European Journal of Pharmacology, 60 (1979) 67--77
67
© Elsevier/North-Holland Biomedical Press
ANTAGONISM OF CHOLECYSTOKININ-LIKE PEPTIDES BY OPIOID PEPTIDES, MORPHINE OR TETRODOTOXIN * GERHARD ZETLER
Institut fiir Pharmakologie der Medizinischen Hochschule Liibeck, Ratzeburger Allee 160, D-2400 Liibeck, Federal Republic of Germany Received 29 March 1979, revised MS received 3 July 1979, accepted 21 August 1979
G. ZETLER, Antagonism of cholecystokinin-like peptides by opioid peptides, morphine or tetrodotoxin, European J. Pharmacol. 60 (1979) 67--77. Morphine, ~-endorphin, Met-enkephalin, and Leu-enkephalin antagonized intestinal actions of cholecystokinin octapeptide (CCK-8), caerulein, and pentagastrin in a manner partly suggesting physiologically competitive antagonism. Further, these acidic peptides (CCK-8, caerulein, pentagastrin) were much more sensitive to the actions of opioids than was angiotensin. Tetrodotoxin also caused changes in the concentration-effect curves, but these were different from the shifts due to the opioids and differentiated between CCK-8, caerulein, and pentagastrin. Naloxone did not modify the response to CCK-8 and caerulein, but completely abolished the antagonistic influence of the opioids. The potencies of morphine and the opioid peptides as antagonists of CCK-8, were of nearly the s a m e order of magnitude. This and the presence in gut and brain of both CCK-like and opioid peptides suggests the hypothesis that these two groups of peptides interact on both myenteric and central nervous system receptors, and thus are directly involved in the regulation of both intestinal motility and satiety. Cholecystokinin Caerulein
Pentagastrin Substance P
Opioid peptides
1. Introduction
Gastrin-like and/or cholecystokinin (CCK)like immunoreactive substances exist in central and peripheral nervous structures (Vanderhaeghen et al., 1975; Dockray, 1976; Uvn~-Wallensten et al., 1977; Dockray et al., 1978). Furthermore, immunoreactivity and bioactivity implicating the COOH-terminal octapeptide (CCK-8) of CCK was found in Fc, the long-known subfraction of crude
* Preliminary reports were presented to the Seminar on Neural Peptides and Proteins, sponsored by the European Society for Neurochemistry and the Belgian Society for Neurology, Brussels, December 9, 1978 (Acta neurol, belg. 79, 60, 1979) and to the German Pharmacological Society (Naunyn-Schmiedeberg's Arch. Pharmacol. 307, R 51, 1979).
Enkephalins
~-Endorphin
Satiety
substance P obtained from brain (Zetler et al., 1978, 1979). Furthermore, like the chemically related peptides caerulein and pentagastrin, CCK and CCK-8 indirectly stimulate the longitudinal muscle of dog and guinea-pig small intestine (Hedner and Rorsman, 1968; Hedner, 1970; Vizi et al., 1972, 1973; Yau and Farrar, 1973; Stewart and Burks, 1977). The indirect nature of this postganglionic action on myenteric neurons was evidenced by the fact that the action was antagonized by tetrodotoxin (TTX) and morphine (but not hexamethonium), and by the fact that these peptides, CCK-8, caerulein and pentagastrin, induce the release of acetylcholine from the intestinal tissue (Vizi, 1973; Vizi et al., 1972, 1973). Similarly, Fc was also found to be antagonized by morphine, but not by hexamethonium, and to release acetylcholine
68 from the guinea-pig ileum (Zetler, 1966). As is the case with morphine, the opioid peptides Met-enkephalin and Leu-enkephalin have been found to reduce or inhibit the release of acetylcholine from electrically stimulated guinea-pig ileum (Waterfield et al., 1977). This leads to the hypothesis that the opioid peptides, Met-enkephalin, Leuenkephalin and ~-endorphin should antagonize the smooth-muscle stimulatory actions of CCK-8, caerulein and pentagastrin. The following is an a t t e m p t to evaluate this hypothesis and to elucidate the mechanism partly by the use of TTX. The influence of TTX on concentration-effect curves of the peptides was found useful in differentiating between CCK-8 and similar peptides having qualitatively identical sensitivity to opioids. Angiotensin II amide (angiotensin), a peptide structure different from that of the above-mentioned acidic peptides and which causes release of acetylcholine from myenteric neurons (review: Gross, 1971) was used as a control.
2. Materials and methods 2.1. Isolated guinea-pig ileum Pieces of terminal ileum of freshly killed guinea pigs (300-500 g) were suspended in 3 ml of Tyrode solution maintained at 32°C and bubbled with 95% O2 + 5% CO2. The solution contained the following salts (mM): NaC1 137, KC1 2.68, NaHCO3 11.9, NaH2PO4 0.42, CaC12 1.35, MgC12 0.49, glucose 5.6. Contractions of the longitudinal muscle were recorded using an isotonic transducer (resting load 250 mg) and a Helcoscriptor He 16. Freshly prepared solutions of drugs and peptides (kept in siliconized flasks to minimize adsorption onto glass) were added to the organ bath using micropipettes. The drug concentrations were such that microliter doses could be used. To prevent tachyphylaxis, the following recovery times (min) were allowed b e t w e e n addition of agonists: CCK-8 (10),
G. ZETLER substance P (5), caerulein {10), angiotensin (15), pentagastrin (15) and carbachol (3). When the contraction elicited by a given agonist contained fast and slow components, only the fast c o m p o n e n t was evaluated since it is indicative of the indirect, i.e., neurogenic part of the mechanism of action of such compounds as angiotensin (Godfraind et al., 1966). 2.2. Field-stimulated mouse vas deferens Vasa deferentia of NMRI mice (35-40 g) were prepared and suspended as described by Hughes et al. (1975). The contractions were recorded isometrically (100-150 mg resting tension). Field stimulation was applied every 30 sec, using t w o platinum ring electrodes (one rectangular pulse, 50 V, duration 0.10.15 msec). 2.3. Estimation o f antagonist activity The results presented in figs. 1, 3 and 4 as well as tables 3 (exception: ~-endorphin; see below) and 4 were obtained from individual experiments using two successive sigmoid concentration-response curves (non-cumulative dosage) and expressing responses as percentages of the maximum (obtained from the first of each pair of curves). The second curve was c o m m e n c e d after full recovery had been achieved (about 60 min after the first). Antagonists were added to the organ bath subsequent to complete recovery from the first curve and 15 min prior to the start of the second curve. The antagonist was also replenished immediately after the washout of an agonist. After linearization of a given curve b y log-probit transformation its regression was calculated b y the m e t h o d of least squares. The regression function yielded the ECs0 and the 'normalized' slope (Zetler and Kampmann, 1979). The m e t h o d of Van den Brink and Lien (1977) was used to calculate the pA2 value (from the shift of the second concentration-response curve) and the pD2' value (when there was, additionally or solely,
OPIOID P E P T I D E S A N T A G O N I Z E C H O L E C Y S T O K I N I N
a depression of the second curve). Because of the very time-consuming protocol (see 2.1.), the method of Arunlakshana and Schild (1959) was used only for estimation of the pA2 and pA~0 in the experiments used to prepare fig. 2 and table 2. In order to save drugs, the pA2 values of fl-endorphin (table 3) were estimated using the method of Lockett and Bartlet (1956) which avoids complete concentration-response curves of the agonist. This procedure was justified since the opioids were found to shift the curves of CCK-8 and related peptides in a parallel fashion. To obtain optimal antagonist effects, the contact time was 10 min for morphine and 2 min for the opioid peptides.
69
2.5. Drugs and materials The C-terminal octapeptide of cholecystokinin (CCK-8; Squibb, SQ 19844), caerulein diethylammonium hydrate (Farmitalia), substance P, Leu- and Met-enkephalin, ~-endorphin (Bachem, Bubendorf, Switzerland), angiotensin (ValS-angiotensin-II-Aspl-~-amid, Ciba), pentagastrin (Bachem, Bubendorf, Switzerland), tetrodotoxin (Sankyo), carbachol (Doryl, Merck), and morphine sulfate (commercial) were used in these experiments. Naloxone hydrochloride (Narcan, Endo Laboratories) was a gift from Prof. Herz, Miinchen.
3. Results
2.4. Statistics Multiple means were compared by analysis of variance using Bartlett's test and the Scheff6 test (cf. Sachs, 1978). The WilcoxonMann-Whitney method was applied in the case of non-homogeneous variances. In general P < 0.05 (two-tailed) was set as threshold of statistical significance.
TABLE 1 ECs0 values and slopes as calculated from the normal concentration-response curves to the agonists. Geometric means and 95% c o n f i d e n c e ranges (n = 20 per group). EDs0 (nM)
Slope
CCK-8
O.8 0.61-1.04
1.8 1.66-2.04
Caerulein
0.75 0.53-1.06
2.05 1.69-2.48
Pentagastrin
32 I 23.1-46.1
1.9 1.68-2.12
Angiotensin
1.5 I 1.20-1.76
2.2 l 1.91-2.50
1 Difference f r o m CCK-8 statistically significant (P
67
© Elsevier/North-Holland Biomedical Press
ANTAGONISM OF CHOLECYSTOKININ-LIKE PEPTIDES BY OPIOID PEPTIDES, MORPHINE OR TETRODOTOXIN * GERHARD ZETLER
Institut fiir Pharmakologie der Medizinischen Hochschule Liibeck, Ratzeburger Allee 160, D-2400 Liibeck, Federal Republic of Germany Received 29 March 1979, revised MS received 3 July 1979, accepted 21 August 1979
G. ZETLER, Antagonism of cholecystokinin-like peptides by opioid peptides, morphine or tetrodotoxin, European J. Pharmacol. 60 (1979) 67--77. Morphine, ~-endorphin, Met-enkephalin, and Leu-enkephalin antagonized intestinal actions of cholecystokinin octapeptide (CCK-8), caerulein, and pentagastrin in a manner partly suggesting physiologically competitive antagonism. Further, these acidic peptides (CCK-8, caerulein, pentagastrin) were much more sensitive to the actions of opioids than was angiotensin. Tetrodotoxin also caused changes in the concentration-effect curves, but these were different from the shifts due to the opioids and differentiated between CCK-8, caerulein, and pentagastrin. Naloxone did not modify the response to CCK-8 and caerulein, but completely abolished the antagonistic influence of the opioids. The potencies of morphine and the opioid peptides as antagonists of CCK-8, were of nearly the s a m e order of magnitude. This and the presence in gut and brain of both CCK-like and opioid peptides suggests the hypothesis that these two groups of peptides interact on both myenteric and central nervous system receptors, and thus are directly involved in the regulation of both intestinal motility and satiety. Cholecystokinin Caerulein
Pentagastrin Substance P
Opioid peptides
1. Introduction
Gastrin-like and/or cholecystokinin (CCK)like immunoreactive substances exist in central and peripheral nervous structures (Vanderhaeghen et al., 1975; Dockray, 1976; Uvn~-Wallensten et al., 1977; Dockray et al., 1978). Furthermore, immunoreactivity and bioactivity implicating the COOH-terminal octapeptide (CCK-8) of CCK was found in Fc, the long-known subfraction of crude
* Preliminary reports were presented to the Seminar on Neural Peptides and Proteins, sponsored by the European Society for Neurochemistry and the Belgian Society for Neurology, Brussels, December 9, 1978 (Acta neurol, belg. 79, 60, 1979) and to the German Pharmacological Society (Naunyn-Schmiedeberg's Arch. Pharmacol. 307, R 51, 1979).
Enkephalins
~-Endorphin
Satiety
substance P obtained from brain (Zetler et al., 1978, 1979). Furthermore, like the chemically related peptides caerulein and pentagastrin, CCK and CCK-8 indirectly stimulate the longitudinal muscle of dog and guinea-pig small intestine (Hedner and Rorsman, 1968; Hedner, 1970; Vizi et al., 1972, 1973; Yau and Farrar, 1973; Stewart and Burks, 1977). The indirect nature of this postganglionic action on myenteric neurons was evidenced by the fact that the action was antagonized by tetrodotoxin (TTX) and morphine (but not hexamethonium), and by the fact that these peptides, CCK-8, caerulein and pentagastrin, induce the release of acetylcholine from the intestinal tissue (Vizi, 1973; Vizi et al., 1972, 1973). Similarly, Fc was also found to be antagonized by morphine, but not by hexamethonium, and to release acetylcholine
68 from the guinea-pig ileum (Zetler, 1966). As is the case with morphine, the opioid peptides Met-enkephalin and Leu-enkephalin have been found to reduce or inhibit the release of acetylcholine from electrically stimulated guinea-pig ileum (Waterfield et al., 1977). This leads to the hypothesis that the opioid peptides, Met-enkephalin, Leuenkephalin and ~-endorphin should antagonize the smooth-muscle stimulatory actions of CCK-8, caerulein and pentagastrin. The following is an a t t e m p t to evaluate this hypothesis and to elucidate the mechanism partly by the use of TTX. The influence of TTX on concentration-effect curves of the peptides was found useful in differentiating between CCK-8 and similar peptides having qualitatively identical sensitivity to opioids. Angiotensin II amide (angiotensin), a peptide structure different from that of the above-mentioned acidic peptides and which causes release of acetylcholine from myenteric neurons (review: Gross, 1971) was used as a control.
2. Materials and methods 2.1. Isolated guinea-pig ileum Pieces of terminal ileum of freshly killed guinea pigs (300-500 g) were suspended in 3 ml of Tyrode solution maintained at 32°C and bubbled with 95% O2 + 5% CO2. The solution contained the following salts (mM): NaC1 137, KC1 2.68, NaHCO3 11.9, NaH2PO4 0.42, CaC12 1.35, MgC12 0.49, glucose 5.6. Contractions of the longitudinal muscle were recorded using an isotonic transducer (resting load 250 mg) and a Helcoscriptor He 16. Freshly prepared solutions of drugs and peptides (kept in siliconized flasks to minimize adsorption onto glass) were added to the organ bath using micropipettes. The drug concentrations were such that microliter doses could be used. To prevent tachyphylaxis, the following recovery times (min) were allowed b e t w e e n addition of agonists: CCK-8 (10),
G. ZETLER substance P (5), caerulein {10), angiotensin (15), pentagastrin (15) and carbachol (3). When the contraction elicited by a given agonist contained fast and slow components, only the fast c o m p o n e n t was evaluated since it is indicative of the indirect, i.e., neurogenic part of the mechanism of action of such compounds as angiotensin (Godfraind et al., 1966). 2.2. Field-stimulated mouse vas deferens Vasa deferentia of NMRI mice (35-40 g) were prepared and suspended as described by Hughes et al. (1975). The contractions were recorded isometrically (100-150 mg resting tension). Field stimulation was applied every 30 sec, using t w o platinum ring electrodes (one rectangular pulse, 50 V, duration 0.10.15 msec). 2.3. Estimation o f antagonist activity The results presented in figs. 1, 3 and 4 as well as tables 3 (exception: ~-endorphin; see below) and 4 were obtained from individual experiments using two successive sigmoid concentration-response curves (non-cumulative dosage) and expressing responses as percentages of the maximum (obtained from the first of each pair of curves). The second curve was c o m m e n c e d after full recovery had been achieved (about 60 min after the first). Antagonists were added to the organ bath subsequent to complete recovery from the first curve and 15 min prior to the start of the second curve. The antagonist was also replenished immediately after the washout of an agonist. After linearization of a given curve b y log-probit transformation its regression was calculated b y the m e t h o d of least squares. The regression function yielded the ECs0 and the 'normalized' slope (Zetler and Kampmann, 1979). The m e t h o d of Van den Brink and Lien (1977) was used to calculate the pA2 value (from the shift of the second concentration-response curve) and the pD2' value (when there was, additionally or solely,
OPIOID P E P T I D E S A N T A G O N I Z E C H O L E C Y S T O K I N I N
a depression of the second curve). Because of the very time-consuming protocol (see 2.1.), the method of Arunlakshana and Schild (1959) was used only for estimation of the pA2 and pA~0 in the experiments used to prepare fig. 2 and table 2. In order to save drugs, the pA2 values of fl-endorphin (table 3) were estimated using the method of Lockett and Bartlet (1956) which avoids complete concentration-response curves of the agonist. This procedure was justified since the opioids were found to shift the curves of CCK-8 and related peptides in a parallel fashion. To obtain optimal antagonist effects, the contact time was 10 min for morphine and 2 min for the opioid peptides.
69
2.5. Drugs and materials The C-terminal octapeptide of cholecystokinin (CCK-8; Squibb, SQ 19844), caerulein diethylammonium hydrate (Farmitalia), substance P, Leu- and Met-enkephalin, ~-endorphin (Bachem, Bubendorf, Switzerland), angiotensin (ValS-angiotensin-II-Aspl-~-amid, Ciba), pentagastrin (Bachem, Bubendorf, Switzerland), tetrodotoxin (Sankyo), carbachol (Doryl, Merck), and morphine sulfate (commercial) were used in these experiments. Naloxone hydrochloride (Narcan, Endo Laboratories) was a gift from Prof. Herz, Miinchen.
3. Results
2.4. Statistics Multiple means were compared by analysis of variance using Bartlett's test and the Scheff6 test (cf. Sachs, 1978). The WilcoxonMann-Whitney method was applied in the case of non-homogeneous variances. In general P < 0.05 (two-tailed) was set as threshold of statistical significance.
TABLE 1 ECs0 values and slopes as calculated from the normal concentration-response curves to the agonists. Geometric means and 95% c o n f i d e n c e ranges (n = 20 per group). EDs0 (nM)
Slope
CCK-8
O.8 0.61-1.04
1.8 1.66-2.04
Caerulein
0.75 0.53-1.06
2.05 1.69-2.48
Pentagastrin
32 I 23.1-46.1
1.9 1.68-2.12
Angiotensin
1.5 I 1.20-1.76
2.2 l 1.91-2.50
1 Difference f r o m CCK-8 statistically significant (P
