Proc. Nati. Acad. Sci. USA Vol. 82, pp. 1860-1863, March 1985 Neurobiology

,u and K opioids inhibit transmitter release by different mechanisms (synaptic potentials/potassium conductance/calcium action potentials/dynorphin/morphine) E. CHERUBINI AND R. A. NORTH Neuropharmacology Laboratory, Massachusetts Institute of Technology, 56-245, Cambridge, MA 02139 Communicated by Hamish N. Munro, November 13, 1984

ABSTRACT The actions ofvarious opioids were examined myenteric plexus neurones when they are excited by a brief on calcium action potentials in the cell somata of guinea pig electrical field stimulus (6, 19). In such assays, all agonists myenteric neurones and on the release of acetylcholine at inhibit the release of AcCho, but the ,u and K receptors synapses onto these cells. The opioids morphine, normor- responsible for this action can be differentiated by their phine, and [D-Ala2, MePhe4, Mete(O)]enkephalin-ol caused sensitivity to blockade by naloxone (6, 19) and 3-funal- membrane hyperpolarizations resulting from an increase in trexamine (/3-FNA) (26-29). The purpose of the present potassium conductance; opioids that are more selective agon- experiments was to investigate the ionic mechanisms ists for the Kc receptor subtype (dynorphin, tifluadom, through which ,- and K-receptor agonists might inhibit the U50488H) did not. Conversely, calcium action potentials were release ofAcCho. The amount ofAcCho released from a few depressed or abolished by the K opioids but were not affected by morphine and [D-Ala2, MePhe4, Met(O)5]enkephalin-ol. presynaptic nerves was assayed by recording the amplitude Both groups of opioids caused presynaptic inhibition of acetyl- of the excitatory postsynaptic potential (EPSP). The cell choline release in the myenteric plexus, depressing the ampli- bodies of these presynaptic nerves are contained also within tude of the fast excitatory postsynaptic potential. The the myenteric plexus; the actions of ;u and K agonists on the presynaptic inhibition caused by [D-Ala2, MePhe , Met(O)s]- properties of the soma membrane were therefore compared. enkephalin-ol, morphine, and normorphine, but not that caused by the K opioids, was prevented by pretreatment with MATERIALS AND METHODS the selective ,u site-directed irreversible antagonist ,B- Intracellular recordings were made with microelectrodes funaltrexamine. Furthermore, the presynaptic inhibitory ac- containing potassium chloride (3 M) from 98 neurones in tion of morphine and [D-Ala2, MePhe4, Met(O)s]enkephalin- myenteric ganglia dissected from the ileal wall of adult ol, but not that of the K-receptor agonists, was reversibly guinea pigs. A piece of tissue comprising several ganglia blocked by barium. The results suggest that presynaptic adherent to the longitudinal muscle layer was superfused inhibition caused by IA receptor activation probably results with the following physiological salt solution at 37°C: 117 from an increase in potassium conductance, whereas mM NaCl/4.7 mM KCl/1.2 mM NaH2PO4/1.2 mM K-receptor agonists may depress the release of acetylcholine by MgCl2/2.5 mM CaCl2/25 mM NaHCO3/11 mM glucose, directly reducing calcium entry into the nerve terminals. gassed with 95% 02/5% C02 (pH 7.4). Drugs were applied by changing this solution to one that differed only in its There are three major classes ofopioid peptides, represented content of the drug. The superfusing solution was pumped at by the enkephalins, ,B-endorphin, and dynorphin. They are 1-2 ml-min-'; the ratio of flow rate to bath volume ensured synthesized independently and have distinct distributions in complete exchange of the solution in 1 min. The period of the mammalian nervous system (1-4). Several types of drug application was 2-5 min, during which time any effect opioid receptor have also been distinguished, the best doc- reached steady state; 15-30 min was allowed for washing umented of which are now termed ,u, 8, and K (5-9). It is between drug applications. EPSPs were evoked by applying generally thought that these receptors are cell-surface mol- a single pulse (typically 1-ms duration) of electric current to ecules that recognize one or more of the opioid peptides and that the peptide binding leads to a change in function of the the presynaptic nerves entering the ganglion, by means of a cell. One important functional change is a reduction in the saline-filled micropipette. In some experiments, tbe EPSPs amount of transmitter released by the neurone when it is were mimicked by applying AcCho iontophoretically from a excited, but the ionic mechanisms that underlie this may be third micropipette positioned within 5 ,m of the membrane different for the different receptors. For example, occupa- of the neurone from which the intracellular recording was tion of the A-receptor subtype leads to an increase in made. Full details of these techniques have been published membrane potassium conductance in several mammalian (29, 30). neurones (10-14), and this may reduce transmitter release by The following drugs (and their sources) were used. Ac- shortening the duration of the presynaptic action potential Cho chloride (Sigma), dynorphin A (Peninsula Laborato- (15). On the other hand, the K-receptor agonist dynorphin ries, San Carlos, CA), morphine sulphate (Mallinckrodt), has been found to reduce calcium action potentials in somata normorphine hydrochloride (National Institute on Drug of cultured dorsal root ganglion cells without affecting potas- Abuse), [D-Ala2, MePhe4, Met(O)5]enkephalin-ol (desig- sium conductance (16, 17). nated FK33824, Sandoz Pharmaceutical), trans-(+)-3,4- The guinea pig myenteric plexus contains all three types of dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzene- opioid receptor (6-9, 18-21) as well as several endogenous acetamide methanesulphonate (designated U50488H, Up- ligands (22-25). Pharmacological assays of the receptors in john), tifluadom (Sandoz Pharmaceutical), 6-(P-fumaram- this tissue generally depend upon the ability ofthe agonist to ate)methyl ester of naltrexamine ([,B-funaltrexamine inhibit the release of acetylcholine (AcCho) from the ,B-FNA), P. S. Portoghese and A. E. Takemori, Univ. of Minnesota], tetrodotoxin (TTX, Sigma). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: /3-FNA, ,B-funaltrexamine; EPSP, excitatory post- in accordance with 18 U.S.C. §1734 solely to indicate this fact. synaptic potential; AcCho, acetylcholine. 1860 Downloaded by guest on September 25, 2021 Neurobiology: Cherubini and North Proc. Natl. Acad. Sci. USA 82 (1985) 1861 A

1/ t \, a m " Control1\~~~~~~~--4FK33824 Wash Dynorphin Wash

Control P-FNA Wash FK33824 Wash Dynorphin Wash

B

Control Tifluadom (1 PM) Naloxone , -, 110 mV (100 nM) (3 PM) 5 ms FIG. 1. Both ,u- and K-receptor agonists depress the cholinergic EPSP; ,-FNA selectively blocks the action of the p-receptor agonist. Each trace is four averaged fast EPSPs evoked by single-pulse stimuli (repeated at 0.05 Hz) to the presynaptic nerve entering the ganglion. (A) Superfusion with FK33824 (30 nM), dynorphin (50 nM), and P-FNA (300 nM) each caused a reversible depression of the EPSP. After washing out the 3-FNA, the effect of the P-receptor agonist FK33824 was lost, though the action of dynorphin remained. (B) The depression of the fast EPSP by another K-receptor agonist (tifluadom) was reversed by naloxone. RESULTS (100 nM), 48.4 ± 5.7% (n = 5). The depression of the EPSP Depression of the EPSP. Morphine (10 nM to 1 AM), by the K agonists was unaffected by barium, even in the same normorphine (100 nM to 5 riM), and FK33824 (10-100 nM) cells in which barium prevented the action of normorphine depressed the EPSP amplitude. An example of the effect of (Fig. 2). Pretreatment with f3-FNA did not block the effect of FK33824 is shown in Fig. 1. Normorphine was applied to 22 K agonists, even when it blocked the depressant action of neurones; the mean depression of the EPSP caused by 1 morphine on the same EPSP. 0-FNA itself reversibly de- AM pressed the EPSP, as would be expected for a K agonist. The was 32.6 ± 1.9% (n = 12; this and other values are means ± SEM for the number of observations shown). The effect of depressant action of the K agonists was also blocked by normorphine and FK33824 was readily blocked by naloxone naloxone (100 nM to 1 MM). Nicotinic depolarizations (1-100 nM) (n = 7); it was also irreversibly blocked by evoked by iontophoretic application of AcCho were not affected K P-FNA (n = 6). The P-FNA (200-300 nM) was applied for by the agonists. 20-30 min and washed out for 30 min prior to testing with Depression of the Calcium Action Potential. The finding normorphine or FK33824. The inhibitory action of morphine that barium was able to discriminate between the effects of was still blocked 6 hr after exposure to p-FNA. The action of dynorphin and FK33824 on the EPSP suggested that the K normorphine was presynaptic because normorphine did not agonists might inhibit AcCho release by an action that did affect depolarizations that mimicked the EPSP produced by not involve an increase in potassium conductance. Another iontophoretic application of AcCho to the cell membrane (n possible mechanism of action is a direct reduction in inward = 5). calcium currents close to the release sites. If it is assumed Morphine and normorphine also hyperpolarize some that calcium entry in the cell soma has the same sensitivity to neurones in the myenteric plexus by increasing the mem- opioids as calcium entry close to the AcCho release site, brane potassium conductance (13); it was possible that such then a study of the calcium action potential in the soma may a potassium conductance increase occurring at or near the provide information about the mechanism of action of AcCho release sites might contribute to the presynaptic opioids on AcCho release. This assumption is likely to be inhibition. We tested this by adding a low concentration of valid in the myenteric plexus because the somata of the barium to the superfusing solution; barium (200-300 AuM) neurones that supply the cholinergic presynaptic terminals prevented the hyperpolarizing action of morphine recorded are themselves within the myenteric ganglia. Therefore, we at the cell soma, as it has previously been shown to do in observed action potentials in the cell somata in the presence neurones of the rat locus coeruleus (15). These concentra- of tetrodotoxin; after addition of tetrodotoxin, the action tions of barium were chosen because they did not change the potentials were reversibly abolished by calcium ion removal shape of the intracellularly recorded action potential and had or by addition of divalent cations such as cobalt (30, 32, 33). Dynorphin and tifluadom depressed or even abolished the a small effect (less than 5-mV or no on depolarization) effect calcium action potentials without having any consistent the postsynaptic membrane potential. Barium completely effect on the resting potential (10 of 22 neurones). prevented the depressive action of morphine on the EPSP (n The possibility that a potassium conductance increase = 3). contributed to the depression of the calcium action potential Dynorphin and the other K-receptor agonists also revers- was tested by repeating the experiments with cesium chlo- ibly depressed the EPSP (EPSPs in 34 neurones were tested ride in the recording microelectrodes; this also delayed with both morphine or FK33824 and a K agonist). The repolarization of the action potential and prolonged its percentage depression of the EPSP caused by some selected duration from about 2 ms to several hundred milliseconds. In concentrations of the K agonists were: tifluadom (300 nM), these circumstances also, the K agonists greatly reduced the 51.6 ± 4.3% (n = 6); U50488H (10 AM), 46.6 ± 5.8% (n = duration and amplitude of the action potential (Fig. 3). This 5); dynorphin (30 nM), 32.0 ± 3.7% (n = 3); and dynorphin effect was seen in 22 of 42 neurones. The effective concen- Downloaded by guest on September 25, 2021 1862 Neurobiology: Cherubini and North Proc. Natl. Acad. Sci. USA 82 (1985) A Control

-I q % Control Normorphine (5 PM) Control Dynorphin (80 nM)

B Barium (300 pM) 10 mV 5 me

N. ( D h0 Control Normorphine (5 PM) Control Dynorphin (80 nMl) FIG. 2. Barium selectively prevents the presynaptic inhibition by normorphine. (A) Both normorphine (5 AM) and dynorphin (80 nM) depressed the EPSP by approximately the same amount. (B) After addition of barium (300 kLM), the effect of normorphine was lost but the depression by dynorphin remained. Barium itself depolarized the cell by 3-4 mV and slightly prolonged the EPSP; the EPSPs were evoked after repolarizing the membrane to its control potential. trations were dynorphin (10-30 nM), tifluadom (300 nM), tions of [D-Ala2-Met5]enkephalinamide had an effect similar U50488H (5-10 ,M), and P-FNA (200-300 nM), Percentage to that which we found with dynorphin (35). That action also reductions in action potential duration for some K agonist was observed in the presence of barium, suggesting that it concentrations were: tifluadom (300 nM), 43.1 ± 6.8% (n = resulted from a direct depression of calcium entry rather 10); tifluadom (1 PM), 53.3 ± 12.5% (n = 3); and than an effect secondary to a potassium-conductance in- dynorphin (30 nM), 60.4 ± 14.4% (n = 5). Morphine had no crease. The effect was correlated with an inhibition of effect on the duration of the action potential (see also ref. substance P release from the same cell cultures, measured 34). The shortening of the action potential by dynorphin was by radioimmunoassay. A similar depression of the calcium very similar to that caused by cobalt, indicating that it action potential has been reported in amphibian probably resulted from a reduction of calcium entering Rohon-Beard neurones, but again the agonist used was through voltage-dependent channels. Naloxone (1-10 gM) likely to be nonselective at the concentration used (36). In prevented the action of the K agonists (n = 3). the light of the present findings, it is possible that these effects result from activation of receptors of the K subtype. DISCUSSION Werz and MacDonald recently communicated that action The present findings suggest that the mechanism by which potentials in mouse dorsal root ganglion cells in culture were opioids inhibit AcCho release at interneuronal cholinergic depressed by opioids and suggested that ,t and 8 ligands synapses in the myenteric plexus is not common to ,u- and might affect a potassium conductance and K agonists might K-receptor agonists. The potassium conductance that is depress calcium entry (16, 17). Thus, the present findings increased by activation of ,u receptors is remarkably sensi- Werz and but also extend them tive to barium (ref. 15 and unpublished data); the finding that confirm those of MacDonald the depression of the EPSP by morphine and FK33824 is in two ways. First, our classification of the receptor type prevented by barium suggests that a potassium conductance depends not only on selectivity of agonists but on the use of increase on presynaptic fibers may be involved in the action the selective antagonist 13-FNA; second, we were able to of these compounds. In contrast, the K agonists, but not estimate transmitter release directly from the amplitude of morphine or FK33824, markedly inhibited the entry of the EPSP. calcium into myenteric neurones in conditions in which Kandel (37) has distinguished two primary mechanisms by potassium conductance increases were eliminated. which drugs or transmitters might act presynaptically to Earlier studies on the action potential of immature chick inhibit release of the same or a different transmitter. The first dorsal root ganglion cells showed that rather high concentra- is direct modulation of calcium entry; an example of a A B

Normorphine U50488-H (10 pM) U50488-H (10 pM) Tifluadom A -FNA (1 P1M) Naloxone (10 pM) (300 nM) (300 nM) I 20 mV 20 mV 800 me I 280 ma

FIG. 3. The calcium action potential is reduced by K- but not by ,u-receptor agonists. The action potentials were greatly prolonged because the recording electrode contained cesium chloride. (A) Four superimposed traces are shown in each record. The one marked by the arrow was taken during superfusion with the drug indicated; the filled circle marks the control action potential, and the asterisk marks the action potential after washing out the drug. The unmarked traces show the effect of the agonist before they reached maximum. Notice that tifluadom and P-FNA completely abolished the action potential in the same neurone in which normorphine was without effect. The actual order of application of drugs to this cell was normorphine, tifluadom, and ,-FNA. (B) In another cell U50488H shortened the action potential, and this action was much reduced in the concomitant presence of naloxone. The symbols beside the three superimposed traces have the same meaning as in A. Downloaded by guest on September 25, 2021 Neurobiology: Cherubini and North Proc. Natl. Acad. Sci. USA 82 (1985) 1863 substance that appears to act in this way is y-aminobutyric 8. Chavkin, C., James, I. F. & Goldstein, A. (1982) Science 215, acid (acting on B-type receptors) (38, 39). The present 413-415. results suggest that dynorphin, acting on K receptors, be- 9. Corbett, A. D., Paterson, S. J., McKnight, A. T., Magnan, J. & Kosterlitz, H. W. (1982) Nature (London) 299, 79-81. longs to this class. The second is indirect modulation of 10. Williams, J. T., Egan, T. M. & North, R. A. (1982) Nature transmitter release, by which is meant a change in calcium (London) 299, 74-76. entry that occurs secondarily as a result of an altered 11. Pepper, C. M. & Henderson, G. (1980) Science 209, 394-396. potassium conductance. For example, 5-hydroxytryptamine 12. Yoshimura, M. & North, R. A. (1983) Nature (London) 305, appears to increase transmitter release by reducing a potas- 529-530. sium conductance at certain Aplysia synapses (40). Mor- 13. Morita, K. & North, R. A. (1981) Brain Res. 242, 145-150. phine (acting on ,u receptors) (14, 15) and catecholamines 14. Williams, J. T. & North, R. A. (1984) Mol. Pharmacol. 26, (acting on a2 receptors) (41, 42) reduce calcium entry into rat 489-497. locus coeruleus neurones by increasing potassium conduct- 15. North, R. A. & Williams, J. T. (1983) Br. J. Pharmacol. 80, ance; such an action at transmitter release sites would cause 225-228. 16. Werz, M. A. & MacDonald, R. L. (1982) Brain Res. 239, presynaptic inhibition. 315-321. The three families of opioid peptides are synthesized from 17. MacDonald, R. L. & Werz, M. A. (1983) Soc. Neurosci. distinct precursors (1-3). Three main subtypes of receptor Abstr. 9, 1129. have been described, and the suggestion has been made that 18. Goldstein, A. & James, I. F. (1984) Mol. Pharmacol. 25, these receptors might bind preferentially the agonist ligands 343-348. derived from one particular family. Neurotransmitters such 19. James, I. F. & Goldstein, A. (1984) Mol. Pharmacol. 25, as noradrenaline, AcCho, and y-aminobutyric acid also each 337-342. act on more than one receptor subtype; distinct patterns of 20. Leslie, F. M., Chavkin, C. & Cox, B. (1980) J. Pharmacol. membrane conductance can be associated with each Exp. Ther. 214, 395-411. change 21. Gintzler, A. R. & Scalisi, J. A. (1982) Brain Res. 238, 254-259. of the individual subtypes. Our experiments show that 22. Watson, S. J., Akil, H., Ghazarossian, V. E. & Goldstein, A. distinct functional consequences can follow activation of (1981) Proc. NatI. Acad. Sci. USA 76, 1260-1268. different subtypes of a neuropeptide receptor. Furthermore, 23. Tachibana, S., Araki, K., Ohya, S. & Yoshida, S. (1982) activation of both ,u or K subtypes of the opioid receptor Nature (London) 295, 339-341. leads to a reduction of the amount of transmitter released by 24. Vincent, S. R., Dalsgaard, C.-J., Schultzberg, M., Hokfelt, T., the nerve cell, but this is brought about by a different Christensson, I. & Terenius, L. (1984) Neuroscience 11, intermediate step. Where both g and K receptors coexist on 973-987. the same nerve fibers, as seems to be the case in the 25. Schultzberg, M., Hokfelt, T., Nilsson, G., Terenius, L., myenteric plexus (Fig. 1), the possibility is raised that ,u and Rehfeld, J. F., Brown, M., Elde, R. P., Goldstein, M. & Said, S. (1980) Neuroscience 5, 689-744. K agonists might act synergistically to inhibit neurotrans- 26. Ward, S. J., Portoghese, P. S. & Takemori, A. E. (1982) Eur. mitter release. The different mechanisms of action of the J. Pharmacol. 80, 377-384. different classes of opioids could prove favorable for the 27. Ward, S. J., Portoghese, P. S. & Takemori, A. E. (1982) Eur. development of therapeutic agents which combine agonist J. Pharmacol. 85, 163-170. actions at both , and K receptors. 28. Portoghese, P. S., Larson, D. L., Sayre, L. M., Fries, D. S. & Takemori, A. E. (1982) J. Med. Chem. 23, 234-236. This work was made possible only by the generous gift of 3-FNA 29. Ward, S. J., Portoghese, P. S. & Takemori, A. E. (1983) J. from Drs. P. S. Portoghese and A. E. Takemori. Dr. John Williams Pharmacol. Exp. Ther. 220, 494-498. made valuable suggestions throughout the course of the experi- 30. Nishi, S. & North, R. A. (1973) J. Physiol. (London) 231, ments. This work was supported by grants from the U.S. Depart- 471-491. ment of Health and Human Services, DA03160 and AM/NS 32979. 31. North, R. A. & Tokimasa, T. (1982) J. Physiol. (London) 333, 151-156. 1. Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, 32. Hirst, G. D. S. & Spence, I. (1973) Nature (London) 243, A. C. Y., Cohen, S. N. & Numa, S. (1981) Nature (London) 54-56. 278, 423-427. 33. North, R. A. (1973) Br. J. Pharmacol. 49, 709-711. 2. Noda, M., Furutani, Y., Takahashi, H., Toyosato, M., Hirose, 34. Cherubini, E., Morita, K. & North, R. A. (1984) Br. J. T., Inayama, S., Nakanishi, S. & Numa, S. (1982) Nature Pharmacol. 81, 617-622. 202-206. 35. Mudge, A. W., Leeman, S. E. & Fischbach, G. D. (1979) (London) 295, Proc. Natl. Acad. Sci. USA 76, 526-530. 3. Kakidani, H., Furutani, Y., Takahashi, H., Noda, M., 36. Bixby, J. L. & Spitzer, N. C. (1983) J. Neurosci. 3, 1014-1018. Morimoto, Y., Hirose, T., Asai, M., Inayama, S., Nakanishi, 37. Kandel, E. R. (1981) Nature (London) 293, 697-700. S. & Numa, S. (1982) Nature (London) 298, 245-249. 38. Dunlap, K. (1981) Br. J. Pharmacol. 74, 579-585. 4. Hollt, V. (1983) Trends Neurosci. 6, 21-23. 39. Cherubini, E. & North, R. A. (1984) Br. J. Pharmacol. 82, 5. Lord, J. A. H., Waterfield, A. A., Hughes, J. & Kosterlitz, 101-105. H. W. (1977) Nature (London) 267, 495-500. 40. Klein, M. & Kandel, E. R. (1980) Proc. NatI. Acad. Sci. USA 6. Chavkin, C. & Goldstein, A. (1981) Nature (London) 291, 77, 6912-6917. 591-593. 41. Williams, J. T., Henderson, G. & North, R. A. (1984) 7. Chavkin, C. & Goldstein, A. (1981) Proc. Natl. Acad. Sci. Neuroscience, in press. USA 78, 6543-6547. 42. Williams, J. T. & North, R. A. (1984) Neuroscience, in press. Downloaded by guest on September 25, 2021