density of receptor on muscle membrane being increased from a-bungarotoxin. 6/,um2 in normal diaphragm to 38//um2. Junctional receptors were also

Transcription

1 J. Physiol. (1975), 250, pp With 5 text-figure. Printed in Great Britain EFFECTS OF CHRONIC TREATMENT WITH VARIOUS NEUROMUSCULAR BLOCKING AGENTS ON THE NUMBER AND DISTRIBUTION OF ACETYLCHOLINE RECEPTORS IN THE RAT DIAPHRAGM BY C. C. CHANG, SING-TAI CHUANG AND M. C. HUANG From the Pharmacological Institute, College of Medicine, National Taiwan Universty, Taipei, Taivwan 100, Republic of China (Received 31 December 1974) SUMMARY 1. Acetylcholine receptors in the end-plate and non-end-plate areas of the rat diaphragm, after treating the animal with hemicholinium-3, cc- or f,- bungarotoxin in vivo, were studied by their specific binding of labelled a-bungarotoxin. 2. Subcutaneous injection of maximum tolerable doses of hemicholinium-3 (50,ug/kg) twice daily for 7 days increased the number of extrajunctional receptors along the whole length of muscle fibre, the approximate density of receptor on muscle membrane being increased from 6/,um2 in normal diaphragm to 38//um2. Junctional receptors were also increased in number from 2.2 x 107 to 2-8 x 107 per end-plate. 3. Five days after denervation, there were approximately 153/ m2 extrajunctional receptors and the number of receptors on the end-plate was increased by 220 %. 4. Intrathoracic injection of f-bungarotoxin (50 gtg/kg) also increased the density of extrajunctional receptors to approximately 104/gm2, and the number of end-plate receptors by 140 % in 5 days. The neuromuscular block was extensive and prolonged. 5. [3H]Diacetyl a-bungarotoxin (150 gg/kg) injected into thoracic cavity caused complete neuromuscular blockade for 12 hr. At 24 hr, the synaptic transmission was restored in 80 % of the junctions with less than 10 % end-plate receptors freed, whereas the safety factor for transmission in normal diaphragm was 3-5. Extrajunctional receptors appeared to increase within 24 hr. This increase continued despite the restoration of neuromuscular transmission, and the receptor density at 5 days was approximately 51/gm2. The number of junctional receptors, however, was not increased. Repeated injection of the toxin gave the same result. 6-2

2 162 C. C. CHANG, SING-TAI CHUANG AND M. C. HUANG 6. It is concluded that the numbers of junctional and extrajunctional acetylcholine receptors are regulated in different ways, and the possible role of acetylcholine is discussed. INTRODUCTION It is well known that in focally innervated skeletal muscles, the acetylcholine-sensitive sites are largely restricted to end-plate region (Kuffler, 1943; Axelsson & Thesleff, 1959; Miledi, 1960; Albuqerque & McIsaac, 1970; Peper & McMahan, 1972). The main distribution of acetylcholine receptors in this region is further substantiated by the binding of iso-topically labelled a-bungarotoxin (Lee, Tseng & Chiu, 1967; Miledi & Potter, 1971; Barnard, Wieckowski & Chiu, 1971; Berg, Kelly, Sargent, Williamson & Hall, 1972; Hartzell & Fambrough, 1972; Fambrough & Hartzell, 1972; Chang, Chen & Chuang, 1973 a), a specific irreversible binding agent of acetylcholine receptors on the end-plate (Chang & Lee, 1963). After section of the motoneurone, supersensitivity occurs in the denervated skeletal muscle by an increase in sensitivity to acetylcholine at nonend-plate regions (Axelsson & Thesleff, 1959; Miledi, 1960; Albuqerque & Mclsaac, 1970; L0mo & Rosenthal, 1972), and by an increase and spreading of ac-bungarotoxin-binding sites along the whole length of muscle fibre (Lee et al. 1967; Miledi & Potter, 1971; Berg et al. 1972; Hartzell & Fambrough, 1972; Chang et al. 1973a). In contrast to denervation, when rats were treated chronically with neostigmine to inhibit acetylcholinesterase for a prolonged period, muscle weakness developed (Roberts & Thesleff, 1969) and the number of acetylcholine receptors on end-plate, as well as the release of the neurotransmitter, was decreased (Chang, Chen & Chuang, 1973b; Fambrough, Drachman & Satyamurti, 1973). Attempts were therefore made to follow the changes in the number of receptors on end-plates, as well as on the non-end-plate zone, during defective neuromuscular transmission induced by various pharmacological agents with different modes of action. The effects of hemicholinium-3 which interferes with neuromuscular transmission primarily by inhibition of acetylcholine synthesis at low doses (Schueler, 1960),,fl-bungarotoxin, which blocks the release mechanism of neurotransmitter (Chang, Chen & Lee, 1973c; Chang & Huang, 1974) and ac-bungarotoxin were compared with the effect of denervation on the rat diaphragm. METHODS Assay of neuromuscular transmission Both hemidiaphragms, with phrenic nerves attached, were isolated from normal or drug treated Long-Evans rats of either sex weighing about g, and bathed in 50 ml. Tyrode solution (composition in m : NaCl, 137; KCl, 2-7; CaCl21 8;

3 REGULATION OF ACh RECEPTOR 163 MgCl2, 1-1; NaHCO3, 11-9: NaH2 P04, 0-33 and glucose, 11-2) at 370 C, oxygenated with 95% % CO2. The phrenic nerve was stimulated with a single supramaximal rectangular pulse every 10 sec. The diaphragm muscle was also stimulated (pulse width, 0-5 msec) directly with a pair of electrodes placed parallel to, and on either side of the preparation, as close to the rib end as possible. Involvement of nerve terminals in the above direct stimulation was negligible. The tension developed was recorded isometrically on a Grass polygraph with the aid of forcetransducer (FT. 03). The ratio of tension evoked by stimulation of nerve to that evoked by direct stimulation of muscle was taken to indicate the extent of neuromuscular transmission of the drug-treated preparation. The diaphragms from normal animals usually gave the same amplitude of contraction by these two modes of stimulation. None of the agents used had any direct effect on the muscle. Assay of acetylcholine receptors The numbiar of acetylcholine receptors on hemidiaphragm was estimated by the irreversible binding of N,O-di[3H]acetyl a-bungarotoxin (400 mc/m-mole) as previously described (Chang et al. 1973a). Hemidiaphragms were incubated with the labelled toxin at 1-25 x 10-v M for 2 hr at 370 C, a condition known to saturate the receptor (Chang et al a), and then washed for 5 hr with twelve changes of Tyrode solution to remove non-specifically bound label. Each muscle was stretched on a paper board, dried and cut parallel to the end-plate zone into ten or three segments. In the latter case, the end-plates were included ill the central segment. Radioactivities in each segment were counted by liquid scintillation spectrometry. One dpm corresponded to 7-5 x 108 toxin-binding sites. Density of the extrajunctional acetylcholine receptor was calculated by assuming a homogeneous distribution on the surface of 10,000 muscle fibres (Krnjevicd & Miledi, 1958) of 30,m diameter and 15 mm length. Drug treatment Hemicholinium-3 (Aldrich Chem. Co.), 50,ug/kg, was administered subcutaneously twice daily for 7 days. This was almost a maximal dose tolerated by the rat without causing severe neuromuscular blockade leading to respiratory paralysis. N,O-di[3H]acetyl a-bungarotoxin, 150,ug/kg, dissolved in 1 0 ml. normal saline was injected into the thoracic cavity through lower intercostal space. The animals were kept in upright posture in a restricted cage for a few hours in order to facilitate the contact of diaphragm with toxin. In other groups of animals, two additional doses (50,ug/kg) of the toxin were given at 30 and 72 hr after the first dose. Most of the rats survived after this treatment.,8-bungarotoxin (Chang et al. 1973c), 50,ug/kg, was administered into the thoracic cavity as in the case of [3H]cc-bungarotoxin. About one half of the rats so treated died of respiratory failure 1 or 2 days after treatment. Since intrathoracic administration affected both sides of the diaphragm similarly, both hemidiaphragms were used for the assay of toxin-binding sites. RESULTS Relation between the number of end-plate receptors and neuromuscular transmission The diaphragm isolated from normal rats was treated with nm [3H]a-bungarotoxin for 1-2 hr to obtain various extents of neuromuscular blockade. As illustrated in Fig. 1, occupation by the toxin of end-plate

4 164 C. C. CHANG, SING-TAI CHUANG AND M. C. HUANG receptors up to 40% had no visible effect on the neuromuscular transmission when tested by single twitches. When about 5 % blockade occurred, 63 % of the receptors were found to be occupied by the toxin. Thereafter, increasing receptor occupation from 63 to 82 % caused transmission blockade in about 75 % of synapses and complete paralysis occurred when 95 % of the receptors were occupied. This result indicates that the safety factor in most of the neuromuscular junctions of the rat diaphragm for single twitches is 3-5. A similar relationship has been reported for the mouse diaphragm (Barnard et al. 1971). 100 _ 0 (U 80 - U 0 60 a, 40 E 0 z 20 0 ō~~ Receptor occupied (%) Fig. 1. Relation between receptor occupation by a-bungarotoxin and neuromuscular blockade. The normal isolated rat diaphragm was treated in vitro at 370 C with nm (03-1.0,ugfml.) [3H]acetyl ez-bungarotoxin for various times to obtain 0, 5, 75 and 100 % blockade of contraction elicited by single nerve stimulation. The diaphragm was washed for 5 hr and the end-plate receptor bound with the labelled toxin was compared with that at saturation obtained by treating the diaphragm with 125 nm [3H]toxin for 2 hr. Mean ± S.E. (n = 4-5). Effect of hemicholinium-3 Rats were sacrificed 14 hr after the last injection of the 7 day treatment. The diaphragms isolated from the treated rats responded to a single stimulus of the phrenic nerve with contraction % smaller than the normal, and no sustained tetanus was observed on stimulation with train (10 see) of pulses at 50 Hz, indicating that the synaptic transmission was affected by this hemicholinium treatment. Labelling with [3H]a-bungaro-

5 REGULATION OP ACh RECEPTOR16 toxin revealed that significant diffuse toxin-binding had appeared along the whole length of the muscle fibre (Fig. 2). The calculated density of extrajunctional receptors increased from about 6/#sM2 in normal to 38//%m2. The specific binding at the end-plates (segment nos. 5-7) after subtracting that at the non-end-plate zone was increased by about 30 %.The number of toxin-binding sites per end-plate was calculated to be X 107 (s.ie., n = 4) against 2* x 107 (n = 4) -for the normal diaphragm. There was a two- to threefold increase in the total number of E EV100 I 50 2 Semn nme Fig 2 Efecofheicoliiu-3.Ras wretrate wth 0 g/k hmi in s~~~~~~egmentsn.57 numberefrmtecnaledo. -,oto; Fig 2.Efeto hemicholinium--rete. Reat±s~: wer traewih40)gghei acetylcholine receptors. This experiment indicates that, when the neuromuscular transmission is made insufficient by inhibition of the synthesis of neurotransmitter, there are increases of acetylcholine receptors not only at the motor end-plate, but also in the non-end-plate zone. Effect of fl-bunigarotoxin andi denervation Several hours after injection of 50 /,tg/kg f6-bungarotoxin into the thoracic cavity, all of the rats showed severe dyspnoea which lasted for about 2 days. About ~50 % of the treated rats survived. The diaphragms isolated 5 days after toxin injection was found to be still under the influence of fl-toxin and the contractile response elicited by indirect single stimulation

6 166 C. C. CHANG, SING-TAI CHUANG AND M. C. HUANG was only about % (S.D., n = 4) of untreated control. Fibrillation of the diaphragm was also evident. The binding sites for labelled ac-bungarotoxin increased to a much greater extent than that in the animals treated with hemicholinium-3 or c-bungarotoxin and was only slightly less than that produced after denervation for the same period (Fig. 3). The receptor densities in the non-end-plate areas after treatment with f-bungarotoxin and denervation were 104//tm2 and 153/#tm2, respectively. In both cases, E bo 800 o j Control fi-bungaro- Denervated toxin Fig. 3. Induction of acetylcholine receptors by fl-bungarotoxin or after denervation. Observations made 5 days after denervation (n = 4) by cutting the phrenic nerve in the neck, or intrathoracic injection of 50 /.Zg/kg fl-bungarotoxin (n = 10). The diaphragm was cut into three segments, the middle one containing end-plates (shaded column) and the central tendon side on the left. Mean + s.e. the number of receptors in the end-plate area increased more markedly than the number in the non-end-plate area of the muscle. If one assumes that the density of extrajunctional receptors is the same along the length of muscle fibre, the data in Fig. 3 may indicate that the number of acetylcholine receptors at the subsynaptic area and its immediate vicinity have increased after f8-bungarotoxin treatment and denervation by 140 and 220 %, respectively Effect of [3H]ac-bungarotoxin About 3 hr after intrathoracic administration of 150,tg/kg[3H]ccbungarotoxin, dyspnoea occurred and lasted for about 12 hr among most

7 REGULATION OF ACh RECEPTOR 167 of the treated rats. The neuromuscular transmission at 4-9 hr after toxin injection was found to be completely blocked and the receptors on the end-plate area were saturated with the labelled toxin. The concentration of labelled toxin in blood at 9 hr after injection was less than 50 ng/ml. and no paralysis of limbs or neck was noticed. Most of the animals survived and, 24 hr after injection, the neuromuscular transmission upon single stimulation was restored to % of normal. Tetanic stimulation at 50 Hz also gave a rather well sustained contraction. The radioactivity E o Days Fig. 4. Effect of az-bungarotoxin. [3H]acetyl a-bungarotoxin (150/ig/kg) was injected into thoracic cavity at zero time. Diaphragms were isolated 1, 3 and 5 days after injection and further treated in vitro with the labelled toxin to saturate the receptors. Ordinate is the radioactivity in the central segment with end-plate (0-0) or in the outside segments (0-0). Interrupted line (A) denotes the difference between the toxin bindings of central and outside segments except the data for day 1 which was obtained from the radioactivities without further treatment with the labelled toxin in vitro. Means ± S.E. (n = 5-10). remaining bound to end-plate zone of the diaphragm, however, declined by only 8-4 %. Saturation of the receptor in vitro with the labelled toxin did not increase the radioactivity above the control value, indicating that no significant new receptor had appeared. Two days after toxin-injection, neuromuscular transmission of the diaphragm was fully restored, but remained very vulnerable to further treatment with ac-bungarotoxin so that the time needed to induce complete paralysis upon addition of the toxin (1,tg/ml.) in vitro was shortened from 160 min in the normal to 40 min. Up to 5 days after toxin injection, the time needed to cause paralysis was still shorter (85 min). This result is in accord with the long

8 168 C. C. CHANG, SING-TAI CHUANG AND M. C. HUANG half-time (7.5 days) of the toxin bound to the junctional receptors (Chang & Huang, 1975). The toxin binding sites at the two outside segments of non-end-plate area began to increase as early as 1 day after toxin treatment, and continued to increase during the period of 5 day observation (Fig. 4). The density of extrajunctional receptor was calculated to be 51/fIm2 5 days after toxin injection, a value slightly higher than that obtained by hemicholiniumtreatment but much lower than that found after denervation or fl-bungarotoxin treatment. Interestingly, however, the binding sites on the central Soo 400- C E 300 bo Control a-bungarotoxin a-bungaro- toxin One dose Three doses Fig. 5. Effect of cc-bungarotoxin. Rats were treated with single dose (150,ug/ kg) or repeatedly treated at time 0, 30, and 72 hr with 150, 50 and 50 jug/kg [3H]acetyl a-bungarotoxin, respectively, and sacrificed 5 days after the first treatment (n = 8). The diaphragms were saturated with the labelled toxin in vitro. Note that the difference in the number of toxin-binding sites between the end-plate and non-end-plate areas was not changed. Mean ± S.E. segment did not increase until 2 days after toxin treatment. This result may suggest that blockade with cz-bungarotoxin causes generation of extrajunctional acetylcholine receptors beginning at the peripheral muscle tendon region. The number of toxin-binding sites on the synaptic area, as computed by subtracting the number on the non-end-plate zone from that on the end-plate zone, was found to be constant (Fig. 4), indicating that, in contrast to the extrajunctional site, the acetylcholine receptor on the endplate was not increased by blockade with c-bungarotoxin. In other experiments, rats were treated with two additional doses (50,tg/kg) of the labelled az-bungarotoxin at 30 and 72 hr after the first injection of 150,ug/kg of the toxin in order to attain more prolonged neuromuscular insufficiency. The

9 REGULATION OF ACh RECEPTOR16169 increase in the number of toxin binding sites on the non-end-plate region, however, was not significantly greater than that obtained by single treatment with the toxin, and again no increase in the number of endplate receptors was observed (Fig. 5). DISCUSSION The present experiments show that all of the three neuromuscular blocking agents, hemicholinium-3, x- and fi-bungarotoxins, caused changes in the binding sites for labelled az-bungarotoxin. Since non-specific binding of az-bungarotoxin which is reversible (Chang et al a) was excluded by washing, the radioactivity of the treated diaphragms may be regarded as proportional to the number of acetylcholine receptors in the muscle. Treatment with fl-bungarotoxin, which caused the most extensive and prolonged blockade of neuromuscular transmission, induced changes very similar to those that follow surgical denervation. Spontaneous fibrillation of the muscle was evident, and an increase in the number of ac-bungarotoxin-binding sites occurred at both end-plate and non-end-plate zones. These effects of /3-bungarotoxin might be the result of ultrastructural changes of nerve terminals induced by this toxin (Chen & Lee, 1970; Tsai, 1975). Denervation-like changes of acetylcholine supersensitivity by botulinum toxin and fi-bungarotoxin have been reported (Thesleff, 1960; Hoffmann & Thesleff, 1972). In addition to the increase in the number of receptors, denervation and fi-bungarotoxin-treatment also increase the turnover of the end-plate receptors (Chang & Huangr, 1975). The previous studies showing that the sensitivity to iontophoretic application of acetylcholine at end-plate was not significantly increased after denervation (_Miledi, 1960; Albuquerque & Mclsaac, 1970) are not in accord with the increase in the number of receptor sites at the end-plate in our experiments. This might be due to poor resolution in the iontophoretic method, or alternatively the ion conductance modulator which mediates the effect of acetylcholine-receptor interaction (Albuquerque, Barnard, Chiu, Lapa, Dolly, Jansson, Daly & Witkop, 1973) may have not increased in proportion to the number of receptors. Repeated injection with hemicholinium-3 also induced an increase in the number of ca-bungarotoxin-binding sites in both end-plate and non-end plate areas of the diaphragm. The effect, however, was much smaller than that of denervation or /3-bungarotoxin-treatment. Although the dose of hemicholinium-3 used was the highest one tolerated by the rat, the extent of neuromuscular blockade was less than that induced by local application of fl-bungarotoxin or denervation. It is also possible that the difference may be due to different mechanisms involved in the effects of these agents.

10 170 C. C. CHANG, SING-TAI CHUANG AND M. C. HUANG The effect elicited by treatment with c-bungarotoxin appeared to be different from that of other agents or denervation, in that the number of receptors at the end-plate, or in its vicinity, was not increased. Since a-bungarotoxin does not cause any inhibition of acetylcholine release (Chang & Lee, 1963), or change in the motor nerve terminal (Tsai, 1975), the increase in the number of extrajunctional receptors by a-bungarotoxin could not be due to a deficiency of any neurotrophic substances. The increase in the number of extrajunctional receptors by a-bungarotoxin is likely to be the result of muscle inactivity per se which has been considered by various authors as one of the causes of denervation supersensitivity (Drachman & Witzke, 1972; Drachman, 1974; L0mo & Rosenthal, 1972; Cohen & Fischbach, 1973; Jones & Vrbovai, 1970). Indeed, disuse alone may also cause some spread of end-plate receptors (Fishbach & Robbins, 1971). The muscle inactivity caused by c-bungarotoxin may thus increase the number of acetylcholine receptors beginning at the muscle-tendon area, while the receptor induction in the end-plate area was retarded by the intact neurotrophic influence. It is to be noted that, while a-bungarotoxin-treatment caused neuromuscular insufficiency for only the first day, the increase in the number of extrajunctional receptors continued for the 5 days of the observation period. A similar increase in acetylcholine sensitivity was observed after treatment with marcaine by Libelius, Sonesson, Stamenovic & Thesleff (1970). It is interesting in this respect that the inhibition by actinomycin D of the increase in acetylcholine sensitivity (Grampp, Harris & Thesleff 1972) and also the inhibition of increase in the number of acetylcholine receptors (Chang & Tung, 1974) were effective only when the inhibitor was administered within 24 hr after denervation. It may be inferred that the effect triggered by muscle inactivity takes 4-5 days to become fully developed despite subsequent restoration of neuromuscular transmission. The increase in the number of extrajunctional receptors induced by a-bungarotoxin- or hemicholinium-treatment appeared to be smaller than that induced by denervation or f8-bungarotoxin. Since the repetitive administration of z-bungarotoxin did not significantly further increase the change, the difference between these blocking agents may not be due simply to the extent of neuromuscular block attained. Whether or not the motor nerve is intact seems to be intimately related to the extent of induction of acetylcholine receptor. It may be suggested that more than one mechanism is involved in this control. Denervation is known to cause changes in many electro-physiological properties of muscle membranes in addition to an increase in extrajunctional acetylcholine sensitivity (Thesleff, 1974), and possible mechanisms for these neurotrophic interactions have been discusssed recently (Drachman, 1974; Albuquerque

11 REGULATION OF ACh RECEPTOR 171 Warnick, Sansone & Onur, 1974). By comparison of the effect of denervation, fl-bungarotoxin and hemicholinium on one hand, with that of azbungarotoxin on the other, it seems likely that the number of acetylcholine receptors at the end-plate is regulated by mechanisms different from that which regulates extrajunctional receptors. Findings such as the increase in the number of end-plate receptors by hemicholinium, but not by x- bungarotoxin, and the decrease by neostigmine (Chang et al. 1973b; Fambrough et at. 1973) may suggest that the concentration of acetylcholine in the synaptic cleft is one of the determining factors. It is possible that this acts as a self-regulatory mechanism to maintain the neuromuscular transmission. Another finding worth mentioning is the change in the safety margin for neuromuscular transmission. In the normal diaphragm, occupation of about 80 % of the synaptic receptors by ac-bungarotoxin was shown to cause neuromuscular blockade in the majority of junctions. At 24 hr after injection of ac-bungarotoxin, less than 10 % of the receptor was freed whereas neuromuscular transmission was restored in about 80 % of the junctions, indicating that the efficiency of receptor was increased. It remains to be elucidated whether this is due to an increase of transmitter release or to increased efficiency of the ion conductance modulator. The authors are grateful to Mr T. P. Hsu for his assistance in the preparation of the manuscript. This work was supported by grants from the National Science Council, Republic of China. REFERENCES ALBUQUERQUE, E. X., BARNARD, E. A., CHiu, T. H., LAPA, A. J., DoLLY, J. O., JANSSON, S. E., DALY, J. & Wnr'op, B. (1973). Acetylcholine receptor and ion conductance modulator sites at the murine neuromuscular junction: Evidence from specific toxin reactions. Proc. natn. Acad. Sci. U.S.A. 70, ALBUQuERQulE, E. X. & McIsA~c, R. J. (1970). Fast and slow mammalian muscle after denervation. Expl Neurol. 26, ALBuQUERQuE, E. X., WARNICK, J. E., SANsoNE, F. M. & ONAR, R. (1974). The effects of vinblastin and cholchicine on neural regulation of muscle. Ann. N.Y. Acad. Sci. 228, AXELSSON, J. & THsLEIFF, S. (1959). A study of supersensitivity in denervated mammalian skeletal muscle. J. Phyaiol. 147, BARNARD, E. A., WIEcKowsK, J. & C=ir, T. H. (1971). Cholinergic receptor molecules and cholinesterase molecules at mouse skeletal muscle junctions. Nature, Lond. 234, BERG, D. K., KELLY, R. B., SARGENT, P. B., WILLIAMSON, P. &THTAT-T, Z. W. (1972). Binding of a-bungarotoxin to acetylcholine receptors in mammalian muscle. Proc. natn. Acad. Sci. U.S.A. 69, CHANG, C. C., CHEN, T. F. & CmLANG, S. T. (1973a). N, O-Di and N,N,O-tri[3H] acetyl a-bungarotoxins as specific labelling agents of cholinergic receptors. Br. J. Pharmac. 47,

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