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Inhibition by neurosurugatoxin of nicotine-induced antinociception
360
Brain Research, 375 (1986) 360- 362 Elsevier
BRE 21577
Inhibition by neosurugatoxin of nicotine-induced antinociception SHIZUO YAMADA 1, YOSHIYUKI KAGAWA1, NORIYASU TAKAYANAGIl, EIICHI HAYASHI 1, KUNIRO TSUJI 2 and TAKUO KOSUGE 2
Departments of 1Pharmacologyand 2MedicinalChemistry of Natural Products, Shizuoka Collegeof Pharmaceutical Sciences, Shizuoka (Japan) (Accepted February llth, 1986)
Key words: neosurugatoxin - - nicotine antinociception - - [3H]nicotine binding - - mecamylamine - - pempidine
We have found a selective inhibition of nicotine-induced antinociception in mice by neosurugatoxin (NSTX, 0.4-3.8 nmol/kg), a neurotoxin with a high affinity for ganglionic nicotinic receptors (ED50 = 0.65 nmol/kg). The toxin also reduced specific [3H]nicotine binding in mouse brain membranes (IC50 = 95 nM). The anti-nicotinic activity of NSTX was markedly greater than that of mecamylamine and pempidine. These data indicate that NSTX may block functional nicotinic cholinoceptors possibly in the central nervous system. It has b e e n shown that neosurugatoxin (NSTX), isolated from the J a p a n e s e ivory mollusc (Babylonia japonica) 4 exerted a blocking action at nicotinic receptors in autonomic ganglia 2'3. W e have found NSTX to be a selective inhibitor of [3H]nicotine binding sites in the rat brain 2'1°. The affinity of N S T X was at least 3 orders of magnitude higher than that of hexamethonium. In addition, R a p i e r et ai. 7 have rep o r t e d that low concentrations of N S T X blocked 1,1-dimethyl-4-phenylpiperazinium (DMPP)-evoked [3H]dopamine release from rat striatal synaptosomes p r e l o a d e d with [3H]dopamine. Thus, this toxin appears to be a p o t e n t antagonist of nicotinic receptors in the central nervous system (CNS) as well as in autonomic ganglia. It is well known that nicotine causes centrally m e d i a t e d effects such as antinociception, hypothermia, t r e m o r , antidiuresis, hypotension and bradycardia 5'6'9. W e have recently investigated the action of N S T X on nicotine-induced antinociception in mice and have confirmed its high affinity for the functional nicotinic cholinoceptors. Male mice (16-25 g) were used through the study. The nicotine-induced antinociception in mice was measured according to the tail-flick m e t h o d described previously 8'9. Mice were placed in a plastic
restraint cage and their tails immersed, to a constant depth, in a water bath maintained constant at 48.0 + 0.3 °C. The time between immersion and the animal withdrawing its tail was recorded. A control nociceptive response remains r e m a r k a b l y constant when repeatedly d e t e r m i n e d over a 60-min period. The timecourse of antinociception was d e t e r m i n e d by measuring tail-flick activity at 0.5, 2, 3, 5, 10 and 15 min after an s.c. injection of nicotine (8/~mol/kg) to mice. A maximum latency of 10 s was imposed if no response occurred within that time. The antinociceptive response was expressed as a percentage of maximum possible effect ( M P E , % ) which was calculated from latency times (s) according to the formula: M P E = [ ( t e s t - c o n t r o l ) / ( 1 0 - control)] × 100. Mice were pretreated (s.c.) with either saline or different doses of NSTX, m e c a m y l a m i n e , p e m p i d i n e and hexamethonium 10 min before nicotine administration. The mouse forebrain m e m b r a n e s (800-900 # g of protein) were incubated with 6 nM [3H]nicotine (71.2 Ci/mmol; N E N ) in 20 m M Tris-HCl buffer at 37 °C as d e s c r i b e d previously 2'1°. A f t e r 5 min, the reaction was terminated by dilution and subsequent rapid filtration under vacuum through W h a t m a n G F / B glass fiber filters, and each filter was rinsed 4 times with 4
Correspondence: S. Yamada, Department of Pharmacology, Shizuoka College of Pharmaceutical Sciences, 2-2-1 Oshika, Shizuoka 422, Japan. 0006-8993/86/$03.50 ~ 1986 Elsevier Science Publishers B.V. (Biomedical Division)
361 ml of ice-cold buffer. Tissue-bound radioactivity was extracted from the filters and kept overnight in 6 ml of scintillation fluid and counted. Specific binding was defined as the difference in binding determined in the absence and presence of 10/~M (-)-nicotine. The ability of NSTX and cholinergic antagonists to inhibit specific [3H]nicotine binding was estimated by IC50 values, which are the molar concentration of unlabeled drugs for displacing 50% of the specific binding. Statistical analysis of data was performed using a double-tailed Student's t-test. As illustrated in Fig. 1, nicotine (8 /~mol/kg) exerted an antinociceptive effect in mice. The maxmum antinociceptive effect (85%) was observed at 2 min after nicotine administration, and this effect disappeared completely within 15 min. Pretreatment with NSTX (0.4-3.8 nmol/kg) antagonized effectively the antinociceptive effect in mice elicited by nicotine (Fig. 1). A dose-response (inhibition) relationship was established for NSTX by measuring nicotine-induced antinociception at the time (2 min) of maximum effect. As shown in Table I, NSTX at doses of 0.4, 1.3 and 3.8 nmol/kg produced a dose-dependent (31%, 74% and 92% respectively) inhibition of nicotine-induced antinociception. The EDs0 value of NSTX was found to be 0.65 nmol/kg. NSTX (1.3 nmol/kg) at the dose which inhibited nicotine-induced antinociception had no effect on tail-
/
./
50
L I0
15
Time c,~,~ after nicotine
Fig. 1. Effect of NSTX on the nicotine-induced antinociception in mice. Mice received an s.c. injection of nicotine (8 #mol/kg)
10 min after pretreatment with saline (O) or NSTX (O 0.4, 'A 1.3, • 3.8 nmol/l~g,s.c.). The antinociceptiveresponse to nicotine was expressed as MPE, as described in the text. The insert shows a dose-response (inhibition) curve for NSTX at 2 min after nicotine administration. Each point represents the mean + S.E.M. of 6-14 determinations.
TABLE I Effect of neosurugatoxin (NSTX), mecamylamine, pempidine and hexamethonium on the nicotine-induced antinociception in mice
Mice received nicotine (8/~mol/kg, s.c.) 10 min after each drug treatment. The antinociceptive response at 2 rain after nicotine administration was expressed as maximum possible effect (MPE), as described in the text. Each value represents the mean + S.E.M. of 5-14 determinations. Drugs
MPE (%)
Saline
85.4 + 10.1 58.6 + 12.3" 22.0 + 7.2** 6.9 + 3.1"* 3.2 + 1.5"* 14.9 + 6.4** 47.8 + 11.3"*
NSTX (nmol/kg)
0.4 1.3 3.8 Mecamylamine ~mol/kg) 4.9 Pempidine ~mol/kg) 16 Hexamethonium (,umol/kg) 18
Significantly different from control (saline) value; * P < 0.05, • * P < 0.001.
flick activity when given alone, suggesting no agonistic activity. The antinicotinic action of NSTX was compared with that of classical nicotinic antagonists. Mecamylamine (4.9 /~mol/kg) and pempidine (16 /~mol/kg), which are secondary and tertiary nicotinic antagonists, inhibited the nicotine-induced antinociception by 96% and 83%, respectively. On the other hand, the nicotine effect was partially (44%) antagonized by hexamethonium (18/~mol/kg). Thus, the antinicotinic activity of NSTX in mice was markedly greater than that of mecamylamine and pempidine. NSTX (1.3 nmol/kg) was ineffective in inhibiting the morphine-induced antinociception when given 10 min before morphine (5/~mol/kg, s.c.; % MPE at 7 min after morphine; control, 61.7 + 13.1; NSTX, 63.1 + 8.9(n = 6)). The specific [3H]nicotine binding in mouse brain membranes was markedly inhibited by low concentrations (1 nM-3/~M) of NSTX, and its IC50 value was 95 + 11 nM (n = 5). Mecamylamine, pempidine and hexamethonium were much weaker inhibitors of brain [3H]nicotine binding as demonstrated by the IC50 values of 589 ___ 183,207 + 35 and 144 + 21/~M (n = 4), respectively. The potency of inhibition of brain [3H]nicotine binding by these antagonists in mice agrees well with that in rat brain 2'~°. Previous studies have shown that NSTX may be a neurotoxin with a selective affinity for ganglionic nicotinic receptors 2. In the present study, low doses of
362 NSTX blocked nicotine-induced antinociception in mice in a dose-related manner, without affecting the morphine-induced response. Compared to NSTX, mecamylamine and pempidine, hexamethonium, a quaternary nicotinic antagonist which does not readily penetrate the brain, was a relatively weaker inhibitor of the nicotinic response in vivo. Mansner 5 and Tripathi et al. 9 have shown that nicotine-induced antinociception in mice may be mediated through central nicotinic receptors because there was a good correlation between the time-course of antinociception and brain levels after an s.c. administration of nicotine. Taken together, these observations indicate that the antagonism by NSTX and nicotinic antagonists of nicotine-induced antinociception in mice may result from a blockade of the functional nicotinic receptors possibly in the CNS. The inhibition of nicotine-induced antinociception in mice by NSTX seems to be associated with that of brain [3H]nicotine binding in vitro. The higher affinity of the toxin for brain [3H]nicotine binding sites as compared to mecamylamine, pempidine and hexa-
methonium has also been demonstrated in mice as well as in rats 2,1°. Thus, the present study may be the first report of the in vivo antagonism by NSTX of nicotinic receptor function. In addition to our data, Rapier et al. 7 have recently reported that low concentrations of NSTX inhibited selectively the DMPP-induced release of [3H]dopamine from rat striatal synaptosomes preloaded with [3H]dopamine. The correlation between the NSTX inhibition of [3H]nicotine binding and interaction with the functional nicotinic receptors in the CNS may have a significant implication for nicotinic receptor studies, since there is an apparent dissociation between biochemical and pharmacological effects of a-bungarotoxin, an antagonist of neuromuscular nicotinic receptors, in the brain 1. In conclusion, we have demonstrated a correlation between the biochemical and pharmacological behavior of NSTX in the nicotinic cholinergic system.
1 Brown, D.A., Neurotoxin and the ganglionic (C6) type of nicotinic receptors. In B. Ceccarelli and F. Clementi (Eds.), Advances in Cytopharmacology, Raven Press, New York, 1979, pp. 225-230. 2 Hayashi, E., Isogai, M., Kagawa, Y., Takayanagi, N. and Yamada, S., Neosurugatoxin, a specific antagonist of nicotinic acetylcholine receptors, J. Neurochem., 42 (1984) 1491-1494. 3 Hayashi, E. and Yamada, S,, Pharmacological studies on surugatoxin, the toxic principle from Japanese ivory mollusc (Babylonia japonica), Br. J. Pharmacol., 53 (1975) 207-215. 4 Kosuge, T., Tsuji, K., Hirai, K., Yamaguchi, K., Okamoto, T. and Iitaka, Y., Isolation and structure determination of a new marine toxin neosurugatoxin, from the Japanese ivory shell, Babylonia japonica, Tetrahedron Lett., 22 (1981) 3417-3420. 5 Mansner, R., Relationship between some central effects of nicotine and its brain levels in the mouse, Ann. Med. Exp. Biol. Fenn., 50 (1972) 205-212.
6 Porsius, A.J. and Van Zwieten, P.A., Central actions of some cholinergic drugs (arecaidine esters) and nicotine on blood pressure and heart rate of cats, Prog. Brain Res., 47 (1977) 131-135. 7 Rapier, C., Harrison, R., Lunt, G.G. and Wonnacott, S., Neosurugatoxin blocks nicotinic acetylcholine receptors in the brain, Neurochern. Int., 7 (1985) 389-396. 8 Sewell, R.D.E. and Spencer, P.S.J., Modification of the antinociceptive activity of narcotic agonists and antagonists by intraventricular injection of biogenic amines in mice, Br. J. Pharmacol., 51 (1974) 140P-141P. 9 Tripathi, H.L., Martin, B.R. and Aceto, M.D., Nicotineinduced antinociception in rats and mice: correlation with nicotine brain levels. J. Pharmacol. Exp. Ther., 221 (1982) 91-96. 10 Yamada, S., Isogai, M., Kagawa, Y., Takayanagi, N., Hayashi, E., Tsuji, K. and Kosuge, T., Brain nicotinic acetylcholine receptors: biochemical characterization by neosurugatoxin, Mol. Pharmacol., 28 (1985) 120-127.
We would like to thank to Dr. N.W. Pedigo, Department of Pharmacology, University of Kentucky, for his helpful suggestions.
Brain Research, 375 (1986) 360- 362 Elsevier
BRE 21577
Inhibition by neosurugatoxin of nicotine-induced antinociception SHIZUO YAMADA 1, YOSHIYUKI KAGAWA1, NORIYASU TAKAYANAGIl, EIICHI HAYASHI 1, KUNIRO TSUJI 2 and TAKUO KOSUGE 2
Departments of 1Pharmacologyand 2MedicinalChemistry of Natural Products, Shizuoka Collegeof Pharmaceutical Sciences, Shizuoka (Japan) (Accepted February llth, 1986)
Key words: neosurugatoxin - - nicotine antinociception - - [3H]nicotine binding - - mecamylamine - - pempidine
We have found a selective inhibition of nicotine-induced antinociception in mice by neosurugatoxin (NSTX, 0.4-3.8 nmol/kg), a neurotoxin with a high affinity for ganglionic nicotinic receptors (ED50 = 0.65 nmol/kg). The toxin also reduced specific [3H]nicotine binding in mouse brain membranes (IC50 = 95 nM). The anti-nicotinic activity of NSTX was markedly greater than that of mecamylamine and pempidine. These data indicate that NSTX may block functional nicotinic cholinoceptors possibly in the central nervous system. It has b e e n shown that neosurugatoxin (NSTX), isolated from the J a p a n e s e ivory mollusc (Babylonia japonica) 4 exerted a blocking action at nicotinic receptors in autonomic ganglia 2'3. W e have found NSTX to be a selective inhibitor of [3H]nicotine binding sites in the rat brain 2'1°. The affinity of N S T X was at least 3 orders of magnitude higher than that of hexamethonium. In addition, R a p i e r et ai. 7 have rep o r t e d that low concentrations of N S T X blocked 1,1-dimethyl-4-phenylpiperazinium (DMPP)-evoked [3H]dopamine release from rat striatal synaptosomes p r e l o a d e d with [3H]dopamine. Thus, this toxin appears to be a p o t e n t antagonist of nicotinic receptors in the central nervous system (CNS) as well as in autonomic ganglia. It is well known that nicotine causes centrally m e d i a t e d effects such as antinociception, hypothermia, t r e m o r , antidiuresis, hypotension and bradycardia 5'6'9. W e have recently investigated the action of N S T X on nicotine-induced antinociception in mice and have confirmed its high affinity for the functional nicotinic cholinoceptors. Male mice (16-25 g) were used through the study. The nicotine-induced antinociception in mice was measured according to the tail-flick m e t h o d described previously 8'9. Mice were placed in a plastic
restraint cage and their tails immersed, to a constant depth, in a water bath maintained constant at 48.0 + 0.3 °C. The time between immersion and the animal withdrawing its tail was recorded. A control nociceptive response remains r e m a r k a b l y constant when repeatedly d e t e r m i n e d over a 60-min period. The timecourse of antinociception was d e t e r m i n e d by measuring tail-flick activity at 0.5, 2, 3, 5, 10 and 15 min after an s.c. injection of nicotine (8/~mol/kg) to mice. A maximum latency of 10 s was imposed if no response occurred within that time. The antinociceptive response was expressed as a percentage of maximum possible effect ( M P E , % ) which was calculated from latency times (s) according to the formula: M P E = [ ( t e s t - c o n t r o l ) / ( 1 0 - control)] × 100. Mice were pretreated (s.c.) with either saline or different doses of NSTX, m e c a m y l a m i n e , p e m p i d i n e and hexamethonium 10 min before nicotine administration. The mouse forebrain m e m b r a n e s (800-900 # g of protein) were incubated with 6 nM [3H]nicotine (71.2 Ci/mmol; N E N ) in 20 m M Tris-HCl buffer at 37 °C as d e s c r i b e d previously 2'1°. A f t e r 5 min, the reaction was terminated by dilution and subsequent rapid filtration under vacuum through W h a t m a n G F / B glass fiber filters, and each filter was rinsed 4 times with 4
Correspondence: S. Yamada, Department of Pharmacology, Shizuoka College of Pharmaceutical Sciences, 2-2-1 Oshika, Shizuoka 422, Japan. 0006-8993/86/$03.50 ~ 1986 Elsevier Science Publishers B.V. (Biomedical Division)
361 ml of ice-cold buffer. Tissue-bound radioactivity was extracted from the filters and kept overnight in 6 ml of scintillation fluid and counted. Specific binding was defined as the difference in binding determined in the absence and presence of 10/~M (-)-nicotine. The ability of NSTX and cholinergic antagonists to inhibit specific [3H]nicotine binding was estimated by IC50 values, which are the molar concentration of unlabeled drugs for displacing 50% of the specific binding. Statistical analysis of data was performed using a double-tailed Student's t-test. As illustrated in Fig. 1, nicotine (8 /~mol/kg) exerted an antinociceptive effect in mice. The maxmum antinociceptive effect (85%) was observed at 2 min after nicotine administration, and this effect disappeared completely within 15 min. Pretreatment with NSTX (0.4-3.8 nmol/kg) antagonized effectively the antinociceptive effect in mice elicited by nicotine (Fig. 1). A dose-response (inhibition) relationship was established for NSTX by measuring nicotine-induced antinociception at the time (2 min) of maximum effect. As shown in Table I, NSTX at doses of 0.4, 1.3 and 3.8 nmol/kg produced a dose-dependent (31%, 74% and 92% respectively) inhibition of nicotine-induced antinociception. The EDs0 value of NSTX was found to be 0.65 nmol/kg. NSTX (1.3 nmol/kg) at the dose which inhibited nicotine-induced antinociception had no effect on tail-
/
./
50
L I0
15
Time c,~,~ after nicotine
Fig. 1. Effect of NSTX on the nicotine-induced antinociception in mice. Mice received an s.c. injection of nicotine (8 #mol/kg)
10 min after pretreatment with saline (O) or NSTX (O 0.4, 'A 1.3, • 3.8 nmol/l~g,s.c.). The antinociceptiveresponse to nicotine was expressed as MPE, as described in the text. The insert shows a dose-response (inhibition) curve for NSTX at 2 min after nicotine administration. Each point represents the mean + S.E.M. of 6-14 determinations.
TABLE I Effect of neosurugatoxin (NSTX), mecamylamine, pempidine and hexamethonium on the nicotine-induced antinociception in mice
Mice received nicotine (8/~mol/kg, s.c.) 10 min after each drug treatment. The antinociceptive response at 2 rain after nicotine administration was expressed as maximum possible effect (MPE), as described in the text. Each value represents the mean + S.E.M. of 5-14 determinations. Drugs
MPE (%)
Saline
85.4 + 10.1 58.6 + 12.3" 22.0 + 7.2** 6.9 + 3.1"* 3.2 + 1.5"* 14.9 + 6.4** 47.8 + 11.3"*
NSTX (nmol/kg)
0.4 1.3 3.8 Mecamylamine ~mol/kg) 4.9 Pempidine ~mol/kg) 16 Hexamethonium (,umol/kg) 18
Significantly different from control (saline) value; * P < 0.05, • * P < 0.001.
flick activity when given alone, suggesting no agonistic activity. The antinicotinic action of NSTX was compared with that of classical nicotinic antagonists. Mecamylamine (4.9 /~mol/kg) and pempidine (16 /~mol/kg), which are secondary and tertiary nicotinic antagonists, inhibited the nicotine-induced antinociception by 96% and 83%, respectively. On the other hand, the nicotine effect was partially (44%) antagonized by hexamethonium (18/~mol/kg). Thus, the antinicotinic activity of NSTX in mice was markedly greater than that of mecamylamine and pempidine. NSTX (1.3 nmol/kg) was ineffective in inhibiting the morphine-induced antinociception when given 10 min before morphine (5/~mol/kg, s.c.; % MPE at 7 min after morphine; control, 61.7 + 13.1; NSTX, 63.1 + 8.9(n = 6)). The specific [3H]nicotine binding in mouse brain membranes was markedly inhibited by low concentrations (1 nM-3/~M) of NSTX, and its IC50 value was 95 + 11 nM (n = 5). Mecamylamine, pempidine and hexamethonium were much weaker inhibitors of brain [3H]nicotine binding as demonstrated by the IC50 values of 589 ___ 183,207 + 35 and 144 + 21/~M (n = 4), respectively. The potency of inhibition of brain [3H]nicotine binding by these antagonists in mice agrees well with that in rat brain 2'~°. Previous studies have shown that NSTX may be a neurotoxin with a selective affinity for ganglionic nicotinic receptors 2. In the present study, low doses of
362 NSTX blocked nicotine-induced antinociception in mice in a dose-related manner, without affecting the morphine-induced response. Compared to NSTX, mecamylamine and pempidine, hexamethonium, a quaternary nicotinic antagonist which does not readily penetrate the brain, was a relatively weaker inhibitor of the nicotinic response in vivo. Mansner 5 and Tripathi et al. 9 have shown that nicotine-induced antinociception in mice may be mediated through central nicotinic receptors because there was a good correlation between the time-course of antinociception and brain levels after an s.c. administration of nicotine. Taken together, these observations indicate that the antagonism by NSTX and nicotinic antagonists of nicotine-induced antinociception in mice may result from a blockade of the functional nicotinic receptors possibly in the CNS. The inhibition of nicotine-induced antinociception in mice by NSTX seems to be associated with that of brain [3H]nicotine binding in vitro. The higher affinity of the toxin for brain [3H]nicotine binding sites as compared to mecamylamine, pempidine and hexa-
methonium has also been demonstrated in mice as well as in rats 2,1°. Thus, the present study may be the first report of the in vivo antagonism by NSTX of nicotinic receptor function. In addition to our data, Rapier et al. 7 have recently reported that low concentrations of NSTX inhibited selectively the DMPP-induced release of [3H]dopamine from rat striatal synaptosomes preloaded with [3H]dopamine. The correlation between the NSTX inhibition of [3H]nicotine binding and interaction with the functional nicotinic receptors in the CNS may have a significant implication for nicotinic receptor studies, since there is an apparent dissociation between biochemical and pharmacological effects of a-bungarotoxin, an antagonist of neuromuscular nicotinic receptors, in the brain 1. In conclusion, we have demonstrated a correlation between the biochemical and pharmacological behavior of NSTX in the nicotinic cholinergic system.
1 Brown, D.A., Neurotoxin and the ganglionic (C6) type of nicotinic receptors. In B. Ceccarelli and F. Clementi (Eds.), Advances in Cytopharmacology, Raven Press, New York, 1979, pp. 225-230. 2 Hayashi, E., Isogai, M., Kagawa, Y., Takayanagi, N. and Yamada, S., Neosurugatoxin, a specific antagonist of nicotinic acetylcholine receptors, J. Neurochem., 42 (1984) 1491-1494. 3 Hayashi, E. and Yamada, S,, Pharmacological studies on surugatoxin, the toxic principle from Japanese ivory mollusc (Babylonia japonica), Br. J. Pharmacol., 53 (1975) 207-215. 4 Kosuge, T., Tsuji, K., Hirai, K., Yamaguchi, K., Okamoto, T. and Iitaka, Y., Isolation and structure determination of a new marine toxin neosurugatoxin, from the Japanese ivory shell, Babylonia japonica, Tetrahedron Lett., 22 (1981) 3417-3420. 5 Mansner, R., Relationship between some central effects of nicotine and its brain levels in the mouse, Ann. Med. Exp. Biol. Fenn., 50 (1972) 205-212.
6 Porsius, A.J. and Van Zwieten, P.A., Central actions of some cholinergic drugs (arecaidine esters) and nicotine on blood pressure and heart rate of cats, Prog. Brain Res., 47 (1977) 131-135. 7 Rapier, C., Harrison, R., Lunt, G.G. and Wonnacott, S., Neosurugatoxin blocks nicotinic acetylcholine receptors in the brain, Neurochern. Int., 7 (1985) 389-396. 8 Sewell, R.D.E. and Spencer, P.S.J., Modification of the antinociceptive activity of narcotic agonists and antagonists by intraventricular injection of biogenic amines in mice, Br. J. Pharmacol., 51 (1974) 140P-141P. 9 Tripathi, H.L., Martin, B.R. and Aceto, M.D., Nicotineinduced antinociception in rats and mice: correlation with nicotine brain levels. J. Pharmacol. Exp. Ther., 221 (1982) 91-96. 10 Yamada, S., Isogai, M., Kagawa, Y., Takayanagi, N., Hayashi, E., Tsuji, K. and Kosuge, T., Brain nicotinic acetylcholine receptors: biochemical characterization by neosurugatoxin, Mol. Pharmacol., 28 (1985) 120-127.
We would like to thank to Dr. N.W. Pedigo, Department of Pharmacology, University of Kentucky, for his helpful suggestions.