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Enzyme activities in skeletal muscle after contractile activity in vitro
Int.
3.
Biochem., 1974, Vol. 5, pp. 487 to 494. Pergamon Press. Printed in Great Britain
ENZYME
ACTIVITIES
IN SKELETAL
CONTRACTILE
ACTIVITY
S.-L. BOSTROM, Department
G. HtiGBERG
of Zoophysiology,
AND
University
MUSCLE
487
AFTER
IJV VITRO R. G. JOHANSSON
of Gotehorg,
Goteborg,
Sweden
(Receivedrg October 1973) ABSTRACT I. Changes in muscle enzyme activities following isometric and isotonic contractile activity of electrically stimulated plantaris longus from frog were studied in vitro. The effect of contractile activity under hypoxic conditions and the time-course of changes in enzyme activities after exercise were also investigated. z. Isometric contractile work caused decreased activities in creatine phosphokinase, phosphorylase and phosphofructokinase and increased activities in hexokinase and cytochrome oxidase. The activity levels of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydroqenase, pyruvate kinase, lactate dehydrogenase and fumarase were not significantly different from control values. 3. After isotonic contraction no significant changes in creatine phosphokinase, phosphorylase and phosphofructokinase activities were seen. Besides hexokinase and cytochrome oxidase, fumarase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were also increased. 4. There were no significant differences between the enzyme response obtained after isometric contractions in deoxygenated or oxygenated medium. .5. After a recovery period for 5 hours following isometric work the activities of hexokmase, the hexose monophosphate shunt dehydrogenases, fumarase and cytochrome These declined to control values after 20 hours reoxidase showed increased activities. covery except for hexokinase and 6-phosphogluconate dehydrogenase. The activity levels of creatine phosphokinase, phosphorylase and phosphofructokinase were progressively Pyruvate kinase and lactate dehydrogenase also decreased during the recovery period. showed decreased activity values after zo hours recovery.
ADAPTATION of skeletal muscle to prolonged exercise is followed by an increase in the activities of enzymes related to muscle metabolism (Holloszy, 1967; Kraus & Kirsten, 1969). The major feature of this enzyme adaptation is an increase in the Single capacity of aerobic metabolism. bouts of exercise have also been shown to induce increases in muscle enzyme activities (Kendrick-Jones & Perry, 1965; Lamb, Peter, Jeffress & Wallace, rg6g; Bostriim, Fahlen, Hjalmarsson & Johansson, r 974-a). Little is known about the biochemical mechanisms involved in the adaptation process. It is difficult to decide whether the response in enzyme activity levels is controlled by intracellular mechanisms or if it is a response to humoral or neuronal factors. It could be thought that changes in enzyme activities may be mediated through the
endocrine system since changes in plasma concentrations of many hormones have been found during and after physical exercise (Sutton, Young, Lazarus, Hickie & Maksvytis, Hartley, Mason, Hogan, Jones, 1969; Kotchen, Mougey, Wherry, Pennington & Ricketts, 1972). Contractile properties (Eccles, Eccles & Kozak, 1962) as well as metabolic characteristics (Yellin, 1967) of muscle fibers are also suggested to be under neuronal control. As most of the foregoing studies of physical activity on skeletal muscle enzymes are obtained in vivo it was considered of interest to find out if skeletal muscle exercised in an in vitro system would give a similar response in activity of enzymes of significance for muscle metabolism. In this study, isolated frog plantaris longus was electrically isometric stimulated to maximum and
488
BosTRiiM et al.
isotonic contractile activity. The time-scale of enzyme activity changes during a recovery period following the contractile work was also examined. MATERIALS AND METHODS Kana tenlporaria were kept in water at 5’ C before use. Plantaris longus muscles were dissected out and mounted in a muscle holder immersed in frog-Ringer solution containing 5 mM glucose gassed with 100% oxygen. The muscles were allowed to equilibrate for 30 minutes before onset of the electrical stimulation. The muscles were directly stimulated longitudinally once every 3 seconds for 5 hours by stimuli of supramaximal intensity. The duration of each shock was 20 msec. Paired muscles were used as controls. Each experimental group included o animals. The muscles were stimulated to contract isometricallv and isotonicallv. The force disnlacement transducer used for isometric recording of muscle tension was a Grass FT 03. Isotonic measurements were obtained with a differential transformer. The output potential from both transducers was recorded on a Grass Polygraph. The control muscles were stretched to a similar length as the stimulated muscles and were kept in separate chambers. The frog-Ringer solution was exchanged every IO minutes for both stimulated and control muscles. In two series the muscles were given a recovery period of 5 hours and 20 hours before examining the enzvme activities. During this neriod both stimulated and control muscles”were slill mounted and the frog-Ringer solution was exchanged every r 5 minutes. In one of the series the muscles were forced to contract under hypoxic conditions. The frogRinger solution was gassed I hour before and during the experiment with IOO % nitrogen. The oxygen partial pressure was 30 mm Hg in the deoxygenated frog-Ringer solution compared to 345 mm Hg in the oxygenated normally-used The oxygen tension was measured medium. polarographically &h a Radiometer (Copenhagen) gas monitor PHM 27GM. The preparation of homogenates and methods for the‘ determination of the maximal enzyme activities of hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase, lactate dehydrogenase, fumarase and cvtochrome oxidase have been described recently (Bostrom, Hogberg, & Johansson, I g73a). The method described bv Oscai 1sr Holloszv (1971) was used for determination of creatine phosphokinase. Total phosphorylasc activity was analyzed according to Bass, Brdiczka, Eyer, Hofer & Pette (1969) and phosphofructokinase was assayed as described by Bostrom, Hogberg & Johansson (rg73b). Protein was measured by the method of Lowry, Rosebrough, Farr & Randall (1951).
Int.
3. Biochem.
RESULTS Isolated frog plantaris longus muscles were stimulated continously to contractile work for 5 hours by stimuli of supramaximal intensity. Activities of various enzymes of significance for muscle metabolism were then assayed. The specific enzyme activities are given in per cent compared with unstimulated paired control muscles. The muscles were contracting all the time although the contraction amplitude was successively reduced during the experiment. The water content of the stimulated muscles was about 10% higher than in the controls. In FIG. I the effects of isometric contractile work on enzyme activities are shown. A marked drop in the creatine phosphokinase, and phosphofructokinase phosphorylase activities occurred. On the other hand, an increase in hexokinase and cytochrome oxidase activities was found. The activity levels of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase, lactate dehydrogenase and fumarase were unaffected by the exercise. A rather different enzyme response was obtained after repeated isotonic contractions (FIG. I). No significant decrease in the activity levels of creatine phosphokinase, phosphorylase and phosphofructokinase was now seen. The rise in the oxidative capacity was more pronounced following isotonic work. Thus, the increase in cytochrome oxidase activity was 130%. Besides hexokinase and fumarase the activity levels of glucose-6-phosphate dehydrogenase and 6phosphogluconate dehydrogenase were also elevated. As also found after isometric work there was no change in the activities of pyruvate kinase and lactate dehydrogenase. The effect of contractile activity on enzyme activities wfas also studied under hypoxic conditions. As shown in FIG. I there was no significant difference between isometric work in deoxygenated and oxygenated media. A more marked decline in creatine phosphokinase, phosphorylase and phosphofructokinase activities could, however, be seen. Studies were also done on changes in enzyme activities during a recovery period following isometric work. In FIG. 2 results are
1974,
5
ENZYMATIC
1
CREATINE PHOSPHOKINASE
150
ADAPTATION
TO
CONTRACTILE
PHOSPHORYLASE HEXOKINASE
489
ACTIVITY
GLUCOSE-66-PHOSPHOPHOSPHATE DE- GLUCONATE MHYDROGENASE HYDROGENASE
25
LACTATE l?%ETEDEHYDROGENASE
FUMARASE
CYTOCHROME OXIDASE
6 a150 5
0
*125 8
I
q NO q 5H
n
RECOVERY
m 20 H RECOVERY
RECOVERY
I.-Activities of enzymes in various metabolic pathways in plantaris longus from frog stimulated electrically to isometric and isotonic contractile activity in vitro and isometric contractions in deoxygenAll results are expressed as nanomoles of substrate utilized per minute per mg. of protein ated medium. The control value is shown by the and are given as percentages of the appropriate control value. horizontal broken line. The significance was calculated with paired Student’s t-test and significant values (p to.05) are marked with an asterisk. In all experiments plantaris longus was stimulated to contractile act&ity for 5 hours. FIG.
BOSTRijM
490
I
CREATINE ~OS~INASE
Int. J. Biochem.
et cd.
PHOSPHORYLASE
GLUCOSE-6 6-PHOSFWPHWWW DE- GLUCONATE HYOROGENASE MHYDROGENASE
HEXOKiNASE
LACTATE DEHYDROGENASE
FUMPRhSE %.%JROME
225 200 175
D
ISOMETRIC
fl
iSOMETRICI CONTRACTION
CONTRACTION
q ISOTONIC
CO~RA~ION
IN DEOXYGENATED MEDIUM
FIG. z.-Activities of enzymes in various metabolic pathways in plantaris longus from frog stimulated electrically to isometric contractile activity in z&a followed by recovery periods of varying length. All results are expressed as nanomoles of substrate utilized per minute per mg. of protein and are given as percentages of the appropriate control value. The control value is shown by the horizontal broken line. The significance was calculated with paired Student’s t-test and significant values (I ~0.05) are marked with an asterisk. In all experiments plantaris longus was stimulated to contractile activity for 5 hours.
‘974, 5
ENZYMATIC
ADAPTATION
presented on enzyme activities found immediately after contractile work and after 5 hours and 20 hours recovery of the muscles in oxygenated frog-Ringer solution. Creatine phosphokinase, phosphorylase and phosphofructokinase activities showed decreased activities immediately after the contractile work and this decrease was successively more marked during the recovery period. The oxidative enzymes fumarase and cytochrome oxidase showed the highest activity values 5 hours after the exercise period and these were decreased to control values after 20 hours. Also the a.ctivities of hexokinase and the hexose monophosphate shunt dehydrogenases were increased after 5 hours recovery. This increase remained for hexokinase and 6phosphogluconate dehydrogenase also after 20 hours. The activities of pyruvate kinase and lactate dehydrogenase were unchanged 5 hours but decreased 20 hours after the exercise period. DISCUSSION It is well known that a correlation exists between muscle enzyme activities and levels of musclar activity both between various animal species and within a species (Crabtree & Newsholrne, 1972 ; Bostrijm & Johansson, 1972). Rats subjected to different training programs adapt to the contractile activity by showing an increased capacity of muscle mitochondria to oxidize pyruvate and fatty acids (Holloszy, 1967; Mole, Oscai & Holloszy, 197 I). This increased capacity is reflected in increased enzyme activities. Thus, changes in maximal enzyme activities in vitro are considered to reflect an altered metabolic flux (Holloszy, 1971; Gumaa & McLean, I 97 I ) . The work of Schimke, Ganschow, Doyle & Arias (1968) suggests that the increase in enzyme activities following physical activity is due to synthesis of new enzyme protein. Earlier investigations have also shown an increased incorporation of amino acids into protein and mRNA formation in the isolated guinea-pig heart in response to an increased pressure load (Schreiber, Oratz & Rothschildt, 1967; Schreiber, Oratz, Evans, Shaver & Rothschildt, 1968).
TO CONTRACTILE
ACTIVITY
49’
Our experiments show that isometric as well as isotonic contractile activity of skeletal muscle in vitro cause changes in Isotonic and isometric enzyme activities. contraction are special physiological characteristics in the laboratory. However, when an animal is performing normal muscular activity it is not possible to distinguish between the two types of contraction. Thus, it seemed interesting to study whether these different types of contraction in vitro influenced the enzyme response uniformly. The results show that isometric and isotonic contraction are followed by different changes in enzyme activities. The isometric contraction resulted in increased activities of hexokinase and cytochrome oxidase and decreased activities of phosphorylase, creatine phosphokinase and phosphofructokinase. The activity levels of pyruvate kinase and lactate dehydrogenase remained unchanged. On the other hand the isotonic contraction was followed by not only a rise in the activities of hexokinase and cytochrome oxidase but also of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Moreover, the activities of creatine phosphokinase, phosphorylase and phosphofructokinase were not decreased as observed after isometric contraction. It thus seems that isotonic contraction was more effective in inducing enhanced enzyme activities. When skeletal muscle was made to contract under isometric conditions with reduced oxygen tension the enzyme activity response was about the same as seen after isometric work in oxygenated medium despite the fact that the oxygen tension was reduced to ro”/b of the control value. It is probable that during the strenuous activity of the isolated muscle the oxygen demand may be greater than can be supplied to the muscle by diffusion and thus an intracellular hypoxia may occur in muscles contracting in the oxygenated medium. After intermittent ischemia of rat skeletal muscle for 5 days, increased activities of hexokinase and the hexose monophosphate dehydrogenases were also found (Bostrdm et al., 1974). Muscle myoglobin, cytochrome and succinate oxidase showed increased values in guinea-pigs kept
492
BOSTRijM
at high altitudes (Tappan & Reynafarje, I 957 ; Tappan, Reynafarje, Potter & Hurtado, 1957). Thus, exercise and hypoxia as well as &hernia influence the enzyme activities partly in the same manner. A lowered oxygen tension in the muscle could be the common factor causing changes in enzyme activities. On the other hand, the cytochrome oxidase activity has been shown to be related to the oxygen tension with decreased activity following decreased oxygen tension (Hakami & Pious, 1967 ; Simon & Robin, I 970). It is obvious that the type and intensity of exercise has to be considered when studying the presence or absence of enzyme changes. However, when examining the adaptation of muscle to contractile activity it is also of importance to permit determination of the appearance and duration of the enzyme response. It is clear that different enzymes do not necessarily respond qualitatively and Thus, quantitively in the same way. immediately after acute exercise of rats in viva (Bostriim et al., 1974”) and stimulation to contractile activity in vitro the muscle enzyme activity patterns are not the same as found after some hours’ recovery. This is illustrated by the activities of glucose6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, unaffected immediately after exercise but significantly increased 24 hours after exhaustive work in viva and after 5 hours recovery in this in vitro experiment. The most marked increase in enzyme activities was found after 5 hours recovery of the frog muscle when hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, fumarase and cytochrome oxidase showed the highest The activities of phosphorylase, levels. creatine phosphokinase and phosphofructokinase decreased progressively during the recovery period. After 20 hours recovery almost all enzyme activities were declining. This may be explained by morphological disintegration of the muscle cells. Changes in enzyme activities similar to these demonstrated in this in vitro study are also found after exhaustive swimming of rats and partly after stimulation of the isolated smooth muscle taenia coli (Bostrom
et cd.
Int. J. Biochem.
The et al., Ig73a; Bostrom et al., Ig74a). activities of phosphorylase, creatine phosphokinase and phosphofructokinase were significantly reduced after isometric contractile activity of frog muscle in vitro. The decline in phosphofructokinase activity is in agreement with results obtained immediately after acute exercise in viva. On the other hand, the phosphofructokinase activity 24 hours after acute exercise showed increased values. A decrease in the activity of phosphorylase has been reported immediately after exercise by Edgerton, Simpson, Banard & Peter (I 970). They showed that the phosphorylase activity was decreasing with increasing duration of exercise. It has been reported that an increase in the specific creatine phosphokinase activity in the isolated frog sartorius muscle could be demonstrated in response to repeated isotonic (Kendrick-Jones & Perry, 1965) as well as isometric contraction (Kendrick-Jones & Perry, 1967). On the other hand, Oscai & Holloszy (1971) found no increase in creatine phosphokinase activity after isometric contraction of the same muscle. Hajek & Perry (1967) have shown in isolated frog muscle that increases occur in hexokinase, lactate dehydrogenase, aldolase and phosphoglycerate kinase activities after isometric contraction, pyruvate kinase activity remaining unThese authors found that the changed. glucose present in the Ringer solution played a significant role in stimulating the increase in hexokinase activity. The rapid increase in this enzyme might follow the higher influx of glucose into the muscle cell (Ruderman, Houghton & Hems, 197 I) which might be one of the initial metabolic changes following contractile activity. In rat skeletal muscle the myosin-ATPase activity found immediately after exercise has been shown to increase or decrease, dependent on the type and duration of the exercise (Makarova, I 958). Similar changes in enzyme activities, as obtained in this study, are also found after intermittent long-term stimulation of fast rabbit muscle in situ (Pette, Smith, Staudte & Vrbova, 1973). Increased activity levels of hexokinase and citrate synthetase and a decline in the phosphorylase, triosephosphate isomerase and lactate dehydrogenase activities
‘974, 5
ENZYMATIC ADAPTATION
were observed. A decrease in the activities of creatine phosphokinase and adenylate kinase was also found. It is to be observed that enzyme activity changes obtained after long-term electrical stimulation are similar to those observed in cross-innervation experiments (Dubowitz, 1967) but differ partly from those produced by endurance training (Holloszy, 1971). Also after nerve section of the rat diaphragm, hexokinase and the hexosemonophosphate dehydrogenases show increases in enzyme activities, whereas a small rise in lactate dehydrogenase activity is also indicated (Turner & Manchester, 1972). By comparison, total phosphorylase activity shows a steady decline to low values. From the results obtained it is quite clear that marked enzyme activity changes take place in frog skeletal muscle following contractile activity in vitro. Most changes in enzyme activities are similar to those observed some hours after one single bout of exhaustive exercise (Bostrom et al., rg74a). The findings also indicate that increases in enzyme activities can occur in the absence of hormones and nerve function. In vivo experiments .by Gollnick & Ianuzzo (1972) have shown that changes in enzyme activities can occur in hormone-deficient animals. It has also been found that glycogen degradation occurred during training in rats almost completely devoid of normal hormonal regulation (Gollnick, Soule, Taylor, Williams & Ianuzzo, 1970). Gollnick and co-workers also suggested that glycogenolysis can be stimulated by local metabolites or lack of oxygen supply. Our data indicate that the enzyme adaptation in skeletal muscle that occurs following contractile activity in vitro might be induced by local factors. This enzyme adaptation may be a secondary response in order to maintain the ratio between the maximal capacity of enzymes and the flux in the pathway and hence ensure an excess capacity (Scrutton & Utter, 1968). ACKNOWLEDGEMENTS We are grateful to Mrs. Gunilla Rydgren for excellent technical assistance. This work was supported by grants from the Swedish Natural
TO CONTRACTILE
ACTIVITY
493
Science Research Council and the Faculty science, University of Giiteborg.
of
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Physiol. Scar&., go, 544-554. BOSTR~M. S. L.. FAHL~N. M.. HTALMARSSON.A.. & JOHANSSON, R. (rg74b)( ‘ Muscle en&me activities after repeated ischemia ‘, Int. 3.
Biochem., 5, 359-363. BOSTR~M, S. L., H~GBERG, G., & JOHANSSON, R. ‘ Changes in enzyme activities in (r973a), smooth muscle after contractile activity in vitro ‘, Int. 3. Biochem., 4, 415-420. BOSTRBM, S.-L., H~GBERG, G., & .TOHANSSON,R. (Icmb), ‘ Enzyme response to contractile activitv in isolated smooth muscle : Effects of cycloheximide and insulin ‘, Int. J. Biochem.,
4¶543_547.
BOSTR~M, S.-L., & JOHANSSON,R. (1g72), ‘Enzyme activity patterns in white and red muscle of the eel (A&&la anguilla) at different developmental stages ‘, camp. Biochem. Physiol., 42, 533-542. CRABTREE, B., & NEWSHOLME, E. A. (Ig72), ‘The activities ofphosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase and the glycerol 3-phosphate dehydrogenases in muscles from vertebrates and invertebrates ‘, Biochem. J.,
x26,49-58.
DUBOWITZ, V. (Ig67), ‘ Cross-innervated mammalian skeletal muscle : histochemical, physiological and biochemical observations ‘, 3.
Physiol., Lond., 193, 481-496. ECCLES,J. C., ECCLES, R. M., & KOZAK, W. (rg62), ‘ Further investigations on the influence ofmotor neurons on the speed of muscle contraction ‘,
3.
Physiol., Lond., 163, 324-339.
EDGERTON, V. R., SIMPSON, D., BANARD, R. J., & PETER, J. B. (Ig7o), ‘ Phosphorylase activity in acutely exercised muscle ‘, Nuture, Lond.,
225, 866-867. GOLLNICK, P. D., & IANUZZO, C. D. (rg72), ‘ Hormonal deficiencies and the metabolic adaptations of rats to training ‘, Am. 3. Physiol., 223,
278-282. GOLLNICK, P. D., SOULE, R. G., TAYLOR, WILLIAMS, C., & IANUZZO, C. D. ‘Exercise-induced glycogenolysis and in the rat : hormonal influence ‘, Am. 3.
2x9, 729-733.
A. W., (rg7o), lipolysis
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GUMAA, K. A., & MCLEAN, P. (rg7r), ‘ Factors controlling the flux of glucose through the pentose phosphate pathway ‘, Postgrad. Med. J., (June suppl.) 47, 403-406.
BOSTRGM
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HAJEK, I., & PERRY, S. V. (rg67), ‘ Enzyme adaptation in isolated muscle ‘, Biochem. J., 105,45~-46~. HAKAMI, N., & PIOUS, D. A. (1967) ‘ Regulation of cytochrome oxidase in human cells in culture ‘, Nature, Lond., 216, Io87-Iogo. HARTLEY, L. H., MASON, .l. W., HOGAN, R. P., JONES, L. G., KOTCHEN, T. A.,.MOUGE;, E. H.; WHERRY. F. E.. PENNINGTON,L. L.. & RICKETTS. P. T. (1672), “Multiple hormonal responses to graded exercise in relation to physical training’, 3. A@. Physiol., 33, 602-606. HOLLOSZY, J. 0. (1g67), ‘ Biochemical adaptations in muscle. Effects of exercise on mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle ‘, 3. Biol. Chem., 242, 2278-2282. HOLLOSZY, .J. 0. (197 I), ‘Biochemical adaptations to endurance exercise in skeletal muscle ‘, in Muscle metabolism during exercise, (ed. Pernow & Saltin) pp. 51-61. New York : Plenum Press. KENDRICK-JONES, J., & PERRY, S. V. (rg65), ‘ Enzymatic adaptation to contractile activity in skeletal muscle ‘, A’ature, Lond., 208, 10681070. KENDRICK-JONES, J., & PERRY, S. V. (rg67), ‘ Protein synthesis and enzyme response to contractile activity in skeletal muscle ‘, Nature New Biology, 237, jo-5 I. KRAUS. H.. & KIRSTEN. R. (1~164). ‘ Die Wirkune ““. von Schwimmund Lauftraming auf die cellulare Funktion und Struktur des Muskels ‘, PJliigers Archiv Euro& 3. P/zysiol., 308,57-59. LAMB, D. R., PETER, J. B., JEFFRESS, R. N., & WALLACE, H. A. (rg6g), ‘ Glycogen, hexokinase and glycogen synthetase adaptations to exercise ’ Am. 3. Physiol., 217, 1628-1632. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., & RANDALL, R. J. (rg5r), ‘ Protein measurement with the Folin phenol reagent ‘, 3. Biol. Chem., 193, 265-275. MAKAROVA, A. F. (Ig58), ‘ Effect of various types of muscular activity on the ATPase activity of myosine ‘, Ukr. Biokhim.
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239. MOLF.. P. A.. OSCAI, L. B., & HOLLOSZY, 1. 0. (rgjr), ‘ Adaptation to exercise. Incre&e in levels of oalmitvl CoA svnthetase. carnitine palmityltransferase and palmityl ’ CoA dehydrogenase and in the capacity to oxidize fatty acids ‘,3. Clin. InUest., 50, 2323-2330. OSCAI, L. B., & HOLLOSZY, J. 0. frg7r), ‘ Biochemical adaptations in muscle. II. Response of mitochondrial adenosine triphosphatase, creatine phosphokinase, and adenylatd kinase activities in skeletal muscle to exercise ‘, 3. Biol. Chem., 246, 6968-6972.
et d.
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PETTE, D., SMITII, M. E., STAUDTE, H. W., & VRBOVA, G. (rg78), ‘ Effects of long-term electrical stimulation on some contractile and metabolic characteristics of fast rabbit muscles ‘, P’iigers Archiv. Euro& 3. Physiol., 338, 257-272. RUDF,RMAN, N. B., HOUGHTON, C. R. S., & HEMS, R. (IgTr), ‘Evaluation of the isolated perfused rat hindquarter for the study of muscle metabolism ‘, Biochem. J., 124, 639-65 I. SCHIMKE, R. T., GANSCHOW, R., DOYLE, D., & ARIAS, I. M. (tg68), ‘ Regulation of protein turnover in mammalian tissues ‘, Fedn. Proc. Fedn. Am. Sots. Exp. Biol., 27, 1223: SCHREIBER. S. S.. ORATZ. M.. EVANS. C.. SHAVER. E., & ROTHS&HII.DT, M. A. (rg68), ‘Effect of acute overload on cardiac muscle mRNA ‘, Am. 3. Physiol., 215, I 250-1259. SCHREIBER, S. S., ORATZ. M.. & ROTHSCHII_~T. M. A. (rg67),’ ‘ Effect’ of acute overload on protein synthesis in cardiac muscle microsomes ‘, Am. 3. Physiol., 213, 1552-1555. SCRUTTON, M. C., & UTTER, M. F. (1968), ‘ The regulation of glycolysis and gluconeogenesis in animal tissues ‘, A. Rev. Biochem., ~7. 24+zo2. SIMON, L. M., & ‘ROBIN, E. D. (rg7,jt ‘ Changes in heart and skeletal muscle cytochrome oxidase activity during anaerobiosis in the freshwater turtle, Pseudemys scribta elecans ‘, Comb. Biochem. Physiol., 37, 437-443. _ SUTTON, J. R., YOUNG, J. D., LAZARUS, L., HICKIE, J. B., & MAKSVYTIS, J. (rg6g), ‘ The hormonal response to physical exercise ‘, Australas. Ann. Med.. 18. 84-40. TAPPAN, D. V., & REYNAFA~JE, B. D. (1957)) ‘ Tissue pigment manifestations of adaptation to high altitudes ‘, Am. 3. Physiol., ego, gg-x0.7. TAPPAN, D. V., REYNAFARJE, B. D., POTTER, V. R., & HURTADO, A. (ig57), ‘ Alternations in enzymes and metabolites resulting from adaptation to low oxygen tensions ‘, Am. 3. Physiol., 19% 99-98. TURNER. L. V.. & MANCHESTER. K. L. (1472). \ -, ,, ‘ Effects of denervation on the glycogen content and on the activities of enzymes of glucose and glycogen metabolism in rat diaphragm muscle ‘, Biochem. .7., 128, 78q-80 I. YELLIN, H-( ;g67); i Neural regulation of enzymes in muscle fibers of red and white muscle ‘, Expl. Neural., 19, 92-103.
Key 1l’ord Index : Enzyme activities, muscle, contractile activity in vitro.
skeletal
3.
Biochem., 1974, Vol. 5, pp. 487 to 494. Pergamon Press. Printed in Great Britain
ENZYME
ACTIVITIES
IN SKELETAL
CONTRACTILE
ACTIVITY
S.-L. BOSTROM, Department
G. HtiGBERG
of Zoophysiology,
AND
University
MUSCLE
487
AFTER
IJV VITRO R. G. JOHANSSON
of Gotehorg,
Goteborg,
Sweden
(Receivedrg October 1973) ABSTRACT I. Changes in muscle enzyme activities following isometric and isotonic contractile activity of electrically stimulated plantaris longus from frog were studied in vitro. The effect of contractile activity under hypoxic conditions and the time-course of changes in enzyme activities after exercise were also investigated. z. Isometric contractile work caused decreased activities in creatine phosphokinase, phosphorylase and phosphofructokinase and increased activities in hexokinase and cytochrome oxidase. The activity levels of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydroqenase, pyruvate kinase, lactate dehydrogenase and fumarase were not significantly different from control values. 3. After isotonic contraction no significant changes in creatine phosphokinase, phosphorylase and phosphofructokinase activities were seen. Besides hexokinase and cytochrome oxidase, fumarase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were also increased. 4. There were no significant differences between the enzyme response obtained after isometric contractions in deoxygenated or oxygenated medium. .5. After a recovery period for 5 hours following isometric work the activities of hexokmase, the hexose monophosphate shunt dehydrogenases, fumarase and cytochrome These declined to control values after 20 hours reoxidase showed increased activities. covery except for hexokinase and 6-phosphogluconate dehydrogenase. The activity levels of creatine phosphokinase, phosphorylase and phosphofructokinase were progressively Pyruvate kinase and lactate dehydrogenase also decreased during the recovery period. showed decreased activity values after zo hours recovery.
ADAPTATION of skeletal muscle to prolonged exercise is followed by an increase in the activities of enzymes related to muscle metabolism (Holloszy, 1967; Kraus & Kirsten, 1969). The major feature of this enzyme adaptation is an increase in the Single capacity of aerobic metabolism. bouts of exercise have also been shown to induce increases in muscle enzyme activities (Kendrick-Jones & Perry, 1965; Lamb, Peter, Jeffress & Wallace, rg6g; Bostriim, Fahlen, Hjalmarsson & Johansson, r 974-a). Little is known about the biochemical mechanisms involved in the adaptation process. It is difficult to decide whether the response in enzyme activity levels is controlled by intracellular mechanisms or if it is a response to humoral or neuronal factors. It could be thought that changes in enzyme activities may be mediated through the
endocrine system since changes in plasma concentrations of many hormones have been found during and after physical exercise (Sutton, Young, Lazarus, Hickie & Maksvytis, Hartley, Mason, Hogan, Jones, 1969; Kotchen, Mougey, Wherry, Pennington & Ricketts, 1972). Contractile properties (Eccles, Eccles & Kozak, 1962) as well as metabolic characteristics (Yellin, 1967) of muscle fibers are also suggested to be under neuronal control. As most of the foregoing studies of physical activity on skeletal muscle enzymes are obtained in vivo it was considered of interest to find out if skeletal muscle exercised in an in vitro system would give a similar response in activity of enzymes of significance for muscle metabolism. In this study, isolated frog plantaris longus was electrically isometric stimulated to maximum and
488
BosTRiiM et al.
isotonic contractile activity. The time-scale of enzyme activity changes during a recovery period following the contractile work was also examined. MATERIALS AND METHODS Kana tenlporaria were kept in water at 5’ C before use. Plantaris longus muscles were dissected out and mounted in a muscle holder immersed in frog-Ringer solution containing 5 mM glucose gassed with 100% oxygen. The muscles were allowed to equilibrate for 30 minutes before onset of the electrical stimulation. The muscles were directly stimulated longitudinally once every 3 seconds for 5 hours by stimuli of supramaximal intensity. The duration of each shock was 20 msec. Paired muscles were used as controls. Each experimental group included o animals. The muscles were stimulated to contract isometricallv and isotonicallv. The force disnlacement transducer used for isometric recording of muscle tension was a Grass FT 03. Isotonic measurements were obtained with a differential transformer. The output potential from both transducers was recorded on a Grass Polygraph. The control muscles were stretched to a similar length as the stimulated muscles and were kept in separate chambers. The frog-Ringer solution was exchanged every IO minutes for both stimulated and control muscles. In two series the muscles were given a recovery period of 5 hours and 20 hours before examining the enzvme activities. During this neriod both stimulated and control muscles”were slill mounted and the frog-Ringer solution was exchanged every r 5 minutes. In one of the series the muscles were forced to contract under hypoxic conditions. The frogRinger solution was gassed I hour before and during the experiment with IOO % nitrogen. The oxygen partial pressure was 30 mm Hg in the deoxygenated frog-Ringer solution compared to 345 mm Hg in the oxygenated normally-used The oxygen tension was measured medium. polarographically &h a Radiometer (Copenhagen) gas monitor PHM 27GM. The preparation of homogenates and methods for the‘ determination of the maximal enzyme activities of hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase, lactate dehydrogenase, fumarase and cvtochrome oxidase have been described recently (Bostrom, Hogberg, & Johansson, I g73a). The method described bv Oscai 1sr Holloszv (1971) was used for determination of creatine phosphokinase. Total phosphorylasc activity was analyzed according to Bass, Brdiczka, Eyer, Hofer & Pette (1969) and phosphofructokinase was assayed as described by Bostrom, Hogberg & Johansson (rg73b). Protein was measured by the method of Lowry, Rosebrough, Farr & Randall (1951).
Int.
3. Biochem.
RESULTS Isolated frog plantaris longus muscles were stimulated continously to contractile work for 5 hours by stimuli of supramaximal intensity. Activities of various enzymes of significance for muscle metabolism were then assayed. The specific enzyme activities are given in per cent compared with unstimulated paired control muscles. The muscles were contracting all the time although the contraction amplitude was successively reduced during the experiment. The water content of the stimulated muscles was about 10% higher than in the controls. In FIG. I the effects of isometric contractile work on enzyme activities are shown. A marked drop in the creatine phosphokinase, and phosphofructokinase phosphorylase activities occurred. On the other hand, an increase in hexokinase and cytochrome oxidase activities was found. The activity levels of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase, lactate dehydrogenase and fumarase were unaffected by the exercise. A rather different enzyme response was obtained after repeated isotonic contractions (FIG. I). No significant decrease in the activity levels of creatine phosphokinase, phosphorylase and phosphofructokinase was now seen. The rise in the oxidative capacity was more pronounced following isotonic work. Thus, the increase in cytochrome oxidase activity was 130%. Besides hexokinase and fumarase the activity levels of glucose-6-phosphate dehydrogenase and 6phosphogluconate dehydrogenase were also elevated. As also found after isometric work there was no change in the activities of pyruvate kinase and lactate dehydrogenase. The effect of contractile activity on enzyme activities wfas also studied under hypoxic conditions. As shown in FIG. I there was no significant difference between isometric work in deoxygenated and oxygenated media. A more marked decline in creatine phosphokinase, phosphorylase and phosphofructokinase activities could, however, be seen. Studies were also done on changes in enzyme activities during a recovery period following isometric work. In FIG. 2 results are
1974,
5
ENZYMATIC
1
CREATINE PHOSPHOKINASE
150
ADAPTATION
TO
CONTRACTILE
PHOSPHORYLASE HEXOKINASE
489
ACTIVITY
GLUCOSE-66-PHOSPHOPHOSPHATE DE- GLUCONATE MHYDROGENASE HYDROGENASE
25
LACTATE l?%ETEDEHYDROGENASE
FUMARASE
CYTOCHROME OXIDASE
6 a150 5
0
*125 8
I
q NO q 5H
n
RECOVERY
m 20 H RECOVERY
RECOVERY
I.-Activities of enzymes in various metabolic pathways in plantaris longus from frog stimulated electrically to isometric and isotonic contractile activity in vitro and isometric contractions in deoxygenAll results are expressed as nanomoles of substrate utilized per minute per mg. of protein ated medium. The control value is shown by the and are given as percentages of the appropriate control value. horizontal broken line. The significance was calculated with paired Student’s t-test and significant values (p to.05) are marked with an asterisk. In all experiments plantaris longus was stimulated to contractile act&ity for 5 hours. FIG.
BOSTRijM
490
I
CREATINE ~OS~INASE
Int. J. Biochem.
et cd.
PHOSPHORYLASE
GLUCOSE-6 6-PHOSFWPHWWW DE- GLUCONATE HYOROGENASE MHYDROGENASE
HEXOKiNASE
LACTATE DEHYDROGENASE
FUMPRhSE %.%JROME
225 200 175
D
ISOMETRIC
fl
iSOMETRICI CONTRACTION
CONTRACTION
q ISOTONIC
CO~RA~ION
IN DEOXYGENATED MEDIUM
FIG. z.-Activities of enzymes in various metabolic pathways in plantaris longus from frog stimulated electrically to isometric contractile activity in z&a followed by recovery periods of varying length. All results are expressed as nanomoles of substrate utilized per minute per mg. of protein and are given as percentages of the appropriate control value. The control value is shown by the horizontal broken line. The significance was calculated with paired Student’s t-test and significant values (I ~0.05) are marked with an asterisk. In all experiments plantaris longus was stimulated to contractile activity for 5 hours.
‘974, 5
ENZYMATIC
ADAPTATION
presented on enzyme activities found immediately after contractile work and after 5 hours and 20 hours recovery of the muscles in oxygenated frog-Ringer solution. Creatine phosphokinase, phosphorylase and phosphofructokinase activities showed decreased activities immediately after the contractile work and this decrease was successively more marked during the recovery period. The oxidative enzymes fumarase and cytochrome oxidase showed the highest activity values 5 hours after the exercise period and these were decreased to control values after 20 hours. Also the a.ctivities of hexokinase and the hexose monophosphate shunt dehydrogenases were increased after 5 hours recovery. This increase remained for hexokinase and 6phosphogluconate dehydrogenase also after 20 hours. The activities of pyruvate kinase and lactate dehydrogenase were unchanged 5 hours but decreased 20 hours after the exercise period. DISCUSSION It is well known that a correlation exists between muscle enzyme activities and levels of musclar activity both between various animal species and within a species (Crabtree & Newsholrne, 1972 ; Bostrijm & Johansson, 1972). Rats subjected to different training programs adapt to the contractile activity by showing an increased capacity of muscle mitochondria to oxidize pyruvate and fatty acids (Holloszy, 1967; Mole, Oscai & Holloszy, 197 I). This increased capacity is reflected in increased enzyme activities. Thus, changes in maximal enzyme activities in vitro are considered to reflect an altered metabolic flux (Holloszy, 1971; Gumaa & McLean, I 97 I ) . The work of Schimke, Ganschow, Doyle & Arias (1968) suggests that the increase in enzyme activities following physical activity is due to synthesis of new enzyme protein. Earlier investigations have also shown an increased incorporation of amino acids into protein and mRNA formation in the isolated guinea-pig heart in response to an increased pressure load (Schreiber, Oratz & Rothschildt, 1967; Schreiber, Oratz, Evans, Shaver & Rothschildt, 1968).
TO CONTRACTILE
ACTIVITY
49’
Our experiments show that isometric as well as isotonic contractile activity of skeletal muscle in vitro cause changes in Isotonic and isometric enzyme activities. contraction are special physiological characteristics in the laboratory. However, when an animal is performing normal muscular activity it is not possible to distinguish between the two types of contraction. Thus, it seemed interesting to study whether these different types of contraction in vitro influenced the enzyme response uniformly. The results show that isometric and isotonic contraction are followed by different changes in enzyme activities. The isometric contraction resulted in increased activities of hexokinase and cytochrome oxidase and decreased activities of phosphorylase, creatine phosphokinase and phosphofructokinase. The activity levels of pyruvate kinase and lactate dehydrogenase remained unchanged. On the other hand the isotonic contraction was followed by not only a rise in the activities of hexokinase and cytochrome oxidase but also of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Moreover, the activities of creatine phosphokinase, phosphorylase and phosphofructokinase were not decreased as observed after isometric contraction. It thus seems that isotonic contraction was more effective in inducing enhanced enzyme activities. When skeletal muscle was made to contract under isometric conditions with reduced oxygen tension the enzyme activity response was about the same as seen after isometric work in oxygenated medium despite the fact that the oxygen tension was reduced to ro”/b of the control value. It is probable that during the strenuous activity of the isolated muscle the oxygen demand may be greater than can be supplied to the muscle by diffusion and thus an intracellular hypoxia may occur in muscles contracting in the oxygenated medium. After intermittent ischemia of rat skeletal muscle for 5 days, increased activities of hexokinase and the hexose monophosphate dehydrogenases were also found (Bostrdm et al., 1974). Muscle myoglobin, cytochrome and succinate oxidase showed increased values in guinea-pigs kept
492
BOSTRijM
at high altitudes (Tappan & Reynafarje, I 957 ; Tappan, Reynafarje, Potter & Hurtado, 1957). Thus, exercise and hypoxia as well as &hernia influence the enzyme activities partly in the same manner. A lowered oxygen tension in the muscle could be the common factor causing changes in enzyme activities. On the other hand, the cytochrome oxidase activity has been shown to be related to the oxygen tension with decreased activity following decreased oxygen tension (Hakami & Pious, 1967 ; Simon & Robin, I 970). It is obvious that the type and intensity of exercise has to be considered when studying the presence or absence of enzyme changes. However, when examining the adaptation of muscle to contractile activity it is also of importance to permit determination of the appearance and duration of the enzyme response. It is clear that different enzymes do not necessarily respond qualitatively and Thus, quantitively in the same way. immediately after acute exercise of rats in viva (Bostriim et al., 1974”) and stimulation to contractile activity in vitro the muscle enzyme activity patterns are not the same as found after some hours’ recovery. This is illustrated by the activities of glucose6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, unaffected immediately after exercise but significantly increased 24 hours after exhaustive work in viva and after 5 hours recovery in this in vitro experiment. The most marked increase in enzyme activities was found after 5 hours recovery of the frog muscle when hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, fumarase and cytochrome oxidase showed the highest The activities of phosphorylase, levels. creatine phosphokinase and phosphofructokinase decreased progressively during the recovery period. After 20 hours recovery almost all enzyme activities were declining. This may be explained by morphological disintegration of the muscle cells. Changes in enzyme activities similar to these demonstrated in this in vitro study are also found after exhaustive swimming of rats and partly after stimulation of the isolated smooth muscle taenia coli (Bostrom
et cd.
Int. J. Biochem.
The et al., Ig73a; Bostrom et al., Ig74a). activities of phosphorylase, creatine phosphokinase and phosphofructokinase were significantly reduced after isometric contractile activity of frog muscle in vitro. The decline in phosphofructokinase activity is in agreement with results obtained immediately after acute exercise in viva. On the other hand, the phosphofructokinase activity 24 hours after acute exercise showed increased values. A decrease in the activity of phosphorylase has been reported immediately after exercise by Edgerton, Simpson, Banard & Peter (I 970). They showed that the phosphorylase activity was decreasing with increasing duration of exercise. It has been reported that an increase in the specific creatine phosphokinase activity in the isolated frog sartorius muscle could be demonstrated in response to repeated isotonic (Kendrick-Jones & Perry, 1965) as well as isometric contraction (Kendrick-Jones & Perry, 1967). On the other hand, Oscai & Holloszy (1971) found no increase in creatine phosphokinase activity after isometric contraction of the same muscle. Hajek & Perry (1967) have shown in isolated frog muscle that increases occur in hexokinase, lactate dehydrogenase, aldolase and phosphoglycerate kinase activities after isometric contraction, pyruvate kinase activity remaining unThese authors found that the changed. glucose present in the Ringer solution played a significant role in stimulating the increase in hexokinase activity. The rapid increase in this enzyme might follow the higher influx of glucose into the muscle cell (Ruderman, Houghton & Hems, 197 I) which might be one of the initial metabolic changes following contractile activity. In rat skeletal muscle the myosin-ATPase activity found immediately after exercise has been shown to increase or decrease, dependent on the type and duration of the exercise (Makarova, I 958). Similar changes in enzyme activities, as obtained in this study, are also found after intermittent long-term stimulation of fast rabbit muscle in situ (Pette, Smith, Staudte & Vrbova, 1973). Increased activity levels of hexokinase and citrate synthetase and a decline in the phosphorylase, triosephosphate isomerase and lactate dehydrogenase activities
‘974, 5
ENZYMATIC ADAPTATION
were observed. A decrease in the activities of creatine phosphokinase and adenylate kinase was also found. It is to be observed that enzyme activity changes obtained after long-term electrical stimulation are similar to those observed in cross-innervation experiments (Dubowitz, 1967) but differ partly from those produced by endurance training (Holloszy, 1971). Also after nerve section of the rat diaphragm, hexokinase and the hexosemonophosphate dehydrogenases show increases in enzyme activities, whereas a small rise in lactate dehydrogenase activity is also indicated (Turner & Manchester, 1972). By comparison, total phosphorylase activity shows a steady decline to low values. From the results obtained it is quite clear that marked enzyme activity changes take place in frog skeletal muscle following contractile activity in vitro. Most changes in enzyme activities are similar to those observed some hours after one single bout of exhaustive exercise (Bostrom et al., rg74a). The findings also indicate that increases in enzyme activities can occur in the absence of hormones and nerve function. In vivo experiments .by Gollnick & Ianuzzo (1972) have shown that changes in enzyme activities can occur in hormone-deficient animals. It has also been found that glycogen degradation occurred during training in rats almost completely devoid of normal hormonal regulation (Gollnick, Soule, Taylor, Williams & Ianuzzo, 1970). Gollnick and co-workers also suggested that glycogenolysis can be stimulated by local metabolites or lack of oxygen supply. Our data indicate that the enzyme adaptation in skeletal muscle that occurs following contractile activity in vitro might be induced by local factors. This enzyme adaptation may be a secondary response in order to maintain the ratio between the maximal capacity of enzymes and the flux in the pathway and hence ensure an excess capacity (Scrutton & Utter, 1968). ACKNOWLEDGEMENTS We are grateful to Mrs. Gunilla Rydgren for excellent technical assistance. This work was supported by grants from the Swedish Natural
TO CONTRACTILE
ACTIVITY
493
Science Research Council and the Faculty science, University of Giiteborg.
of
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Physiol. Scar&., go, 544-554. BOSTR~M. S. L.. FAHL~N. M.. HTALMARSSON.A.. & JOHANSSON, R. (rg74b)( ‘ Muscle en&me activities after repeated ischemia ‘, Int. 3.
Biochem., 5, 359-363. BOSTR~M, S. L., H~GBERG, G., & JOHANSSON, R. ‘ Changes in enzyme activities in (r973a), smooth muscle after contractile activity in vitro ‘, Int. 3. Biochem., 4, 415-420. BOSTRBM, S.-L., H~GBERG, G., & .TOHANSSON,R. (Icmb), ‘ Enzyme response to contractile activitv in isolated smooth muscle : Effects of cycloheximide and insulin ‘, Int. J. Biochem.,
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BOSTR~M, S.-L., & JOHANSSON,R. (1g72), ‘Enzyme activity patterns in white and red muscle of the eel (A&&la anguilla) at different developmental stages ‘, camp. Biochem. Physiol., 42, 533-542. CRABTREE, B., & NEWSHOLME, E. A. (Ig72), ‘The activities ofphosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase and the glycerol 3-phosphate dehydrogenases in muscles from vertebrates and invertebrates ‘, Biochem. J.,
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EDGERTON, V. R., SIMPSON, D., BANARD, R. J., & PETER, J. B. (Ig7o), ‘ Phosphorylase activity in acutely exercised muscle ‘, Nuture, Lond.,
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HAJEK, I., & PERRY, S. V. (rg67), ‘ Enzyme adaptation in isolated muscle ‘, Biochem. J., 105,45~-46~. HAKAMI, N., & PIOUS, D. A. (1967) ‘ Regulation of cytochrome oxidase in human cells in culture ‘, Nature, Lond., 216, Io87-Iogo. HARTLEY, L. H., MASON, .l. W., HOGAN, R. P., JONES, L. G., KOTCHEN, T. A.,.MOUGE;, E. H.; WHERRY. F. E.. PENNINGTON,L. L.. & RICKETTS. P. T. (1672), “Multiple hormonal responses to graded exercise in relation to physical training’, 3. A@. Physiol., 33, 602-606. HOLLOSZY, J. 0. (1g67), ‘ Biochemical adaptations in muscle. Effects of exercise on mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle ‘, 3. Biol. Chem., 242, 2278-2282. HOLLOSZY, .J. 0. (197 I), ‘Biochemical adaptations to endurance exercise in skeletal muscle ‘, in Muscle metabolism during exercise, (ed. Pernow & Saltin) pp. 51-61. New York : Plenum Press. KENDRICK-JONES, J., & PERRY, S. V. (rg65), ‘ Enzymatic adaptation to contractile activity in skeletal muscle ‘, A’ature, Lond., 208, 10681070. KENDRICK-JONES, J., & PERRY, S. V. (rg67), ‘ Protein synthesis and enzyme response to contractile activity in skeletal muscle ‘, Nature New Biology, 237, jo-5 I. KRAUS. H.. & KIRSTEN. R. (1~164). ‘ Die Wirkune ““. von Schwimmund Lauftraming auf die cellulare Funktion und Struktur des Muskels ‘, PJliigers Archiv Euro& 3. P/zysiol., 308,57-59. LAMB, D. R., PETER, J. B., JEFFRESS, R. N., & WALLACE, H. A. (rg6g), ‘ Glycogen, hexokinase and glycogen synthetase adaptations to exercise ’ Am. 3. Physiol., 217, 1628-1632. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., & RANDALL, R. J. (rg5r), ‘ Protein measurement with the Folin phenol reagent ‘, 3. Biol. Chem., 193, 265-275. MAKAROVA, A. F. (Ig58), ‘ Effect of various types of muscular activity on the ATPase activity of myosine ‘, Ukr. Biokhim.
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PETTE, D., SMITII, M. E., STAUDTE, H. W., & VRBOVA, G. (rg78), ‘ Effects of long-term electrical stimulation on some contractile and metabolic characteristics of fast rabbit muscles ‘, P’iigers Archiv. Euro& 3. Physiol., 338, 257-272. RUDF,RMAN, N. B., HOUGHTON, C. R. S., & HEMS, R. (IgTr), ‘Evaluation of the isolated perfused rat hindquarter for the study of muscle metabolism ‘, Biochem. J., 124, 639-65 I. SCHIMKE, R. T., GANSCHOW, R., DOYLE, D., & ARIAS, I. M. (tg68), ‘ Regulation of protein turnover in mammalian tissues ‘, Fedn. Proc. Fedn. Am. Sots. Exp. Biol., 27, 1223: SCHREIBER. S. S.. ORATZ. M.. EVANS. C.. SHAVER. E., & ROTHS&HII.DT, M. A. (rg68), ‘Effect of acute overload on cardiac muscle mRNA ‘, Am. 3. Physiol., 215, I 250-1259. SCHREIBER, S. S., ORATZ. M.. & ROTHSCHII_~T. M. A. (rg67),’ ‘ Effect’ of acute overload on protein synthesis in cardiac muscle microsomes ‘, Am. 3. Physiol., 213, 1552-1555. SCRUTTON, M. C., & UTTER, M. F. (1968), ‘ The regulation of glycolysis and gluconeogenesis in animal tissues ‘, A. Rev. Biochem., ~7. 24+zo2. SIMON, L. M., & ‘ROBIN, E. D. (rg7,jt ‘ Changes in heart and skeletal muscle cytochrome oxidase activity during anaerobiosis in the freshwater turtle, Pseudemys scribta elecans ‘, Comb. Biochem. Physiol., 37, 437-443. _ SUTTON, J. R., YOUNG, J. D., LAZARUS, L., HICKIE, J. B., & MAKSVYTIS, J. (rg6g), ‘ The hormonal response to physical exercise ‘, Australas. Ann. Med.. 18. 84-40. TAPPAN, D. V., & REYNAFA~JE, B. D. (1957)) ‘ Tissue pigment manifestations of adaptation to high altitudes ‘, Am. 3. Physiol., ego, gg-x0.7. TAPPAN, D. V., REYNAFARJE, B. D., POTTER, V. R., & HURTADO, A. (ig57), ‘ Alternations in enzymes and metabolites resulting from adaptation to low oxygen tensions ‘, Am. 3. Physiol., 19% 99-98. TURNER. L. V.. & MANCHESTER. K. L. (1472). \ -, ,, ‘ Effects of denervation on the glycogen content and on the activities of enzymes of glucose and glycogen metabolism in rat diaphragm muscle ‘, Biochem. .7., 128, 78q-80 I. YELLIN, H-( ;g67); i Neural regulation of enzymes in muscle fibers of red and white muscle ‘, Expl. Neural., 19, 92-103.
Key 1l’ord Index : Enzyme activities, muscle, contractile activity in vitro.
skeletal