European Journal of Pharmacology 377Ž. 1999 21±28 www.elsevier.nlrlocaterejphar Differential antagonism of the rate-decreasing effects of k-opioid receptor agonists by naltrexone and norbinaltorphimine 1 Kelly R. Powell ), Stephen G. Holtzman Department of Pharmacology, Emory UniÕersity School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA Received 28 January 1999; received in revised form 17 May 1999; accepted 26 May 1999 Abstract Eight k-opioid receptor agonists were examined for their effects in squirrel monkeys responding under a fixed interval 3-min schedule of stimulus termination. Six of these k-opioid receptor agonists decreased dose-dependently the total number of responses and with an order of potency consistent with k-opioid receptor interaction. Three of these k-opioid receptor agonists, bremazocine, U69,593 ÄwŽ.Ž.5a,7a,8b - q -N-w 7-Ž.Ž.. 1-pyrrolidinyl -1-oxaspiro 4,5 dec-8-ylx benzeneacetamide4 and enadoline, were evaluated following pretreat- ment with 1.0 mgrkg of naltrexone or 3.0 mgrkg of norbinaltorphimine. The effects of the three agonists were antagonized significantly by naltrexone, but only those of bremazocine and U69,593 were antagonized significantly by norbinaltorphimine. Statistical analysis of the data averaged over six monkeys revealed that naltrexone was significantly more potent than norbinaltorphimine at antagonizing enadoline and U69,593, but naltrexone and norbinaltorphimine were equipotent at antagonizing bremazocine. Moreover, naltrexone was 8-fold more potent at antagonizing U69,593 and enadoline than at antagonizing bremazocine. These results suggest that under these conditions the effects of U69,593 and enadoline may be mediated, in part, by a different receptor population, perhaps a subtype of k-opioid receptors, from the one that mediates the effects of bremazocine. q 1999 Elsevier Science B.V. All rights reserved. Keywords: k-Opioid receptor;Ž. Squirrel monkey ; Fixed interval; Naltrexone; Nor-binaltorphimine X 1. Introduction 5r,9r.Ž-5,9-dimethyl-2- 1-tetrahydrofurfuryl-2 -hydro-6,7- benzomorphan.x4 , but not ethylketocyclazocine and tiflu- Since the discovery of drugs that act selectively at adom, increase plasma levels of dihydroxyphenylacetic k-opioid receptors scientists have attempted to distinguish acid and homovanillic acidŽ. Iyengar et al., 1986 . In the mechanisms of action underlying the analgesic and addition, whereas ethylketocyclazocine- and tifluadom-in- unwanted side effects of these drugs. The hypothesis driv- duced decreases in plasma levels of corticosterone and ing this line of investigation is that more selective k-opioid thyroid stimulating hormone are blocked with the opioid receptor agonists can be developed that provide clinically antagonist quadazocine, U50,488- and MR-2034-induced useful analgesic effects without the unwanted side effects decreases are not. Furthermore, there are at least two or abuse potential. To this end, functional differences studies in mice in which antinociceptive cross-tolerance between k-opioid receptor agonists have been examined in between k-opioid receptor agonists was not observed: various physiological and behavioral assays, suggesting the U69,593ÄwŽ.Ž. 5a,7a,8 b - q -N-w 7-Ž. 1-pyrrolidinyl -1- existence of subtypes of the k-opioid receptor. For exam- oxaspiroŽ. 4,5 dec-8-yl .x benzene-acetamide4 and brema- ple, the k-opioid receptor agonists U50,488 Äwtrans- zocineŽ. Horan and Porreca, 1993 , and U50,488 and nalox- Ž." 3,4-dichloro-N-methyl-N-w 2-Ž. 1-pyrrolidinyl cyclo- one benzoylhydrazoneŽ. Gistrak et al., 1989 . hexylx benzeneacetamide4Ä and MR-2034 wŽ.y -a-l Žr, Traditionally, receptor-selective antagonists have been used to differentiate among receptors mediating the in vivo effects of various types of opioids Ž.m vs. k vs. d . The ) Corresponding author. Tel.: q1-404-727-0356; fax: q1-404-727- lack of antagonists selective for proposed subtypes of the 0365; E-mail: [email protected] k-opioid receptor has been a major impediment to the 1 This research was supported in part by Grant DA 00541, individual post-doctoral fellowship F32 DA05709 to K.R.P., and Research Scientist differentiation and characterization of those receptors. Award K05 DA00008 to S.G.H., all from the National Institute on Drug Nonetheless, important behavioral distinctions among k- Abuse, NIH. opioid receptor agonists have been demonstrated using the 0014-2999r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S0014-2999Ž. 99 00394-5 22 K.R. Powell, S.G. HoltzmanrEuropean Journal of Pharmacology 377() 1999 21±28 non-selective opioid antagonistsŽ antagonists that are capa- lidinyl. -cyclohexylxwx benzo b thiophene-4-acetamide xŽ Clark ble of antagonizing m- andror d- andror k-opioid recep- et al., 1989.Ž , spiradoline VonVoigtlander and Lewis, tor agonists. that are currently available. For example, in 1988.Ž , U50,488 VonVoigtlander et al., 1983 . , and U69,593 mice the antinociceptive effects of U69,593 are antago- Ž.Lahti et al., 1985 , represent two chemical families and a nized by the reported selectiveŽ. De Costa et al., 1989 range of relative affinities for k-opioid receptorsŽ see Nock k-opioid receptor antagonist Ž.y -UPHITÄ 1S,2S-trans-2- et al., 1988a; France et al., 1994. In order to establish that isothiocyanato-4,5-dichloro-N-methyl-N-w 2-Ž. 1-pyrrolidinyl k-opioid receptors mediate the dependent measure in the cyclohexylx benzeneacetamide4 , but not by the non-selec- present study, the rank order of potencies for the kappa tiveŽ. Dykstra et al., 1987; Pitts and Dykstra, 1994 opioid agonists in this study were compared to the potency order receptor antagonist quadazocine, whereas the antinocicep- of the same drugs in different procedures. Similar rank tive effects of bremazocine are antagonized by quada- orders of potency across procedures is suggestive that the zocine, but not by Ž.y -UPHIT Ž Horan et al., 1991, 1993 . effects are mediated by the same population of receptors. In these same studies, the antinociceptive effects of wD-Pen2 , In addition, antagonism of the effects of these k-opioid D-Pen5 x enkephalin Ž.d-opioid receptor agonist and Tyr-D- receptor agonists by the selective k-opioid receptor antago- Ala-Gly-NMe-Gly-ol Ž.m-opioid receptor agonist are an- nist norbinaltorphimine should rule out non-k-opioid re- tagonized by quadazocine at doses 3-fold less than the ceptors as the site of action for these effects. Finally, we dose required to antagonize bremazocine. Additionally, in compared the ability of the non-selective opioid receptor pigeons, lower doses of the non-selective opioid antago- antagonist naltrexone to antagonize the effects of three of nists naltrexone and naloxone are required to antagonize the k-opioid receptor agonists. If these k-opioid receptor the rate-decreasing and discriminative stimulus effects of agonists produce their effects through a homogenous re- bremazocine and enadoline than are required to antagonize ceptor population, the potency of a given antagonist should these effects of U50,488Ž Mattox et al., 1994; Picker, not differ across agonists. 1994. Furthermore, naltrexone differs in its ability to antagonize the rate-decreasing effects of bremazocine com- pared to U50,488 in ratsŽ. Pitts et al., 1996 . The selective 2. Materials and methods k-opioid receptor antagonist norbinaltorphimine Ž.Portoghese et al., 1987 antagonizes the antinociceptive 2.1. Subjects effects of U69,593 and U50,488, but not those of brema- zocine, enadoline, and ethylketocyclazocine in rhesus Ž. monkeysŽ. Butelman et al., 1993 . Similarly, the non-selec- Six squirrel monkeys Saimiri sciureus with previous tive opioid antagonist naltrexone is much more potent at experience on a fixed interval stimulus termination sched- antagonizing the antinociceptive effects of U50,488 and ule and previous exposure to various opioid drugs were U69,593 than at antagonizing the effects of bremazocine used in the present study. All monkeys were drug-free for and enadoline in rhesus monkeysŽ. Ko et al., 1998 . To- at least 1 year prior to this experiment. Monkeys were gether, these reports suggest that non-selective antagonists given food and water ad libitum in the home cages and might be useful in differentiating behavioral effects medi- received fresh fruit, peanuts, or a nutritional supplement ated by different receptor subtypes. daily. The facility for housing the animals was accredited Along this line, some investigators have suggested that by the American Association for Accreditation of Labora- the differences in the pharmacological profiles of these tory Animal Care and care of the animals conformed to the antagonists against different k-opioid receptor agonists National Institutes of Health Guide for the Care and Use of represent activity at different subtypes of the k-opioid Laboratory Animals. receptorŽ. Horan et al., 1993; Ko et al., 1998 . Based on that assumption and in order to provide further evidence 2.2. Apparatus for k-opioid receptor subtypes, the purpose of the current study was to compare the antagonism by naltrexone and The monkeys were restrained in a small primate chair norbinaltorphimine of the effects of various k-opioid re- that was placed inside a ventilated, sound-attenuating ceptor agonists in squirrel monkeys responding under a chamber provided with white noise during the experimen- fixed interval schedule of stimulusrshock termination. This tal sessions. The chamber was illuminated by a house light stimulus termination procedure was chosen based on our that was mounted on the rear wall of the chamber. A observations that it is sensitive to the k-opioid receptor- shaved portion of the monkey's tail was secured beneath mediated rate-decreasing effects of k-opioid receptor ago- two brass electrodes and held in place by a Plexiglas stock; nistsŽ. Jones and Holtzman, 1994 . The k-opioid receptor the electrodes were connected to a current generatorŽ Med agonists that were examined, bremazocineŽ Romer et al., Associates, Fairfield, VT. One response lever was mounted 1980.Ž , enadoline Boyle et al., 1990 .