Restoration of transmitter release in botulinum-poisoned skeletal muscle

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Restoration of transmitter release in botulinum-poisoned skeletal muscle

HAKAN LUNDH*, STUART G. CULL-CANDY**, STEFAN LEANDER* AND STEPHEN THESLEFF*** Department t~fPharmacology, University oJ Lund, Lund (Sweden)

(Accepted March 18th, 1976)

Botulinum toxin (BoTx) blocks acetylcholine (ACh) release from motor nerve terminals without affecting impulse conduction or ACh synthesis (see review by Simpson13). Since transmitter release is a calcium requiring process s it was of interest to examine the effect of calcium ions on the release process in BoTx-poisoned nerve terminals. In the present study we have examined the effects of the calcium ionophore A-23187 on spontaneous transmitter release and of tetraethylammonium (TEA) and guanidine on neurally evoked release. The results show that the aforementioned drugs, which are all believed to increase the intracellular concentration of calcium in nerve terminals, restore transmitter release to almost normal levels. The experiments were made in vitro on the extensor digitorum longus (EDL) muscle of adult male Sprague-Dawley rats with a body weight of about 180 g. BoTx type A was given as a single injection of 0.25 ml s.c. into the anterolateral region of the right hirtdleg. The toxin produced complete paralysis of the leg within 18 h which lasted for at least 3 weeks. At 2-9 days after BoTx the EDL muscle was removed from animals under ether anaesthesia and placed in a constant temperature bath (29 °C for mechanical recording and 37 °C for intracellular recording) and perfused with oxygenated medium as described by Liley 9. The nerve to the muscle was stimulated at 0.1 Hz by the use of a glass capillary suction electrode and supramaximal (8 V) current pulses of 0.05 msec duration. Isometric twitch tension was recorded with a Grass FT-03 transducer connected to an ink-writing oscillograph. The resting tension of the muscle was adjusted to give maximal twitch response. Conventional intracellular microelectrodes were used to record spontaneous transmitter release as miniature endplate potentials (mepps) and neurally evoked transmitter release as endplate potentials (epps). The calcium ionophore A-23187 (Eli Lilly Co.) was dissolved in ethanol and added to the bathing solution to give a final concentration of 10--:' M. TEA was used as the chloride and guanidine as the hydrochloride.

* Graduate student. Department of Pharmacology, University of Lund. ** Recipient of a European Fellowship of the Royal Society and the Science Research Council of U.K. Present address: Dept. of Biophysics, University College London, England. *** Professor, Department of Pharmacology, University of Lund.

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Spontaneous transmitter release. The mean 4- S.E.M. frequency of mepps 2-9 days after injection of BoTx was 0.5 4- 0.34 (39 fibres; 27 muscles), which compared with 7.0 4- 1.47 (6 fibres; 4 muscles) in normal muscle. Raising the extracellular calcium concentration [Ca]0 from 2 to 16 m M failed to affect the frequency of mepps recorded in BoTx poisoned muscles but increased 4-6 fold the frequency of mepps recorded in normal muscle, as shown in Fig. I A. In the presence of the calcium ionophore, which is believed to allow the passage of calcium ions across biological membranes 12, perfusion of the endplate region with 15 m M calcium caused a large increase in mepp frequency in BoTx-poisoned muscles, and when the pipette was withdrawn the frequency returned to the initial low value as shown in Fig. 1B. Under these conditions the amplitude distribution of mepps changed from the bimodal distribution seen in BoTx-poisoned muscles TM to a normal Gaussian one. When the intracellular entry of the ion is facilitated by the use of the calcium ionophore, high [Ca]0 causes increased release of ACh quanta from BoTx-poisoned terminals. Neuromuscular transmission. Several lines of evidence suggest that the entry of calcium ions through the presynaptic membrane is directly correlated with evoked release of transmitter 5 from normal nerve terminals. In BoTx-poisoned muscles neuromuscular transmission was blocked. Intracellular recording revealed the presence of occasional epps with small amplitude, consisting presumably of a single quantum of ACh (Fig. 2A). Increasing the [Ca]0 from 4 to 16 m M increased mean quantum content (m) of epps about 6-fold but was in no instance sufficient to restore neuromuscular transmission. The increase in m was less than that produced in normal unpoisoned muscles (about 33-fold) by a similar change in [Ca]0. It is possible to enhance the entry of Ca2+ions into the nerve terminal by prolonging the duration of the nerve

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Fig. 1. A: mean mepp frequencies recorded in different concentrations of [Ca]0 at two endplates in normal muscle (open circles) and at 4 endplates in muscles 2~, days after BoTx (closed circles). B: the frequency of mepps at a BoTx-poisoned endplate in the presence of the calcium ionophore A23187. Between the arrows the endplate is superfused with 15 mM calcium.

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Fig. 2. A: endplate potentials of quantal size and many failures of release in response to stimulaticn at 0.5 Hz in a muscle 2 days after BoTx poisoning; [Ca]04 mM. B: the same fibre after the addition of 0.5 mM TEA. action potential1, 6. This can be achieved by the use of TEA which blocks the depolarization-induced increase in potassium conductanceT, s. In concentrations of 0.2-3.2 m M TEA restored neuromuscular transmission in BoTx poisoned muscle in a dosedependent manner. Fig. 2B shows the same epp as in Fig. 2A but in the presence of 0.5 m M TEA; note that the addition of TEA abolished all failures of transmitter release and markedly increased epp amplitude. An analysis of mean quantum content (m) of epps showed that 0.2 m M TEA increased m 6 times; 0.4 m M increased it 12 times and 0.6 m M increased it 23 times. Twitch tensions similar to those in normal unpoisoned muscles were recorded, as shown in Fig. 3. TEA at 0.2-2.0 mM failed to alter mepp frequency at BoTx-poisoned endplates and, similarly, elevating the level of [Ca]0 from 2 to 10 m M failed to increase mepp frequency in the presence of TEA (2 mM), showing that the effect of the drug was restricted to evoked transmitter release. Guanidine acts presynaptically in normal muscle to increase the amount of ACh released by nerve impulses but fails to affect the spontaneous release of ACh 4,~°. It appears to act in a manner different from that of TEA since it has little effect on the time course of the action potential 4. As shown in Fig. 3, guanidine 0.2-3.2 m M was able to restore neuromuscular transmission in BoTx-poisoned muscles. As with TEA, guanidine, at the time when it restored transmission, failed to affect spontaneous transmitter release. BoTx-treated nerve terminals are refractory to several procedures, such as depolarization and nerve stimulation, which, by liberating intracellular calcium or allowing the entry of calcium ions, cause an increase in the release of transmitter from normal terminals a. However, when calcium ions in excess of normal were allow-

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Fig. 3. Isometric twitch tensions of EDL muscles in vitro stimulated at 0.1 Hz through their motor nerve. The values are means ± S.E.M. of 5 muscles. Open and closed circles are from BoTxpoisoned muscles (2-5 days after poisoning) and show, respectively, that TEA and guanidine, in a dose-dependent fashion, increase twitch tension and restore neuromuscular transmission to about normal level. The value indicated with the triangle is from normal unpoisoned muscles. [Ca]0 4 raM. ed to enter the nerve terminal, b y the use o f the calcium i o n o p h o r e , a massive release o f A C h q u a n t a occurred. Similarly, when T E A p r o l o n g e d the d u r a t i o n o f the nerve t e r m i n a l a c t i o n p o t e n t i a l a n d t h e r e b y increased the a m o u n t o f calcium which entered the t e r m i n a l a m a r k e d increase in the phasic release o f t r a n s m i t t e r occurred, n e u r o m u s c u l a r t r a n s m i s s i o n being restored. G u a n i d i n e h a d a similar a c t i o n on n e u r o m u s c u l a r transmission. H o w e v e r , its m o d e o f a c t i o n on the release process is u n k n o w n . Several investigators have shown t h a t g u a n i d i n e inhibits the b i n d i n g a n d u p t a k e o f d i v a l e n t cations to subcellular organelles such as mitochondria2, u . It is t h e r e f o r e possible t h a t g u a n i d i n e interferes with the intracellular b i n d i n g o f calcium in nerve t e r m i n a l s a n d t h e r e b y p r o l o n g s a n d enhances the effect o f the calcium which enters the t e r m i n a l d u r i n g the nerve a c t i o n potential. The results we have o b t a i n e d indicate t h a t after BoTx p o i s o n i n g the t r a n s m i t t e r release m e c h a n i s m is intact b u t t h a t it requires a higher t h a n usual level o f intracellular calcium to p r o d u c e activation. Once the i n t r a c e l l u l a r level o f calcium is raised to this level (by the use o f the calcium i o n o p h o r e , by the influx o f calcium d u r i n g a p r o l o n g e d a c t i o n p o t e n t i a l or by the use o f guanidine), an a p p r o x i m a t e l y n o r m a l level o f t r a n s m i t t e r release occurs. The study was a i d e d b y G r a n t BT0-14X-3112 f r o m the Swedish M e d i c a l R e s e a r c h C o u n c i l a n d by a g r a n t f r o m the M u s c u l a r D y s t r o p h y A s s o c i a t i o n o f A m e r i c a Inc. T h e calcium i o n o p h o r e A-23187 was a gift from Eli Lilly Co.

1 BAKER, P. F., Transport and metabolism of calcium ions in nerve, Progr. Biophys. molec. Biol., 24

(1972) 177-223. 2 DAVIDOFF, F., Effects of guanidine derivatives on mitochondrial function, J, biol. Chem., 249

(1974) 6406-6415. 3 HARRIS, A. J., AND MILEDI, R., The effect of type D botulinum toxin on frog neuromuscular junctions, J. Physiol. (Lond.), 217 (1971) 497-515.

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