E'~ropean Journal of Pharmacology, 212 (1992) 283- 286 ~.9 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/S05.00
283
EJP 21013 Short communication
The ~-opioid activi~ of ~-opioid receptor agonist compounds in the guinea pig ileum I l o n a P. B c r z c t e i - G u r s k e a n d L a w r e n c e Toll SRI International, Menlo Park, CA, U.S.A. Received 12 September 1991, revised MS received 27 December 1991, accepted 7 January 1992
On the basis of their in vivo activity and binding affinity, nalorphinc and (--)SKF 10,047 were classified as mixed agonist/antagonist compounds. However, in isolated tissue preparations without a selective antagonist to block their agonist effect, the characterization of these compounds and the determination of their antagonist activity were ve~' difficult. Nor-binaltorphimine, a selective ~-opioid receptor antagonist, was used in the longitudinal muscle preparations of the guinea pig ileum to block the n-agonist activity of nalorphine and ( --)SKF 10,047. In the absence of their ~-agonist activity, we were able to determine the p.-antagonist activity using the p.-sclcctive agonist DAMGO ([D-Ala2,N-Me-Phc4,Gly-olS]enkcphalin). The pA 2 values for nalorphinc and ( - ) S K F 1.0,047 were 7.50 and 7.69, respectively. Opioid receptor agonist/antagonist (mixed); DAMGO ([D-AIa2,N-Me-Phe4,GIy5-ol]enkephalin); Nor-binaltorphiminc; Ileum (guinea-pig)
1,. Introduction
Martin and coworkers (Martin et al., 1976) recognized early that a single opioid compound might have divergent activities on different receptors (i.e., it could block a biological response mediated by one receptor type yet evoke a distinct biok)gical response mediated by a second receptor type). These mixed a g o n i s t / antagonists, of which nalorphine is perhaps prototypic, tend to have high binding affinity at both p.- and K-opioid receptors and are potent agonists on the g3inea pig ileum preparation (Magnan et al., 1982). They fail to display antinociceptive action in many a:aimal test procedures but effectively reduce abdominal constrictions induced by various chemical irritants (e.g., phenylquinone, acctylcholine, and bradykinin) in the so-called writhing test (Shaw et al., 1988). In humans, such compounds have analgesic activity equivalent to or better than morphine yet precipitate withdrawal in addicts (Keats and Telford, 1956). One attraction of these dualists is their likelihood of having a low physical dependence capacity; this is essentially true of nalorphine, although the drug cannot be used
Correspondence to: I.P. Berzctci-Gurske, SRI International, Department of Ncuroscicnce, Building LA, Room 105, 333 Ravcqswood Avenue, Menlo Park, C'A 94025, U.S.A. Tel. 1.415.859 3354, fax 1.J,15.859 3153.
clinically because of its undesirable psychotomimctic side effects (Telford ct al., 1961). Although several studies suggested its ~-antagonist activity (Unna, 1943; Telford et al., 1961) and partial K-agonist activity (Leandcr, 1983), nalorphine has also bccn suggested to bc a /.t-agonist ( Z i m m e r m a n ct al., 1987). No detailed in vitro analysis has been performed because of the lack of a specific K-antagonist compound. With the recent introduction of the selective K-antagonist norbinaltorphiminc (Nor-BNI) by Portoghese et al. (1987), the antagonist effect of mixed agonists/antagonists on the longitudinal muscle of the guinea pig ileum can be studied. The compounds selected for our study were nalorphine, ( - )SKF 10,047, and (+)cthylketocyclazocine (EKC).
2. Materials and methods
2.1. Longitudinal muscle strip of guinea pig ileum Male Hartley guinea pigs weighing 350-400 g were decapitated and the small intestine removed. The longitudinal muscle with the myentcric plexus attached was gently separated from the underlying circular muscle by the method of Paton and Vizi (1969). The muscle strips were mounted in an 8-ml water-jacketed organ bath containing Krebs bicarbonate solution of the following composition (in mM): NaCI 118; CaCl 2
284
2.5; KCI 4.7; NaHCO3 25; K H 2 P O 4 1.2; MgSO 4 1.2 and glucose 11.5. The tissues were kept at 37°C and bubblcd with 5% CO 2 in oxygen. An initial tension of 1.0 g was applied to the strips. The tissues were electrically stimulated for 60 min before the start of the experiments. Field electrical stimulation was applied, delivered through platinum wire electrodes positioned at the top and bottom of the organ bath, and kept at a fixed distance (3.5 cm apart). The uppcr clcctrodc was a ring 4 mm in diameter. The parameters of rectangular stimuli wcrc supramaximal voltage, 1 ms impulse duration at 0.1 Hz. A Grass S-88 electrostimulator was used. The electrically induced twitches were recorded by using an isometric transducer (Metrigram) coupled to a multichanncl polygraph (Grass 7D). 2.2. Kinetics The agonist potencies of compounds were determined from concentration rcsponsc curves and characterized by the ICs0 values. Agonists were prescnt for 3 - 6 rain or until maximal inhibitory effect was produccd. Dose response curves were constructed in a cumulative fashion for the ~-agonists because of slow onset and washout of agonist activity. The percentage of inhibition of the stimulation-induced contraction produced by each agonist was plotted against the log agonist concentration. IC5, is defined as the concentration of the agonist that produces 50% inhibition of the contraction. Antagonists wcrc present for at least 30 rain. Antagonist activities were calculated for each single tissue from full concentration response curves before and after the addition of a single antagonist concentration. D R (dose ratio) is the shift of the agonist concentration response curve in the presence of the antagonist. At least three different concentrations wcrc used, and only one antagonist concentration was tested on each tissue, pA~_ values were determined from Schild plots (Arunlakshana and Schild, 1959). Each individual point was plotted, and pAz was determined by using a statisticaI least-square regression analysis program package (R. Barlow, U.K.). 2.3. Materials Drugs were obtained from commercial sources, except for the following: Nor-binaltorphimine dihydrochloride from RBI; nalorphinc hydrochloridc, (_+)ethylketocyclazocine-succinatc, D A M G O ([D-AIa2,N-Me Phe4,Gly-ol5]enkephalin]), ( - ) S K F 10,047.HC1, CTAP-NH 2 (D-Phc-Cys-Tyr-D-Trp-Arg-Thr-Pen-ThrNH2), U 69,593 (5a,7tr,8/3)-(+)-N-methyl-N-[7-(l-pyrrolidinyl)-l-oxa-spiro-(4,5)-dec-8-yl], and morphine sulfate from NIDA.
3. Results
The longitudinal lnusclc of the guinea pig ileum is known to contain p~- and ~-opioid receptors (Chavkin and Goldstcin, 1981). To determine whether the ICs0 values measured in the guinea pig ileum came from the activation of b~- or ~-receptors, experiments were conducted in the presence of either a 100-nM concentration of the selective ~-reccptor antagonist ( ~ A P (Kramcr ct al., 1988) or 211 nM of the selective ~-reccptot antagonist Nor-BNI. The ICs0 wdue of the ~,-selectivc compound D A M G O was increased with a dose ratio of approximately 5 by administration of CTAP, whereas the IC~0s of the u-selective compounds U 69,593, ethylketocyclazocine, nalorphinc, and SKF 10,047 were not increased. Surprisingly, the affinities of the x-agonists apparently increased in the presence of ( ~ A P , having dose ratios less than 1.0. This however turned out to be an artifact of the experimental protocol. IC.s0 values decrease in both control and Cl'AP-treated tissues after prolonged incubations. This indicated that the ileum becomes more sensitive with incrcascd time in the tissue bath. When experiments were conducted in the presence of the ~:-sclcctivc antagonist Nor-BNI, D A M G O and morphine had only a 1.7- and 1.05-fold shift, respccliveIy, while the ~selective compound U 69,593 had a 360-fold shift. Table 1 contains the results of both experiments, showing that, in addition to U 69,593, nalorphine, SKI: 10,047, and cthylkctocyclazocine arc all apparently acting through the ~:-receptors in the guinea pig ileam. The rank order of ~-agonist potency is: ethylketocyciazocine > U 69,593 > SKF 10,047 > nalorphinc. In the next set of experiments, the ~¢-agonist activity of nalorphine, e~:hylketocyclazocine and SKF 10,047 has been blocked with 20 nM Nor-BNI, which was present throughout the experiment in the Krebs solu-
'FABI,E 1 IC~0 and dose ratio values of various opioid agonists with CTAP and Nor-BNI on guinea pig ileum preparation. Means + S.E. Compound
DAMGO Morphine U69,593 (-.-)Ethylketocyclazocinc Nalorphinc (-)SKF10,047
IC'5t~(nM)
8.25_* 2.0 (13) 24.75:- 2.4 (4) 1.66= 0.63 (12) 11.44___ 0.14 (8) 29.2 ± 15 115) 10.5 + 3.9 (7)
Dose ratio with (;TAP (100 nM)
Nor-BNI (20 nM)
4.98±0.3 (4) 4.27 (2) 0.68+0.11 (4) 0.60_+0.09 (4) 0.911 (2) 11.44 (2)
1.74.-: 0.14 (6) 1.05 (2) 363.11 +97 (8) 61.2 j: 8.8 (8) 643.0 (2) 273.1 (2)
285 TABLE 2 p~,~ values and Schild slope for the antagonists nalorphine and ( - ) S K F 10,047 on the guinea pig ileum using the/z-selective agonist D A M G O in the presence of 20 nM Nor-BNI. Antagonist
pA 2 value
Schild slope
Nalorphine ( - ) S K F 10,047 ( ± )Ethylketocyclazocinc Naloxonc
7.49 7.69 ~ 8.53 ~
- 0.873 - 1.216 - 0.99
a There was no shift in thc presence of (_+.)ethylketocyclazocinc. ~ The experiment with naloxone was without Nor-BNI.
tion. Under these conditions, only ethylketocyclazocine maintained significant agonist activity, which allowed us to determine the ability of SKI" 10,047 and nalorphine to antagonize the /x-agonist D A M G O with 20 nM Nor-BN1 present in the tissue bath. The concentration response curves for the /x-agonist D A M G O were conducted in the presence of various concentrations of SKF 10,047 and nalorphine to determine the pA2 vzduc for their antagonist activity. Table 2 contains both pA 2 values as well as the slope of the Schild plot. As a comparison, the pA~ value and Schild slope of naloxone, determined in the absence of Nor-BNI, are also shown in table 2. The rank order of the /Xantagonist potency is: naloxone > SKF 10,047 > nalorphine. Although the binding affinity at the /xopioid receptor is almost the same for naloxone and nalorphine, there is a 10-fold difference in the /xantagonist potency. The binding results of cthylkctocyclazocine show equal affinity at /X- and K-receptors (Magnan c t a l . , 1982). Because it still had substantial agonist activity in the prcscnce of Nor-BN1, we planned an experiment to determine whether ethylketoc3'clazocine would also activate the/x-receptors if the number of K-receptors was reduced. A concentration response curve was made with ethylketocyclazocine in the presence of 20 nM Nor-BNI; then the tissue was incubated with 100 nM CTAP for 30 min, and the ethylketocyclazocinc concentration response curve was repeated. In the presence of CTAP in addition to Nor-BNI, there was a further 2-fold shift in the ICs0 value for ethylketocyclazocine, proving that, under these conditions, ethylketocyclazocine was also activating thc /x-receptors of the guinea pig ileum preparation.
4. Discussion
Nalorphine was the first narcotic antagonist made available for medical use in 1952. Its activity in animals a'~; a morphine antagonist was demonstrated by Unna (1943). Keats and Telford (1956) demonstrated that it
was equipotent with morphine as an analgesic in the clinic. As a mixed agonist/antagonist compound, nalorphine was studied in the guinea pig ileum by Gyang and Kosterlitz in 1966 and by Kosterlitz and Watt in 1968. By the single-dose method, they were able to show the morphine antagonist activity of this compound as well as that of SKF 10,047. A later study on the rat vas deferens, which does not contain K-opioid receptors, also showed that nalorphine worked as a /x-antagonist. However, both these studies are incompletc. A Schild plot to determine the type of antagonism cannot be constructed by the single-dose method. Furthermore, although the rat vas deferens has no K-receptors, this tissue apparently has very few /x-receptors, for even morphine is an antagonist in this preparation (Wuster et al., 1980). With the introduction of the highly selective K-antagonist compound Nor-BNI, the/x-activity of compounds acting as full or partial agonists at the K-opioid receptors could be studied fully for the first time. As a whole, the results shown in table 1 are not surprising. The potencies of /x-agonists D A M G O and morphine are reduced by the /x-antagonist C I A P but not by the K-antagonist Nor-BNI. Conversely, the 'Kagonists' U 69,593, ethylketocyclazocinc, nalorphine, and SKF 10,047 are inhibited by Nor-BNI but not by CTAP. Interestingly, unlike the other agonists, EKC is still a potent compound even in the presence of 20 nM Nor-BNI (ICs0 value ~ 27 nM). The other K-agonists had no agonist activity in the presence of Nor-BNI when used at nanomolar concentrations. These data correspond nicely to the results found in /3-FNA treated tissue, another '/x-less' preparation. In fl-FNA treated tissue the potencies of nalorphine, SKF 10,047 and ethylketoc3'clazocine do not greatly change, though the IC.~0 of morphine and D A M G O shift significantly to the right. Again this indicates that the agonist activity of ethylketocyclazocinc, nalorphine, and SKF 10,047 is through the K-receptor in the GPI (Hayes et al., 1985, and unpublished observation of the authors). A lack of agonist activity in the presence of Nor-BNI made possible the examination of these compounds as antagonists of the /x-agonist DAMGO. Under these conditions, both nalorphine and SKF 10,047 were potent /x-antagonists, with pA 2 values approximately one-tenth that of naloxone. It is interesting that naloxone, SKF 10,047, and nalorphine all have comparable /x-binding affintites (Magnan et al., 1982) but naloxone is a more potent antagonist. This may be due to a small amount of intrinsic activity in nalorphine and SKF 10,047. If they were very low efficacy agonists they may not manifest agonist activity in the GPI but would require high receptor occupancy to antagonize the activity of DAMGO. Perhaps if a tissue was found with more spare receptors these compound would act as agonists. This may explain the observation by Zimmer-
286
man et al. (1987) that nalorphine can act as a #-agonist in the acetic acid writhing test in mice. Because cthylkctocyclazocine maintained significant agonist activity in the presence of Nor-BNI, its ability to act as a /x-antagonist could not bc detcrmined. Furthermore, the question remained as to whether the residual activity was duc to ~-agonism not blocked by 20 nM Nor-BNI or to /x-agonism, now revcaled by blocking of ~:-receptors. When the ileum was incubated with CTAP in addition to Nor-BNI, the 1C50 of cthylkctocyclazocine was shifted further to the right. This indicates that ethylkctocyclazocine can act as a ~zagonist in the GPI. In conclusion, the availability of the selective ~:antagonist Nor-BNI permits the study of compounds with high affinit3, at both g- and x-opioid receptors at #-receptors on the GP1. Of the ~(-agonist compounds tested, nalorphinc and SKF 10,047 possessed potent p.-antagonist activity in the GPI, however the '~: agonist' ethylketo-cyclazocinc is apparently also an agonist at the ~-receptor.
Acknowledgements This work was supported by Grant DA06682 to I,.T. from the National Institute on Drug Abuse. We thank Valorie Wcizcr for her competent technical assistance and Rosic McCormick for typing the manuscript.
References Arunlakshana, O. and II.O. Schild, 1959, Somc quantitative uses of drug antagonists, Br. J. Pharmacol. 14, 48. Chavkin, C. and A. Goldstcin, 1981, Demonstration of a specific dynorphin receptor in guinea pig ileum myenteric plexus, Nature 291,591. Gyang, E.A. and tI.W. Kostcrlitz, 1966, Agonist and antagonisl actions of morphine-like drugs on the guinea-pig isolated ileum, Br. J. Pharmacol. 27, 514.
Hayes, A.G., M.J. Shcchan anti M.B. Thyers, 1985, Determination of the receptor selectivity of opioid agonists in the guinea-pig ileum and mouse vas defercns by use of /3-funaltrcxamine, Br. J. Pharmacol. 86, 899. Keats, A.S. and J. Telford, 1956, Nalorphine, a potent analgesic in man, J. Pharmacol. Exp. Ther. 117, I90. Kosterlitz, H.W. and A.J. Watt, 1968, Kinetic parameters of narcotic agonists and antagonists, with particular reference to N-alIylnoroxymorphone (naloxone), Br. J. Pharmacol. Chexnother. 33, 266. Kramer, T., W. Kazmierski, J. Shook. V. Hruby and T. Burks, 1988. A novel peptidic mu opioid antagonist with exceptional potency and specificity, in: National Institute on Drug Abuse Research Monograph, Problems of Drug Dependence, Vol. 90, cd. I. llarris (DHIIS publication) p. 47. t.eander, J.D., 1983, Evidence that nalorphine, butorphanol and oxilorphan are partial agonists at a K-opioid receptor, Eur. J. Pharmacol. 86, 467. Magnan, J., S.J. Paterson, A. Tavani and H.W. Kostcrlitz, 1982, The binding spectrum of narcotic analgesic drugs with different agonist and antagonist properties, Naunyn-Schrniedeb. Arch. Pharmacol. 319, 197. Martin, W.R., C.G. Eades, J.A. Thompson, R.E. ltuppler ~nd P.E. Gilbert, 1976, Thc effects of morphine- and nalorphinc-like drugs in the m~ndcpcndent and morphine-dependent chronic spir~al dog, J. Pharmacol. Exp. Thor. 197, 517. Paton, W.D.M. and E.S. Vizi, 1969, "l'hc inhibitory action of noradrenaline and adrenaline ou acctylcholinc output on guinea-pig hmgitudinaI muscle strip, Br. J. Pharmacol. 35, 10. Portoghese; P.S., A.W. Lipkowski and A.E. Takemori, I987, Binaltorphimine and norbinaltorphimine, potent and selective ~-opioid receptor antagonists, Life Sci. 40, 1287. Shaw, J.S., J.D. Ronrke and K.M. Burns, 1988, Differential scnsitivily of antinociccptivc lests to opioid agonists and partial agonists, Br. J. Pharmacol. 95, 578. Telford, 5., C.N. PapadOpoulos and A.S. Keats, 1961, Morphine antagonists as analgesics, J. Pharmacoi. Exp. Ther. 133, 116. Unna, K., 1943, Antagonistic effect of N-allylnormorphine upon m~rphine, J. Pharmacol. Exp. Ther. 79, 27. Wiistcr, M., R. Schulz and A. llerz, 1980, Opioid agonists and antagonists: Action on multiple opiate receptors, in: t:~ndogenous and Exogenous Opiate Agonists and Antagonists, cd. E.L. Way (Pergamon Press, New York) p. 75. Zirnmerman, D.M., J.D. Leander, J.K. Reel and M.D. tlynes, I987, Use of /3-funaltrcxamine t~) dctcrmine mu opioid receptor involvement in the analgesic activity of various opioid ligands, J. Pharmacol. Exp. Thcr. 241, 374.
283
EJP 21013 Short communication
The ~-opioid activi~ of ~-opioid receptor agonist compounds in the guinea pig ileum I l o n a P. B c r z c t e i - G u r s k e a n d L a w r e n c e Toll SRI International, Menlo Park, CA, U.S.A. Received 12 September 1991, revised MS received 27 December 1991, accepted 7 January 1992
On the basis of their in vivo activity and binding affinity, nalorphinc and (--)SKF 10,047 were classified as mixed agonist/antagonist compounds. However, in isolated tissue preparations without a selective antagonist to block their agonist effect, the characterization of these compounds and the determination of their antagonist activity were ve~' difficult. Nor-binaltorphimine, a selective ~-opioid receptor antagonist, was used in the longitudinal muscle preparations of the guinea pig ileum to block the n-agonist activity of nalorphine and ( --)SKF 10,047. In the absence of their ~-agonist activity, we were able to determine the p.-antagonist activity using the p.-sclcctive agonist DAMGO ([D-Ala2,N-Me-Phc4,Gly-olS]enkcphalin). The pA 2 values for nalorphinc and ( - ) S K F 1.0,047 were 7.50 and 7.69, respectively. Opioid receptor agonist/antagonist (mixed); DAMGO ([D-AIa2,N-Me-Phe4,GIy5-ol]enkephalin); Nor-binaltorphiminc; Ileum (guinea-pig)
1,. Introduction
Martin and coworkers (Martin et al., 1976) recognized early that a single opioid compound might have divergent activities on different receptors (i.e., it could block a biological response mediated by one receptor type yet evoke a distinct biok)gical response mediated by a second receptor type). These mixed a g o n i s t / antagonists, of which nalorphine is perhaps prototypic, tend to have high binding affinity at both p.- and K-opioid receptors and are potent agonists on the g3inea pig ileum preparation (Magnan et al., 1982). They fail to display antinociceptive action in many a:aimal test procedures but effectively reduce abdominal constrictions induced by various chemical irritants (e.g., phenylquinone, acctylcholine, and bradykinin) in the so-called writhing test (Shaw et al., 1988). In humans, such compounds have analgesic activity equivalent to or better than morphine yet precipitate withdrawal in addicts (Keats and Telford, 1956). One attraction of these dualists is their likelihood of having a low physical dependence capacity; this is essentially true of nalorphine, although the drug cannot be used
Correspondence to: I.P. Berzctci-Gurske, SRI International, Department of Ncuroscicnce, Building LA, Room 105, 333 Ravcqswood Avenue, Menlo Park, C'A 94025, U.S.A. Tel. 1.415.859 3354, fax 1.J,15.859 3153.
clinically because of its undesirable psychotomimctic side effects (Telford ct al., 1961). Although several studies suggested its ~-antagonist activity (Unna, 1943; Telford et al., 1961) and partial K-agonist activity (Leandcr, 1983), nalorphine has also bccn suggested to bc a /.t-agonist ( Z i m m e r m a n ct al., 1987). No detailed in vitro analysis has been performed because of the lack of a specific K-antagonist compound. With the recent introduction of the selective K-antagonist norbinaltorphiminc (Nor-BNI) by Portoghese et al. (1987), the antagonist effect of mixed agonists/antagonists on the longitudinal muscle of the guinea pig ileum can be studied. The compounds selected for our study were nalorphine, ( - )SKF 10,047, and (+)cthylketocyclazocine (EKC).
2. Materials and methods
2.1. Longitudinal muscle strip of guinea pig ileum Male Hartley guinea pigs weighing 350-400 g were decapitated and the small intestine removed. The longitudinal muscle with the myentcric plexus attached was gently separated from the underlying circular muscle by the method of Paton and Vizi (1969). The muscle strips were mounted in an 8-ml water-jacketed organ bath containing Krebs bicarbonate solution of the following composition (in mM): NaCI 118; CaCl 2
284
2.5; KCI 4.7; NaHCO3 25; K H 2 P O 4 1.2; MgSO 4 1.2 and glucose 11.5. The tissues were kept at 37°C and bubblcd with 5% CO 2 in oxygen. An initial tension of 1.0 g was applied to the strips. The tissues were electrically stimulated for 60 min before the start of the experiments. Field electrical stimulation was applied, delivered through platinum wire electrodes positioned at the top and bottom of the organ bath, and kept at a fixed distance (3.5 cm apart). The uppcr clcctrodc was a ring 4 mm in diameter. The parameters of rectangular stimuli wcrc supramaximal voltage, 1 ms impulse duration at 0.1 Hz. A Grass S-88 electrostimulator was used. The electrically induced twitches were recorded by using an isometric transducer (Metrigram) coupled to a multichanncl polygraph (Grass 7D). 2.2. Kinetics The agonist potencies of compounds were determined from concentration rcsponsc curves and characterized by the ICs0 values. Agonists were prescnt for 3 - 6 rain or until maximal inhibitory effect was produccd. Dose response curves were constructed in a cumulative fashion for the ~-agonists because of slow onset and washout of agonist activity. The percentage of inhibition of the stimulation-induced contraction produced by each agonist was plotted against the log agonist concentration. IC5, is defined as the concentration of the agonist that produces 50% inhibition of the contraction. Antagonists wcrc present for at least 30 rain. Antagonist activities were calculated for each single tissue from full concentration response curves before and after the addition of a single antagonist concentration. D R (dose ratio) is the shift of the agonist concentration response curve in the presence of the antagonist. At least three different concentrations wcrc used, and only one antagonist concentration was tested on each tissue, pA~_ values were determined from Schild plots (Arunlakshana and Schild, 1959). Each individual point was plotted, and pAz was determined by using a statisticaI least-square regression analysis program package (R. Barlow, U.K.). 2.3. Materials Drugs were obtained from commercial sources, except for the following: Nor-binaltorphimine dihydrochloride from RBI; nalorphinc hydrochloridc, (_+)ethylketocyclazocine-succinatc, D A M G O ([D-AIa2,N-Me Phe4,Gly-ol5]enkephalin]), ( - ) S K F 10,047.HC1, CTAP-NH 2 (D-Phc-Cys-Tyr-D-Trp-Arg-Thr-Pen-ThrNH2), U 69,593 (5a,7tr,8/3)-(+)-N-methyl-N-[7-(l-pyrrolidinyl)-l-oxa-spiro-(4,5)-dec-8-yl], and morphine sulfate from NIDA.
3. Results
The longitudinal lnusclc of the guinea pig ileum is known to contain p~- and ~-opioid receptors (Chavkin and Goldstcin, 1981). To determine whether the ICs0 values measured in the guinea pig ileum came from the activation of b~- or ~-receptors, experiments were conducted in the presence of either a 100-nM concentration of the selective ~-reccptor antagonist ( ~ A P (Kramcr ct al., 1988) or 211 nM of the selective ~-reccptot antagonist Nor-BNI. The ICs0 wdue of the ~,-selectivc compound D A M G O was increased with a dose ratio of approximately 5 by administration of CTAP, whereas the IC~0s of the u-selective compounds U 69,593, ethylketocyclazocine, nalorphinc, and SKF 10,047 were not increased. Surprisingly, the affinities of the x-agonists apparently increased in the presence of ( ~ A P , having dose ratios less than 1.0. This however turned out to be an artifact of the experimental protocol. IC.s0 values decrease in both control and Cl'AP-treated tissues after prolonged incubations. This indicated that the ileum becomes more sensitive with incrcascd time in the tissue bath. When experiments were conducted in the presence of the ~:-sclcctivc antagonist Nor-BNI, D A M G O and morphine had only a 1.7- and 1.05-fold shift, respccliveIy, while the ~selective compound U 69,593 had a 360-fold shift. Table 1 contains the results of both experiments, showing that, in addition to U 69,593, nalorphine, SKI: 10,047, and cthylkctocyclazocine arc all apparently acting through the ~:-receptors in the guinea pig ileam. The rank order of ~-agonist potency is: ethylketocyciazocine > U 69,593 > SKF 10,047 > nalorphinc. In the next set of experiments, the ~¢-agonist activity of nalorphine, e~:hylketocyclazocine and SKF 10,047 has been blocked with 20 nM Nor-BNI, which was present throughout the experiment in the Krebs solu-
'FABI,E 1 IC~0 and dose ratio values of various opioid agonists with CTAP and Nor-BNI on guinea pig ileum preparation. Means + S.E. Compound
DAMGO Morphine U69,593 (-.-)Ethylketocyclazocinc Nalorphinc (-)SKF10,047
IC'5t~(nM)
8.25_* 2.0 (13) 24.75:- 2.4 (4) 1.66= 0.63 (12) 11.44___ 0.14 (8) 29.2 ± 15 115) 10.5 + 3.9 (7)
Dose ratio with (;TAP (100 nM)
Nor-BNI (20 nM)
4.98±0.3 (4) 4.27 (2) 0.68+0.11 (4) 0.60_+0.09 (4) 0.911 (2) 11.44 (2)
1.74.-: 0.14 (6) 1.05 (2) 363.11 +97 (8) 61.2 j: 8.8 (8) 643.0 (2) 273.1 (2)
285 TABLE 2 p~,~ values and Schild slope for the antagonists nalorphine and ( - ) S K F 10,047 on the guinea pig ileum using the/z-selective agonist D A M G O in the presence of 20 nM Nor-BNI. Antagonist
pA 2 value
Schild slope
Nalorphine ( - ) S K F 10,047 ( ± )Ethylketocyclazocinc Naloxonc
7.49 7.69 ~ 8.53 ~
- 0.873 - 1.216 - 0.99
a There was no shift in thc presence of (_+.)ethylketocyclazocinc. ~ The experiment with naloxone was without Nor-BNI.
tion. Under these conditions, only ethylketocyclazocine maintained significant agonist activity, which allowed us to determine the ability of SKI" 10,047 and nalorphine to antagonize the /x-agonist D A M G O with 20 nM Nor-BN1 present in the tissue bath. The concentration response curves for the /x-agonist D A M G O were conducted in the presence of various concentrations of SKF 10,047 and nalorphine to determine the pA2 vzduc for their antagonist activity. Table 2 contains both pA 2 values as well as the slope of the Schild plot. As a comparison, the pA~ value and Schild slope of naloxone, determined in the absence of Nor-BNI, are also shown in table 2. The rank order of the /Xantagonist potency is: naloxone > SKF 10,047 > nalorphine. Although the binding affinity at the /xopioid receptor is almost the same for naloxone and nalorphine, there is a 10-fold difference in the /xantagonist potency. The binding results of cthylkctocyclazocine show equal affinity at /X- and K-receptors (Magnan c t a l . , 1982). Because it still had substantial agonist activity in the prcscnce of Nor-BN1, we planned an experiment to determine whether ethylketoc3'clazocine would also activate the/x-receptors if the number of K-receptors was reduced. A concentration response curve was made with ethylketocyclazocine in the presence of 20 nM Nor-BNI; then the tissue was incubated with 100 nM CTAP for 30 min, and the ethylketocyclazocinc concentration response curve was repeated. In the presence of CTAP in addition to Nor-BNI, there was a further 2-fold shift in the ICs0 value for ethylketocyclazocine, proving that, under these conditions, ethylketocyclazocine was also activating thc /x-receptors of the guinea pig ileum preparation.
4. Discussion
Nalorphine was the first narcotic antagonist made available for medical use in 1952. Its activity in animals a'~; a morphine antagonist was demonstrated by Unna (1943). Keats and Telford (1956) demonstrated that it
was equipotent with morphine as an analgesic in the clinic. As a mixed agonist/antagonist compound, nalorphine was studied in the guinea pig ileum by Gyang and Kosterlitz in 1966 and by Kosterlitz and Watt in 1968. By the single-dose method, they were able to show the morphine antagonist activity of this compound as well as that of SKF 10,047. A later study on the rat vas deferens, which does not contain K-opioid receptors, also showed that nalorphine worked as a /x-antagonist. However, both these studies are incompletc. A Schild plot to determine the type of antagonism cannot be constructed by the single-dose method. Furthermore, although the rat vas deferens has no K-receptors, this tissue apparently has very few /x-receptors, for even morphine is an antagonist in this preparation (Wuster et al., 1980). With the introduction of the highly selective K-antagonist compound Nor-BNI, the/x-activity of compounds acting as full or partial agonists at the K-opioid receptors could be studied fully for the first time. As a whole, the results shown in table 1 are not surprising. The potencies of /x-agonists D A M G O and morphine are reduced by the /x-antagonist C I A P but not by the K-antagonist Nor-BNI. Conversely, the 'Kagonists' U 69,593, ethylketocyclazocinc, nalorphine, and SKF 10,047 are inhibited by Nor-BNI but not by CTAP. Interestingly, unlike the other agonists, EKC is still a potent compound even in the presence of 20 nM Nor-BNI (ICs0 value ~ 27 nM). The other K-agonists had no agonist activity in the presence of Nor-BNI when used at nanomolar concentrations. These data correspond nicely to the results found in /3-FNA treated tissue, another '/x-less' preparation. In fl-FNA treated tissue the potencies of nalorphine, SKF 10,047 and ethylketoc3'clazocine do not greatly change, though the IC.~0 of morphine and D A M G O shift significantly to the right. Again this indicates that the agonist activity of ethylketocyclazocinc, nalorphine, and SKF 10,047 is through the K-receptor in the GPI (Hayes et al., 1985, and unpublished observation of the authors). A lack of agonist activity in the presence of Nor-BNI made possible the examination of these compounds as antagonists of the /x-agonist DAMGO. Under these conditions, both nalorphine and SKF 10,047 were potent /x-antagonists, with pA 2 values approximately one-tenth that of naloxone. It is interesting that naloxone, SKF 10,047, and nalorphine all have comparable /x-binding affintites (Magnan et al., 1982) but naloxone is a more potent antagonist. This may be due to a small amount of intrinsic activity in nalorphine and SKF 10,047. If they were very low efficacy agonists they may not manifest agonist activity in the GPI but would require high receptor occupancy to antagonize the activity of DAMGO. Perhaps if a tissue was found with more spare receptors these compound would act as agonists. This may explain the observation by Zimmer-
286
man et al. (1987) that nalorphine can act as a #-agonist in the acetic acid writhing test in mice. Because cthylkctocyclazocine maintained significant agonist activity in the presence of Nor-BNI, its ability to act as a /x-antagonist could not bc detcrmined. Furthermore, the question remained as to whether the residual activity was duc to ~-agonism not blocked by 20 nM Nor-BNI or to /x-agonism, now revcaled by blocking of ~:-receptors. When the ileum was incubated with CTAP in addition to Nor-BNI, the 1C50 of cthylkctocyclazocine was shifted further to the right. This indicates that ethylkctocyclazocine can act as a ~zagonist in the GPI. In conclusion, the availability of the selective ~:antagonist Nor-BNI permits the study of compounds with high affinit3, at both g- and x-opioid receptors at #-receptors on the GP1. Of the ~(-agonist compounds tested, nalorphinc and SKF 10,047 possessed potent p.-antagonist activity in the GPI, however the '~: agonist' ethylketo-cyclazocinc is apparently also an agonist at the ~-receptor.
Acknowledgements This work was supported by Grant DA06682 to I,.T. from the National Institute on Drug Abuse. We thank Valorie Wcizcr for her competent technical assistance and Rosic McCormick for typing the manuscript.
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