Accelerated degradation of junctional acetylcholine receptor-α-bungarotoxin complexes in denervated rat diaphragm

Brain Research, 233 (1982) 133-142 Elsevier BiomedicalPress

133

ACCELERATED DEGRADATION OF JUNCTIONAL ACETYLCHOLINE RECEPTOR-a-BUNGAROTOXIN COMPLEXES IN DENERVATED RAT DIAPHRAGM

R. S. BRETT, S. G. YOUNKIN, M. KONIECZKOWSKIand R. M. SLUGG Case Western Reserve University, School of Medicine, Department of Pharmacology, Cleveland, OH 44106 (U.S.A.)

(Accepted October 9th, 1981) Key words: turnover--junctional acetylcholinereceptors -- denervation

SUMMARY [125I]a-bungarotoxin was administered to rats in vivo to label acetylcholine receptors in innervated diaphragm, 5-day denervated diaphragm, or diaphragm which had been denervated immediately before labeling. The rate of degradation of junctional toxin-receptor complexes was followed by sacrificing animals at various times after labeling. The rate of degradation of junctional toxin-receptor complexes was significantly faster in 5-day denervated left hemidiaphragm (t 1/2 = 2.0 days) than in innervated left hemidiaphragrn (t 1/2 -- 10.7 days). The rate of degradation of junctional toxin-receptor complexes in left hemidiaphragm denervated at the time of labeling was essentially identical to that in innervated muscle for 3 days but then increased to a significantly more rapid rate (t 1/2 ---- 3.7 days in the period 3-13 days after denervation and labelling). These findings support the concept that continuous innervation is needed to maintain the metabolic stability of junctional acetylcholine receptors.

INTRODUCTION Acetylcholine receptors (AChR) are distributed over the entire surface of embryonic muscle fibers. As these fibers become innervated, the number of AChR increases in junctional membrane and decreases in extrajunctional membrane so that adult skeletal muscle fibers have a very high density of AChR in junctional membrane and a low density in extrajunctional membranel,4,5,7,17,is. Junctional AChR differ from extrajunctional AChR in that junctional AChR have a half-life of at least 6 days whereas embryonic AChR and the AChR which appear in the extrajunctional

134 membrane of adult muscle after denervation have a half-life of less than 1 day2,~,s,9, 11,t3,16,20. TO evaluate the concept that continuous innervation is needed to maintain the metabolic stability of junctional AChR, we examined junctional AChR in innervated and denervated rat diaphragms using a method for the in vivo labeling of AChR with [lzSI]a-bungarotoxin similar to that described by Berg and Hall 3. If continuous innervation is needed to maintain the metabolic stability of junctional AChR then denervation would be expected to increase the rate of degradation of junctional toxinreceptor complexes. Our results, which have been presented in abstract form6, indicate that denervation dramatically increases the rate of degradation of junctional toxinreceptor complexes. MATERIALS AND METHODS

Experiments were performed on 250-350 g male Wistar rats. The left hemidiaphragm was denervated by sectioning the intrathoracic portion of the left phrenic nerve after exposing it with a glass hook inserted through a small incision in the thorax. AChR were labeled in vivo by injecting 1.0 ml of [125I]a-bungarotoxin (1.0 #g/ml in rat Ringer's solution, 10-20/~Ci//~g, New England Nuclea0 into the left thoracic cavity and maintaining the animals in a vertical position overnight. The rats tolerated this procedure well and virtually all animals survived. AChR in the left hemidiaphragm were usually labeled slightly better than those in the right hemidiaphragm with this procedure (see Table I). AChR were labeled in vitro by incubating segments 0.5 cm wide for 2 h at room temperature in mammalian Ringer's solution containing 1.5 #g/ml [125I]a-bungarotoxin. At appropriate times after labeling AChR in vivo, diaphragms were removed and washed overnight (4 changes of solution containing 0.15 M NaCI, 0.4 mM EGTA, 0.02 M Tris-HCl, ph 7.4) to remove unbound toxin. A number of segments of equal width were then dissected by cutting parallel to the muscle fibers. Muscle in the region 3 mm anterior or posterior to the insertion of the phrenic nerve into the diaphragm was discarded because the pattern of innervation in that region is complex and endplates are not confined to the central portion of the muscle fibers. One segment was always taken from the anterior region of the light innervated hemidiaphragm and 2-4 segments of the same width were taken from the left hemidiaphragm. In the initial experimentation in which innervated or 5-day denervated left hemidiaphragms were examined 5 days after labeling, 4 segments were taken from the left hemidiaphragm. In subsequent experimentation in which 5-day denervated left hemidiaphragms were examined 1, 3, 5 and 8 days after labeling, 2 segments were taken from the left hemidiaphragm. In the experimentation in which the left hemidiaphragm was denervated and the diaphragm immediately labeled, 4 segments were again taken from the left hemidiaphragm. In one series of experiments the spatial distribution of AChR in the segment between 3 and 13 mm anterior to the point of phrenic nerve entry was examined in

135 innervated and 5-day denervated left hemidiaphragms 5 days after labelling in vivo. In those experiments (Fig. 1) each segment was dissected into 5 one-mm-wide strips (strips 2-6, Fig. 1) by cutting across the muscle fibers and parallel to the phrenic nerve (contained in strip 3). The areas of muscle between the central tendon and strip 2 and between strip 6 and the costal o~igin of the diaphragm (designated 1 and 7 respectively) were also taken for analysis. In all of the experimentation in which animals were denervated for 5 days and then labeled, at least two strips were obtained from each segment by cutting across the muscle fibers and parallel to the phrenic nerve. One strip contained the phrenic nerve and virtually all of the endplates. The other strip was an adjacent strip of equal width dissected from the central tendon side and contained essentially no endplates. The toxin bound to the strip on the central tendon side of the phrenic nerve was subtracted from that bound to the endplate-bearing strip to correct for toxin bound to extrajunctional AChR in the endplate-bearing strip. This correction was substantial 1 day after labeling denervated muscle (61 ~ of total binding to the endplate-bearing strip) but became less important by 3 days after labeling (25 ~ of total binding to the endplate-bearing strip). The correction for extra-junctional toxin-binding was trivial in innervated segments ( < 3 9/0) or in denervated segments in which toxin-receptor complexes had been allowed to decay for 5 days or more ( < 9 9/0). The fact that there was very little toxin bound to the strips on the central tendon side of the nerve under these circumstances showed that these strips were essentially endplate-free. In the experiments in which the left hemidiaphragm was denervated and the diaphragms immediately labeled, whole segments were counted without dividing the muscle into strips because no correction for binding to extra-junctional AChR was necessary. Segments and strips of hemidiaphragm were counted with a gamma counter (Beckman Biogamma II) in almost all experiments. In a few of the early experiments muscle was solubilized and counted using conventional liquid scintillation spectrometry. To minimize the effect of animal to animal variability in toxin binding the junctional toxin bound to each denervated left hemidiaphragm segment was expressed as a fraction of the junctional toxin bound to the innervated right hemidiaphragm segment from the same animal. The mean and S.E. of the fractional binding at each time after labeling was then computed and multiplied by the mean right sided binding at the same time after labeling (shown in Fig. 2) to give normalized values for left-sided toxin binding. In some experiments the toxin bound to endplate AChR was evaluated by microdissecting labeled endplates as described by Robbins et al. 21. Segments of innervated and 5-day denervated left hemidiaphragm labelled in vitro or for 1 day in vivo were washed overnight to remove unbound toxin (4 changes of a solution containing 0.15 M NaCI, 0.4 mM EGTA, 0.02 M Tris-HCl, pH 7.4) and fixed for 90 min in 4 ~o glutaraldehyde. The fixed segments were then placed in 5.0 ml of water, dissociated into single fibers with a Polytron (Brinkmann) operated at low speed, and stained 1 h for acetylcholinesteraselL Stained endplates (mean length ---- 80 #m) and immediately adjacent extrajunctional pieces of similar size were then dissected from at least 20 single fibers of each muscle, measured for length, and counted in a gamma counter

136 (Beckman, Biogamma II). A correction for the small amount of toxin bound to the perijunctional region of the endplate-bearing piece was then made on a per length basis by subtracting extrajunctional binding from the total binding in the endplatebearing pieces. The correction for perijunctional toxin binding was never more than 20 % of the total toxin-binding in the endplate-bearing piece (Table 1I). The absolute amount of toxin bound to endplate AChR was calculated on the basis of the commercially stated toxin concentration and was determined by counting aliquots of toxin at the time of experimentation. RESULTS

[125I]a-bungarotoxin was administered in vivo to normal rats and to animals whose left hemidiaphragm had been denervated for 5 days. Five days later the spatial distribution of toxin-receptor complexes was examined in the innervated and denervated hemidiaphragms. The results of this experiment are shown in Fig. 1. Because of the rapid rate of degradation of extrajunctional toxin-receptor complexes virtually all of the toxin remaining bound to denervated muscle was associated with the more slowly degraded junctional AChR in the endplate region (compare innervated and denervated muscle, Fig. 1). In Fig. 1 it can be seen that 5 days after in vivo labeling, the

Strip number I

2

3

i

567

4 i

i

t

i

12

Inn. I0 Ira

. . . . . 5-day den

i

[

8

I

-

6

Distance from central tendon (ram)

Fig. 1. Spatial distribution of toxin-receptor complexes remaining in innervated and 5-day denervated rat hemidiaphragms 5 days after in vivo administration of [l~sI]a-bungarotoxin. The spatial distribution of toxin-receptor complexes in innervated or denervated left hemidiaphragm was evaluated by removing the segment between 3 and 13 mm anterior to the point at which the phrenic nerve enters the muscle and dissecting it into 5 one-mm-wide strips (strips 2-6 on abscissa) by cutting parallel to the intramuscular phrenic nerve. The wider areas of muscle between the central tendon and strip 2 and between strip 6 and the costal origin of the diaphragm (designated 1 and 7 respectively) were also taken for analysis. The toxin (fmoles) bound to strips cut at various distances from the intramuscular phrenic nerve is plotted at the midpoint of each strip.

137 TABLE I

[lz5IJa-bungarotoxin bound to diaphragm muscles 5 days after in vivo application (fmol/ep-containing region) Numbers are the mean ± S.E. Numbers in parentheses refer to the number of animals and the number of hemidiaphragm segments examined.

Expt. no.

1 2 3 Mean

Innervated Rats

Rats denervated 5 days prior to toxin application

Innervated left hemidiaphragm

Innervated Right hemidiaphragm

Denervated left hemidiaphragm

Denervated right hemidiaphragm

10.8 13.2 14.7 12.6

10.0 10.6 11.6 10.6

0.9 3.6 2.5 2.2

9.3 11.8 11.0 10.8

:k 0.6 :k 1.9 ::k 0.9 ± 0.6

(6,24) (6,12) (4,16) (16,52)

± 4:k :k

1.4 1.6 1.2 0.8

(6,6) (6,6) (4,4) (16,16)

:k :t: :k :k

0.1 0.8 0.3 0.2

(5,20) (6,12) (6,24) (17,56)

4:k ± :k

2.1 2.7 1.5 1.2

(5,5) (6,6) (6,6) (17,17)

toxin bound to the endplate region of denervated hemidiaphragms was only 21 ~o of that bound to the endplate region of innervated hemidiaphragms (P < 0.001 by Student's t-test). This result was reproducible. Table I shows that, in each of 3 experiments, the toxin bound to the endplate region of denervated left hemidiaphragms was dramatically less than that bound to the endplate region of innervated left hemidiaphragms. The mean toxin bound to the endplate region of denervated hemidiaphragms was 17~ of that bound to the endplate region of innervated hemidiaphragms (P < 0.001 by Student's t-test). The toxin bound to the endplate region of right innervated hemidiaphragms was essentially the same in normal animals and in those whose left hemidiaphragms had been denervated (Table I). Three possible explanations may be offered to account for the decrease in junctional toxin binding after denervation: (1) denervation decieases the number of junctional toxin binding sites; (2) denervation produces changes in muscle which interfere with the in vivo labeling of junctional AChR by [l~sI]a-bungarotoxin; (3) denervation causes an increase in the rate of degradation of junctional toxin-receptor complexes. These possibilities are not mutually exclusive so we examined each of them. To measure the number of junctional AChR present in denervated muscle, one must distinguish the junctional AChR in endplates from extrajunctionai AChR. We did this by microdissecting endplates from single muscle fibers using a method suggested to us by Robbins et al. 21. Segments of innervated and 5-day denervated hemidiaphragm were dissected, labeled with [125I]a-btmgarotoxin under saturating conditions, washed to remove unbound toxin, fixed in glutaraldehyde, dissociated into single fibers using a Polytron (Brinkman) operated at low speed, and the dissociated fibers stained for acetylcholinesterase. The stained endplates from at least 20 fibers of each segment were then dissected (mean length = 80 #m), pooled, and counted. A small correction was made for the toxin bound to extrajunetional AChR as described in Materials and Methods. Denervation for 5 days did not reduce junctional toxin-

138 TABLE II

[1251]a-bungarotoxin bound to junctional AChR in innervated and 5-day denervated muscle Numbers sh~,vn are the mean ~. S.E.

Junctional toxin binding ( M ;< 1017 ,;12517aBT/EP)

Innervated muscle labeled in vitro 5-Day-denervated muscle labeled in vitro Innervated muscle labeled in vivo 5-Day denervated muscle labeled in vivo

Correctionrequired for extrajunctionalbinding binding in + EP segment (%)

Number of muscles examined

Number of fibersexamined

1.51 -k_ 0.12

6.4

7

210

1.85 ± 0.14

14.9

7

179

0.44 ± 0.11

0.0

5

332

0.23 :~: 0.03

20.0

6

272

binding as measured by this technique. Table II shows that innervated muscle bound 1.51 x 10-17 M a-bungarotoxin per endplate and that 5-day denervated muscle bound 1.85 x 10-17 M per endplate. The apparent increase in junctional AChR in the denervated muscle was not quite significant (P = 0.09) in this series of experiments. This result indicated that the profound decrease in junctional toxin-binding in denervated muscle shown in Table I did not occur because of a loss of junctional AChR. To determine if denervation produces changes which interfere with the in vivo labelling of AChR, hemidiaphragms were labeled in vivo and 1 day after labeling, segments were removed and the junctional AChR in single fibers assessed as described above. Table II shows that one day after in vivo labeling innervated muscle bound 0.44 )< 10-17 M of toxin per endplate and that 5-day denervated muscle bound 0.23 x 10-7 M per endplate. It appeals, therefore, that denervation produces changes in muscle which interfere with the in vivo labeling of junctional AChR. The decreased binding of toxin to denervated junctional AChR may reflect competition between extraj unctional and junctional AChR for available toxin or may be the result of impaired diffusion of toxin to the junctional area. In addition, accelerated degradation of junctional toxinreceptor complexes in the denervated muscle during the one day before measurement may have contributed to the reduction in junctional toxin-receptor complexes. A denervation-induced interference with in vivo labeling of junctional AChR does not, however, fully account for the reduction in junctional toxin-receptor complexes shown in Table 1. One day after in vivo labeling, 5-day denervated muscle contained 52 ~ of the junctional toxin-receptor complexes found in innervated muscle (Table II). By 5 days after labeling, denervated muscle contained only 17 ~ of the junctional toxinreceptor complexes found in innervated muscle (Table I). This result suggests that junctional toxin-receptor complexes are degraded more rapidly in denervated than in innervated muscle. To confirm this, innervated and 5-day denervated rat hemidiaphragms were labeled in vivo and toxin binding was examined 1, 3, 5 and 8 days after

139

:30.0

Junctional toxin receptor complexes in inhraqm; i"1/2 : 10.7 days

A.

I0.0 1 ~

50 3.0

Itn

1.0

I

0.5

~

JunchonoL toxin-receptor complexes in mmidiaphragm; 1l/2 = H.9 days

duncttonal toxin-receptor "complexes m left hero,diaphragm denervated for 5 days ~ T before~m administration; 1'1/2= 2 0 days i

~

E LI-

50.0

g. Junctional toxin-receptor complexes in inmidiophragm; t l / 2 = It 9days

I0.0 50 3,0

~ldlophragm

denervated ,madministration ;

mediatelyh ~ ~ n

tl,'2 (day 5- 3}= 3.7days

Days after in vivo administration of 125I-~BT Fig. 2. The rate of degradation of junctional toxin-receptor complexes in innervated and denervated rat hemidiaphragms. A" rate of degradation of junctional toxin-receptor complexes in innervated right hemidiaphragrn, innervated left hemidiaphragrn, and left hemidiaphragm denervated 5 days prior to toxin administration. B: rate of degradation of junctional toxin-receptor complexes in innervated right hemidiaphragm and left hemidiaphragm denervated immediately before toxin administration. A single segment was examined in each innervated right hemidiaphragm. The number of right hemidiaphragms examined at 1, 3, 5, 8 and 13 days was 16, 17, 38, 15 and 3 respectively. Two segments were examined in each innervated left hemidiaphragm, and a total of 12 segments from 6 muscles was examined at each time following toxin administration. The number of segments examined in left hemidiaphragms denervated 5 days prior to toxin administration was as follows: day 1, 10 segments from 5 muscles; day 3, 12 segments from 6 muscles; day 5, 56 segments from 17 muscles; day 8, 10 segments from 5 muscles. The number of segments examined in left hemidiaphragms denervated immediately before toxin administration was as follows: day 1, 20 segments from 5 muscles; day 3, 20 segments from 5 muscles; day 5, 20 segments from 5 muscles; day 8, 20 segments from 5 muscles; day 13, 12 segments from 3 muscles. Points represent the mean 4- S.E. of the toxin bound to diaphragmatic segments at various times after labeling. The values for toxin bound to left denervated muscle were normalized with respect to the toxin bound to the innervated right side as described in the text.

140 labeling (6, 8, 10 and 13 days after denervation). The toxin bound to junctional AChR was calculated as the difference between the total toxin bound to a central endplatecontaining muscle strip and that bound to an immediately adjacent endplate-free strip on the central tendon side. This correction for extrajunctional toxin binding was substantial 1 day after labeling (61 ~ of total binding to the endplate-containing strip), became less important by 3 days (25 %o of total binding to the endplate-containing strip), and was trivial at later times. The results of this experiment are shown in Fig. 2A. The degradation of junctional toxin-receptor complexes in innervated muscle occurred at a rate similar to that reported previous[y3,S,11,13,20and was essentially the same in muscle of the left and right sides (t 1,/2 -~ 10.7 days in innervated left hemidiaphragms; t 1,/2 = 11.9 days in innervated right hemidiaphragms). The degradation of junctional toxin-receptor complexes in denervated left hemidiaphragms, however, was markedly accelerated (t 1,/2 ~ 2.0 days; P < 0.001 with respect to either left or right innervated hemidiaphragms). The foregoing results suggest that two factors account for the reduction of junctional toxin-receptor complexes in denervated muscle shown in Table I: decreased binding of toxin to junctional AChR and accelerated degradation of junctional toxinreceptor complexes. In order to examine the effect of denervation on the degradation of junctional AChR independently of alterations in toxin binding, we carried out the in vivo labeling procedure in a separate group of animals immediately following denervation of the left hemidiaphragm. This experiment had the additional important advantage that no correction for toxin binding to extrajunctional AChR was necessary because junctional AChR were labeled before the emergence of extrajunctional AChR. The results are shown in Fig. 2B. The degradation of junctional toxin-receptor complexes in denervated left hemidiaphragms was apparently identical to that in innervated hemidiaphragms during the first 3 days following denervation but subsequently increased to a significantly more rapid rate with a t l/2 of 3.7 days (P -< 0.005 with respect to either left or right innervated hemidiaphragms). DISCUSSION Taken together, the results shown in Fig. 2 provide strong evidence that denervation increases the rate of degradation of junctional toxin-receptor complexes. Our results (Fig. 2B) indicate that the accelerated degradation of junctional toxinrecepto~ complexes does not begin immediately, is just detectable 5 days after denervation, and only becomes obvious 8-13 days after denervation. In a previous study Berg and Hail a concluded that denervation had no effect on junctional toxinreceptor complexes, but these authors concentrated on the first 5 days after denervation, and this probably accounts for their failure to detect any effect of denervation on junctional toxin-receptor complexes. Berg and Halla did report on one series of experiments in which they examined junctional toxin-receptor complexes in 5-day denervated muscle 5 days after labeling in vivo (10 days after denervation). Although they do not comment on it, their results (Table 1, p. 779 of ref. 3) show a significant decrease in toxin-receptor complexes in denervated muscle under these conditions.

141 In recent experiments conducted independently of ours, Loring and Salpeter 15 used EM autoradiography to evaluate junctional A C h R in denervated muscle. These authors found that the density of junctional A C h R is essentially the same in innervated and 8-day denervated rat sternomastoid. They then evaluated the turnover of junctional A C h R in innervated and 8-day denervated muscle by blocking A C h R with cold bungarotoxin and following the appearance of new junctional AChR. Using this methodology, they found that denervation accelerates the turnover of junctional A C h R in rat sternomastoid in much the same way as reported here in rat diaphragm. One of the most striking features of junctional A C h R in adult innervated skeletal muscle is their metabolic stability. The long-half life of junctional A C h R could be a fixed characteristic of adult skeletal muscle or could reflect a continuing influence of innervation. The fact that denervation accelerates the rate of degradation of junctional toxin-receptor complexes supports the concept that continuous innervation is needed to maintain the stability of junctional AChR. The density of junctional AChR, on the other hand, has been shown in previous studies 1°,14,15,19 to be relatively independent of continuous innervation. The present results imply, therefore, that the density and turnover of junctional A C h R are to some extent independently regulated. The influence of nerve on the stability of junctional A C h R could be mediated by the activity set up in muscle by nerve or by a t r o p h i c factor(s) delivered to muscle by nerve. In a previous study ~2 we described a factor which increased the extrajunctional A C h sensitivity of denervated organ-cultured adult skeletal muscle and provided evidence that this factor had the characteristics expected of a t r o p h i c factor (i.e. presence in nerve, movement by axonal transport, and release from nerve). It is interesting to speculate that the increase in extrajunctional ACh sensitivity observed in that study might have been due to stabilization of A C h R by a neural factor which normally stabilizes junctional AChR.

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142 10 Frank, E., Gautvik, K. and Sommerschild, H., Cholinergic receptors at denervated mammalian motor endplates, Acta physioL scand., 95 (1975) 66-76. 11 Heinemann, S., Merlie, J. and Lindstrom, J., Modulation of acetylcholine receptor in rat diaphragm by anti-receptor sera, Nature (Lond.), 274 (1978) 65-68. 12 Karnovsky, M. J. and Roots, L., A 'direct-coloring' thiocholine method for cholinesterase, J. Histochem. Cytochem., 12 (1964) 219-221. 13 Linden, D. C. and Fambrough, D. M., Biosynthesis and degradation of acetylcholine receptors in rat skeletal muscles. Effects of electrical stimulation, Neuroscience, 4 (1979) 527-538. 14 Loring, R. G. and Salpeter, M. M., 1-125-a-bungarotoxin binding to denervated muscle: a survey study using light and EM autoradiography, Neurosci. Abstr., 4 (1978) 604. 15 Loring, R. H. and Salpeter, M. M., Denervation increases turnover rate of junctional acetylcholine receptors, Proc. nat. Acad. Sci. (U.S.A.), 77 (1980) 2293-2297. 16 Merlie, J. P., Changeux, J. P. and Gros, F., Acetylcholine receptor degradation measured by pulse chase labeling, Nature (Lond.j, 264 (1976) 74-76. 17 Miledi, R., Junctional and extrajunctional ACh receptors in skeletal muscle fibers, J. Physiol. (Lond.), 151 (1960)24-30. 18 Miledi, R. and Potter, L. T., The acetylcholine receptors in muscle fibers, Nature (Lond.), 233 (1971) 599-603. 19 Porter, C. W. and Barnard, E. A., Distribution and density of cholinergic receptors at the motor endplates of a denervated mouse muscle, Exp. NeuroL, 48 (1975) 542-556. 20 Reiness, C. G., Weinberg, C. B. and Hall, Z. W., Antibody to acetylcholine receptor increases degradation of junctional and extrajunctional receptors in adult muscle, Nature (Lond.), 274 (1978) 68-70. 21 Robbins, N., Olek, A., Kelly, S. S., Takach, P. and Christopher, M., Quantitative study of motor endplates in muscle fibers dissociated by a single procedure, Proc. roy. Soc. B, (1981) in press. 22 Younkin, S. G., Brett, R. S., Davey, B. and Younkin, L. H., Substances moved by axonal transport and released by nerve stimulation have an innervation-like effect on muscle, Science, 200 (1978) 1292-1295.