Brain Research 884 2000 184–191 www.elsevier.com locate bres
Research report
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Selective opioid agonist and antagonist competition for [ H]-naloxone binding in amphibian spinal cord
Leslie C. Newman, David R. Wallace, Craig W. Stevens
Department of Pharmacology and Physiology , Oklahoma State University, College of Osteopathic Medicine, 1111 W. 17th Street, Tulsa, OK 74107,
USA Accepted 12 September 2000
Abstract
Opioids elicit antinociception in mammals through three distinct types of receptors designated as m, k and d. However, it is not clear what type of opioid receptor mediates antinociception in non-mammalian vertebrates. Radioligand binding techniques were employed to
characterize the sites of opioid action in the amphibian, Rana pipiens. Naloxone is a general opioid antagonist that has not been
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characterized in Rana pipiens. Using the non-selective opioid antagonist, [ H]-naloxone, opioid binding sites were characterized in amphibian spinal cord. Competitive binding assays were done using selective opioid agonists and highly-selective opioid antagonists.
Naloxone bound to a single-site with an affinity of 11.3 nM and 18.7 nM for kinetic and saturation studies, respectively. A B value of
max
2725 fmol mg protein in spinal cord was observed. The competition constants K of unlabeled m, k and d ranged from 2.58 nM to 84
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mM. The highly-selective opioid antagonists yielded similar K values ranging from 5.37 to 31.1 nM. These studies are the first to
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examine opioid binding in amphibian spinal cord. In conjunction with previous behavioral data, these results suggest that non-mammalian vertebrates express a unique opioid receptor which mediates the action of selective m, k and d opioid agonists.
2000 Elsevier Science
B.V. All rights reserved.
Theme : Neurotransmitters, modulators, transporters, and receptors
Topic : Opioid receptors
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Keywords : Amphibian; [ H]-Naloxone; Antinociception; Opioid; b-FNA; nor-BNI; NTI
1. Introduction effects of a number of opioid agonists in amphibians have
been well characterized using the acetic acid test [22]. The It is known that opioids produce antinociception in
antinociception produced by opioid agonists in amphibians mammals and analgesia in humans through the activation
was shown to be opioid receptor mediated as it was of one or more distinct types of opioid receptors. Evidence
significantly blocked by the general opioid antagonists, for the multiplicity of opioid receptors in mammals
naloxone and naltrexone [32,35]. Selective m, k, and d mediating
antinociception originated
with behavioral
opioid agonists elicit consistent and potent antinociception studies [16], was validated by radioligand binding studies
following systemic or central administration in Rana [8,14] and was further confirmed with the identification of
pipiens [31,32,36]. Interestingly, the relative antinocicep- genes for three distinct types of opioid receptors [26].
tive potency of selective m, k and d opioid ligands in Whereas the multiplicity of opioid receptors in mam-
amphibians and rodents is highly correlated in both mals is certain, it has not been shown that the opioid
systemic and intraspinal administration studies [31,32]. actions in non-mammalian vertebrates are mediated by
Based on these findings, differences in the opioid receptor more than one type of opioid receptor. The antinociceptive
proteins between mammals and amphibians would not be expected.
Recently, data from behavioral studies in amphibians
Corresponding author. Tel.: 11-918-561-8234; fax: 11-918-561-
employing selective m, k and d opioid ligands as well as
8412. E-mail address
: scraigosu-com.okstate.edu C.W. Stevens.
highly-selective opioid antagonists administered intraspi-
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 6 7 - X
L .C. Newman et al. Brain Research 884 2000 184 –191
185
nally produced a surprising finding: highly-selective an- 11,11-dimethyl-2,6-methano-3-benzazocin-8-ol hydrochlo-
tagonists for m, k, and d opioid receptors were not ride
bremazocine, 17,179-bisCyclopropylmethyl-
selective in amphibians [34]. That is, the m-selective 6, 69, 7, 79-tetrahydro-4, 5, 49, 59-diepoxy-6, 69-imino[7,79 -
antagonist, b-funaltrexamine b-FNA, prevented the an- bimorphinan]-3,39,14,149-tetrol dihydrochloride nor-binal-
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tinociceptive effects of m, k, and d opioid agonists with the torphimine, [D-Ala ]-deltorphin-II, dynorphin A-1-13
same unexpected finding observed for the d-selective and
17-Cyclopropylmethyl-6,7-dehydro-4,5-epoxy-3,14- antagonist, naltrindole NTI, and the k-selective antago-
dihydroxy-6,7,29,39-indolomorphinan hydrochloride nal- nist, nor-binaltorphimine nor-BNI [34].
trindole were obtained from Research Biochemicals Inter- Previous binding studies using amphibian brain tissue
national Natick, MA. 1-4-[aR-a-2S,5R-4-Allyl- have shown predominantly one k-like opioid binding site
2,5-dimethyl- 1- piperazinyl-3-methoxybenzyl]-N ,N-dieth-
with few sites characterized as m or d opioid binding sites ylbenzamide SNC-80 was obtained from Tocris Cookson
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[1,29]. It has been determined that this opioid binding site Ballwin, MO. [ H]-Naloxone 1.78 TBq mmol; 48 Ci
in amphibians is so uniquely different from mammalian mmol was purchased from Amersham Arlington Heights,
opioid receptors that some authors call it as a ‘non-m, IL. All drugs were mixed with buffer 50 mM Tris HCl
non-d, non-k’ opioid receptor [17]. No studies thus far with 100 mM NaCl.
have examined a full complement of selective m, k, and d opioid ligands, nor have they used highly selective opioid
2.2. Tissue preparation antagonists in competitive binding assays using an am-
phibian model. Frogs were decapitated and whole spinal cord prepara-
Recent binding studies in amphibian brain tissue using tions were obtained by expulsion out the rostral end of the
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[ H]-naloxone yielded interesting results with the selective vertebral column using a saline filled syringe inserted into
antagonists. All three selective antagonists possessed near- the caudal end. Tissue was stored at -708C until used in the
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ly identical K values in their competition for [ H]-nalox- tissue homogenate binding assay. Spinal cord tissues had a
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one binding [19]. These studies may suggest that either wet weight average of approximately 75 mg. On the day of
there are three promiscuous receptors that bind several the experiment, spinal cord tissue was thawed and
opioid classes or that there is a single binding site homogenized in approximately 100 volumes weight of 50
mediating antinociception for multiple opioids. mM Tris HCl with 1 mM sodium EDTA, pH 7.4. Pellets
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In the present study, a full characterization of [ H]- were obtained by centrifugation of the homogenate at 400
naloxone binding was performed in amphibian spinal cord rpm 29 g at 48C for 15 min followed by 14,500 rpm
tissue using kinetic, saturation and competition analyses to 24,000 g at 48C for 15 min. The resulting pellet was
provide a pharmacological correlate to intraspinal be- suspended in 50 mM Tris HCl with 100 mM NaCl, pH 7.4
havioral data obtained in Rana pipiens as well as for and rehomogenized for immediate use in the binding
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comparison to [ H]-naloxone binding in brain tissue. A assay. This working buffer included 100 mM NaCl for the
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number of m-, k- and d-selective opioid agonists were used optimization of [ H]-naloxone binding. Protein analysis
to compete with naloxone binding. Finally, the highly- was determined according to the Bradford method using
selective m opioid antagonist, b-FNA [38], the d-selective bovine serum albumin BSA as the standard BioRad,
antagonist, NTI [25] and the k-selective antagonist, nor- Richmond, CA.
BNI [39], were assayed against naloxone binding. 2.3. Binding assay
2. Materials and methods Experiments were performed in triplicate and the re-
