Neuromuscular Effects of Some Opioid Agonists and Antagonists*
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NEUROMUSCULAR EFFECTS OF SOME OPIOID AGONISTS AND ANTAGONISTS* Krishnaswami RAMABADRAN, Meng Kwoon SIM and Kwok Chan LUN** Departments of Pharmacology, **Social Medicine and Public Health, Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore 0511 Accepted August 21, 1982 Abstract-The effects of morphine, pethidine, (-)naloxone, N-methylnaloxone, naltrex one and levallorphan on acetylcholine-induced contraction of the toad rectus were studied. The drugs were shown to inhibit the contraction, and their inhibitory effect was suggested to be partly mediated via a peripheral opiate binding site. The depression of acetylcholine-induced contraction by levallorphan and dextrallorphan might indicate possible involvement of stereospecific binding sites, as the latter required a significantly higher concentration to produce the same magnitude of depression. Statistical analysis of the slope of the computer-plotted dose-response of acetylcholine in the presence and absence of each of the opioids indicates that these drugs can be classified into four categories. Morphine and naltrexone each formed a class of its own; (-)naloxone, N- methylnaloxone and pethidine formed another class; levallorphan and dextrallorphan formed the fourth class. The classification of the opioids into four categories reveals the possible existence of multiple opiate binding sites on the skeletal muscle. The significance of each of the sub-types of binding sites in the contraction of skeletal muscle and the mechanism by which it affects the contraction remains to be investigated. Of late, the neuromuscular action of the opioid (12). However, whether the morphine has been of interest to pharma depressant effect of opiate drugs on neuro cologists. At concentrations of 10-8 to 10-5 muscular transmission involved the mediation M, the drug has been found to depress of stereospecific sites or not is still con cholinergic transmission in the smooth muscle troversial (8, 13, 14). We attempt to address of experimental animals (1-5). At higher this problem by studying the effect of opiate concentrations (10-3 to 10-5 M), the same agonists, antagonists, stereoisomer of one of effect was also observed in the skeletal the antagonists and N-methylnaloxone on the muscle (6-8). Frank (9) suggested the Ach-induced contractions of the toad rectus existence of opiate receptors in the skeletal abdominis. muscle of the frog. From his work on frog's skeletal muscle (10) and sciatic nerve (11), Materials and Methods he attributed the marked difference in the Rectus muscle preparation: The rectus effective concentrations of morphine in the muscle from toads weighing between 30 to two type of muscular preparations to the 40 g was used. The tissue of approximately difference in sensitivity of the receptors to 3 cm length was mounted and stretched with a weight of 10 g in a 10 ml organ bath at room " A preliminary communication of this paper was temperature (28-30°C) containing Frog presented at the Third Western Pacific and South East Asian Regional Meeting of Pharmacologists Ringer solution bubbled with air. It was in Bangkok in May 1982. allowed to equilibrate for 1 hr before drug exposure. During the equilibration period, naloxone hydrobromide [(MRZ 2593) gift the bath solution was replaced frequently. from Dr. H. Merz, Boehringer Ingelheim, Contractile responses were recorded on a F.R.G.], levallorphan tartrate and dextral Washington Oscillograph recorder (Model lorphan hydrobromide (gift from Dr. W. 400 M D1) through an isotonic lever Haefely, Hoffmann-La Roche Ltd., Switzer transducer (Model T2) and a strain gauge land). coupler (Model FC1 17). Statistical analysis: The dose-response The responses of the rectus to 4, 8, 16, relations were linearly plotted, and the 32x10-6 M of acetylcholine in the absence responses at 16 x 10-6 M of Ach were and presence of each increasing concen predicted using the regression lines. The tration (10, 20, 40, 80, 160, 320x10-6 M ) difference in these predicted responses in the of each opioid were determined. The bracket absence and presence of opioid was noted ing technique was employed. Following each for each of the different concentrations of the Ach-induced response, the organ bath was opioid used. The experiment was replicated drained, and the preparation was washed five or six times for each concentration of the three times with 10 ml of the Ringer. In all opioid, and hence, five or six sets of differ cases, the recovery of the responses after ences were available for each concentration. washing was more than 95%. The pH of the When regressed against the concentrations of Frog Ringer was 6.5, and dissolutions of the the opioids, the replicated differences pro drugs even at concentrations 320x10-6 M vided a comparison between opioids for slope did not alter the pH of this solution. For the and position of the regression lines. All Ringer containing Ach or Ach plus the statistical calculations were carried out on a opioid, the contact time with the preparation computer (TRS-80 model II). was one min. Prior to exposure to the Ringer containing Ach plus the respective opioid, Results the preparation was exposed to the Ringer There was a general trend that the opioid containing the corresponding opioid for 2 min. agonists (morphine and pethidine) and Similarly, 2 min exposure to plain Ringer was antagonists (naloxone, N methylnaloxone, employed before adding the Ringer con naltrexone and levallorphan) produced an taining Ach. For each set of dose-response inhibition of Ach-induced contractions of the experiments, the rectus muscle was alter toad rectus in a competitive manner. Morphine nately exposed to increasing concentration of did not produce a dose-dependent inhibition. Ach and the corresponding combination of Table 1 shows the averaged differences in Ach and one of the doses of opioid. For a response height (obtained at 16 x 10-6 M Ach dose of each opioid, five or six pairs of dose in the absence and presence of each opioid) response relations were obtained using five and the varying concentrations of the opioids. or six rectus muscles. For each opioid, a linear regression of Drugs: The following drugs were used: differences in heights against concentration Acetylcholine iodide (BDH chemicals, of the opioid used was carried out. The England), morphine hydrochloride (United different regression lines were then compared Pharmaceutical Works Ltd., Holland), for differences in slope and position as shown pethidine hydrochloride (Govt. Pharm. in Fig. 1 . Laboratory, Singapore), (-)naloxone hydro The slopes of the regression lines were chloride and naltrexone hydrochloride (gift compared using the t-test. Table 2 gives a from Endo Laboratories, U.S.A.), N-methyl matrix of the t-values obtained. It can be seen Table 1. Averaged differences in response height and varying concentrations of opioids regression lines. However, when tested for position, the pair of regression lines for levallorphan and dextrallorphan was found to be significantly different (t=13.8; d.f.=56), viz., each unit increase in concentration of the opioid produced different magnitudes of responses. On the other hand, (-)naloxone was found to be grouped with N-methyl naloxone and pethidine. Interestingly, the regression lines obtained for N-methyl naloxone and pethidine did not differ from each other either in slope or position (t=0.04; Fig. 1. Regression lines of differences in responses d.f.=69). It appeared, therefore, that these (height) against concentrations of opioids. 0 MOR: Morphine, x NLX: Naltrexone, 0 NAL=(-) opioids produced the same magnitude of Naloxone, • PET: Pethidine, / MRZ=N-methyl depressant effect on the Ach-induced con naloxone, A DEX=Dextrallorphan, A LEV: Leval tractions. lorphan Discussion that morphine and (-)naltrexone each forms The results obtained clearly show that both a class of its own, the slopes of their re the opioid agonist (pethidine) and antagonists gression lines being significantly different [(-)naloxone, N-methylnaloxone, naltrexone from the rest of the opioids. Among the other and levallorphan] in adequate concentrations opioids, levallorphan and dextrallorphan did can inhibit acetylcholine induced con not significantly differ in the slopes of their tractions of the toad rectus. The inhibitory 76 effect of the opioid agonists might be partly of multiple opiate binding sites on the mediated via a peripheral opiate binding site. skeletal muscle. The significance of each of This proposal is in agreement with the results the sub-types of binding sites and the of other investigators which showed that mechanism by which it affects the contraction morphine and pethidine blocked the muscle remains to be investigated. action potential in frog's sartorius muscle Acknowledgements: This research project fibers (9, 15) and that pethidine depressed the was supported by the Singapore Turf Club. twitch response to nerve stimulation in frog The gifts mentioned in the "Methods" sciatic nerve-sartorius muscle preparation section are gratefully acknowledged. We (12). However, it is not known at present wish to thank Miss Mui Eng Chua and Mr. whether the depressant action of morphine Philip Chua for their expert technical and other opioid agonists involve stereo assistance and Mrs. Lim Li Lin for secretrial specific binding sites. This aspect requires assistance. further analysis with stereoisomers such as levorphanol and dextrorphan and ( References methadone and d-methadone. 1) Paton, W.D.M.: The action of morphine and On the other hand, high concentrations of related substances on contraction and on the opioid antagonists such as (-)naloxone, acetylcholine output of coaxially stimulated N methylnaloxone, naltrexone and leval guinea-pig ileum. Br. J. Pharmacol. Chemother. 12, 119-127 (1957) lorphan also produced inhibition of acetyl 2) Schaumann, W.: Inhibition by morphine on the choline-induced responses. This might be release of acetylcholine from the intestine of partly due to the phenomenon of the "oploid guinea-pig. Br. J. Pharmacol. Chemother. 12, binding site mediated event", as high concen 115-116 (1957) 3) Kosterlitz, H.W.