EXPERIMENTAL
NEUROLOGY
52,
Glycolysis KENNETH
R.
40-48 (1976)
in Skeletal
WAGNER,
of Neurology
CAROL
and Pediatrics,
Medicine, Baltimore, Received
Regeneration
R. MAX, L. KOSKI
STEPHEN AND
Departments
Muscle
University
Maryland
’
M.
GROLLMAN,
of Maryland
School
EDWIN
of
21201
February lo,1976
The myotoxic local anesthetic Marcaine causes widespread destruction of skeletal muscle followed by complete regeneration. To identify the metabolic adaptations that underlie muscle regeneration, we have assessed the glycolytic capacity of Marcaine-treated muscles. There was no change in the activities of adenylate kinase or creatine kinase. A 30% increase was observed in hexokinase activity and a 20 to 307% decrease in phosphofrucmkinase, pyruvate kinase, a-glycerophosphate dehydrogenase, and lactate dehydrogenase activities in rat anterior tibia1 muscles. The latter enzyme activities returned essentially to control values by about Day 11 after drug injection. A 605% decrease was noted in total glycogen phosphorylase activity, follow4 by a return to control by Day 28. Lactate production by intact extensor digitorum muscles in vitro was unaffected by Marcaine administration, suggesting that the decrease in enzyme activities was not sufficient to impair overall glycolytic activity. Thus glycolysis is maintained to a much greater extent than oxidative metabolism and may fulfill the energy requirements during MarCaine-induced regeneration of skeletal muscle.
INTRODUCTION Skeletal muscle possessesa remarkable capacity for regeneration following administration of myotoxic drugs, mincing and autoimplantation, and ischemia (1-3, 10). Under each of these circumstances an initial period of muscle degeneration occurs, followed by regeneration. A fundamental question concerns the nature of the energy sources that permit survival of degenerating muscles and support subsequent muscle regeneration. We have attempted to answer this question by surveying the major 1 Supported by grants from the National Institutes of He&h (NS-11342-Ol), the Muscular Dystrophy Associations of America, Inc., and the Frank G. Bressler Fund. We thank Ms. B. Pasko for preparation of the typescript and Drs. C. Sumbilla and P. T. Ozand for helpful comments. 40 Copyright 1976 by Academic Press, Inc. All rights oQ reproduction in any form reserved.
GLYCOLYSIS
IN
MUSCLE
REGENERATION
41
energy-yielding metabolic pathways in muscle undergoing degeneration and regeneration following administration of a myotoxic local anesthetic, Marcaine. In a previous report ( 1 1), we described loss of oxidative metabolism in Marcaine-treated muscles and enhanced activity of the hexosemonophosphate shunt, In the present communication, we present evidence which suggests that glycolysis survives Marcaine-induced degeneration. A preliminary report of these data has been presented (IS). MATERIALS
AND
METHODS
The materials used and their sources were: enzymes and Sigma ; [ 3H] mannitol and [ UJ4C] glucose-l-P Amersham Searle POPOP, New England Nuclear ; Marcaine (bupivacaine) , Laboratories. Adult, male, Sprague-Dawley rats, weighing 200 to 250 g, chased from Charles River Breeding Laboratories, Inc., and and Purina Rat Chow ad lib. Extracellular
substrates, ; PPO and Winthrop were purfed water
Space and Lactate Production
Treatment of Muscles. For in vitro studies with intact muscles, the extensor digitorum longus muscle was employed, because its small size permits diffusion of substrates and metabolites to a greater extent than the larger anterior tibia1 muscle (9). On three successive days, a 0.9% NaCl solution containing Marcaine (2.0 mg) plus hyaluronidase (Sigma, 300 units) was injected into the extensor digitorum longus after surgical exposure, and the incision was sutured. The contralateral extensor digitorum longus was surgically exposed and injected with 0.9% NaCl. At 1, 3, and 6 days after the third injection, the muscles were excised, passively stretched on stainless-steel frames (4)) and incubated in Robinson’s medium (16) containing mannitol (1 nlM) and glucose (1 mg/ml) in 25-ml Erlenmeyer flasks (4). Incubation was carried out at 37°C with continuous gassing with a mixture of O2 and CO, (95 : 5). Extvacelhlar Space. This parameter was assessed in the in vitro system described above using [ 3H ] mannitol (4). Lactate Production. At 0, 30, 60, and 90-min intervals during the incubation samples of the medium were removed. The lactate produced was measured by spectrophotometric assay (6) and reported as pmoles/hr/g fresh weight of muscles. Muscles lost about 5% of their fresh weight after 90 min of incubation. The initial weight was employed for the calculations. Lactate production was linear with respect to muscle size in the range used in this study (60 to 150 mg).
42
WAGNER
ET
AL.
Enzyme Analyses Treatment of Muscles. An 0.9% NaCl solution containing Marcaine (2.0 mg) plus hyaluronidase (Sigma, 300 units), was injected into the anterior tibia1 muscle of rats (3, 11)) with the contralateral anterior tibia1 muscles serving as controls and receiving injections of 0.9% NaCl. At 1, 3, 5, 7, 12, 19 and 28 days after three daily injections of Marcaine plus hyaluronidase or of 0.9% NaCl, the rats were decapitated and the muscles were removed, weighed, and minced with scissors in ice-cold 0.05 M Tris-HCl, pH 7.6, containing 0.20 mM dithiothreitol. The finely minced muscles were then homogenized (1: 10, w/v) by hand, using a Tenbroeck homogenizer. The homogenate was centrifuged at 18,000g for 20 min at 4°C. Enzymes were assayed in the resulting supernatants. Enzyme Assays. The following enzymes were assayed by spectrophotometric procedures : hexokinase (EC 2.7.1.1.) ( 17)) phosphofructokinase (EC 2.7.1.11) (8, 17), py ruvate kinase (EC 2.7.1.40) ( 12)) cytoplasmic a-glycerophosphate dehydrogenase (EC 2.7.1 .S) (5)) creatine kinase (EC 2.7.3.2) (6)) adenylate kinase (EC 2.7.4.3) (6)) and lactate dehydrogenase (EC 1.1.1.27) (6). Creatine kinase was assayed in the presence of 5’-AMP to inhibit adenylate kinase ( 13). Glycogen phosphorylase (EC 2.4.1.1 j was measured in the direction of glycogen synthesis in the presence and absence of S-AMP, using glycogen and [U-14C]glucose-1-P as substrates. The assay is a modification of a method for glycogen synthetase determination (15 ; B. I. Brown, personal communication). Phosphorylase data are given as activity in the presence of 5’-AMP. Protein was determined by the method of Lowry et al. (7). Expression of Data, Enzyme activities were linear with respect to time and protein concentration under our assay conditions. Enzyme data are exTABLE Enzyme
Activities
Enzyme
Number Animals
Phosphorylase Hexokinase Phosphofructokinase or-glycerophosphate dehydrogenase Pyruvate kinase Lactate dehydrogenase Adenylate kinase Creatine kinase a values
are mean
f
1 in Control
SEM.
24 16 36 37 37 12 37 37
Muscles of
Specific activity” (nmoles/min/mg protein) 1402.70 26.20 723.00 931.00 5155.00 259.20 1430.00 579.00
& 56.22 zk 0.88 f 38.00 f 35.00 i 299.00 f 10.90 i 72.00 i 35.00
GLYCOLYSIS
IN
MUSCLE
TABLE Effect Days after third Marcaine injection 1 3 6
Number animals
of Marcaine
of
2
on Extracellular Extracellular
Marcaine-treated (ml/100 9 3 3
74.44 60.67 49.67
Space” P
space
g fresh
f 13.86b f 9.29 h 3.79
Q Experimental procedures as given in digitorum muscles (N = 16), extracellular b Numbers are g + SEM.
43
REGENERATION
Control weight) 56.22 49.00 51.00
f 9.23 f 5.57 f 8.00
the text. For space is 31.69
pressed as average percentage of contralateral tive control values are given in Table 1.
NEUROLOGY
52,
Glycolysis KENNETH
R.
40-48 (1976)
in Skeletal
WAGNER,
of Neurology
CAROL
and Pediatrics,
Medicine, Baltimore, Received
Regeneration
R. MAX, L. KOSKI
STEPHEN AND
Departments
Muscle
University
Maryland
’
M.
GROLLMAN,
of Maryland
School
EDWIN
of
21201
February lo,1976
The myotoxic local anesthetic Marcaine causes widespread destruction of skeletal muscle followed by complete regeneration. To identify the metabolic adaptations that underlie muscle regeneration, we have assessed the glycolytic capacity of Marcaine-treated muscles. There was no change in the activities of adenylate kinase or creatine kinase. A 30% increase was observed in hexokinase activity and a 20 to 307% decrease in phosphofrucmkinase, pyruvate kinase, a-glycerophosphate dehydrogenase, and lactate dehydrogenase activities in rat anterior tibia1 muscles. The latter enzyme activities returned essentially to control values by about Day 11 after drug injection. A 605% decrease was noted in total glycogen phosphorylase activity, follow4 by a return to control by Day 28. Lactate production by intact extensor digitorum muscles in vitro was unaffected by Marcaine administration, suggesting that the decrease in enzyme activities was not sufficient to impair overall glycolytic activity. Thus glycolysis is maintained to a much greater extent than oxidative metabolism and may fulfill the energy requirements during MarCaine-induced regeneration of skeletal muscle.
INTRODUCTION Skeletal muscle possessesa remarkable capacity for regeneration following administration of myotoxic drugs, mincing and autoimplantation, and ischemia (1-3, 10). Under each of these circumstances an initial period of muscle degeneration occurs, followed by regeneration. A fundamental question concerns the nature of the energy sources that permit survival of degenerating muscles and support subsequent muscle regeneration. We have attempted to answer this question by surveying the major 1 Supported by grants from the National Institutes of He&h (NS-11342-Ol), the Muscular Dystrophy Associations of America, Inc., and the Frank G. Bressler Fund. We thank Ms. B. Pasko for preparation of the typescript and Drs. C. Sumbilla and P. T. Ozand for helpful comments. 40 Copyright 1976 by Academic Press, Inc. All rights oQ reproduction in any form reserved.
GLYCOLYSIS
IN
MUSCLE
REGENERATION
41
energy-yielding metabolic pathways in muscle undergoing degeneration and regeneration following administration of a myotoxic local anesthetic, Marcaine. In a previous report ( 1 1), we described loss of oxidative metabolism in Marcaine-treated muscles and enhanced activity of the hexosemonophosphate shunt, In the present communication, we present evidence which suggests that glycolysis survives Marcaine-induced degeneration. A preliminary report of these data has been presented (IS). MATERIALS
AND
METHODS
The materials used and their sources were: enzymes and Sigma ; [ 3H] mannitol and [ UJ4C] glucose-l-P Amersham Searle POPOP, New England Nuclear ; Marcaine (bupivacaine) , Laboratories. Adult, male, Sprague-Dawley rats, weighing 200 to 250 g, chased from Charles River Breeding Laboratories, Inc., and and Purina Rat Chow ad lib. Extracellular
substrates, ; PPO and Winthrop were purfed water
Space and Lactate Production
Treatment of Muscles. For in vitro studies with intact muscles, the extensor digitorum longus muscle was employed, because its small size permits diffusion of substrates and metabolites to a greater extent than the larger anterior tibia1 muscle (9). On three successive days, a 0.9% NaCl solution containing Marcaine (2.0 mg) plus hyaluronidase (Sigma, 300 units) was injected into the extensor digitorum longus after surgical exposure, and the incision was sutured. The contralateral extensor digitorum longus was surgically exposed and injected with 0.9% NaCl. At 1, 3, and 6 days after the third injection, the muscles were excised, passively stretched on stainless-steel frames (4)) and incubated in Robinson’s medium (16) containing mannitol (1 nlM) and glucose (1 mg/ml) in 25-ml Erlenmeyer flasks (4). Incubation was carried out at 37°C with continuous gassing with a mixture of O2 and CO, (95 : 5). Extvacelhlar Space. This parameter was assessed in the in vitro system described above using [ 3H ] mannitol (4). Lactate Production. At 0, 30, 60, and 90-min intervals during the incubation samples of the medium were removed. The lactate produced was measured by spectrophotometric assay (6) and reported as pmoles/hr/g fresh weight of muscles. Muscles lost about 5% of their fresh weight after 90 min of incubation. The initial weight was employed for the calculations. Lactate production was linear with respect to muscle size in the range used in this study (60 to 150 mg).
42
WAGNER
ET
AL.
Enzyme Analyses Treatment of Muscles. An 0.9% NaCl solution containing Marcaine (2.0 mg) plus hyaluronidase (Sigma, 300 units), was injected into the anterior tibia1 muscle of rats (3, 11)) with the contralateral anterior tibia1 muscles serving as controls and receiving injections of 0.9% NaCl. At 1, 3, 5, 7, 12, 19 and 28 days after three daily injections of Marcaine plus hyaluronidase or of 0.9% NaCl, the rats were decapitated and the muscles were removed, weighed, and minced with scissors in ice-cold 0.05 M Tris-HCl, pH 7.6, containing 0.20 mM dithiothreitol. The finely minced muscles were then homogenized (1: 10, w/v) by hand, using a Tenbroeck homogenizer. The homogenate was centrifuged at 18,000g for 20 min at 4°C. Enzymes were assayed in the resulting supernatants. Enzyme Assays. The following enzymes were assayed by spectrophotometric procedures : hexokinase (EC 2.7.1.1.) ( 17)) phosphofructokinase (EC 2.7.1.11) (8, 17), py ruvate kinase (EC 2.7.1.40) ( 12)) cytoplasmic a-glycerophosphate dehydrogenase (EC 2.7.1 .S) (5)) creatine kinase (EC 2.7.3.2) (6)) adenylate kinase (EC 2.7.4.3) (6)) and lactate dehydrogenase (EC 1.1.1.27) (6). Creatine kinase was assayed in the presence of 5’-AMP to inhibit adenylate kinase ( 13). Glycogen phosphorylase (EC 2.4.1.1 j was measured in the direction of glycogen synthesis in the presence and absence of S-AMP, using glycogen and [U-14C]glucose-1-P as substrates. The assay is a modification of a method for glycogen synthetase determination (15 ; B. I. Brown, personal communication). Phosphorylase data are given as activity in the presence of 5’-AMP. Protein was determined by the method of Lowry et al. (7). Expression of Data, Enzyme activities were linear with respect to time and protein concentration under our assay conditions. Enzyme data are exTABLE Enzyme
Activities
Enzyme
Number Animals
Phosphorylase Hexokinase Phosphofructokinase or-glycerophosphate dehydrogenase Pyruvate kinase Lactate dehydrogenase Adenylate kinase Creatine kinase a values
are mean
f
1 in Control
SEM.
24 16 36 37 37 12 37 37
Muscles of
Specific activity” (nmoles/min/mg protein) 1402.70 26.20 723.00 931.00 5155.00 259.20 1430.00 579.00
& 56.22 zk 0.88 f 38.00 f 35.00 i 299.00 f 10.90 i 72.00 i 35.00
GLYCOLYSIS
IN
MUSCLE
TABLE Effect Days after third Marcaine injection 1 3 6
Number animals
of Marcaine
of
2
on Extracellular Extracellular
Marcaine-treated (ml/100 9 3 3
74.44 60.67 49.67
Space” P
space
g fresh
f 13.86b f 9.29 h 3.79
Q Experimental procedures as given in digitorum muscles (N = 16), extracellular b Numbers are g + SEM.
43
REGENERATION
Control weight) 56.22 49.00 51.00
f 9.23 f 5.57 f 8.00
the text. For space is 31.69
pressed as average percentage of contralateral tive control values are given in Table 1.
