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The sequence of changes in cholinesterase activity during reinnervation of muscle
l&329-336
EXPERIMENTALNEUROLOGY
The
Sequence
of
During
(1965)
Changes
in Cholinesterase
Reinnervation
of Muscle
LLOYD GUTH AND WILLIAM
C. BROWN’
Laboratory of Neuroanatonaical Sciences, National Institute and Blindness, National Institutes of Health, Public Health of Health, Education, and Welfare, Bethesda, Received
February
Activity
16,
of Neurological Service, U. S. Maryland
Diseases
Department
1965
The following evidence supports the view that cholinesterase (ChE) activity of muscle is regulated by its nerve supply: denervation produces a more rapid decrease in ChE than in total protein, and tenotomy produces loss of protein without change in ChE. The present study concerns changes in ChE during regeneration of nerve fibers into denervated muscle. In twenty rats the nerve to the sternomastoid was crushed at the muscle. Alternate, frozen, serial, crosssections of muscle were stained histochemically or assayed quantitatively for ChE. Sole-plate ChE was distinguished from background or non-sole-plate ChE by comparing regions of muscle having sole plates with regions of muscle lacking them. Background ChE and sole-plate ChE both decreased to 68% of normal at 1 week postoperatively and increased gradually to 93% of normal by 6 weeks. The ratio of background ChE to sole-plate ChE was constant at 1, 3, and 6 weeks, indicating a similar rate of reappearance of enzyme at both sites. Inasmuch as reinnervation of the muscle was probably complete at 2 weeks, the subsequent increase in ChE probably reflects a slow rate of synthesis rather than a slow addition of newly reinnervated muscle fibers. This slow, gradual synthesis following reinnervation contrasts markedly with the rapid decrease in ChE (40% in 3 days) following denervation. Total protein showed no similar gradual restoration, being about 93% of normal 1, 3, and 6 weeks postoperatively.
Introduction Denervation of a muscle results in a characteristic atrophy and loss of protein (7). However, it cannot be concluded that the innervation exerts a specific regulatory function over protein metabolism, for muscle atrophy can alsobe produced by disuse (2 ) , immobilization (4)) or tenotomy (2,4), i.e., without alteration of the innervation. Nevertheless, there is evidence 1 The authors gratefully acknowledge the statistical assistance Mosimann (Biometrics Branch, NINDB) and the many helpful R. Wayne Albers (Laboratory of Neurochemistry, NINDB). 329
of Dr. James E. suggestions of Dr.
330
GUTH
AND
BROWN
that the nerve is specifically related to at least one muscle protein: cholinesterase. First of all, denervation produces a more prompt decrease in cholinesterase than in other muscle proteins. Secondly, although both tenotomy and denervation result in a rather similar decrease in total protein, tenotomy has no effect on cholinesterase activity of muscle (5). It has been shown that denervation produces an equal decrease in ChE at the sole plate and in regions containing no sole plates (5). Thus it appears that the ChE is primarily muscular rather than neural in localization, and that the regulatory effect of the nerve is exerted throughout the muscle fiber and not merely at the site of the nerve terminations. Considering that the cell nucleus plays a key role in the regulation of protein synthesis, and recognizing that the skeletal muscle fiber is a multinucleated cell characterized by aggregation of nuclei at the sole plate, it consequently seems pertinent to inquire whether reinnervated muscle will form cholinesterase at the sole-plate region prior to forming it at other regions. In the present experiment we have determined the changes in sole-plate and in background ChE activity at intervals of 1, 3, and 6 weeks after crushing the nerve to the sternomastoid muscle. In an effort to understand the mechanism by which the nerve regulates the ChE metabolism of muscle we have compared the rate of disappearance of ChE following denervation with the rate of reappearance following reinnervation, and the changes in sole-plate ChE with those of background ChE. Materials
and
Methods
Operative Procedure. Random-bred female, Osborne-Mendel rats ( 175200 g) were anesthetized with chloral hydrate (400 mg/kg). A midline incision was made in the ventral aspect of the neck and the sternomastoid muscle exposed. The nerve to this muscle was crushed by means of a fine forceps (nonserrated, blade width less than 0.5 mm). The operation was performed unilaterally, half of the operations being done on the right and half on the left sides. Eight rats were killed at 7 days, seven at 21 days, and six at 42 days postoperatively. Histological Procedure. The muscles of the operated side and the contralateral unoperated side were removed, weighed, stretched to approximately normal length in a U-shaped glass frame, and frozen rapidly by immersion in cold (-50 C) N-methylbutane. Frozen cross-sections of the muscle were cut at 28 p. Serial sections were taken in groups of four. The first and fourth sections
CHOLINESTERASE
IN
MUSCLE
331
were placed on a glass slide for cholinesterase histochemistry, and the second and third were combined in a single ‘test tube for quantitative biochemical analysis. In this way the two sections on the slide served as histological controls for the sections in the test tube. The succeeding fourteen sections were discarded, following which the procedure was repeated. This sequence was followed from the rostra1 to the caudal end of the muscle so that the entire length of the muscle was surveyed biochemically and histochemically at approximately OS-mm intervals. The slides were fixed in unbuffered 3% glutaraldehyde and stained for cholinesterase by a thiolacetic acid procedure ( 10). The quantitative analysis of the sections in the test tubes was by adaptation of Ellman’s (3) method to a microchemical scale (5). The sections were incubated in phosphate-buffered acetylthiocholine, and the thiocholine resulting from the esterase action was converted to a colored product by addition of dithiobisnitrobenzoate. Optical density was determined spectrophotometritally. Protein determinations were done by Lowry’s method (9). The histochemical and biochemical methods outlined here have recently been described in detail (5). Results
Total Cholinesterase Activity of the Muscle. In the discontinuous serial section method that we employed, two sections were studied quantitatively, two histochemically and fourteen discarded. Consequently quantitative determinations were made on two of eighteen sections or one-ninth of the muscle. Total cholinesterase activity was computed by obtaining the sum of the activity of all twenty samples from each muscle and multiplying by nine. The total protein content of the muscle was obtained similarly. The specific activity was obtained by dividing the ChE activity by the protein content for each muscle. These measures are summarized in Table 1. The ChE activity decreased to approximately 66% of normal at 1 week postoperatively and increased to about 90% of normal by 6 weeks. The total protein showed no such gradual restoration, being about 93% of normal at 1, 3, and 6 weeks. The specific activity was normal by 6 weeks only because both the protein and the ChE values were equally diminished at this time. Because the operation produced differential changes in ChE and protein, it seems more meaningful to describe the changes in ChE per muscle and not on the basis of total protein (i.e., specific activity). Background and Sole-plate ChE Activity. The total ChE activity may be considered as representing the contribution of the background ChE
332
TOTAL
GUTH
CONTENT
Time One
crushing Three
Normal
6.43 23.76 0.275
0 The specific activity vidually for each animal.
after
week
Oper.
100
BROWN
TABLE 1 ACTIVITY (MICROMOLES/MUS~LE/HOUR) (mg/MuscLE) OF THE STERNOMASTOID
CHOLINESTERASE
Total ChE activity Total protein Specific activitya
AND
Oper.
the average
PROTEIN
to sternomastoid
weeks
Six weeks
Normal
8.24 26.77 0.314
9.58 25.71 0.3 79
represents
nerve
AND TOTAL MUSCLE
Oper.
10.33 28.52 0.3 73
of the specific
Normal
10.65 23.12 0.462
activities
calculated
11.60 24.88 0.466 indi-
r
25
50
75 NUMBER
too
125
OF SOLE
PLATES
150
175
200
1. Cholinesterase activity of tissue sections plotted as a function of the number of sole plates counted in the adjacent histochemically-stained preparations. This graph has been prepared from the data of one rat whose sternomastoid nerve was crushed unilaterally 7 days previously. Inasmuch as the sole-plate counts were prepared from tissue sections, the abscissa actually represents the number of sole-plate fragments that were counted rather than the number of entire sole plates. Note the units for the ordinate: millimicromoles per sample per hour. In Figs. 3, 4, 6 and Table 1 of our previous communication (5) these units were incorrectly desribed as micromoles rather than as millimicromoles per sample per hour. FIG.
CHOLINESTERASE
IN
333
MUSCLE
(i.e., not associated with sole plates) and sole-plate ChE activity. The background activity was determined as follows. For both muscles of each animal, a scatter graph was prepared in which the ChE activity of each test tube was plotted as a function of the number of sole plates counted in the adjacent histochemically-stained sections. A typical graph is presented in Fig. 1. Fourteen of the twenty-four graphs appeared perfectly linear, and ten showed a slight tendency to flatten at values greater ,than 100 sole plates. Linear regression lines (of ChE activity on sole plates) were comTABLE 2 CHOLINESTERASE ACTIVITY (MICROMOLES/MUSCLE/HOUR) OF THE STERNOMASTOID MUSCLE Time
after
One week Oper. Background ChE activity Total sole-plate activity % Change background ChEa % Change sole-plate ChEa Background Ratio
nerve
Three
Normal
4.55 1.88
crushing
6.91 2.67
Oper.
weeks
Six weeks
Normal
Oper.
Normal
7.22 3.11
7.54 3.11
8.13 3.47
5.66 2.58
66 70
to sternomastoid
78 88
93 92
ChEa
of sole-p!ate
ChE
2.64
2.63
2.26
2.49
2.47
a The percentage change in background activity, the percentage change activity and the ratio of background to sole-plate activity was calculated for each animal. The averages obtained from these figures are presented
2.44 in sole-plate individually in the table.
puted therefore for each muscle using only those quantitative measures of ChE activity corresponding to sections having 100 sole plates or less. The Y-intercept of the regression line (i.e., the point at which the line crosses the vertical axis) is a measure of ChE activity for sections exhibiting no sole plates. This value is taken as the background or non-sole-plate activity. The sole-plate activity was obtained by subtraction of background from total ChE activities: Total
ChE = background
activity + sole-plate activity.
Changes in Background and Sole-Plate Activity During Reinnervation. The background and sole-plate ChE activity decreased similarly (66% and 70%) at 1 week postoperatively (Table 2). By six weeks the average ChE activity had returned to 92% and 93% of normal. At this time three of the six animals on which the average was based exhibited normal ChE activity, and the others had not yet attained the normal level.
334
GUTH
AND
BROWN
To determine whether the sole-plate ChE was being restored at a faster rate than the background ChE (the former was 88% of normal and the latter 78% of normal at 3 weeks) we calculated the ratio of background to sole-plate ChE for each muscle (Table 2). If the sole plate ChE increased more rapidly than the background ChE, then the value of this ratio would have to decrease. There were no significant differences between operated and normal muscles at 1, 3, or 6 weeks postoperatively or between operated muscles alone at 1, 3, and 6 weeks. In normal muscle, the total ChE, sole-plate ChE and background ChE all exhibited a gradual increase between I and 6 weeks (Tables 1 and 2 ) . The total protein showed no comparable trend, and consequently the specific activity of ChE in normal muscles was higher at 6 weeks than at 1 week (Table 1) . These changes in unoperated muscles may be related to the gradual increase in age and maturation of the rats in the 6-week postoperative interval since the wet weights of the normal muscles averaged 228, 235, and 249 mg at 1, 3, and 6 weeks respectively. Discussion
The changes in ChE in denervated muscle (5) and in reinnervated muscle show certain similarities as well as certain differences. The rate of loss of background and of sole-plate ChE in denervated muscle is identical (5). Similarly in the present study we found no differences in the rate of reappearance of background and sole-plate ChE activity following reinnervation. However, the decrease in ChE activity following denervation is complete by 3 days postoperatively, whereas the restoration of ChE activity in reinnervated muscle is not quite complete by 6 weeks postoperatively. Such a gradual increase in ChE of reinnervated muscle could reflect either of two mechanisms. Upon reinnervation of an individual muscle fiber, ChE synthesis could be a rapid process. The gradual increase of ChE per whole muscle would in this case result from a slow addition of newly innervated muscle fibers. Alternatively, a rather rapid reinnervation of all muscle fibers followed by a slow and gradual synthesis of ChE would effect the same result. To distinguish between these alternatives it is necessary to make an estimate of the rate of nerve regeneration. In the present experiment the nerve fibers were crushed at a distance of less than 3 mm from the sole plates. According to measurements of the initial rate of regeneration of crushed peripheral nerve (8) we can conclude that the majority of nerve fibers reached their termination within 1 week. The delay in the establishment of a mature or functional synapse probably adds no more
CHOLINESTERASE
IN
335
MUSCLE
than 2 weeks to the process (6). Consequently the marked increase in ChE activity that we observed between 3 and 6 weeks postoperatively probably reflects a slow synthesis within individual muscle fibers rather than the gradual addition of newly innervated fibers. It is well known that not all fibers of injured nerves regenerate; there is generally a loss of a small proportion of the fibers (11). This fact might explain the observation that even after 6 weeks, the ChE had returned to only 93% of normal. Also, it has been observed that following a crushing injury of nerve, there is an increase in the proportion of doubly innervated muscle fibers (1). If some nerve factor regulated the total level of muscle ChE then such hyperneuritization might be expected to result in a supranormal ChE content. This was not observed in the present experiment, but experiments are under way to determine the ChE content of muscles in which the degree of hyperneuritization is better controlled. For the present it appears as though the presence of the nerve merely enables the muscle to ultilize fully its intrinsic potentiality for synthesizing the enzyme. The observation that the ratio of background ChE to sole-plate ChE remained constant (and normal) at 1, 3, and 6 weeks permits the conclusion only that no difference was observed between the rate of reappearance of background and sole-plate ChE. It does not permit us to infer that the nerve influence acts simultaneously at both sites. If the influence of the nerve occurred at one site several days prior to the other, and if the ensuing steps in the synthesis of protein were slow (occupying several weeks, for example) then no measure of the rate of formation of end-product would be expected to reflect changes in the more rapidly occurring steps in the process. The rate of formation of background ChE vs. sole-plate ChE would be pertinent to this question only if we were certain that the neurotrophic factor acted at the rate limiting step in the synthesis of the enzyme. Only after studying the influence of innervation on each of the steps involved, will we be able to determine the mechanism by which the nerve regulates the level of cholinesterase activity of muscle. References 1.
2.
3.
BERNSTEIN, J. J., and L. GUTR. 1961. Nonselectivity in muscular connections following nerve regeneration in 4: 262-275. ECCLES, J. C. 1944. Investigations on muscle atrophies tenotomy. J. Physiol. London 103: 253-266. ELLMAN, G. L., K. D. COURTNEY, V ANDRES, JR., and 1961. A new and rapid calorimetric determination of tivity. Biochem. Pharmacol. 7: 88-95.
establishment of nexothe rat. Erptl. Neural. arising
from
disuse
and
R. M. FEATHERST~NE. acetylcholinesterase ac-
336 4.
5.
6. 7. 8. 9. 10. 11.
GUTH
AND
BROWN
FISCHER, E., and V. W. RAMSEY. 1946. Changes in protein content and in some physicochemical properties of the protein during muscular atrophies of various types. Am. J. Physiol. 145: 571-582. GUTH, L., R. W. ALBERS, and W. C. BROWN. 1964. Quantitative changes in cholinesterase activity of denervated muscle fibers and sole plates. Exptl. Neural. lo: 236-250. GUI-MANN, E. 1942. Factors affecting recovery of motor function after nerve lesions. 1. Ncurol. Neurosurg. Psyckiat. 5: 81-95. HINES, H. M., and J. D. THOMSON. 1956. Changes in muscle and nerve following motor neuron denervation. Am. J. Physical Med. 35: 35-57. JACOBSON, S., and L. GUTH. 1965. An electrophysiological study of the early stages of peripheral nerve regeneration. Exptl. Neural. 11: 48-60. LOWRY, 0. H., N. J. ROSEBROTJGH, A. L. FARR, and R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Ckem. 193: 265-275. MTJMENTHALER, M., and W. K. ENGEL. 1961. Cytological localization of cholinesterase in developing chick embryo skeletal muscle. Acta Anat. 47: 274-299. “Degeneration and Regeneration of the Nervous RAM~N Y CAJAL, S. 1928. System” [R. M. May, trans. and ed.1, Vol. 1, p. 276. Oxford Univ. Press, London and New York.
EXPERIMENTALNEUROLOGY
The
Sequence
of
During
(1965)
Changes
in Cholinesterase
Reinnervation
of Muscle
LLOYD GUTH AND WILLIAM
C. BROWN’
Laboratory of Neuroanatonaical Sciences, National Institute and Blindness, National Institutes of Health, Public Health of Health, Education, and Welfare, Bethesda, Received
February
Activity
16,
of Neurological Service, U. S. Maryland
Diseases
Department
1965
The following evidence supports the view that cholinesterase (ChE) activity of muscle is regulated by its nerve supply: denervation produces a more rapid decrease in ChE than in total protein, and tenotomy produces loss of protein without change in ChE. The present study concerns changes in ChE during regeneration of nerve fibers into denervated muscle. In twenty rats the nerve to the sternomastoid was crushed at the muscle. Alternate, frozen, serial, crosssections of muscle were stained histochemically or assayed quantitatively for ChE. Sole-plate ChE was distinguished from background or non-sole-plate ChE by comparing regions of muscle having sole plates with regions of muscle lacking them. Background ChE and sole-plate ChE both decreased to 68% of normal at 1 week postoperatively and increased gradually to 93% of normal by 6 weeks. The ratio of background ChE to sole-plate ChE was constant at 1, 3, and 6 weeks, indicating a similar rate of reappearance of enzyme at both sites. Inasmuch as reinnervation of the muscle was probably complete at 2 weeks, the subsequent increase in ChE probably reflects a slow rate of synthesis rather than a slow addition of newly reinnervated muscle fibers. This slow, gradual synthesis following reinnervation contrasts markedly with the rapid decrease in ChE (40% in 3 days) following denervation. Total protein showed no similar gradual restoration, being about 93% of normal 1, 3, and 6 weeks postoperatively.
Introduction Denervation of a muscle results in a characteristic atrophy and loss of protein (7). However, it cannot be concluded that the innervation exerts a specific regulatory function over protein metabolism, for muscle atrophy can alsobe produced by disuse (2 ) , immobilization (4)) or tenotomy (2,4), i.e., without alteration of the innervation. Nevertheless, there is evidence 1 The authors gratefully acknowledge the statistical assistance Mosimann (Biometrics Branch, NINDB) and the many helpful R. Wayne Albers (Laboratory of Neurochemistry, NINDB). 329
of Dr. James E. suggestions of Dr.
330
GUTH
AND
BROWN
that the nerve is specifically related to at least one muscle protein: cholinesterase. First of all, denervation produces a more prompt decrease in cholinesterase than in other muscle proteins. Secondly, although both tenotomy and denervation result in a rather similar decrease in total protein, tenotomy has no effect on cholinesterase activity of muscle (5). It has been shown that denervation produces an equal decrease in ChE at the sole plate and in regions containing no sole plates (5). Thus it appears that the ChE is primarily muscular rather than neural in localization, and that the regulatory effect of the nerve is exerted throughout the muscle fiber and not merely at the site of the nerve terminations. Considering that the cell nucleus plays a key role in the regulation of protein synthesis, and recognizing that the skeletal muscle fiber is a multinucleated cell characterized by aggregation of nuclei at the sole plate, it consequently seems pertinent to inquire whether reinnervated muscle will form cholinesterase at the sole-plate region prior to forming it at other regions. In the present experiment we have determined the changes in sole-plate and in background ChE activity at intervals of 1, 3, and 6 weeks after crushing the nerve to the sternomastoid muscle. In an effort to understand the mechanism by which the nerve regulates the ChE metabolism of muscle we have compared the rate of disappearance of ChE following denervation with the rate of reappearance following reinnervation, and the changes in sole-plate ChE with those of background ChE. Materials
and
Methods
Operative Procedure. Random-bred female, Osborne-Mendel rats ( 175200 g) were anesthetized with chloral hydrate (400 mg/kg). A midline incision was made in the ventral aspect of the neck and the sternomastoid muscle exposed. The nerve to this muscle was crushed by means of a fine forceps (nonserrated, blade width less than 0.5 mm). The operation was performed unilaterally, half of the operations being done on the right and half on the left sides. Eight rats were killed at 7 days, seven at 21 days, and six at 42 days postoperatively. Histological Procedure. The muscles of the operated side and the contralateral unoperated side were removed, weighed, stretched to approximately normal length in a U-shaped glass frame, and frozen rapidly by immersion in cold (-50 C) N-methylbutane. Frozen cross-sections of the muscle were cut at 28 p. Serial sections were taken in groups of four. The first and fourth sections
CHOLINESTERASE
IN
MUSCLE
331
were placed on a glass slide for cholinesterase histochemistry, and the second and third were combined in a single ‘test tube for quantitative biochemical analysis. In this way the two sections on the slide served as histological controls for the sections in the test tube. The succeeding fourteen sections were discarded, following which the procedure was repeated. This sequence was followed from the rostra1 to the caudal end of the muscle so that the entire length of the muscle was surveyed biochemically and histochemically at approximately OS-mm intervals. The slides were fixed in unbuffered 3% glutaraldehyde and stained for cholinesterase by a thiolacetic acid procedure ( 10). The quantitative analysis of the sections in the test tubes was by adaptation of Ellman’s (3) method to a microchemical scale (5). The sections were incubated in phosphate-buffered acetylthiocholine, and the thiocholine resulting from the esterase action was converted to a colored product by addition of dithiobisnitrobenzoate. Optical density was determined spectrophotometritally. Protein determinations were done by Lowry’s method (9). The histochemical and biochemical methods outlined here have recently been described in detail (5). Results
Total Cholinesterase Activity of the Muscle. In the discontinuous serial section method that we employed, two sections were studied quantitatively, two histochemically and fourteen discarded. Consequently quantitative determinations were made on two of eighteen sections or one-ninth of the muscle. Total cholinesterase activity was computed by obtaining the sum of the activity of all twenty samples from each muscle and multiplying by nine. The total protein content of the muscle was obtained similarly. The specific activity was obtained by dividing the ChE activity by the protein content for each muscle. These measures are summarized in Table 1. The ChE activity decreased to approximately 66% of normal at 1 week postoperatively and increased to about 90% of normal by 6 weeks. The total protein showed no such gradual restoration, being about 93% of normal at 1, 3, and 6 weeks. The specific activity was normal by 6 weeks only because both the protein and the ChE values were equally diminished at this time. Because the operation produced differential changes in ChE and protein, it seems more meaningful to describe the changes in ChE per muscle and not on the basis of total protein (i.e., specific activity). Background and Sole-plate ChE Activity. The total ChE activity may be considered as representing the contribution of the background ChE
332
TOTAL
GUTH
CONTENT
Time One
crushing Three
Normal
6.43 23.76 0.275
0 The specific activity vidually for each animal.
after
week
Oper.
100
BROWN
TABLE 1 ACTIVITY (MICROMOLES/MUS~LE/HOUR) (mg/MuscLE) OF THE STERNOMASTOID
CHOLINESTERASE
Total ChE activity Total protein Specific activitya
AND
Oper.
the average
PROTEIN
to sternomastoid
weeks
Six weeks
Normal
8.24 26.77 0.314
9.58 25.71 0.3 79
represents
nerve
AND TOTAL MUSCLE
Oper.
10.33 28.52 0.3 73
of the specific
Normal
10.65 23.12 0.462
activities
calculated
11.60 24.88 0.466 indi-
r
25
50
75 NUMBER
too
125
OF SOLE
PLATES
150
175
200
1. Cholinesterase activity of tissue sections plotted as a function of the number of sole plates counted in the adjacent histochemically-stained preparations. This graph has been prepared from the data of one rat whose sternomastoid nerve was crushed unilaterally 7 days previously. Inasmuch as the sole-plate counts were prepared from tissue sections, the abscissa actually represents the number of sole-plate fragments that were counted rather than the number of entire sole plates. Note the units for the ordinate: millimicromoles per sample per hour. In Figs. 3, 4, 6 and Table 1 of our previous communication (5) these units were incorrectly desribed as micromoles rather than as millimicromoles per sample per hour. FIG.
CHOLINESTERASE
IN
333
MUSCLE
(i.e., not associated with sole plates) and sole-plate ChE activity. The background activity was determined as follows. For both muscles of each animal, a scatter graph was prepared in which the ChE activity of each test tube was plotted as a function of the number of sole plates counted in the adjacent histochemically-stained sections. A typical graph is presented in Fig. 1. Fourteen of the twenty-four graphs appeared perfectly linear, and ten showed a slight tendency to flatten at values greater ,than 100 sole plates. Linear regression lines (of ChE activity on sole plates) were comTABLE 2 CHOLINESTERASE ACTIVITY (MICROMOLES/MUSCLE/HOUR) OF THE STERNOMASTOID MUSCLE Time
after
One week Oper. Background ChE activity Total sole-plate activity % Change background ChEa % Change sole-plate ChEa Background Ratio
nerve
Three
Normal
4.55 1.88
crushing
6.91 2.67
Oper.
weeks
Six weeks
Normal
Oper.
Normal
7.22 3.11
7.54 3.11
8.13 3.47
5.66 2.58
66 70
to sternomastoid
78 88
93 92
ChEa
of sole-p!ate
ChE
2.64
2.63
2.26
2.49
2.47
a The percentage change in background activity, the percentage change activity and the ratio of background to sole-plate activity was calculated for each animal. The averages obtained from these figures are presented
2.44 in sole-plate individually in the table.
puted therefore for each muscle using only those quantitative measures of ChE activity corresponding to sections having 100 sole plates or less. The Y-intercept of the regression line (i.e., the point at which the line crosses the vertical axis) is a measure of ChE activity for sections exhibiting no sole plates. This value is taken as the background or non-sole-plate activity. The sole-plate activity was obtained by subtraction of background from total ChE activities: Total
ChE = background
activity + sole-plate activity.
Changes in Background and Sole-Plate Activity During Reinnervation. The background and sole-plate ChE activity decreased similarly (66% and 70%) at 1 week postoperatively (Table 2). By six weeks the average ChE activity had returned to 92% and 93% of normal. At this time three of the six animals on which the average was based exhibited normal ChE activity, and the others had not yet attained the normal level.
334
GUTH
AND
BROWN
To determine whether the sole-plate ChE was being restored at a faster rate than the background ChE (the former was 88% of normal and the latter 78% of normal at 3 weeks) we calculated the ratio of background to sole-plate ChE for each muscle (Table 2). If the sole plate ChE increased more rapidly than the background ChE, then the value of this ratio would have to decrease. There were no significant differences between operated and normal muscles at 1, 3, or 6 weeks postoperatively or between operated muscles alone at 1, 3, and 6 weeks. In normal muscle, the total ChE, sole-plate ChE and background ChE all exhibited a gradual increase between I and 6 weeks (Tables 1 and 2 ) . The total protein showed no comparable trend, and consequently the specific activity of ChE in normal muscles was higher at 6 weeks than at 1 week (Table 1) . These changes in unoperated muscles may be related to the gradual increase in age and maturation of the rats in the 6-week postoperative interval since the wet weights of the normal muscles averaged 228, 235, and 249 mg at 1, 3, and 6 weeks respectively. Discussion
The changes in ChE in denervated muscle (5) and in reinnervated muscle show certain similarities as well as certain differences. The rate of loss of background and of sole-plate ChE in denervated muscle is identical (5). Similarly in the present study we found no differences in the rate of reappearance of background and sole-plate ChE activity following reinnervation. However, the decrease in ChE activity following denervation is complete by 3 days postoperatively, whereas the restoration of ChE activity in reinnervated muscle is not quite complete by 6 weeks postoperatively. Such a gradual increase in ChE of reinnervated muscle could reflect either of two mechanisms. Upon reinnervation of an individual muscle fiber, ChE synthesis could be a rapid process. The gradual increase of ChE per whole muscle would in this case result from a slow addition of newly innervated muscle fibers. Alternatively, a rather rapid reinnervation of all muscle fibers followed by a slow and gradual synthesis of ChE would effect the same result. To distinguish between these alternatives it is necessary to make an estimate of the rate of nerve regeneration. In the present experiment the nerve fibers were crushed at a distance of less than 3 mm from the sole plates. According to measurements of the initial rate of regeneration of crushed peripheral nerve (8) we can conclude that the majority of nerve fibers reached their termination within 1 week. The delay in the establishment of a mature or functional synapse probably adds no more
CHOLINESTERASE
IN
335
MUSCLE
than 2 weeks to the process (6). Consequently the marked increase in ChE activity that we observed between 3 and 6 weeks postoperatively probably reflects a slow synthesis within individual muscle fibers rather than the gradual addition of newly innervated fibers. It is well known that not all fibers of injured nerves regenerate; there is generally a loss of a small proportion of the fibers (11). This fact might explain the observation that even after 6 weeks, the ChE had returned to only 93% of normal. Also, it has been observed that following a crushing injury of nerve, there is an increase in the proportion of doubly innervated muscle fibers (1). If some nerve factor regulated the total level of muscle ChE then such hyperneuritization might be expected to result in a supranormal ChE content. This was not observed in the present experiment, but experiments are under way to determine the ChE content of muscles in which the degree of hyperneuritization is better controlled. For the present it appears as though the presence of the nerve merely enables the muscle to ultilize fully its intrinsic potentiality for synthesizing the enzyme. The observation that the ratio of background ChE to sole-plate ChE remained constant (and normal) at 1, 3, and 6 weeks permits the conclusion only that no difference was observed between the rate of reappearance of background and sole-plate ChE. It does not permit us to infer that the nerve influence acts simultaneously at both sites. If the influence of the nerve occurred at one site several days prior to the other, and if the ensuing steps in the synthesis of protein were slow (occupying several weeks, for example) then no measure of the rate of formation of end-product would be expected to reflect changes in the more rapidly occurring steps in the process. The rate of formation of background ChE vs. sole-plate ChE would be pertinent to this question only if we were certain that the neurotrophic factor acted at the rate limiting step in the synthesis of the enzyme. Only after studying the influence of innervation on each of the steps involved, will we be able to determine the mechanism by which the nerve regulates the level of cholinesterase activity of muscle. References 1.
2.
3.
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