1 nmol/I 1 p.mol/l); controls and CTOP-NH2 alone, n - 18. P values refer to either the comparison with controls (*) or the comparison between the experimentally observed effect of the combination with the theoretical curve for additive effects ( + ). *'+ P < 0.05. ** P ~<0.01.
237
tx-opioid receptor antagonism has been demonstrated in intestinal mucosa (for review, see Kromer, 1993). It suggests a dual modulatory opioid system intrinsic to the colonic submucosal plexus.
Acknowledgements I like to thank Mrs. B. Heller for expert technical assistance and Dr. D. Hauschke for biometrical advice.
References Gulya, K., J.T. Pelton, V.J. Hruby and H.I. Yamamura, 1986, Cyclic somatostatin octapeptide analogues with high affinity and selectivity toward mu opioid receptors, Life Sci. 38, 2221.
Kromer, W., 1991, Voltage-clamp experiments reveal receptor typedependent modulation of chloride secretion in the guinea pig colonic mucosa by intestinal opioids, Naunyn-Schmiedeb. Arch. Pharmacol. 344, 360. Kromer, W., 1993, Gastrointestinal effects of opioids, in: Handbook of Exp. Pharmacology 104/If, eds. A. Herz, H. Akil, E.J. Simon (Springer, Heidelberg) p. 163. 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 receptor, Eur. J. Pharmacol. 109, 281. Nakayama, S., K. Taniyama, S. Matsuyama, N. Ohgushi, K. Tsunekawa and C. Tanaka, 1990, Regulatory role of enteric and K opioid receptors in the release of acetylcholine and norepinephrine from guinea pig ileum, J. Pharmacol. Exp. Ther. 254, 792. P6ch, G. and S. Holzmann, 1980, Quantitative estimation of overadditive and underadditive drug effects by means of theoretical, additive dose-response curves, J. Pharmacol. Meth. 4, 179.
235
EJP 21167 Short communication
Synergistic opioid inhibition of colonic C1- secretion by K-opioid receptor agonism plus/x-opioid receptor antagonism Wolfgang Kromer Department of Pharmacology, Byk Gulden Pharmaceuticals, Byk-Gulden-Strasse 2, D-7750 Konstanz, Germany Received 22 July 1992, revised MS received 6 November 1992, accepted 10 November 1992
On the basis of the novel concept of a dual prosecretory/antisecretory intestinal opioid system, a voltage-clamp experiment was performed with guinea-pig colonic mucosa, and net anion (CI-) secretion was measured after stimulation with prostaglandin E 1 (PGE 0 plus theophylline. Synergism between K-opioid receptor agonism by U69593 and ~-opioid receptor antagonism by CTOP-NH 2 (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) was observed. Opioids; Receptor subtypes; C1 secretion (intestinal)
1. Introduction
It has recently been shown that submucosal K-opioid receptors inhibit and /x-opioid receptors enhance prostaglandin E x (PGE~) plus theophylline-stimulated net chloride secretion in guinea-pig colonic mucosa in vitro (Kromer, 1991). T h e / x - o p i o i d receptor antagonist C T O P - N H 2 (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-ThrNH2; Gulya et al., 1986) also impairs net water secretion (enteropooling) in the rat jejunum in vivo. The antisecretory effect of C T O P - N H 2 was prevented by prior administration of high concentrations of naloxone, both in vivo and in vitro (Kromer and Beubler, in preparation). These data suggested a dual prosecret o r y / a n t i s e c r e t o r y role of intestinal opioid systems and prompted the present experiments to test for an overadditive, antisecretory effect of concomitant activation of K-opioid receptors and blockade of/x-opioid receptors.
2. Materials and methods
2.1. Experimental system and conditions Mucosal preparations with adherent submucosal plexus, freed of circular and longitudinal muscle layers, were obtained from male guinea-pigs (350-450 g body
Correspondence to: W. Kromer, Byk Gulden, Byk-Gulden-Strasse 2, D-7750 Konstanz, Germany. Tel. 49-7531/84-2628.
weight) and mounted in Ussing-type chambers (see Kromer, 1991). Short circuit current (Isc) was measured as an indicator of net anion secretion in the presence of 100 /xmol/1 amiloride after stimulation with 1 / x m o l / l P G E 1 plus 100 /xmol/1 theophylline. Amiloride was used to block Na + fluxes. U n d e r these conditions, the attribution of changes in I sc to changes in C I - secretion is based on control experiments performed in the absence of C I - and has been discussed elsewhere (Kromer, 1991). Experimental conditions were as described by Kromer (1991), except for the composition of the buffer, which was (mmol/l): NaC1 113.6, KC1 5.4, CaC12 1.2, MgCI 2 1.2, N a z H P O 4 2.4, N a H 2 P O 4 0.6, N a H C O 3 21, glucose 10, p H 7.4, 37°C, and gassed with 95% O 2 plus 5% CO 2. 2.2. Experimental protocol After 60 min of equilibration, drugs were administered in a single experiment in the following order: amiloride, followed 7.5 rain later by C T O P - N H 2 , followed 2.5 min later by the K-opioid receptor agonist U69593 (Lahti et al., 1985), followed 10 min later by theophylline, and immediately thereafter by P G E 1. Amiloride was applied to the mucosal side and PGE1 to the serosal side. The other compounds were applied to either side. Isc recordings were made every 2.5 min. The difference between the mean value of four recordings after PGE~ and the basal value immediately before stimulation was taken as AIsc (see fig. 1). 1 /xA = 0.037/xEq × h - ~ × cm 2. A concentration-inhibition curve was made for the K-opioid receptor agonist U69593 in the absence or
236 presence of the /z-opioid receptor antagonist CTOPN H 2, 100 nmol/l. C T O P - N H 2 alone was tested in parallel. In addition, U69593- and CTOP-NH2-free control experiments were performed. For each concentration of the K-opioid receptor agonist, an intraindividual comparison was made, with random allocation of the different experimental conditions to adjacent colonic segments of each animal.
2.3. Calculation and statistical analysis The existence of synergism between U69593 and C T O P - N H 2 was demonstrated according to the method of P6ch and Holzmann (1980), by expressing the C T O P - N H 2 concentration in terms of the equipotent concentration of U69593. This transformed value was then added to the concentration of U69593 used in the combination experiment with CTOP-NH2, and the expected effect of this additive concentration was read from the concentration-inhibition curve of U69593 and compared to the combined effect actually observed. The statistical significance of synergistic effects was tested with the one-sample t-test, one-sided. This test rejects the Ho hypothesis which claims correspondence between the empirical distribution and the theoretical mean value for additive synergism. All of the other comparisons were assessed with Student's t-test for paired samples, two-sided.
2.4. Drugs Amiloride hydrochloride, PGEt, U69593 (5a,7a, 8/3)( - )-N-methyl-N-(7-[ 1-pyrrolidinyl]- 1-oxaspiro[4,5]dec-8-yl)benzeneacetamide (all Sigma, FRG), CTOPNH2 (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NHz; Peninsula, USA) and theophylline (Byk Gulden, F R G ) were used. Aqueous solutions were prepared except for P G E 1, for which an ethanolic stock solution was made and further diluted with water.
4. Discussion The present work clearly demonstrates that ~-opioid receptors constitute a prosecretory counterpart to the antisecretory function of K-opioid receptors in guineapig colonic mucosa with adherent submucosal plexus. It should be noted, however, that Nakayama et al. (1990) reported the presynaptic inhibition of acetylcholine release via K-opioid receptors, but the inhibition of both acetylcholine and noradrenaline release via /z-opioid receptors in the guinea-pig ileum in vitro. While the inhibitory modulation of noradrenaline release by p.-opioid receptors under conditions that cause stimulation of sympathetic nerve endings in the mucosa may well explain the antisecretory action of the #opioid receptor antagonist C T O P - N H 2 in the present study, the inhibition of acetylcholine release via #opioid receptors may relate to an additional antisecretory component of /z-opioid receptor activation possibly missed under the present experimental conditions. The method of P6ch and Holzmann (1980), used to demonstrate overadditive effects, i.e., synergism, required a C T O P - N H 2 concentration that caused a significant but nevertheless small effect on its own. Lower (10 n m o l / l ) or higher (1 p.mol/l) concentrations of C T O P - N H 2 had either no significant or too pronounced an inhibitory effect on their own. Therefore, synergism could not be demonstrated between these C T O P - N H 2 concentrations and U69593 (not shown). The maximum effect achieved at 1 /~mol/l U69593 (fig. 1) may represent the total antisecretory efficacy of the intestinal opioid system under the experimental conditions used. In conclusion, this is the first time that a synergistic antisecretory effect of K-opioid receptor agonism and
'E 4 .u
3. Results
[]
[]
~7
o-2 2
IH v
C T O P - N H 2 shifted the concentration-inhibition curve of U69593 in a non-parallel fashion to the left, without significantly affecting the maximum effect (fig. 1). The response to the two compounds was more pronounced than theoretically expected on the basis of individual effects (middle curve). Absolute values for lsc observed in controls (Co.; column) were 3.85 + 0.42, corresponding to Alsc values (differences to individual, basal values) of 6.29 _+ 1.73 (mean + S.E.M.; /zEq × h -1 × cm-2). The AIsc value for C T O P - N H 2 alone (100 n m o l / 1 ) was 4.23 _+ 1.95, corresponding to an equieffective concentration of U69593 of 22 nmol/l.
[]
.
10r
r
T i ilrriT9
i
i r irlu,8
r
i'l
1111117
i
i , ,lilt,6
Co.
- log (mol/I)
Fig. 1. Change in PGE 1 plus theophylline-stimulated lsc (ordinate) elicited by either U69593 alone (v) or in combination with CTOPNH2, 100 nmol/l (ll). The abscissa refers to the concentration of U69593 (-log mol/l). © represents the effect of 100 nmol/l CTOPNH 2 alone. The middle curve (D) indicates the theoretical curve for additive effects, the column represents controls (Co.) without U69593 or CTOP-NH2. n = 7 (U69593, 0.1 nmol/l) or 9 (U69593, 1 nmol/I 1 p.mol/l); controls and CTOP-NH2 alone, n - 18. P values refer to either the comparison with controls (*) or the comparison between the experimentally observed effect of the combination with the theoretical curve for additive effects ( + ). *'+ P < 0.05. ** P ~<0.01.
237
tx-opioid receptor antagonism has been demonstrated in intestinal mucosa (for review, see Kromer, 1993). It suggests a dual modulatory opioid system intrinsic to the colonic submucosal plexus.
Acknowledgements I like to thank Mrs. B. Heller for expert technical assistance and Dr. D. Hauschke for biometrical advice.
References Gulya, K., J.T. Pelton, V.J. Hruby and H.I. Yamamura, 1986, Cyclic somatostatin octapeptide analogues with high affinity and selectivity toward mu opioid receptors, Life Sci. 38, 2221.
Kromer, W., 1991, Voltage-clamp experiments reveal receptor typedependent modulation of chloride secretion in the guinea pig colonic mucosa by intestinal opioids, Naunyn-Schmiedeb. Arch. Pharmacol. 344, 360. Kromer, W., 1993, Gastrointestinal effects of opioids, in: Handbook of Exp. Pharmacology 104/If, eds. A. Herz, H. Akil, E.J. Simon (Springer, Heidelberg) p. 163. 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 receptor, Eur. J. Pharmacol. 109, 281. Nakayama, S., K. Taniyama, S. Matsuyama, N. Ohgushi, K. Tsunekawa and C. Tanaka, 1990, Regulatory role of enteric and K opioid receptors in the release of acetylcholine and norepinephrine from guinea pig ileum, J. Pharmacol. Exp. Ther. 254, 792. P6ch, G. and S. Holzmann, 1980, Quantitative estimation of overadditive and underadditive drug effects by means of theoretical, additive dose-response curves, J. Pharmacol. Meth. 4, 179.