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Regulation of increased acid proteinase in denervated skeletal muscle
Regulation
of Increased Acid Proteinase Skeletal Muscle JACK
Department
of
NICLAUGHLIN
AKD
H.
BRUCE
Pharmacology alld ToxicologJj, Uxizwsity dledicille and Delztistry, Rochester, Nezv Receizled
July
17, 1975;
rezlisiort
rcccizvd
in Denervated
BOSMANN of
York August
*
Rochester 14642
School
of
22, 1975
Acid proteolytic enzyme activity was determined in homogenates of control and denervated rat extensor digitorum longus muscle. With either undenatured or [3H]acetylated hemoglobin (pH 3.5) as substrate, total enzyme activity in muscles denervated near the point of nerve entry was consistently increased 48 and 72 hr after surgery. A series of ‘near-far’ experiments was performed to determine if the length of nerve stump left attached to the denervated muscle would affect the time course of the elevation in proteolytic activity, but these experiments did not result in differences that could be attributed to the level of nerve section. Acid proteinase, presumably important in degradative processes, is clearly under some form of neural control. Whether the motor nerve exerts this influence by controlling the level of muscular activity or by additional means remains to be established.
INTRODUCTION Numerous studies have indicated that proteolytic enzyme activity at acid pH is increased in atrophic conditions of skeletal muscle, including denervation. While most work with denervated muscle has been concerned with long-term effects, several reports have demonstrated that increased acid proteolytic activity is an early consequence of denervation (3, 6, 9, 10). An important question is whether this early effect on proteolytic activity is primarily determined by changes in muscle activity or by some additional, extra-impulse factors. Hijek, Gutmann, and Syrovl (3) have reported that the increased proteolytic activity is dependent on the length of nerve stump left attached to the denervated muscle, evidence of metabolic control inde1 Dr. McLaughlin is a postdoctoral fellow of the Muscular Dystrophy Associations of America. Dr. Bosmann is a Scholar of the Leukemia Society of America. This study was supported in part by a grant from the Muscular Dystrophy Associations of America. 276 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
DENERVATED
MUSCLE
277
pendent of impulse transmission. The purpose of the present study was to assess in more detail the relative contributions of muscle activity and factors other than activity to neural control of muscle acid proteinase. METHODS Tissz(e Preparation. Experiments were carried out on extensor digitorum longus muscles of adult male Long Evans or adult female Holtzman rats. Animals, heavily anesthetized with ether, were subjected to one of the three following surgical procedures: (a) The extensor digitorum longus of either the left or the right leg was denervated by transecting the nerve at the knee just external to the peroneal muscle group. A sham operation was performed on the opposite leg and its extensor digitorum longus served as the control muscle. (b) The sciatic nerve of one leg was transected in the upper thigh at a level near the head of the femur, while on the opposite leg the nerve supplying extensor digitorum longus was transected at the knee as above. Appropriate sham operations were made on each leg. (c) The sciatic nerve of each leg was transected in the upper thigh. Additionally, in one leg the nerve was transected near extensor digitorum longus, while a sham operation was performed on the other leg. In this last procedure the ‘control’ and experimental extensor digitorum longus muscles thus differed only in the length of severed nerve stump left attached to the muscle. No antibiotics were given; the incisions were sutured, and the animals were maintained for various intervals on a regular laboratory diet with ad lib access to water. Animals were killed by a sharp blow on the neck, and the extensor digitorum longus of each leg was removed and placed in a beaker of icecold saline. Each muscle was then minced with scissors in 25 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 measurement of acid proteolytic enzyme activity by Assay 1, but was diluted two-fold with 0.1% Triton X-100 for use in Assay 2. Acid Proteolytic Activity: Assay 1. Proteolytic activity at pH 3.5 was assayed by the method of Anson (1) using dialyzed and lyophilized bovine hemoglobin (Sigma Chemical Co., Type I) as substrate. To 1.0 ml of 2.5% (w/v) hemoglobin was added 1.0 ml of a solution of 1.35 M acetic acid and 0.02 M ammonium sulfate, followed by the addition of 0.5 ml of the muscle homogenate. This mixture was incubated for 2 hr at 37 C in a Dubnoff metabolic shaker. Incubation was terminated by placing the assay tubes in an ice-water bath and adding 1.0 ml of cold 17.5% (w/v) trichloracetic acid to the tubes. After 15 min the tubes were centrifuged at
278
~~CLAUGHLIN
AND
BOSM.~NN
1500 g for 10 min. The supernatant was removed to new test tubes and recentrifuged. An aliquot of this supernatant was then assayed by a slight modification of the method of Lowry, Rosebrough, Farr, and Randall (5)) using bovine serum albumin as an arbitrary standard. Controls consisted of usual assays in which water was substituted for the hemoglobin solution, usual assays in which 0.1% Triton X-100 was substituted for the muscIe homogenate, and assays with and without hemoglobin which were immediately terminated with trichloracetic acid. These control assays corrected for contributions from trichloracetic acid soluble substances present in the hemoglobin solution and in the unincubated muscle homogenates, as well as for enzyme activity directed against endogenous substrates. Total proteolytic activity is expressed as mg of albumin equivalents released/muscle/ hr. All assays were performed in triplicate. Assay 2. Proteolytic activity at pH 3.5 was measured using as substrate [3H] acetylated bovine hemoglobin prepared by the method of Hille, Barrett, Dingle, and Fell (4). This assay, described in detail previously (6), monitors the enzymatic production of trichloracetic acid soluble radioactivity. Total activity is here expressed as counts per min (cpm) x 10ml;/ muscle/hr. All assays were performed in quintuplicate. Product formation for both Assay 1 and Assay 2 was linear with respect to time of incubation and to the quantity of added muscle homogenate. Statistic-s. There was considerable variation among animals in the values of enzyme activities, but no consistent differences were found in individual control animals when the right and left muscles were compared. In the comparisons of experimental and control muscles, the absolute value of the control muscle was subtracted from that of the experimental muscle of each animal. The median and range of values for the indicated number of experimental and control muscle pairs 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-rants test ( 11) . RESULTS An early elevation in acid proteolytic activity of muscle homogenates after denervation has been well-documented (3, 6, 9, 10). Our data for extensor digitorum Iongus muscles denervated near the point of nerve entry need only be summarized. With either hemoglobin (Assay 1) or [3H]acetylated hemoglobin (Assay 2) as substrate, acid proteinase in the denervated muscle homogenates was consistently increased 48 and 72 hr after surgery. At 72 hr after denervation, the average increases (n = 10) were 3.570 (Assay 1) and 20% (Assay 2). N o si g ni fi cant differences between denervated and control muscle enzyme activities were found 2-l hr after dener&on,
DEKERX’ATED
TABLE Acid
Proteolytic
1
Activity in Holtzman Rat Extensor Digitorum ‘Single-Far’ and ‘Single-Near’ Denervation’ Near
1 day (6) 2 days 3 days
(6) (6)
279
MUSCLE
2.09 2.17 2.17
(1.40-2.89) (1.83-2.83) (1.64-2.98)
Far 1.96 2.16 2.18
Longus
Following
Differences
(1.43-2.68) (1.60-2.71) (1.53-2.45)
(-0.40 to +0.57) ( - 0.24 to +0.49) (-0.13 to f0.53)
n Denervation was carried out using procedure (b), described in Methods. Extensor digitorum longus with a short nerve stump is referred to as “near” and extensor digitorum longus with the longer nerve as “far”. Values are the medians and ranges of the absolute values of muscles and the ranges of the individual differences obtained from the number of animals inside parentheses. Units of enzyme activity are mg albumin equivalents degraded. muscle-l. hr-‘. Differences are not significant.
“Near-far” experiments were conducted to determine if the length of nerve stump left attached to denervated muscle affected the time course of the development of increased acid proteinase activity, In experiments with Holtzman rats, the sciatic nerve of one side was transected in the upper thigh, while on the opposite side the nerve supplying extensor digitorum longus was cut at the knee. The results of proteolytic assays (undenatured hemoglobin, substrate) on one, two, and three day denerrated and control extensor digitorum longus homogenates are presented in Table 1. Total acid proteolytic activity, corrected for endogenous activity (activity in the absence of added hemoglobin), was not significantly different when muscles with short and long nerve stumps were compared. In Table 2 are presented the results of experiments with Long Evans rats in which the sciatic nerve on each side was transected in the upper thigh. On the experimental side the nerve was cut near the muscle at the TABLE Acid
Proteolytic
Activity in Long Evans Rat Extensor ing “Double-Far” and “Single-Near” Near
1 day (6) 2 days 3 days
(5) (3)
2
4.82 (4.13-5.19) 5.61 (4.88-6.65) 5.73 (4.92-6.81)
Far 4.74 (3.85-5.17) 5.89 (4.72-6.72) 5.79 (5.49-7.40)
Digitorum Denervationn
Longus
Follow-
Differences (-0.76 (-0.28 (-0.05
to +0.96) to +0.16) to -0.59)
Q Denervation was carried out using procedure (c), described in Methods. Extensor digitorum longus with a short attached nerve stump is referred to as.“near”, and exValues are as given in Table 1. tensor digitorum longus with the longer nerve as “far”. IJnits are cpm . lO-‘~rn~cle-~~ hr-‘. Differences are nol significant.
280
MCLAUGHLIN
AND
BOSMANN
knee, while a sham operation was performed on the ‘control’ side. The data do not show any significant differences in enzymatic activities between experimental and control muscles at 24, 48, or 72 hr after surgery. DISCUSSION Because changes in muscle wt and protein concentration that accompany denervation complicate selection of an appropriate base on which to refer enzyme activity data, acid proteolytic activity in the present report was expressed on a whole muscle basis. The results confirm previous reports that muscle proteolytic activity assayed at acid pH with hemoglobin as substrate is elevated soon after denervation (3, 6, 9, 10). Qualitatively similar results were obtained in numerous parallel assays with both undenatured and [ 3H] acetylated bovine hemoglobin as substrate. The greater increase observed with undenatured hemoglobin as substrate is consistent with the suggestion of Park and Pennington (8) that the trichloracetic acid soluble breakdown products analyzed by the method of Lowry et al. (5) are largely free amino acids. The present experiments place the onset of increased proteolytic activity some time between 24 and 48 hr after “near” denervation. Pluskal and Pennington (9) reported slightly increased proteolytic activity in homogenates of rat extensor digitorum longus denervated for 24 h r by cuttmg ’ the sciatic nerve. The role of muscle acid proteolytic enzymes either in general protein catabolism or in the biochemical regulation of membrane systems of muscle is not well understood, but could prove to be important in the production of the various physiological and chemosensitive alterations characteristic of denervated muscle (6). Hijek et al. (3) presented data indicating that 21 hr after surgery proteolytic activity (hemoglobin substrate, pH 3.5) in extensor digitorum longus denervated near the muscle was about 130% of the activity in the contralateral extensor digitorum longus which had been denervated by cutting the sciatic nerve in the thigh. The authors concluded that “nervous nonimpulse mechanisms” regulate the rate of muscle protein degradation. Because of the important implications of such phenomena, we attempted in two series of experiments to differentiate between the effects of muscle inactivity and the effects of other “neural” factors on the development of the postdenervation increase in muscle proteolytic activity. One experimental design (Table 1) was similar to that of HAjek et al. (3), except that sham operations were included to control for the known effects of trauma and ischemia on acid hydrolase activities (2, 7). In the other experimental paradigm (Table 2) the sciatic nerve of each side was cut in the upper thigh. On the experimental side the nerve was additionally cut near extensor digitorum longus, while a sham
DENERVATED
MUSCLE
281
second operation was performed on the contralateral “control” leg. The experimental and control muscles were thus equated in terms of general disuse and differed only in the length of the attached nerve stump. No differences between experimental and control muscles were found 24, 48, or 72 hr after surgery using either “near-far” technique. As discussed previously, increased acid proteinase activity in singly denervated muscles could be easily demonstrated 48 and 72 hr after nerve section. With regard to the inability to confirm the observations of Hajek et al. (3)) it should be pointed out that in that study 2% KC1 muscle homogenate supernatants were used as the enzyme source rather than the whole Triton X-100 homogenates used in the present study. Furthermore, Hdjek et al. (3) did not correct for proteolytic activity directed against endogenous muscle proteins. In the present study assay conditions were optimized for the degradation of hemoglobin. The use of other substrates and the optimization of proteolytic activity directed against particular endogenous substrates should give a more complete understanding of the early changes in denervated muscle. We have begun, therefore, to characterize in some detail early changes in the autolytic capacity of homogenates of denervated muscle. The results closely parallel those of the present report. Trichloracetic acid soluble material enzymatically released by muscle homogenates in the absence of exogenous substrates was analyzed both by the method of Lowry et al. (5) and by the sensitive and specific tyrosine assay method of Waalkes and Undenfriend ( 12). Autolytic activity in homogenates of denervated extensor digitorum longus measured with either technique was found to have consistently increased 48 and 72 hr after surgery. “Near-far” experiments of the type described in the present report again proved negative in the sense that no influence of nerve stump length could be demonstrated on the time course of the increase in autolytic activity. While the failure to demonstrate any “near-far” differences suggests that the most important determinant of the early increase in acid proteinase in extensor digitorum longus muscle is the reduced level of muscular activity, alternate possibilities should be considered. The most favored experimental circumstance for demonstrating an influence of the distal nerve stump length on the development of denervation-produced alterations in muscle is the situation in which the onset of the alteration is preceded by a lag phase during which no change is observed, and is followed by a period of abrupt, rapid change. The rate of increase in muscle acid proteinase that follows denervation could be too slow to demonstrate “nearfar” differences. Moreover, nonimpulse mediated effects could be restricted to particular proteolytic enzymes and obscured by the fact that in crude muscle homogenates, acid proteolytic activity most likely represents the concerted action of a number of protein and peptide hydrolase enzymes,
282
MCLAUGHLIN
AND
BOSMANN
REFERENCES 1. ANSON, M. L. 1938. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J. Gen. Physiol. 22 : 79-89. 2. ARCANGELI, P., P. DEL SOLDATO, TT. DIGIESI, and F. MELANI. 1973. Changes in the activities of lysosomal enzymes in striated muscle following ischemia. Life Sri. 12: 3. HAJEK,
4.
5. 6.
7.
8. 9. 10. 11. 12.
Part
II:
13-23.
I., E. GUTMANN, and I. SYROV~. 1964. Proteolytic activity in denervated and reinervated muscle. Physiol. Bohcrr~osluv 13 : 32-38. HILLE, M. B., -4. J. BARRETT, J. T. DIXGLE, and H. B. FELL. 1970. Microassay for cathepsin D shows an unexpected effect of cycloheximide on limb-bone rudiments in organ culture. ExP. Cell. Rcs. 61 : 470-471. LOWRY, 0. H., H. J. ROSEBROUGII, .4. L. FARR, and R. J. RCTDALL. 1951. Protein measurement with the Folin phenoi reagent. J. Bid. Chrm. 193 : 26.5375. MCLAUGHLIN, J., L. G. ABOOD, and H. B. BOS~~AKN. 1974. Early elevations of glycosidase, acid phosphatase, and acid proteolytic enzyme activity in denervated skeletal muscle. Exp. Newel. 42: 541-554. NAKAHARA, M. 1971. Lysosomal enzyme activities in muscle with traumatically induced edema. Pharmacology 6 : 365-370. PARK, D. C., and R. J. PENNINGTON. 1967. Proteinase activity in muscle particles. Enzym. Biol. Clin. 8 : 149-160. PLUSXAL, M. G., and R. J. PENNINGTON. 1973. Peptide hydrolase activities in denervated muscle. Biochem. Sol-. Tram. 1 : 1307-1310. POLLACK, M. S., and J. W. C. BIRD. 1968. Distribution and particle properties of acid hydrolases in denervated muscle. ,4rlt. J. Phjviol. 215 : 71&722. SIEGEL, S. 1956. “Nonparametric Statistics for the Behavioral Sciences.” McGra\vHill Book Co., Inc., New York. &‘AALXES, T. P., and S. UNDESFRIEXD. 1957. A fluorometric method for the estimation of tyrosine in plasma and tissues. J. Lab. Clint. Jltd. 50: 733-736.
of Increased Acid Proteinase Skeletal Muscle JACK
Department
of
NICLAUGHLIN
AKD
H.
BRUCE
Pharmacology alld ToxicologJj, Uxizwsity dledicille and Delztistry, Rochester, Nezv Receizled
July
17, 1975;
rezlisiort
rcccizvd
in Denervated
BOSMANN of
York August
*
Rochester 14642
School
of
22, 1975
Acid proteolytic enzyme activity was determined in homogenates of control and denervated rat extensor digitorum longus muscle. With either undenatured or [3H]acetylated hemoglobin (pH 3.5) as substrate, total enzyme activity in muscles denervated near the point of nerve entry was consistently increased 48 and 72 hr after surgery. A series of ‘near-far’ experiments was performed to determine if the length of nerve stump left attached to the denervated muscle would affect the time course of the elevation in proteolytic activity, but these experiments did not result in differences that could be attributed to the level of nerve section. Acid proteinase, presumably important in degradative processes, is clearly under some form of neural control. Whether the motor nerve exerts this influence by controlling the level of muscular activity or by additional means remains to be established.
INTRODUCTION Numerous studies have indicated that proteolytic enzyme activity at acid pH is increased in atrophic conditions of skeletal muscle, including denervation. While most work with denervated muscle has been concerned with long-term effects, several reports have demonstrated that increased acid proteolytic activity is an early consequence of denervation (3, 6, 9, 10). An important question is whether this early effect on proteolytic activity is primarily determined by changes in muscle activity or by some additional, extra-impulse factors. Hijek, Gutmann, and Syrovl (3) have reported that the increased proteolytic activity is dependent on the length of nerve stump left attached to the denervated muscle, evidence of metabolic control inde1 Dr. McLaughlin is a postdoctoral fellow of the Muscular Dystrophy Associations of America. Dr. Bosmann is a Scholar of the Leukemia Society of America. This study was supported in part by a grant from the Muscular Dystrophy Associations of America. 276 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
DENERVATED
MUSCLE
277
pendent of impulse transmission. The purpose of the present study was to assess in more detail the relative contributions of muscle activity and factors other than activity to neural control of muscle acid proteinase. METHODS Tissz(e Preparation. Experiments were carried out on extensor digitorum longus muscles of adult male Long Evans or adult female Holtzman rats. Animals, heavily anesthetized with ether, were subjected to one of the three following surgical procedures: (a) The extensor digitorum longus of either the left or the right leg was denervated by transecting the nerve at the knee just external to the peroneal muscle group. A sham operation was performed on the opposite leg and its extensor digitorum longus served as the control muscle. (b) The sciatic nerve of one leg was transected in the upper thigh at a level near the head of the femur, while on the opposite leg the nerve supplying extensor digitorum longus was transected at the knee as above. Appropriate sham operations were made on each leg. (c) The sciatic nerve of each leg was transected in the upper thigh. Additionally, in one leg the nerve was transected near extensor digitorum longus, while a sham operation was performed on the other leg. In this last procedure the ‘control’ and experimental extensor digitorum longus muscles thus differed only in the length of severed nerve stump left attached to the muscle. No antibiotics were given; the incisions were sutured, and the animals were maintained for various intervals on a regular laboratory diet with ad lib access to water. Animals were killed by a sharp blow on the neck, and the extensor digitorum longus of each leg was removed and placed in a beaker of icecold saline. Each muscle was then minced with scissors in 25 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 measurement of acid proteolytic enzyme activity by Assay 1, but was diluted two-fold with 0.1% Triton X-100 for use in Assay 2. Acid Proteolytic Activity: Assay 1. Proteolytic activity at pH 3.5 was assayed by the method of Anson (1) using dialyzed and lyophilized bovine hemoglobin (Sigma Chemical Co., Type I) as substrate. To 1.0 ml of 2.5% (w/v) hemoglobin was added 1.0 ml of a solution of 1.35 M acetic acid and 0.02 M ammonium sulfate, followed by the addition of 0.5 ml of the muscle homogenate. This mixture was incubated for 2 hr at 37 C in a Dubnoff metabolic shaker. Incubation was terminated by placing the assay tubes in an ice-water bath and adding 1.0 ml of cold 17.5% (w/v) trichloracetic acid to the tubes. After 15 min the tubes were centrifuged at
278
~~CLAUGHLIN
AND
BOSM.~NN
1500 g for 10 min. The supernatant was removed to new test tubes and recentrifuged. An aliquot of this supernatant was then assayed by a slight modification of the method of Lowry, Rosebrough, Farr, and Randall (5)) using bovine serum albumin as an arbitrary standard. Controls consisted of usual assays in which water was substituted for the hemoglobin solution, usual assays in which 0.1% Triton X-100 was substituted for the muscIe homogenate, and assays with and without hemoglobin which were immediately terminated with trichloracetic acid. These control assays corrected for contributions from trichloracetic acid soluble substances present in the hemoglobin solution and in the unincubated muscle homogenates, as well as for enzyme activity directed against endogenous substrates. Total proteolytic activity is expressed as mg of albumin equivalents released/muscle/ hr. All assays were performed in triplicate. Assay 2. Proteolytic activity at pH 3.5 was measured using as substrate [3H] acetylated bovine hemoglobin prepared by the method of Hille, Barrett, Dingle, and Fell (4). This assay, described in detail previously (6), monitors the enzymatic production of trichloracetic acid soluble radioactivity. Total activity is here expressed as counts per min (cpm) x 10ml;/ muscle/hr. All assays were performed in quintuplicate. Product formation for both Assay 1 and Assay 2 was linear with respect to time of incubation and to the quantity of added muscle homogenate. Statistic-s. There was considerable variation among animals in the values of enzyme activities, but no consistent differences were found in individual control animals when the right and left muscles were compared. In the comparisons of experimental and control muscles, the absolute value of the control muscle was subtracted from that of the experimental muscle of each animal. The median and range of values for the indicated number of experimental and control muscle pairs 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-rants test ( 11) . RESULTS An early elevation in acid proteolytic activity of muscle homogenates after denervation has been well-documented (3, 6, 9, 10). Our data for extensor digitorum Iongus muscles denervated near the point of nerve entry need only be summarized. With either hemoglobin (Assay 1) or [3H]acetylated hemoglobin (Assay 2) as substrate, acid proteinase in the denervated muscle homogenates was consistently increased 48 and 72 hr after surgery. At 72 hr after denervation, the average increases (n = 10) were 3.570 (Assay 1) and 20% (Assay 2). N o si g ni fi cant differences between denervated and control muscle enzyme activities were found 2-l hr after dener&on,
DEKERX’ATED
TABLE Acid
Proteolytic
1
Activity in Holtzman Rat Extensor Digitorum ‘Single-Far’ and ‘Single-Near’ Denervation’ Near
1 day (6) 2 days 3 days
(6) (6)
279
MUSCLE
2.09 2.17 2.17
(1.40-2.89) (1.83-2.83) (1.64-2.98)
Far 1.96 2.16 2.18
Longus
Following
Differences
(1.43-2.68) (1.60-2.71) (1.53-2.45)
(-0.40 to +0.57) ( - 0.24 to +0.49) (-0.13 to f0.53)
n Denervation was carried out using procedure (b), described in Methods. Extensor digitorum longus with a short nerve stump is referred to as “near” and extensor digitorum longus with the longer nerve as “far”. Values are the medians and ranges of the absolute values of muscles and the ranges of the individual differences obtained from the number of animals inside parentheses. Units of enzyme activity are mg albumin equivalents degraded. muscle-l. hr-‘. Differences are not significant.
“Near-far” experiments were conducted to determine if the length of nerve stump left attached to denervated muscle affected the time course of the development of increased acid proteinase activity, In experiments with Holtzman rats, the sciatic nerve of one side was transected in the upper thigh, while on the opposite side the nerve supplying extensor digitorum longus was cut at the knee. The results of proteolytic assays (undenatured hemoglobin, substrate) on one, two, and three day denerrated and control extensor digitorum longus homogenates are presented in Table 1. Total acid proteolytic activity, corrected for endogenous activity (activity in the absence of added hemoglobin), was not significantly different when muscles with short and long nerve stumps were compared. In Table 2 are presented the results of experiments with Long Evans rats in which the sciatic nerve on each side was transected in the upper thigh. On the experimental side the nerve was cut near the muscle at the TABLE Acid
Proteolytic
Activity in Long Evans Rat Extensor ing “Double-Far” and “Single-Near” Near
1 day (6) 2 days 3 days
(5) (3)
2
4.82 (4.13-5.19) 5.61 (4.88-6.65) 5.73 (4.92-6.81)
Far 4.74 (3.85-5.17) 5.89 (4.72-6.72) 5.79 (5.49-7.40)
Digitorum Denervationn
Longus
Follow-
Differences (-0.76 (-0.28 (-0.05
to +0.96) to +0.16) to -0.59)
Q Denervation was carried out using procedure (c), described in Methods. Extensor digitorum longus with a short attached nerve stump is referred to as.“near”, and exValues are as given in Table 1. tensor digitorum longus with the longer nerve as “far”. IJnits are cpm . lO-‘~rn~cle-~~ hr-‘. Differences are nol significant.
280
MCLAUGHLIN
AND
BOSMANN
knee, while a sham operation was performed on the ‘control’ side. The data do not show any significant differences in enzymatic activities between experimental and control muscles at 24, 48, or 72 hr after surgery. DISCUSSION Because changes in muscle wt and protein concentration that accompany denervation complicate selection of an appropriate base on which to refer enzyme activity data, acid proteolytic activity in the present report was expressed on a whole muscle basis. The results confirm previous reports that muscle proteolytic activity assayed at acid pH with hemoglobin as substrate is elevated soon after denervation (3, 6, 9, 10). Qualitatively similar results were obtained in numerous parallel assays with both undenatured and [ 3H] acetylated bovine hemoglobin as substrate. The greater increase observed with undenatured hemoglobin as substrate is consistent with the suggestion of Park and Pennington (8) that the trichloracetic acid soluble breakdown products analyzed by the method of Lowry et al. (5) are largely free amino acids. The present experiments place the onset of increased proteolytic activity some time between 24 and 48 hr after “near” denervation. Pluskal and Pennington (9) reported slightly increased proteolytic activity in homogenates of rat extensor digitorum longus denervated for 24 h r by cuttmg ’ the sciatic nerve. The role of muscle acid proteolytic enzymes either in general protein catabolism or in the biochemical regulation of membrane systems of muscle is not well understood, but could prove to be important in the production of the various physiological and chemosensitive alterations characteristic of denervated muscle (6). Hijek et al. (3) presented data indicating that 21 hr after surgery proteolytic activity (hemoglobin substrate, pH 3.5) in extensor digitorum longus denervated near the muscle was about 130% of the activity in the contralateral extensor digitorum longus which had been denervated by cutting the sciatic nerve in the thigh. The authors concluded that “nervous nonimpulse mechanisms” regulate the rate of muscle protein degradation. Because of the important implications of such phenomena, we attempted in two series of experiments to differentiate between the effects of muscle inactivity and the effects of other “neural” factors on the development of the postdenervation increase in muscle proteolytic activity. One experimental design (Table 1) was similar to that of HAjek et al. (3), except that sham operations were included to control for the known effects of trauma and ischemia on acid hydrolase activities (2, 7). In the other experimental paradigm (Table 2) the sciatic nerve of each side was cut in the upper thigh. On the experimental side the nerve was additionally cut near extensor digitorum longus, while a sham
DENERVATED
MUSCLE
281
second operation was performed on the contralateral “control” leg. The experimental and control muscles were thus equated in terms of general disuse and differed only in the length of the attached nerve stump. No differences between experimental and control muscles were found 24, 48, or 72 hr after surgery using either “near-far” technique. As discussed previously, increased acid proteinase activity in singly denervated muscles could be easily demonstrated 48 and 72 hr after nerve section. With regard to the inability to confirm the observations of Hajek et al. (3)) it should be pointed out that in that study 2% KC1 muscle homogenate supernatants were used as the enzyme source rather than the whole Triton X-100 homogenates used in the present study. Furthermore, Hdjek et al. (3) did not correct for proteolytic activity directed against endogenous muscle proteins. In the present study assay conditions were optimized for the degradation of hemoglobin. The use of other substrates and the optimization of proteolytic activity directed against particular endogenous substrates should give a more complete understanding of the early changes in denervated muscle. We have begun, therefore, to characterize in some detail early changes in the autolytic capacity of homogenates of denervated muscle. The results closely parallel those of the present report. Trichloracetic acid soluble material enzymatically released by muscle homogenates in the absence of exogenous substrates was analyzed both by the method of Lowry et al. (5) and by the sensitive and specific tyrosine assay method of Waalkes and Undenfriend ( 12). Autolytic activity in homogenates of denervated extensor digitorum longus measured with either technique was found to have consistently increased 48 and 72 hr after surgery. “Near-far” experiments of the type described in the present report again proved negative in the sense that no influence of nerve stump length could be demonstrated on the time course of the increase in autolytic activity. While the failure to demonstrate any “near-far” differences suggests that the most important determinant of the early increase in acid proteinase in extensor digitorum longus muscle is the reduced level of muscular activity, alternate possibilities should be considered. The most favored experimental circumstance for demonstrating an influence of the distal nerve stump length on the development of denervation-produced alterations in muscle is the situation in which the onset of the alteration is preceded by a lag phase during which no change is observed, and is followed by a period of abrupt, rapid change. The rate of increase in muscle acid proteinase that follows denervation could be too slow to demonstrate “nearfar” differences. Moreover, nonimpulse mediated effects could be restricted to particular proteolytic enzymes and obscured by the fact that in crude muscle homogenates, acid proteolytic activity most likely represents the concerted action of a number of protein and peptide hydrolase enzymes,
282
MCLAUGHLIN
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