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The actions of three diaminopyridines on the chick biventer cervicis muscle
European Journal o f Pharmacology, 44 (1977) 303--309 © Elsevier/North-Holland Biomedical Press
303
THE ACTIONS OF THREE DIAMINOPYRIDINES ON THE CHICK BIVENTER CERVICIS MUSCLE ALAN L. HARVEY and IAN G. MARSHALL
The Department o f Physiology and Pharmacology, University o f Strathclyde, Glasgow, G1 1XW, Scotland Received 12 February 1977, revised MS received 27 April 1977, accepted 28 April 1977
A.L. HARVEY and I.G. MARSHALL, The action o f three diaminopyridines on the chick biventer cervicis muscle,European J. Pharmacol. 44 (1977) 303--309. The effects of 2,3-, 2,6- and 3,4-diaminopyridine were investigated on the isolated chick biventer cervicis muscle preparation. All three compounds reversed tubocurarine blockade and augmented twitch height in indirectly stimulated preparations. Less twitch augmentation was observed in directly stimulated preparations. 3,4-Diaminopyridine was the most effective of the compounds in facilitating neuromuscular transmission, but exhibited less convulsant activity than 4-aminopyridine. 3,4- and 2,3-diaminopyridine in high concentrations caused contractures that were inhibited by erabutoxin b or by ~-bungarotoxin. It is suggested that the diaminopyridines increase both evoked and spontaneous acetylcholine release. Diaminopyridines
Chick biventer cervicis muscle
1. Introduction 2-, 3- and 4-aminopyridine were initially shown to augment responses in skeletal muscle preparations by Fastier and McDowall (1958). Subsequently the anti-curare action of these c o m p o u n d s has been studied by Lemeignan and Lechat (1967) and by Bowman et al. {1976,1977). In a recent study of the effect of aminopyridines on adrenergic transmission, Johns et al. (1976) tested 3,4-diaminopyridines 3,4Diaminopyridine was as active as 4-aminopyridine in augmenting adrenergic transmission in the rabbit vas deferens preparation. As there is no published work on the effects of
Neuromuscular transmission
Acetylcholine release
the diaminopyridines on neuromuscular transmission, we studied 2,3- (left), 2,6- (right) and 3,4-diaminopyridine (middle) (fig. 1) under the same experimental conditions as described in our previous paper on the aminopyridines (Bowman et al., 1977). An a t t e m p t was also made to test the hypothesis that a c o m p o n e n t of the aminopyridines' action may involve calcium ions (Bowman et al., 1977). The diaminopyridines were compared with compounds such as caffeine and nystatin whose action is known to involve calcium ions. As 4-aminopyridine also exhibits convulsant activity (Fastier and McDowall, 1958), the convulsant activity of 3,4-diaminopyridine was tested.
NH 2
2. Materials and m e t h o d s ~-N ~
~NH 2
.
NH 2
Fig. 1. Formulae of the diaminopyridines tested. 2,3AP, 2,3-diaminopyridine (left); 2,6AP, 2,6-diaminopyridine (right); 3,4AP, 3,4-diaminopyridine (middle).
2.1. Chick biventer cervicis muscle
The biventer cervicis muscles from chicks aged 2--10 days were m o u n t e d in Krebs-Henseleit (1932) solution maintained at 32°C
304 and gassed with oxygen containing 5% carbon dioxide (Ginsborg and Warriner, 1960).
2.1.1. Indirectly stimulated preparations The nerve within the tendon was stimulated by ring electrodes at a frequency of 0.1 Hz with rectangular pulses of 0.2 msec duration, and of strength greater than that required to elicit a maximal twitch. For the construction of dose--response curves, each preparation was used for only one addition of the diaminopyridine. In some experiments the nerve stimulation was periodically stopped and acetylcholine (5 X 10 -4 M) or carbachol (2 X 10 -s M) was added to the tissue bath. Acetylcholine and carbachol were allowed to remain in contact with the tissues for 30 sec and 90 sec respectively. 2.1.2. Directly stimulated preparations Preparations were set up and stimulated as described for indirect stimulation. Neuromuscular transmission was then inhibited by the irreversible snake venom toxin erabutoxin b {4pg/ml) (Tamiya and Arai, 1966). After abolition of neuromuscular transmission the ring electrodes were moved into contact with the muscle which was then stimulated at a frequency of 0.1 Hz with rectangular pulses of 1 msec duration and of strength greater than that required to elicit a maximal twitch. For the construction of dose--response curves, each preparation was used for only one addition of the diaminopyridine. 2.2. Convulsant activity Compounds were injected intraperitoneally into male mice (30--40 g) and the time from injection to death by convulsion was measured. Each dose level was tested in 5--6 animals.
2.3. Drugs The drugs used were 4-aminopyridine, 2,3-, 2,6- and 3,4-diaminopyridine, nystatin and
A.L. HARVEY, I.G. MARSHALL {+)-tubocurarine chloride (Sigma), acetylcholine chloride, caffeine and carbachol chloride (British Drug Houses), ~-bungarotoxin (Boehringer Ingelheim), A23187 (Lilly) and erabutoxin b (supplied by Professor N. Tamiya, Department of Chemistry, Tohoku University, Japan). Drugs were dissolved in 0.9% saline, except nystatin which was dissolved in 1% ethanol and A23187 which was dissolved in ethanol.
2.4. Statistics In the dose--response curves each concentration was tested on six individual preparations and the points on the curves represent the mean + standard error. Differences between means were tested by Student's non paired t-test, values of p < 0.05 being regarded as significant.
3. Results
3.1. Reversal o f (+)-tubocurarine neuromuscular blockade 3,4-Diaminopyridine was the most effective of the compounds tested in reversing the blockade of indirectly stimulated preparations induced by (+)-tubocurarine. Within 15 min 3,4-diaminopyridine (10 -s M) completely restored twitches depressed to 30% of control by (+)-tubocurarine (5 X 10 -6 M). 10 -4 M 3,4Diaminopyridine could restore twitches abolished by 2 X 10 -s M (+)-tubocurarine (fig. 2). The concentration of (+)-tubocurarine had to be increased 10-fold to re,establish neuromuscular blockade in the continued presence of 3,4-diaminopyridine. 2,3- and 2,6
D I A M I N O P Y R I D I N E S A N D CHICK M U S C L E
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Fig. 2. Reversal o f (+)-tuboeurarine blockade by 3,4d i a m i n o p y r i d i n e . The preparation was stimulated indirectly, 2 × 10 -s M (+)otuboeurarine was added at the left arrow, and 10 -4 M 3 , 4 - d i a m i n o p y r i d i n e was
added at the right arrow. 3.2. Augmentation o f twitches in indirectly stimulated preparations All three c o m p o u n d s augmented maximal twitch height in indirectly stimulated preparations. Again 3,4-diaminopyridine was the most p o t e n t of the three c o m p o u n d s tested, producing 3 2 5 + 48% increases in twitch tension at 10 -3 M, whereas 2,6
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Fig. 3. A u g m e n t a t i o n of directly and indirectly elicited twitches by 2,3-diaminopyridine (left), 2,6-diaminopyridine (middle) and 3,4-diaminopyridine (right panel). C o n c e n t r a t i o n is m o l a r (abscissa) and twitch a u g m e n t a t i o n is expressed as percentage increase f r o m control twitch height (ordinate). The results with direct stimulation (m) and with indirect stimulation (e) are shown as m e a n + S.E.M. o f 6 determinations. Error bars are indicated unless smaller than the symbols.
elicited by acetylcholine (5 × 10 -4 M) or by carbachol (10 -s M). Fig. 4 shows the result of a typical experiment with 3,4-diaminopyridine. 3.4. Augmentation o f twitches in directly stim ula ted preparations All three c o m p o u n d s produced significantly (p < 0.05) less augmentation of directly elicited twitches than of indirectly elicited 5 min I
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Fig. 4. The effect o f 3,4-diaminopyridine on responses to acetylcholine and carbachol. T o p panel: control responses to indirect stimulation and to 5 x 1 0 - 4 M acetylcholine (left arrows) and 2 × 10 -s M carbachol (right arrows). B o t t o m panel: 1 0 - 4 M 3,4-diaminopyridine was added at the left arrow. Responses to 5 × 10 -4 M a c e t y l c h o l i n e (middle arrows) and 2 × 10 -s M carbachol (right' arrows) were t h e n o b t a i n e d in the presence o f 3,4-diaminopyridine.
306
twitches (fig. 3). The largest augmentation (50 + 8%) was produced by 3,4-diaminopyridine (10 -2 M). In the directly stimulated preparations no contracture was observed at 10-3M 3,4-diaminopyridine, although at 1 0 - 2 M 3,4-diaminopyridine a very slowly developing contracture of immediate onset was occasionally observed. The contracture generally reached the equivalent of approximately 25% of maximal twitch height to direct stimulation in 10 min.
3.5. Effect o f caffeine on indirectly and directly stimulated preparations Caffeine (10 -4 to 10 -3 M) was tested, 5 × 1 0 - 4 M and 10-3M proving effective in augmenting both indirectly and directly elicited twitches to similar degrees. 1 0 - 3 M caffeine produced a small contracture (ca. 10% maximal twitch height) in both indirectly and directly stimulated preparations. Caffeine (5 × 10 -4 M) produced no reversal of neuromuscular block by (+)-tubocurarine (1--2 ×10 -s M). In the continued presence of caffeine (5 × 10 -4 M), 3,4-diaminopyridine (10-4--10 .3 M) remained an effective reversing agent.
3.6. Effect o f nystatin and A 2 3 1 8 7 on indirectly and directly stimulated preparations Neither nystatin (10-6--10 -s M) nor A23187 (5--10 pg/ml) produced augmentation of either indirectly or directly elicited twitches.
A.L. H A R V E Y , I.G. M A R S H A L L
3.8. Effect o f 3,4-diaminopyridine on [3-bungarotoxin-induced neuromuscular blockade 3,4-Diaminopyridine produced a temporary reversal of the neuromuscular block induced by ~-bungarotoxin (2 pg/ml). The degree and duration of the reversal was dependent upon the time of addition and the concentration of the diaminopyridine. When added at around 95% twitch blockade, 3,4-diaminopyridine (10-3 M) restored twitch height to around 20% greater than control within 3 min. When the same concentration of 3,4-diaminopyridine was added 4 min and 30 min after complete twitch blockade, the twitches returned to around 75% and less than 5% of control respectively. In these three situations the times for the /3-bungarotoxininduced block to redevelop fully in the continued presence of 3,4-diaminopyridine were 35, 20 and 1 min respectively (fig. 5). 3,4-Diaminopyridine at a concentration of 10-4M was less effective at reversing blockade induced by fl-bungarotoxin. When added at about 95% twitch blockade, 1 0 - 4 M 3,4diaminopyridine restored twitches to around 40% of control and twitch blockade was re-established after 25 min (fig. 5).
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3.7. Effects o f sodium hydroxide The diaminopyridines in the largest concentrations used caused a rise in pH of 1 unit in the bathing solution. Sodium hydroxide in a concentration (2.5 × 10 -2 N) sufficient to produce the same increase in pH, produced up to 10% increase in twitch tension in indirectly stimulated preparations. No contractures were observed.
Fig. 5. E f f e c t o f 3 , 4 - d i a m i n o p y r i d i n e o n b l o c k a d e by ~ - b u n g a r o t o x i n . 2 p g / m l f l - b u n g a r o t o x i n was a d d e d at the t i m e s i n d i c a t e d by t h e l e f t - h a n d arrows and 3,4d i a m i n o p y r i d i n e was a d d e d at t h e r i g h t - h a n d arrows ( u p p e r arrow: 10 -4 M; 3 l o w e r arrows: 10 -3 M).
DIAMINOPYRIDINES AND CHICK MUSCLE
The contractures observed with 10 -3 M 3,4diaminopyridine in indirectly stimulated preparations (section 3.2.) was reduced in the presence of /~-bungarotoxin, and was completely absent when the diaminopyridine was added 30 min after the complete development of ~-bungarotoxin blockade. Postjunctional sensitivity, assessed by the response to carbachol (5 × 10 -2 M) remained intact in the presence of fl-bungarotoxin and 3,4-diaminopyridine.
3.9. Convulsant action of 3,4-diaminopyridine The convulsant activity of the most p o t e n t neuromuscular facilitatory c o m p o u n d 3,4-diaminopyridine was compared with that of 4-aminopyridine in the mouse. Both c o m p o u n d s caused death from convulsions, b u t the pattern of the s y m p t o m s was different. 4-Aminopyridine caused piloerection shortly after injection, and the terminal strychnine-like convulsion was preceded by a short period of tremor, four-leg jumping, and mad running. In contrast, 3,4-diaminopyridine in equiactive doses caused a long period of tremor and intense salivation and the animals experienced several minor convulsions before the terminal convulsion. Plots of time to death against dose of ami30
20
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Fig 6. T h e l e t h a l e f f e c t s o f 4 - a m i n o p y r i d i n e a n d 3,4d i a m i n o p y r i d i n e w h e n given t o m i c e i.p. A m i n o p y r i dine dose is in m g / k g (abscissa) a n d t i m e t o d e a t h was m e a s u r e d in rain ( o r d i n a t e ) . T h e results w i t h 4 - a m i n o p y r i d i n e ( e ) a n d 3 , 4 - d i a m i n o p y r i d i n e (0) are s h o w n as m e a n + S.E.M. o f a t least 5 d e t e r m i n a t i o n s . E r r o r bars are i n d i c a t e d unless smaller t h a n t h e s y m b o l s .
307
nopyridine (fig. 6) show 3,4-diaminopyridine to be less effective as a convulsant than 4-aminopyridine. Additional evidence for the greater convulsant activity of 4-aminopyridine is that this c o m p o u n d caused death at a dose of 20 mg/kg whereas 3,4-diaminopyridine was ineffective at this dose.
4. Discussion
The actions exhibited by the diaminopyridines were essentially similar to those found with the aminopyridines in our previous study (Bowman et al., 1977). Thus, the diaminopyridines augmented indirectly elicited twitches of the chick biventer cervicis muscle considerably more effectively than they augmented directly elicited twitches. Therefore in this preparation, the main site of action must be at the neuromuscular junction. The c o m p o u n d s did n o t augment responses to acetylcholine or carbachol, indicating that their action is n o t mediated by an anticholinesterase action or by an increased postjunctional sensitivity. We propose that the diaminopyridines increase the evoked release of acetylcholine. The contractures produced by 10 -3 M 3,4diaminopyridine were absent in preparations in which the acetylcholine receptors were irreversibly blocked. This indicated that the diaminopyridine contracture involves either a direct or indirect activation of acetylcholine receptors. However, the contracture to 3,4diaminopyridine was reduced or abolished in the presence of the prejunctionally active fl-bungarotoxin. Since the sensitivity of the postjunctional membrane was intact in the presence of fl-bungarotoxin, this result indicated that 3,4-diaminopyridine does n o t directly activate the receptors. Thus we suggest that, like the aminopyridines, the contracture caused b y 3,4-diaminopyridine in indirectly stimulated preparations is due to an increased spontaneous release of acetylcholine. As discussed previously (Bowman et al.,
308
1977), most of the actions of the aminopyridines are explicable in terms of the known action of the c o m p o u n d s to inhibit potassium conductance in nerve (Pelhate and Pichon, 1974; Schauf et al., 1976; Yeh et al., 1976a,b; Ulbricht and Wagner, 1976) and muscle (Gillespie and Hutter, 1975). At the neuromuscular junction, inhibition of potassium conductance will prolong the nerve terminal action potential, producing a greater release of acetylcholine and hence a larger and longer endplate potential which will lead to a greater muscle contraction (Benoit and Mambrini, 1970). In muscle T-tubules a prolongation of the falling phase of the muscle action potential will lead to increased twitch tension because of a longer period of Ca 2÷ release from the sarcoplasmic reticulum (Taylor et al., 1972). However, there are two findings of the present study that are difficult to explain by an effect on potassium conductance. They are the increase in spontaneous release of acetylcholine and the slow contracture produced by 3,4-diaminopyridine in the presence of erabutoxin b. As intracellular calcium ion concentration is known to be an important factor in regulating spontaneous transmitter release (Miledi and Thies, 1971; Alnaes et al., 1974; Crawford and Fettiplace, 1971), it is tempting to invoke an action via calcium ions as a component of the action of the aminopyridines. Calcium ions could also be involved in the contracture produced by 3,4-diaminopyridine in directly stimulated preparations. The actions of 3,4-diaminopyridine differed from those of caffeine which releases calcium from the sarcoplasmic reticulum (Bianchi, 1961; Herz and Weber, 1965), from those of nystatin which increases membrane calcium permeability (Crawford and Fettiplace, 1971) and from those of A23187, a calcium ionophore (Statham and Duncan, 1976). The aminopyridines could act by releasing calcium from binding or storage sites within the nerve terminal or muscle, b u t the most likely explanation of the increased spontaneous transmitter release is depolarization
A.L. HARVEY, I.G. MARSHALL
of the nerve terminals. Recently Yeh et al. (1976a) have observed depolarization of membranes in the squid giant axon. The observation that the prejunctional block produced by fl-bungarotoxin may be temporarily reversed by 3,4-diaminopyridine is of interest. Some reversal was noted in preparations in which twitches were completely blocked by the toxin. Presumably when twitches have been abolished, the fl-bungarotoxin has diffused through the entire tissue such that the endplate potential in each muscle fibre is reduced below the contraction threshold. The reversing action of 3,4-diaminopyridine could be due to a large augmentation of transmission in a few fibres unaffected by the toxin, the contractions of which would normally be below the mechanical threshold of the recording system. An alternative explanation of the observed effects is that, although all the neuromuscular juntions are depressed by the ~-bungarotoxin, the blocking action takes some considerable time to become irreversible (Chang et al., 1973. The diaminopyridines exhibited essentially the same actions as the aminopyridines and there is no evidence indicating differences in mechanisms of action. 3,4-Diaminopyridine proved to be slightly more effective in facilitating neuromuscular transmission than was 4-aminopyridine (Bowman et al., 1977). Although both 3,4-diaminopyridine and 4-aminopyridine exhibited convulsant activity when tested in the mouse, 3,4-diaminopyridine was much the weaker of the two c o m p o u n d s and its margin of safety for use as an anticurare or facilitatory agent may be greater than that of 4-aminopyridine. References Alnaes, E., U. Meiri, H. Rahaminoff and R. Rahaminoff, 1974, Possible role of mitochondria in transmitter release, J. Physiol. 241, 30P. Benoit, P.R. and J. Mambrini, 1970, Modification of transmitter release by ions which prolong the presynaptic action potential, J. Physiol. 210, 681. Bianchi, C.P., 1961, The effect of caffeine on radio-
DIAMINOPYRIDINES AND CHICK MUSCLE calcium movement in frog sartorius, J. Gen. Physiol. 4 4 , 8 4 5 . Bowman, W.C., A.L. Harvey and I.G. Marshall, 1976, Facilitatory actions of aminopyridines on neuromuscular transmission, J. Pharm. Pharmacol. 28, Suppl. 79P. Bowman, W.C., A.L. Harvey and I.G. Marshall, 1977, The actions of aminopyridines on avian muscle, Naunyn-Schiedeb. Arch. Pharmacol. 297, 99. Chang, C.C., T.F. Chen and C.Y. Lee, 1973, Studies of the presynaptic effect of ~-bungarotoxin on neuromuscular transmission, J. Pharmacol. Exptl. Therap. 184,339. Crawford, A.C. and R. Fettiplace, 1971, A method for altering the intracellular calcium concentration, J. Physiol. 217, 20P. Fastier, F.N. and M.A. McDowall, 1958, A comparison of the pharmacological properties of the three isomeric aminopyridines, Aust. J. Expl. Biol. Med. Sci. 3 6 , 3 6 5 . Gillespie, J.I. and O.F. Hutter, 1975, The actions of 4-aminopyridine on the delayed potassium current in skeletal muscle fibres, J. Physiol. 252, 70P. Ginsborg, B.L. and J. Warriner, 1960, The isolated chick biventer cervicis nerve-muscle preparation, Brit. J. Pharmacol. Chemotherap. 15,410. Herz, R. and A. Weber, 1965, Caffeine inhibition of Ca uptake by muscle reticulum, Federation Proc. 24,208. Johns, A., D.S. Golko, P.A. Lauzon and D.M. Paton, 1976, The potentiating effects of 4-aminopyridine on adrenergic transmission in the rabbit vas deferens, European J. Pharmacol. 38, 71. Krebs, H.A. and K. Henseleit, 1932, Untersuchungen fiber die Harnstoffbildung im TierkSrper, HoppeSeylers Z. Physiol. Chem. 210, 33.
309 Lemeignan, M. and P. Lechat, Sur l'action anticurare des aminopyridines, C.R. Acad. Sci. (Paris) (D) 264, 169. Miledi, R. and R. Thies, 1971, Tetanic and posttetanic rise in frequency of miniature endplate potentials in low calcium solutions, J. Physiol. 212,245. Pelhate, M. and Y. Pibhon, 1974, Selective inhibition of potassium current in the giant axon of the cockroach, J. Physiol. 242, 90P. Schauf, C.L., C.A. Colton, J.S. Colton and F.A. Davis, 1976, Aminopyridines and sparteine as inhibitors of membrane potassium conductance: Effects on Myxicola giant axons and the lobster neuromuscular junction, J. Pharmacol. Exptl. Therap. 197, 414. Statham, H.E. and C.J. Duncan, 1976, The action of ionophores at the frog neuromuscular junction, Life Sci. 17, 1401. Tamiya, N. and H. Arai, 1966, Studies on sea snake venoms, Biochem. J. 99, 624. Taylor, S.R., H. Preiser and A. Sandow, 1972, Action potential parameters affecting excitation--contraction coupling, J. Gen. Physiol. 59,421. Ulbricht, W. and H.-H. Wagner, 1976, Block of potassium channels of the nodal membrane by 4-aminopyridine and its partial removal on depolarization, Pfltigers Arch. 367, 77. Yeh, J.Z., G.S. Oxford, C.H. Wu and T. Narahashi, 1976a, Interactions of aminopyridines with potassium channels of squid axon membranes, Biophysical J. 16, 77. Yeh, J.Z., G.S. Oxford, C.H. Wu and T. Narahashi, 1976b, Dynamics of aminopyridine block of potassium channels in squid axon membranes, J. Gen. Physiol. 68,519.
303
THE ACTIONS OF THREE DIAMINOPYRIDINES ON THE CHICK BIVENTER CERVICIS MUSCLE ALAN L. HARVEY and IAN G. MARSHALL
The Department o f Physiology and Pharmacology, University o f Strathclyde, Glasgow, G1 1XW, Scotland Received 12 February 1977, revised MS received 27 April 1977, accepted 28 April 1977
A.L. HARVEY and I.G. MARSHALL, The action o f three diaminopyridines on the chick biventer cervicis muscle,European J. Pharmacol. 44 (1977) 303--309. The effects of 2,3-, 2,6- and 3,4-diaminopyridine were investigated on the isolated chick biventer cervicis muscle preparation. All three compounds reversed tubocurarine blockade and augmented twitch height in indirectly stimulated preparations. Less twitch augmentation was observed in directly stimulated preparations. 3,4-Diaminopyridine was the most effective of the compounds in facilitating neuromuscular transmission, but exhibited less convulsant activity than 4-aminopyridine. 3,4- and 2,3-diaminopyridine in high concentrations caused contractures that were inhibited by erabutoxin b or by ~-bungarotoxin. It is suggested that the diaminopyridines increase both evoked and spontaneous acetylcholine release. Diaminopyridines
Chick biventer cervicis muscle
1. Introduction 2-, 3- and 4-aminopyridine were initially shown to augment responses in skeletal muscle preparations by Fastier and McDowall (1958). Subsequently the anti-curare action of these c o m p o u n d s has been studied by Lemeignan and Lechat (1967) and by Bowman et al. {1976,1977). In a recent study of the effect of aminopyridines on adrenergic transmission, Johns et al. (1976) tested 3,4-diaminopyridines 3,4Diaminopyridine was as active as 4-aminopyridine in augmenting adrenergic transmission in the rabbit vas deferens preparation. As there is no published work on the effects of
Neuromuscular transmission
Acetylcholine release
the diaminopyridines on neuromuscular transmission, we studied 2,3- (left), 2,6- (right) and 3,4-diaminopyridine (middle) (fig. 1) under the same experimental conditions as described in our previous paper on the aminopyridines (Bowman et al., 1977). An a t t e m p t was also made to test the hypothesis that a c o m p o n e n t of the aminopyridines' action may involve calcium ions (Bowman et al., 1977). The diaminopyridines were compared with compounds such as caffeine and nystatin whose action is known to involve calcium ions. As 4-aminopyridine also exhibits convulsant activity (Fastier and McDowall, 1958), the convulsant activity of 3,4-diaminopyridine was tested.
NH 2
2. Materials and m e t h o d s ~-N ~
~NH 2
.
NH 2
Fig. 1. Formulae of the diaminopyridines tested. 2,3AP, 2,3-diaminopyridine (left); 2,6AP, 2,6-diaminopyridine (right); 3,4AP, 3,4-diaminopyridine (middle).
2.1. Chick biventer cervicis muscle
The biventer cervicis muscles from chicks aged 2--10 days were m o u n t e d in Krebs-Henseleit (1932) solution maintained at 32°C
304 and gassed with oxygen containing 5% carbon dioxide (Ginsborg and Warriner, 1960).
2.1.1. Indirectly stimulated preparations The nerve within the tendon was stimulated by ring electrodes at a frequency of 0.1 Hz with rectangular pulses of 0.2 msec duration, and of strength greater than that required to elicit a maximal twitch. For the construction of dose--response curves, each preparation was used for only one addition of the diaminopyridine. In some experiments the nerve stimulation was periodically stopped and acetylcholine (5 X 10 -4 M) or carbachol (2 X 10 -s M) was added to the tissue bath. Acetylcholine and carbachol were allowed to remain in contact with the tissues for 30 sec and 90 sec respectively. 2.1.2. Directly stimulated preparations Preparations were set up and stimulated as described for indirect stimulation. Neuromuscular transmission was then inhibited by the irreversible snake venom toxin erabutoxin b {4pg/ml) (Tamiya and Arai, 1966). After abolition of neuromuscular transmission the ring electrodes were moved into contact with the muscle which was then stimulated at a frequency of 0.1 Hz with rectangular pulses of 1 msec duration and of strength greater than that required to elicit a maximal twitch. For the construction of dose--response curves, each preparation was used for only one addition of the diaminopyridine. 2.2. Convulsant activity Compounds were injected intraperitoneally into male mice (30--40 g) and the time from injection to death by convulsion was measured. Each dose level was tested in 5--6 animals.
2.3. Drugs The drugs used were 4-aminopyridine, 2,3-, 2,6- and 3,4-diaminopyridine, nystatin and
A.L. HARVEY, I.G. MARSHALL {+)-tubocurarine chloride (Sigma), acetylcholine chloride, caffeine and carbachol chloride (British Drug Houses), ~-bungarotoxin (Boehringer Ingelheim), A23187 (Lilly) and erabutoxin b (supplied by Professor N. Tamiya, Department of Chemistry, Tohoku University, Japan). Drugs were dissolved in 0.9% saline, except nystatin which was dissolved in 1% ethanol and A23187 which was dissolved in ethanol.
2.4. Statistics In the dose--response curves each concentration was tested on six individual preparations and the points on the curves represent the mean + standard error. Differences between means were tested by Student's non paired t-test, values of p < 0.05 being regarded as significant.
3. Results
3.1. Reversal o f (+)-tubocurarine neuromuscular blockade 3,4-Diaminopyridine was the most effective of the compounds tested in reversing the blockade of indirectly stimulated preparations induced by (+)-tubocurarine. Within 15 min 3,4-diaminopyridine (10 -s M) completely restored twitches depressed to 30% of control by (+)-tubocurarine (5 X 10 -6 M). 10 -4 M 3,4Diaminopyridine could restore twitches abolished by 2 X 10 -s M (+)-tubocurarine (fig. 2). The concentration of (+)-tubocurarine had to be increased 10-fold to re,establish neuromuscular blockade in the continued presence of 3,4-diaminopyridine. 2,3- and 2,6
D I A M I N O P Y R I D I N E S A N D CHICK M U S C L E
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Fig. 2. Reversal o f (+)-tuboeurarine blockade by 3,4d i a m i n o p y r i d i n e . The preparation was stimulated indirectly, 2 × 10 -s M (+)otuboeurarine was added at the left arrow, and 10 -4 M 3 , 4 - d i a m i n o p y r i d i n e was
added at the right arrow. 3.2. Augmentation o f twitches in indirectly stimulated preparations All three c o m p o u n d s augmented maximal twitch height in indirectly stimulated preparations. Again 3,4-diaminopyridine was the most p o t e n t of the three c o m p o u n d s tested, producing 3 2 5 + 48% increases in twitch tension at 10 -3 M, whereas 2,6
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Fig. 3. A u g m e n t a t i o n of directly and indirectly elicited twitches by 2,3-diaminopyridine (left), 2,6-diaminopyridine (middle) and 3,4-diaminopyridine (right panel). C o n c e n t r a t i o n is m o l a r (abscissa) and twitch a u g m e n t a t i o n is expressed as percentage increase f r o m control twitch height (ordinate). The results with direct stimulation (m) and with indirect stimulation (e) are shown as m e a n + S.E.M. o f 6 determinations. Error bars are indicated unless smaller than the symbols.
elicited by acetylcholine (5 × 10 -4 M) or by carbachol (10 -s M). Fig. 4 shows the result of a typical experiment with 3,4-diaminopyridine. 3.4. Augmentation o f twitches in directly stim ula ted preparations All three c o m p o u n d s produced significantly (p < 0.05) less augmentation of directly elicited twitches than of indirectly elicited 5 min I
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Fig. 4. The effect o f 3,4-diaminopyridine on responses to acetylcholine and carbachol. T o p panel: control responses to indirect stimulation and to 5 x 1 0 - 4 M acetylcholine (left arrows) and 2 × 10 -s M carbachol (right arrows). B o t t o m panel: 1 0 - 4 M 3,4-diaminopyridine was added at the left arrow. Responses to 5 × 10 -4 M a c e t y l c h o l i n e (middle arrows) and 2 × 10 -s M carbachol (right' arrows) were t h e n o b t a i n e d in the presence o f 3,4-diaminopyridine.
306
twitches (fig. 3). The largest augmentation (50 + 8%) was produced by 3,4-diaminopyridine (10 -2 M). In the directly stimulated preparations no contracture was observed at 10-3M 3,4-diaminopyridine, although at 1 0 - 2 M 3,4-diaminopyridine a very slowly developing contracture of immediate onset was occasionally observed. The contracture generally reached the equivalent of approximately 25% of maximal twitch height to direct stimulation in 10 min.
3.5. Effect o f caffeine on indirectly and directly stimulated preparations Caffeine (10 -4 to 10 -3 M) was tested, 5 × 1 0 - 4 M and 10-3M proving effective in augmenting both indirectly and directly elicited twitches to similar degrees. 1 0 - 3 M caffeine produced a small contracture (ca. 10% maximal twitch height) in both indirectly and directly stimulated preparations. Caffeine (5 × 10 -4 M) produced no reversal of neuromuscular block by (+)-tubocurarine (1--2 ×10 -s M). In the continued presence of caffeine (5 × 10 -4 M), 3,4-diaminopyridine (10-4--10 .3 M) remained an effective reversing agent.
3.6. Effect o f nystatin and A 2 3 1 8 7 on indirectly and directly stimulated preparations Neither nystatin (10-6--10 -s M) nor A23187 (5--10 pg/ml) produced augmentation of either indirectly or directly elicited twitches.
A.L. H A R V E Y , I.G. M A R S H A L L
3.8. Effect o f 3,4-diaminopyridine on [3-bungarotoxin-induced neuromuscular blockade 3,4-Diaminopyridine produced a temporary reversal of the neuromuscular block induced by ~-bungarotoxin (2 pg/ml). The degree and duration of the reversal was dependent upon the time of addition and the concentration of the diaminopyridine. When added at around 95% twitch blockade, 3,4-diaminopyridine (10-3 M) restored twitch height to around 20% greater than control within 3 min. When the same concentration of 3,4-diaminopyridine was added 4 min and 30 min after complete twitch blockade, the twitches returned to around 75% and less than 5% of control respectively. In these three situations the times for the /3-bungarotoxininduced block to redevelop fully in the continued presence of 3,4-diaminopyridine were 35, 20 and 1 min respectively (fig. 5). 3,4-Diaminopyridine at a concentration of 10-4M was less effective at reversing blockade induced by fl-bungarotoxin. When added at about 95% twitch blockade, 1 0 - 4 M 3,4diaminopyridine restored twitches to around 40% of control and twitch blockade was re-established after 25 min (fig. 5).
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3.7. Effects o f sodium hydroxide The diaminopyridines in the largest concentrations used caused a rise in pH of 1 unit in the bathing solution. Sodium hydroxide in a concentration (2.5 × 10 -2 N) sufficient to produce the same increase in pH, produced up to 10% increase in twitch tension in indirectly stimulated preparations. No contractures were observed.
Fig. 5. E f f e c t o f 3 , 4 - d i a m i n o p y r i d i n e o n b l o c k a d e by ~ - b u n g a r o t o x i n . 2 p g / m l f l - b u n g a r o t o x i n was a d d e d at the t i m e s i n d i c a t e d by t h e l e f t - h a n d arrows and 3,4d i a m i n o p y r i d i n e was a d d e d at t h e r i g h t - h a n d arrows ( u p p e r arrow: 10 -4 M; 3 l o w e r arrows: 10 -3 M).
DIAMINOPYRIDINES AND CHICK MUSCLE
The contractures observed with 10 -3 M 3,4diaminopyridine in indirectly stimulated preparations (section 3.2.) was reduced in the presence of /~-bungarotoxin, and was completely absent when the diaminopyridine was added 30 min after the complete development of ~-bungarotoxin blockade. Postjunctional sensitivity, assessed by the response to carbachol (5 × 10 -2 M) remained intact in the presence of fl-bungarotoxin and 3,4-diaminopyridine.
3.9. Convulsant action of 3,4-diaminopyridine The convulsant activity of the most p o t e n t neuromuscular facilitatory c o m p o u n d 3,4-diaminopyridine was compared with that of 4-aminopyridine in the mouse. Both c o m p o u n d s caused death from convulsions, b u t the pattern of the s y m p t o m s was different. 4-Aminopyridine caused piloerection shortly after injection, and the terminal strychnine-like convulsion was preceded by a short period of tremor, four-leg jumping, and mad running. In contrast, 3,4-diaminopyridine in equiactive doses caused a long period of tremor and intense salivation and the animals experienced several minor convulsions before the terminal convulsion. Plots of time to death against dose of ami30
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Fig 6. T h e l e t h a l e f f e c t s o f 4 - a m i n o p y r i d i n e a n d 3,4d i a m i n o p y r i d i n e w h e n given t o m i c e i.p. A m i n o p y r i dine dose is in m g / k g (abscissa) a n d t i m e t o d e a t h was m e a s u r e d in rain ( o r d i n a t e ) . T h e results w i t h 4 - a m i n o p y r i d i n e ( e ) a n d 3 , 4 - d i a m i n o p y r i d i n e (0) are s h o w n as m e a n + S.E.M. o f a t least 5 d e t e r m i n a t i o n s . E r r o r bars are i n d i c a t e d unless smaller t h a n t h e s y m b o l s .
307
nopyridine (fig. 6) show 3,4-diaminopyridine to be less effective as a convulsant than 4-aminopyridine. Additional evidence for the greater convulsant activity of 4-aminopyridine is that this c o m p o u n d caused death at a dose of 20 mg/kg whereas 3,4-diaminopyridine was ineffective at this dose.
4. Discussion
The actions exhibited by the diaminopyridines were essentially similar to those found with the aminopyridines in our previous study (Bowman et al., 1977). Thus, the diaminopyridines augmented indirectly elicited twitches of the chick biventer cervicis muscle considerably more effectively than they augmented directly elicited twitches. Therefore in this preparation, the main site of action must be at the neuromuscular junction. The c o m p o u n d s did n o t augment responses to acetylcholine or carbachol, indicating that their action is n o t mediated by an anticholinesterase action or by an increased postjunctional sensitivity. We propose that the diaminopyridines increase the evoked release of acetylcholine. The contractures produced by 10 -3 M 3,4diaminopyridine were absent in preparations in which the acetylcholine receptors were irreversibly blocked. This indicated that the diaminopyridine contracture involves either a direct or indirect activation of acetylcholine receptors. However, the contracture to 3,4diaminopyridine was reduced or abolished in the presence of the prejunctionally active fl-bungarotoxin. Since the sensitivity of the postjunctional membrane was intact in the presence of fl-bungarotoxin, this result indicated that 3,4-diaminopyridine does n o t directly activate the receptors. Thus we suggest that, like the aminopyridines, the contracture caused b y 3,4-diaminopyridine in indirectly stimulated preparations is due to an increased spontaneous release of acetylcholine. As discussed previously (Bowman et al.,
308
1977), most of the actions of the aminopyridines are explicable in terms of the known action of the c o m p o u n d s to inhibit potassium conductance in nerve (Pelhate and Pichon, 1974; Schauf et al., 1976; Yeh et al., 1976a,b; Ulbricht and Wagner, 1976) and muscle (Gillespie and Hutter, 1975). At the neuromuscular junction, inhibition of potassium conductance will prolong the nerve terminal action potential, producing a greater release of acetylcholine and hence a larger and longer endplate potential which will lead to a greater muscle contraction (Benoit and Mambrini, 1970). In muscle T-tubules a prolongation of the falling phase of the muscle action potential will lead to increased twitch tension because of a longer period of Ca 2÷ release from the sarcoplasmic reticulum (Taylor et al., 1972). However, there are two findings of the present study that are difficult to explain by an effect on potassium conductance. They are the increase in spontaneous release of acetylcholine and the slow contracture produced by 3,4-diaminopyridine in the presence of erabutoxin b. As intracellular calcium ion concentration is known to be an important factor in regulating spontaneous transmitter release (Miledi and Thies, 1971; Alnaes et al., 1974; Crawford and Fettiplace, 1971), it is tempting to invoke an action via calcium ions as a component of the action of the aminopyridines. Calcium ions could also be involved in the contracture produced by 3,4-diaminopyridine in directly stimulated preparations. The actions of 3,4-diaminopyridine differed from those of caffeine which releases calcium from the sarcoplasmic reticulum (Bianchi, 1961; Herz and Weber, 1965), from those of nystatin which increases membrane calcium permeability (Crawford and Fettiplace, 1971) and from those of A23187, a calcium ionophore (Statham and Duncan, 1976). The aminopyridines could act by releasing calcium from binding or storage sites within the nerve terminal or muscle, b u t the most likely explanation of the increased spontaneous transmitter release is depolarization
A.L. HARVEY, I.G. MARSHALL
of the nerve terminals. Recently Yeh et al. (1976a) have observed depolarization of membranes in the squid giant axon. The observation that the prejunctional block produced by fl-bungarotoxin may be temporarily reversed by 3,4-diaminopyridine is of interest. Some reversal was noted in preparations in which twitches were completely blocked by the toxin. Presumably when twitches have been abolished, the fl-bungarotoxin has diffused through the entire tissue such that the endplate potential in each muscle fibre is reduced below the contraction threshold. The reversing action of 3,4-diaminopyridine could be due to a large augmentation of transmission in a few fibres unaffected by the toxin, the contractions of which would normally be below the mechanical threshold of the recording system. An alternative explanation of the observed effects is that, although all the neuromuscular juntions are depressed by the ~-bungarotoxin, the blocking action takes some considerable time to become irreversible (Chang et al., 1973. The diaminopyridines exhibited essentially the same actions as the aminopyridines and there is no evidence indicating differences in mechanisms of action. 3,4-Diaminopyridine proved to be slightly more effective in facilitating neuromuscular transmission than was 4-aminopyridine (Bowman et al., 1977). Although both 3,4-diaminopyridine and 4-aminopyridine exhibited convulsant activity when tested in the mouse, 3,4-diaminopyridine was much the weaker of the two c o m p o u n d s and its margin of safety for use as an anticurare or facilitatory agent may be greater than that of 4-aminopyridine. References Alnaes, E., U. Meiri, H. Rahaminoff and R. Rahaminoff, 1974, Possible role of mitochondria in transmitter release, J. Physiol. 241, 30P. Benoit, P.R. and J. Mambrini, 1970, Modification of transmitter release by ions which prolong the presynaptic action potential, J. Physiol. 210, 681. Bianchi, C.P., 1961, The effect of caffeine on radio-
DIAMINOPYRIDINES AND CHICK MUSCLE calcium movement in frog sartorius, J. Gen. Physiol. 4 4 , 8 4 5 . Bowman, W.C., A.L. Harvey and I.G. Marshall, 1976, Facilitatory actions of aminopyridines on neuromuscular transmission, J. Pharm. Pharmacol. 28, Suppl. 79P. Bowman, W.C., A.L. Harvey and I.G. Marshall, 1977, The actions of aminopyridines on avian muscle, Naunyn-Schiedeb. Arch. Pharmacol. 297, 99. Chang, C.C., T.F. Chen and C.Y. Lee, 1973, Studies of the presynaptic effect of ~-bungarotoxin on neuromuscular transmission, J. Pharmacol. Exptl. Therap. 184,339. Crawford, A.C. and R. Fettiplace, 1971, A method for altering the intracellular calcium concentration, J. Physiol. 217, 20P. Fastier, F.N. and M.A. McDowall, 1958, A comparison of the pharmacological properties of the three isomeric aminopyridines, Aust. J. Expl. Biol. Med. Sci. 3 6 , 3 6 5 . Gillespie, J.I. and O.F. Hutter, 1975, The actions of 4-aminopyridine on the delayed potassium current in skeletal muscle fibres, J. Physiol. 252, 70P. Ginsborg, B.L. and J. Warriner, 1960, The isolated chick biventer cervicis nerve-muscle preparation, Brit. J. Pharmacol. Chemotherap. 15,410. Herz, R. and A. Weber, 1965, Caffeine inhibition of Ca uptake by muscle reticulum, Federation Proc. 24,208. Johns, A., D.S. Golko, P.A. Lauzon and D.M. Paton, 1976, The potentiating effects of 4-aminopyridine on adrenergic transmission in the rabbit vas deferens, European J. Pharmacol. 38, 71. Krebs, H.A. and K. Henseleit, 1932, Untersuchungen fiber die Harnstoffbildung im TierkSrper, HoppeSeylers Z. Physiol. Chem. 210, 33.
309 Lemeignan, M. and P. Lechat, Sur l'action anticurare des aminopyridines, C.R. Acad. Sci. (Paris) (D) 264, 169. Miledi, R. and R. Thies, 1971, Tetanic and posttetanic rise in frequency of miniature endplate potentials in low calcium solutions, J. Physiol. 212,245. Pelhate, M. and Y. Pibhon, 1974, Selective inhibition of potassium current in the giant axon of the cockroach, J. Physiol. 242, 90P. Schauf, C.L., C.A. Colton, J.S. Colton and F.A. Davis, 1976, Aminopyridines and sparteine as inhibitors of membrane potassium conductance: Effects on Myxicola giant axons and the lobster neuromuscular junction, J. Pharmacol. Exptl. Therap. 197, 414. Statham, H.E. and C.J. Duncan, 1976, The action of ionophores at the frog neuromuscular junction, Life Sci. 17, 1401. Tamiya, N. and H. Arai, 1966, Studies on sea snake venoms, Biochem. J. 99, 624. Taylor, S.R., H. Preiser and A. Sandow, 1972, Action potential parameters affecting excitation--contraction coupling, J. Gen. Physiol. 59,421. Ulbricht, W. and H.-H. Wagner, 1976, Block of potassium channels of the nodal membrane by 4-aminopyridine and its partial removal on depolarization, Pfltigers Arch. 367, 77. Yeh, J.Z., G.S. Oxford, C.H. Wu and T. Narahashi, 1976a, Interactions of aminopyridines with potassium channels of squid axon membranes, Biophysical J. 16, 77. Yeh, J.Z., G.S. Oxford, C.H. Wu and T. Narahashi, 1976b, Dynamics of aminopyridine block of potassium channels in squid axon membranes, J. Gen. Physiol. 68,519.