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Agonist action of neostigmine on acetylcholine receptors of cultured mammalian muscle
378
Brain Research, 172 (1979) 378-~1 © Elsevier/North-HollandBiomedical Press
Agonist action of neostigmine on acetylcholine receptors of cultured mammalian muscle
ROBERT J. BLOCH and WILLIAM B. STALLCUP Neurobiology and Developmental Biology Laboratories, The Salk Institute, San Diego, Calif. 92112 (u.s.A.)
(Accepted April 26th, 1979)
Thirty years ago, Riker and Wescoe reported that neostigmine had effects on skeletal muscle that could not be explained by its inhibition of acetylcholinesterase9. To account for these effects, they suggested that neostigmine and acetylcholine have 'a common chemical basis of action '9. In more modern terms, they proposed that neostigmine, like acetylcholine, is an agonist of the acetylcholine receptor. We have used molecular techniques to investigate this proposal. To assay the ability of neostigmine to induce opening of the acetylcholine receptor (AChR) ion channel, we measured the rates o f N a + flux into cultured mouse muscle cells. These cells, termed BC3H-1 (ref. 10), respond to cholinergic agonists by increasing their permeability to Na ÷ (ref. 8). To obtain independent estimates of the affinity ofneostigmine for AChR, we also measured its effects on the binding of radiolabeled a-bungarotoxin ([125I]aBT) to BCaH-1 cells. Patrick et al. v have shown that this binding is specific for the AChR on the cell surface. Our results, presented below, strongly support the idea that neostigmine is an agonist of the nicotinic AChR of mammalian skeletal muscle. Neostigmine salts were purchased from Sigma. Neostigmine bromide (lot 117C-0229) was estimated by Sigma to be ~99.8 ~o pure using thin-layer chromatography and chemical analysis. Neostigmine methylsulfate (lot 106C-0347) was estimated to be 98-99 ~ pure. The bromide salt was therefore used for most experiments. The interaction of neostigmine with AChR results in an increased permeability of BC3H-1 cells to [22Na+] (Fig. 1). The initial velocity of neostigmine-stimulated [22Na+] influx shows a sigmoid dependence on neostigmine concentration, with an apparent half-maximum at 3 × 10-4 M. Neostigmine also stimulates [22Na+] flux into primary rat skeletal muscle myotubes (not shown). Influx is blocked by D-tubocurarine, and is similar whether the bromide or methylsulfate salt of neostigmine is used. Thus, influx seems to be stimulated by direct interaction of the neostigmine cation with AChR. In addition to stimulating receptor-mediated Na + flux, neostigmine also inhibits the binding of [125I]aBT to AChR. When BC3H-I cells are preincubated with varying concentrations of neostigmine for 10 min prior to addition of [125I]aBT, toxin binding
379
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NEOSTIGMINE. (MxIO-3) Fig. 1. Dependence of [~"Na+]flux into BCzH-1 cells on neostigmine concentration. Assay of initial rates of [2'ZNa+]influx was as described 12 except that MgSO4 and Na2HPO4 were omitted from the assay buffer. The bromide salt of neostigmine (Sigma) was used.
is inhibited 50% by about 7 × 10-5 M neostigmine (Fig. 2; mean zk S.D. in 3 experiments = 9 ~: 2 × 10 -5 M). However, if neostigmine is added to cells at the same time as [lzsI]aBT, with no prior exposure of cells to the drug, it is a less potent inhibitor of toxin binding (Table I; 50 ~ inhibition at 2-3 × 10-4 M). In its ability to block toxin binding more effectively after preincubation with cells, neostigmine resembles carbachol, but differs from o-tubocurarine (Table I). The increased efficacy with preincubation of inhibition of aBT binding has been interpreted to be due to drug-induced I00 85 80
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Neos~igmine (MxlO -4) Fig. 2. Inhibition of [125I]a-bungarotoxin binding to BCzH-I cells by increasing concentrations of neostigmine. Assay of the initial rates of [I~5Ila-BT binding to differentiated cultures of BCsH-1 cells was performed exactly as described 7. Cultures were preincubated for 10 rain with the concentration of neostigmine bromide noted before reaction with 10-8 M [I~I]aBT for 5 rain. Maximum initial rate of binding (100 ~ value) was determined by subtracting the value obtained in the presence of 10-4 M o-tubocurarine from the value obtained in the absence of any additional drugs.
380 TABLE I
Effect of Prehtcubation on Neostigmine Inhibition of a-Bungarotoxin Binding The initial rates of [~24I]aBT binding to differentiated cultures of BC3H-I cells were determined as described in the legend to Fig. 2, except that [a25I]aBT concentration was 6 × 10-8 M and reaction times were 30 sec. These conditions were used to minimize the amount of agonist-induced A C h R desensitization occurring during the reaction with [~zSI]aBT. Samples in the column 'no preincubation' were those in which [125I]aBT and the cholinergic drug were added simultaneously, without a prior exposure of the cells to drug. Values are the mean ~- S.D. followed by the number of determinations in parentheses.
Drug
Concentration ( M)
No preincubation With 10 min preincubation (pmol/dish/30 see × 102)
0 D-Tubocurarine Carbachol Neostigmine Neostigmine
-5 × 10-7 3 x 10 5 10-4 2 × 10 4
7.62 3.96 5.60 5.41 4.33
-t- 0.26 ~ 0.37 i 0.45 ± 0.39 ± 0.20
(4) (4) (3) (4) (4)
7.49 :t_ 0.46 4.09 ~ 0.22 2.72-t-0.38 3.70 :~ 0.14 2.95 ± 0.35
(4) (4) (4) (3) (4)
receptor desensitization3,5,13-1L Thus, interaction of neostigmine appears capable of desensitizing AChR. It is noteworthy that the difference in the rates of [lzSI]aBT binding with and without preincubation with neostigmine is not as great as it is in the case of carbachol (Table 1). In another experiment, we estimated that the maximum rate of [22Na+] influx stimulated by neostigmine was only about 20 % of that stimulated by carbachol (not shown). Thus, though it can interact with all exposed toxin binding sites of BC3H-1 cells (Fig. 2), neostigmine is a less potent agonist of AChR than is carbachol. This may be due to an ion channel-blocking effect of neostigmine, similar to those of cationic anesthetics6, to shorter channel open times of the neostigmine-AChR complex or to other factors. Despite this difference between neostigmine and carbachol, our data clearly indicate that these 2 drugs are very similar. Neostigmine, like carbachol 7, blocks pzsI]aBT binding to AChR of cultured cells and stimulates the flux of [22Na+] into cells with about the same affinity. This flux stimulation, like that by carbachol 2, obeys sigmoid kinetics and is inhibited by D-tubocurarine. Finally, neostigmine, like carbachol 8,5,18-15, desensitizes AChR. Neostigmine also induces the contraction of denervated muscle 9, desensitizes muscle in situ 9, blocks the interaction of [125I]aBT with Aplysia neurons x and of [3H]acetylcholine with AChR of Torpedo electroplax11, and depolarizes cultured embryonic chick skeletal muscle a. Considered together, these data clearly indicate that neostigmine is an agonist of the AChR of mammalian muscle. We thank J. H. Steinbach, S. Sine and S. Heinemann for their useful suggestions. This work was supported by a fellowship to R.J.B. from the American Heart Association, San Diego Chapter, by grants and fellowships to W.B.S. from the NIH (1 RO1 CA NS 21791-O1A1), the NSF (BNS 76-01548), the Alfred P. Sloan Foundation
381 (BR 1873) and the N a t i o n a l F o u n d a t i o n Basil O ' C o n n e r (5-60), by a grant f r o m the Samuel Roberts N o b l e F o u n d a t i o n o f A r d m o r e , Okla., and by grants to S. H e i n e m a n n f r o m the N I H (2 R01 N S 11549) and the M u s c u l a r D y s t r o p h y Associations o f America.
1 Carpenter, D. O., Greene, L. A., Shain, W. and Vogel, Z., Effects of eserine and neostigmine on the interaction of a-bungarotoxin with Aplysia acetylcholine receptors, Molec. Pharmaeol., 12 (1976) 999-1006. 2 Catterall, W. A., Sodium transport by the acetylcholine receptor of cultured muscle cells, J. biol. Chem., 250 (1975) 1776-1781. 3 Colquhoun, D. and Rang, H. P., Effects of inhibitors on the binding ofiodinated a-bungarotoxin to acetylcholine receptors in rat muscle, Molec. Pharmacol., 12 (1976) 519 535. 4 Harvey, A. L. and Dryden, W. F., Depolarization, desensitization and the effects of tubocurarine and neostigmine in cultured skeletal muscle, Europ. J. PharmaeoL, 27 (1974) 5-13. 5 Lee, T., Witzemann, V., Schlimerlik, M. and Raftery, M. A., Cholinergic ligand-induced affinity changes in Torpedo ealifornica acetylcholine receptor, Arch. Biochem. Biophys., 183 (1977) 57-63. 6 Neher, E. and Steinbach, J. H., Local anaesthetics transiently block currents through single acetylcholine-receptor channels, J. Physiol. (Lond.), 277 (1978) 153-176. 7 Patrick, J., McMillan, J., Wolfson, H. and O'Brien, J. C. Acetylcholine receptor metabolism in a nonfusing muscle cell line, J. biol. Chem., 252 (1977) 2143-2153. 8 Patrick, 3. and Stallcup, W. B., Immunological distinction between acetylcholine receptor and the a-bungarotoxin-binding component on sympathetic neurons, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 4689 4692. 9 Riker, W. F., Jr. and Wescoe, W. C., The direct action of prostigmine on skeletal muscle: its relationship to the choline esters, J. PharmaeoL exp. Ther., 88 (1946) 58-66. 10 Schubert, D., Harris, A. J., Devine, C. E. and Heinemann, S., Characterization of a unique muscle cell line, J. Cell Biol. 61 (1974) 398M13. 11 Seifert, S. A. and Eldefrawi, M. E., Affinity of myasthenia drugs to acetylcholinesterase and acetylcholine receptor, Biochem. Med., 10 (1974) 258-265. 12 Stallcup, W. B. and Cohn, M., Electrical properties ofa clonal cell line as determined by measurement of ion fluxes, Exp. Cell Res., 98 (1976) 277 284. 13 Weber, M., David-Pfeuty, T. and Changeux, J. P., Regulation of binding properties of the nicotinic receptor protein by cholinergic ligands in membrane fragments from Torpedo marmorata, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 3443-3447. 14 Weiland, G., Georgia, B., Lappi, S., Chignell, C. F. and Taylor, P., Kinetics of agonist-mediated transitions in state of the cholinergic receptor, J. biol. Chem., 252 (1977) 7648-7656. 15 Weiland, G., Georgia, B., Wee, V. T., Chignell, C. F. and Taylor, P., Ligand interactions with cholinergic receptor-enriched membrane from Torpedo : influence of agonist exposure on receptor properties, Molec. Pharmacol., 12 (1976) 1091-1105.
Brain Research, 172 (1979) 378-~1 © Elsevier/North-HollandBiomedical Press
Agonist action of neostigmine on acetylcholine receptors of cultured mammalian muscle
ROBERT J. BLOCH and WILLIAM B. STALLCUP Neurobiology and Developmental Biology Laboratories, The Salk Institute, San Diego, Calif. 92112 (u.s.A.)
(Accepted April 26th, 1979)
Thirty years ago, Riker and Wescoe reported that neostigmine had effects on skeletal muscle that could not be explained by its inhibition of acetylcholinesterase9. To account for these effects, they suggested that neostigmine and acetylcholine have 'a common chemical basis of action '9. In more modern terms, they proposed that neostigmine, like acetylcholine, is an agonist of the acetylcholine receptor. We have used molecular techniques to investigate this proposal. To assay the ability of neostigmine to induce opening of the acetylcholine receptor (AChR) ion channel, we measured the rates o f N a + flux into cultured mouse muscle cells. These cells, termed BC3H-1 (ref. 10), respond to cholinergic agonists by increasing their permeability to Na ÷ (ref. 8). To obtain independent estimates of the affinity ofneostigmine for AChR, we also measured its effects on the binding of radiolabeled a-bungarotoxin ([125I]aBT) to BCaH-1 cells. Patrick et al. v have shown that this binding is specific for the AChR on the cell surface. Our results, presented below, strongly support the idea that neostigmine is an agonist of the nicotinic AChR of mammalian skeletal muscle. Neostigmine salts were purchased from Sigma. Neostigmine bromide (lot 117C-0229) was estimated by Sigma to be ~99.8 ~o pure using thin-layer chromatography and chemical analysis. Neostigmine methylsulfate (lot 106C-0347) was estimated to be 98-99 ~ pure. The bromide salt was therefore used for most experiments. The interaction of neostigmine with AChR results in an increased permeability of BC3H-1 cells to [22Na+] (Fig. 1). The initial velocity of neostigmine-stimulated [22Na+] influx shows a sigmoid dependence on neostigmine concentration, with an apparent half-maximum at 3 × 10-4 M. Neostigmine also stimulates [22Na+] flux into primary rat skeletal muscle myotubes (not shown). Influx is blocked by D-tubocurarine, and is similar whether the bromide or methylsulfate salt of neostigmine is used. Thus, influx seems to be stimulated by direct interaction of the neostigmine cation with AChR. In addition to stimulating receptor-mediated Na + flux, neostigmine also inhibits the binding of [125I]aBT to AChR. When BC3H-I cells are preincubated with varying concentrations of neostigmine for 10 min prior to addition of [125I]aBT, toxin binding
379
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NEOSTIGMINE. (MxIO-3) Fig. 1. Dependence of [~"Na+]flux into BCzH-1 cells on neostigmine concentration. Assay of initial rates of [2'ZNa+]influx was as described 12 except that MgSO4 and Na2HPO4 were omitted from the assay buffer. The bromide salt of neostigmine (Sigma) was used.
is inhibited 50% by about 7 × 10-5 M neostigmine (Fig. 2; mean zk S.D. in 3 experiments = 9 ~: 2 × 10 -5 M). However, if neostigmine is added to cells at the same time as [lzsI]aBT, with no prior exposure of cells to the drug, it is a less potent inhibitor of toxin binding (Table I; 50 ~ inhibition at 2-3 × 10-4 M). In its ability to block toxin binding more effectively after preincubation with cells, neostigmine resembles carbachol, but differs from o-tubocurarine (Table I). The increased efficacy with preincubation of inhibition of aBT binding has been interpreted to be due to drug-induced I00 85 80
I
I
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I
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I
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2 ~)
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Neos~igmine (MxlO -4) Fig. 2. Inhibition of [125I]a-bungarotoxin binding to BCzH-I cells by increasing concentrations of neostigmine. Assay of the initial rates of [I~5Ila-BT binding to differentiated cultures of BCsH-1 cells was performed exactly as described 7. Cultures were preincubated for 10 rain with the concentration of neostigmine bromide noted before reaction with 10-8 M [I~I]aBT for 5 rain. Maximum initial rate of binding (100 ~ value) was determined by subtracting the value obtained in the presence of 10-4 M o-tubocurarine from the value obtained in the absence of any additional drugs.
380 TABLE I
Effect of Prehtcubation on Neostigmine Inhibition of a-Bungarotoxin Binding The initial rates of [~24I]aBT binding to differentiated cultures of BC3H-I cells were determined as described in the legend to Fig. 2, except that [a25I]aBT concentration was 6 × 10-8 M and reaction times were 30 sec. These conditions were used to minimize the amount of agonist-induced A C h R desensitization occurring during the reaction with [~zSI]aBT. Samples in the column 'no preincubation' were those in which [125I]aBT and the cholinergic drug were added simultaneously, without a prior exposure of the cells to drug. Values are the mean ~- S.D. followed by the number of determinations in parentheses.
Drug
Concentration ( M)
No preincubation With 10 min preincubation (pmol/dish/30 see × 102)
0 D-Tubocurarine Carbachol Neostigmine Neostigmine
-5 × 10-7 3 x 10 5 10-4 2 × 10 4
7.62 3.96 5.60 5.41 4.33
-t- 0.26 ~ 0.37 i 0.45 ± 0.39 ± 0.20
(4) (4) (3) (4) (4)
7.49 :t_ 0.46 4.09 ~ 0.22 2.72-t-0.38 3.70 :~ 0.14 2.95 ± 0.35
(4) (4) (4) (3) (4)
receptor desensitization3,5,13-1L Thus, interaction of neostigmine appears capable of desensitizing AChR. It is noteworthy that the difference in the rates of [lzSI]aBT binding with and without preincubation with neostigmine is not as great as it is in the case of carbachol (Table 1). In another experiment, we estimated that the maximum rate of [22Na+] influx stimulated by neostigmine was only about 20 % of that stimulated by carbachol (not shown). Thus, though it can interact with all exposed toxin binding sites of BC3H-1 cells (Fig. 2), neostigmine is a less potent agonist of AChR than is carbachol. This may be due to an ion channel-blocking effect of neostigmine, similar to those of cationic anesthetics6, to shorter channel open times of the neostigmine-AChR complex or to other factors. Despite this difference between neostigmine and carbachol, our data clearly indicate that these 2 drugs are very similar. Neostigmine, like carbachol 7, blocks pzsI]aBT binding to AChR of cultured cells and stimulates the flux of [22Na+] into cells with about the same affinity. This flux stimulation, like that by carbachol 2, obeys sigmoid kinetics and is inhibited by D-tubocurarine. Finally, neostigmine, like carbachol 8,5,18-15, desensitizes AChR. Neostigmine also induces the contraction of denervated muscle 9, desensitizes muscle in situ 9, blocks the interaction of [125I]aBT with Aplysia neurons x and of [3H]acetylcholine with AChR of Torpedo electroplax11, and depolarizes cultured embryonic chick skeletal muscle a. Considered together, these data clearly indicate that neostigmine is an agonist of the AChR of mammalian muscle. We thank J. H. Steinbach, S. Sine and S. Heinemann for their useful suggestions. This work was supported by a fellowship to R.J.B. from the American Heart Association, San Diego Chapter, by grants and fellowships to W.B.S. from the NIH (1 RO1 CA NS 21791-O1A1), the NSF (BNS 76-01548), the Alfred P. Sloan Foundation
381 (BR 1873) and the N a t i o n a l F o u n d a t i o n Basil O ' C o n n e r (5-60), by a grant f r o m the Samuel Roberts N o b l e F o u n d a t i o n o f A r d m o r e , Okla., and by grants to S. H e i n e m a n n f r o m the N I H (2 R01 N S 11549) and the M u s c u l a r D y s t r o p h y Associations o f America.
1 Carpenter, D. O., Greene, L. A., Shain, W. and Vogel, Z., Effects of eserine and neostigmine on the interaction of a-bungarotoxin with Aplysia acetylcholine receptors, Molec. Pharmaeol., 12 (1976) 999-1006. 2 Catterall, W. A., Sodium transport by the acetylcholine receptor of cultured muscle cells, J. biol. Chem., 250 (1975) 1776-1781. 3 Colquhoun, D. and Rang, H. P., Effects of inhibitors on the binding ofiodinated a-bungarotoxin to acetylcholine receptors in rat muscle, Molec. Pharmacol., 12 (1976) 519 535. 4 Harvey, A. L. and Dryden, W. F., Depolarization, desensitization and the effects of tubocurarine and neostigmine in cultured skeletal muscle, Europ. J. PharmaeoL, 27 (1974) 5-13. 5 Lee, T., Witzemann, V., Schlimerlik, M. and Raftery, M. A., Cholinergic ligand-induced affinity changes in Torpedo ealifornica acetylcholine receptor, Arch. Biochem. Biophys., 183 (1977) 57-63. 6 Neher, E. and Steinbach, J. H., Local anaesthetics transiently block currents through single acetylcholine-receptor channels, J. Physiol. (Lond.), 277 (1978) 153-176. 7 Patrick, J., McMillan, J., Wolfson, H. and O'Brien, J. C. Acetylcholine receptor metabolism in a nonfusing muscle cell line, J. biol. Chem., 252 (1977) 2143-2153. 8 Patrick, 3. and Stallcup, W. B., Immunological distinction between acetylcholine receptor and the a-bungarotoxin-binding component on sympathetic neurons, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 4689 4692. 9 Riker, W. F., Jr. and Wescoe, W. C., The direct action of prostigmine on skeletal muscle: its relationship to the choline esters, J. PharmaeoL exp. Ther., 88 (1946) 58-66. 10 Schubert, D., Harris, A. J., Devine, C. E. and Heinemann, S., Characterization of a unique muscle cell line, J. Cell Biol. 61 (1974) 398M13. 11 Seifert, S. A. and Eldefrawi, M. E., Affinity of myasthenia drugs to acetylcholinesterase and acetylcholine receptor, Biochem. Med., 10 (1974) 258-265. 12 Stallcup, W. B. and Cohn, M., Electrical properties ofa clonal cell line as determined by measurement of ion fluxes, Exp. Cell Res., 98 (1976) 277 284. 13 Weber, M., David-Pfeuty, T. and Changeux, J. P., Regulation of binding properties of the nicotinic receptor protein by cholinergic ligands in membrane fragments from Torpedo marmorata, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 3443-3447. 14 Weiland, G., Georgia, B., Lappi, S., Chignell, C. F. and Taylor, P., Kinetics of agonist-mediated transitions in state of the cholinergic receptor, J. biol. Chem., 252 (1977) 7648-7656. 15 Weiland, G., Georgia, B., Wee, V. T., Chignell, C. F. and Taylor, P., Ligand interactions with cholinergic receptor-enriched membrane from Torpedo : influence of agonist exposure on receptor properties, Molec. Pharmacol., 12 (1976) 1091-1105.