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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 asm,kandd. However, it is not clear what type of opioid receptor mediates antinociception in non-mammalian vertebrates. Radioligand binding techniques were employed to characterize the site(s) 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_maxvalue of 2725 fmol / mg protein in spinal cord was observed. The competition constants (K ) of unlabeledi m,kanddranged from 2.58 nM to 84

mM. The highly-selective opioid antagonists yielded similar K values ranging from 5.37 to 31.1 nM. These studies are the first to_i 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 selectivem,kanddopioid 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: 1

1-918-561-employing selective m, k and dopioid ligands as well as 8412.

E-mail address: scraig@osu-com.okstate.edu (C.W. Stevens). highly-selective opioid antagonists administered

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 -bis(Cyclopropylmethyl)-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 ofm,k, anddopioid 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 asmor dopioid 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, andd

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-i tissue homogenate binding assay. Spinal cord tissues had a 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 ofm-,k- andd-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

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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 50ml of buffer (for Drugs used include naltrexone hydrochloride, b-funal- total binding) or 50ml 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

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Branch, Rockville, MD). Dermorphin and [D-Pen , D- complex and the binding reaction was terminated by rapid

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Pittsburgh, PA). Specific binding was defined as the 2.7. Data analysis difference between non-specific binding (measured in the

presence of excess concentrations (10mM) 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

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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 10mM 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 ) andD

which point further binding was blocked by the addition of density (Bmax) data. In competition experiments, the con-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 byi

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

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[ H]-naloxone to determine receptor density (Bmax) and the nonlinear least-squares curve-fitting by GraphPad apparent affinity (K ). Nonspecific binding was defined byD Prism (version 3.00, San Diego, CA) and are based on the 10 mM naltrexone. Binding reactions proceeded as de- statistical F-test.

scribed in the binding assay.

2.6. Competition studies 3. Results

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Competition binding experiments were performed using 3.1. Kinetics of [ H]-naloxone binding

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[ H]-naloxone (10 nM) with increasing concentrations

(15) of unlabeled ligand (0.01 nM–100 mM). 10 mM Kinetic analysis was performed to determine the time naltrexone was used to define nonspecific binding. Binding needed to attain the condition of steady-state as well as the reactions proceeded as described in the binding assay. rate constants for association and dissociation. Kinetic

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Table 1

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Kinetically and experimentally derived affinity and density parameters for [ H]-naloxone binding in Rana pipiens spinal cord

Kinetic analysis Saturation analysis

Parameters Statistics Parameters Statistics

a

kobs 0.458160.1822 F value 0.1467, K 18.75D 619.55 nM F value 1.591,

P50.9523 P50.2285

a d

koff 0.242960.1607 F value 0.06451, Bmax 272561055 –

P50.9377

b

kon 0.02152 –

c

KD 11.29 nM –

d

Bmax10906145 –

a 21

min .

b 21 21

mol min .

kobs2koff koff

c _{]]] ]}

KD values were calculated from rate constant on / off values where kon5_{[radioligand]} and KD5_k .

on d

fmol / mg protein.

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analyses of [ H]-naloxone (10 nM) binding in Rana [ H]-naloxone were determined. Saturation data for spinal

pipiens spinal cord homogenates are shown in Fig. 1. cord tissue is shown in Fig. 2. Scatchard analysis of these Association studies (Fig. 1A) in the spinal cord yielded a data is shown in the inset. The experimentally derived KD

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kobs (observed association rate) value of 0.3505 min and Bmax from saturation analysis were found to be 18.75 while dissociation (Fig. 1B) results yielded a koff(dissocia- nM and 2725 fmol / mg protein, respectively. Kinetic and

21 3

tion rate constant) value of 0.2429 min . Nonspecific saturation data for [ H]-naloxone are summarized in Table binding represented 25% of total binding. These rate 1. These data were best fit to a one site binding model as constants yielded a KD value of 11.29 nM. Statistical determined by the F-test.

analysis of the comparison between one and two site

models yielded a best fit for the one site model (see Table 3.3. Competition analysis 1 for results of F-test and significance).

In order to clarify drug interaction with particular

3.2. Saturation studies receptor types, inhibition experiments were performed with

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selective opioid ligands using [ H]-naloxone as the label. The properties of naloxone binding sites were studied Fig. 3 shows these results with Fig. 3A depicting

competi-3

over an extended range of concentrations of [ H]-naloxone tion withm agonists, Fig. 3B showing competition withk

(0.5–70 nM) where apparent affinity and density data for ligands and Fig. 3C, competition withdreceptor agonists.

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Fig. 2. Saturation analysis of [ H]-naloxone in spinal cord tissue homogenates. The membrane preparation was incubated with various concentrations of

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spinal cord K values in the amphibian where a correlationi

value of 0.786 was obtained. Additional competition studies with increasing concentrations (0.01 nM–100mM)

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of selective antagonists against [ H]-naloxone (10 nM) were performed. These results are shown in Fig. 5 with a summary of the K values shown in Table 3. In the case ofi

all competitive ligands, the data were best fit to a one site model as determined by the F-test.

4. Discussion

4.1. Opioid action in amphibians

The study of opioid receptor expression in phylogen-etically different species has played a significant role in the understanding of opioid receptor pharmacology [5,20]. It is widely recognized that three distinct receptors mediate the effects of opioids in mammals. However, previous be-havioral and binding studies in Rana pipiens suggest the possibility of a single opioid receptor which may mediate the actions of m, k and d opioids [19,34]. The present results are the first to document the binding characteristics

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of [ H]-naloxone in Rana pipiens spinal cord homoge-nates. Furthermore, the present data are the first to use highly-selective opioid antagonists in a competitive bind-ing assay usbind-ing central nervous system tissue from a non-mammalian vertebrate species.

4.2. Naloxone binding affinity and density

Numerous mammalian binding studies have shown that opioid agonists elicit antinociception through m, k and d

opioid receptors in both brain and spinal cord tissue homogenates [3,9,11,15,21,24,33,41]. Kinetic analysis of

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[ H]-naloxone in Rana pipiens spinal cord tissue resulted

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in a KD value of 11.29 nM. [ H]-Naloxone binding was

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saturable in amphibian spinal cord tissue, yielding a K

Fig. 3. Inhibition of 10 nM [ H]-naloxone binding with various unlabeled _D

opioid receptor ligands in Rana pipiens spinal cord tissue homogenates. _{value of 18.75 nM. The kinetic and saturation K values}

D

(A) depicts competition withmagonists, (B) shows competition with k _{were not statistically different. This similarity in K}

D

ligands and (C) withd agonists. Aliquots of tissue homogenates were

values, together with the linear transformation of the incubated with radioligand in the presence of various concentrations (0.01

binding data, is suggestive of binding to a single, nonin-nM–100 mM) of cold competitor. Data was normalized to aid

com-teractive site but does not rule out binding to several parisons defining the smallest value in the data set as 0% and the largest

value as 100% of specific binding. K values for these competitors arei _{different sites with a similar affinity. However, analysis of}

shown in Table 2. Data points are the means of one representative _{the data show a best fit to a single site as indicated by the} experiment in triplicate determinations, which was repeated three times. 3

F-test. Comparable high affinity for [ H]-diprenorphine, a

general opioid antagonist, binding to a single site was also seen in Rana pipiens brain tissue homogenates [18]. The density of opioid binding sites was 2725 fmol / mg protein Percent specific binding was measured over a range of in the amphibian spinal cord. This density value in Rana concentrations (0.01 nM–100mM) of cold competitor. For pipiens brain tissue studies as well as values in other

Table 2

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Competition of [ H]-naloxone (10 nM) binding by selective opioid receptor ligands in Rana pipiens spinal cord

a b

Drug Type K (nM)i 95% CI Hill 95% CI

bremazocine k 2.58 (1.50–4.42) 20.5461 (20.78 to20.31)

naloxone m,d,k 15.4 (7.97–29.95) 20.2643 (20.40 to20.12)

morphine m 728 (515–1029) 21.109 (21.5 to20.76)

fentanyl m 223 (137–362) 20.9327 (21.3 to20.54)

dynorphin k 4252 (2005–9018) 21.209 (22.0 to20.37)

dermorphin m 1870 (285–12,250) 20.3797 (20.98 to 0.22)

CI977 k 7755 (3207–18,750) 21.231 (22.3 to20.20)

SNC-80 d 25050 (3064–204,800) 20.9404 (21.4 to20.66)

DPDPE d 12320 (4673–32,470) 20.6799 (21.2 to20.13)

deltorphin d 84910 (32,000–225,300) 20.3432 (20.90 to 0.21)

a

95% confidence interval.

b

Hill slope.

4.3. Competitive binding with selectivem, k andd studies, bremazocine has been shown to attenuate

mor-opioid ligands phine analgesia in frog spinal cord without demonstrating

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agonist activity [2]. This potent competition of [ H]-nalox-In assays of expressed mammalian opioid receptors, one binding by bremazocine was also seen in other

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naloxone preferentially interacts withmbinding sites (KD5 amphibian binding studies using [ H]-naloxone [7,19,30] 3.9 nM) but also has significant affinity for k-opioid as well as in this lab with Chinese hamster ovary (CHO) receptors (KD516 nM) and a lesser affinity for d-opioid membrane preparations expressing the human m-opioid

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receptors (KD595 nM) [27]. [ H]-Naloxone, through receptor (unpublished data). Affinity values for the com-competition analysis, has been useful in the determination petitors in spinal cord tissue were similar to those observed of receptor affinities of type-selective opioids in mammals in brain where the corresponding K values were found toi

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[23,28,37]. The K values for [ H]-naloxone binding ini be highly correlated (see Fig. 4). Additionally, in be-frog spinal cord ranged from 2.58 nM for bremazocine to havioral studies in this lab, systematically administered 84mM for deltorphin. As is shown in Table 2,dreceptor bremazocine showed partial agonist / antagonist properties ligands and mostkreceptor agents were weak competitors as it significantly blocked the antinociception produced by

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of [ naloxone binding. The strong competition of [ H]-naloxone binding by bremazocine is interesting as it is classified as a k-selective agonist in mammalian studies [10], but has been considered a non-selective antagonist in previous binding studies [4,40]. Additionally, in behavioral

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Fig. 5. Competition of 10 nM [ H]-naloxone binding with increasing concentrations (0.01 nM–10mM) of selective antagonists in Rana pipiens spinal cord.b-funaltrexamine is am-selective antagonist, naltrindole is d-selective and nor-binaltorphimine is ak-selective antagonist. Data was normalized to aid comparisons defining the smallest value in the data set Fig. 4. Correlation plot of Rana pipiens spinal cord K values versusi as 0% and the largest value as 100% of specific binding. Data points are

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Table 3

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Competition of [ H]-naloxone (10 nM) binding by selective antagonists in Rana pipiens spinal cord

a b c

fentanyl following systemic administration (data not that the opioid receptors of the amphibian differ from those shown). In examining average K values, the overall trendi of the mammal. However, alternative explanations may of binding in Rana pipiens shows an affinity series of include a unique physical arrangement of multiple

re-m.k.d. This affinity profile is consistent with the relative ceptors as opioid receptor heterodimers have been reported affinity of naloxone for m, k and d receptors [27]. The [12]. Additionally, in another amphibian (Rana cates-agonist K values showed a high degree of correlationi biana), partial m-,k- and d-like opioid receptor sequences between brain and spinal cord as is shown in Fig. 4. This were cloned [13].

consistency in K values between brain and spinal cord hasi In conclusion, Rana pipiens represents a unique non-also been observed in the rat [15]. mammalian model for which there is a well-established behavioral assay for testing antinociception elicited by 4.4. Competition binding with highly-selective opioid opioid ligands. Further studies employing radiolabeled

antagonists selective agonists are needed to fully characterize the sites

of opioid binding in the amphibian and are near comple-The finding that naloxone bound to a single high-affinity tion. Finally, the ultimate determination of the number and site in amphibian spinal cord and thatm,k anddopioids type of distinct opioid receptors in amphibians will come could complete with naloxone may be suggestive of a from receptor cloning studies that are currently in progress single-type of opioid receptor binding site. To further test in our lab and elsewhere.

this hypothesis, the selective opioid antagonists were employed. In mammals these highly selective m, k and d

antagonists affect the binding of opioid agonists only at

Acknowledgements their respective receptors [38,39]. As mentioned above,

behavioral studies revealed a lack of selectivity of these

3 _{Support for this research was provided by the National}

antagonists in Rana pipiens and binding studies with [

H]-Institutes of Health-National Institute of Drug Abuse grant naloxone in brain yielded nearly identical K values for thei

(DA 12448). Portions of these studies were previously selective antagonists [19,34]. Interestingly, the three

selec-reported at the International Narcotic Research Confer-tive antagonists also yielded similar K values in Ranai

ence, July, 1999.

pipiens spinal cord tissue (Fig. 5). K values for thei

selective antagonists in mammals have been determined using selective ligands for the m, k and d cloned opioid receptors. In cell lines expressing the m-opioid receptor,

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