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Response of rat skeletal muscle to neural application of batrachotoxin or tetrodotoxin: Effect on soluble proteins
EXPERIMENTAL
NEUROLOGY
74,447-457
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Response of Rat Skeletal Muscle to Neural Application of Batrachotoxin or Tetrodotoxin: Effect on Soluble Proteins K. K. WAN AND R. J. ESOEGMAN' Department of Pharmacology
Queen’s University, Kingston. Ontario K’L 3N6, Canada
Received February 27, 1981; revision received June 2, 1981 Batrachotoxin (BTX) when applied to a nerve results in a block of impulse transmission and axonal, transport whereas tetrodotoxin (TTX) blocks only impulse transmission. After chronic application of these toxins to the rat tibia1 nerve for 20 to 40 days, the total soluble protein content and the activity of lactate dehydrogenase (LDH), creatine kinase (CK), pyruvate kinase (PK), as well as the heme content, decreased in the soleus muscle. The response after denervation was similar to but greater than that obtained with BTX. Gel electrophoresis of the enzymes showed a change in the isoenzyme pattern of LDHl and LDHS. PK-Ml and PK-M2 in the soleus after BTX or denervation. In sharp contrast to BTX and denervation, TTX caused only a slight reduction in LDH 1. No change was observed in the soleus creatine kinase isoenzyme pattern after denervation or neural application of BTX or TTX.
INTRODUCTION Fast and slow skeletal muscles show differences in their energy requirement (27). This disparity is reflected in different metabolic enzyme patterns. Certain enzymes, such as NAD-linked glycerol phosphate dehydrogenase and lactic acid dehydrogenase (LDH), are characteristically present in different amounts in fast and slow muscles (25, 27). The LDH isoenzyme pattern also differs in fast and slow muscle. Thus fast muscle contains mainly the M type and slow’muscle the H type (12, 27). Muscle metabolic enzyme activity changes after cross reinnervation (9, Abbreviations: BTX-batrachotoxin, TTX-tetrodotoxin. LDH-lactic dehydrogenase (EC 1.1.1.27). PK- pyruvate kinase (EC 2.7.1.40), CK-creatine kinase (EC 2.7.3.2). ’ This work was supported by a grant (R. J. B.) and a studentship (K. K. W.) from the Canadian Muscular Dystrophy Association. BTX was a generous gift from Dr. E. X. Albuquerque. Address reprint requests to Dr. Boegman. 447 0014-4886/81/110447-llSO2.00/0 copyIi#llt Q 1981 by Aadcmic .Ulti&SolrrpoductiollillUlyt~rclcmd.
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12, 24, 30), altered impulse acitivty (28), denervation ( 11, 13, 14, 20, 29) or neural application of toxins (5, 16). This suggests that metabolic enzymes in muscle are under some form of neural trophic control. The nature of this neural influence, however, remains unresolved. It was proposed that muscle contractile activity and/or neurotrophic factors play a role in the neural regulation of muscle structure and function (14). To distinguish between the role of contractile activity and axonal transport of trophic factors in influencing certain muscle enzymes, we made use of batrachotoxin (BTX), which blocks both axonal transport and impulse activity, and tetrodotoxin (TTX), which blocks only impulse activity (6, 3 l-33). In this communication, we report the effect on rat muscle LDH, pyruvate kinase (PK), and creatine kinase (CK) after neural application of these toxins. MATERIALS
AND METHODS
A total of 82 female Sprague-Dawley rats weighing 300 to 350 g were used. The tibia1 and peroneal nerves were injected with toxin or vehicle as reported (3 l-33). Individual muscles from five to eight animals were weighed, pooled, and a 10% (w/v) homogenate in buffered 0.3 M sucrose solution prepared as described (32, 33). Denervation was carried out by removing a 3-mm segment of the sciatic nerve. The homogenate was subjected to differential centrifugation to remove connective tissue, mitochondria, and the microsomal fraction. The soluble supernatant obtained after centrifugation at 78,000 g was used for enzyme assays. Protein was determined as described by Lowry et al. (19). LDH activity was assayed according to Amador et al. (2), PK activity as described by Harano et al. (15), and CK activity according to Desjarlais et al. (6). Heme protein in the high-speed supernatant was measured by the Soret band (22). CK, LDH, and heme proteins were measured on the same day of the preparation and PK was measured after storing at -20°C. Gel electrophoresis of LDH and CK was carried out in 1% agarose using the LKB Multiphor system. Agarose was dissolved in a medium containing Tris, 0.1 M, glycine 0.05 M, and magnesium acetate, 0.03 M, at pH 8.5. The same medium was used in the electrode compartments. Electrophoresis was at 4°C for 5 to 6 h at a constant voltage of 15 V/cm. Polyacrylamide gel electrophoresis of PK was according to Imamura and Tanaka ( 18) in a Pharmacia GE-4 II electrophoresis apparatus at 4°C with a separating gel containing 5% acrylamide and 0.14% NJ’-methylenebisacrylamide. Samples for electrophoresis were reconstituted from a lyophilized powder of the soluble protein fractions. Chemicals and enzymes used in the enzyme assays and elctrophoresis were obtained from Sigma. Other reagents used were of analytical grade. Statistical analysis was by Student’s t test.
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RESULTS Soluble Protein. The data obtained from the experimental muscle were compared with the contralateral sham-operated control muscle. The control muscle was not significantly different from unoperated normal muscle. Consistent with the observation that the denervated soleus undergoes atrophy (10, 33), the total soluble protein content of this muscle decreased to 59% and 42% of control at 20 and 40 days after denervation, respectively (Fig. 1). Neurally applied BTX also caused a decrease in soluble protein content of the soleus (83% and 63% of control at 20 to 40 days). TTX, however, caused no significant change in the soluble protein content of the soleus. Twenty days after denervation a 30% decrease in the soluble protein from the soleus muscle was noted and BTX resulted in a 13% decrease 12s
SW
r
* -----------F--------
.-r ...* ..... .... .... .,,.*... . . L TX BTX TTX BTX DN TTX ITX t
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. . .. . .. . . .. . . .. . . .. . . .. . ..*. . .. . . . .. . . .. . . .. . . .. . . .. .. .. . . *. .... . .. . . . .. . .. . .. .*. . .. .. . ...
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20 DAYS
I B
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FIG. 1. Soluble protein from the soleus muscle homogenate after denervation (DN) or neural application of tetrodotoxin (TTX) or batrachotoxin (BTX). Results are presented as percentages of contralateral controls and are the mean + SE with N = 6.
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and TTX had no effect (Fig. 1). This is consistent with our earlier report on toxin-induced changes in the extensor digitorum longus muscle of the rat (6, 32). Enzyme Activity of the Soluble Supernatant. The relative LDH, PK, and CK activities and Soret band absorption of muscle supernatants are shown in Table 1. The total LDH activity in the muscle supernatant decreased after denervation, or neural application of TTX or BTX. In the 40-day experiments, LDH activities were 30,78, and 48% respectively, of control values (Table 1). The LDH activity of the extensor digitorum longus decreased to 40, 90, and 79% of control 20 days after denervation or application of TTX and BTX, respectively. The total CK activity in soleus after denervation or neural application of TTX and BTX decreased with respect to the sham-operated control muscle (Table 1). At 20 days, TTX and BTX did not cause a significant alteration in the CK of the extensor digitorum longus muscle. However, denervation resulted in a decrease of 30% compared with control. The total PK activity decreased after denervation or neural application of BTX, being 37 and 42% respectively, of control at 40 days. Neural application of TTX, however, did not result in a significant change during the 40-day period (Table 1). The trend in PK activity of the extensor digitorum after TABLE
1
Specific Enzyme Activities in the Soluble Supernatant of the Soleus Muscle after Denervation (DN) and Neural Application of BTX or TTX” Days after treatment 20
40
Sample
LDH (mol lactatc/mg proteinjmin)
CK (mol NADP/mg protein/mitt)
Control DN
1.64 f 0.05 0.74 k 0.02 (45)
12.3 k 0.1 8.3 + 0.2 (67)
5.03 * 0.11 2.64 f 0.16 (53)
0.216 0.163
k 0.035 f 0.025 (75)
Cantrol Tl-X
1.89 + 0.03 1.56 + 0.05 (83)
16.1 + 0.6 12.7 k 0.6 (81)
2.72 + 0.60 2.84 + 0.43 (111).
0.247 0.214
+ 0.012 k 0.017 (87)
Control BTX
1.48 f 0.06 0.89 + 0.11 (60)
10.2 5 0.7 7.2 k 0.3 (71)
5.15 * 0.09 4.23 f 0.40 (82)
0.258 zt 0.027 0.226 f 0.027 (84)
Contml DN
1.82 * 0.12 0.54 * 0.01 (30)
17.2 f 0.4 5.3 f 0.2 (31)
6.79 f 0.08 2.53 f 0.06 (37)
0.230 2 0.016 0.187 * 0.011 (81)
Control TTX
1.43 + 0.07 1.12 * 0.04 (78)
11.6 + 0.4 9.3 * 0.2 (80)
2.73 f 0.06 2.97 f 0.60 (108)’
0.263 0.150
f f
Control BTX
1.25 + 0.01 0.60 k 0.01 (48)
18.5 f 0.1 12.1 k 0.3 (65)
3.71 k 0.09 1.54 + 0.20 (42)
0.153 0.068
* 0.047 f 0.029 (43)
PK (mol NADH/ m g pmtein/min)
’ The results were fmm paired analyses in each experiment. Valuea arc mean f SE and the percentage all are significantly different (P < 0.05-0.01) exacpt (‘). N = 4 to 6.
Soret band (A4221 m g protein/ml)
0.055 0.021 (57)
is given in parentheses;
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denervation or application of neurotoxin was similar to that seen with the soleus. As noted by other investigators (12, 24, 27), the LDH activity of the extensor digitorum longus was greater than that of the soleus (Table 2). The CK activity in the extensor digitorum longus was twofold greater, whereas the PK activity in the extensor digitorum longus was sevenfold higher (Table 2). An oxidative muscle like the soleus has considerably more myoglobin than a glycolytic muscle such as the extensor digitorum longus (27). We also observed that the heme protein content measured by the Soret band is higher in the soleus than in the extensor digitorum longus (Table 2). After neural application of BTX or TTX, the soleus heme protein content decreased to 43 and 57% respectively of control (Table 1). Denervation also caused a decrease in heme protein but surprisingly the magnitude was smaller than that observed after neural application of toxin. In contrast to the soleus, the extensor digitorum longus showed an increase in heme content to 195 and 122% of control 20 days after denervation or BTX treatment, respectively. Electrophoresis of Enzymes from the Soluble Supernatant. LDH isoenzymes from the soluble fractions of the neurally treated soleus muscle homogenates were separated by agarose gel electrophoresis. The gels were deliberately overloaded with sample in order to show any difference in the isoenzyme pattern. The characteristic five LDH bands (8) were seen in all preparations with the H isoenzyme (LDHl) toward the anode and the M isoenzyme (LDHS) at the cathode (Fig. 2). In the soleus, the faster migrating H bands predominated whereas in the extensor, it was the M type. After denervation, LDHl and LDH2 progressively decreased in intensity compared with control. The decrease was especially marked after 40 days. BTX also caused a reduction in LDHI isoenzyme intensity occurred after BTX but less so than after denervation. Interestingly, BTX at 40 days also TABLE
2
Comparison of Soluble Proteins in Normal Extensor Digitorum Longus and Soleus”
LDH CK PK Soret band
Extensor
Soleus
409 233 686 63
100 100 100 100
0 The soleus values were set at 100 and extensor values as a percentage of the soleus.
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FIG. 3. Agarose gel electrophoresis of creatine kinase isoenzymes from the soluble protein fraction of the soleus. The enzyme activities were visualized by layering on top of the gel a standard mixture in 20% sucrose of the substrate as used in the enzymatic assay [see Desjarlais et al. (7)]. After incubating 15 min at 37°C and drying at 45”C, the gel was viewed and photographed under uv light at 360 nm. l-normal unoperted soleus, 2 and 3-control and DN-40,4 and S--control and TTX-40.6 and ‘I-control and BTX-40, H-rat heart supernatant. and B-rat brain supernatant. All muscle samples contained 12.56 + 0.03 pg protein. Designations as in Fig. 2.
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FIG. 4. Polyacrylamide gel electrophoresis of pyruvate kinase (PK) isoenzymes from the soluble fraction of a soleus homogenate. Visualization of the enzyme activity was as described by lmamura and Tanaka (18). A: I-unoperated normal control, 2 and 3-control and DN20. 4 and S-control and DN-40, 6-5 units PK (Sigma). B: I and 2-control and TTX-20, 3 and 4-control and TTX-40, rat liver supernatant, rat kidney supernatant. C: I and 2control and BTX-I2,3 and 4-control and BTX-20.5 and 6-control and BTX-40. All muscle samples contained 12.56 f 0.03 pg protein. Designations as in Fig. 2.
caused a marked decrease in LDHS. In sharp contrast to denervation and BTX, TTX caused only a slight reduction in LDHl at 40 days. Creatine kinase, which in muscle is found as the MM or MB isoenzyme
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4 (Continued)
(4) did not change markedly in the electrophoretic profile after denervation or neurotoxin treatment (Fig. 3). Pyruvate kinase is widely distributed in mammalian tissues (15) and several different PK isozymes are present. Electrophoretic and immunological studies have established two major groups of PK isozymes ( 18,2 1). Skeletal muscle has Ml whereas brain and heart have Ml and M2 (18, 34). Two intermediate forms, designated M3 and M4, are found in smooth muscle ( 18, 34). The PK isozymes diminished in intensity 20 and 40 days after denervation (Fig. 4) and TTX had no effect on the isoenzyme pattern, whereas BTX caused a decrease in PK after 40 days (Fig. 4). The extent of the isoenzyme decrease is not entirely clear from the electrophoretogram; however, the decrease in isoenzyme pattern corresponds with the decrease in total PK activity as shown in Table 1. DISCUSSION We showed that some of the enzymes involved in energy metabolism in the soleus and extensor digitorum longus muscles are altered after neural application of BTX or to a lesser degree after application of TTX. The’ response seen with BTX was similar to that obtained after denervation. All experimental procedures resulted in muscle inactivity; however, only BTX and denervation blocked axonal transport. Axonal transport therefore appears to play a role in the long-term regulation of certain energy metabolizing enzymes in the soleus.
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The activity and isoenzyme pattern of LDH, CK, and PK in muscle are altered by cross innervation, changes in the impulse pattern to the muscle, or during differentiation and dedifferentiation as well as denervation (9, 11, 12, 20, 23, 24, 28-30). This suggests that these enzymes are under some form of neurotrophic control. The factors performing a trophic function in regulating the enzyme activity are not well defined. However, several processes could play a role. These would include a change in protein metabolism or in general cellular metabolic regulation of the muscle, or possibly alterations in the integrity of cellular membranes leading to enzyme leakage. The latter is supported by the finding that there is an increase in enzyme efflux from muscle and/or change in the enzyme activity and isoenzyme pattern following denervation, blockade of axonal transport, and in certain neuromuscular diseases (1, 3, 4, 15-l 8, 26, 34). The increase in heme content of the extensor digitorum longus after denervation or BTX treatment suggests a shift to more oxidative metabolism (7). Alternatively the increased heme content could be due to a decrease in actomyosin content resulting in an increased ratio of heme protein to total muscle protein. In summary, an intact axonal transport system appears to play a greater role in influencing the total activity of lactate dehydrogenase, creatine kinase, and pyruvate kinase in the predominantly oxidative slow soleus muscle than does muscle contractile activity. REFERENCES 1. ALBERTS, M. C., AND F. J. SAMAHA. 1974. Serum pyruvate kinase in muscle diseases and carrier states. Neurology (Minneapolis) 4: 462-464. 2. AMADOR, E., L. E. DORFMAN, AND W. E. C. WACKER. 1963. Serum LDH activity: an analytical assessment of current assays. Clin. Chem. 9: 391-399. 3. ANAND, R., AND A. E. EMERY. 1980. Calcium stimulated enzyme efflux from human skeletal muscle. Rex Commun. Chem. Pathol. Pharmacol. 28: 541-550. 4. BERGMEYER, H. U. (Ed.) 1974. Methods ofEnzymatic Analysis, 2nd ed., Vol. 1. Academic Press, New York. 5. BOEGMAN, R. J., AND T. W. OLIVER. 1980. Neural influence on muscle hydrolase activity. Life Sci. 27: 1339-1344. 6. BOEGMAN, R. J., AND K. K. WAN. 1980. The nature of the neural influence on Ca-uptake by sarcoplasmic reticulum. In. F. L. SEIGEL, Eds., Calcium-Binding Proteins: Structure and Function. Elsevier/North-Holland, Amsterdam. 7. DESJARLAIS, F., L. G. MORIN, AND R. DAIGNEAULT. 1980. In search of optimum conditions for the measurement of creatine kinase activity: a critical review of nineteen formulations. Cfin. Biochem. 13: 116-121. 8. DIXON, M., AND E. C. WEBB. 1979. Enzymes, 3rd ed. pp. 640-645. Academic Press, New York. 9. DUBOWITZ, V. 1967. Cross-innervated mammalian skeletal muscle: histochemical, physiological and biochemical observations. J. Physiol. (London) 193: 48 l-496. 10. GOLDSPINK, D. 1976. The effects of denervation on protein turnover of rat skeletal muscle. Biochem.
J. 156: 7 I-80.
11. GUTH, L., AND P. K. WATSON. 1967. The influence of innervation on the soluble proteins of slow and fast muscle of the rat. Exp. Neurol. 17: 107-l 17. 12. GUTH, L., P. K. WATSON, AND W. C. BROWN. 1968. Effects of cross-reinnervation on
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25.
26. 27. 28. 29.
30. 3 I.
32.
33. 34.
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some chemical properties of red and white muscles of rat and cat. Exp. Neural. 20: 52-69. GUTH, L. 1968. “Trophic” influences of nerve on muscle. Physiol. Rev. 48: 645-687. GUTMANN, E. 1976. Neurotrophic relations. Annu. Rev. Physiol. 2% 177-216. HARANO, Y., R. ADAIR, R. J. VIGNOS, M. MILLER, AND J. KOWAL. 1973. Pyruvate kinase isoenzymes in progressive muscular dystrophy and in acute myocardial infarction. Metabolism 22: 493-498. HOFFMANN, W. W., AND J. H. PEACOCK. 1973. Postjunctional change induced by partial interruption of axoplasmic flow in motor nerves. Exp. Neural. 41: 345-356. HOOSHMAND, H. 1975. Serum LDH isoenzymes in neuromuscular diseases. Dis. Nervous System 36: 607-61 I. IMAMURA, K., AND T. TANAKA. 1972. Multimolecular forms of pyruvate kinase from rat and other mammalian tissues. J. Eiochem. (Tokyo) 71: 1043-1051. LOWRY, 0. H., N. J. ROSENBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193: 265-275. MARGARETH, A., G. SALVIATI, S. DIMAURO, AND G. TURATI. 1972. Early biochemical consequences of denervation in fast and slow skeletal muscles and their relationship to neural control over muscle differentiation. Biochem. J. 126: 1099-l 1 IO. MARIE, J., A. KAHN, AND P. BOVIN. 1976. Pyruvate kinase isozymes in man. Hum. Genet. 31: 35-45. MCPHERSON, A., AND J. TOKUNAGA. 1967. The effects of cross-innervation on the myoglobin concentration of tonic and phasic muscles. J. Physiol. (London) 188: 121- 129. MIRANDA, A. F., H. SOMER, AND S. DIMAURO. 1979. Pages 453-473 in A. MAURO et al. Eds., Muscle Regeneration. Raven Press, New York. MOMMAERTS, W. F. H. M., K. SERAYDARIAN, M. SUH, C. J. C. KEAN, AND A. J. BULLER. 1977. The conversion of some biochemical properties of mammalian skeletal muscles following cross-reinnervation. Exp. Neurol. 55: 637-653. OPIE, L., AND E. A. NEWSHOLME. 1967. The activities of fructose 1,6-diphosphatase, phosphofructokinase and phosphoenolpyruvate carboxykinase in white muscle and red muscle. B&hem. .I. 103: 391-398. PENNINGTON, R. J. T. 1980. Clinical biochemistry of muscular dystrophy. Brit. Med. Bull. 36: 123-126. PETTE, D., AND H. W. STAUDTE. 1973. Differences between red and white muscles. In J. KEUL, Ed., Limiting Factors of Physical Performance. Thieme, Stuttgart. PETTE, D., M. E. SMITH, H. W. STAUDTE, AND G. VRBOVA. 1973. Effects of long term electrical stimulation on some contractile and metabolic characteristics of fast rabbit muscles. Pffigers Arch. 338: 257-272. ROBBINS, N., AND D. CARLSON. 1979. Early changes in muscle G6P-DH after denervation: locus and dependence on nerve stump length. Brain Res. 177: 145-156. ROMANUL, F. C. A., AND J. P. VAN DER MEULEN. 1972. Slow and fast muscle after cross innervation. Arch. Neural. (Chicago) 17: 387-402. WAN, K. K., AND R. J. BOEGMAN. 1980. Calcium uptake by muscle sarcoplasmic reticulum following neural application of batrachotoxin or tetrodotoxin. FEBS Lett. 112: 163-167. WAN, K. K., AND R. J. BOEGMAN. 1980. Changes in rat muscle sarcoplasmic reticulum following neural application of batrachotoxin or tetrodotoxin. Exp. Neurol. 70: 475486. WAN, K. K., AND R. J. BOEGMAN. 1981. Response of rat skeletal muscle to neural application of batrachotoxin or tetrodotoxin: effect on sarcoplasmic reticulum. Exp. Neural. 74: 439-446. ZATZ, M., L. J. SHAPIRO, D. S. CAMPION, E. ODA, AND M. M. KABACK. 1978. Serum PK and CPK in progressive dystrophies. J. Neurof. Sci. 36: 349-362.
NEUROLOGY
74,447-457
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Response of Rat Skeletal Muscle to Neural Application of Batrachotoxin or Tetrodotoxin: Effect on Soluble Proteins K. K. WAN AND R. J. ESOEGMAN' Department of Pharmacology
Queen’s University, Kingston. Ontario K’L 3N6, Canada
Received February 27, 1981; revision received June 2, 1981 Batrachotoxin (BTX) when applied to a nerve results in a block of impulse transmission and axonal, transport whereas tetrodotoxin (TTX) blocks only impulse transmission. After chronic application of these toxins to the rat tibia1 nerve for 20 to 40 days, the total soluble protein content and the activity of lactate dehydrogenase (LDH), creatine kinase (CK), pyruvate kinase (PK), as well as the heme content, decreased in the soleus muscle. The response after denervation was similar to but greater than that obtained with BTX. Gel electrophoresis of the enzymes showed a change in the isoenzyme pattern of LDHl and LDHS. PK-Ml and PK-M2 in the soleus after BTX or denervation. In sharp contrast to BTX and denervation, TTX caused only a slight reduction in LDH 1. No change was observed in the soleus creatine kinase isoenzyme pattern after denervation or neural application of BTX or TTX.
INTRODUCTION Fast and slow skeletal muscles show differences in their energy requirement (27). This disparity is reflected in different metabolic enzyme patterns. Certain enzymes, such as NAD-linked glycerol phosphate dehydrogenase and lactic acid dehydrogenase (LDH), are characteristically present in different amounts in fast and slow muscles (25, 27). The LDH isoenzyme pattern also differs in fast and slow muscle. Thus fast muscle contains mainly the M type and slow’muscle the H type (12, 27). Muscle metabolic enzyme activity changes after cross reinnervation (9, Abbreviations: BTX-batrachotoxin, TTX-tetrodotoxin. LDH-lactic dehydrogenase (EC 1.1.1.27). PK- pyruvate kinase (EC 2.7.1.40), CK-creatine kinase (EC 2.7.3.2). ’ This work was supported by a grant (R. J. B.) and a studentship (K. K. W.) from the Canadian Muscular Dystrophy Association. BTX was a generous gift from Dr. E. X. Albuquerque. Address reprint requests to Dr. Boegman. 447 0014-4886/81/110447-llSO2.00/0 copyIi#llt Q 1981 by Aadcmic .Ulti&SolrrpoductiollillUlyt~rclcmd.
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Inc.
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WAN AND BOEGMAN
12, 24, 30), altered impulse acitivty (28), denervation ( 11, 13, 14, 20, 29) or neural application of toxins (5, 16). This suggests that metabolic enzymes in muscle are under some form of neural trophic control. The nature of this neural influence, however, remains unresolved. It was proposed that muscle contractile activity and/or neurotrophic factors play a role in the neural regulation of muscle structure and function (14). To distinguish between the role of contractile activity and axonal transport of trophic factors in influencing certain muscle enzymes, we made use of batrachotoxin (BTX), which blocks both axonal transport and impulse activity, and tetrodotoxin (TTX), which blocks only impulse activity (6, 3 l-33). In this communication, we report the effect on rat muscle LDH, pyruvate kinase (PK), and creatine kinase (CK) after neural application of these toxins. MATERIALS
AND METHODS
A total of 82 female Sprague-Dawley rats weighing 300 to 350 g were used. The tibia1 and peroneal nerves were injected with toxin or vehicle as reported (3 l-33). Individual muscles from five to eight animals were weighed, pooled, and a 10% (w/v) homogenate in buffered 0.3 M sucrose solution prepared as described (32, 33). Denervation was carried out by removing a 3-mm segment of the sciatic nerve. The homogenate was subjected to differential centrifugation to remove connective tissue, mitochondria, and the microsomal fraction. The soluble supernatant obtained after centrifugation at 78,000 g was used for enzyme assays. Protein was determined as described by Lowry et al. (19). LDH activity was assayed according to Amador et al. (2), PK activity as described by Harano et al. (15), and CK activity according to Desjarlais et al. (6). Heme protein in the high-speed supernatant was measured by the Soret band (22). CK, LDH, and heme proteins were measured on the same day of the preparation and PK was measured after storing at -20°C. Gel electrophoresis of LDH and CK was carried out in 1% agarose using the LKB Multiphor system. Agarose was dissolved in a medium containing Tris, 0.1 M, glycine 0.05 M, and magnesium acetate, 0.03 M, at pH 8.5. The same medium was used in the electrode compartments. Electrophoresis was at 4°C for 5 to 6 h at a constant voltage of 15 V/cm. Polyacrylamide gel electrophoresis of PK was according to Imamura and Tanaka ( 18) in a Pharmacia GE-4 II electrophoresis apparatus at 4°C with a separating gel containing 5% acrylamide and 0.14% NJ’-methylenebisacrylamide. Samples for electrophoresis were reconstituted from a lyophilized powder of the soluble protein fractions. Chemicals and enzymes used in the enzyme assays and elctrophoresis were obtained from Sigma. Other reagents used were of analytical grade. Statistical analysis was by Student’s t test.
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RESULTS Soluble Protein. The data obtained from the experimental muscle were compared with the contralateral sham-operated control muscle. The control muscle was not significantly different from unoperated normal muscle. Consistent with the observation that the denervated soleus undergoes atrophy (10, 33), the total soluble protein content of this muscle decreased to 59% and 42% of control at 20 and 40 days after denervation, respectively (Fig. 1). Neurally applied BTX also caused a decrease in soluble protein content of the soleus (83% and 63% of control at 20 to 40 days). TTX, however, caused no significant change in the soluble protein content of the soleus. Twenty days after denervation a 30% decrease in the soluble protein from the soleus muscle was noted and BTX resulted in a 13% decrease 12s
SW
r
* -----------F--------
.-r ...* ..... .... .... .,,.*... . . L TX BTX TTX BTX DN TTX ITX t
25-
. . .. . .. . . .. . . .. . . .. . . .. . ..*. . .. . . . .. . . .. . . .. . . .. . . .. .. .. . . *. .... . .. . . . .. . .. . .. .*. . .. .. . ...
sir
I
12 DAYS
20 DAYS
I B
40 DAYS
FIG. 1. Soluble protein from the soleus muscle homogenate after denervation (DN) or neural application of tetrodotoxin (TTX) or batrachotoxin (BTX). Results are presented as percentages of contralateral controls and are the mean + SE with N = 6.
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and TTX had no effect (Fig. 1). This is consistent with our earlier report on toxin-induced changes in the extensor digitorum longus muscle of the rat (6, 32). Enzyme Activity of the Soluble Supernatant. The relative LDH, PK, and CK activities and Soret band absorption of muscle supernatants are shown in Table 1. The total LDH activity in the muscle supernatant decreased after denervation, or neural application of TTX or BTX. In the 40-day experiments, LDH activities were 30,78, and 48% respectively, of control values (Table 1). The LDH activity of the extensor digitorum longus decreased to 40, 90, and 79% of control 20 days after denervation or application of TTX and BTX, respectively. The total CK activity in soleus after denervation or neural application of TTX and BTX decreased with respect to the sham-operated control muscle (Table 1). At 20 days, TTX and BTX did not cause a significant alteration in the CK of the extensor digitorum longus muscle. However, denervation resulted in a decrease of 30% compared with control. The total PK activity decreased after denervation or neural application of BTX, being 37 and 42% respectively, of control at 40 days. Neural application of TTX, however, did not result in a significant change during the 40-day period (Table 1). The trend in PK activity of the extensor digitorum after TABLE
1
Specific Enzyme Activities in the Soluble Supernatant of the Soleus Muscle after Denervation (DN) and Neural Application of BTX or TTX” Days after treatment 20
40
Sample
LDH (mol lactatc/mg proteinjmin)
CK (mol NADP/mg protein/mitt)
Control DN
1.64 f 0.05 0.74 k 0.02 (45)
12.3 k 0.1 8.3 + 0.2 (67)
5.03 * 0.11 2.64 f 0.16 (53)
0.216 0.163
k 0.035 f 0.025 (75)
Cantrol Tl-X
1.89 + 0.03 1.56 + 0.05 (83)
16.1 + 0.6 12.7 k 0.6 (81)
2.72 + 0.60 2.84 + 0.43 (111).
0.247 0.214
+ 0.012 k 0.017 (87)
Control BTX
1.48 f 0.06 0.89 + 0.11 (60)
10.2 5 0.7 7.2 k 0.3 (71)
5.15 * 0.09 4.23 f 0.40 (82)
0.258 zt 0.027 0.226 f 0.027 (84)
Contml DN
1.82 * 0.12 0.54 * 0.01 (30)
17.2 f 0.4 5.3 f 0.2 (31)
6.79 f 0.08 2.53 f 0.06 (37)
0.230 2 0.016 0.187 * 0.011 (81)
Control TTX
1.43 + 0.07 1.12 * 0.04 (78)
11.6 + 0.4 9.3 * 0.2 (80)
2.73 f 0.06 2.97 f 0.60 (108)’
0.263 0.150
f f
Control BTX
1.25 + 0.01 0.60 k 0.01 (48)
18.5 f 0.1 12.1 k 0.3 (65)
3.71 k 0.09 1.54 + 0.20 (42)
0.153 0.068
* 0.047 f 0.029 (43)
PK (mol NADH/ m g pmtein/min)
’ The results were fmm paired analyses in each experiment. Valuea arc mean f SE and the percentage all are significantly different (P < 0.05-0.01) exacpt (‘). N = 4 to 6.
Soret band (A4221 m g protein/ml)
0.055 0.021 (57)
is given in parentheses;
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451
denervation or application of neurotoxin was similar to that seen with the soleus. As noted by other investigators (12, 24, 27), the LDH activity of the extensor digitorum longus was greater than that of the soleus (Table 2). The CK activity in the extensor digitorum longus was twofold greater, whereas the PK activity in the extensor digitorum longus was sevenfold higher (Table 2). An oxidative muscle like the soleus has considerably more myoglobin than a glycolytic muscle such as the extensor digitorum longus (27). We also observed that the heme protein content measured by the Soret band is higher in the soleus than in the extensor digitorum longus (Table 2). After neural application of BTX or TTX, the soleus heme protein content decreased to 43 and 57% respectively of control (Table 1). Denervation also caused a decrease in heme protein but surprisingly the magnitude was smaller than that observed after neural application of toxin. In contrast to the soleus, the extensor digitorum longus showed an increase in heme content to 195 and 122% of control 20 days after denervation or BTX treatment, respectively. Electrophoresis of Enzymes from the Soluble Supernatant. LDH isoenzymes from the soluble fractions of the neurally treated soleus muscle homogenates were separated by agarose gel electrophoresis. The gels were deliberately overloaded with sample in order to show any difference in the isoenzyme pattern. The characteristic five LDH bands (8) were seen in all preparations with the H isoenzyme (LDHl) toward the anode and the M isoenzyme (LDHS) at the cathode (Fig. 2). In the soleus, the faster migrating H bands predominated whereas in the extensor, it was the M type. After denervation, LDHl and LDH2 progressively decreased in intensity compared with control. The decrease was especially marked after 40 days. BTX also caused a reduction in LDHI isoenzyme intensity occurred after BTX but less so than after denervation. Interestingly, BTX at 40 days also TABLE
2
Comparison of Soluble Proteins in Normal Extensor Digitorum Longus and Soleus”
LDH CK PK Soret band
Extensor
Soleus
409 233 686 63
100 100 100 100
0 The soleus values were set at 100 and extensor values as a percentage of the soleus.
452
WAN
AND
BOEGMAN
FIG. 3. Agarose gel electrophoresis of creatine kinase isoenzymes from the soluble protein fraction of the soleus. The enzyme activities were visualized by layering on top of the gel a standard mixture in 20% sucrose of the substrate as used in the enzymatic assay [see Desjarlais et al. (7)]. After incubating 15 min at 37°C and drying at 45”C, the gel was viewed and photographed under uv light at 360 nm. l-normal unoperted soleus, 2 and 3-control and DN-40,4 and S--control and TTX-40.6 and ‘I-control and BTX-40, H-rat heart supernatant. and B-rat brain supernatant. All muscle samples contained 12.56 + 0.03 pg protein. Designations as in Fig. 2.
454
WAN
AND
BOEGMAN
FIG. 4. Polyacrylamide gel electrophoresis of pyruvate kinase (PK) isoenzymes from the soluble fraction of a soleus homogenate. Visualization of the enzyme activity was as described by lmamura and Tanaka (18). A: I-unoperated normal control, 2 and 3-control and DN20. 4 and S-control and DN-40, 6-5 units PK (Sigma). B: I and 2-control and TTX-20, 3 and 4-control and TTX-40, rat liver supernatant, rat kidney supernatant. C: I and 2control and BTX-I2,3 and 4-control and BTX-20.5 and 6-control and BTX-40. All muscle samples contained 12.56 f 0.03 pg protein. Designations as in Fig. 2.
caused a marked decrease in LDHS. In sharp contrast to denervation and BTX, TTX caused only a slight reduction in LDHl at 40 days. Creatine kinase, which in muscle is found as the MM or MB isoenzyme
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PROTEINS
455
4 (Continued)
(4) did not change markedly in the electrophoretic profile after denervation or neurotoxin treatment (Fig. 3). Pyruvate kinase is widely distributed in mammalian tissues (15) and several different PK isozymes are present. Electrophoretic and immunological studies have established two major groups of PK isozymes ( 18,2 1). Skeletal muscle has Ml whereas brain and heart have Ml and M2 (18, 34). Two intermediate forms, designated M3 and M4, are found in smooth muscle ( 18, 34). The PK isozymes diminished in intensity 20 and 40 days after denervation (Fig. 4) and TTX had no effect on the isoenzyme pattern, whereas BTX caused a decrease in PK after 40 days (Fig. 4). The extent of the isoenzyme decrease is not entirely clear from the electrophoretogram; however, the decrease in isoenzyme pattern corresponds with the decrease in total PK activity as shown in Table 1. DISCUSSION We showed that some of the enzymes involved in energy metabolism in the soleus and extensor digitorum longus muscles are altered after neural application of BTX or to a lesser degree after application of TTX. The’ response seen with BTX was similar to that obtained after denervation. All experimental procedures resulted in muscle inactivity; however, only BTX and denervation blocked axonal transport. Axonal transport therefore appears to play a role in the long-term regulation of certain energy metabolizing enzymes in the soleus.
456
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The activity and isoenzyme pattern of LDH, CK, and PK in muscle are altered by cross innervation, changes in the impulse pattern to the muscle, or during differentiation and dedifferentiation as well as denervation (9, 11, 12, 20, 23, 24, 28-30). This suggests that these enzymes are under some form of neurotrophic control. The factors performing a trophic function in regulating the enzyme activity are not well defined. However, several processes could play a role. These would include a change in protein metabolism or in general cellular metabolic regulation of the muscle, or possibly alterations in the integrity of cellular membranes leading to enzyme leakage. The latter is supported by the finding that there is an increase in enzyme efflux from muscle and/or change in the enzyme activity and isoenzyme pattern following denervation, blockade of axonal transport, and in certain neuromuscular diseases (1, 3, 4, 15-l 8, 26, 34). The increase in heme content of the extensor digitorum longus after denervation or BTX treatment suggests a shift to more oxidative metabolism (7). Alternatively the increased heme content could be due to a decrease in actomyosin content resulting in an increased ratio of heme protein to total muscle protein. In summary, an intact axonal transport system appears to play a greater role in influencing the total activity of lactate dehydrogenase, creatine kinase, and pyruvate kinase in the predominantly oxidative slow soleus muscle than does muscle contractile activity. REFERENCES 1. ALBERTS, M. C., AND F. J. SAMAHA. 1974. Serum pyruvate kinase in muscle diseases and carrier states. Neurology (Minneapolis) 4: 462-464. 2. AMADOR, E., L. E. DORFMAN, AND W. E. C. WACKER. 1963. Serum LDH activity: an analytical assessment of current assays. Clin. Chem. 9: 391-399. 3. ANAND, R., AND A. E. EMERY. 1980. Calcium stimulated enzyme efflux from human skeletal muscle. Rex Commun. Chem. Pathol. Pharmacol. 28: 541-550. 4. BERGMEYER, H. U. (Ed.) 1974. Methods ofEnzymatic Analysis, 2nd ed., Vol. 1. Academic Press, New York. 5. BOEGMAN, R. J., AND T. W. OLIVER. 1980. Neural influence on muscle hydrolase activity. Life Sci. 27: 1339-1344. 6. BOEGMAN, R. J., AND K. K. WAN. 1980. The nature of the neural influence on Ca-uptake by sarcoplasmic reticulum. In. F. L. SEIGEL, Eds., Calcium-Binding Proteins: Structure and Function. Elsevier/North-Holland, Amsterdam. 7. DESJARLAIS, F., L. G. MORIN, AND R. DAIGNEAULT. 1980. In search of optimum conditions for the measurement of creatine kinase activity: a critical review of nineteen formulations. Cfin. Biochem. 13: 116-121. 8. DIXON, M., AND E. C. WEBB. 1979. Enzymes, 3rd ed. pp. 640-645. Academic Press, New York. 9. DUBOWITZ, V. 1967. Cross-innervated mammalian skeletal muscle: histochemical, physiological and biochemical observations. J. Physiol. (London) 193: 48 l-496. 10. GOLDSPINK, D. 1976. The effects of denervation on protein turnover of rat skeletal muscle. Biochem.
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11. GUTH, L., AND P. K. WATSON. 1967. The influence of innervation on the soluble proteins of slow and fast muscle of the rat. Exp. Neurol. 17: 107-l 17. 12. GUTH, L., P. K. WATSON, AND W. C. BROWN. 1968. Effects of cross-reinnervation on
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some chemical properties of red and white muscles of rat and cat. Exp. Neural. 20: 52-69. GUTH, L. 1968. “Trophic” influences of nerve on muscle. Physiol. Rev. 48: 645-687. GUTMANN, E. 1976. Neurotrophic relations. Annu. Rev. Physiol. 2% 177-216. HARANO, Y., R. ADAIR, R. J. VIGNOS, M. MILLER, AND J. KOWAL. 1973. Pyruvate kinase isoenzymes in progressive muscular dystrophy and in acute myocardial infarction. Metabolism 22: 493-498. HOFFMANN, W. W., AND J. H. PEACOCK. 1973. Postjunctional change induced by partial interruption of axoplasmic flow in motor nerves. Exp. Neural. 41: 345-356. HOOSHMAND, H. 1975. Serum LDH isoenzymes in neuromuscular diseases. Dis. Nervous System 36: 607-61 I. IMAMURA, K., AND T. TANAKA. 1972. Multimolecular forms of pyruvate kinase from rat and other mammalian tissues. J. Eiochem. (Tokyo) 71: 1043-1051. LOWRY, 0. H., N. J. ROSENBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193: 265-275. MARGARETH, A., G. SALVIATI, S. DIMAURO, AND G. TURATI. 1972. Early biochemical consequences of denervation in fast and slow skeletal muscles and their relationship to neural control over muscle differentiation. Biochem. J. 126: 1099-l 1 IO. MARIE, J., A. KAHN, AND P. BOVIN. 1976. Pyruvate kinase isozymes in man. Hum. Genet. 31: 35-45. MCPHERSON, A., AND J. TOKUNAGA. 1967. The effects of cross-innervation on the myoglobin concentration of tonic and phasic muscles. J. Physiol. (London) 188: 121- 129. MIRANDA, A. F., H. SOMER, AND S. DIMAURO. 1979. Pages 453-473 in A. MAURO et al. Eds., Muscle Regeneration. Raven Press, New York. MOMMAERTS, W. F. H. M., K. SERAYDARIAN, M. SUH, C. J. C. KEAN, AND A. J. BULLER. 1977. The conversion of some biochemical properties of mammalian skeletal muscles following cross-reinnervation. Exp. Neurol. 55: 637-653. OPIE, L., AND E. A. NEWSHOLME. 1967. The activities of fructose 1,6-diphosphatase, phosphofructokinase and phosphoenolpyruvate carboxykinase in white muscle and red muscle. B&hem. .I. 103: 391-398. PENNINGTON, R. J. T. 1980. Clinical biochemistry of muscular dystrophy. Brit. Med. Bull. 36: 123-126. PETTE, D., AND H. W. STAUDTE. 1973. Differences between red and white muscles. In J. KEUL, Ed., Limiting Factors of Physical Performance. Thieme, Stuttgart. PETTE, D., M. E. SMITH, H. W. STAUDTE, AND G. VRBOVA. 1973. Effects of long term electrical stimulation on some contractile and metabolic characteristics of fast rabbit muscles. Pffigers Arch. 338: 257-272. ROBBINS, N., AND D. CARLSON. 1979. Early changes in muscle G6P-DH after denervation: locus and dependence on nerve stump length. Brain Res. 177: 145-156. ROMANUL, F. C. A., AND J. P. VAN DER MEULEN. 1972. Slow and fast muscle after cross innervation. Arch. Neural. (Chicago) 17: 387-402. WAN, K. K., AND R. J. BOEGMAN. 1980. Calcium uptake by muscle sarcoplasmic reticulum following neural application of batrachotoxin or tetrodotoxin. FEBS Lett. 112: 163-167. WAN, K. K., AND R. J. BOEGMAN. 1980. Changes in rat muscle sarcoplasmic reticulum following neural application of batrachotoxin or tetrodotoxin. Exp. Neurol. 70: 475486. WAN, K. K., AND R. J. BOEGMAN. 1981. Response of rat skeletal muscle to neural application of batrachotoxin or tetrodotoxin: effect on sarcoplasmic reticulum. Exp. Neural. 74: 439-446. ZATZ, M., L. J. SHAPIRO, D. S. CAMPION, E. ODA, AND M. M. KABACK. 1978. Serum PK and CPK in progressive dystrophies. J. Neurof. Sci. 36: 349-362.