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A novel opioid peptide, leumorphin, acts as an agonist at the κ opiate receptor
Life Sciences, Vol. 32, pp. 2769-2775 Printed in the U.S.A.
Pergamon Press
A NOVEL OPIOID PEPTIDE, LEUMORPHIN, ACTS AS AN AGONIST AT THE K OPIATE RECEPTOR Mitsuaki Suda, Kazuwa Nakao, Takaaki Yoshimasa, Yoshio Ikeda, Makoto Sakamoto, Noboru Yanaihara, Shosaku Numa & Hiroo Imura 2nd Division, Department of Medicine and Department of Medical Chemistry (S.N.), Kyoto University School of Medicine, Kyoto 606, Shizuoka College of Pharmacy, Shizuoka and National I n s t i t u t e for Physiological Sciences, Okazaki (N.Y.), Japan. (Received in final form April 6, 1983)
Summary The primary structure of the common precursor of porcine Bneo-endorphin and dynorphin (preproenkephalin B) has shown the existence of a t h i r d leucine-enkephalin (leu-enkephalin) sequence with a C-terminal extension of 24 amino acids. This nonacosapeptide, named leumorphin, was approximately 70 times more potent than leuenkephalin in i n h i b i t i n g the contraction of the myenteric plexuslongitudinal muscle preparation of the guinea pig ileum. This action of leumorphin, l i k e those of B-neo-endorphin and dynorphin, was antagonized less e f f e c t i v e l y by naloxone than that of leuenkephalin, but more e f f e c t i v e l y by Mr2266, an antagonist r e l a t i v e l y specific for the K type opiate receptor. The i n h i b i t o r y action of leumorphin or B-neo-endorphin on the contraction of the guinea pig ileum muscle s t r i p was reduced in a dose-dependent manner by pretreatment with dynorphin and vice versa. Leumorphin as well as Bneo-endorphin and dynorphin i ~ i ~ e contraction of the rabbit vas deferens which is known to have only the K type opiate receptor. This action was also e f f e c t i v e l y antagonized by Mr2266. I t is concluded that leumorphin has potent opioid a c t i v i t y and acts at the K receptor, l i k e other opioid peptides derived from preproenkephalin B. The primary structure of the common precursor of B-neo-endorphin and. dynorphin has recently been deduced from the nucleotide sequence of cloned DNA complementary to the porcine hypothalamic mRNAencoding the precursor ( I ) . This precursor protein, designated preproenkephalin B, contains a t h i r d leucineenkephalin (leu-enkephalin) sequence with a C-terminal extension of 24 amino acids. This nonacosapeptide is designated leumorphin. Leumorphin, B-neoendorphin and dynorphin are a l l flanked by paired basic amino acid residues, Lys-Arg. This suggests that these peptides are derived from preproenkephalin B by proteolytic processing. Both dynorphin and B-neo-endorphin are known to exert a potent i n h i b i t o r y effect on the contraction of the guinea pig ileum (2,3), but the action of leumorphin is s t i l l unknown. The present study was performed to elucidate whether or not synthetic porcine leumorphin has opioid a c t i v i t y . Since i t is known that there are multiple opiate receptors, the receptor s e l e c t i v i t y of leumorphin was also examined.
Address correspondence to Kazuwa Nakao, M.D., Second Division of Internal Medicine, Kyoto University School of Medicine, 54 Kawahara-cho, Sho~oin, Sakyoku, Kyoto 606, JAPAN. 0024-3205/83/242769-07503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
2770
Leumorphin is a < Opiate Receptor Ligand
Vol. 32, No. 24, 1983
Materials and Methods I.
Compounds
Porcine leumorphin was synthesized by a solid phase method (4). Dynorphin was obtained from Takeda Chemical Industries, Ltd., leu-enkephalin and methionine-enkephalin (met-enkephalin) from Daiichi Pharmaceutical Co., Ltd., and G- and B-neo-endorphin from Dr. H. Matsuo. Opiate antagonists, Mr2266, (-)2-(3-furylmethyl)-5,9-diethyl-2-hydroxy-6,7-benzomorphan and naloxone hydrochloride were provided by Sandoz Ltd. and Endo Laboratories Inc., respectively. 2.
Bioassay procedure
Male Hartley guinea pigs weighing 300-500 g and male Japanese white rabbits weighing 2.5-4 kg were used. The myenteric plexus-longitudinal muscle s t r i p was prepared as described by Paton and Zar (5). The s t r i p was suspended in a 10 ml organ bath which contained Krebs solution (millimolar concentrations: NaCl 118, KCl 4.75, CaCl2 2.54, KH2PO4 1.19, MgSO4 1.2, NaHCO3 3.25, glucose 11), kept at 36°C and bubbled with 95% 02 and 5% CO2. The rabbit vas deferens was prepared as previously described (6). The bath f l u i d of the vas deferens was Krebs solution without magnesium ions. The resting tension of the myenteric plexus-longitudinal muscle s t r i p and the vas deferens was maintained at I g. The intramural nerves were stimulated through two platinum electrodes with supramaximal rectangular pulses of 1.0 ms duration at 0.1Hz. The e l e c t r i c a l l y induced t w i t c h - l i k e contractions of the s t r i p were recorded isometrically by means of a strain gauge transducer and an ink-writing pen oscillograph. The concentration giving 50 percent i n h i b i t i o n (IC50) of the e l e c t r i c a l l y stimulated muscle twitch was determined by testing at three or more concentrations giving 20 to 80 percent i n h i b i t i o n and then interpolating by l o g - l i n e a r regression analysis. The apparent dissociation constants (Ke) of naloxone and Mr2266 against opioids were determined by the "single dose" method of Kosterlitz and Watt (7). Details of the precedure were published previously (8). For testing the interaction between leumorphin and dynorphin, dose-effect curves of leumorphin were determined in the presence of dynorphin and vice versa. The tissues were pretreated with one agonist for 2.5 min prior to the ~enge with the other, as described by Huidobro-Toro (9). Results Electrical stimulation of the intramural nerves of the guinea pig ileum preparation caused t w i t c h - l i k e contraction. The addition of leumorphin s i g n i f i c a n t l y depressed the e l e c t r i c a l l y evoked contraction of the muscle s t r i p
Fig. I
Effect of porcine leumorphin on the elect r i c a l l y evoked contraction of the myent e r i c plexus-longitudinal muscle preparation of the guinea pig ileum. Leumorphin at a final concentration of 30 nM was added at the point shown by a closed c i r c l e .
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Leumorphin is a K Opiate Receptor Ligand
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TABLE I IC50 Values of Opioid Agonists Tested with Either the Guinea Pig Ileum Myenteric Plexus-Longitudinal Muscle Preparation or the Rabbit Vas Deferens. The IC50 Values Were Determined by Testing the Effects of Three or More Doses and by Interpolating the Linear Regression Line on the Semi-Logarithmic Plot. Values Are Means + Standard Errors of 3-6 Experiments. IC50 (nM) Agonist
Rabbit vas deferens
Guinea pig ileum
Leu-enkephalin
520
L 68
Inactive*
Met-enkephalin
135
L 18
Inactive*
Leumorphin
8.92 +
1.43
98 +
7.7
m-Neo-endorphin
8.68 +
1.21
104 +
8.7
B-Neo-endorphin
26.7
Dynorphin
+ 3.3
1.24 ~
200 + 13.4
0.18
61 ~
8.3
* No s i g n i f i c a n t i n h i b i t i o n at a concentration of 10-4 M.
(Fig. I). Other opioid peptides also inhibited the contraction. The ICso values are listed in Table I. The order of inhibitory potency was: dynorphin> leumorphin= ~-neo-endorphin >B-neo-endorphin >met-enkephalin >leu-enkephalin. The effects of opiate antagonists on the opioid peptide-induced inhibition of the contraction of the guinea pig myenteric plexus-longitudinal muscle strip were studied. As shown in Table II, leumorphin, ~- and B-neo-endorphin and dynorphin were less sensitive to the antagonistic action of naloxone (high Ke values)than met- and leu-enkephalin which had low Ke values. On the other hand, Mr2266, an antagonist relatively specific for the ~ receptor, was as effective as naloxone in antagonizing the inhibitory action of the opioid peptides on the guinea pig ileum muscle strip. Thus, the relative effectiveness
TABLE H Naloxone and Mr2266 Sensitivities of Opioid Agonists Tested with the Myenteric Plexus-Longitudinal Muscle Preparation of the Guinea Pig Ileum.
Agonist
Naloxone Ke (nM)
Mr2266 Ke (nM)
Relative effectiveness of Naloxone to Mr2266
Leu-enkephalin
2.46 + 0.26
2.42 + 0.45
0.98
Met-enkephalin
2.48 + 0.67
3.33 + 0.19
1.34
Leumorphin
16.6
+ 1.8
4.50 + 0.60
0.27
~-Neo-endorphin
13.8
~ 1.1
2.84 ~ 0.48
0.21
B-Neo-endorphin
14.4
~ 0.9
3.34 ~ 0,22
0.23
Dynorphin
15.1
+ 1.5
4.45 + 0.43
0.29
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Leumorphin is a K Opiate Receptor Ligand
Vol. 32, No. 24, 1983
lOO, -r o II" ZL
0 Z
50 ¸
_o I.-
E _z o
o,1 OPIOIDS
( nM
)
Fig. 2 Relationships between doses of porcine leumorphin ( • ) or dynorphin ( • ) and percent i n h i b i t i o n of the e l e c t r i c a l l y evoked contraction of the guinea pig ileum. Effect of the pretreatment with 0.3 nM dynorphin on the i n h i b i t o r y action of graded doses of leumorphin is shown by closed triangles. Dynorphin per se at a concentration of 0.3 nM did not i n h i b i t the muscular twitching by more t--han 30%. Symbols represent the means of 3-6 determinations. of naloxone to Mr2266, a measure of the opiate receptor s p e c i f i c i t y , in antagonizing the action of leumorphin, neo-endorphins and dynorphin was 3 to 5 times less than that for met- and leu-enkephalin. The a b i l i t y of porcine leumorphin to i n h i b i t the e l e c t r i c a l l y induced contraction of the guinea pig ileum was markedly reduced in a dose-related manner by pretreatment with dynorphin, although dynorphin per se behaved as an opiate agonist. As shown in Fig. 2, the IC50 of leumorphin increased more than
a
b
•
•
Fig. 3 Effects of porcine leumorphin, ~- and B-neo-endorphin of the e l e c t r i c a l l y evoked contraction of the rabbit vas deferens. The l e f t part of this figure shows the effect of addition of 150 nM leumorphin (a) and 200 nM naloxone (b). The middle and r i g h t parts show the effect of addition of 200 nM ~-neoendorphin and 500 nM B-neo-endorphin, respectively.
Vol. 32, No. 24, 1983
Leumorphin is a < Opiate Receptor Ligand
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3-fold by pretreatment with 0.3 nM dynorphin. Likewise, in the pretreatment of 2 nM leumorphin the IC50 for dynorphin increased 4-fold. The electrically evoked contraction of the rabbit vas deferens was also inhibited by the addition of leumorphin, ~- and B-neo-endorphin (Fig. 3) and dynorphin. Since the inhibitory action of opioid peptides changes with a lapse of time in this preparation (6), the IC50 values were compared from 4 to 5 h after the suspension of the tissue. As shown in Table I, dynorphin was the most potent, followed by leumorphin, ~- and B-neo-endorphin in that order, whereas leu- and met-enkephalin had no significant effect. Mr2266 was more effective than naloxone in antagonizing the inhibitory action of these opioid peptides on the rabbit vas deferens. However, the Ke values of opiate antagonists could not be determined due to the rapid development of supersensitivities of the rabbit vas deferens to opioid peptides, as previously reported for
agonists (6). In contrast to the results obtained with the guinea pig ileum preparation, pretreatment of the rabbit vas deferens with dynorphin for more than 2 h prior to the challenge with leumorphin rendered the vas more sensitive to leumorphin and vice versa. However, pretreatment with these opioid peptides did not s i g n T f - i - c a ~ a f f e c t the sensitivity of the vas to met- and leu-enkephalin, and u receptor agonists.
Discussion I t has been known that ~-neo-endorphin (10) and dynorphin (2) are more potent in inhibiting the contraction of the guinea pig ileum muscle induced by electrical stimulation than met- and leu-enkephalin. The present study demonstrates that leumorphin, a novel opioid peptide present in the molecule of preproenkephalin B, is as active as ~-neo-endorphin in inhibiting the electrically stimulated muscle contraction of the guinea pig ileum, although a l i t t l e less potent than dynorphin. Since Martin et al (11) f i r s t postulated three different opiate receptors, the concept of multiple opiate receptors has been accepted by many i n v e s t i - gators. Recent studies have established that dynorphin is a specific endogenous ligand for the K type opiate receptor (9,12,13). The present study demonstrates that leumorphin, ~- and B-neo-endorphin also bind to the K opiate receptor, because these peptides inhibited the electrically stimulated contraction of the rabbit vas deferens. Rabbit vas deferens has been considered to have only the K receptor, since K receptor agonists such as dynorphin and ketocyclazocine inhibit its contraction, whereas ~, 6 and o receptor agonists, such as morphine, B-endorphin, enkephalins and N-allylnorcyclazocine have no effect. Additional evidence that leumorphin, ~- and B-neo-endorphin act as K receptor agonists is that Mr2266, a relatively specific K receptor antagonist (13,14,15), is more effective than naloxone, a ~ receptor antagonist, in antagonizing the actions of leumorphin, ~- and B-neo-endorphin on the guinea pig ileum and the rabbit vas deferens. In addition, the naloxone Ke value, which is a measure of receptor selectivity (7), for leumorphin, ~- and B-neoendorphin and dynorphin in the action on the guinea pig ileum was almost the same, 13 to 17 nM, of magnitude as that of other K receptor agonists (13,16). Further evidence supporting the contention that leumorphin acts as an agonist at the K receptor is the observation that pretreatment of the rabbit vas deferens with dynorphin rendered the vas more sensitive to leumorphin and vice versa. This phenomenon has previously been reported for other K agonists TBTf3]-7~n contrast to the effect on the rabbit vas deferens, the pretreatment of a low dose of dynorphin reduced the sensitivity of the guinea pig ileum to leumorphin and ~-neo-endorphin in a dose-related manner and vice versa. This
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Leumorphin is a K Opiate Receptor Ligand
Vol. 32, No. 24, 1983
type of competitive inhibition is commonly observed with antagonists that act reversibly at the same receptor site (17) and all narcotic analgesic drugs have dual agonist and antagonist actions on the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum (18). Taking all these results into consideration, i t is concluded that leumorphin, e- and 6-neo-endorphin and dynorphin act as agonists at the ~ type opiate receptor. In contrast, leu-enkephalin which corresponds to the f i r s t five amino acids of leumorphin, ~- and B-neo-endorphin and dynorphin binds to the ~ and p opiate receptors but to the K receptor. Although i t is s t i l l unknown how preproenkephalin B is processed in a variety of tissues and whether leu-enkephalin is derived from this precursor, our present observations along with other reports suggest that leumorphin, ~- and B-neo-endorphin and dynorphin with their K type opiate receptor constitute a unique opioid peptide system that plays an important physiological role. Acknowledgements We are indebted to Professor H. Matsuo, Department of Biochemistry, Miyazaki Medical College, Miyazaki, Japan, for the generous supply of e- and 6neo-endorphin, to Drs. M. Fujino and M. Wakimasu, Central Research Division, Takeda Chemical Industries, Osaka, Japan, for the generous supply of dynorphin, to Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan, for the g i f t of met- and leu-enkephalin, to Sandoz Ltd. for the g i f t for Mr2266 and to Endo Laboratories Inc. for the g i f t of naloxone. We also thank Dr. K. Kurahashi, Department of Pharmacology, Kyoto University for the valuable technical advice, Drs. S. Oki and I. Tanaka for the valuable discussions and Misses Y. Mitsuda, K. Horii, K. Kitamura and F. Yamauchi for preparing the manuscript. This work was supported in part by research grants from the Japanese Ministry of Health and Welfare, from the Japanese Ministry of Education, Science and Culture and from the Naito Foundation. References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
H. KAKIDANI, Y. FURUTANI, H. TAKAHASHI, M. NODA, Y. MORIMOTO, T. HIROSE, M. ASAI, S. INAYAMA, S. NAKANISHI and S. NUMA, Nature (London) 298 245249 (1982). A. GOLDSTEIN, W. FISCHLI, L.I. LOWNEY, M. HUNKAPILLER and L. HOOD, Proc. Natl. Acad. Sci. U.S.A. 78 7219-7223 (1981). N. MINAMINO, K. KANGAWA,-N. CHINO, S. SAKAKIBARA and H. MATSUO, Biochem. Biophys. Res. Commun. 99 864-870 (1981). Yo YAMAMOTO,C. YANAIHT~I~A, Y. KATSUMARU, T. MOCHIZUKI, A. TOBE, M. EGAWA, H. IMURA, S. NUMAand N. YANAIHARA, Regul. Peptides in press (1983). W.D.M. PATONand M. ABOOZAR, J. Physiol.(London) 194 13-33 (1968). T. OKA, K. NEGISHI, M. SUDA, T. MATSUMIYA, T. INAZ~nd M. UEKI, Eur. J. Pharmacol. 73 235-236 (1980). H.W. KOSTER]]TTZand A.J. WATT, Br. J. Pharmac. Chemother. 33 266-276 (1968). T. OKA, K. NEGISHI and M. SUDA, Neurotransmitter Receptors: Biological Aspect and Physiological Significnace, H.I. Yamamura and H. Yoshida (Eds.), John Wiley and Sons, New York, in press (1981). J.P. HUIDOBRO-TORO, K. YOSHIMURA, N.M. LEE, H.H. LOH and E.L. WAY, Eur. J. Pharmacol. 72 265-266 (1981). K. KANGAWA,-]~. MINAMINO, N. CHINO, S. SAKAKIBARA and H. MATSUO, Biochem. Biophys. Res. Commun. 99 871-878 (1981). W.R. MARTIN, Life Sci__28 1547-1554 (1981). C. CHAVKIN, I.F. JAMES a ~ A . GOLDSTEIN, Science 215 413-415 (1982). T. OKA, K. NEGESHI, M. SUDA, A. SAWA, M. FUJINO an~T-f~. WAKIMASU, Eur. J. Pharmacol. 77 137-141 (1982).
Vol. 32, No. 24, 1983
14. 15. 16. 17. 18.
Leumorphin is a K Opiate Receptor Ligand
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J.A.H. LORD, A.A. WATERFIELD, J. HUGHESand H.W. KOSTERLITZ, Nature (London) 267 495-499 (1977). D. ROEMER~. BUESCHER, R.C. HILL, R. MAURER, l . J . PETCHER, H.B.A. WELLE, H.C.C.K. BAKEL and A.M. AKKERMAN, Life Sci. 27 971-978 (1980). M. HUTCHINSON, H.W. KOSTERLITZ, F.M. LESLIE a ~ A . A . WATERFIELD, Br. J. Pharmacol. 55 541-546 (1975). A.G. GILMAN-~-S.E. MAYERand K.L. MELMON, The Pharmacological Basis of Therapeutics, A.G. Gilman, L.S. Goodman and A. Gilman (Eds.), pp. 28-39, Macmillan, New York (1980). E.A. GYANGand H.W. KOSTERLITZ, Br. J. Pharmacol. Chemother. 27 514-527 (1966).
Pergamon Press
A NOVEL OPIOID PEPTIDE, LEUMORPHIN, ACTS AS AN AGONIST AT THE K OPIATE RECEPTOR Mitsuaki Suda, Kazuwa Nakao, Takaaki Yoshimasa, Yoshio Ikeda, Makoto Sakamoto, Noboru Yanaihara, Shosaku Numa & Hiroo Imura 2nd Division, Department of Medicine and Department of Medical Chemistry (S.N.), Kyoto University School of Medicine, Kyoto 606, Shizuoka College of Pharmacy, Shizuoka and National I n s t i t u t e for Physiological Sciences, Okazaki (N.Y.), Japan. (Received in final form April 6, 1983)
Summary The primary structure of the common precursor of porcine Bneo-endorphin and dynorphin (preproenkephalin B) has shown the existence of a t h i r d leucine-enkephalin (leu-enkephalin) sequence with a C-terminal extension of 24 amino acids. This nonacosapeptide, named leumorphin, was approximately 70 times more potent than leuenkephalin in i n h i b i t i n g the contraction of the myenteric plexuslongitudinal muscle preparation of the guinea pig ileum. This action of leumorphin, l i k e those of B-neo-endorphin and dynorphin, was antagonized less e f f e c t i v e l y by naloxone than that of leuenkephalin, but more e f f e c t i v e l y by Mr2266, an antagonist r e l a t i v e l y specific for the K type opiate receptor. The i n h i b i t o r y action of leumorphin or B-neo-endorphin on the contraction of the guinea pig ileum muscle s t r i p was reduced in a dose-dependent manner by pretreatment with dynorphin and vice versa. Leumorphin as well as Bneo-endorphin and dynorphin i ~ i ~ e contraction of the rabbit vas deferens which is known to have only the K type opiate receptor. This action was also e f f e c t i v e l y antagonized by Mr2266. I t is concluded that leumorphin has potent opioid a c t i v i t y and acts at the K receptor, l i k e other opioid peptides derived from preproenkephalin B. The primary structure of the common precursor of B-neo-endorphin and. dynorphin has recently been deduced from the nucleotide sequence of cloned DNA complementary to the porcine hypothalamic mRNAencoding the precursor ( I ) . This precursor protein, designated preproenkephalin B, contains a t h i r d leucineenkephalin (leu-enkephalin) sequence with a C-terminal extension of 24 amino acids. This nonacosapeptide is designated leumorphin. Leumorphin, B-neoendorphin and dynorphin are a l l flanked by paired basic amino acid residues, Lys-Arg. This suggests that these peptides are derived from preproenkephalin B by proteolytic processing. Both dynorphin and B-neo-endorphin are known to exert a potent i n h i b i t o r y effect on the contraction of the guinea pig ileum (2,3), but the action of leumorphin is s t i l l unknown. The present study was performed to elucidate whether or not synthetic porcine leumorphin has opioid a c t i v i t y . Since i t is known that there are multiple opiate receptors, the receptor s e l e c t i v i t y of leumorphin was also examined.
Address correspondence to Kazuwa Nakao, M.D., Second Division of Internal Medicine, Kyoto University School of Medicine, 54 Kawahara-cho, Sho~oin, Sakyoku, Kyoto 606, JAPAN. 0024-3205/83/242769-07503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
2770
Leumorphin is a < Opiate Receptor Ligand
Vol. 32, No. 24, 1983
Materials and Methods I.
Compounds
Porcine leumorphin was synthesized by a solid phase method (4). Dynorphin was obtained from Takeda Chemical Industries, Ltd., leu-enkephalin and methionine-enkephalin (met-enkephalin) from Daiichi Pharmaceutical Co., Ltd., and G- and B-neo-endorphin from Dr. H. Matsuo. Opiate antagonists, Mr2266, (-)2-(3-furylmethyl)-5,9-diethyl-2-hydroxy-6,7-benzomorphan and naloxone hydrochloride were provided by Sandoz Ltd. and Endo Laboratories Inc., respectively. 2.
Bioassay procedure
Male Hartley guinea pigs weighing 300-500 g and male Japanese white rabbits weighing 2.5-4 kg were used. The myenteric plexus-longitudinal muscle s t r i p was prepared as described by Paton and Zar (5). The s t r i p was suspended in a 10 ml organ bath which contained Krebs solution (millimolar concentrations: NaCl 118, KCl 4.75, CaCl2 2.54, KH2PO4 1.19, MgSO4 1.2, NaHCO3 3.25, glucose 11), kept at 36°C and bubbled with 95% 02 and 5% CO2. The rabbit vas deferens was prepared as previously described (6). The bath f l u i d of the vas deferens was Krebs solution without magnesium ions. The resting tension of the myenteric plexus-longitudinal muscle s t r i p and the vas deferens was maintained at I g. The intramural nerves were stimulated through two platinum electrodes with supramaximal rectangular pulses of 1.0 ms duration at 0.1Hz. The e l e c t r i c a l l y induced t w i t c h - l i k e contractions of the s t r i p were recorded isometrically by means of a strain gauge transducer and an ink-writing pen oscillograph. The concentration giving 50 percent i n h i b i t i o n (IC50) of the e l e c t r i c a l l y stimulated muscle twitch was determined by testing at three or more concentrations giving 20 to 80 percent i n h i b i t i o n and then interpolating by l o g - l i n e a r regression analysis. The apparent dissociation constants (Ke) of naloxone and Mr2266 against opioids were determined by the "single dose" method of Kosterlitz and Watt (7). Details of the precedure were published previously (8). For testing the interaction between leumorphin and dynorphin, dose-effect curves of leumorphin were determined in the presence of dynorphin and vice versa. The tissues were pretreated with one agonist for 2.5 min prior to the ~enge with the other, as described by Huidobro-Toro (9). Results Electrical stimulation of the intramural nerves of the guinea pig ileum preparation caused t w i t c h - l i k e contraction. The addition of leumorphin s i g n i f i c a n t l y depressed the e l e c t r i c a l l y evoked contraction of the muscle s t r i p
Fig. I
Effect of porcine leumorphin on the elect r i c a l l y evoked contraction of the myent e r i c plexus-longitudinal muscle preparation of the guinea pig ileum. Leumorphin at a final concentration of 30 nM was added at the point shown by a closed c i r c l e .
Vol. 32, No. 24, 1983
Leumorphin is a K Opiate Receptor Ligand
2771
TABLE I IC50 Values of Opioid Agonists Tested with Either the Guinea Pig Ileum Myenteric Plexus-Longitudinal Muscle Preparation or the Rabbit Vas Deferens. The IC50 Values Were Determined by Testing the Effects of Three or More Doses and by Interpolating the Linear Regression Line on the Semi-Logarithmic Plot. Values Are Means + Standard Errors of 3-6 Experiments. IC50 (nM) Agonist
Rabbit vas deferens
Guinea pig ileum
Leu-enkephalin
520
L 68
Inactive*
Met-enkephalin
135
L 18
Inactive*
Leumorphin
8.92 +
1.43
98 +
7.7
m-Neo-endorphin
8.68 +
1.21
104 +
8.7
B-Neo-endorphin
26.7
Dynorphin
+ 3.3
1.24 ~
200 + 13.4
0.18
61 ~
8.3
* No s i g n i f i c a n t i n h i b i t i o n at a concentration of 10-4 M.
(Fig. I). Other opioid peptides also inhibited the contraction. The ICso values are listed in Table I. The order of inhibitory potency was: dynorphin> leumorphin= ~-neo-endorphin >B-neo-endorphin >met-enkephalin >leu-enkephalin. The effects of opiate antagonists on the opioid peptide-induced inhibition of the contraction of the guinea pig myenteric plexus-longitudinal muscle strip were studied. As shown in Table II, leumorphin, ~- and B-neo-endorphin and dynorphin were less sensitive to the antagonistic action of naloxone (high Ke values)than met- and leu-enkephalin which had low Ke values. On the other hand, Mr2266, an antagonist relatively specific for the ~ receptor, was as effective as naloxone in antagonizing the inhibitory action of the opioid peptides on the guinea pig ileum muscle strip. Thus, the relative effectiveness
TABLE H Naloxone and Mr2266 Sensitivities of Opioid Agonists Tested with the Myenteric Plexus-Longitudinal Muscle Preparation of the Guinea Pig Ileum.
Agonist
Naloxone Ke (nM)
Mr2266 Ke (nM)
Relative effectiveness of Naloxone to Mr2266
Leu-enkephalin
2.46 + 0.26
2.42 + 0.45
0.98
Met-enkephalin
2.48 + 0.67
3.33 + 0.19
1.34
Leumorphin
16.6
+ 1.8
4.50 + 0.60
0.27
~-Neo-endorphin
13.8
~ 1.1
2.84 ~ 0.48
0.21
B-Neo-endorphin
14.4
~ 0.9
3.34 ~ 0,22
0.23
Dynorphin
15.1
+ 1.5
4.45 + 0.43
0.29
2772
Leumorphin is a K Opiate Receptor Ligand
Vol. 32, No. 24, 1983
lOO, -r o II" ZL
0 Z
50 ¸
_o I.-
E _z o
o,1 OPIOIDS
( nM
)
Fig. 2 Relationships between doses of porcine leumorphin ( • ) or dynorphin ( • ) and percent i n h i b i t i o n of the e l e c t r i c a l l y evoked contraction of the guinea pig ileum. Effect of the pretreatment with 0.3 nM dynorphin on the i n h i b i t o r y action of graded doses of leumorphin is shown by closed triangles. Dynorphin per se at a concentration of 0.3 nM did not i n h i b i t the muscular twitching by more t--han 30%. Symbols represent the means of 3-6 determinations. of naloxone to Mr2266, a measure of the opiate receptor s p e c i f i c i t y , in antagonizing the action of leumorphin, neo-endorphins and dynorphin was 3 to 5 times less than that for met- and leu-enkephalin. The a b i l i t y of porcine leumorphin to i n h i b i t the e l e c t r i c a l l y induced contraction of the guinea pig ileum was markedly reduced in a dose-related manner by pretreatment with dynorphin, although dynorphin per se behaved as an opiate agonist. As shown in Fig. 2, the IC50 of leumorphin increased more than
a
b
•
•
Fig. 3 Effects of porcine leumorphin, ~- and B-neo-endorphin of the e l e c t r i c a l l y evoked contraction of the rabbit vas deferens. The l e f t part of this figure shows the effect of addition of 150 nM leumorphin (a) and 200 nM naloxone (b). The middle and r i g h t parts show the effect of addition of 200 nM ~-neoendorphin and 500 nM B-neo-endorphin, respectively.
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Leumorphin is a < Opiate Receptor Ligand
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3-fold by pretreatment with 0.3 nM dynorphin. Likewise, in the pretreatment of 2 nM leumorphin the IC50 for dynorphin increased 4-fold. The electrically evoked contraction of the rabbit vas deferens was also inhibited by the addition of leumorphin, ~- and B-neo-endorphin (Fig. 3) and dynorphin. Since the inhibitory action of opioid peptides changes with a lapse of time in this preparation (6), the IC50 values were compared from 4 to 5 h after the suspension of the tissue. As shown in Table I, dynorphin was the most potent, followed by leumorphin, ~- and B-neo-endorphin in that order, whereas leu- and met-enkephalin had no significant effect. Mr2266 was more effective than naloxone in antagonizing the inhibitory action of these opioid peptides on the rabbit vas deferens. However, the Ke values of opiate antagonists could not be determined due to the rapid development of supersensitivities of the rabbit vas deferens to opioid peptides, as previously reported for
agonists (6). In contrast to the results obtained with the guinea pig ileum preparation, pretreatment of the rabbit vas deferens with dynorphin for more than 2 h prior to the challenge with leumorphin rendered the vas more sensitive to leumorphin and vice versa. However, pretreatment with these opioid peptides did not s i g n T f - i - c a ~ a f f e c t the sensitivity of the vas to met- and leu-enkephalin, and u receptor agonists.
Discussion I t has been known that ~-neo-endorphin (10) and dynorphin (2) are more potent in inhibiting the contraction of the guinea pig ileum muscle induced by electrical stimulation than met- and leu-enkephalin. The present study demonstrates that leumorphin, a novel opioid peptide present in the molecule of preproenkephalin B, is as active as ~-neo-endorphin in inhibiting the electrically stimulated muscle contraction of the guinea pig ileum, although a l i t t l e less potent than dynorphin. Since Martin et al (11) f i r s t postulated three different opiate receptors, the concept of multiple opiate receptors has been accepted by many i n v e s t i - gators. Recent studies have established that dynorphin is a specific endogenous ligand for the K type opiate receptor (9,12,13). The present study demonstrates that leumorphin, ~- and B-neo-endorphin also bind to the K opiate receptor, because these peptides inhibited the electrically stimulated contraction of the rabbit vas deferens. Rabbit vas deferens has been considered to have only the K receptor, since K receptor agonists such as dynorphin and ketocyclazocine inhibit its contraction, whereas ~, 6 and o receptor agonists, such as morphine, B-endorphin, enkephalins and N-allylnorcyclazocine have no effect. Additional evidence that leumorphin, ~- and B-neo-endorphin act as K receptor agonists is that Mr2266, a relatively specific K receptor antagonist (13,14,15), is more effective than naloxone, a ~ receptor antagonist, in antagonizing the actions of leumorphin, ~- and B-neo-endorphin on the guinea pig ileum and the rabbit vas deferens. In addition, the naloxone Ke value, which is a measure of receptor selectivity (7), for leumorphin, ~- and B-neoendorphin and dynorphin in the action on the guinea pig ileum was almost the same, 13 to 17 nM, of magnitude as that of other K receptor agonists (13,16). Further evidence supporting the contention that leumorphin acts as an agonist at the K receptor is the observation that pretreatment of the rabbit vas deferens with dynorphin rendered the vas more sensitive to leumorphin and vice versa. This phenomenon has previously been reported for other K agonists TBTf3]-7~n contrast to the effect on the rabbit vas deferens, the pretreatment of a low dose of dynorphin reduced the sensitivity of the guinea pig ileum to leumorphin and ~-neo-endorphin in a dose-related manner and vice versa. This
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Leumorphin is a K Opiate Receptor Ligand
Vol. 32, No. 24, 1983
type of competitive inhibition is commonly observed with antagonists that act reversibly at the same receptor site (17) and all narcotic analgesic drugs have dual agonist and antagonist actions on the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum (18). Taking all these results into consideration, i t is concluded that leumorphin, e- and 6-neo-endorphin and dynorphin act as agonists at the ~ type opiate receptor. In contrast, leu-enkephalin which corresponds to the f i r s t five amino acids of leumorphin, ~- and B-neo-endorphin and dynorphin binds to the ~ and p opiate receptors but to the K receptor. Although i t is s t i l l unknown how preproenkephalin B is processed in a variety of tissues and whether leu-enkephalin is derived from this precursor, our present observations along with other reports suggest that leumorphin, ~- and B-neo-endorphin and dynorphin with their K type opiate receptor constitute a unique opioid peptide system that plays an important physiological role. Acknowledgements We are indebted to Professor H. Matsuo, Department of Biochemistry, Miyazaki Medical College, Miyazaki, Japan, for the generous supply of e- and 6neo-endorphin, to Drs. M. Fujino and M. Wakimasu, Central Research Division, Takeda Chemical Industries, Osaka, Japan, for the generous supply of dynorphin, to Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan, for the g i f t of met- and leu-enkephalin, to Sandoz Ltd. for the g i f t for Mr2266 and to Endo Laboratories Inc. for the g i f t of naloxone. We also thank Dr. K. Kurahashi, Department of Pharmacology, Kyoto University for the valuable technical advice, Drs. S. Oki and I. Tanaka for the valuable discussions and Misses Y. Mitsuda, K. Horii, K. Kitamura and F. Yamauchi for preparing the manuscript. This work was supported in part by research grants from the Japanese Ministry of Health and Welfare, from the Japanese Ministry of Education, Science and Culture and from the Naito Foundation. References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
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