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Amphetamine inhibition of α-bungarotoxin binding at the mouse neuromuscular junction
Life Sciences Vol . 20, pp . 1495-1500, Printed in the U.S .A .
1977 .
Pergamon Press
AMPHETAMINE INHIBITION OF a-BUNGAROTOXIN BINDING AT THE MOUSE NEUROMUSCULAR JUNCTION Kenneth A. Skau and Michael C. Gerald Division of Pharmacology, College of Pharmacy Ohio State University Columbus, Ohio
43210
(Received in final form February 14, 1977) Summary Amphetamine inhibited neuromuscular transmission and prevented the irreversible blockade produced by a-bungarotoxin (a-BGT) in the isolated mouse phrenic nerve-diaphragm preparation. Similarly amphetamine (1 .35 X 10 -4 to 3 X 10 - 3M) inhibited the binding of 125Ia-BGT to mouse hemidiaphragms in a concentration-dependent manner ; (+)-amphetamine was found to be twice as potent as its (-)-isomer with respect to inhibition of 1251-a-BGT binding . It is suggested that amphetamine binds to the nicotinic, cholinergic receptor of skeletal muscle and may produce weakness and paralysis in amphetamine overdosage . Toxic doses of amphetamine produce muscle weakness and arm and leg paralysis in humans (1-3), and hindlimb paresis in several species of laboratory animals (4-6) . The mechanism for this weakness and paralysis may involve cen tral and peripheral components, such as drug-induced cerebral damage and/or inhibition of neuromuscular function . Amphetamine reversibly blocks neuromuscular transmission in the nerve-stimulated, isolated rat phrenic nervediaphragm ; nerve conduction and the ability of the muscle to contract upon direct stimulation remain unimpaired (7-10) . We have recently suggested that amphetamine blockade is postsynaptic and resembles the blockade produced by curare-like drugs [(+)-tubocurarine] (10) . In this report we present evidence that amphetamine inhibits neuromuscular transmission in the mouse phrenic nerve-diaphragm preparation and prevents the binding of a-bungarotoxin (a-BGT) to the postsynaptic, nicotinic, cholinergic receptor . Methods Muscle Contraction Studies . Male albino ICR mice (Lab Supply, Indianapolis, Indiana), weighing 28-33g were used in this study. Animals were stunned by a blow to the head and sacrificed by cervical dislocation. The left hemidiaphragm with its phrenic nerve was removed as described for the rat by Bülbring (11) and mounted in a 20 ml organ bath in oxygenated Krebs' solution at 32 0C . Details of the procedure and composition of the Krebs' solution have been previously reported (9) . The phrenic nerve was passed through a pair of electrodes and stimulated at 0.2 Hz with 5 volt rectangular wave pulses of 0 .4 msec duration . * To whom reprint requests should be addressed.
1495
1496
Amphetamine Inhibition a-Bungarotoxin
Vol . 20, No . 9, 1977
Binding Studies . Left and right hemidiaphragms were removed as described above and preincubated at 34 0C for 2 h in 0 .5 ml of Krebs' solution containing (+)amphetamine, (-)-amphetamine, (+)-tubocurarine [(+)TC], or in the absence of drug ; 1251-a-BGT* was then added to the bath for 0 .5 h . Tissues were washed first at 5 min intervals for 60 min with 5 ml aliquots of Krebs' solution containing (+)TC (10 -414), then washed overnight in 25 ml of the same solution ; this procedure was designed to remove residual toxin and reduce nonspecifically bound toxin . The amount of 125 ,-a-BGT bound to the hemidiaphragms was determined by counting in a Gamma Set 500 (ICN, Antwerp, Belgium) gamma counter at 70% efficiency . Specifically bound toxin was defined as the binding of 1251-a-BGT that could be prevented by preincubation for 2 h with unlabeled aBGT (1 Fig/ml) ; nonspecific binding was not prevented by preincubation with aBGT and was subtracted from total binding . Results & Discussion Muscle Contraction Studies . This series of experiments was designed to determine whether amphetamine inhibits neuromuscular transmission in the mouse diaphragm as it does in rat preparations and to locate the site of the blockade . As seen in Fig . 1, (+)-amphetamine inhibited nerve-stimulated muscle contractions in the mouse diaphragm in a concentration-dependent manner . The concentrations that produce inhibition of neuromuscular transmission in the mouse preparation are in the same range as those concentrations that inhibit rat preparations (9) . As was observed in the rat preparation, the blockade was easily reversed upon washing the tissue with Krebs' solution, and there was a transient enhancement of contractions preceding neuromuscular blockade, although this effect was less consistent in the mouse preparation . In similarly prepared tissues, amphetamine prevented the neuromuscular blocking effects of a-BGT . This law molecular weight protein has been shown to bind specifically and covalently to the nicotinic receptor of skeletal muscle producing irreversible blockade of neuromuscular transmission (12) . Pretreatment of tissues with drugs that bind to the nicotinic receptor, such as (+)TC, decamethonium, succinylcholine, and carbachol, protect the receptor and inhibit binding of a-BGT (13) . In our studies, a-BGT (1 hg/ml), completely blocked neuromuscular transmission in 40 min ; nerve-stimulated muscle contractions did not return even after repeated tissue washings at 5-10 min intervals for 2 h . By contrast, tissues pretreated with a neuromuscular blocking concentration of (+)-amphetamine (8 .1 X 10 -4 14) were protected against irreversible a-BGT blockade (Fig . 2) . Nerve-stimulated muscle contractions returned to predrug levels within 15 min when washed at 5 min intervals . (-)-Amphetamine also was observed to inhibit neuromuscular contractions and protect against a-BGT, although this isomer was somewhat less potent than the (+)-enantiomer . These results suggest that amphetamine may be binding to the cholinergic, nicotinic receptor of muscle and thus inhibiting the ability of a-BGT to bind to these receptors . Binding Studies . The next series of experiments were designed to quantitatively study the interaction of amphetamine at the neuromuscular junction . To avoid the inherent problems associated with the study of muscle contractions, such as fatigue of the muscle and the presence of spare receptors, we examined the ability of amphetamine to inhibit the specific binding of 125 1-a-BGT to mouse hemidiaphragms . (+)TC, used as a standard reference inhibitor of 12 5I-aBGT binding (11), reduced binding of the labeled toxin by 14 .3-87 .5% at concentrations of 10 - M to 10-314 . * a-BGT, . supplied by the Miami Serpentarium, was labeled with 1251 to a specific activity of 14 Ci/mmole by the New England Nuclear Corp .
Vol . 20, No . 9, 1977
W
Amphetamine Inhibition a-Bungarotozin
1497
100
J W z
Q
m
75
z0 w M
~_
LL
0 0
TIME (min.)
Fig. 1
(+)-Amphetamine Inhibition of Neuromuscular Transmission in the Mouse Phrenic Nerve-Diaphragm. Each point represents the mean % of pre-drug tension ± S .E .M. of 3 or 4 tissues . Control, 0 ;concentrations of (+)-amphetamine : O , 2.7 X 10 -4M ; O , 4.05 X 10 -ß+M ; / , 5.4 X 10 -ß+M ; A , 8 .1 X 10 -4M .
Fig.
2
Protection b + -Am hetamine ainat Irreversible a-BGT Neuromuscular Block ade . (+)-Amphetamine (8 .1 X 10 -4M) was added to the bath of a nerve-stimulated mouse diaphragm producing blockade of neuromuscular transmission . a-BGT (1 i=g/ml) was added to the bath for 40 min. With the commencement of washing (w), nerve-stimulated muscle contractions returned to predrug levels within 15 min. In tissues treated with a-BGT, in the absence of amphetamine, total neuromuscular blockade was effected in 40 min and no signs of recovery were noted even after 2 hours of washing at 5-10 min intervals .
1498
Amphetamine Inhibition a-Bungarotoxin
Vol . 20, No . 9, 1977
The effects of (+)- and (-)-amphetamine on toxin binding are shown in Fig . 3 . The amphetamine isomers inhibited specific binding of the labeled toxin in a concentration-dependent manner and showed stereochemical differences ; concentrations producing 50% inhibition of toxin binding were 3 .38 X 10 - 4M for the (+)-isomer and 6 .98 X 10 - 4M for (-)-amphetamine . We have previously reported that a similar 2-fold difference in potency exists between the amphetamine isomers for another aspect of neuromuscular transmission- inhibition of tetanus and posttetanic potentiation in the rat phrenic nerve-diaphragm preparation (10) . Methamphetamine, ephedrine, and phenethylamine also inhibited 125,E-BGT binding in this system (data not shown) .
90 6 .0 80 70
5 .5
û
N
i
60 50
5 .0
4.5
40
Z
30
Z
o~r 20 4.0 10
1X10 -3 3x10 -4 1x10 -4
AMPHETAMINE (M)
'
3x10-3
Fig . 3 Inhibition of Binding of 1251-cX-BGT by Amphetamine Isomers . Best fit lines were generated by linear regression analysis and do not deviate from parallelity . Ordinate : percent of control binding concentration of of 125 ,-(z-BGT expressed as probits . Abscissa : amphetamine (log scale) : 0----o , (+)-amphetamine (r = 0 .82)o- --o, (-)-amphetamine (r = 0 .81) . At 50% inhibition of 125I(X-BGT binding (probit 5), the (+) isomer is 2 .06 times more potent than the (-)-isomer . N = 4 at each point .
Vol . 20, No . 9, 1977
Amphetamine Inhibition a-Bungarotoxin
1499
It has been suggested that the muscle concentration of amphetamine in rats pretreated with lethal doses is twice the concentration needed to block isolated nerve-muscle preparations (8) . Such in vitro concentrations, however, are not easily extrapolated to muscle concentrations or _in vivo dosages . Since individuals abusing amphetamines often employ very high doses of the drug (14), it is possible that the weakness and paralysis associated with amphetamine overdosage may be, in part, the result of peripheral neuromuscular blockade . The results presented herein suggest that amphetamine binds to the nicotinic, cholinergic receptor of skeletal muscle, and support the concept that amphetamine blocks neuromuscular transmission in vitro by a curare-like antidepolarizing blockade . Acknow ledgments This work was supported by USPHS grant DA-01477 . K .A .S . i s a predoctoral fellow of the American Foundation for Pharmaceutical Education . Amphetamine isomers were generously provided by Smith Kline & French Laboratories .
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 .
A . POTELIAKHOFF and B . C . ROUGHTON, Brit . Med . J . _1 26-27 (1956) . 0 . L . GERICKE, J . Amer . Med . Assoc . 12 8 1098-1099 (1945) . R . GREENWOOD and R . S . PEACHY, Brit . Med . J . _ 1 742-744 (1957) . M . G . RARDINGE and D . I . PETERSON, J . Pharmac Exp . Ther . 145 47-51 (1964) . W . E . EHRICH and H . B . KRUMBHAAR, Ann . Int . Med . 10 1874-1888 (1937) . R . HÖHN and L . LASAGNA, Psychopharmacologia _1 210-220 (1960) . E . G . ANDERSON and A . AMMANN, J . Pharmac . Exp . Ther . _140 179-182 (1963) . D . I . PETERSON, M . G . HARDINGE, and B . E . TILTON, J . Pharmac . Exp . Ther . 14 6 175-179 (1964) . M . C . GERALD and S . Y . HSU, Neuropharmacology 14 115-123 (1975) . K . A . SKAU and M. C . GERALD, The Pharmacologist 18 194 (1976) . E . BÜLBRING, Br . J . Pharmac . 1 38-61 (1946) . C . C . CHANG and C . Y . LEE, Arch . Int . Pharmacodyn . 144 241-257 (1963) . D . COLQUHOUN and H . P . RANG, Mol . Pharmacol . 12 519-535 (1976) . J . C . KRAMER, V . S . FISHMAN, and D . C . LITTLEFIELD, J . Amer . Med . Assoc . 20 1 305-309 (1967) .
1977 .
Pergamon Press
AMPHETAMINE INHIBITION OF a-BUNGAROTOXIN BINDING AT THE MOUSE NEUROMUSCULAR JUNCTION Kenneth A. Skau and Michael C. Gerald Division of Pharmacology, College of Pharmacy Ohio State University Columbus, Ohio
43210
(Received in final form February 14, 1977) Summary Amphetamine inhibited neuromuscular transmission and prevented the irreversible blockade produced by a-bungarotoxin (a-BGT) in the isolated mouse phrenic nerve-diaphragm preparation. Similarly amphetamine (1 .35 X 10 -4 to 3 X 10 - 3M) inhibited the binding of 125Ia-BGT to mouse hemidiaphragms in a concentration-dependent manner ; (+)-amphetamine was found to be twice as potent as its (-)-isomer with respect to inhibition of 1251-a-BGT binding . It is suggested that amphetamine binds to the nicotinic, cholinergic receptor of skeletal muscle and may produce weakness and paralysis in amphetamine overdosage . Toxic doses of amphetamine produce muscle weakness and arm and leg paralysis in humans (1-3), and hindlimb paresis in several species of laboratory animals (4-6) . The mechanism for this weakness and paralysis may involve cen tral and peripheral components, such as drug-induced cerebral damage and/or inhibition of neuromuscular function . Amphetamine reversibly blocks neuromuscular transmission in the nerve-stimulated, isolated rat phrenic nervediaphragm ; nerve conduction and the ability of the muscle to contract upon direct stimulation remain unimpaired (7-10) . We have recently suggested that amphetamine blockade is postsynaptic and resembles the blockade produced by curare-like drugs [(+)-tubocurarine] (10) . In this report we present evidence that amphetamine inhibits neuromuscular transmission in the mouse phrenic nerve-diaphragm preparation and prevents the binding of a-bungarotoxin (a-BGT) to the postsynaptic, nicotinic, cholinergic receptor . Methods Muscle Contraction Studies . Male albino ICR mice (Lab Supply, Indianapolis, Indiana), weighing 28-33g were used in this study. Animals were stunned by a blow to the head and sacrificed by cervical dislocation. The left hemidiaphragm with its phrenic nerve was removed as described for the rat by Bülbring (11) and mounted in a 20 ml organ bath in oxygenated Krebs' solution at 32 0C . Details of the procedure and composition of the Krebs' solution have been previously reported (9) . The phrenic nerve was passed through a pair of electrodes and stimulated at 0.2 Hz with 5 volt rectangular wave pulses of 0 .4 msec duration . * To whom reprint requests should be addressed.
1495
1496
Amphetamine Inhibition a-Bungarotoxin
Vol . 20, No . 9, 1977
Binding Studies . Left and right hemidiaphragms were removed as described above and preincubated at 34 0C for 2 h in 0 .5 ml of Krebs' solution containing (+)amphetamine, (-)-amphetamine, (+)-tubocurarine [(+)TC], or in the absence of drug ; 1251-a-BGT* was then added to the bath for 0 .5 h . Tissues were washed first at 5 min intervals for 60 min with 5 ml aliquots of Krebs' solution containing (+)TC (10 -414), then washed overnight in 25 ml of the same solution ; this procedure was designed to remove residual toxin and reduce nonspecifically bound toxin . The amount of 125 ,-a-BGT bound to the hemidiaphragms was determined by counting in a Gamma Set 500 (ICN, Antwerp, Belgium) gamma counter at 70% efficiency . Specifically bound toxin was defined as the binding of 1251-a-BGT that could be prevented by preincubation for 2 h with unlabeled aBGT (1 Fig/ml) ; nonspecific binding was not prevented by preincubation with aBGT and was subtracted from total binding . Results & Discussion Muscle Contraction Studies . This series of experiments was designed to determine whether amphetamine inhibits neuromuscular transmission in the mouse diaphragm as it does in rat preparations and to locate the site of the blockade . As seen in Fig . 1, (+)-amphetamine inhibited nerve-stimulated muscle contractions in the mouse diaphragm in a concentration-dependent manner . The concentrations that produce inhibition of neuromuscular transmission in the mouse preparation are in the same range as those concentrations that inhibit rat preparations (9) . As was observed in the rat preparation, the blockade was easily reversed upon washing the tissue with Krebs' solution, and there was a transient enhancement of contractions preceding neuromuscular blockade, although this effect was less consistent in the mouse preparation . In similarly prepared tissues, amphetamine prevented the neuromuscular blocking effects of a-BGT . This law molecular weight protein has been shown to bind specifically and covalently to the nicotinic receptor of skeletal muscle producing irreversible blockade of neuromuscular transmission (12) . Pretreatment of tissues with drugs that bind to the nicotinic receptor, such as (+)TC, decamethonium, succinylcholine, and carbachol, protect the receptor and inhibit binding of a-BGT (13) . In our studies, a-BGT (1 hg/ml), completely blocked neuromuscular transmission in 40 min ; nerve-stimulated muscle contractions did not return even after repeated tissue washings at 5-10 min intervals for 2 h . By contrast, tissues pretreated with a neuromuscular blocking concentration of (+)-amphetamine (8 .1 X 10 -4 14) were protected against irreversible a-BGT blockade (Fig . 2) . Nerve-stimulated muscle contractions returned to predrug levels within 15 min when washed at 5 min intervals . (-)-Amphetamine also was observed to inhibit neuromuscular contractions and protect against a-BGT, although this isomer was somewhat less potent than the (+)-enantiomer . These results suggest that amphetamine may be binding to the cholinergic, nicotinic receptor of muscle and thus inhibiting the ability of a-BGT to bind to these receptors . Binding Studies . The next series of experiments were designed to quantitatively study the interaction of amphetamine at the neuromuscular junction . To avoid the inherent problems associated with the study of muscle contractions, such as fatigue of the muscle and the presence of spare receptors, we examined the ability of amphetamine to inhibit the specific binding of 125 1-a-BGT to mouse hemidiaphragms . (+)TC, used as a standard reference inhibitor of 12 5I-aBGT binding (11), reduced binding of the labeled toxin by 14 .3-87 .5% at concentrations of 10 - M to 10-314 . * a-BGT, . supplied by the Miami Serpentarium, was labeled with 1251 to a specific activity of 14 Ci/mmole by the New England Nuclear Corp .
Vol . 20, No . 9, 1977
W
Amphetamine Inhibition a-Bungarotozin
1497
100
J W z
Q
m
75
z0 w M
~_
LL
0 0
TIME (min.)
Fig. 1
(+)-Amphetamine Inhibition of Neuromuscular Transmission in the Mouse Phrenic Nerve-Diaphragm. Each point represents the mean % of pre-drug tension ± S .E .M. of 3 or 4 tissues . Control, 0 ;concentrations of (+)-amphetamine : O , 2.7 X 10 -4M ; O , 4.05 X 10 -ß+M ; / , 5.4 X 10 -ß+M ; A , 8 .1 X 10 -4M .
Fig.
2
Protection b + -Am hetamine ainat Irreversible a-BGT Neuromuscular Block ade . (+)-Amphetamine (8 .1 X 10 -4M) was added to the bath of a nerve-stimulated mouse diaphragm producing blockade of neuromuscular transmission . a-BGT (1 i=g/ml) was added to the bath for 40 min. With the commencement of washing (w), nerve-stimulated muscle contractions returned to predrug levels within 15 min. In tissues treated with a-BGT, in the absence of amphetamine, total neuromuscular blockade was effected in 40 min and no signs of recovery were noted even after 2 hours of washing at 5-10 min intervals .
1498
Amphetamine Inhibition a-Bungarotoxin
Vol . 20, No . 9, 1977
The effects of (+)- and (-)-amphetamine on toxin binding are shown in Fig . 3 . The amphetamine isomers inhibited specific binding of the labeled toxin in a concentration-dependent manner and showed stereochemical differences ; concentrations producing 50% inhibition of toxin binding were 3 .38 X 10 - 4M for the (+)-isomer and 6 .98 X 10 - 4M for (-)-amphetamine . We have previously reported that a similar 2-fold difference in potency exists between the amphetamine isomers for another aspect of neuromuscular transmission- inhibition of tetanus and posttetanic potentiation in the rat phrenic nerve-diaphragm preparation (10) . Methamphetamine, ephedrine, and phenethylamine also inhibited 125,E-BGT binding in this system (data not shown) .
90 6 .0 80 70
5 .5
û
N
i
60 50
5 .0
4.5
40
Z
30
Z
o~r 20 4.0 10
1X10 -3 3x10 -4 1x10 -4
AMPHETAMINE (M)
'
3x10-3
Fig . 3 Inhibition of Binding of 1251-cX-BGT by Amphetamine Isomers . Best fit lines were generated by linear regression analysis and do not deviate from parallelity . Ordinate : percent of control binding concentration of of 125 ,-(z-BGT expressed as probits . Abscissa : amphetamine (log scale) : 0----o , (+)-amphetamine (r = 0 .82)o- --o, (-)-amphetamine (r = 0 .81) . At 50% inhibition of 125I(X-BGT binding (probit 5), the (+) isomer is 2 .06 times more potent than the (-)-isomer . N = 4 at each point .
Vol . 20, No . 9, 1977
Amphetamine Inhibition a-Bungarotoxin
1499
It has been suggested that the muscle concentration of amphetamine in rats pretreated with lethal doses is twice the concentration needed to block isolated nerve-muscle preparations (8) . Such in vitro concentrations, however, are not easily extrapolated to muscle concentrations or _in vivo dosages . Since individuals abusing amphetamines often employ very high doses of the drug (14), it is possible that the weakness and paralysis associated with amphetamine overdosage may be, in part, the result of peripheral neuromuscular blockade . The results presented herein suggest that amphetamine binds to the nicotinic, cholinergic receptor of skeletal muscle, and support the concept that amphetamine blocks neuromuscular transmission in vitro by a curare-like antidepolarizing blockade . Acknow ledgments This work was supported by USPHS grant DA-01477 . K .A .S . i s a predoctoral fellow of the American Foundation for Pharmaceutical Education . Amphetamine isomers were generously provided by Smith Kline & French Laboratories .
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 .
A . POTELIAKHOFF and B . C . ROUGHTON, Brit . Med . J . _1 26-27 (1956) . 0 . L . GERICKE, J . Amer . Med . Assoc . 12 8 1098-1099 (1945) . R . GREENWOOD and R . S . PEACHY, Brit . Med . J . _ 1 742-744 (1957) . M . G . RARDINGE and D . I . PETERSON, J . Pharmac Exp . Ther . 145 47-51 (1964) . W . E . EHRICH and H . B . KRUMBHAAR, Ann . Int . Med . 10 1874-1888 (1937) . R . HÖHN and L . LASAGNA, Psychopharmacologia _1 210-220 (1960) . E . G . ANDERSON and A . AMMANN, J . Pharmac . Exp . Ther . _140 179-182 (1963) . D . I . PETERSON, M . G . HARDINGE, and B . E . TILTON, J . Pharmac . Exp . Ther . 14 6 175-179 (1964) . M . C . GERALD and S . Y . HSU, Neuropharmacology 14 115-123 (1975) . K . A . SKAU and M. C . GERALD, The Pharmacologist 18 194 (1976) . E . BÜLBRING, Br . J . Pharmac . 1 38-61 (1946) . C . C . CHANG and C . Y . LEE, Arch . Int . Pharmacodyn . 144 241-257 (1963) . D . COLQUHOUN and H . P . RANG, Mol . Pharmacol . 12 519-535 (1976) . J . C . KRAMER, V . S . FISHMAN, and D . C . LITTLEFIELD, J . Amer . Med . Assoc . 20 1 305-309 (1967) .