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Effects of a new cholinolytic drug of tropanes and its optical isomers on central muscarinic and nicotinic receptors
PharmacologicalResearH~.Vol.32. No. 3. 1995
105
EFFECTS OF A NEW CHOLINOLYTIC DRUG OF TROPANES AND ITS OPTICAL ISOMERS ON CENTRAL MUSCARINIC AND NICOTINIC RECEPTORS z. G. GAO, L. WANG, C. G. LIU and Q. K. ZHANG
Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 27, Taiping Road, Beijhlg 100850, People's Republic of China Accepted 27 July 1995 2o~-(2',2'-disubstituted-2'-hydroxy-ethoxy)tropane (2~-DHET), a new cholinolytic, is a racemic tertiary amine with two chiral carbonic atoms. It has four optical isomers whose absolute configurations are IR-2o~-2'S, IR-2o~-2'R, IS-2o~-2'R and IS-2c~-2'S. These compounds showed both antimuscarinic and antinicotinic activity, blocking both muscarinic and nicotinic receptors. Central muscarinic receptors, rather than nicotinic receptors, have a stereoselective specificity to these compounds. The 2'R configurations are more suitable to the stereostructure of the binding site of muscarinic receptors than the 2'S configuration. © 1995 The Italian Pharmacological Society KEYWORDS:muscarinic receptors, nicotinic receptors, optical isomers.
INTRODUCTION
MATERIALS AND METHODS
Stereoselectivity means that the 'active' enantiomer should be about one to two orders more potent than the 'inactive' enantiomer [1]. It is known that many recepto.rs show stereoselectivity to isomers of their ligands [2]. It has been found that muscarinic receptors have many racemic ligands whose enantiomers possess different affinities [2]. Antagonist binding site of muscarinic receptors are asymmetrical, and hence generally capable of discriminating optical isomers of chiral drug. Thus analysis of binding and/or pharmacological properties of stereoisomers provide information about binding site geometry [2]. A new cholinolytic, 2o¢-(2',2'-disubstituted-2'-hydroxy-ethoxy) tropane(2~x-DHET), synthesized at our institute, is a racemic tertiary amine with two chiral carbonic atoms in its molecular structure (Fig. 1). There are thus four optical isomers of different configurations, named 1R2a-2'S, 1R-2a-2'R, 1S-2~x-2'R and 1S-2o~-2'S, as identified by X-ray diffraction and nuclear magnetic resonance. Previous experiments in our laboratory had demonstrated that 2o~-DHET had anti-soman activity and it had no selectivity for muscarinic subtypes. We have now investigated the binding profiles of 2tx-DHET and its four isomers to muscarinic receptors and nicotinic receptors of rat forebrain and their pharmacological activities to central muscarinic and nicotinic receptors.
Membrane preparations The procedure used was similar to that of Yamamura [3]. Male or female Wistar rats, weighing 180-220 g, were decapitated. Forebrains were rapidly removed and homogenized in 10 volumes (w/v) of ice-cold 0.32 M sucrose in a glass homogenizer. The whole homogenate was centrifuged at 1000g for 10 min at 4°C. The pellet was discarded and supernatant was recentrifuged at 20 000g for 30 min at 4°C. The supernatant was poured out and the pellet (P_,) was suspended in four volumes of sodium potassium phosphate buffer (pH 7.4, 4°C). Then /:'2 was rehomogenized in a glass homogenizer and stored at -20°C. P,_ was used within 2 weeks; it has been demonstrated that there were no significant changes in the binding characteristics of P_, stored in this condition for 1 month. Proteins were determined by the method of Lowry et al. [4] using bovine serum albumin as a standard.
Correspondenceto: Z. G. Gao. 1043-6618/951090105-05/$12.00/0
Binding assay To assay the specific binding of [3H]QNB, 0.1 mg of P_, was incubated at 35°C for 3 0 m i n with 0.01-1.0 nM of [3H]QNB in 1 ml of a 50 mM sodium potassium phosphate buffer solution in the absence or presence of I /2M unlabelled atropine to determine the total and non-specific binding, respectively. The incubation was stopped by addition of 3 ml ice-cold buffer solution. Bound [3H]QNB was separated from the unbound ligand by vacuum filtration using Hongguang 49 glass filters (Shanghai, China). The filters were washed three times with 9 ml of an ice-cOld
©1995The ItalianPharmacologicalSociety
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Pharmacological Research. I/ol. 32, No. 3, 1995
buffer solution. Every determination of binding was performed in duplicate. Then the filters were placed in vials containing 3 ml scintillating liquid (containing PPO 0.3% and POPOP 0.3%) and maintained for over 10h at room temperature. The radioactivity was assayed by a liquid scintillating counter (LKB 1215 Rack Beta II model) at a counting efficiency of 36%. The binding of 2 a - D H E T and its isomers and other agents tested to muscarinic receptors was determined by their ability to displace [3H]QNB. Protein (0.1 mg) was incubated with 0.5 nM of [3H]QNB in the presence of 2ot-DHET and its isomers and other agents. To assay the binding of 2a-DHET and its isomers to nicotinic receptors, 0.2 mg of protein was incubated with 5 nM of [3H]nicotine in 1 ml of Tris-HCI buffer (pH 8.0) at 4°C for 60 min in the presence of tested drugs. The samples were then filtered through Hongguang 49 glass filters presoaked with 0.05% polyethylenimine solution for 5 h. The remaining procedures were similar to the binding assay of muscarinic receptors.
Arecoline-induced tremor in mice and the antagonistic effects of drugs Arecoline (8 mg kg -~, s.c.) induced tremor in all 20 control mice in 10 min. Five doses of 2 a - D H E T and its isomers were injected i.p. 15 min before arecoline (8 mg kg -~, s.c.) to estimate the EDso values of the antagonist effect of these compounds. There were 10 Shanghai mice, weighing 18-22 g in each dose-group.
Nicotine-induced convulsion in mice and the antagonistic effects of drugs The procedure used was similar to that of Niu et al. [5]. After injection of 0.8 mg kg -~ of nicotine free base into the caudal vein of mice, tetanic convulsion in all 20 control mice was observed. The methods to estimate the antagonistic effect of 2 a - D H E T and its four optical isomers against nicotine-induced convulsion were the same as for arecoline-induced tremor, but the tested drugs were injected into the caudal vein. EDs0 values of these compounds against arecolineinduced tremor and nicotine-induced convulsion were calculated from the probability-logarithm method [6].
Materials [3H]QNB
(43.3 Ci mmol -I)
and
[3H]nicotine
(78.4Ci mmol -~) were purchased from Amersham, 2o'-DHET and its isomers were synthesized at our institute. Hexamethonium, atropine, gallamine were from Sigma, arecoline from EMK, nicotine freebase from Merk-Schuchard (Munich, Germany). An automatic high-speed refrigerated centrifuge of Hitachi 20 PR-5 and a DTQ II-multitute cell collector of Zhejiang, China were used.
RESULTS
Binding profiles of 2 o~-DHET and its isomers to muscarinic receptors o f rat forebrain As shown in Fig. 2, atropine, a classic muscarinic antagonist, was potent to displace specific binding of [3H]QNB, but other nonmuscarinic agents, such as hexamethonium, mecamylamine and nicotine, only displace less than 15% of specific binding of [3H]QNB, even at the concentrations as high as 10/.tM (data not shown). It was demonstrated that the isomer with IR-2oc-2'R configuration was the most potent and the isomer with 1R-2o~-2'S configuration was the least potent among the four isomers in displacing specific binding of [3H]QNB. Values of ICs~ were calculated from Rodbard and Frazier [7] and those of the inhibitory equilibrium dissociation constant (K0 were calculated from Cheng-Prusoff's [8] equation and are listed in Table 1. The results show that central muscarinic receptors have different affinities to these agents, the order of potency to displace [3H]QNB was IR-2oc2'R> 1S-2 o¢-2'R>2 o~-DHET> 1S-2 ¢x-2'S> 1R-2 ¢z-2'S.
Effects of 2at-DHET and its optical isomers on [3H]nicotine binding To determine the binding specificity of these compounds, experiments were performed with [3H]nicotinc. 2~-DHET and its optical isomers at concentrations of 10/.tM, which resulted in >80% inhibition of [3H]QNB binding, had no effect on reversible [3H]nicotine binding.
Pharmacological effects o f 2 ot-DHET and its four optical isomers on central muscarinic and nicotinic receptors As shown in Fig. 3, arecoline-induced tremor in mice can be antagonized by 2 a - D H E T and its four
Table I Effects of 2 a - D H E T and its isomers on rat and mouse central muscarinic and nicotinic receptors
Ki (riM)* EDso against arecoline (mg kg-~)t EDso against nicotine (mg kg-k):l:
lR-2ct-2'R
1S-2ct-2'R
2 ct-DHET
1S-2¢t-2'S
1R-2ot-2'S
6.1+0.62 0.51+0.07 3.1+0.29
6.8_+0.80 0.72+0.08 3.8+0.76
26.4+2.85 1.2+0.21 3.1+0.29
114.5+10.70 2.9+0.89 3.3+0.41
381.5+27.40 5.6+1.1 I 2.7+0.71
Results are expressed as m e a n ~ . *rat forebrain, n--4. ?areeoline (8 mg kg-~ s.c.) -induced mouse tremor. :~nicotine (0.8 mg kg-~, i.v.) -induced mouse convulsion.
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Pharmacological Rese,:trch, Vol. 32. No. 3. 1995
H3C"-N
OH
2a-DHET
OH /=~
2.1
4r--~ ~ -CH3 i'
N- CH3
a//f~-~ I
© 1R-2a-2'S H3C 1 - ' ~
1R-2a-2"R
H3C-..N
4
~CH2~~
@ 1S-2a-2~R
IS-2a-2'S
Fig. 1. Molecular structure of 2cr-DHET and its optical isomers. The [~]~ values were -11.40, +32.40, +11.60 and -31.76 for I R-2/z-2'S, I R-2o~-2'R, I S-2a-2'R and 1S-2a-2'S, respectively. The optical purity of the four optical isomers were 100%.
o
100
.~.
\\\ \ ~6o~
~\~
?
80
\ \\
60
40
~2 o ~
,1:I
.,~ 9.0 8
o -10
I
-9
-8
-7
-6
-5
--4
Log [M] Fig. 2. Competitive curves of atropine ((3), 2a-DHET (V), IR-2cr-2'S (U), 1R-2/z-2'R (Q), 1S-2ot-2'R (~7) and IS-2/~2'S ([3) at rat forebrain muscarinic receptors. Membrane (0.1 mg) of rat forebrain was incubated with 0.5riM of [3H]QNB at 35°C in l ml of 50mM sodium potassium phosphate buffer (pH 7.4) for 30 min in the presence of atropine, 2a-DHET and its isomers (n=4). optical isomers. The order of their antagonizing potency was 1R-2~z-2'R>IS-2o~-2'R>2ot-DHET>IS2/z-2'S> I R-2 ~-2'S. The EDs0 values were summarized in Table I. The isomer with IR-2o~-2'R configuration
0
2
4
6
8
10
Dose (ragkg-1) Fig. 3. Dose-response curves of 2~x-DHET (V), 1R-2o~-2'S (I--1), IR-2o~-2'R (O), IS-2or-2'R (O) and 1S-2o~-2'S (~7) on arecoline-induced tremor. Different doses of 2a,-DHET and its optical isomers were injected i.p. 15 min before arecoline (8 mg kg-', s.c.). Results were from three experiments.
was about 11 times stronger than the 1R-2o~-2'S isomer. The antagonistic effects of 2 a - D H E T and its isomers against nicotine-induced convulsion in mice were completely different (Fig. 4). There was: no sig-
Pharmacological Research. Vol. 32, No. 3, 1995
108
O
100
80
0
60
':i,'?
8 40
.~ 9.0
I
Z 0
1.5
3.0 4.5 Dose (mg kg-1)
I
6.0
Fig. 4. Dose-response curves of 2a-DHET (O), IR-2a-2'S (O), 1R-2a-2'R (V), IS-2a-2'R (Y) and 1S-2a-2'S (IS]) on nicotine-induced convulsion. Different doses of 2a-DHET and its optical isomers were injected i.v. 15min before nicotine (0.Smg kg-I, i.v.). Results were from three experiments.
nificant difference in EDso values (Table I) to prevent nicotine-induced convulsion among these agents. This suggested that, unlike the central muscarinic receptors, central nicotinic receptors had no stereoselectivity to these compounds.
DISCUSSION Binding assays showed that central muscarinic receptor have a stereoselectivity to 2 a - D H E T and its optical isomers and the results of functional experiments confirmed this. As for the central muscarinic receptors, the relative potency, judged by K~ and EDs0, of 2tr-DHET and its isomers ranks the same order, i.e. 1R-2 a-2'R> 1S-2 a-2'R> 2 a - D H E T > 1S-2 a-2'S> l R2a-2'S. The relationship between LnKi and EDso values against arecoline was linear (r=-0.953, P<0.01). The pharmacological potency of 2 a - D H E T and its isomers to central muscarinic receptors thus corresponds with their affinities to muscarinic receptors. The higher their affinities, the more potent their pharmacological effects. It is possible that the binding site is equivalent to the physiologically and pharmacologically relevant site. It was found from binding assays and functional experiments that the carbon-2' configuration is more important for the antimuscarinic activities of 2aD H E T than the tropane ring configuration. This result is similar to that of 2-substituted tropane esters and 3substituted quinuclidinyl ethers [5, 9]. It is suggested that the R configuration of carbon-2' is more suitable to the stereochemical structure of the binding site of
central muscarinic receptors than that of the S configuration. In contrast to central muscarinic receptors, central nicotinic receptors have no stereoselectivity to these compounds, as the differences of their EDso values against nicotine-induced convulsion were minimal. These results are similar to that of 8018 and its four optical isomers [5]. It is suggested that binding site of central nicotinic receptors may interact with other parts of the molecular structure of these compounds. Central muscarinic receptor is a monomeric receptor with seven consecutive transmembrane regions [I0]. It stereoselectivity comes from the arrangement of its key groups in spacial direction. On the other hand, available evidence suggests that the neuronal nicotinic receptors are composed of a and fl subunits [11]. As in the muscle nicotinic receptor, the agonist recognition site of neuronal nicotinic receptors is located on a subunit, but the total number of subunits of the neuronal receptor and the subunit stoichiometry is at present uncertain [12]. The difference in their antinicotinic activity and their ability to displace the specific binding of [3H]nicotine reveals that it is possible that 2 a - D H E T and its isomers do not act at the recognition site of central nicotinic receptors. Molecular biology studies have elucidated that there are at least 10 nicotinic receptor genes in the brain, namely: o'2, a3, a4, a5, a6, a7 and f12, f13, ,84, f15. Of these, a2, a3 and a 4 can combine with f12 or f14 subunits to form at least six functional a-bugarotoxin insensitive nicotinic receptors [12, 13]. o~7 nicotinic receptor is sensitive to a-bugarotoxin, coinjection of f12, f13 or f14 cDNA, along with a7 cDNA, did not result in functional responses with properties any different than those expressed in oocytes injected with a7 cDNA alone [12]. Thus, in oocytes at least, a7 subunits apparently cannot coassemble with known neuronal fl subunits to form hetero-oligomers that have functional properties distinct from those c~7 homo-oligomers [12]. The f13 gene does not form functional nicotinic receptors when coinjected as mRNA with a2, a3 or a 4 genes [14]. The rat a'5 and a'6 genes, likewise do not participate in the formation of functional nicotinic channels in oocytes when injected in combination with several other a and fl genes as mRNA [15]. The sensitivity of heterologously expressed nicotinic receptors to nicotinic receptor antagonist, a-bugarotoxin, is dependent to the nature of the subunit composition [12]. These data indicate that the structure and function of central nicotinic receptors are more complex than that of muscarinic receptors, and it is necessary to further characterize brain nicotinic receptors with both molecular biology techniques and new specific ligands. In summary, the present study demonstrated that 2ot-DHET and its isomers had both antimuscarinic and antinicotinic activities. Muscarinic receptors had stereoselectivity to these compounds, but nicotinic receptors had no stereoselectivity to these compounds.
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Pharnuwological Research, Vol. 32. No. 3. 1995
The difference in their antinicotinic activity and their ability to displace the specific binding of [3H]nicotine reveals that it is possible that 2 a - D H E T and its isomers do not act at the recognition site of central nicotinic receptors.
7. 8.
9.
REFERENCES 10. 1. Hoyer D. Implications of stereoselectivity in radioligand binding studies. Trends Pharmacol Sci 1986; 7: 227-30. 2. Waelbroek M, Tastenoy M, Camus J, Feifel R, Mutschler E, Strohman C, Tacke R, Lambrecht G, Christophe J. Stereoselectivity of the interaction of muscarinic antagonists with their receptors. Trends Pharmacol Sci 1989: 10(Suppl): 65-69. 3. Yamamura HI, Snyder SH. Muscarinic cholinergic binding in rat brain. Proc Nat Acad Sci USA 1974; 71: 1725-9. 4. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folinphenol reagent. J Biol Chem 195 l: 193: 265-75. 5. Niu WZ, Zhao DL, Liu CG. The effects of a new cholinolytic-8018- and its optical isomers on central muscarinic and nicotinic receptors. Arch lnt Pharmacodyn 1990; 304: 64-74. 6. Bliss CI. The determination of the dosage-mortality
11.
12. 13.
14.
15.
curve from small numbers. Quart J Pharm Pharmacol 1938; 11: 192-216. Rodbard D, Frazier GR. Statistical analysis of radioligand assay data. Methods Enzymol 1975; 37: 3-22. Cheng YC, Prusoff WH. Relationship between the inhibition constant (K~) and the concentration of inhibitor which causes 50 per cent inhibition (/5~) of an enzymatic reaction. Biochem Pharmacol 1973; 22: 3099-108. Atkison ER, Mcritchie DD, Shoer LF, Harris, LS, Archer S, Aceto MA, Pearl J, Luduena FP. Parasympatholytic (anticholinergic) esters of the isomeric 2-tropanols. I. glycolates. J M e d Chem 1977; 20: 1612-17. Wess J. Molecular basis of muscarinic acetylcholine receptor function. Trends Pharmacol Sci 1993; 14: 308-13. Whiting P, Lindstrom J. Purification and characterization of nicotinic acetylcholine receptor from rat brain. Proc Natl Acad Sci USA 1987; 84: 595-9. Sargent PB. The diversity of neuronal nicotinic acetylcholine receptors. Annu Rev Neurosci 1993; 16: 403-43. Anderson DJ, Arneric SP. Nicotinic receptor binding of [3H]cytisine, [3H]nicotine and [3H]methylcarbamylcholine in rat brain. E u r J Pharmacol 1994; 253: 261-7. Deneris ES, Boulter J, Swanson LW, Patrick J, Heinemann S. f12: a new member of nicotinic acetylcholine receptor gene family is expressed in brain. J Biol. Chem 1989; 264: 6268-72. Boulder J, O'Shea-Greenfield A, Duvoisin RM, Connolly JG, Wada E. o¢3, a5, and f14: three members of the rat neuronal nicotinic acetylcholine receptorrelated gene cluster. J Biol Chem 1990; 265: 4472-82.
105
EFFECTS OF A NEW CHOLINOLYTIC DRUG OF TROPANES AND ITS OPTICAL ISOMERS ON CENTRAL MUSCARINIC AND NICOTINIC RECEPTORS z. G. GAO, L. WANG, C. G. LIU and Q. K. ZHANG
Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 27, Taiping Road, Beijhlg 100850, People's Republic of China Accepted 27 July 1995 2o~-(2',2'-disubstituted-2'-hydroxy-ethoxy)tropane (2~-DHET), a new cholinolytic, is a racemic tertiary amine with two chiral carbonic atoms. It has four optical isomers whose absolute configurations are IR-2o~-2'S, IR-2o~-2'R, IS-2o~-2'R and IS-2c~-2'S. These compounds showed both antimuscarinic and antinicotinic activity, blocking both muscarinic and nicotinic receptors. Central muscarinic receptors, rather than nicotinic receptors, have a stereoselective specificity to these compounds. The 2'R configurations are more suitable to the stereostructure of the binding site of muscarinic receptors than the 2'S configuration. © 1995 The Italian Pharmacological Society KEYWORDS:muscarinic receptors, nicotinic receptors, optical isomers.
INTRODUCTION
MATERIALS AND METHODS
Stereoselectivity means that the 'active' enantiomer should be about one to two orders more potent than the 'inactive' enantiomer [1]. It is known that many recepto.rs show stereoselectivity to isomers of their ligands [2]. It has been found that muscarinic receptors have many racemic ligands whose enantiomers possess different affinities [2]. Antagonist binding site of muscarinic receptors are asymmetrical, and hence generally capable of discriminating optical isomers of chiral drug. Thus analysis of binding and/or pharmacological properties of stereoisomers provide information about binding site geometry [2]. A new cholinolytic, 2o¢-(2',2'-disubstituted-2'-hydroxy-ethoxy) tropane(2~x-DHET), synthesized at our institute, is a racemic tertiary amine with two chiral carbonic atoms in its molecular structure (Fig. 1). There are thus four optical isomers of different configurations, named 1R2a-2'S, 1R-2a-2'R, 1S-2~x-2'R and 1S-2o~-2'S, as identified by X-ray diffraction and nuclear magnetic resonance. Previous experiments in our laboratory had demonstrated that 2o~-DHET had anti-soman activity and it had no selectivity for muscarinic subtypes. We have now investigated the binding profiles of 2tx-DHET and its four isomers to muscarinic receptors and nicotinic receptors of rat forebrain and their pharmacological activities to central muscarinic and nicotinic receptors.
Membrane preparations The procedure used was similar to that of Yamamura [3]. Male or female Wistar rats, weighing 180-220 g, were decapitated. Forebrains were rapidly removed and homogenized in 10 volumes (w/v) of ice-cold 0.32 M sucrose in a glass homogenizer. The whole homogenate was centrifuged at 1000g for 10 min at 4°C. The pellet was discarded and supernatant was recentrifuged at 20 000g for 30 min at 4°C. The supernatant was poured out and the pellet (P_,) was suspended in four volumes of sodium potassium phosphate buffer (pH 7.4, 4°C). Then /:'2 was rehomogenized in a glass homogenizer and stored at -20°C. P,_ was used within 2 weeks; it has been demonstrated that there were no significant changes in the binding characteristics of P_, stored in this condition for 1 month. Proteins were determined by the method of Lowry et al. [4] using bovine serum albumin as a standard.
Correspondenceto: Z. G. Gao. 1043-6618/951090105-05/$12.00/0
Binding assay To assay the specific binding of [3H]QNB, 0.1 mg of P_, was incubated at 35°C for 3 0 m i n with 0.01-1.0 nM of [3H]QNB in 1 ml of a 50 mM sodium potassium phosphate buffer solution in the absence or presence of I /2M unlabelled atropine to determine the total and non-specific binding, respectively. The incubation was stopped by addition of 3 ml ice-cold buffer solution. Bound [3H]QNB was separated from the unbound ligand by vacuum filtration using Hongguang 49 glass filters (Shanghai, China). The filters were washed three times with 9 ml of an ice-cOld
©1995The ItalianPharmacologicalSociety
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Pharmacological Research. I/ol. 32, No. 3, 1995
buffer solution. Every determination of binding was performed in duplicate. Then the filters were placed in vials containing 3 ml scintillating liquid (containing PPO 0.3% and POPOP 0.3%) and maintained for over 10h at room temperature. The radioactivity was assayed by a liquid scintillating counter (LKB 1215 Rack Beta II model) at a counting efficiency of 36%. The binding of 2 a - D H E T and its isomers and other agents tested to muscarinic receptors was determined by their ability to displace [3H]QNB. Protein (0.1 mg) was incubated with 0.5 nM of [3H]QNB in the presence of 2ot-DHET and its isomers and other agents. To assay the binding of 2a-DHET and its isomers to nicotinic receptors, 0.2 mg of protein was incubated with 5 nM of [3H]nicotine in 1 ml of Tris-HCI buffer (pH 8.0) at 4°C for 60 min in the presence of tested drugs. The samples were then filtered through Hongguang 49 glass filters presoaked with 0.05% polyethylenimine solution for 5 h. The remaining procedures were similar to the binding assay of muscarinic receptors.
Arecoline-induced tremor in mice and the antagonistic effects of drugs Arecoline (8 mg kg -~, s.c.) induced tremor in all 20 control mice in 10 min. Five doses of 2 a - D H E T and its isomers were injected i.p. 15 min before arecoline (8 mg kg -~, s.c.) to estimate the EDso values of the antagonist effect of these compounds. There were 10 Shanghai mice, weighing 18-22 g in each dose-group.
Nicotine-induced convulsion in mice and the antagonistic effects of drugs The procedure used was similar to that of Niu et al. [5]. After injection of 0.8 mg kg -~ of nicotine free base into the caudal vein of mice, tetanic convulsion in all 20 control mice was observed. The methods to estimate the antagonistic effect of 2 a - D H E T and its four optical isomers against nicotine-induced convulsion were the same as for arecoline-induced tremor, but the tested drugs were injected into the caudal vein. EDs0 values of these compounds against arecolineinduced tremor and nicotine-induced convulsion were calculated from the probability-logarithm method [6].
Materials [3H]QNB
(43.3 Ci mmol -I)
and
[3H]nicotine
(78.4Ci mmol -~) were purchased from Amersham, 2o'-DHET and its isomers were synthesized at our institute. Hexamethonium, atropine, gallamine were from Sigma, arecoline from EMK, nicotine freebase from Merk-Schuchard (Munich, Germany). An automatic high-speed refrigerated centrifuge of Hitachi 20 PR-5 and a DTQ II-multitute cell collector of Zhejiang, China were used.
RESULTS
Binding profiles of 2 o~-DHET and its isomers to muscarinic receptors o f rat forebrain As shown in Fig. 2, atropine, a classic muscarinic antagonist, was potent to displace specific binding of [3H]QNB, but other nonmuscarinic agents, such as hexamethonium, mecamylamine and nicotine, only displace less than 15% of specific binding of [3H]QNB, even at the concentrations as high as 10/.tM (data not shown). It was demonstrated that the isomer with IR-2oc-2'R configuration was the most potent and the isomer with 1R-2o~-2'S configuration was the least potent among the four isomers in displacing specific binding of [3H]QNB. Values of ICs~ were calculated from Rodbard and Frazier [7] and those of the inhibitory equilibrium dissociation constant (K0 were calculated from Cheng-Prusoff's [8] equation and are listed in Table 1. The results show that central muscarinic receptors have different affinities to these agents, the order of potency to displace [3H]QNB was IR-2oc2'R> 1S-2 o¢-2'R>2 o~-DHET> 1S-2 ¢x-2'S> 1R-2 ¢z-2'S.
Effects of 2at-DHET and its optical isomers on [3H]nicotine binding To determine the binding specificity of these compounds, experiments were performed with [3H]nicotinc. 2~-DHET and its optical isomers at concentrations of 10/.tM, which resulted in >80% inhibition of [3H]QNB binding, had no effect on reversible [3H]nicotine binding.
Pharmacological effects o f 2 ot-DHET and its four optical isomers on central muscarinic and nicotinic receptors As shown in Fig. 3, arecoline-induced tremor in mice can be antagonized by 2 a - D H E T and its four
Table I Effects of 2 a - D H E T and its isomers on rat and mouse central muscarinic and nicotinic receptors
Ki (riM)* EDso against arecoline (mg kg-~)t EDso against nicotine (mg kg-k):l:
lR-2ct-2'R
1S-2ct-2'R
2 ct-DHET
1S-2¢t-2'S
1R-2ot-2'S
6.1+0.62 0.51+0.07 3.1+0.29
6.8_+0.80 0.72+0.08 3.8+0.76
26.4+2.85 1.2+0.21 3.1+0.29
114.5+10.70 2.9+0.89 3.3+0.41
381.5+27.40 5.6+1.1 I 2.7+0.71
Results are expressed as m e a n ~ . *rat forebrain, n--4. ?areeoline (8 mg kg-~ s.c.) -induced mouse tremor. :~nicotine (0.8 mg kg-~, i.v.) -induced mouse convulsion.
107
Pharmacological Rese,:trch, Vol. 32. No. 3. 1995
H3C"-N
OH
2a-DHET
OH /=~
2.1
4r--~ ~ -CH3 i'
N- CH3
a//f~-~ I
© 1R-2a-2'S H3C 1 - ' ~
1R-2a-2"R
H3C-..N
4
~CH2~~
@ 1S-2a-2~R
IS-2a-2'S
Fig. 1. Molecular structure of 2cr-DHET and its optical isomers. The [~]~ values were -11.40, +32.40, +11.60 and -31.76 for I R-2/z-2'S, I R-2o~-2'R, I S-2a-2'R and 1S-2a-2'S, respectively. The optical purity of the four optical isomers were 100%.
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60
40
~2 o ~
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-8
-7
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Log [M] Fig. 2. Competitive curves of atropine ((3), 2a-DHET (V), IR-2cr-2'S (U), 1R-2/z-2'R (Q), 1S-2ot-2'R (~7) and IS-2/~2'S ([3) at rat forebrain muscarinic receptors. Membrane (0.1 mg) of rat forebrain was incubated with 0.5riM of [3H]QNB at 35°C in l ml of 50mM sodium potassium phosphate buffer (pH 7.4) for 30 min in the presence of atropine, 2a-DHET and its isomers (n=4). optical isomers. The order of their antagonizing potency was 1R-2~z-2'R>IS-2o~-2'R>2ot-DHET>IS2/z-2'S> I R-2 ~-2'S. The EDs0 values were summarized in Table I. The isomer with IR-2o~-2'R configuration
0
2
4
6
8
10
Dose (ragkg-1) Fig. 3. Dose-response curves of 2~x-DHET (V), 1R-2o~-2'S (I--1), IR-2o~-2'R (O), IS-2or-2'R (O) and 1S-2o~-2'S (~7) on arecoline-induced tremor. Different doses of 2a,-DHET and its optical isomers were injected i.p. 15 min before arecoline (8 mg kg-', s.c.). Results were from three experiments.
was about 11 times stronger than the 1R-2o~-2'S isomer. The antagonistic effects of 2 a - D H E T and its isomers against nicotine-induced convulsion in mice were completely different (Fig. 4). There was: no sig-
Pharmacological Research. Vol. 32, No. 3, 1995
108
O
100
80
0
60
':i,'?
8 40
.~ 9.0
I
Z 0
1.5
3.0 4.5 Dose (mg kg-1)
I
6.0
Fig. 4. Dose-response curves of 2a-DHET (O), IR-2a-2'S (O), 1R-2a-2'R (V), IS-2a-2'R (Y) and 1S-2a-2'S (IS]) on nicotine-induced convulsion. Different doses of 2a-DHET and its optical isomers were injected i.v. 15min before nicotine (0.Smg kg-I, i.v.). Results were from three experiments.
nificant difference in EDso values (Table I) to prevent nicotine-induced convulsion among these agents. This suggested that, unlike the central muscarinic receptors, central nicotinic receptors had no stereoselectivity to these compounds.
DISCUSSION Binding assays showed that central muscarinic receptor have a stereoselectivity to 2 a - D H E T and its optical isomers and the results of functional experiments confirmed this. As for the central muscarinic receptors, the relative potency, judged by K~ and EDs0, of 2tr-DHET and its isomers ranks the same order, i.e. 1R-2 a-2'R> 1S-2 a-2'R> 2 a - D H E T > 1S-2 a-2'S> l R2a-2'S. The relationship between LnKi and EDso values against arecoline was linear (r=-0.953, P<0.01). The pharmacological potency of 2 a - D H E T and its isomers to central muscarinic receptors thus corresponds with their affinities to muscarinic receptors. The higher their affinities, the more potent their pharmacological effects. It is possible that the binding site is equivalent to the physiologically and pharmacologically relevant site. It was found from binding assays and functional experiments that the carbon-2' configuration is more important for the antimuscarinic activities of 2aD H E T than the tropane ring configuration. This result is similar to that of 2-substituted tropane esters and 3substituted quinuclidinyl ethers [5, 9]. It is suggested that the R configuration of carbon-2' is more suitable to the stereochemical structure of the binding site of
central muscarinic receptors than that of the S configuration. In contrast to central muscarinic receptors, central nicotinic receptors have no stereoselectivity to these compounds, as the differences of their EDso values against nicotine-induced convulsion were minimal. These results are similar to that of 8018 and its four optical isomers [5]. It is suggested that binding site of central nicotinic receptors may interact with other parts of the molecular structure of these compounds. Central muscarinic receptor is a monomeric receptor with seven consecutive transmembrane regions [I0]. It stereoselectivity comes from the arrangement of its key groups in spacial direction. On the other hand, available evidence suggests that the neuronal nicotinic receptors are composed of a and fl subunits [11]. As in the muscle nicotinic receptor, the agonist recognition site of neuronal nicotinic receptors is located on a subunit, but the total number of subunits of the neuronal receptor and the subunit stoichiometry is at present uncertain [12]. The difference in their antinicotinic activity and their ability to displace the specific binding of [3H]nicotine reveals that it is possible that 2 a - D H E T and its isomers do not act at the recognition site of central nicotinic receptors. Molecular biology studies have elucidated that there are at least 10 nicotinic receptor genes in the brain, namely: o'2, a3, a4, a5, a6, a7 and f12, f13, ,84, f15. Of these, a2, a3 and a 4 can combine with f12 or f14 subunits to form at least six functional a-bugarotoxin insensitive nicotinic receptors [12, 13]. o~7 nicotinic receptor is sensitive to a-bugarotoxin, coinjection of f12, f13 or f14 cDNA, along with a7 cDNA, did not result in functional responses with properties any different than those expressed in oocytes injected with a7 cDNA alone [12]. Thus, in oocytes at least, a7 subunits apparently cannot coassemble with known neuronal fl subunits to form hetero-oligomers that have functional properties distinct from those c~7 homo-oligomers [12]. The f13 gene does not form functional nicotinic receptors when coinjected as mRNA with a2, a3 or a 4 genes [14]. The rat a'5 and a'6 genes, likewise do not participate in the formation of functional nicotinic channels in oocytes when injected in combination with several other a and fl genes as mRNA [15]. The sensitivity of heterologously expressed nicotinic receptors to nicotinic receptor antagonist, a-bugarotoxin, is dependent to the nature of the subunit composition [12]. These data indicate that the structure and function of central nicotinic receptors are more complex than that of muscarinic receptors, and it is necessary to further characterize brain nicotinic receptors with both molecular biology techniques and new specific ligands. In summary, the present study demonstrated that 2ot-DHET and its isomers had both antimuscarinic and antinicotinic activities. Muscarinic receptors had stereoselectivity to these compounds, but nicotinic receptors had no stereoselectivity to these compounds.
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The difference in their antinicotinic activity and their ability to displace the specific binding of [3H]nicotine reveals that it is possible that 2 a - D H E T and its isomers do not act at the recognition site of central nicotinic receptors.
7. 8.
9.
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