Nicotinic receptor binding of [3H]cytisine, [3H]nicotine and [3H]methylcarbamylcholine in rat brain

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European Journal of Pharmacology253 (1994) 261-267

Nicotinic receptor binding of [3H]cytisine, [3H]nicotine and [3H]methylcarbamylcholine in rat brain David J. Anderson *, Stephen P. Arneric Neuroscience Research, Pharmaceutical Discovery, Abbott Laboratories, Abbott Park, IL 60064, USA

(Received 25 October 1993, accepted 30 November 1993)

Abstract

Three radiolabeled nicotinic receptor agonists were examined for their binding characteristics and for inhibition by cholinergic compounds in order to distinguish possible differential affinities for subtypes of neuronal nicotinic acetylcholine receptors. K D and Bma~ values for [3H]cytisine, [3H]methylcarbamylcholine and [3H]nicotine were determined from Scatchard analysis using an enriched whole-brain membrane fraction from male Sprague-Dawley rats. Respective K D values were 0.15, 1.07 and 0.89 nM while Bmax values were 99, 64 and 115 fmol/mg protein respectively. All three ligands fit a one-site model of receptor-ligand interaction. Concentration-inhibition curves were used to determine K i values for 16 cholinergic compounds. The rank order of potencies for displacement of the three ligands was: (-)-cytisine > (-)-nicotine > (-)-lobeline = methylcarbamylcholine > 1,l-dimethyl-4-phenylpiperazinium, ( + )-nicotine, dihydro-/~-erythroidine, ( + )-nornicotine > carbachol > arecoline >> oxotremorine, tetrahydroaminoacridine, AF102B >> (-)-cotinine > RS86 = heptylphysostigmine. Correlations of the affinities of these compounds determined with the three ligands were very near to unity. In contrast, there was a negative correlation of affinities for [3H]cytisine compared to affinities for the muscarinic receptor agonist, [3H]oxotremorine-M, and the muscarinic receptor antagonist, [3H]quinuclidinyl benzoate. Using membranes from whole rat brain yields data suggesting that all three nicotinic ligands bind to the same nicotinic acetylcholine receptor subtype, and are unable to distinguish subtypes of neuronal nicotinic acetylcholine receptor at the level examined. Key words: [3H]Cytisine; [3H]Nicotine; [3H]Methylcarbamylcholine; Nicotinic acetylcholine receptor, neuronal; (Rat)

1. Introduction Interest in neuronal nicotinic acetylcholine receptors has increased due to evidence that their numbers are reduced in Alzheimer's disease (Wang et al., 1987; Araujo et al., 1988). This, coupled with the potential role of nicotine in learning and memory (Castellano, 1976; Levin, 1992), has given impetus to an emerging understanding of their nature and role in the brain. Molecular biology studies have elucidated that there are at least ten nicotinic receptor genes in the brain, namely: or2, a3, a4, a5, a6, oL7 and/32,/33,/34 and/35 (see H e i n e m a n n et al., 1991; Deneris et al., 1991; Sargent, 1993, for reviews). In situ hybridization studies have demonstrated that the m R N A s for a number of

* Corresponding author. Tel. (708) 937-5830, fax (708) 937-9195. Elsevier Science B.V. SSDI 0014-2999(93)E0874-R

these subunits are widely distributed in rat brain in a broadly overlapping pattern with regional variations in density (Luetje et al., 1990a; Deneris et al., 1991). O f these, a2, a 3 and a 4 can combine with /32 or /34 subunits in X e n o p u s oocytes to form at least six functional a-bungarotoxin insensitive nicotinic receptors. Each of these subtypes of receptor is a ligand-gated ion channel with unique pharmacological properties that have been characterized with the use of electrophysiology for toxin sensitivity and agonist response (Papke et al., 1989; Papke, 1993). Recent expression studies in X e n o p u s oocytes (Luetje and Patrick, 1991) and in vitro studies in the rat habenulo-interpeduncular system (Mulle et al., 1991) have demonstrated differences in the rank order of potency of ( - ) - n i c o t i n e and ( - ) cytisine at nicotinic receptor subtypes. This diversity of agonist sensitivity raises the possibility of distinguishing receptor subtypes by labeling with ligands of differing

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D.J. Anderson, S.P. Arneric / European Journal of Pharmacology 253 (1904) 261-267

selectivity and then determining pharmacological profiles for a number of reference compounds. Since little is known about the pharmacological properties of nicotinic receptors in the brain or their function, it was of interest to compare three available nicotinic receptor ligands for possible distinctions in subtype selectivity. Neuronal nicotinic receptors have classically been defined with the nicotinic receptor agonists, [3H]nicotine (Lippiello and Fernandes, 1986) and [3H]methylcarbamylcholine (Abood and Grassi, 1986; Boksa and Quirion, 1987). These ligands suffer from limitations that restrict their utility. [3H]Nicotine is light sensitive and has been shown to produce a wide range of results depending on a large number of assay variables which can lead to difficulties in the interpretation of data (Wonnacott, 1987). [3H]Methylcarbamylcholine has a rapid off-rate which makes filtration assays sensitive to time and rate variations in procedure, and has lot-to-lot variations in nonspecific binding (unpublished observations). The introduction of a new tritiated nicotinic receptor agonist, [3H]cytisine (Pabreza et al., 1991), presented an opportunity to utilize a high affinity (K D = 0.1 riM) ligand that is very selective for the nicotinic receptor. Using a classical pharmacological approach, an attempt to uncover neuronal nicotinic receptor subtypes was made by examining the rank ordered potencies of a variety of cholinergic agents to interact with the binding sites labeled by [3H]cytisine, [3H]nicotine and [3H]methylcarbamylcholine. K o and Bmax values for [3 H]cytisine, [ 3H]methylcarbamylcholine and [3H]nicotine were determined from saturation binding isotherm analysis using a membrane enriched fraction from whole brains of male Sprague-Dawley rats. Subsequently, the affinities of reference compounds for nicotinic receptors were determined against each ligand from concentration-inhibition curves. Due to crossover of affinities of certain compounds for muscarinic and nicotinic acetylcholine receptors, the compounds were evaluated for binding to muscarinic receptors labeled by [3H]oxotremorine-M, an agonist, and [3H]quinuclidinyl benzilate, an antagonist.

2. Materials and methods

2.1. Membrane fraction preparation Membrane enriched fractions were prepared using a modification of Enna and Snyder (1975). Whole brains from male Sprague-Dawley rats (250-400 g) were homogenized in 15 volumes of 0.32 M sucrose and centrifuged at 1000 × g for 10 min. Supernatants were centrifuged at 20000 × g for 20 min at 4°C. P2 pellets were homogenized with a Polytron for 10 s at a setting of 6 in 15 volumes ice-cold H20 and spun at 8000 × g

for 20 rain. The supernatant and loose buffy coat were spun at 40000 × g for 20 rain. The membrane pellet was washed with ice-cold H 2 0 and recentrifuged before storing at -80°C. Prior to use, pellets were slowly thawed, washed with buffered salt solution (BSS; 120 mM NaCI, 5 mM KCI, 2 mM Cacl 2, 2 mM MgC12 and 50 mM Tris-C1, pH 7.4, 4°C), and resuspended in 30 volumes of buffer. 2.2. Nicotinic receptor binding Pilot studies were performed to adapt the published methodology to bind [3H]cytisine, [3H]nicotine and [3H]methylcarbamylcholine to membranes from wholebrain preparations using one uniform protocol/buffer condition. The conditions, including protein concentrations, were optimized such that the resultant ligand bound did not significantly differ from that published to be optimal for each ligand. Incubation (i.e. association) times and ligand concentrations were found to be the two [east tolerant variables and as such, were not changed appreciably from that of the original methods. Binding of [3H]cytisine to nicotinic receptors was accomplished using an adaptation of the method of Pabreza et al. (1991). Quadruplicate aliquots of homogenate containing 100-150/xg of protein were added to polystyrene minitubes containing test compounds and [3H]cytisine in a final volume of 500 ml and were incubated for 75 rain at 4°C. Parallel determinations of nonspecific binding were incubated in the presence of 10/xM (-)-nicotine in duplicate. For saturation binding isotherms, twelve concentrations of [3H]cytisine from 0.05 to 5 nM were used. Competition studies were done with seven log dilutions of drugs in duplicate in the presence of 0.6 nM [3H]cytisine. Bound radioactivity was isolated by vacuum filtration onto No. 32 glass fiber filters (Schleicher & Scheull, Keene, NH) using an automatic cell harvester (Skatron, Lier, Norway). The filters were prerinsed with 0.5% polyethylenimine just prior to sample filtration to reduce nonspecific binding and then were rapidly rinsed with 10 ml of ice-cold BSS. Filters were counted in 3 ml of Ecolume (ICN, Covina, CA). Protein values were determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. [3H]Nicotine binding was determined as above with the exception that the incubation time was 90 min (Lippiello and Fernandes, 1986). Saturation studies were carried out with 0.1-11 nM [3H]nicotine while competition studies were done with 3 nM [3H]nicotine. [3H]Methylcarbamylcholine binding was accomplished with 0.3-9 nM [3H]methylearbamyleholine for saturation curves and with 3 nM [3H]methylcarbamylcholine for competition studies. Incubation time was 60 min (Boksa and Quirion, 1987).

D.J. Anderson, S.P. Arneric/ European Journal of Pharmacology 253 (1994) 261-267 2.3. Muscarinic receptor binding

Binding of [3H]quinuclidinyl benzilate to muscarinic receptors was done essentially as previously described (Luthin and Wolfe, 1984). Briefly, washed membranes were polytroned at a setting of 7 for 5 s in 50 mM Na-KPO 4 buffer, p H 7.4 at 25°C, and were diluted to a final concentration of 20/xg of protein per tube. Incubations were carried out at 25°C for 60 rain in a final volume of 500 m| utilizing 0.007-0.55 nM of [3H]quinuclidinyl benzilate for saturation studies and 0.12 nM of ligand in concentration-inhibition studies. Atropine at 1 /zM was present to determine nonspecific binding. For filtration, ice-cold 50 mM NaKPO 4 buffer was used. [3H]Oxotremorine-M binding (Birdsall et al., 1978) was done in 20 mM NaKPO 4 buffer, p H 7.4, at 25°C for 45 min with 130 /xg protein per tube. Concentrations of 0.05-5 nM [3H]oxotremorine-M were used for saturation studies while 1.5 nM was used for competition studies. Samples were collected on filters prerinsed with 0.5% polyethylenimine using ice-cold 0.9% NaC1 as the rinse buffer. 2.4. Data analysis

Bmax and K D values were determined from nonlinear least squares regression analysis using InPlot (GraphPad Software, San Diego, CA); Scatchard analysis data were analyzed for both one-site and two-site fits using L I G A N D (Munson, 1987). IC50 values were calculated with a four-parameter logistics program in R S / 1 (BBN, Cambridge, MA) and K i values for unlabeled drugs were determined using the equation, K i = IC50/(1 +[ligand]/K o) (Cheng and Prusoff, 1973). Log-normally distributed K D and g i values were converted to logarithmic form before averages and S.E.s were determined (Fleming et al., 1972). Averages were expressed as antilogarithms and logarithmic S.E.s were multiplied by the arithmetic averages to calculate S.E.M. (DeLean et al., 1982). Correlation coefficients of K i values were determined by linear regression analysis in InPlot. 2.5. Materials

[3H]( -)-Cytisine (42 Ci/mmol), [3H](-)-nicotine (64 C i / m m o l ) , [3H]methylcarbamylcholine (87 C i / mmol) and [3H]oxotremorine-M (87 C i / m m o l ) were purchased from Dupont-NEN (Boston, MA). [3H]QNB (44 C i / m m o l ) was purchased from Amersham (Arlington Heights, IL). (-)-Cytisine, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), (+)-nicotine bitartrate, ( - ) - l o b e l i n e hydrochloride, carbachol chloride, arecoline hydrobromide, (_+)-nornicotine, bovine serum albumin, atropine sulfate and ( - ) - c o t i n i n e were purchased

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from Sigma Chemical (St. Louis, MO). Oxotremorine methiodide (OXO-M) and ( - )-nicotine bitartrate were purchased from RBI (Natick, MA). Methylcarbamylcholine hydrochloride, RS86 (2-ethyl-8-methyl-2,8-diazaspiro[4,5]decan-l,3-dion hydrochloride) and AF 102B ( ( + ) cis-2-methyl-spiro(1,3-oxathiolane-5,3') quinuclidine oxalate) were synthesized at Abbott Labs. Dihydro-fl-erythroidine hydrochloride was the generous gift of Dr. Paul Anderson of Merck Research Laboratories (West Point, PA).

3. Results

Of the three nicotinic receptor agonists tested, [3H]cytisine displayed the highest affinity for the neuronal nicotinic receptor (Table 1). It had a significantly higher affinity of 0.145 + 0.011 nM ( P < 0.05, Fig. 1A) as compared to K D values of 0.89 + 0.06 and 1.07 _+ 0.13 nM for [3H]nicotine (Fig. 1B) and [3H]methylcarbamylcholine (Fig. 1C), respectively. A Bmax value of 99 +__11 f m o l / m g protein for [3H]cytisine was comparable to that obtained for [3H]nicotine, 1 1 5 + 6 fmol/mg. The Bm~x obtained for [3H]methylcarbamylcholine was significantly lower at 64 + 5 f m o l / m g ( P < 0.05). Analysis of saturation curves for [3H]oxotremorineM, a muscarinic receptor agonist, resulted in a two-site best fit with resultant K D values of 0.30 + 0.04 and 5.39 + 0.23 nM and Bmax values of 27 + 8 and 355 + 20 f m o l / m g (n = 3). The muscarinic receptor antagonist [3H]quinuclidinyl benzilate had a K D of 0.123 + 0.014 nM and a Bma~ of 2203 + 66 f m o l / m g (n = 4). A series of compounds with nicotinic or muscarinic receptor activity were tested against single concentrations of the above radioligands to assess their affinities for nicotinic and muscarinic receptors. For each ligand, a submaximal concentration was chosen for inhibition

Table 1 Binding parameters of nicotinic receptor ligands in whole rat brains. Saturation studies in enriched membrane preparations from whole brains of male Sprague-Dawley rats were conducted under similar conditions Ligand KD (nM) B,~, (fmol/mg) r 2 [3H]Cytisine 0.145___0.011 99.1+ 11.1 0.980 [3HlNicotine 0.89 _+0.06 114.5_+5.7 0.993 [3H]Methyl1.07 +0.13 63.8+_ 4.7 a 0.986 carbamylcholine a

Twelve concentrations of ligand in quadruplicate were used: 0.05-5 nM [3H]cytisine(n = 7), 0.1-8 nM [3H]methylcarbamylcholine(n = 6) and 0.3-11 nM [3H]nicotine(n = 5). Nonspecificbinding was determined in the presence of 10 /zM (-)-nicotine. K o _+S.E. values in nM and Bmax _+S.E. values in fmol/mg protein were determined from nonlinear regression analysis (Inplot) as were the squares of the correlation coefficients(r2). a p < 0.05 compared to [3H]nicotine.

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studies. Nicotinic receptor agonists and the competitive nicotinic receptor antagonist, dihydro-#-erythroidine, were included as well as muscarinic receptor agonists and the cholinesterase inhibitors, tacrine and heptylphysostigmine (HPS). As can be seen in Table 2, there is very good agreement between K i values of the compounds against all three nicotinic ligands. The Hill

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slopes of the inhibition curves were all near to one (0.995 _+ 0.015; the mean of 100 values from 7 experiments). The rank order of potency at nicotinic receptors was: ( - )-cytisine > ( - )-nicotine > ( - )-lobeline = methylcarbamylcholine > 1,1-dimethyl-4-phenylpiperazinium, (+)-nicotine, dihydro-#-erythroidine, ( + ) nornicotine > carbachol > arecoline >> oxotremorine, tacrine, AF102B >> ( - ) - c o t i n i n e > RS86 = heptylphysostigmine. At the muscarinic receptors, all the compounds with muscarinic receptor agonist activity except AF102B, namely methylcarbamylcholine, carbachol, arecoline, oxotremorine and RS86, displayed Hill slopes significantly less than one, consistent with literature values (Freedman et al., 1988). Correlations of affinities of the reference compounds for the various radioligands were determined from plots in Figs. 2A, 2B, 2C and 3. The correlation of the compounds for the three nicotinic receptor radioligands are all very near to one (r 2 = 0.994-0.998) with a slope of nearly unity (0.972-1.001) indicating that all three ligands bind to the same population of receptors. When nicotinic receptor inhibition is compared to muscarinic receptor inhibition, it is apparent that different receptors are involved since the pharmacological profiles produce a poor correlation (r 2 = 0.31) and a negative slope (Fig. 3). Fig. 3 also suggests that the compounds fall into clusters of those that are selective for nicotinic receptors (K i < 100 nM against [3H]cytisine and K i > 1000 nM against [3H]oxotremorine-M) and those that have muscarinic selectivity ( K i < 200 nM against [3H]oxotremorine-M and K i > 10000 nM against [3H]cytisine) with relatively nonspecific compounds, e.g. carbachol and arecoline, falling in between. It also should be noted that methylcarbamylcholine, while selective for nicotinic receptors, still retains some (324 nM) muscarinic receptor activity.

4. Discussion The considerably greater binding affinity of [3H]cytisine for nicotinic acetylcholine receptors corn-

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Fig. 1. Binding of radiolabeled nicotinic receptor agonists to rat brain nicotinic receptors. Saturation isotherms of binding to rat brain membranes. Specific binding was determined with twelve concentrations of radioligand assayed in quadruplicate and nonspecificbinding in the presence of 10 /~M (-)-nicotine determined in duplicate. Closed symbols represent specific binding (total minus nonspecific) and open symbolsrepresent nonspecificbinding. Vertical bars represent S.E.M. of four replicates. Inserts show the Scatchard transformations of the data for illustration purposes only. K o and Bmax values were determined from untransformed data using nonlinear least squares regression analysis. These plots are representative of 5-7 separate determinations. (A) [3H]cytisinebinding; (B) [3H]nicotine binding; (C) [3Hlmethylcarbamylcholinebinding.

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D.J. Anderson, S.P. Arneric / European Journal of Pharmacology 253 (1994) 261-267 Table 2 Relative affinities of cholinergic reference compounds for acetylcholine receptors in rat brain Drug

Nicotinic [ 3H]MCC K i (nM)

(-)-Cytisine (-)-Nicotine ( - )-Lobeline MCC DMPP (+)-Nicotine DH/3E (±)-Nornicotine Carbachol Arecoline Tacrine AFI02B (-)-Cot±nine RS86 HPS

0.14 ± 0.01 0.66 + 0.04 1.13 ± 0.15 1.66 ± 0.23 7.95 + 0.82 11,3 ± 1.0 17.4 ± 1.2 15.2 ± 0.6 57.6 ± 11.8 66.9 ± 2.8 12 633 ± 1323 29 633 ± 1773 > 200000 > 1000000 > 1000 000

Muscarinic [ 3H]Cytisine K i (nM) 0.16 ± 0.02 0.58 ± 0.04 1.47 + 0.03 1.48 ± 0.12 8.18 ± 0.48 10.8 ± 0.5 14.8 ± 0.5 16.5 ± 0.5 31,0 ± 2.4 95,9 ± 7.3 17 715 ± 1 263 29121 ± 1502 > 200000 > 1000000 > 1000 000

[ 3H]Nicotine K i (nM) 0,12 ± 0.01 0.60 ± 0.02 1,49 ± 0.07 1,75 ± 0.19 8,71 ± 0.66 12.5 ± 0.2 13.9 ± 0.9 13.1 ± 1.0 34.7 ± 0.5 115.0 ± 2.7 16 187 ± 741 22 677 ± 1288 > 1000000 > 1000000 > 1000 000

[ 3H]QNB K i (nM) > 400000 > 200 000 3 407 ± 56 407 ± 32410 ± 94277± >200000 129313± 18784± 4221± 2 302 ± 1 846 ± > 1 000000 1981 ± 1470 ±

[ 3H]OXO-M K i (nM)

87 5 962 1!261 5565 6038 3 936 264 48 181 185 102

> 400 000 46657 ± 1347 2 388 ± 52 324 ± 7 2525 ± 121 14768± 555 > 150000 47386 ± 290 9.9 ± 0.8 17.3 + 0.2 1 147 ± 58 167 ± 4 > 1000000 107 ± 2 522 ± 97

Membranes were incubated with submaximal concentrations of indicated radioligands and 7 concentrations of each drug. Nonspecific binding was determined in the presence of 10 /zM (-)-nicotine for nicotinic receptor radioligands or 1 ~M atropine for muscarinic receptor radioligands. Values are the means + S.E.M. from 3-6 separate determinations. Slopes were not significantly different from unity for the nicotinic ligands. For the muscarinic ligands [3H]quinuclidinyl benzoate ([3H]QNB) and [3H]oxotremorine-M (OXO-M), slopes were significantly less than one for methylcarbamylcholine (MCC), carbachol, oxotremorine, arecoline and RS86. Abbreviations: 1,1-dimethyl-4-phenylpiperazinium (DMPP); dihydro-/3-erythroidine (DH/3E); heptylphysostigmine (HPS).

pared to that of [3H]nicotine or [3H]methylcarbamylcholine contributes to its effectiveness as a ligand for binding studies. As expected, unlabeled cytisine has the highest affinity for nicotinic receptors of all the compounds tested. It also has a million-fold selectivity for nicotinic over muscarinic receptors. In contrast, methylcarbamylcholine displays considerable affinity for muscarinic receptors. Although there is 200-fold selectivity for nicotinic receptors, this modest selectivity could be a confounding factor in its use to discriminate novel nicotinic receptor agonists or to characterize nicotinic receptors. Unfortunately, using membranes from whole rat brain yields data suggesting that all three nicotinic ligands bind to the same nicotinic receptor subtype, and are unable to distinguish subtypes of neuronal nicotinic receptors at the level examined. Thus, use of conventional receptor binding methods with whole-brain preparations to screen for subtypes of nicotinic receptors is not feasible with these currently available ligands. This technical issue may be resolved by performing binding studies with brain regions or cell lines that express specific subtypes of nicotinic receptors. The lower Bmax observed for [3H]methylcarbamylcholine could be the result of binding to a subpopulation of nicotinic receptors. However, a more likely explanation is that the value is art±factually low due to loss of specific binding during filtration. [3H]Methylcarbamylcholine is very sensitive to filtration conditions due to its rapid rate of dissociation (tl/: off) of 3 min (Boksa and Quirion, 1987). [3H]Cytisine and [3H]nico-

tine on the other hand have respective off-rates of 13 and 15 min (Pabreza et al., 1991; Lippiello and Fernandes, 1986), which present an advantage for filtration assays. Interestingly, recent evidence from an immunoprecipitation study (Flores et al., 1991) demonstrated that [3H]cytisine binding is associated with the a4f12 receptor subtype in isolated nAChRs from rat brain. In light of the fact that approximately 90% of the neuronal nicotinic receptors in the brain have been shown to be a4~2 (Whiting et al., 1991), it is not surprising that this a-bungarotoxin and neuronal bungarotoxin-insensitive receptor subtype (Leutje et al., 1990b) is that which is readily detected in binding studies. It may be that other subtypes of nicotinic receptors are labeled by the three ligands, but that any possible distinctions are simply overwhelmed by the predominance of the a4/32 subtype. It also may be that none of the available radioligands are appropriate for studying subtypes other than a4/32 since it has been suggested (Flores et al., 1991) that high affinity [3H]cytisine binding is exclusively associated with the a4132 subtype in isolated receptors from membrane preparations. However, this would not preclude the binding of [3H]cytisine to other receptors subtypes of lower affinity which could be lost in the isolation procedure at the level of homogenate binding. Leutje and Patrick (1991) have reported that nicotinic receptors expressed in Xenopus oocytes show differential sensitivities to nicotinic receptor agonists. Differences in depolarizing responses were seen with

D.J. Anderson, S.P. Arneric / European Journal o f Pharmacology 253 (1994) 261-267

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-3

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Fig. 3. Correlations of the K i values of reference compounds determined at nicotinic receptors using [3H]cytisine vs. Ki values at muscarinic receptors using [3H]oxotremorine-M (OXO-M). Values plotted are the means of 3-6 independent determinations. nist tested. However, when the a-subunits were coexpressed with /32, ( - ) - c y t i s i n e was nearly inactive, being the lowest in the rank order. In fact, with a3/32-expressing oocytes where ( - ) - n i c o t i n e and ( - ) - c y t i s i n e do not appear to have agonist activity, ( - ) - c y t i s i n e was an effective blocker of the acetylcholine response while ( - ) - n i c o t i n e had no effect. Consistent with the idea that ( - ) - c y t i s i n e may act as an antagonist, it has been recently demonstrated that ( - ) - c y t i s i n e blocks in vivo nicotinically mediated increases in cortical blood flow elicited by electrical stimulation of the basal forebrain, while ( - ) - n i c o t i n e dramatically enhances the response (Linville et al., 1993). It is not possible from this study to determine whether ( - ) - c y t i s i n e is functionally an agonist or an antagonist. However, the demonstration here that ( - )-cytisine has a higher affinity for nicotinic receptors than ( - ) - n i c o t i n e or methylcarbamylcholine is consistent with the idea that ( - ) - c y t i s i n e may be an antagonist at the a4/32 subtype where it has essentially no agonist activity. In light of the report by Leutje and Patrick (1991) that ( - ) - c y t i s i n e may be acting as an antagonist when c~-subunits are associated with /32 subunits, ( - ) - c y t i s i n e would a priori block the response to ( - ) - n i c o t i n e or acetylcholine at a4/32 receptors in an analogous situation to a3/32 receptors. Also, if ( - ) - c y t i s i n e does act as an antagonist at this subtype, then it is still conceivable that lower affinity binding may occur at other receptors since ( - ) - c y t i s i n e is a very potent agonist at/34-containing receptor subtypes. Since the rank order and K i values for the reference compounds tested are very similar for the three radioligands, it appears that all three ligands bind to the same population of receptors even though they do so with different affinities and with apparently different densities. However, since a whole-brain membrane

D.J. Anderson, S.P. Arneric / European Journal of Pharmacology 253 (1994) 261-267

preparation was used in the present study, it is possible that regional differences in selectivity would not be readily apparent. Given the molecular and pharmacological diversity of nicotinic receptors and the regional distribution of mRNAs of subunits, it may yet be possible to delineate receptor subtypes using binding techniques. However, this may require the development of subtype-selective ligands or the identification of tissues that selectively express subtypes of nicotinic receptors in order to discriminate differences.

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