Early elevations of glycosidase, acid phosphatase, and acid proteolytic enzyme activity in denervated skeletal muscle

EXPERIMENTAL

NEUROLOGY

42, 541-554 (1974)

Early Elevations of Glycosidase, Acid Phosphatase, Proteolytic Enzyme Activity in Denervated Skeletal

LEO G. ABOOD, AND H. BRUCE BOSMANN

JACK MCLAUGHLIN, Center versity

for

of

Brain

Rochester,

Research School

and Acid Muscle

and

of

Departmelzt Medicine

Received

of

Pharmacology and Rochester,

alLd Dentistry,

September

1

Toxicology, New York

Uni-

14642

29,1973

Evidence is presented that denervation of rat extensor digitorum longus is rapidly followed by selective increases in the activity of glycosidase, acid phosphatase, and acid proteolytic enzymes. A novel double-denervation paradigm was used to demonstrate that the elevation in at least two enzyme activities, p-n-mannosidase and a+fucosidase is influenced by something in, or some process mediated by, the nerve. The nature of this elevation in enzyme activity points to a sensitive, neurally influenced control mechanism established in the normal relationship between nerve and muscle, but which is upset by denervation. The possible relationships of increased acid hydrolytic enzyme activity to general muscle atrophy is discussed, and a hypothesis is presented that the early metabolic changes triggered by denervation result in the altered membrane properties of denervated muscle.

INTRODUCTION Physiological and chemosensitive properties of muscle membranes are rapidly altered following denervation (2, 3, 18, 29). An understanding of the molecular basis of these membrane alterations would at least require detailed information about the biochemical structure and metabolism of the surface membranes of both normal and denervated muscle. This information is presently not completely available, but some studies have suggested that membrane biochemistry is altered following denervation. An increased incorporation of szP, and(2-3H) glycerol into all glycerophosphatides of membranes isolated from denervated gastronemius muscle of frog and rat has been demonstrated (10). An increased binding of 1 Dr. McLaughlin is a postdoctoral fellow of the Muscular Dystrophy Associations of America. Dr. Bosmann is a Research Career Development Awardee of N.I.G.M.S. This study was supported in part by Grants CA-13220 and MH-08034. We thank Dr. Ryo Tanaka, Mr. Douglas L. Rosene, Mr. David Turriff, and Mr. Kenneth R. Case for advice and encouragement throughout the course of this work. 541 Copyright 0

All

rights

of

1974 by Academic Press,Inc. reproduction in any form reswed.

542

MCLAUGHLIN,

ABOOD,

AND

BOSMANN

ar-bungarotoxin to isolated membranes of denervated rat leg muscle (19) and of calcium to a sarcolemmal preparation from denervated guinea pig gastrocnemius muscle (35) has been reported. In less direct evidence, increased amounts of hematoside (GM3 ganglioside) were found in whole muscle preparations of denervated gastrocnemius muscle of cat, rabbit, and rat (27). There are reports of an increased incorporation of radioactively labeled leucine into a proteolipid fraction derived from diaphragm muscle following denervation (26) and an increased amount of proteinbound sialic acid found in denervated rat skeletal muscle (5, 12). The biochemical evidence along with extensive electrophysiological and pharmacological data indicates that the surface membranes are altered by denervation. The biochemical regulation of macromolecular constituents of animal cell membranes must depend to a great extent on the quantitative and qualitative balance between synthesis and degradation. Acid hydrolases are a large family of enzymes collectively capable of degrading various macromolecules including proteins, lipids, nucleic acids, and complex members of this degradative enzyme carbohydrates. The glycosidases, family, are primarily lysosomal hydrolases thought to be responsible for the hydrolysis of bonds between sugars and amino acids, between sugars and lipid moieties, and between adjacent sugars in glycoproteins, glycolipids, glycosaminoglycans, oligo- and polysaccharides (9). As carbohydrate-containing macromolecules (glycoconjugates) have been found in bullfrog sarcolemma preparations (1) and in rat skeletal muscle (5, 12), glycosidases would be expected to participate in the metabolism of muscle surface membrane glycoconjugates. Sudden alterations in the activity of some of these enzymes could have sudden and drastic physiological consequences. This report demonstrates that denervation of rat extensor results in a rapid elevation of glycosidase, digitorum longus muscle (EDL) acid phosphatase, and acid proteolytic activities. The time course of this elevation of enzyme activities was influenced by the length of the distal nerve stump, suggesting that the effect cannot be entirely attributed to muscle disuse. A link between this elevation of enzyme activities and alterations of muscle membrane function is possible, but it remains to be established. In any event, the activity of some muscle enzymes likely involved in membrane catabolism was found to be under neural control. Enzymes studied were N-acetyl-p-D-glucosaminidase (E C 3.2.1.30)) U-Dglucosidase (E C 3.2.1.20), /3-n-glucosidase (E C 3.2.1.21), N-acetyl-p-ngalactosaminidase (E C 3.2.1.-), a-n-galactosidase (E C 3.2.1.22), P-Dgalactosidase (E C 3.2.1.23)) a-L-fucosidase (E C 3.2.1.--) , P-r.-fucosidase (E C 3.2.1.-), a-o-mannosidase (E C 3.2.1.24), p-n-mannosidase (E C

ENZYMES

3.2.1.25), activity.

acid phosphatase

IN

(E

MATERIALS

DENERVATED

MUSCLE

C 3.1.3.2),

AND

acid and neutral

543 proteolytic

METHODS

Tissue Preparation. Adult (275-325 g) male rats of the Long-Evans hooded strain were anesthetized with sodium secobarbital (50 mg/kg, ip) , The EDL of the left or right hind leg was denervated by transecting the nerve just external to the peroneal muscle group. A sham operation was performed on the opposite leg and its EDL served as a control. In “near-far” experiments, the sciatic nerve of each side was transected in the upper thigh at a level near the head of the femur. On the experimental side the nerve was transected near the EDL as described above while a sham operation was performed on the opposite control leg. The control and experimental EDL thus differed only in the length of the severed nerve stump left attached to the muscle. In “tenotomy” experiments, the EDL of one side was detached from the lateral epicondyle of the femur; the EDL of the opposite sham-operated side again served as the control. No antibiotics were administered; the incisions were sutured closed, and the animals were maintained for varying intervals on a regular laboratory diet with ad lib. access to water. Animals were killed by a sharp blow on the neck and the EDL of each leg was removed and transferred to a beaker of ice-cold saline solution. Each muscle was blotted twice on filter paper and weighed. Muscles were then minced with scissors in 20 vol of 0.1% (v/v) Triton X-100 and thoroughly homogenized for 30 strokes with a Ten Broeck homogenizer held in an ice water bath. This homogenate was used directly for the assay of acid phosphatase, neutral proteolytic, and glycosidase activities, but was diluted S-fold with 0.1% Triton X-100 for measurement of acid protoeolytic activity. Protein. Protein was determined by a slight modification of the method of Lowry et al. (24) using bovine serum albumin as a standard. Glycosidases and Acid Phosphatase. The activity of the various glycosidases or acid phosphatase at pH 4.4 was determined using the appropriate p-nitrophenyl derivative (8) in the following manner. A 100 ~1 portion of a 0.1% Triton X-100 muscle homogenate (containing about 0.60.7 mg protein) was incubated at 37 C with 6.0 coles of the p-nitrophenyl derivative (the final volume was 1.100 ml, 0.05 M in citrate, adjusted to pH 4.4) for 0.5 hr for measurement of acid phosphatase activity, 1.0 hr for ‘p-N-acetylglucosaminidase, and 3.0 hr for the other glycosidases. The reaction was terminated by the addition of 1.0 ml 0.4 M glycine:NaOH buffer, pH 10.5. The reaction mixtures were centrifuged at 5OOOg for 10

544

MCLAUGHLIN,

ABOOD,

AND

BOSMANN

min, and the optical density of the released p-nitrophenolate ion in the supernatant was measured at 420 nm. From these data and a standard p-nitrophenol curve, the nanomoles of exogenous suibstrate hydrolyzed per hour was calculated. Controls consisted of assays in which glass distilled water was substituted for the p-nitrophenyl derivative substrate or in which 0.1% Triton X-100 was substituted for the muscle homogenate. These control values were subtracted from the appropriate average value of triplicate assays to correct for light scattering contriibutions from the tissue and absorbance from the p-nitrophenyl derivatives. The substrates used (Pierce Chemical Co.) were p-nitrophenyl phosphate, p-nitrophenyl-N-acetyl-P-Dglucosaminide, p-nitrophenyl-p-n-glucopyranoside, p-nitrophenyl-a-n-glucopyranoside, g-nitrophenyl-N-acetyl-p-D-galactosaminide, p-nitrophenyla-D-galactoypranoside, p-nitrophenyl-/3-L-fucopyranoside, p-nitrophenyl$n-mannopyranoside, and p-nitrophenyl-a-n-mannopyranoside. Proteolytic Activity. Proteolytic activity at acid pH was measured by a modified method of Anson (6) and Hille et al. (20) as previously described (7). 3H-Acetylated hemoglobin was prepared by the method of Hille et al. (20). 3H-Acetic anhydride (specific activity 400 Ci/mole; New England Nuclear) was reacted with Type I beef blood hemoglobin (Sigma Chem. Co.). The purified 3H-acetylated hemoglobin specific activity was 372 cpm/pmole based on molecular weight of 68,000. Routinely, 50 ~1 (115 pg 3H-acetylated hemoglobin) were added per assay. One hundred pl of diluted muscle homogenate (about 0.100 to 0.150 mg protein) were added to 100 ~1 of a solution of 1.35 acetic acid and 0.02 M ammonium sulfate, pH 3.4. A portion of 50 ~1 of 3H-acetylated hemoglobin was added as the substrate. This mixture was incubated for 1 hr at 37C in a Dubnoff metabolic shaker. The reaction was terminated by placing the assay tubes in an ice water bath and adding 100 pl of 2.5% hemoglobin and 50 pl of 60% trichloroacetic acid. The precipitated protein was removed by centrifugation at 5OOOg for 5 min and an aliquot of the supernatant fluid was plated on a glass fiber filter; the radioactivity was determined by counting in a liquid scintillation counter. Activity is expressed as picomoles of hemoglobin degraded per hour per milligram of enzyme protein. Suitable blanks consisting of 0.1% TX-100 or boiled enzyme (10 min at 1OOC) were added in place of the enzyme extract and incubated simultaneously. All experiments were performed in quintuplicate. Neutral proteolytic activity was analyzed as above except that 100 ~1 of 0.1 M phosphate buffer (pH 7.4) were substituted as the buffer. Product formation for all assays was linear with respect to time and was proportional to the quantity of added muscle extract. In all instances activity was eliminated by boiling the homogenate prior to assay, was negligible in unincubated or immediately terminated reaction mixtures,

ENZYMES

IN

DENERVATED

MUSCLE

54.5

and was greatly reduced in reaction mixtures incubated in an ice water bath. Statistics. There was considerable variation among animals in the values of some enzyme activities, but no differences were found in individual control animals when the right and left muscles were compared. In the comparison of experimental and control muscles, the absolute value of the control muscle was subtracted from that of the experimental muscle of each TABLE WET

WEIGHT

Animal

A20 A21 A22 A23 A24 A25 A34 A35 A36 A41 A42 A43 A46 A47 A48 Mean

f

SEM

0.187

a D refers to denervated not statistically significant. wet weight of muscle.

1

AND PROTEIN CONCENTRATION DENERVATED FOR 3 DAYS@

OF EDL

Wet

Protein

wt

(g)

concentration

D

C

D

C

0.214 0.185 0.182 0.185 0.161 0.189 0.185 0.216 0.150 0.180 0.174 0.173 0.181 0.189 0.234

0.191 0.182 0.182 0.190 0.152 0.183 0.185 0.222 0.144 0.180 0.170 0.181 0.179 0.186 0.236

135 118 126 122 116 112 130 123 118 135 123 134 128 151 125

132 127 122 119 124 118 130 120 135 136 122 149 140 136 133

k

0.006

0.184

z!= 0.006

EDL, while C refers Protein concentration

to the control is milligrams

126 f

3

130 f

2

muscle. Differences are of protein per gram of

animal. The median and range of values for the indicated number of experimental and control muscles are given, as well as the range of the difference values for individual animals. Two-tailed levels of significance were determined using the Wilcoxon matched-pairs signed-ranks test (33). RESULTS The data in Table 1 indicate that no significant change in either the wet weight or protein concentration (milligrams of protein per gram of wet

546

MCLAUGHLIN,

ABOOD, TABLE

ENZYMES

SHOWING

INCKEASED

ACTIVITY

AND 2

2 OK 3 DAYS AFTER

D b-n,-Mannosidase

(EC

1 day@) 2 days(g)* 3 days(l2)*

5.0 (4.4-6.0) 9.1(8.2-12.2) 24.6(17.4-27.1)

a-n-Glucosidase

(EC

Acid

(EC

a-L-Fucosidase

a-n-Galactosidase

B-n-Galactosidase 1 day(O) 2 days(O) 3 daysO)t Proteolytic, 1 day@)

2 days(8)t 3 days(l2)*

to +0.2) to +6.3) to +20.1)

6.4(4.8-8.0) 6.2 (5.3-7.1) 7.5 (6.1-8.0)

(-1.0 (+a.2 ($2.0

to +0.7) to +1.5) to +3.7)

266 (200-284) 277(245-317) 271(223-303)

(-27 (-25 (+16

to +17) to +76) to +147)

3.3 (2.7-6.5) (EC

1 day(O) 2 days(O) 3 days@)t

(-0.3 (f2.1 ($11.7

3.2.1.-)

1 day(O) 2 days(O) 3 days(lO)*

5.0(4.5-5.8) 5.2 (4.7-6.4) 6.0(4.5-8.0)

3.1.3.2)

259 (192-290) 296(220-350) 311(300-443) (EC

Differences

3.2.1.20)

6.3 (5.0-7.8) 6.8(5.8-8.0) 10.2 (8.2-11.0)

1 day@) 2 days(d) 3 days(Vt

C

DENERVATION~

3.2.1.25)

1 day(lO) 2 days(8)* 3 days(9)* phosphatase

BOSMANN

2.5(1.74.4)

(+a.1

to +4.4)

3.2.1.22) -

5.8(4.8-6.6) (EC

-

-

-

4.9(4.4-5.7)

(0.0 to +1.4)

3.2.1.23) -

13.8(12.1-21.5) pH 3.4 (cathepsin-like, 659(495-795) 656(591-791) 702 (597-857)

12.4(10.0-16.2)

(+a.7

to +5.3)

(-36 (-4 (+71

to +51) to f138) to +191)

EC 3.4.4.23) 636 (52 l-759) 648 (495-756) 553 (489-784)

a Units for acid phosphatase and glycosidase activities are nmoles h-1 mg-1, and pmoles Hb* hr-‘.rng-r for proteolytic activity. Values are medians and ranges of the absolute values of denervated (D) and control (C) muscles and range of the individual differences obtained from the number of animals indicated inside parentheses. Significance is as follows: *P < 0.01; t P < 0.05.

ENZYMES

IN

DENERVATED

547

MUSCLE

weight of muscle) of denervated EDL had occurred even 3 days after denervation. The range of values for muscles from normal animals (data not shown) was similar to the range found for control muscles and no gross trend towards hypertrophy of the control muscle was detected. The elevation of various enzyme activities to be described subsequently cannot likely be due to a simple, generalized alteration in muscle protein concentration. The effect of denervation on the various enzymes activities studied is presented in Tables 2 and 3. No significant difference between any denervated and control EDL enzyme activity studied was found 24 hr after denervation, but 48 hr after denervation the activities of at least three enzymes, ,8-n-mannosidase, a-D-glucosidase, and acid proteolytic activity were significantly elevated in homogenates of denervated EDL. P-DMannosidase activity increased from a mean specific activity value of nmoles per hr per mg to 9.5 -C 0.5 (SEM) within 5.2 f 0.2 (SEM) a 24 hr peribd. Additional studies confirmed that this increase in activity is an event of abrupt onset (first detected 33-36 hr after “near” denerTABLE ENZYMES

3

NOT SHOWING SIGNIFICANTLY INCREASED 3 DAYS AFTER DENERVATION~ D

C

cy-D-Mannosidase (EC 3.2.1.24) 1 day(O) 2 days(3) 3 days(l5)

2.0(1.5-2.1) 1.3(0.7-2.9)

1 day@) 2 days(S) 3 days(6)

(EC

Differences

-

2.0(1.7-2.7) 1.9(0.6-3.4)

N-Acetyl+-D-glucosaminidase

ACTIVITY

(-0.4 (-0.5

3.2.1.30) 169(148-177) 155(138-161) 158(139-183)

165(154-179) 148(131-163) 160(131-200)

(-4 (-13 (-13

N-Acetyl-B-D-galactosaminidase (EC 3.2.1-) 1 day@) -

2 days(O)

3 days(ll)

2 days(O) 3 days(6) a Units

and symbols

15.7(13.3-20.0)

to +6) to +8) to +20)

-

-

15.9(13.6-22.2)

Proteolytic, pH 7.4 1 day(O)

to +0.7) to +l.l)

(-2.2

to +2.2)

-

-

-

-

-

-

23.7(13.047.9) as given

21.5(13.7-37.5) in

Table 2.

(-2.0

toI+10.4)

548

MCLAUGHLIN,

ABOOD,

AND

BOSMANN

vation) and rapid development ; it followed a definite lag period of no change in enzyme activity. Three days after denervation the various enzymes studied could be separated into two groups, one which showed significantly elevated activities and one which did not. In addition to the enzyme activities mentioned above, a-D-galactosidase, /3-n-galactosidase, a-r.-fucosidase, and acid phosphatase activities were elevated. However, a-n-mannosidase, N-acetyl-p-n-glucosaminidase, N-acetyl-p-b-galactosaminidase, and neutral proteolytic activities were not significantly elevated at this time. P-L-fucosidase and p-n-glucosidase activities were two low to be reliably assayed in this system. No enzyme activity studied was found to have decreased following denervation. TABLE P-D-MANNOSIDASE

Animal A41

A42

A43

a All amounts of extract control EDL, respectively.

4

ASSAYS ON MIXTURES OF EXTRACTS DENERVATED AND CONTROL EDLa Extract

(mg)

0.680 0.686 0.340

D alone C alone D + 0.343

0.598 0.580 0.299

D alone C alone D + 0.290

0.644 0.752 0.322

D alone C alone D + 0.376

are as protein.

FROM

nmoles

C

C

C

~-DAY

per hr

13.5 4.2 8.6 (found) 8.9 (theoretical) 15.3 3.9 9.6 (found) 9.6 (theoretical) 14.0 4.1 9.1 (found) 9.1 (theoretical)

D and C are abreviations

for denervated

and

The experiments summarized in Table 4 were performed to determine whether some alteration in the concentration of activators, inhibitors, cofactors, etc., could have led to the differences in enzyme activities found after denervation. The data clearly show that P-n-mannosidase activity was essentially additive between extracts of control and 3-day denervated EDL. This would not be the case if factors other than activity were being measured. Similar conclusions were reached by others for different enzymes (32, 34), but none of these experiments or our own would be sufficient to determine whether some stable complex had been formed between enzymes and effecters- (23).

ENZYMES

IN

DENERVATED

TABLE INFLUENCE

549

MUSCLE

5

OF DISTAL NERVESTUMP LENGTH ONTHE DEVELOPMENT GLYCOSIDASE ACTIVITY FOLLOWING DENERVATION"

OF INCREASED

Far

Near

Differences

j3-D-Mannosidase 2 days@)*

10.5(8.4-12.5)

8.9(7.4-10.4)

(+0.7

to +2.7)

3.2(2.44.6)

(fO.2

to +1.q

cr+Fucosidase

2 days(6)t

4.2 (3.0-H)

a As described in Materials and Methods, the sciatic nerve of each side was transected in the upper thigh. In addition, the nerve supplying the EDL was transected near the knee on one side and a sham second operation was performed on the opposite leg. EDL with a short attached nerve stump is referred to as “near”, and EDL with the long nerve stump as “far”. Assays of enzyme activities were performed 48 hr after surgery. Units and symbols of significance are as given in Table 2.

The experiments presented in Table 5 show that the length of the distal nerve stump affected postdenervation elevations in enzyme activity. At 48 hr after surgery values for both “near” and “far” EDL enzyme activities were greater than control values, but the specific activities of enzymes in the near EDL were consistently greater than activities in the far EDL. Moreover, the magnitude of the differences between near and far EDL activities were in each case greater than any differences found between the left and right EDL of control animals or between the l-day denervated and control EDL. Lastly, the data in Table 6 show that tenotomy alone did not result in any significant differences in the specific activity of either P-D-mannosidase or a-L-fucosidase assayed 48 hr after surgery. TABLE

6

EFFECTOFTENOTOMYONACTIVITYOF&D-MANNOSIDASE AND&L-FUCOSIDASE~ T

C

Differences

p-D-Mannosidase 2 days(6)

5.6(5.2-7.0)

5.8(.5.3-6.9)

(-

4.2 (3.64.6)

4.0(3.5-4.3)

(-0.1

0.4 to +0.2)

a-L-Fucosidase 2 days(6)

0 Assays of glycosidase activities were given in Table 2. T refers to tenotomized

performed 48 hr following surgery. EDL and C refers to the control

to +0.8) Units are as muscle.

550

MCLAUGHLIN,

ABOOD,

AND

BOSMANN

DISCUSSION The experiments presented here established that some but not all acid hydrolytic enzyme activities of rat EDL are rapidly elevated following denervation. When extracts of denervated and control EDL were mixed, the enzyme activity present in the extracts was additive, indicating that the concentration of effecters is not the basis of the increased enzyme activities. All muscles were thoroughly homogenized in Triton X-100 and thus a differential liberation of enzymes from some bound, less active state is unlikely. Protein concentration did not change significantly 2 and even 3 days after denervation and a generalized loss in protein cannot be invoked to explain the increase in enzyme activities. Changes in enzyme dependence on pH, temperature, and other such factors could have occurred after denervation, but experiments we have performed and published reports of others (32, 34) have in each case been negative on this point. A more likely hypothesis is that the rapid increase in enzyme activity is simply due to a larger quantity of enzymes present, perhaps because synthesis of the various enzymes has increased. Conceivably, some hydrolases were preferentially spared from a more generalized minor loss in protein, but the rather selective, large, and unequal increasesin particular enzyme activities seemmore consistent with the fact that acid hydrolases are not a genetically linked group. It should be pointed out that the substrates used in these experiments are synthetic and their similarity to natural endogenous substrates has not yet been determined. If the increase in enzyme activities is in fact due to the presence of more enzyme, one would like to know the source(s) of this increase. Canonico on the basis of biochemical evidence obtained in zonal and Bird (ll), centrifugation experiments, suggestedthat rat skeletal muscle homogenates contain two groups of lysosome-like particles with different cellular origins. One group was postulated to have been derived from connective tissue components, e.g., macrophages, fibroblasts, leukocytes, and epithelial cells, and the other from muscle cells. The present data, of course, provide no information as to the cellular, extracellular, or intracellular distribution of elevated enzyme activities. Numerous studies have indicated that acid hydrolytic enzyme activities are elevated in somepathological conditions of skeletal muscle (21, 31, 36), including denervation. Denervation studies have been concerned with longterm effects of nerve section and have not been specific as to muscle type. The few exceptions to this generalization are noteworthy. Pollack and Bird (32) demonstrated an increase in cathepsin D, ribonuclease, and aryl sulfatase activities in rat leg muscle three days after section of the sciatic nerve. Hajek, Gutmann, and Syrovy (17) reported that acid proteolytic activity (denatured hemoglobin as substrate) was increased 24 hr after

ENZYMES

IN

DENERVATED

MUSCLE

551

denervation of rat EDL. In their experiment the activity of one EDL, denervated by cutting the motor nerve at its point of entry into the muscle, was compared with the activity of the contralateral EDL which had been denervated by cutting the sciatic nerve in the thigh. Using a similar, but more sensitive (20)) proteolytic assay we were unable at 24 hr to demonstrate significant differences between experimental and control EDL activitis with the single “near” cut paradigm. However, Hajek, Gutmann, and Syrovy (17) did report that acid proteolytic activity present in 3 day denervated muscles was 120 + 6.8% of control values and this is in very good agreement with our value of 124%. The “near-far” experiments reported here indicate that something present in or some process mediated by the nerve influences the postdenervation elevation of enzyme activities. Both experimental and control muscles were denervated by section of the sciatic nerve and were thus equated in terms of disuse and differed only in the length of the attached nerve stump. If this factor is centrifugally transported by the nerve, the velocity of propagation is clearly in the range of fast axoplasmic transport (30) (at least 120 mm per day, exact value not yet established). These findings need not imply that the regulation of these enzymes is completely unrelated to the functional demands placed on EDL. Nor is the possibility excluded that impulse conduction and the availability of the postulated neural factor are related. Tenotomy, skeletal fixation, and other procedures which produce varying measures and types of disuse can bring about some denervation-like changes, especially those related to general muscle atrophy (16). Elevations in cathepsin D, aryl sulfatase, and p-n-glucuronidase activity in 7-day tenotomized rat leg muscle have been reported (32) ; P-n-glucosidase activity was elevated in rat gastrocnemius muscle immobilized for 2 days by skeletal fixation (28). We have not extensively investigated to what extent these procedures might mimic our biochemical findings in denervated muscle, and such comparisons would be difficult to interpret. Neither p-n-mannosidase or U-L- fucosidase activity was elevated in EDL tenotomized for 48 hr, although increased activity after denervation was easily demonstrated at this time. If both EDL muscles were tenotomized but one EDL was also denervated, p-D-mannosidase activity was again markedly elevated in the denervated muscle 2 days after surgery. Again at 2 days, the activity of /3-n-mannosidase present in denervated (but not tenotomized) EDL was much greater than that present in a contralateral tenotomized (but not denervated) muscle. The acid hydrolases studied in the present report may participate in general cellular and perhaps extracellular degradation of macromolecules, particularly the glyccconjugates. Augmented activity of these enzymes may be important in various muscle-wasting disorders (21, 31, 36). However,

552

MCLAUGHLIN,

ABOOD, AND BOSMANN

the early, abrupt, and selective elevation in particular enzyme activities, and the results of the near-far experiments point to a neurally influenced, specific mechanism capable of exerting direct control over the enzymes involved. Whether similar control is exerted over anabolic systems (e.g., glycosyltransferases) is not known. The disruption produced by denervation (and perhaps by other provocative stimuli) may in part be reflected by some of the elevated enzyme activities reported here. One immediately apparent consequence of this disturbance could be the direct or indirect production of structural and functional abnormalities in the external membrane systems. The time course of chemosensitive, electrical, and physiological alterations, in denervated muscle (3, 29) and the dependence of these changes on the length of the distal nerve stump (13, 18, 25) are consistent with this hypothesis. Additional support stems from the observation that colchicine and other agents which arrest axoplasmic flow produce denervation-like membrane alterations (4)) while inhibitors of protein and RNA synthesis delay their appearance (14, 15, 22).

REFERENCES L. G,, K. KURAHASI, E. BRUNNGRABEX, and K. KOKETSU. 1966. Biochemical analysis of isolated bullfrog sarcolemma. Biochim. Biophys. Acfa 112: 330-339. ALBUQUERQUE, E. X., and S. THESLEFF. 1968. A comparative study of membrane properties of innervated and chronically den,ervated fast and slow skeletal muscles of the rat. Acta Physiol. Stand. 73: 471-480. ALBUQUERQUE, E. X., and R. J. MCISAAC. 1970. Fast and slow mammalian muscles after denervation. Exp. Neurol. 26: 183-202. ALBUQUERQUE, E. X., J. E. WARNICK, J. R. TASSE, and F. M. SANSONE. 1972. Effects of vinblastine and colchicine on neural regulation of the fast and slow skeletal muscles of the rat. Exp. Neural. 37: 607-634. ANDREW, C. G., and S. H. APPEL. 1973. Macromolecular characterization of muscle membranes. I. Proteins and sialic acid of normal and denervated muscle. J. Biol. Chew. 2M: 5156-5163. ANSON, M. L. 1938. The estimation of pepsin papain and cathepsin with herno.. globin. J. Gen. Physiol. 22: 79-89. BERNACKI, R. J., and H. B. BOSMANN. 1972. Red cell hydrolases. II. Proteinase activities in human erythrocyte plasma membranes. J. Membrane Biol. 7: l-14. BOSMANN, H. B. 1971. Red cell hydrolases: Glycosidase activities in human erythrocyte plasma membranes. J. Membrane Biol. 4: 113-123. BOSMANN, H. B., and W. D. MERRITT. 1969. Glycoprotein and glycolipid degradation: Glycosidases of guinea pig cerebellum. Physiol. Chem. Phys. 1: 555-567. BUNCH, W., G. KALLSEN, J. BERRY, and C. EDWARDS. 1970. The effect of denervation on incorporation of s2P and [3H] glycerol by the music membrane. J. Newochem. 17 : 613-620. CANONICO, P. G., and J. W. C. BIRD. 1970. Lysosomes in skeletal muscle tissue. Zonal centrifugation evidence for multiple cellular sources. J. Cell Biol. 45: 321-333.

1. ABOOD,

2.

3. 4.

5.

6. 7. 8. 9. 10. 11.

ENZYMES

IN DENERVATED

MUSCLE

5.53

12. COTRUFO, R., and S. H. APPEL. 1973. Effects of denervation on glycoproteins of rabbit gastrocnemius and soleus muscles. Exp. Neurol. 39: 58-69. 13. EMMELIN, N., and L. MALM. 1965. Development of supersensitivity as dependent on the length of degenerating nerve fibers. Quart. J. Expt. Physiol. 50: 142145. 14. FAMBROUGH, D. M. 1970. Acetylcholine sensitivity of muscle fiber membranes: mechanism of regulation by motoneurons. Science 168: 372-373. 15. GRAMP, W., J. B. HARRIS, and S. THESLEFF. 1972. Inhibition of denervation changes in skeletal muscle by blockers of protein synthesis. J. Physiol. (London) 221: 743-754. 16. GUTH, L. 1968. “Trophic” influences of nerve on muscle. Physiol. Rev. 48: 645-687. 17. HAJEK, I., E. GUTMANN, and I. SYROVY. 1964. Proteolytic activity in denervated and reinnervated muscle. Physiol. Bohemoslov. 13: 32-38. 18. HARRIS, J. B., and S. THESLEFF. 1972. Nerve stump length and membrane changes in denervated skeletal muscle. Nature New Biol. 236: 60-61. 19. HARTZELL, H. C., and D. M. FAMBROUGH. 1972. Acetylcholine receptors. Distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity. J. Gen. Physiol. 60: 248-262. 20. HILLE, M. B., A. J. BARRETT, J. T. DINGLE, and H. B. FELL. 1970. Microassay for cathepsin D shows an unexpected effect of cycloheximide on limb-bone rudiments in organ culture. Exp. Cell. Res. 61: 470-472. 21. KAR, N. C., and PEARSON, C. M. 1972. Acid, neutral, and alkaline cathepsins in normal and diseased human muscle. Enzyme 13: 188-196. 22. KIMURA, I., K. TSUKADA, and M. KIMURA. 1972. Relationship between supersensitization to acetylcholine and protein synthesis in denervated rat diaphragm muscle. Jap. J. Pharmacol. 22: 39. 23. KINT, J. A., G. DACREMONT, D. CARTON, E. ORYE, and C. HOOFT. 1973. Mucopolysaccharidosis : secondarily induced abnormal distribution of lysosomal isoenzymes. Science 181: 352-254. 24. LOWRY, 0. H., H. J. ROSEBROUGH,A. L. FARR, and R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Bbl. Chem. 193: 265-275. 2.5. Luco, J. V., and C. EYZAGUIRE. 1955. Fibrillation and hypersensitivity to ACh in denervated muscle : effect of degenerating nerve fibers. 1. Neurophysiol 18 : 65-73. 26. LUNT, G. G., E. STEFANI, and E. DEROBERTIS. 1971. Increased incorporation of [G*H] leucine into a possible “receptor” proteolipid in denervated muscle in vivo. J. Neurochem. 18: 1545-1553. 27. MAX, S. R., P. G. NELSON, and R. 0. BRADY. 1970. The effect of denervation on the composition of muscle gangliosides. J. Neurochem. 17: 1517-1520. 28. MAX, S. R., R. F. MAYER, and L. VOGELSANG. 1971. Lysosomes and disuse atrophy of skeletal muscle. Arch. Biochem. Biophys. 146: 227-232. 29. MILEDI, R., and C. R. SLATER. 1970. On the degeneration of rat neuromuscular junctions after nerve section. J. Physiol. (London) 207: 507-528. 30. OCHS, S. 1972. Fast transport of materials in mammalian nerve fibers. Science 176 : 252-259.

PENNINGTON, R. J. T. 1972. Biochemistry of muscle diseases, pp. 41-67. In “Biochemical Aspects of Nervous Diseases.” J. N. Cumings [Ed.]. Plenum, London. 32. POLLACK, M. S., and J. W. C. BIRD. 1968. Distribution and particle properties of acid hydrolase in denervated muscle. .4mer. J. Physiol. 215: 7X-72!?. 31.

554 33.

MCLAUGHLIN,

ABOOD,

AND

ROSMANN

S. 1956. “Nonparametric Statistics for the Behavioral Sciences.” McGrawHill, New York. 34. SYROVY, I., I. HAJEK, and E. GUTMANN. 1966. Factors affecting the proteolytic activity in denervated muscle. Physiol. Bohemoslov. 15 : 7-13. 35. THORPE, W. R., and P. SEEMAN. 1971. Effect of denervating skeletal muscle on calcium binding by isolated sarcolemma. Exp. Neural. 30: 277-290. 36. WEINSTOCK, I. M., and A. A. IODICE. 1969. Acid hydrolase activity in muscular dystrophy and denervation atrophy, pp. 450-468. In “Lysosomes in Biology and Pathology.” J. T. Dingle and H. B. Fell [Eds.]. North-Holland, Amsterdam. SIEGEL,