Upsala J Med Sci 78: 150-152, 1973 Stereospecific Uptake of Narcotic Analgesics by a Subcellular Fraction of the Guinea-pig Ileum A Preliminary Communication LARS TERENIUS From the Department o f Pharmacology, University of Uppsala, Uppsala, Sweden ABSTRACT Longitudinal muscle of guinea-pig ileum was homogenized. Precipitates from centrifugation were incubated with la- belled dihydromorpbine and competitors. The uptake of label to a high-speed fraction was stereospecific (the analgesics levorphanol and levo-methadone were more active competbrs than their inactive dextro-counterparts). Morphine, normorphine, dextromoramide, the antagonists nalorphine and naloxone inhibited uptake. Codeine was inactive. INTRODUCTION The mode of action of narcotic analgesics remains obscure in spite of the intense work which has been attributed to the problem. I t is even largely unknown a t which site(s) in the CNS they exert their analgesic action. Therefore, the longitudinal muscle of the guinea-pig ileum, which is anatomi- cally well-defined and which is affected by mor- phine-like analgesics in vitro (Schaumann, 1957; Weinstock, 1971) has considerable interest as a model tissue. The present communication analyses the inter- action between narcotic analgesics and subcellular fractions of the longitudinal muscle from guinea- pig ileum. Evidence is provided that there is a specific interaction. Part of this work has been presented a t the Scandinavian Pharmacological Society Meeting, Uppsala, June 1972 (Terenius, 1972). MATERIALS AND METHODS Guinea-pigs of either sex weighing at least 450 g were killed by a blow on the head. In a typical experiment (12-18 incubation flasks, see below) 5 animals were used. The ileum except for 10-15 cm of the terminal part was removed. Longitudi- nal muscle strips were prepared from 10-15 cm Upsalu J Med Sci 78 segments by the method of Rang (1964), carefully avoiding mesenteric fat. The muscle with adhering nerve plexi was finely cut with a pair of scissors. All subsequent operations were at 0” to 4OC. Ten times the weight of ice-cold 0.32 M sucrose in bidistilled water was added. Homogenization was done with an Ultraturrax (Kinematica, Luzern) 2 times for 15 sec at setting 2 with cooling for 2 min inbetween. The homogenate was further dis- integrated in a glass homogenizer with a loose- fitting Teflon pestle at low speed. It was then centrifuged at 2 X 1000 g for 10 min. The super- natant was saved and centrifuged a t 120000 g for 30 min. In a few experiments the 2 x 1000 g supernatant was centrifugated at 10000 g for 30 min, see text. The respective pellets were re- suspended in a small volume of 0.32 M sucrose. Aliquots of the suspension containing about 1 mg protein were added to 4 ml of incubation buffer, NaCl 124 mM, KC1 5 mM, KH2P0, 1.2 mM, CaCl, 0.75 mM, MgSO, 1.3 mM, N-2-hydroxy- ethylpiperazine-N’-2-ethanesulfonic acid “HEPES” 26 mM, pH 8.0 a t 25°C; this buffer is modified from Bradford & Thomas (1969) which in ap- propriate cases also contained non-labelled test substances. After a pre-incubation period of 10 minutes a t 25°C with the test substances, d i h y d r ~ m o r p h i n e - ~ H (specific activity 66.6 mCi/ mg, radiochemical purity 95-98 % ) in 0.2 ml in- cubation buffer was added, and the incubation was continued for 1 hour. The incubation was stopped by cooling the vessels on ice. The content was quantitatively transferred to centrifuge tubes and centrifuged for 30 min a t 120 000 g. Aliquots (0.5 ml) of the supernatant were removed for radioactivity measurement. The remaining super- natant was discarded, and the pellets were rapidly washed with ice-cold buffer. The tubes were im- mediately inversed and allowed to stand in an in- Uptake of narcotic analgesics 151 Table I. Competition experiment showing the stereo- specificity of the uptake of labelled dihydromorphine to the 120 000gparticulate fraction (Expts. 1-2) or the 10 000 gparticulate fraction (Expts. 3-4) of guinea-pig ileum The concentration of dihydromorphine was 0.55 x M, of levorphanol, dextrorphan and of the methadone antipodes 1 . 1 x lo-* M. Each experiment represents one homogenate, each treatment was run in duplicate Uptake (DPM/mg protein) Competitor Expt. 1 2 3 4 Table 11. Effect of various analgesics and analgesic antagonists on the uptake (DPMlmg protein) of labelled dihydromorphine (concentration 0.7 x M ) to a high-speedparticulate fraction of guinea-pig ileum A different homogenate was used for each substance. Means + S . E . M . are given for 3 samples per group Concentration (M) of competitor Substance 0 10-9 10-8 10-7 Morphine 9 8 1 f 5 4 794+50 585+3 508f55 Dextromora- mide 953+46 1 0 1 2 k 2 9 845+34 6 1 5 5 3 7 Normorphine 1 0 6 9 5 1 6 9 0 5 i 6 7 9 8 5 2 0 6 5 8 i 4 8 Nalorphine I 2 9 6 f 4 2 1 0 2 7 5 4 4 9 5 1 k 3 4 570+24 Naloxone 1 1 2 1 1 3 1 805f32 5 7 1 k 3 2 3 9 5 5 1 3 Codeine 1 2 5 6 + 1 2 - 1 2 2 5 5 1 8 1 2 1 9 5 6 2 N o n e 761 844 1 1 1 8 1 2 5 2 Levorphanol 330 346 807 810 Dextrorphan 653 674 1052 1 0 6 4 None 647 759 650 634 1019 1 0 5 6 999 1003 Levo-Methadone 432 424 483 436 665 736 619 626 Dextro- Methadone 565 765 107 742 985 997 847 920 versed position for a t least one hour. The pellets were digested with Soluenea (Packard, Downers Grove, Ill.) and the radioactivity content meas- ured. Protein was determined according to Lowry et al. (1951) using bovine serum albumin as stand- ard. RESULTS Competition experiments were performed in order to establish the specificity of the interaction be- tween dihydromorphine and the high-speed sub- cellular fraction. It was found that the optical antipodes, levorphanol/dextrorphan and levo-/ dextro-methadone differentially inhibited the up- take of dihydromorphine to the high-speed precipi- tate, the analgetically more active levo-antipodes being the more active competitors (Table I, expts. 1 and 2). In experiments 3 and 4 (Table I) is shown that the stereospecific uptake is also present in the 10000 g precipitate. Other narcotic analgesics (morphine, normor- phine and dextromoramide) and the narcotic an- tagonists (nalorphine and naloxone) were active competitors in the test system while codeine was without effect (Table 11). DISCUSSION Morphine-like compounds inhibit the contractions of the longitudinal muscle of the guinea-pig ileum (see Schaumann, 1957; Weinstock, 1971). This tissue should therefore contain morphine receptors and an interaction with such receptors might be observable in simple incubation experiments. I n view of the very strict steric requirements for activity on the ileum (Schaumann e t al., 1953) (which seem identical to those for analgesic ac- tivity) one might expect a similar selectivity in the binding properties of the receptor. The present work shows a stereoselective inter- action with the opiates levorphanol and dextror- phan as competitors and as well with the all synthetic, structurally completely different metha- done antipodes. Those isomers which are more potent on the ileum (Schaumann et al., 1953) are the more active competitors (Table I). Also an- other synthetic analgesic, dextromoramide, showed considerable competitive activity. On the other hand, codeine is practically inactive as a compe- titor (Table 11) as on the ileum preparation (Kos- terlitz & Watt, 1968). All this evidence favors the assumption that the observed interaction is of fundamental importance for the action of mor- phine-like drugs and that it could be ascribed to an interaction with a receptor. I t is also notable that the narcotic antagonists, nalorphine and naloxone, are active as competi- tors (Table 11). Although most narcotic antago- nists including nalorphine have agonistic proper- ties, naloxone is held to be a pure antagonist (also on the ileum preparation, Kosterlitz & Watt, 1968) indicating that the antagonistic activity of nar- cotic antagonists may depend on competition with the analgesics for the receptors. Also normorphine is active as competitor (Table 11), which is of interest since this compound has been implicated Upsala J Med Sci 78 152 L. Terenius in the action of morphine (Beckett et al., 1956). Preliminary experiments with further fractiona- tion (combined with enzymological and morpho- logical examination) indicate that particles rich in acetylcholinesterase and 5’-nucleotidase and not mitochondria are responsible for the uptake. ACKNOWLEDGMENT Technical assistance was given by Mrs Ulla Staav. Nor- morphine was kindly donated by D r E. L. May at N.I.H., B-thesda, Md, USA, naloxone by D r H. W. Kosterlitz, University of Aberdeen, Scotland, and levorphanol and dextrorphan by Hoffmann-La Roche, Basel, Switzerland. The work was supported by the Swedish Medical Re- search Council (Proj. No. K72-14X-3756-01). REFERENCES 1. Beckett, A. H., Casy, A . F. & Harper, N. J.: Anal- gesics and their antagonists: some steric and chemical considerations. J Pharm Pharmacol 8: 874, 1956. 2. Bradford, H. F. & Thomas, A. J.: Metabolism of glucose and glutamate by synaptosomes from mam- malian cerebral cortex. J Neurochem 16: 1495, 1969. 3. Kosterlitz, H. W. & Watt, A. J.: Kinetic parameters of narcotic agonists and antagonists, with particular reference t o N-allylnoroxy-morphone (Naloxone). Brit J Pharmacol 33:266, 1968. 4. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L. & Ran- dall, R. J.: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265, 1951. 5. Rang, H. P.: Stimulant actions of volatile anaesthetics on smooth muscle. Brit J Pharmacol 22:356, 1964. 6. Schaumann, 0.: Morphin und morphinahnlich wirkende Verbindungen. I n Handbuch der experimentellen Phar- makologie, Erganzungswerk (ed. 0. Elchler & A. Farah), vol. 12, pp. 170-189. Springer, Berlin, 1957. 7. Schaumann, O., Jcchum, K. & Schaumann, W.: Anal- getika und Darmmotorik. 11. Wirkung auf den Langs- muskeltonus. Arch Pharmakol 217: 360, 1953. 8. Terenius, L.: Specific uptake of narcotic analgesics by subcellular fractions of the guinea-pig ileum. Acta Pharmacol Toxicol 3I:Suppl. I, 50, 1972. 9. Weinstock, M.: Sites of action of narcotic analgesic drugs: i n peripheral tissues. In Narcotic Drugs, Bio- chemical Pharmacology (ed. D. H. Clouet), pp. 394- 407. Plenum Press, New York. Received December 15, 1972 Address for reprints: L. Terenius Department of Pharmacology University of Uppsala Box 573, 751 23 Uppsala Sweden Upsala J M e d Sci 78