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β-Bungarotoxin elevates diaphragm acetylcholine levels
486
Brain Research, 182 (1980) 486-490 © Elsevier/North-Holland Biomedical Press
fl-Bungarotoxin elevates diaphragm acetylcholine levels CAMERON B. GUNDERSEN, MICHAEL W. NEWTON and DONALD J. JENDEN Department of Pharmacology, UCLA School of Medicine, Los Angeles, Cahlf 90024 (U.S.A.)
(Accepted September 27th, 1979) Key words: acetylcholine - - f l - b u n g a r o t o x i n - diaphragm
fl-Bungarotoxin (fl-Btx) is a presynaptically acting polypeptide neurotoxin 1. In muscle the toxin causes an initial depression and the enhancement of spontaneous and evoked acetylcholine (ACh) release 1,2,4. These stages are followed by a neuromuscular block associated with reduced ACh outputL Among the hypotheses offered to explain the mechanism of action of fl-Btx are: depletion of nerve terminal energy levels 1°,1s, lysis of the nerve endings 16, inhibition of choline (Ch) transportS,14,15, interruption of vesicle recycling 12 or direct action on the release mechanism4,11. Since fl-Btx has phospholipase activity is, several groups have argued for a role of this enzyme activity in mediating the action of the toxin1, lt,l~,ls. While investigating neurochemical correlates of fl-Btx action, we observed a large increase in tissue levels of ACh after stimulation of rat diaphragm muscle treated with fl-Btx. Since previous studies led us to expect normal 4 or decreased 14 levels of ACh in the tissue, the following report describes in more detail the effects of fl-Btx on ACh metabolism in this nerve-muscle preparation. Our initial observations were made on diaphragms subjected to high frequency stimulation of the phrenic nerve. Fan-shaped segments of the left hemidiaphragm of 90-150 g male rats were dissected with the costal margin intact. The tissue was equilibrated for 30 min at 37 °C in 3.5 ml of eserinized (15 #M) Krebs bicarbonate medium with a steady stream of 95 ~ 02-5 ~ CO2. fl-Btx was added to the bath only during the equilibration period. The fl-Btx we used corresponds to the fla fraction 1 and was the gift of Dr. Bruce Howard. Release of ACh into the eserinized Krebs was estimated during subsequent 10 or 15 min collection periods. Stimulation of the preparation was via a suction electrode using square pulses of 4-6 V, 0.1 msec duration at the indicated frequency. After the final collection period the diaphragm tissue was cut free of the costal margin and both pieces of tissue were lightly blotted, placed in 1 M formic acid in acetone (3 : 17, v/v) and weighed. The total wet weight of the tissue (diaphragm plus rib) ranged between 120 and 210 mg. ACh was extracted from the tissue either by homogenization using a polytron or by standing for 20-24 h at 4 °C. A gas chromatographic mass spectrometric assay was used to estimate ACh in all
487 TABLE I Effects o f fl-Btx on ACh release and on tissue ACh levels
Diaphragm strips were treated with and without fl-Btx (1.4 pg/ml). Release of ACh into eserinized (15/~M) Krebs solution was measured during periods of rest (R) (15 min) or stimulation (S) (10 min) in the sequences and at the frequencies indicated. After the last collection period tissue levels of ACh were measured. The results given are for ACh content of the diaphragm portion only. Results are means 4- S.D. Condition (n)
Collection periods (pmol ACh/min) 1
2
3
4
Tissue 5
pmol A Ch
pmol ACh mg wet wt.
10 H z (R,S,S,S,R) Control (3) 1.6±0.5 7.3±0.5 6.74-0.3 6.84-0.3 1.54-0.4 140.54-16.0 2.28±0.30 fl-Btx(5) 1.3±0.4 2.44-0.5* 2.14-0.8" 1.84,0.5" 1.8±0.3 426.4±70.5* 7.094.1.09" 1 Hz (R,S,S,S,R)
Control (3) 1.44,0.3 1.8±0.2 1.84,0.3 1.84,0.3 0.94-0.4 146.14,15.1 fl-Btx (3) 1.3±0.4 1.74,0.6 1.84,0.6 2.44-0.3 1.5±0.4 566.94-48.8*
2.314.0.51 8.314,1.74'
Rest (R,R,R,R)
Control (15) 1.32_0.3 1.2±0.3 1.24,0.3 1.24,0.3 fl-Btx (5) 1.24.0.6 1.24.0.3 1.54,0.5 1.5±0.4
---
126.92-19.3 1.834,0.32 396.84,21.8" 5.384-0.89*
* Values for toxin treated differ from control at P < 0.001. samples6,% The significance of the data was evaluated using the unpaired Students ttest. As indicated in Table I, a 30 min exposure to fl-Btx (1.4/~g/ml) caused the ACh output during successive periods of 10 Hz stimulation to remain at background levels. Concomitantly, ACh levels in the tissues exceed by more than 3 times those of controls. Chang, Chen and LeO did not observe such a change in /3-Btx-treated diaphragms after low frequency stimulation. When we reduced the stimulation frequency to 1 Hz, or omitted stimulation altogether, fl-Btx still caused significant increases in tissue ACh (Table I). A similar response was obtained when the dose of toxin was reduced 10-fold. We performed 4 tests to characterize further the action of fl-Btx: (1) heat treatment of the toxin (15 min at 100 °C, pH 8.0) prevented its inhibitory effects on ACh release, and no changes in tissue ACh were seen; (2) the rise in tissue ACh did not require the presence of eserine. Using the protocol described in Table I with 10 Hz stimulation in the absence of eserine, ACh levels in toxin-treated tissue were 4.80 i 0.60 pmol/mg, while controls were 2.46 ± 0.29 pmol/mg (also see Fig. 1); (3) when rats were pretreated with an intraperitoneal injection of botulinum toxin (100 rat LDs0 for 3 h), the 1ise in tissue ACh normally evoked by fl-Btx was reduced more than 80 ~. This observation is reminiscent of the mutually antagonistic actions of fl-Btx and botulinum toxina; and (4) the toxin-induced rise in tissue ACh levels was prevented when SrCI2 (2 mM) was substituted for CaC12 in the Krebs medium. Strontium cannot replace calcium in maintaining the phospholipase activity of fl-Btx 11.
488 FIGURE
!
10,
7: o
]g 2.0. o')
I
1" m
O.
1.0. 23
8
C 0
15
9
9
9
2 2
B
C
B
C
3P
B
45 TIME
4
C
B
60
(rain)
Fig. 1. Time course o f the increase in tissue A C h levels. A C h levels were m e a s u r e d in freshly excised h e m i d i a p h r a g m s or in tissue treated with or without fl-Btx (0.14 # g / m l ) for the indicated times. Eserine was not present in the K r e b s m e d i u m . T h e n u m b e r o f separate determinations is given, a n d the error bars indicate S.D. ** Difference between toxin treated a n d control is significant at P < 0.05. * Difference between toxin treated a n d control is significant at P <: 0.01.
The time course of the toxin effect was evaluated using fl-Btx (0.14 #g/ml) with no eserine present. Segments of R and L hemidiaphragms were dissected from the same rat. One hemidiaphragm was exposed to toxin while its pair served as a control. Fig. 1 shows that ACh levels in control tissue remain about the same as those in freshly excised R or L hemidiaphragms ('O') time. After 15 min exposure to fl-Btx a slight increase in tissue ACh occurs. This rise becomes highly significant after 30 min. When rat hemidiaphragms are exposed to fl-Btx, a reduction of evoked transmitter release and an increase in tissue ACh can be observed within 30 rain. It is not clear why an earlier group 4 did not record a similar effect on tissue ACh levels. We are currently testing to determine whether the size of the animal, length of exposure to fl-Btx or concentration of cholinesterase inhibitor used (Chang et al. 4 employed a high dose (1 mg/ml) of mipafox) might resolve this difference. We originally anticipated that nerve stimulation would be necessary to initiate the large changes in tissue ACh produced by fl-Btx. Data presented in the last two lines of Fig. 1 and those in Table I show that nerve activation is not necessary. Moreover, the time course for the rise in tissue ACh content and for the depression in ACh output are similar. These data suggest that fl-Btx does not have a sustained inhibitory effect on Ch uptake14; rather that fl-Btx may cause a transient depolarization of nerve terminals13,14 which may inhibit Ch transport. Subsequently, those changes which
489 lead to blockade of t r a n s m i t t e r release a n d an increase in A C h levels m u s t predominate. F r o m the data presented herein we c a n n o t state what the toxin i n d u c e d changes may be. However, it appears that the increased A C h c o n t e n t is n o t a simple consequence of reduced A c h release. First, the q u a n t i t y of A C h i n the tissue exceeded by as m u c h as 300 p m o l the A C h that would otherwise have been released (see Table I). Second, blocking evoked A C h release by other m e a n s (e.g. low Ca 2+ media or b o t u l i n u m toxin) does n o t cause tissue A C h levels to rise 7. A postulate which we are currently investigating is that the toxin-induced rise in tissue A C h levels occurs as a consequence of a n increased i n t r a t e r m i n a l a c c u m u l a t i o n of Ca 2 ~. Kelly a n d Brown 1° originally offered evidence that the toxin might alter Ca 2+ m e t a b o l i s m in the nerve ending. Regardless of its m e c h a n i s m of action, fl-Btx m a y provide a valuable tool for studying the otherwise closely controlled synthesis of A C h that has been observed in n e r v o u s tissue s,17. The authors wish to t h a n k Dr. Bruce H o w a r d for the fl-Btx used in this study. C.G. is the recipient o f a U S P H S G r a n t (NS-05753). Supported in part by U S P H S G r a n t MH-17691.
1 Abe, T., Alema, S. and Miledi, R., Isolation and characterization of presynaptically acting neurotoxins from the venom of Bungarus snakes, Europ. J. Biochem., 8 (1977) 1-12. 2 Chang, C. C. and Lee, C. Y., Isolation of neurotoxins from the venom of Bungarus multicinctus and their modes of neuromuscular blocking action, Arch. Int. Pharmacodyn. Ther., 144 (1963) 241-257. 3 Chang, C. C. and Lee, C. Y., Mutual antagonism between botulinum toxin and fl-bungarotoxin, Nature (Lond.), 243 (1973) 176-177. 4 Chang, C. C., Chen, T. F. and Lee, C. Y., Studies on the presynaptic effect of fl-bungarotoxin on neuromuscular transmission, J. PharmaeoL exp. Ther., 184 (1973) 339-345. 5 Dowdall, M. J., Fohlman, J. P. and Eaker, D., Inhibition of high affinity choline transport in peripheral nerve endings by presynaptic snake venom neurotoxins, Nature (Lond.), 269 (1977) 700-702. 6 Freeman, J. J., Choi, L. and Jenden, D. J., Plasma choline: its turnover and exchange with brain choline, J. Neurochem., 24 (1975) 729-734. 7 Gundersen, C. B. and Jenden, D. J., Botulinum toxin depresses resting acetylcholine output from the rat diaphragm, Trans. Amer. Soc. Neurochem., 10 (1979) 119. 8 Haubrich, D. R. and Chippendale, T. J., Regulation of aeetylcholine synthesis in nervous tissue, Life Sci., 20 (1977) 1465-1478. 9 Jenden, D. J., Roch, M. and Booth, R., Simultaneous measurement of endogenous and deuterium labelled tracer variants of choline and acetylcholine in subpicomole quantities by gas chromatography mass spectrometry, Analyt. Biochem., 55 (1973) 438-448. 10 Kelly, R. B. and Brown, F. R., Biochemical and physiological properties of a purified snake venom neurotoxin which acts presynaptically, J. NeurobioL, 5 (1974) 135-150. 11 Kelly, R. B., Oberg, S. G., Strong, P. N. and Wagner, G. M., fl-Bungarotoxin, a phospholipase that stimulates transmitter release, Cold Spr. Harb. Syrup. quant. Biol., 40 (1975) 117-125. 12 Lassignal, N. L. and Heuser, J. E., Evidence that fl-bungarotoxin arrests synaptic vesicle recycling by blocking coated vesicle formation, Neurosci. Abstr., 3 (1977) 373. 13 Ng, R. and Howard, B. D., Deenergization of nerve terminals by fl-bungarotoxin, Biochemistry, 17 (1978) 4978-4986. 14 Sen, I. and Cooper, J. R., Similarities of fl-bungarotoxin and phospholipase A2 and their mechanism of action, J. Neurochem., 30 (1978) 1369-1375. 15 Sen, I., Grantham, P. A. and Cooper, J. R., Mechanism of action of fl-bungarotoxin on synaptosomal preparations, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 2664-2668.
490 16 Strong, P. N., Heuser, J. E. and Kelly, R. B., Selective enzymatic hydrolysis of phospholipids by fl-bungarotoxin: biochemical and morphological studies. In Z. Hall, R. Kelly and C. F. Fox (Eds.), Cellular Neurobi9logy, A. R. Liss, New York, 1977, pp. 227-249. 17 Tucek, S., Acetyleholine Synthesis in Neurons, J. Wiley, New York, 1978. 18 Wernicke, J. F., Vanker, A. D. and Howard, B. D., The mechanism of action of/~-bungarotoxin, J. Neurochem., 25 (1975) 483-496.
Brain Research, 182 (1980) 486-490 © Elsevier/North-Holland Biomedical Press
fl-Bungarotoxin elevates diaphragm acetylcholine levels CAMERON B. GUNDERSEN, MICHAEL W. NEWTON and DONALD J. JENDEN Department of Pharmacology, UCLA School of Medicine, Los Angeles, Cahlf 90024 (U.S.A.)
(Accepted September 27th, 1979) Key words: acetylcholine - - f l - b u n g a r o t o x i n - diaphragm
fl-Bungarotoxin (fl-Btx) is a presynaptically acting polypeptide neurotoxin 1. In muscle the toxin causes an initial depression and the enhancement of spontaneous and evoked acetylcholine (ACh) release 1,2,4. These stages are followed by a neuromuscular block associated with reduced ACh outputL Among the hypotheses offered to explain the mechanism of action of fl-Btx are: depletion of nerve terminal energy levels 1°,1s, lysis of the nerve endings 16, inhibition of choline (Ch) transportS,14,15, interruption of vesicle recycling 12 or direct action on the release mechanism4,11. Since fl-Btx has phospholipase activity is, several groups have argued for a role of this enzyme activity in mediating the action of the toxin1, lt,l~,ls. While investigating neurochemical correlates of fl-Btx action, we observed a large increase in tissue levels of ACh after stimulation of rat diaphragm muscle treated with fl-Btx. Since previous studies led us to expect normal 4 or decreased 14 levels of ACh in the tissue, the following report describes in more detail the effects of fl-Btx on ACh metabolism in this nerve-muscle preparation. Our initial observations were made on diaphragms subjected to high frequency stimulation of the phrenic nerve. Fan-shaped segments of the left hemidiaphragm of 90-150 g male rats were dissected with the costal margin intact. The tissue was equilibrated for 30 min at 37 °C in 3.5 ml of eserinized (15 #M) Krebs bicarbonate medium with a steady stream of 95 ~ 02-5 ~ CO2. fl-Btx was added to the bath only during the equilibration period. The fl-Btx we used corresponds to the fla fraction 1 and was the gift of Dr. Bruce Howard. Release of ACh into the eserinized Krebs was estimated during subsequent 10 or 15 min collection periods. Stimulation of the preparation was via a suction electrode using square pulses of 4-6 V, 0.1 msec duration at the indicated frequency. After the final collection period the diaphragm tissue was cut free of the costal margin and both pieces of tissue were lightly blotted, placed in 1 M formic acid in acetone (3 : 17, v/v) and weighed. The total wet weight of the tissue (diaphragm plus rib) ranged between 120 and 210 mg. ACh was extracted from the tissue either by homogenization using a polytron or by standing for 20-24 h at 4 °C. A gas chromatographic mass spectrometric assay was used to estimate ACh in all
487 TABLE I Effects o f fl-Btx on ACh release and on tissue ACh levels
Diaphragm strips were treated with and without fl-Btx (1.4 pg/ml). Release of ACh into eserinized (15/~M) Krebs solution was measured during periods of rest (R) (15 min) or stimulation (S) (10 min) in the sequences and at the frequencies indicated. After the last collection period tissue levels of ACh were measured. The results given are for ACh content of the diaphragm portion only. Results are means 4- S.D. Condition (n)
Collection periods (pmol ACh/min) 1
2
3
4
Tissue 5
pmol A Ch
pmol ACh mg wet wt.
10 H z (R,S,S,S,R) Control (3) 1.6±0.5 7.3±0.5 6.74-0.3 6.84-0.3 1.54-0.4 140.54-16.0 2.28±0.30 fl-Btx(5) 1.3±0.4 2.44-0.5* 2.14-0.8" 1.84,0.5" 1.8±0.3 426.4±70.5* 7.094.1.09" 1 Hz (R,S,S,S,R)
Control (3) 1.44,0.3 1.8±0.2 1.84,0.3 1.84,0.3 0.94-0.4 146.14,15.1 fl-Btx (3) 1.3±0.4 1.74,0.6 1.84,0.6 2.44-0.3 1.5±0.4 566.94-48.8*
2.314.0.51 8.314,1.74'
Rest (R,R,R,R)
Control (15) 1.32_0.3 1.2±0.3 1.24,0.3 1.24,0.3 fl-Btx (5) 1.24.0.6 1.24.0.3 1.54,0.5 1.5±0.4
---
126.92-19.3 1.834,0.32 396.84,21.8" 5.384-0.89*
* Values for toxin treated differ from control at P < 0.001. samples6,% The significance of the data was evaluated using the unpaired Students ttest. As indicated in Table I, a 30 min exposure to fl-Btx (1.4/~g/ml) caused the ACh output during successive periods of 10 Hz stimulation to remain at background levels. Concomitantly, ACh levels in the tissues exceed by more than 3 times those of controls. Chang, Chen and LeO did not observe such a change in /3-Btx-treated diaphragms after low frequency stimulation. When we reduced the stimulation frequency to 1 Hz, or omitted stimulation altogether, fl-Btx still caused significant increases in tissue ACh (Table I). A similar response was obtained when the dose of toxin was reduced 10-fold. We performed 4 tests to characterize further the action of fl-Btx: (1) heat treatment of the toxin (15 min at 100 °C, pH 8.0) prevented its inhibitory effects on ACh release, and no changes in tissue ACh were seen; (2) the rise in tissue ACh did not require the presence of eserine. Using the protocol described in Table I with 10 Hz stimulation in the absence of eserine, ACh levels in toxin-treated tissue were 4.80 i 0.60 pmol/mg, while controls were 2.46 ± 0.29 pmol/mg (also see Fig. 1); (3) when rats were pretreated with an intraperitoneal injection of botulinum toxin (100 rat LDs0 for 3 h), the 1ise in tissue ACh normally evoked by fl-Btx was reduced more than 80 ~. This observation is reminiscent of the mutually antagonistic actions of fl-Btx and botulinum toxina; and (4) the toxin-induced rise in tissue ACh levels was prevented when SrCI2 (2 mM) was substituted for CaC12 in the Krebs medium. Strontium cannot replace calcium in maintaining the phospholipase activity of fl-Btx 11.
488 FIGURE
!
10,
7: o
]g 2.0. o')
I
1" m
O.
1.0. 23
8
C 0
15
9
9
9
2 2
B
C
B
C
3P
B
45 TIME
4
C
B
60
(rain)
Fig. 1. Time course o f the increase in tissue A C h levels. A C h levels were m e a s u r e d in freshly excised h e m i d i a p h r a g m s or in tissue treated with or without fl-Btx (0.14 # g / m l ) for the indicated times. Eserine was not present in the K r e b s m e d i u m . T h e n u m b e r o f separate determinations is given, a n d the error bars indicate S.D. ** Difference between toxin treated a n d control is significant at P < 0.05. * Difference between toxin treated a n d control is significant at P <: 0.01.
The time course of the toxin effect was evaluated using fl-Btx (0.14 #g/ml) with no eserine present. Segments of R and L hemidiaphragms were dissected from the same rat. One hemidiaphragm was exposed to toxin while its pair served as a control. Fig. 1 shows that ACh levels in control tissue remain about the same as those in freshly excised R or L hemidiaphragms ('O') time. After 15 min exposure to fl-Btx a slight increase in tissue ACh occurs. This rise becomes highly significant after 30 min. When rat hemidiaphragms are exposed to fl-Btx, a reduction of evoked transmitter release and an increase in tissue ACh can be observed within 30 rain. It is not clear why an earlier group 4 did not record a similar effect on tissue ACh levels. We are currently testing to determine whether the size of the animal, length of exposure to fl-Btx or concentration of cholinesterase inhibitor used (Chang et al. 4 employed a high dose (1 mg/ml) of mipafox) might resolve this difference. We originally anticipated that nerve stimulation would be necessary to initiate the large changes in tissue ACh produced by fl-Btx. Data presented in the last two lines of Fig. 1 and those in Table I show that nerve activation is not necessary. Moreover, the time course for the rise in tissue ACh content and for the depression in ACh output are similar. These data suggest that fl-Btx does not have a sustained inhibitory effect on Ch uptake14; rather that fl-Btx may cause a transient depolarization of nerve terminals13,14 which may inhibit Ch transport. Subsequently, those changes which
489 lead to blockade of t r a n s m i t t e r release a n d an increase in A C h levels m u s t predominate. F r o m the data presented herein we c a n n o t state what the toxin i n d u c e d changes may be. However, it appears that the increased A C h c o n t e n t is n o t a simple consequence of reduced A c h release. First, the q u a n t i t y of A C h i n the tissue exceeded by as m u c h as 300 p m o l the A C h that would otherwise have been released (see Table I). Second, blocking evoked A C h release by other m e a n s (e.g. low Ca 2+ media or b o t u l i n u m toxin) does n o t cause tissue A C h levels to rise 7. A postulate which we are currently investigating is that the toxin-induced rise in tissue A C h levels occurs as a consequence of a n increased i n t r a t e r m i n a l a c c u m u l a t i o n of Ca 2 ~. Kelly a n d Brown 1° originally offered evidence that the toxin might alter Ca 2+ m e t a b o l i s m in the nerve ending. Regardless of its m e c h a n i s m of action, fl-Btx m a y provide a valuable tool for studying the otherwise closely controlled synthesis of A C h that has been observed in n e r v o u s tissue s,17. The authors wish to t h a n k Dr. Bruce H o w a r d for the fl-Btx used in this study. C.G. is the recipient o f a U S P H S G r a n t (NS-05753). Supported in part by U S P H S G r a n t MH-17691.
1 Abe, T., Alema, S. and Miledi, R., Isolation and characterization of presynaptically acting neurotoxins from the venom of Bungarus snakes, Europ. J. Biochem., 8 (1977) 1-12. 2 Chang, C. C. and Lee, C. Y., Isolation of neurotoxins from the venom of Bungarus multicinctus and their modes of neuromuscular blocking action, Arch. Int. Pharmacodyn. Ther., 144 (1963) 241-257. 3 Chang, C. C. and Lee, C. Y., Mutual antagonism between botulinum toxin and fl-bungarotoxin, Nature (Lond.), 243 (1973) 176-177. 4 Chang, C. C., Chen, T. F. and Lee, C. Y., Studies on the presynaptic effect of fl-bungarotoxin on neuromuscular transmission, J. PharmaeoL exp. Ther., 184 (1973) 339-345. 5 Dowdall, M. J., Fohlman, J. P. and Eaker, D., Inhibition of high affinity choline transport in peripheral nerve endings by presynaptic snake venom neurotoxins, Nature (Lond.), 269 (1977) 700-702. 6 Freeman, J. J., Choi, L. and Jenden, D. J., Plasma choline: its turnover and exchange with brain choline, J. Neurochem., 24 (1975) 729-734. 7 Gundersen, C. B. and Jenden, D. J., Botulinum toxin depresses resting acetylcholine output from the rat diaphragm, Trans. Amer. Soc. Neurochem., 10 (1979) 119. 8 Haubrich, D. R. and Chippendale, T. J., Regulation of aeetylcholine synthesis in nervous tissue, Life Sci., 20 (1977) 1465-1478. 9 Jenden, D. J., Roch, M. and Booth, R., Simultaneous measurement of endogenous and deuterium labelled tracer variants of choline and acetylcholine in subpicomole quantities by gas chromatography mass spectrometry, Analyt. Biochem., 55 (1973) 438-448. 10 Kelly, R. B. and Brown, F. R., Biochemical and physiological properties of a purified snake venom neurotoxin which acts presynaptically, J. NeurobioL, 5 (1974) 135-150. 11 Kelly, R. B., Oberg, S. G., Strong, P. N. and Wagner, G. M., fl-Bungarotoxin, a phospholipase that stimulates transmitter release, Cold Spr. Harb. Syrup. quant. Biol., 40 (1975) 117-125. 12 Lassignal, N. L. and Heuser, J. E., Evidence that fl-bungarotoxin arrests synaptic vesicle recycling by blocking coated vesicle formation, Neurosci. Abstr., 3 (1977) 373. 13 Ng, R. and Howard, B. D., Deenergization of nerve terminals by fl-bungarotoxin, Biochemistry, 17 (1978) 4978-4986. 14 Sen, I. and Cooper, J. R., Similarities of fl-bungarotoxin and phospholipase A2 and their mechanism of action, J. Neurochem., 30 (1978) 1369-1375. 15 Sen, I., Grantham, P. A. and Cooper, J. R., Mechanism of action of fl-bungarotoxin on synaptosomal preparations, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 2664-2668.
490 16 Strong, P. N., Heuser, J. E. and Kelly, R. B., Selective enzymatic hydrolysis of phospholipids by fl-bungarotoxin: biochemical and morphological studies. In Z. Hall, R. Kelly and C. F. Fox (Eds.), Cellular Neurobi9logy, A. R. Liss, New York, 1977, pp. 227-249. 17 Tucek, S., Acetyleholine Synthesis in Neurons, J. Wiley, New York, 1978. 18 Wernicke, J. F., Vanker, A. D. and Howard, B. D., The mechanism of action of/~-bungarotoxin, J. Neurochem., 25 (1975) 483-496.