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Noradrenaline augments tetanic potentiation of transmitter release by a calcium dependent process
200
Brain Research, 214 (1981) 200-204 © Elsevier/North-Holland Biomedical Press
Noradrenaline augments tetanic potentiation of transmitter release by a calcium dependent process
HAGAI BERGMAN, SILVIO GLUSMAN, RONALD M. HARRIS-WARRICK, EDWARD A. KRAVITZ, ITZCHAK NUSSINOVITCH and RAMI RAHAMIMOFF* Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, N. Y. 11724 (U.S.A.) and ( H.B., I.N. and R.R.) Department of Physiology, Hebrew University - - Hadassah Medical School, P.O. Box 1172, Jerusalem 91000 (Israel)
(Accepted January 29th, 1981) Key words: neuromuscular synapse - - noradrenaline - - tetanic potentiation - - calcium - - trans-
mitter release
Noradrenaline (25 #M-50/~M) causes an increase in tetanic potentiation and in the augmentation phase of posttetanic potentiation of miniature and plate potential frequency. These effects were observed at both the frog and the rat neuromuscular junctions. The action of noradrenaline on quantal transmitter release depends on the presence of calcium ions in the extracellular medium.
It has been known since the early part of this century that adrenaline or sympathetic nerve stimulation can improve neuromuscular function and restore transmission across fatigued neuromuscular junctions2,13,14. Later these effects were analyzed on the cellular level and it was found that adrenaline and noradrenaline act on the presynaptic nerve terminal to increase the frequency of acetylcholine quanta released spontaneously (miniature end plate potentials - - m.e.p.p.s.) and of the number of quanta liberated by the nerve impulse. The latter effect, together with an increased input resistance of the postsynaptic muscle membrane, can lead to larger end plate potentials (e.p.p.s.) 5-s. In this article we examine the presynaptic action of noradrenaline during high frequency activation of the nerve terminal and report that an augmentation of the tetanic potentiation, seen at both frog and rat neuromuscular junctions, is dependent on the presence of calcium ions in the extracellular medium. Experiments were performed at room temperature on the frog (Rana pipiens) sartorius and the rat (Sabra strain) soleus nerve muscle preparations, bathed in frog and mammalian Ringer solutions with variable calcium concentrations. The standard frog Ringer solution contains 116 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, while the standard mammalian Ringer contains: 138 mM sodium chloride, 5 mM potassium chloride, 1 mM magnesium chloride, 2 mM calcium chloride, 15 mM sodium bicarbonate, 1 mM sodium phosphate monobasic, 11 mM D* To whom correspondence should be addressed.
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Fig. 1. The effect of noradrenaline on tetanic potentiation (TP) and posttetanic potentiation (PTP) of m.e.p.p, frequency at the frog sartorius neuromuscular junction. Ringer solution with 50/~M calcium chloride and 1.0 m M magnesium chloride present throughout the experiment. A: control. B: in the presence of noradrenaline (25/~M). Note the increase in TP and PTP compared to A. C: forty minutes after the noradrenaline was washed out. The increase in m.e.p.p, frequency during and after tetanic stimulation has returned to control values. All the data are from the same neuromuscular junction. The points show the raw data; each point is the m.e.p.p, frequency for a 5 sec period. The continuous lines represent the smoothed data by the moving bin method z~.
glucose. The mammalian Ringer was bubbled continuously with 95 ~o oxygen and 5 carbon dioxide. The pH was adjusted to 7.0 in both Ringer solutions. The synaptic activity was recorded by conventional 3.0 M potassium chloride filled micro-pipettes. To produce tetanic potentiation, the frog motor nerve was stimulated at a rate of 20-50 Hz for 20-40 sec. The mammalian preparation was stimulated at a higher frequency (100 Hz for 40-100 sec) since there are differences in the response to tetanic stimulation between the rat and frog nerve muscle preparations ]z. To prevent cumulative effects of successive tetani, they were separated by intervals of at least 20 min. In some experiments the control tetani were collected before the addition of noradrenaline, while in others, the effect of tetanic stimulation was first examined in noradrenaline containing media and afterwards in the control solutions. To avoid contraction during tetanic stimulation, the experiments were performed in media with [Ca z+] not exceeding 50/~M. Under such conditions e.p.p.s, are rare and the main
202 effect o f tetanic nerve stimulation is an increase in the frequency o f the m.e.p.p.s. 1,8, 4,11. T h e time course o f tetanic a n d posttetanic p o t e n t i a t i o n o f m.e.p.p.s, however, closely resembles t h a t o f the e.p.p.s.3,1°. Since the time o f occurrence o f the m.e.p.p.s. is r a n d o m in nature, a s m o o t h i n g p r o c e d u r e was necessary. In the present experiments the ' m o v i n g bin' technique was used 15. In the r a w d a t a the m.e.p.p, frequency was m e a s u r e d each second. Then an average was p e r f o r m e d for a bin p e r i o d o f 10 seconds a n d the p o i n t was d r a w n in the center o f the bin. Subsequently the w i n d o w for averaging was m o v e d by one second, until the end o f the record. The s m o o t h i n g was d o n e with the aid o f a digital c o m p u t e r . The m a i n experimental observations are illustrated in Figs. 1 a n d 2: tetanic p o t e n t i a t i o n is m o r e effective in the presence o f n o r a d r e n a l i n e (25 y M ) . The degree o f the effect varied s o m e w h a t f r o m p r e p a r a t i o n to p r e p a r a t i o n . I n 20 experiments with the frog p r e p a r a t i o n the m e a n increase in t r a n s m i t t e r release d u r i n g the tetanus was 82 ~ larger in the presence o f n o r a d r e n a l i n e (range o f increase is 20 ~o-296 ~ ) . Similar results were o b t a i n e d in 18 experiments with the rat p r e p a r a t i o n . The facilitatory effect o f n o r a d r e n a l i n e was n o t restricted to the p e r i o d o f stimulation, but occurred also d u r i n g the posttetanic p o t e n t i a t i o n p e r i o d (Figs. 1 and 2).
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T I M E (s) Fig. 2. The effect of noradrenaline on tetanic and posttetanic increase in transmitter release at the rat neuromuscular synapse. Control: tetanic (A) and posttetanic (B) potentiation in a Ringer solution containing 50/~M calcium chloride. Noradrenaline: tetanic (C) and posttetanic (D) potentiation after exposure to noradrenaline (50 pM) added to control Ringer solution. Note that noradrenaline induced increase in transmitter release during and after tetanic stimulation (100 Hz). The m.e.p.p.s, in the absence and presence of noradrenaline were recorded from the same synapse. The motor nerve was stimulated for 100 sec. The change in m.e.p.p, frequency with time is illustrated with the moving bin method. Each point represents the mean frequency of me.e.p.p.s, for 10 sec. The 'move" from point to point is 1 sec. Records A and B for the control and C and D for noradrenaline are continuous (the 'gap' at the beginning and end of each record is due to the moving bin method, which places each average in the center of the respective bin). The basic frequency before the tetani in control and noradrenaline conditions were 0.33 and 0.45 m.e.p.p.s/sec respectively.
203
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Fig. 3. The lack of effect of noradrenaline on tetanic (A, B) and posttetanic (C, D) increase in m.e.p.p.s, frequency in the virtual absence of extracellular calcium. Rat neuromuscular synapse. Control: Ringer solution containing 1 mM EGTA (ethyleneglycol-bis-(B-amino-ethyl ether) N'Ntetra-acetic acid) with no added calcium ions. Noradrenaline (B,D): the same Ringer solution with noradrenaline (50 yM). The figures illustrated are an averaged response of 3 tetani. Tetanic and posttetanic potentiation are not augmented in the presence of noradrenaline when calcium ions are omitted from the extracellular medium. The motor nerve was stimulated for 100 sec. The change in m.e.p.p, frequency with time is illustrated with the moving bin method. Bin of l0 sec with a displacement of 1 sec.
It has been shown recently that this posttetanic potentiation can be subdivided into 4 components 10. Two o f them can easily be demonstrated with m.e.p.p.s.: the augmentation phase (time constant o f about 10 sec) and potentiation (time constant o f hundreds o f seconds) a. At least two separate processes contribute to tetanic and posttetanic potentiationg, 16. The first is the entry o f calcium ions f r o m the extracellular medium (in the augmentation) and the second, is a sodium dependent potentiation o f transmitter release that does not require extracellular calcium (potentiation). To determine which o f these processes is augmented by noradrenaline, we repeated the experiments in a bathing solution with no added calcium ions and with the addition o f 1 m M o f the calcium chelating agent E G T A . (To avoid the deleterious effects found in media with low divalent ion concentrations, at least 1.0 m M magnesium chloride was present in the extracellular Ringer solution.) Nine experiments were done in Ringer solution with E G T A . Fig. 3 shows the averaged response to 3 tetani under such conditions. Tetanic stimulation still produces a potentiation in transmitter release but
204 the degree o f this p o t e n t i a t i o n is n o t increased by n o r a d r e n a l i n e . Therefore, the p o t e n t iating effect o f n o r a d r e n a l i n e on t r a n s m i t t e r release requires an i n w a r d - d i r e c t e d electrochemical g r a d i e n t for calcium across the p r e s y n a p t i c m e m b r a n e . Previous studies with rat d i a p h r a g m n e u r o m u s c u l a r p r e p a r a t i o n s , also show that an i n w a r d - d i r e c t e d calcium g r a d i e n t is necessary to induce an increase in m.e.p.p, frequency by n o r a d r e n a l i n e in u n s t i m u l a t e d p r e p a r a t i o n s 8. The m a g n i t u d e o f t h a t increase in m.e.p.p, frequency at low calcium c o n c e n t r a t i o n s is, however, c o n s i d e r a b l y smaller (in 0.01 m M the increase was 15 ~ , in 0.1 m M it was 30 ~ , a n d in 0.5 m M the increase was 56 ~o)8 t h a n the effects o f n o r a d r e n a l i n e we observe on tetanic a n d posttetanic potentiation. T h e significant p o t e n t i a t i n g action o f n o r a d r e n a l i n e on t r a n s m i t t e r release b y a tetanic nerve s t i m u l a t i o n is a likely p a r t o f the n o r m a l physiological role o f catecholamines in muscle activation, since c a t e c h o l a m i n e s are k n o w n to be liberated in stressful situations when m a x i m a l muscle activation m a y be necessary. W e t h a n k the D i r e c t o r a n d staff o f C o l d Spring H a r b o r L a b o r a t o r y for their hospitality a n d encouragement. The w o r k in C o l d Spring H a r b o r L a b o r a t o r y was s u p p o r t e d b y the R o b e r t s o n F o u n d a t i o n , a n d in J e r u s a l e m by the M u s c u l a r D y s t r o phy A s s o c i a t i o n a n d the U . S . - I s r a e l Binational Science F o u n d a t i o n . E q u i p m e n t in Jerusalem was p u r c h a s e d with the aid o f the Bay F o u n d a t i o n . 1 Del Castillo, J. and Katz, B., Statistical factors involved in neuromuscular facilitation and depression, J. PhysioL (Lond.), 124 (1954) 574-585. 2 Dessy, S. and Grandis, V., Contribution a l'etude de la fatigue. Action de l'adrenaline sur la fonction du muscle, Arch. ital. Biol., 41 (1904) 225-244. 3 Erulkar, S. D. and Rahamimoff, R., The role of calcium ions in tetanic and post-tetanic increase of miniature end plate potential frequency, J. Physiol. (Lond.), 278 (1978) 501-511. 4 Hurlbut, W. P., Longenecker, H. B., Jr. and Mauro, A., Effects of calcium and magnesium on the frequency of miniature end plate potentials during prolonged tetanization, J. Physiol. (Lond.), 219 (1971) 17-38. 5 Jenkinson, D. H., Stamenovic, B. A. and Whittaker, B. D. L., The effect of noradrenaline on the end-plate potential in twitch fibres of the frog, J. Physiol. (Lond.), 195 (1968) 743-754. 6 Krnjevic, K. and Miledi, R., Some effects producedby adrenaline upon neuromuscular propagation in rats, J. Physiol. (Lond.), 141 (1958) 291-304. 7 Kuba, K., Effects of catecholamines on the neuromuscular junction in the rat diaphragm, J. Physiol. (Lond.), 211 (1970) 551-570. 8 Kuba, K. and Tomita, T., Noradrenaline action on nerve terminal in the rat diaphragm, J. Physiol. (Lond.), 217 (1971) 19-31. 9 Lev-Tov, A. and Rahamimoff, R., A study of tetanic and post-tetanic potentiation of miniature end-plate potentials at the frog neuromuscular junction, J. Physiol. (Lond.), in press. 10 Magleby, K. L. and Zengel, J. E., Augmentation: a process that acts to increase transmitter release at the frog neuromuscular junction, J. Physiol. (Lond.), 257 (1976) 449~,70. 11 Miledi, R. and Thies, R., Tetanic and post-tetanic rise in frequency of miniature end plate potentials in low calcium solutions, J. Physiol. (Lond.), 212 (1971) 245-257. 12 Nussinovitch, I., Wendon, L. M. B. and Rahamimoff, R., Tetanic and post-tetanic transmitter release in a mammalian synapse, Israel J. med. Sci., 14 (1978) 1090. 13 •rbe•i• L. A.• Die sympathetische •nnervati•n der Ske•ettmuske•n• Bu••. •nst. Sci.Leshaft.• 6 ( • 923) 194-197.
14 Panella, A., Action du principe actif surrenal sur la fatigue musculaire, Arch. ital. Biol., 43 (1907) 430-463. 15 Rahamimoff, R. and Yaari, Y., Delayed release of transmitter at the frog neuromuscular junction, J. PhysioL (Lond.), 228 (1973) 241-257. 16 Rahamimoff, R., Erulkar, S. D., Lev-Tov, A. and Meiri, H., Intracellular and extracellular calcium ions in transmitter release at neuromuscular synapse, Ann. N. Y. Acad. Sci., 307 (1978) 583-598.
Brain Research, 214 (1981) 200-204 © Elsevier/North-Holland Biomedical Press
Noradrenaline augments tetanic potentiation of transmitter release by a calcium dependent process
HAGAI BERGMAN, SILVIO GLUSMAN, RONALD M. HARRIS-WARRICK, EDWARD A. KRAVITZ, ITZCHAK NUSSINOVITCH and RAMI RAHAMIMOFF* Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, N. Y. 11724 (U.S.A.) and ( H.B., I.N. and R.R.) Department of Physiology, Hebrew University - - Hadassah Medical School, P.O. Box 1172, Jerusalem 91000 (Israel)
(Accepted January 29th, 1981) Key words: neuromuscular synapse - - noradrenaline - - tetanic potentiation - - calcium - - trans-
mitter release
Noradrenaline (25 #M-50/~M) causes an increase in tetanic potentiation and in the augmentation phase of posttetanic potentiation of miniature and plate potential frequency. These effects were observed at both the frog and the rat neuromuscular junctions. The action of noradrenaline on quantal transmitter release depends on the presence of calcium ions in the extracellular medium.
It has been known since the early part of this century that adrenaline or sympathetic nerve stimulation can improve neuromuscular function and restore transmission across fatigued neuromuscular junctions2,13,14. Later these effects were analyzed on the cellular level and it was found that adrenaline and noradrenaline act on the presynaptic nerve terminal to increase the frequency of acetylcholine quanta released spontaneously (miniature end plate potentials - - m.e.p.p.s.) and of the number of quanta liberated by the nerve impulse. The latter effect, together with an increased input resistance of the postsynaptic muscle membrane, can lead to larger end plate potentials (e.p.p.s.) 5-s. In this article we examine the presynaptic action of noradrenaline during high frequency activation of the nerve terminal and report that an augmentation of the tetanic potentiation, seen at both frog and rat neuromuscular junctions, is dependent on the presence of calcium ions in the extracellular medium. Experiments were performed at room temperature on the frog (Rana pipiens) sartorius and the rat (Sabra strain) soleus nerve muscle preparations, bathed in frog and mammalian Ringer solutions with variable calcium concentrations. The standard frog Ringer solution contains 116 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, while the standard mammalian Ringer contains: 138 mM sodium chloride, 5 mM potassium chloride, 1 mM magnesium chloride, 2 mM calcium chloride, 15 mM sodium bicarbonate, 1 mM sodium phosphate monobasic, 11 mM D* To whom correspondence should be addressed.
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120 160 200 21,0 280 320 TOME ( s )
Fig. 1. The effect of noradrenaline on tetanic potentiation (TP) and posttetanic potentiation (PTP) of m.e.p.p, frequency at the frog sartorius neuromuscular junction. Ringer solution with 50/~M calcium chloride and 1.0 m M magnesium chloride present throughout the experiment. A: control. B: in the presence of noradrenaline (25/~M). Note the increase in TP and PTP compared to A. C: forty minutes after the noradrenaline was washed out. The increase in m.e.p.p, frequency during and after tetanic stimulation has returned to control values. All the data are from the same neuromuscular junction. The points show the raw data; each point is the m.e.p.p, frequency for a 5 sec period. The continuous lines represent the smoothed data by the moving bin method z~.
glucose. The mammalian Ringer was bubbled continuously with 95 ~o oxygen and 5 carbon dioxide. The pH was adjusted to 7.0 in both Ringer solutions. The synaptic activity was recorded by conventional 3.0 M potassium chloride filled micro-pipettes. To produce tetanic potentiation, the frog motor nerve was stimulated at a rate of 20-50 Hz for 20-40 sec. The mammalian preparation was stimulated at a higher frequency (100 Hz for 40-100 sec) since there are differences in the response to tetanic stimulation between the rat and frog nerve muscle preparations ]z. To prevent cumulative effects of successive tetani, they were separated by intervals of at least 20 min. In some experiments the control tetani were collected before the addition of noradrenaline, while in others, the effect of tetanic stimulation was first examined in noradrenaline containing media and afterwards in the control solutions. To avoid contraction during tetanic stimulation, the experiments were performed in media with [Ca z+] not exceeding 50/~M. Under such conditions e.p.p.s, are rare and the main
202 effect o f tetanic nerve stimulation is an increase in the frequency o f the m.e.p.p.s. 1,8, 4,11. T h e time course o f tetanic a n d posttetanic p o t e n t i a t i o n o f m.e.p.p.s, however, closely resembles t h a t o f the e.p.p.s.3,1°. Since the time o f occurrence o f the m.e.p.p.s. is r a n d o m in nature, a s m o o t h i n g p r o c e d u r e was necessary. In the present experiments the ' m o v i n g bin' technique was used 15. In the r a w d a t a the m.e.p.p, frequency was m e a s u r e d each second. Then an average was p e r f o r m e d for a bin p e r i o d o f 10 seconds a n d the p o i n t was d r a w n in the center o f the bin. Subsequently the w i n d o w for averaging was m o v e d by one second, until the end o f the record. The s m o o t h i n g was d o n e with the aid o f a digital c o m p u t e r . The m a i n experimental observations are illustrated in Figs. 1 a n d 2: tetanic p o t e n t i a t i o n is m o r e effective in the presence o f n o r a d r e n a l i n e (25 y M ) . The degree o f the effect varied s o m e w h a t f r o m p r e p a r a t i o n to p r e p a r a t i o n . I n 20 experiments with the frog p r e p a r a t i o n the m e a n increase in t r a n s m i t t e r release d u r i n g the tetanus was 82 ~ larger in the presence o f n o r a d r e n a l i n e (range o f increase is 20 ~o-296 ~ ) . Similar results were o b t a i n e d in 18 experiments with the rat p r e p a r a t i o n . The facilitatory effect o f n o r a d r e n a l i n e was n o t restricted to the p e r i o d o f stimulation, but occurred also d u r i n g the posttetanic p o t e n t i a t i o n p e r i o d (Figs. 1 and 2).
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240
T I M E (s) Fig. 2. The effect of noradrenaline on tetanic and posttetanic increase in transmitter release at the rat neuromuscular synapse. Control: tetanic (A) and posttetanic (B) potentiation in a Ringer solution containing 50/~M calcium chloride. Noradrenaline: tetanic (C) and posttetanic (D) potentiation after exposure to noradrenaline (50 pM) added to control Ringer solution. Note that noradrenaline induced increase in transmitter release during and after tetanic stimulation (100 Hz). The m.e.p.p.s, in the absence and presence of noradrenaline were recorded from the same synapse. The motor nerve was stimulated for 100 sec. The change in m.e.p.p, frequency with time is illustrated with the moving bin method. Each point represents the mean frequency of me.e.p.p.s, for 10 sec. The 'move" from point to point is 1 sec. Records A and B for the control and C and D for noradrenaline are continuous (the 'gap' at the beginning and end of each record is due to the moving bin method, which places each average in the center of the respective bin). The basic frequency before the tetani in control and noradrenaline conditions were 0.33 and 0.45 m.e.p.p.s/sec respectively.
203
A 3.20 100 a z BIN :10s 2.t,0 8BIN = 1 s 1.60 COHIROL ~
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0.80
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150
180
210
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0.80
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30
60
90
120
Fig. 3. The lack of effect of noradrenaline on tetanic (A, B) and posttetanic (C, D) increase in m.e.p.p.s, frequency in the virtual absence of extracellular calcium. Rat neuromuscular synapse. Control: Ringer solution containing 1 mM EGTA (ethyleneglycol-bis-(B-amino-ethyl ether) N'Ntetra-acetic acid) with no added calcium ions. Noradrenaline (B,D): the same Ringer solution with noradrenaline (50 yM). The figures illustrated are an averaged response of 3 tetani. Tetanic and posttetanic potentiation are not augmented in the presence of noradrenaline when calcium ions are omitted from the extracellular medium. The motor nerve was stimulated for 100 sec. The change in m.e.p.p, frequency with time is illustrated with the moving bin method. Bin of l0 sec with a displacement of 1 sec.
It has been shown recently that this posttetanic potentiation can be subdivided into 4 components 10. Two o f them can easily be demonstrated with m.e.p.p.s.: the augmentation phase (time constant o f about 10 sec) and potentiation (time constant o f hundreds o f seconds) a. At least two separate processes contribute to tetanic and posttetanic potentiationg, 16. The first is the entry o f calcium ions f r o m the extracellular medium (in the augmentation) and the second, is a sodium dependent potentiation o f transmitter release that does not require extracellular calcium (potentiation). To determine which o f these processes is augmented by noradrenaline, we repeated the experiments in a bathing solution with no added calcium ions and with the addition o f 1 m M o f the calcium chelating agent E G T A . (To avoid the deleterious effects found in media with low divalent ion concentrations, at least 1.0 m M magnesium chloride was present in the extracellular Ringer solution.) Nine experiments were done in Ringer solution with E G T A . Fig. 3 shows the averaged response to 3 tetani under such conditions. Tetanic stimulation still produces a potentiation in transmitter release but
204 the degree o f this p o t e n t i a t i o n is n o t increased by n o r a d r e n a l i n e . Therefore, the p o t e n t iating effect o f n o r a d r e n a l i n e on t r a n s m i t t e r release requires an i n w a r d - d i r e c t e d electrochemical g r a d i e n t for calcium across the p r e s y n a p t i c m e m b r a n e . Previous studies with rat d i a p h r a g m n e u r o m u s c u l a r p r e p a r a t i o n s , also show that an i n w a r d - d i r e c t e d calcium g r a d i e n t is necessary to induce an increase in m.e.p.p, frequency by n o r a d r e n a l i n e in u n s t i m u l a t e d p r e p a r a t i o n s 8. The m a g n i t u d e o f t h a t increase in m.e.p.p, frequency at low calcium c o n c e n t r a t i o n s is, however, c o n s i d e r a b l y smaller (in 0.01 m M the increase was 15 ~ , in 0.1 m M it was 30 ~ , a n d in 0.5 m M the increase was 56 ~o)8 t h a n the effects o f n o r a d r e n a l i n e we observe on tetanic a n d posttetanic potentiation. T h e significant p o t e n t i a t i n g action o f n o r a d r e n a l i n e on t r a n s m i t t e r release b y a tetanic nerve s t i m u l a t i o n is a likely p a r t o f the n o r m a l physiological role o f catecholamines in muscle activation, since c a t e c h o l a m i n e s are k n o w n to be liberated in stressful situations when m a x i m a l muscle activation m a y be necessary. W e t h a n k the D i r e c t o r a n d staff o f C o l d Spring H a r b o r L a b o r a t o r y for their hospitality a n d encouragement. The w o r k in C o l d Spring H a r b o r L a b o r a t o r y was s u p p o r t e d b y the R o b e r t s o n F o u n d a t i o n , a n d in J e r u s a l e m by the M u s c u l a r D y s t r o phy A s s o c i a t i o n a n d the U . S . - I s r a e l Binational Science F o u n d a t i o n . E q u i p m e n t in Jerusalem was p u r c h a s e d with the aid o f the Bay F o u n d a t i o n . 1 Del Castillo, J. and Katz, B., Statistical factors involved in neuromuscular facilitation and depression, J. PhysioL (Lond.), 124 (1954) 574-585. 2 Dessy, S. and Grandis, V., Contribution a l'etude de la fatigue. Action de l'adrenaline sur la fonction du muscle, Arch. ital. Biol., 41 (1904) 225-244. 3 Erulkar, S. D. and Rahamimoff, R., The role of calcium ions in tetanic and post-tetanic increase of miniature end plate potential frequency, J. Physiol. (Lond.), 278 (1978) 501-511. 4 Hurlbut, W. P., Longenecker, H. B., Jr. and Mauro, A., Effects of calcium and magnesium on the frequency of miniature end plate potentials during prolonged tetanization, J. Physiol. (Lond.), 219 (1971) 17-38. 5 Jenkinson, D. H., Stamenovic, B. A. and Whittaker, B. D. L., The effect of noradrenaline on the end-plate potential in twitch fibres of the frog, J. Physiol. (Lond.), 195 (1968) 743-754. 6 Krnjevic, K. and Miledi, R., Some effects producedby adrenaline upon neuromuscular propagation in rats, J. Physiol. (Lond.), 141 (1958) 291-304. 7 Kuba, K., Effects of catecholamines on the neuromuscular junction in the rat diaphragm, J. Physiol. (Lond.), 211 (1970) 551-570. 8 Kuba, K. and Tomita, T., Noradrenaline action on nerve terminal in the rat diaphragm, J. Physiol. (Lond.), 217 (1971) 19-31. 9 Lev-Tov, A. and Rahamimoff, R., A study of tetanic and post-tetanic potentiation of miniature end-plate potentials at the frog neuromuscular junction, J. Physiol. (Lond.), in press. 10 Magleby, K. L. and Zengel, J. E., Augmentation: a process that acts to increase transmitter release at the frog neuromuscular junction, J. Physiol. (Lond.), 257 (1976) 449~,70. 11 Miledi, R. and Thies, R., Tetanic and post-tetanic rise in frequency of miniature end plate potentials in low calcium solutions, J. Physiol. (Lond.), 212 (1971) 245-257. 12 Nussinovitch, I., Wendon, L. M. B. and Rahamimoff, R., Tetanic and post-tetanic transmitter release in a mammalian synapse, Israel J. med. Sci., 14 (1978) 1090. 13 •rbe•i• L. A.• Die sympathetische •nnervati•n der Ske•ettmuske•n• Bu••. •nst. Sci.Leshaft.• 6 ( • 923) 194-197.
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