Materials and methods Experiments were performed in triplicate and the re-
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-
2
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
3
[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
3
[ 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
3
ly identical K values in their competition for [ H]-nalox- tissue homogenate binding assay. Spinal cord tissues had a
i
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
3
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
3
comparison to [ H]-naloxone binding in brain tissue. A assay. This working buffer included 100 mM NaCl for the
3
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