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Naunyn-Schmiedeberg's Arch. Pharmacol. 308, 255-260 (1979)
Pharmacology 9 by Springer-Verlag 1979
Centrally and Peripherally Mediated Inhibition of Intestinal Motility by Opioids Rtidiger Schulz, Michael Wtister, and Albert Herz Institut f/Jr Neuropharmakologie, Max-Planck-Institut fiir Psychiatrie, Kraepelinstrasse 2, D-8000 Mfinchen 40, Federal Republic of Germany
Summary. Intracerebroventricularly injected morphine is 50-fold more potent in arresting intestinal peristalsis in rats, mice or guinea pigs than morphine administered systemically. Using quaternary naloxone as narcotic antagonist, it has been demonstrated that the peripheral pathway of the centrally mediated constipatory effect of morphine does not involve opioid peptidergic mechanisms. Further, this effect is not due to the release of opioid peptides from the pituitary, since hypophysectomy fails to affect the antipropulsive activity of morphine. On the other hand, the intestinal motility can be affected directly by activation of opiate receptors located in the gut. This was best demonstrated with loperamide, which exhibits predominantly a peripheral site of action. Thus, two mechanisms of the action of morphine on gastrointestinal propulsive activity have been demonstrated. One arises in the central nervous system (CNS) and is mediated peripherally not by opioid peptidergic pathways, whereas the other is due to a direct action of morphine on the gut. Key words: Constipation - Quaternary naloxone Morphine - Loperamide - Opiate receptors.
Introduction
Morphine injected intracerebroventricularly (i.c.v.) causes a strong inhibition of the propulsive activity of the gut, whereas an identical dose given systemically fails to affect the motility of the gut (Margolin, 1954; Margolin and Plekss, 1965; Stewart et al., 1977; Parolaro et al., 1977). These findings may suggest that the central nervous system (CNS) mediates the constipating action of morphine by an, as yet unelucidated mechanism. According to Margolin and Plekss (1965) morphine elicits the discharge of an unspecified Send offprint requests to R. Schulz at the above address
neurohumoral substance from the brain into the circulating blood, which subsequently arrests gastrointestinal propulsion. Alternative explanation of the centrally mediated morphine action must take into consideration sympathetic control mechanisms originating from the brain. Although surgical interruption (sympathectomy) of this neuronal pathway seemed to be ineffective in blocking the constipation which follows i.c.v, administered morphine (Margolin, 1963), such neuronal components of the mechanisms under discussion cannot be excluded (Stewart et al., 1977). Of particular interest is any role opioid peptides may play in mediating the central constipatory effect of morphine. The pituitary, for example, contains extremely high concentrations of /%endorphin (Teschemacher, 1978), which may be released under specific conditions, such as stress, into the circulating blood (Guillemin et al., 1977; Rossier et al., 1977). Moreover, morphine given systemically increases the /?-endorphin concentration in the blood (H611t et al., 1978). The opiate receptors in the small intestine (Schulz, 1978), thought to mediate the antidiarrheal properties of opioids (for review see Weinstock, 1971) are probably a target for these circulating opioid peptides. On the other hand, the high enkephalin concentrations in the myenteric plexus (Hughes et al., 1977; Schultzberg et al., 1978) suggest that opioid petides may be involved in mediating the CNS outflow responsible for controlling gut peristalsis. These considerations induced us to investigate whether or not the inhibition of the propulsive activity of the gut induced by i.c.v, injected morphine is mediated by a peripheral opioid peptidergic mechanism. In order to achieve a functional characterization of such mechanisms, we employed quaternary naloxone. This narcotic antagonist should manifest its action predominantly at opiate receptors outside the brain, since it penetrates the blood brain barrier only poorly owing of its cationic charge.
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Naunyn-Schmiedeberg's Arch. Pharmacol. 308 (1979)
Methods Male rats (Sprague Dawley, 150 g), mice (25 g) and guinea pigs (250 g) were starved overnight before treatment. Only one experiment was performed on each animal. Drugs were given intraperitoneally (i.p.), intravenously (i.v.) and intracerebrovent~icularly (i.c.v.). I.c.v. injections (5 ~1) into mice were carried out according to the technique described by Haley and McCormick (1957). Rats and guinea pigs were implanted chronically with guide cannulas into the left anterior horn of the lateral ventricle (for technicaI details see Laschka et al., 1976) 3 days before the i.c.v. injections (10 ~l). The "charcoal meal test" was employed for the examination of gastrointestinal motility. The animals received via a stomach tube a 10 % (w/v) charcoal suspension in a 5 % (w/v) gum arabic solution. Mice were given 0.3 ml of the suspension, whilst rats and guinea pigs received 1.5 ml. Immediately thereafter the drug under investigation was administex,d. In each experiment controls were given saline. After 30rain the animals were decapitated and the complete small intestine was removed. The propulsive activity of the gut was determined by calculating the quotient "A": A=
distance of charcoal meal travelled total length of small intestine
Values A t were from drug-treated animals and A~ from controls. The data are presented as quotients (Q) of A~/A2, so that e~ch result obtained from drug-treated animals is related to a c~ntrol value. Thus, Q = 1 indicated absence of inhibition of gut peristalsis; the lower the quotient, the stronger was the inhibition of propulsive activity. The dose required for an opioid to inhibit the propulsive activity of the gut by 50 % is called the IDs0. Log-probit analysis was used for graphic display of dose-response curves. Tests for analgesia in mice were conducted accoring to the "tailflick" procedure (D'Amour and Smith, 1941). The cut off time was 20 s. The dose of an opiate, which caused a 50 % delay in reaction time (usually 7 s) is called the EDs0. The electrically stimulated longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum, as described by Schulz and Goldstein (1972), served as a bioassay for determination of the activity of narcotic antagonists. Transpharyngeal hypophysectomy of rats was performed under pentobarbita] anaesthesia. Results were evaluated only from those rats in which it was shown that the pituitary had been completely removed (macroscopic examination). The content of tritium in the brain and gut following i.c.v. injection of ~H-loperamide (10 gCi) was determined by scintillation counting. A 306 oxidizer (Packard Instruments) was employed for preparation of the samples. The following drugs were used: morphine hydrochloride (Merck, Darmstadt, FRG), FK 33-824 (D-Ala 2, MePhe 4, Met-(O)5-ol enkephalin) (donated by Dr. Roemer, Sandoz AG, Baste, Switzerland), loperamide, 3H-loperamide (0.1 Ci/mmole) (provided by Dr. Heykants, Janssen Pharmaceutica, Beerse, Belgium), naloxone hydrochloride (provided by Dr. Ferster, Endo Laboratories, Garden City, New York, USA). Quaternary na[oxone (N,Nmeflayl-allyl-noroxymorphone) was obtained by qualernization of the tertiary naloxone precursor (Organikum, 1971). Naloxone hydrochloride was exposed to 5 N NaOH to precipitate the free naloxone base, which was dissolved in nitromethane (1 : 4, g/v)~ After addition of methyliodide (1 :, v/v), the solution was allowed to sit for 2 h. Thereafter, Ihe material was evaporated to dryness using a rotary evaporator. The residue was dissolved in ethanol and purified on thin layer chromatography (silicaget 0.25 ram, Merck, Darmstadt, FRG). The following s