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

2. Materials and methods Experiments were performed in triplicate and the re-

3 ceptor binding reactions were initiated by adding [ H]- 2.1. Drugs naloxone 50 ml to 400 ml of tissue homogenate 0.17 60.08 mg of protein containing either 50 ml of buffer for Drugs used include naltrexone hydrochloride, b-funal- total binding or 50 ml of naltrexone for the determination trexamine, morphine and fentanyl which were obtained of nonspecific binding. The components were incubated from the National Institute on Drug Abuse Drug Supply for 60 min at room temperature in order to equilibrate. Program Mr. Robert Walsh of the Research Technology Unbound ligand was separated from the receptor–ligand 2 Branch, Rockville, MD. Dermorphin and [ D -Pen , D - complex and the binding reaction was terminated by rapid 5 Pen ]-enkephalin DPDPE were obtained from a commer- filtration using a Brandel 24-cell tissue harvester Gaithers- cial source Bachem Bioscience, Prussia, PA. 5R- burg, MD followed by washing 435 ml; 15 s with cold 544a,744a,845b-N-methyl-N-[7-1-pyrrolidinyl-1-oxas- buffer onto Whatman GF B glass-fiber filters which were piro [4,5] dec-8yl]-4-benzofuranacetamide monohydro- pre-soaked for at least 1 h in 0.3 polyethylenimine PEI chloride CI977, Enadoline was obtained from Ms. Carol to decrease nonspecific binding. Radioactivity was counted Germain of Parke-Davis Ann Arbor, MI. 6-6-Ethyl- using a Beckman LS1801 scintillation counter 40–50 1, 2, 3, 4, 5, 6-hexahydro-3-[1-hydroxycyclopropylmethyl]- efficiency with Scintiverse scintillation fluid Fisher, 186 L Pittsburgh, PA. Specific binding was defined as the 2.7. Data analysis difference between non-specific binding measured in the presence of excess concentrations 10 mM of naltrexone Association kinetic analysis involved fitting the data by to block opioid receptor sites and total binding. the one phase exponential association equation or the two phase exponential association equation to determine the 2.4. Kinetic studies best fit. The one phase exponential association equation resulted in the best fit. Dissociation kinetic data were fitted The association component of kinetic analysis involved to one and two phase exponential decay to as before 3 the addition of [ H]-naloxone 10 nM at various time determine the best fit for the data. As with the association points 10 measurements where specific binding was data, the one phase equation was the best fit. For saturation measured. Nonspecific binding was defined by a parallel analysis the data were first fit to the rectangular hyperbolic series of tubes containing 10 mM naltrexone. The dissocia- function followed by linear transformation Scatchard, tion component was accomplished by allowing the bound free versus bound. Analysis of the rectangular radioligand and homogenate to bind to equilibrium at hyperbola was used to obtain apparent affinity K and D which point further binding was blocked by the addition of density B data. In competition experiments, the con- max 10 mM naltrexone at various time points 10 measure- centrations of unlabeled ligand that bound to half of the ments where specific binding was measured. binding sites at equilibrium K were calculated by i GraphPad using the correction of Cheng and Prusoff [6] 2.5. Saturation studies which corrects for the concentration of radioligand as well as the affinity of the radioligand for its binding site. Saturation analysis was performed by measuring specific Competition curves were fitted to one- or two-site binding binding over increasing concentrations 0.5–70 nM of models, to determine to which the data were best fit, using 3 [ H]-naloxone to determine receptor density B and the nonlinear least-squares curve-fitting by GraphPad max apparent affinity K . Nonspecific binding was defined by Prism version 3.00, San Diego, CA and are based on the D 10 mM naltrexone. Binding reactions proceeded as de- statistical F-test. scribed in the binding assay. 2.6. Competition studies

3. Results