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[3H]TRICYCLOPINATE BINDING TO BRAIN MUSCARINIC ACETYLCHOLINE RECEPTORS: A COMPARISON WITH [3H]QUINUCLIDINYL BENZILATE
Pharmacological Research, Vol. 33, No. 4/5, 1996
[3H]TRICYCLOPINATE BINDING TO BRAIN MUSCARINIC ACETYLCHOLINE RECEPTORS: A COMPARISON WITH [3H]QUINUCLIDINYL BENZILATE Z. G. GAO and C. G. LIU Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, P.R. China Accepted 18 March 1996 The purpose of our study was to investigate the binding characteristics of a newly synthesized compound, tricyclopinate, at muscarinic acetylcholine receptors from rat cerebral cortex. This was achieved through the use of radiolabelled quinuclidinyl benzilate and radiolabelled tricyclopinate. Our data demonstrated that the saturation binding parameters of [3H]tricyclopinate (K d=0.10 nM, Bmax=1056 fmol mg−1) were almost identical to those of [3H]quinuclidinyl benzilate (Kd=0.11 nM, B max=1022 fmol mg−1); both ligands fit a one site model of receptor-ligand interaction. Concentration–inhibition curves were used to determine K i values for four antimuscarinic compounds. The rank order of potencies of the antagonists for displacement of the two ligands was: tricyclopinate=quinuclidinyl benzilate>atropine> pirenzepine. The competition binding parameters of [3H]tricyclopinate were similar to those of [3H]quinuclidinyl benzilate. The associate rate constants (K 1) were 0.25 and 0.21 nM−1 min−1 for [3H]tricyclopinate and [3H]quinuclidinyl benzilate, respectively. The dissociation of bound [3H]tricyclopinate from central muscarinic acetylcholine receptors was complete and was modified by the allosteric agent, gallamine. By comparison, only half of the bound [3H]quinuclidinyl benzilate was dissociated from muscarinic acetylcholine receptors and the dissociation of bound [3H]quinuclidinyl benzilate was not modified by gallamine. The dissociation rate constants (K −1 ) were 0.0325 and 0.0072 min−1 for [3H]tricyclopinate and [3H]quinuclidinyl benzilate, respectively. These results showed that the two ligands have different binding characteristics to muscarinic acetylcholine receptors. [3 H]tricyclopinate should be very useful for further study of central muscarinic acetylcholine receptors; it might complement the use of [3H]N-methylscopolamine and [3H]quinuclidinyl benzilate in the study of muscarinic acetylcholine receptors. 1996 The Italian Pharmacological Society KEY WORDS: muscarinic acetylcholine receptors, [ 3H]tricyclopinate, [3 H]quinuclidinyl benzilate, cerebral cortex.
INTRODUCTION Receptors can be characterized at the molecular level by studying their interactions with radiolabelled ligands that exhibit high affinity. The radiolabelled antagonist [3H]quinuclidinyl benzilate exhibits the desired properties and has been used extensively to study the muscarinic acetylcholine receptors from tissues, e.g.heart [1], brain [2, 3] and ileum [4, 5]. However, the binding profile of the [3H]quinuclidinyl benzilate seems to be rather complex. The dissociation of [3 H]quinuclidinyl benzilate from muscarinic acetylcholine receptors in rat cerebral cortex is not complete and can not be modified by gallamine and other allosteric agents [6]. It is impossible to study the interaction of allosteric agents at muscarinic acetylcholine receptors in rat cerebral cortex using [3 H]quinuclidinyl benzilate [6]. At present, only [3 H]Correspondence to: Dr C. G. Liu. 1043–6618/96/040283–07/$25.00/0
methylscopolamine was used when the allosteric modulation of central muscarinic acetylcholine receptors was studied [6, 7]. [3H]Quinuclidinyl benzilate, [3H]-methylquinuclidinyl benzilate, and [3H]N-methylscopolamine bind to an apparently homogeneous population of binding sites in rat cerebral cortex [3, 6, 8, 9]. These ligands suffer from limitations that restrict their utility [9]. Among these non-selective radiolabelled antagonists of muscarinic acetylcholine receptors, [3 H]quinuclidinyl benzilate was used as a standard antagonist for labelling muscarinic acetylcholine receptor binding sites [8, 9]. The binding sites labelled by [3H]Nmethylscopolamine are similar to the binding sites labelled by [3H]quinuclidinyl benzilate [9], but Nmethylscopolamine is a positive-charged quarternary amine; it is not able to penetrate the blood–brain barrier into the central nervous system, and N-methylscopolamine is about one order of magnitude less potent than quinuclidinyl benzilate in inhibiting the 1996 The Italian Pharmacological Society
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Pharmacological Research, Vol. 33, No. 4/5, 1996 O *
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[ H] Tricyclopinate
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Fig. 1. Molecular structure of [3 H]tricyclopinate and [ 3H]quinuclidinyl benzilate.
specific binding of [3H]quinuclidinyl benzilate [9]. So each ligand presently has its disadvantages, it being necessary to develop a ligand that might complement the use of [3H]quinuclidinyl benzilate and [3 H]methylscopolamine in the study of muscarinic acetylcholine receptors. In the course of investigating various antagonists to arecoline and nicotine, and their action on muscarinic and nicotinic acetylcholine receptors, a series of anticholinergics were synthesized at our Institute, which showed both antimuscarinic and antinicotinic activities [10–13]. Among these newly synthesized compounds, tricyclopinate (Fig. 1) was found to be a potent muscarinic acetylcholine receptor antagonist [12]. Previous experiments in our laboratory had shown that tricyclopinate had no selectivity for muscarinic acetylcholine receptor subtypes. In this paper, we report the characteristics of [3 H]tricyclopinate binding to muscarinic acetylcholine receptors in rat brain. The aim of the present study was to develop a new radiolabelled ligand that might complement the use of [3H]quinuclidinyl benzilate and [3H]N-methylscopolamine in studying muscarinic acetylcholine receptors.
MATERIALS AND METHODS
Drugs
[3H]Tricyclopinate (72.2 Ci mmol−1) was synthesized and provided by Dr Deyou Li of our Institute. The purity was determined to be greater than 99% by high performance liquid chromatography and thin layer chromatography. The authenticity of the material was confirmed by chromatography with a
sample of non-radioactive tricyclopinate as standard. High performance liquid chromatography analysis six months after preparation revealed that the radioactivity was almost quantitatively present in one peak with a retention time corresponding to the radioligand. [3H]quinuclidinyl benzilate (43.3 Ci mmol−1) was obtained from Amersham (Arlington Heights, IL, USA). Quinuclidinyl benzilate and tricyclopinate were synthesized at our Institute. Atropine sulphate, nicotine hydrogen tartrate, hexamethonium, pirenzepine and gallamine triethidide were from Sigma Co. (St Louis, MO, USA).
Preparation of rat cerebral cortex homogenates Male or female Wistar rats (180– 220 g) were killed by decapitation. The cerebral cortex was immediately removed and homogenized at 4°C in a glass homogenizer in 50 mM Na+/K + phosphate buffer (pH 7.4). The homogenate was centrifuged for 10 min at 1000 g at 4°C, and the supernatant fraction was centrifuged at 20,000 g for 45 min. The resultant pellet was resuspended in buffer. Proteins were determined by the method of Lowry et al. [14] using bovine serum albumin as a standard.
Muscarinic acetylcholine receptor binding assays To assay the specific binding of [3H]tricyclopinate and [3 H]quinuclidinyl benzilate, homogenates of rat cerebral cortex (0.1 mg protein) were incubated in duplicate with 0.01–1.0 nM of [3 H]tricyclopinate or [3H]quinuclidinyl benzilate for 60 min at 35°C in a final volume of 1 ml 50 mM Na+/K + phosphate buffer (pH 7.4). The reaction was terminated by rapid filtration under vacuum through Hongguang 49 glass filters (Shanghai, China) using a DTQ-II multitude cell harvester (Zhejiang, China), followed by washing of the filters three times with 9 ml of ice-cold 50 mM Na+/K + phosphate buffer. Non-specific binding was determined in the presence of 1 µM atropine. Filters were put into scintillation vials in 3 ml of scintillation fluid [containing PPO (2, 5-diphenyloxazol) 0.3%, POPOP (1,4-bis-5-phenyloxazoyl-2-benzene) 0.03% and radioactivity determined 12 h later. In competition binding studies, rat cerebral cortex homogenates (0.1 mg ml−1) were incubated in duplicate with 0.5 nM of [3 H]tricyclopinate or [3H]quinuclidinyl benzilate in the presence and absence of increasing concentrations of tested drugs. Incubations were carried out in a final volume of 1 ml of buffer at 35°C for 60 min. Nonspecific binding was determined in the presence of 1 µM atropine. Dissociation of the radioligand–receptor complex was initiated by adding a final concentration of 1 µM atropine in the absence and presence of various concentrations of gallamine when the association reached equilibrium. The dissociation time of [3H]tricyclopinate binding was 2 h, and the dissociation time of [3H]quinuclidinyl benzilate binding was more than 5 h.
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Data analysis
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The Kd and B max values were calculated as described by Niu et al. [10]. The Ki values were calculated from the method of Robard and Frazier [15] and ChengPrusoff’s equation [16]. The F-test was used for the analysis of the one and multiple site model. Other significant differences were assessed using the t-test, with a P value less than 0.05 being considered significant.
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As shown in Fig. 3, the rates of association of [3 H]tricyclopinate and [3H]quinuclidinyl benzilate were determined at various concentrations of each of the [3H]-ligands. The biomolecular rate constant for the [3H]tricyclopinate–receptor association (K1 ) is 0.25±0.03 nM−1 min −1, which is similar to the K1 value of [3H]quinuclidinyl benzilate (K 1=0.21± 0.02 nM−1 min−1).
Dissociation of specific [3H]tricyclopinate and [3H]quinuclidinyl benzilate binding As shown in Fig. 4, the dissociation of [3H]tricyclopinate from muscarinic acetylcholine receptors of the cerebral cortex was rapid and monophasic upon addition of 1 µM atropine. The dissociation rate constant (K−1) is 0.0325 min−1. Half-life of dissociation of this complex was calculated to be 17.2±2.8 min (n=4). When atropine and gallamine were added simultaneously, the dissociation of [3H]tricyclopinate remained monophasic but was slowed considerably (Fig. 4). Hence, 30 and 100 µM gallamine increased the half-life to 26.8±3.7 min (n=4) and 34.2±3.3 min (n=4), respectively. These values are significantly different from that obtained in the absence of gallamine (P<0.05). In contrast, a muscarinic antagonist, pirenzepine, did not influence the dissociation of [3 H]tricyclopinate even at the concentration of 300 µM. Therefore, the interaction of gallamine with an allosteric site on the rat brain muscarinic acetylch-
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Rates of association of [3H]tricyclopinate and [3H]quinuclidinyl benzilate at varying radioligand concentrations
0.2 0.4 0.6 0.8 1.0 B (pmol mg–1 protein)
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Saturation isotherms for the binding of [3H]tricyclopinate and [3H]quinuclidinyl benzilate at equilibrium to cerebral cortex membranes are shown in Fig. 2; Scatchard replots of these data were linear (Fig. 2). Analysis of the data reveals that both ligands binding to the receptor fit a one-site model. Respective Bmax values were 1056±47 and 1022±123 fmol mg−1 protein for [3 H]tricyclopinate and [3 H]quinuclidinyl benzilate, while the values of Kd were 0.10±0.01 and 0.11 ±0.01 nM for [3H]tricyclopinate and [3H]quinuclidinyl benzilate, respectively.
Bound (fmol mg
Determination of the number of binding sites for each radioligand
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Fig. 2. Saturation binding of radiolabelled muscarinic acetylcholine receptor antagonists to rat brain muscarinic acetylcholine receptors. Specific binding was determined with six concentrations of radioligand assayed in duplicate and non-specific binding in the presence of 1 µ M atropine determined in duplicate. Solid symbols represent specific binding (total minus non-specific binding). These plots were representative of 3–4 separate determinations. (A) [3H]tricyclopinate binding: (B) [ 3H]quinuclidinyl benzilate binding. The inserts show the Scatchard plots.
oline receptor is readily apparent in these experiments. In contrast to the dissociation of bound [3H]tricyclopinate, when the dissociation time was prolonged to 5 h, only about half of the bound [3H]quinuclidinyl benzilate was dissociated from rat central muscarinic acetylcholine receptors and the dissociation of bound [3H]quinuclidinyl benzilate was not modified by gallamine even when the concentration of gallamine was as high as 1000 µM (Fig. 4). The dissociation rate constant (K −1=0.0074 min −1) was not significantly different from that obtained in the absence of gallamine (K−1=0.0072 min−1) (P>0.05).
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Fig. 3. Association of [3 H]tricyclopinate (A) and [ 3H]quinuclidinyl benzilate (B) binding in rat cerebral cortex. The association rate was determined by incubation of tissues with 0.5 nM of [3H]tricyclopinate or [3H]quinuclidinyl benzilate at 35°C for the times shown, before filtration. The association rate constant, K1, was calculated from the equation K1 =(Kobs−K −1)/[ 3H]-ligand concentration. This experiment was replicated three times.
Fig. 4. Dissociation of [ 3H]tricyclopinate (A) and [3H]quinuclidinyl benzilate (B) binding in rat cerebral cortex and the effects of gallamine on the dissociation. Dissociation of the ligand–receptor complex was induced by 1 µ M atropine alone (d) or simultaneously added with 30 µM (,) or 100 µM (.) gallamine for [3H]tricyclopinate binding (A) and 1000 µM (,) gallamine for [3H]quinuclidinyl benzilate binding (B). Each point represents the mean from four independent experiments performed in duplicate.
Effects of cholinergic and non-cholinergic drugs on specific binding of [3H]tricyclopinate and [3H]quinuclidinyl benzilate
the tested compounds to muscarinic acetylcholine receptors fit a one-site model.
As shown in Fig. 5, the specific binding of [3 H]tricyclopinate and [3 H]quinuclidinyl benzilate was inhibited by the non-radiolabelled quinuclidinyl benzilate, tricyclopinate, atropine and pirenzepine. The K i and nH values of these compounds are summarized in Table I. Other non-muscarinic agents, such as nicotine, hexamethonium, mecamylamine and histamine, at concentrations as high as 10 µM, only displaced less than 15% of the specific binding of [3H]tricyclopinate. These data are similar to those obtained using [3 H]quinuclidinyl benzilate (Fig. 5). Analysis of the competition data suggested that the binding of all of
DISCUSSION An aim of the present study was to develop an additional affinity ligand for muscarinic acetylcholine receptors. The present study has demonstrated that the values of Kd and Bmax of [3H]tricyclopinate and [3H]quinuclidinyl benzilate binding to muscarinic acetylcholine receptors, and the rank order of Ki values for the reference compounds tested are very similar for the two ligands. Since the binding of both
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zilate displaced the binding of [3H]tricyclopinate and [3H]quinuclidinyl benzilate with almost identical nH and Ki values; it appears that both of the two ligands bind to the same population of receptors even though they do so with different binding characteristics. Our experiments showed that the dissociation of [3H]quinuclidinyl benzilate from rat central muscarinic acetylcholine receptors was not complete and could not be modified by gallamine. In contrast, the dissociation of bound [3H]tricyclopinate was complete and could be modified by gallamine. One possible explanation for the data presented in this article is that gallamine binds to an allosteric site as well as to the site to which quinuclidinyl benzilate binds. According to this scheme, quinuclidinyl benzilate must be insensitive to the allosteric interaction that affects the binding of tricyclopinate. Another possibility is that quinuclidinyl benzilate and gallamine interact allosterically, but that the binding of either ligand reduces the affinity of the other to such an extent that ternary complexes can not be demonstrated. It is possible that tricyclopinate might interact with the receptor in a different manner than does quinuclidinyl benzilate. Previous experiments of Ellis and Lenox [6] had investigated the effects of gallamine on the dissociation of [3 H]quinuclidinyl benzilate and [3H]-methylscopolamine from rat forebrain homogenates. They demonstrated that gallamine significantly slowed the dissociation of bound [3H]N-methylscopolamine, but it failed to alter the dissociation rate of bound [3H]quinuclidinyl benzilate. The present results showed the dissociation kinetics of [3H]tricyclopinate as different from that of [3H]quinuclidinyl benzilate. It remains to be seen whether most other muscarinic ligands will be found to behave like [3H]tricyclopinate, [3H]quinuclidinyl benzilate or [3H]N-methylscopolamine in the presence of gallamine. The possible mechanisms of the allosteric regulation of [3H]tricyclopinate by gallamine are that gallamine acts at the allosteric site on muscarinic acetylcholine receptors, the interaction between gallamine and the allosteric binding site induced the conformational change of the receptor, and this made the binding of the [3H]tricyclopinate–receptor complex more stable, so the dissociation of [3H]tricyclopinate from the receptor was slowed. It is possible that gallamine’s effects on [3H]tricyclopinate dissociation are
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Fig. 5. Displacement of specific [3 H]tricyclopinate (A) and [ 3H]quinuclidinyl benzilate (B) binding by various concentrations of tricyclopinate (d), quinuclidinyl benzilate (,), atropine (.) and pirenzipine (h). 0.1 mg of tissue was incubated with 0.5 nM of [ 3H]tricyclopinate or 3 [ H]quinuclidinyl benzilate in 1 ml of Na +−K+ phosphate buffer (pH 7.4) for 60 min at 35°C. Each point represents the mean from four independent experiments performed in duplicate.
ligands to muscarinic acetylcholine receptors fit a one-site model, tricyclopinate and quinuclidinyl ben-
Table I Effects of various compounds on [3 H]tricyclopinate and [3 H]quinuclidinyl benzilate binding to muscarinic acetylcholine receptors in rat cerebral cortex [3 H]Tricyclopinate
Tricyclopinate Quinuclidinyl benzilate Atropine Pirenzepine
[3 H]Quinuclidinyl benzilate
Ki (nM)
nH
K i (nM)
nH
0.22±0.02 0.27±0.04 1.6±0.27 175±15
0.82±0.05 0.84±0.03 0.86±0.11 0.81±0.16
0.37±0.06 0.48±0.04 2.3±0.15 102±8.3
0.83±0.07 0.84±0.09 0.82±0.13 0.80±0.23
Results were expressed as mean±SEM, n=4.
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similar to its effects on [3H]N-methylscopolamine dissociation as described elsewhere [1, 6, 17, 18]. The dissociation rate of quinuclidinyl benzilate is low and can not be modified by gallamine. However, the dissociation of the quinuclidinyl benzilate–receptor complex was increased by the presence of the Nmethyl group in the structure of quinuclidinyl benzilate [19]. It is possible that allosteric effects at the muscarinic receptor are most apparent when the labelling ligand contains a tropate nucleus with a positively charged quarternary amine functional group. It has been demonstrated that allosteric effects of gallamine strongly depend on the ligands used to conduct the binding experiments [6]. For example, the binding of [3H]N-methylscopolamine in brain tissue is much more susceptible to allosteric modification by gallamine than the binding of either [3H]quinuclidinyl benzilate [6] or 3H]pirenzepine [17]. Furthermore, the allosteric characteristics of gallamine also appear to be tissue dependent [1, 6, 17, 18]. Gallamine was most potent in modifying the dissociation rate of [3H]Nmethylscopolamine, followed in a descending order by that of the [3H]atropine, [3H]N-methylquinuclidinyl benzilate and [3H]quinuclidinyl benzilate. Therefore, it appears that there is a dependence of the allosteric interactions of gallamine with muscarinic acetylcholine receptors on both the charge and nucleus of the radiolabelled ligand used [8]. As seen in the present study, when compared to [3H]N-methylscopolamine and [3H]methylquinuclidinyl benzilate, there is also a N-methyl group in the molecular structure of tricyclopinate. It is possible that the N-methyl group is important for gallamine’s allosteric effects. The varying allosteric effects of gallamine exhibited by using different muscarinic ligands might be due to the fact that these ligands might induce specific conformational states of the receptor [20], so that the complexes of the ligands and the receptor might modulate gallamine binding differently, or are modulated differently by gallamine. In summary, our experiments demonstrated that the saturation and competition binding parameters of [3 H]tricyclopinate to brain muscarinic acetylcholine receptors are almost identical to those of [3H]quinuclidinyl benzilate. The dissociation of bound [3 H]quinuclidinyl benzilate was not complete and could not be modified by an allosteric agent, but the dissociation of bound [3H]tricyclopinate was complete and was modified by an allosteric agent. So the newly synthesized [3 H]tricyclopinate should be very useful for further studies of central muscarinic acetylcholine receptors.
ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China.
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REFERENCES 1. Lee NH, El-Fakahany EE. Allosteric antagonists of the muscarinic acetylcholine receptors. Biochem Pharmacol 1991; 42: 199–205. 2. Abood LG, Saraswati M, Lerner-Marmarash N, Hashmi M. Affinity ligands and related agents for brain muscarinic and nicotinic cholinergic receptors. Biochem Pharmacol 1993; 45: 2143–8. 3. Christopoulos A, Mitchelson F. Assessment of the allosteric interactions of the bisquaternary heptane-1,7bis(dimethyl-3'-phthalimidopropyl)ammonium bromide at M1 and M2 muscarinic receptors. Mol Pharmacol 1994; 46: 105–14. 4. Elnatan A, Mitchelson F. The interaction of McN-A343 with muscarine receptors in cardiac and smooth muscle. Biochem Pharmacol 1993; 46: 993–1003. 5. Tucek S, Musilkova J, Nedoma J, Shelkovnikov S, Vorlicek J. Positive cooperativity in the binding of alcuronium and N-methylscopolamine to muscarinic acetylcholine receptors. Mol Pharmacol 1990; 38: 674–80. 6. Ellis J, Lenox RH. Characterization of the interactions of gallamine with muscarinic receptors from brain. Biochem Pharmacol 1985; 34: 2214–7. 7. Leither F, Mohr K, Staschen CM. Interaction of the hexamethonium derivative W84, an allosteric effector at muscarinic cholinoceptors, with rat brain nicotinic binding sites. Pharmacol Toxicol 1990; 67: 317–21. 8. Lee NH, El-Fakahany EE. Influence of ligand choice on the apparent binding profile of gallamine to cardiac muscarinic receptors. Identification of three main types gallamine-muscarinic receptor interactions. J Pharmacol Exp Ther 1988; 246: 829–38. 9. Vickroy TW, Watson M, Yamamura HI, Roeske WR. Muscarinic Cholinergic Binding. In: O’Brien RA, ed. Receptor Binding in Drug Research. New York: Basel, 1986: 297–319. 10. Niu WZ, Zhao DL, Liu CG. The effects of a new cholinolytic—8018—and its optical isomers on the central muscarinic and nicotinic receptors. Arch Int Pharmacodyn 1990; 304: 64–74. 11. Gao ZG, Wang L, Liu CG. Effects of 2 α-DHET and its optical isomers on muscarinic and nicotinic receptors. Life Sci 1995; 56: 461–6. 12. Liu CG, Wang H, Zhao DL, Gao ZG, Cui WY, Zhang SP, Qiao QS, Ran YZ. Tricyclopinate HCl—a new synthetic compound with both muscarinic and nicotinic antagonist activities. In: Clark PBS, Quik M, Thurau K, Adlkofer F, eds. The Effects of Nicotine on Biological Systems II. Birkhauser Verlag, 1994: 20. 13. Gao ZG, Liu CG. Competitive and allosteric binding of 2α -DHET and its optical isomers to rat cardiac muscarinic receptors. Eur J Pharmacol 1995; 289: 369–73. 14. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folinphenol reagent. J Biol Chem 1951; 193: 265–75. 15. Rodbard D, Frazier GR. Statistical analysis of radioligand assay data. Methods Enzymol 1975; 37: 3–22. 16. Cheng YC, Prusoff WH. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 1973; 22: 3099–108. 17. Gillard W, Waelbroeck M, Christophe J. In vitro effects of gallamine on dissociation kinetics of [ 3H]N-methylscopamine and [3H]pirenzepine from rat brain muscarinic receptors. J Recept Res 1986; 6: 47–61. 18. Ellis J, Seidenberg M. Two modulators interact at a common site on cardiac muscarinic receptors. Mol Pharmacol 1992; 42: 638–41.
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19. Waelbroeck M, Tastenoy M, Camus J, Feifel R, Mutscher E, Strohman C, Tack R, Lambrecht G, Christophe J. Stereoselectivity of the interaction of muscarinic antagonists with their receptors. Trends Pharmacol Sci 1989; 10 (Suppl.): 65–9.
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20. Luthin GR, Wolfe BB. [3 H]Pirenziepine and [3 H]quinuclidinyl benzilate binding to brain muscarinic cholinergic receptors. Differences in measured receptor density are not explained by differences in receptor isomerization. Mol Pharmacol 1984; 26: 164–9.
[3H]TRICYCLOPINATE BINDING TO BRAIN MUSCARINIC ACETYLCHOLINE RECEPTORS: A COMPARISON WITH [3H]QUINUCLIDINYL BENZILATE Z. G. GAO and C. G. LIU Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, P.R. China Accepted 18 March 1996 The purpose of our study was to investigate the binding characteristics of a newly synthesized compound, tricyclopinate, at muscarinic acetylcholine receptors from rat cerebral cortex. This was achieved through the use of radiolabelled quinuclidinyl benzilate and radiolabelled tricyclopinate. Our data demonstrated that the saturation binding parameters of [3H]tricyclopinate (K d=0.10 nM, Bmax=1056 fmol mg−1) were almost identical to those of [3H]quinuclidinyl benzilate (Kd=0.11 nM, B max=1022 fmol mg−1); both ligands fit a one site model of receptor-ligand interaction. Concentration–inhibition curves were used to determine K i values for four antimuscarinic compounds. The rank order of potencies of the antagonists for displacement of the two ligands was: tricyclopinate=quinuclidinyl benzilate>atropine> pirenzepine. The competition binding parameters of [3H]tricyclopinate were similar to those of [3H]quinuclidinyl benzilate. The associate rate constants (K 1) were 0.25 and 0.21 nM−1 min−1 for [3H]tricyclopinate and [3H]quinuclidinyl benzilate, respectively. The dissociation of bound [3H]tricyclopinate from central muscarinic acetylcholine receptors was complete and was modified by the allosteric agent, gallamine. By comparison, only half of the bound [3H]quinuclidinyl benzilate was dissociated from muscarinic acetylcholine receptors and the dissociation of bound [3H]quinuclidinyl benzilate was not modified by gallamine. The dissociation rate constants (K −1 ) were 0.0325 and 0.0072 min−1 for [3H]tricyclopinate and [3H]quinuclidinyl benzilate, respectively. These results showed that the two ligands have different binding characteristics to muscarinic acetylcholine receptors. [3 H]tricyclopinate should be very useful for further study of central muscarinic acetylcholine receptors; it might complement the use of [3H]N-methylscopolamine and [3H]quinuclidinyl benzilate in the study of muscarinic acetylcholine receptors. 1996 The Italian Pharmacological Society KEY WORDS: muscarinic acetylcholine receptors, [ 3H]tricyclopinate, [3 H]quinuclidinyl benzilate, cerebral cortex.
INTRODUCTION Receptors can be characterized at the molecular level by studying their interactions with radiolabelled ligands that exhibit high affinity. The radiolabelled antagonist [3H]quinuclidinyl benzilate exhibits the desired properties and has been used extensively to study the muscarinic acetylcholine receptors from tissues, e.g.heart [1], brain [2, 3] and ileum [4, 5]. However, the binding profile of the [3H]quinuclidinyl benzilate seems to be rather complex. The dissociation of [3 H]quinuclidinyl benzilate from muscarinic acetylcholine receptors in rat cerebral cortex is not complete and can not be modified by gallamine and other allosteric agents [6]. It is impossible to study the interaction of allosteric agents at muscarinic acetylcholine receptors in rat cerebral cortex using [3 H]quinuclidinyl benzilate [6]. At present, only [3 H]Correspondence to: Dr C. G. Liu. 1043–6618/96/040283–07/$25.00/0
methylscopolamine was used when the allosteric modulation of central muscarinic acetylcholine receptors was studied [6, 7]. [3H]Quinuclidinyl benzilate, [3H]-methylquinuclidinyl benzilate, and [3H]N-methylscopolamine bind to an apparently homogeneous population of binding sites in rat cerebral cortex [3, 6, 8, 9]. These ligands suffer from limitations that restrict their utility [9]. Among these non-selective radiolabelled antagonists of muscarinic acetylcholine receptors, [3 H]quinuclidinyl benzilate was used as a standard antagonist for labelling muscarinic acetylcholine receptor binding sites [8, 9]. The binding sites labelled by [3H]Nmethylscopolamine are similar to the binding sites labelled by [3H]quinuclidinyl benzilate [9], but Nmethylscopolamine is a positive-charged quarternary amine; it is not able to penetrate the blood–brain barrier into the central nervous system, and N-methylscopolamine is about one order of magnitude less potent than quinuclidinyl benzilate in inhibiting the 1996 The Italian Pharmacological Society
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Fig. 1. Molecular structure of [3 H]tricyclopinate and [ 3H]quinuclidinyl benzilate.
specific binding of [3H]quinuclidinyl benzilate [9]. So each ligand presently has its disadvantages, it being necessary to develop a ligand that might complement the use of [3H]quinuclidinyl benzilate and [3 H]methylscopolamine in the study of muscarinic acetylcholine receptors. In the course of investigating various antagonists to arecoline and nicotine, and their action on muscarinic and nicotinic acetylcholine receptors, a series of anticholinergics were synthesized at our Institute, which showed both antimuscarinic and antinicotinic activities [10–13]. Among these newly synthesized compounds, tricyclopinate (Fig. 1) was found to be a potent muscarinic acetylcholine receptor antagonist [12]. Previous experiments in our laboratory had shown that tricyclopinate had no selectivity for muscarinic acetylcholine receptor subtypes. In this paper, we report the characteristics of [3 H]tricyclopinate binding to muscarinic acetylcholine receptors in rat brain. The aim of the present study was to develop a new radiolabelled ligand that might complement the use of [3H]quinuclidinyl benzilate and [3H]N-methylscopolamine in studying muscarinic acetylcholine receptors.
MATERIALS AND METHODS
Drugs
[3H]Tricyclopinate (72.2 Ci mmol−1) was synthesized and provided by Dr Deyou Li of our Institute. The purity was determined to be greater than 99% by high performance liquid chromatography and thin layer chromatography. The authenticity of the material was confirmed by chromatography with a
sample of non-radioactive tricyclopinate as standard. High performance liquid chromatography analysis six months after preparation revealed that the radioactivity was almost quantitatively present in one peak with a retention time corresponding to the radioligand. [3H]quinuclidinyl benzilate (43.3 Ci mmol−1) was obtained from Amersham (Arlington Heights, IL, USA). Quinuclidinyl benzilate and tricyclopinate were synthesized at our Institute. Atropine sulphate, nicotine hydrogen tartrate, hexamethonium, pirenzepine and gallamine triethidide were from Sigma Co. (St Louis, MO, USA).
Preparation of rat cerebral cortex homogenates Male or female Wistar rats (180– 220 g) were killed by decapitation. The cerebral cortex was immediately removed and homogenized at 4°C in a glass homogenizer in 50 mM Na+/K + phosphate buffer (pH 7.4). The homogenate was centrifuged for 10 min at 1000 g at 4°C, and the supernatant fraction was centrifuged at 20,000 g for 45 min. The resultant pellet was resuspended in buffer. Proteins were determined by the method of Lowry et al. [14] using bovine serum albumin as a standard.
Muscarinic acetylcholine receptor binding assays To assay the specific binding of [3H]tricyclopinate and [3 H]quinuclidinyl benzilate, homogenates of rat cerebral cortex (0.1 mg protein) were incubated in duplicate with 0.01–1.0 nM of [3 H]tricyclopinate or [3H]quinuclidinyl benzilate for 60 min at 35°C in a final volume of 1 ml 50 mM Na+/K + phosphate buffer (pH 7.4). The reaction was terminated by rapid filtration under vacuum through Hongguang 49 glass filters (Shanghai, China) using a DTQ-II multitude cell harvester (Zhejiang, China), followed by washing of the filters three times with 9 ml of ice-cold 50 mM Na+/K + phosphate buffer. Non-specific binding was determined in the presence of 1 µM atropine. Filters were put into scintillation vials in 3 ml of scintillation fluid [containing PPO (2, 5-diphenyloxazol) 0.3%, POPOP (1,4-bis-5-phenyloxazoyl-2-benzene) 0.03% and radioactivity determined 12 h later. In competition binding studies, rat cerebral cortex homogenates (0.1 mg ml−1) were incubated in duplicate with 0.5 nM of [3 H]tricyclopinate or [3H]quinuclidinyl benzilate in the presence and absence of increasing concentrations of tested drugs. Incubations were carried out in a final volume of 1 ml of buffer at 35°C for 60 min. Nonspecific binding was determined in the presence of 1 µM atropine. Dissociation of the radioligand–receptor complex was initiated by adding a final concentration of 1 µM atropine in the absence and presence of various concentrations of gallamine when the association reached equilibrium. The dissociation time of [3H]tricyclopinate binding was 2 h, and the dissociation time of [3H]quinuclidinyl benzilate binding was more than 5 h.
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Data analysis
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The Kd and B max values were calculated as described by Niu et al. [10]. The Ki values were calculated from the method of Robard and Frazier [15] and ChengPrusoff’s equation [16]. The F-test was used for the analysis of the one and multiple site model. Other significant differences were assessed using the t-test, with a P value less than 0.05 being considered significant.
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As shown in Fig. 3, the rates of association of [3 H]tricyclopinate and [3H]quinuclidinyl benzilate were determined at various concentrations of each of the [3H]-ligands. The biomolecular rate constant for the [3H]tricyclopinate–receptor association (K1 ) is 0.25±0.03 nM−1 min −1, which is similar to the K1 value of [3H]quinuclidinyl benzilate (K 1=0.21± 0.02 nM−1 min−1).
Dissociation of specific [3H]tricyclopinate and [3H]quinuclidinyl benzilate binding As shown in Fig. 4, the dissociation of [3H]tricyclopinate from muscarinic acetylcholine receptors of the cerebral cortex was rapid and monophasic upon addition of 1 µM atropine. The dissociation rate constant (K−1) is 0.0325 min−1. Half-life of dissociation of this complex was calculated to be 17.2±2.8 min (n=4). When atropine and gallamine were added simultaneously, the dissociation of [3H]tricyclopinate remained monophasic but was slowed considerably (Fig. 4). Hence, 30 and 100 µM gallamine increased the half-life to 26.8±3.7 min (n=4) and 34.2±3.3 min (n=4), respectively. These values are significantly different from that obtained in the absence of gallamine (P<0.05). In contrast, a muscarinic antagonist, pirenzepine, did not influence the dissociation of [3 H]tricyclopinate even at the concentration of 300 µM. Therefore, the interaction of gallamine with an allosteric site on the rat brain muscarinic acetylch-
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Saturation isotherms for the binding of [3H]tricyclopinate and [3H]quinuclidinyl benzilate at equilibrium to cerebral cortex membranes are shown in Fig. 2; Scatchard replots of these data were linear (Fig. 2). Analysis of the data reveals that both ligands binding to the receptor fit a one-site model. Respective Bmax values were 1056±47 and 1022±123 fmol mg−1 protein for [3 H]tricyclopinate and [3 H]quinuclidinyl benzilate, while the values of Kd were 0.10±0.01 and 0.11 ±0.01 nM for [3H]tricyclopinate and [3H]quinuclidinyl benzilate, respectively.
Bound (fmol mg
Determination of the number of binding sites for each radioligand
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Fig. 2. Saturation binding of radiolabelled muscarinic acetylcholine receptor antagonists to rat brain muscarinic acetylcholine receptors. Specific binding was determined with six concentrations of radioligand assayed in duplicate and non-specific binding in the presence of 1 µ M atropine determined in duplicate. Solid symbols represent specific binding (total minus non-specific binding). These plots were representative of 3–4 separate determinations. (A) [3H]tricyclopinate binding: (B) [ 3H]quinuclidinyl benzilate binding. The inserts show the Scatchard plots.
oline receptor is readily apparent in these experiments. In contrast to the dissociation of bound [3H]tricyclopinate, when the dissociation time was prolonged to 5 h, only about half of the bound [3H]quinuclidinyl benzilate was dissociated from rat central muscarinic acetylcholine receptors and the dissociation of bound [3H]quinuclidinyl benzilate was not modified by gallamine even when the concentration of gallamine was as high as 1000 µM (Fig. 4). The dissociation rate constant (K −1=0.0074 min −1) was not significantly different from that obtained in the absence of gallamine (K−1=0.0072 min−1) (P>0.05).
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Fig. 3. Association of [3 H]tricyclopinate (A) and [ 3H]quinuclidinyl benzilate (B) binding in rat cerebral cortex. The association rate was determined by incubation of tissues with 0.5 nM of [3H]tricyclopinate or [3H]quinuclidinyl benzilate at 35°C for the times shown, before filtration. The association rate constant, K1, was calculated from the equation K1 =(Kobs−K −1)/[ 3H]-ligand concentration. This experiment was replicated three times.
Fig. 4. Dissociation of [ 3H]tricyclopinate (A) and [3H]quinuclidinyl benzilate (B) binding in rat cerebral cortex and the effects of gallamine on the dissociation. Dissociation of the ligand–receptor complex was induced by 1 µ M atropine alone (d) or simultaneously added with 30 µM (,) or 100 µM (.) gallamine for [3H]tricyclopinate binding (A) and 1000 µM (,) gallamine for [3H]quinuclidinyl benzilate binding (B). Each point represents the mean from four independent experiments performed in duplicate.
Effects of cholinergic and non-cholinergic drugs on specific binding of [3H]tricyclopinate and [3H]quinuclidinyl benzilate
the tested compounds to muscarinic acetylcholine receptors fit a one-site model.
As shown in Fig. 5, the specific binding of [3 H]tricyclopinate and [3 H]quinuclidinyl benzilate was inhibited by the non-radiolabelled quinuclidinyl benzilate, tricyclopinate, atropine and pirenzepine. The K i and nH values of these compounds are summarized in Table I. Other non-muscarinic agents, such as nicotine, hexamethonium, mecamylamine and histamine, at concentrations as high as 10 µM, only displaced less than 15% of the specific binding of [3H]tricyclopinate. These data are similar to those obtained using [3 H]quinuclidinyl benzilate (Fig. 5). Analysis of the competition data suggested that the binding of all of
DISCUSSION An aim of the present study was to develop an additional affinity ligand for muscarinic acetylcholine receptors. The present study has demonstrated that the values of Kd and Bmax of [3H]tricyclopinate and [3H]quinuclidinyl benzilate binding to muscarinic acetylcholine receptors, and the rank order of Ki values for the reference compounds tested are very similar for the two ligands. Since the binding of both
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zilate displaced the binding of [3H]tricyclopinate and [3H]quinuclidinyl benzilate with almost identical nH and Ki values; it appears that both of the two ligands bind to the same population of receptors even though they do so with different binding characteristics. Our experiments showed that the dissociation of [3H]quinuclidinyl benzilate from rat central muscarinic acetylcholine receptors was not complete and could not be modified by gallamine. In contrast, the dissociation of bound [3H]tricyclopinate was complete and could be modified by gallamine. One possible explanation for the data presented in this article is that gallamine binds to an allosteric site as well as to the site to which quinuclidinyl benzilate binds. According to this scheme, quinuclidinyl benzilate must be insensitive to the allosteric interaction that affects the binding of tricyclopinate. Another possibility is that quinuclidinyl benzilate and gallamine interact allosterically, but that the binding of either ligand reduces the affinity of the other to such an extent that ternary complexes can not be demonstrated. It is possible that tricyclopinate might interact with the receptor in a different manner than does quinuclidinyl benzilate. Previous experiments of Ellis and Lenox [6] had investigated the effects of gallamine on the dissociation of [3 H]quinuclidinyl benzilate and [3H]-methylscopolamine from rat forebrain homogenates. They demonstrated that gallamine significantly slowed the dissociation of bound [3H]N-methylscopolamine, but it failed to alter the dissociation rate of bound [3H]quinuclidinyl benzilate. The present results showed the dissociation kinetics of [3H]tricyclopinate as different from that of [3H]quinuclidinyl benzilate. It remains to be seen whether most other muscarinic ligands will be found to behave like [3H]tricyclopinate, [3H]quinuclidinyl benzilate or [3H]N-methylscopolamine in the presence of gallamine. The possible mechanisms of the allosteric regulation of [3H]tricyclopinate by gallamine are that gallamine acts at the allosteric site on muscarinic acetylcholine receptors, the interaction between gallamine and the allosteric binding site induced the conformational change of the receptor, and this made the binding of the [3H]tricyclopinate–receptor complex more stable, so the dissociation of [3H]tricyclopinate from the receptor was slowed. It is possible that gallamine’s effects on [3H]tricyclopinate dissociation are
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Fig. 5. Displacement of specific [3 H]tricyclopinate (A) and [ 3H]quinuclidinyl benzilate (B) binding by various concentrations of tricyclopinate (d), quinuclidinyl benzilate (,), atropine (.) and pirenzipine (h). 0.1 mg of tissue was incubated with 0.5 nM of [ 3H]tricyclopinate or 3 [ H]quinuclidinyl benzilate in 1 ml of Na +−K+ phosphate buffer (pH 7.4) for 60 min at 35°C. Each point represents the mean from four independent experiments performed in duplicate.
ligands to muscarinic acetylcholine receptors fit a one-site model, tricyclopinate and quinuclidinyl ben-
Table I Effects of various compounds on [3 H]tricyclopinate and [3 H]quinuclidinyl benzilate binding to muscarinic acetylcholine receptors in rat cerebral cortex [3 H]Tricyclopinate
Tricyclopinate Quinuclidinyl benzilate Atropine Pirenzepine
[3 H]Quinuclidinyl benzilate
Ki (nM)
nH
K i (nM)
nH
0.22±0.02 0.27±0.04 1.6±0.27 175±15
0.82±0.05 0.84±0.03 0.86±0.11 0.81±0.16
0.37±0.06 0.48±0.04 2.3±0.15 102±8.3
0.83±0.07 0.84±0.09 0.82±0.13 0.80±0.23
Results were expressed as mean±SEM, n=4.
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similar to its effects on [3H]N-methylscopolamine dissociation as described elsewhere [1, 6, 17, 18]. The dissociation rate of quinuclidinyl benzilate is low and can not be modified by gallamine. However, the dissociation of the quinuclidinyl benzilate–receptor complex was increased by the presence of the Nmethyl group in the structure of quinuclidinyl benzilate [19]. It is possible that allosteric effects at the muscarinic receptor are most apparent when the labelling ligand contains a tropate nucleus with a positively charged quarternary amine functional group. It has been demonstrated that allosteric effects of gallamine strongly depend on the ligands used to conduct the binding experiments [6]. For example, the binding of [3H]N-methylscopolamine in brain tissue is much more susceptible to allosteric modification by gallamine than the binding of either [3H]quinuclidinyl benzilate [6] or 3H]pirenzepine [17]. Furthermore, the allosteric characteristics of gallamine also appear to be tissue dependent [1, 6, 17, 18]. Gallamine was most potent in modifying the dissociation rate of [3H]Nmethylscopolamine, followed in a descending order by that of the [3H]atropine, [3H]N-methylquinuclidinyl benzilate and [3H]quinuclidinyl benzilate. Therefore, it appears that there is a dependence of the allosteric interactions of gallamine with muscarinic acetylcholine receptors on both the charge and nucleus of the radiolabelled ligand used [8]. As seen in the present study, when compared to [3H]N-methylscopolamine and [3H]methylquinuclidinyl benzilate, there is also a N-methyl group in the molecular structure of tricyclopinate. It is possible that the N-methyl group is important for gallamine’s allosteric effects. The varying allosteric effects of gallamine exhibited by using different muscarinic ligands might be due to the fact that these ligands might induce specific conformational states of the receptor [20], so that the complexes of the ligands and the receptor might modulate gallamine binding differently, or are modulated differently by gallamine. In summary, our experiments demonstrated that the saturation and competition binding parameters of [3 H]tricyclopinate to brain muscarinic acetylcholine receptors are almost identical to those of [3H]quinuclidinyl benzilate. The dissociation of bound [3 H]quinuclidinyl benzilate was not complete and could not be modified by an allosteric agent, but the dissociation of bound [3H]tricyclopinate was complete and was modified by an allosteric agent. So the newly synthesized [3 H]tricyclopinate should be very useful for further studies of central muscarinic acetylcholine receptors.
ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China.
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