BIOCHEMICAI
MEDICINF
Effect
16, 13x-142 (1976,
of Exercise
Training on Adenyl Cyclase Phosphodiesterase in Skeletal Muscle, Heart, and Liver
and
G. LYNIS DOHM, SAM N. PENNINGTON,AND HISHAM BARAKAT Department
of Biochemistry. School of Medicine, Greernille. North Carolina
East Carolina 27834
University.
Received June 24, 1976
Numerous studies have shown that exercise training is accompanied by adaptive changes which increase the capacity of the animal to mobilize (l-3) and oxidize metabolic substrates (4-6). In general these adaptations enhance the capacity for ATP synthesis to cover an increased energy demand in the muscle. The enzymes responsible for mobilization of carbohydrate (phosphorylase) and lipid (triglyceride lipase) are known to exist in active and inactive forms. Since the activation of both phosphorylase and triglyceride lipase is ultimately under the control of cyclic AMP (7), it would seem that adaptive changes in the enzymes which synthesize and degrade cyclic AMP might be as important for the mobilization of fat and carbohydrate during exercise as the levels of phosphorylase and triglyceride lipase. Therefore, we have investigated the effect of exercise training on adenyl cyclase and phosphodiesterase. METHODS Male SpragueDawley rats weighing approximately 220 g at the start of the experiment were housed in individual cages and given water and commercial lab chow ad lib. Rats were divided into two groups: untrained, which remained sedentary in their cages, and trained, which were subjected to running on a locally built treadmill. The training schedule consisted of a daily run, 5 days a week beginning at 15 m/min for 15 min and gradually increasing time and rate to 25 m/min for 25 min, 30 m/min for 40 min, 35 m/min for 50 min, 35 m/min for 60 min, and 35 m/min for 60 min with an 8% grade by the end of weeks 1, 2, 3, 4, and 5, respectively, and maintaining this final rate for weeks 6 and 7 of the experiment. Rats were sacrificed by decapitation, and the heart, gastrocnemius muscle group, and liver were quickly excised and chilled in ice-cold 0.15 M KCl. Heart and liver were homogenized in a medium containing 250 mM sucrose, 10 mM Tris-HCl (pH 7.5). and 1 mM MgC&. The homogeni138 Copyright @ 1576 by Academic Press. Inc. All rights of reproduction in any form reserved.
EFFECT
OF TRAINING
ON
ADENYL
CYCLASE
139
zation medium for muscle consisted of 100 mM KCl, 50 mM Tris (pH 7.5), 5 mM MgSO,, and 1 mM EDTA. Tissues were minced with scissors, homogenized with five strokes in a glass-Teflon homogenizer, followed by a second homogenization in a glass-glass Ten-Broeck homogenizer. Muscle and liver homogenates were filtered through cheesecloth, and the whole homogenates were used for adenyl cyclase and phosphodiesterase assays. For the assay of heart adenyl cyclase, membrane particles were prepared by centrifuging the homogenate at 20008 for 15 min, washing the pellet twice, and suspending in the homogenization medium. Whole heart homogenate was used for the phosphodiesterase assay. Basal adenyl cyclase activity was assayed in a medium consisting of 20 mM Tris-HCl (pH 7.5), 10 mM creatine phosphate (pH 7.5), 10 mM MgCl,, 10 mM theophylline, 1.5 mM [3H]ATP (10&i, Amersham/Searle, Arlington Heights, Illinois) and 10 units of creatine phosphokinase in a total volume of 1.0 ml. Substrate was added to initiate the assay, after which the reaction was incubated at 37” for 10 min and terminated by boiling for 3 min. Cyclic AMP was separated from other labeled nucleotides by the method of Krishna et al. (8), and the 3H-labeled cyclic AMP was counted in a Packard Tri-Carb liquid scintillation counter. Blanks were prepared by adding [3H]ATP after boiling the enzyme and reaction medium. In addition to the blank and basal reactions, adenyl cyclase activity was also determined in the presence of 0.1 mM epinephrine and 10 mM NaF. The phosphodiesterase assay medium contained 35 mM Tris-HCl (pH 7.0), 10 mM MnCl,, and 0.33 mM cyclic AMP in a total volume of 1.0 ml. The reaction was incubated at 37” for 30 min after initiating with enzyme. The assay was terminated by boiling for 2 min, and the disappearance of cyclic AMP was quantitated by high speed liquid chromatography as described by Pennington (9). The conditions listed above were found to give maximal adenyl cyclase and phosphodiesterase activities, and enzyme activities were proportional to time and enzyme concentration. Protein was determined by the biuret procedure of Gornall et al. (10). RESULTS
AND DlSCUSSlON
The effects of exercise training on adenyl cyclase activity in muscle, heart, and liver are shown in Table 1. Training increased the basal adenyl cyclase activity in muscle, depressed basal and epinephrine-stimulated activity in heart, and did not alter adenyl cyclase activity in liver. Epinephrine-stimulated adenyl cyclase activity in muscle tended to be higher in the trained animals but the difference was not statistically significant (0.1 > P > 0.05). Phosphodiesterase was lowered by training in liver but was unchanged in heart and muscle (Table 2). It is now well established that the plasma concentrations of a number of hormones are altered by exercise and physical training (11). Since cyclic
140
DOHM. PENNINGTON
AND BARAKAT
TABLE THE
EFFECT
OF TRAINING IN MUSCLE,
1
ON AL~NYL HEART,
AND
CYCLASE
ACTWIT>
LIVER
Adenyl cyclase activity (pmoles/min/mg
Epinephrine Stimulated
6.42 k 0.66 (9)a 8.91 2 0.72 (9)” P < 0.025
34.72 + 1.31 (9) .34.42 IT 1.67 (9)
12.40 2 0.87 (8) 15.41 2 1.33 (8)
16.9 2 1.7 (8) 12.5 f 0.8 (8)b
60.0 f 4.3 (8) 55.2 f 3.8 (8)
Basal
Heart Untrained Trained
protein)
Fluoride Stimulated
Tissue Muscle Untrained Trained
of
24.7 t 2.7 (8) 18.0 4 1.2 (8)*
P < 0.05
Liver Untrained Trained
P < 0.05
7.06 f 0.47 (9) 7.02 f 0.63 (9)
11.81 k 0.68 (9) 11.37 + 0.79 (7)
30.21 k 1.88 (9) 29.75 t 1.51 (9)
’ Values are means f SEM with the number of observations in parentheses. b Statistically significant difference between untrained and trained.
AMP has come to be recognized as a second messenger mediating a variety of hormonal effects (7), it seems likely that the increased adenyl cyclase activity of muscle and the decreased phosphodiesterase activity of liver might be significant adaptations to endurance training. The decreased adenyl cyclase activity in heart is at variance with the conclusions drawn for muscle and liver. However, this variance may be in TABLE THE
EFFECT
OF TRAINING IN MUSCLE,
2
ON PHOSPHODIESTERASE HEART, AND LIVER
ACTIVITY
Phosphodiesterase activity (nmoles/min/mg of protein) Tissue
Untrained
Muscle Heart Liver
0.83 2 0.07 (12) 1.46 f 0.22 (6) 4.44 f 0.52 (12)
Trained 0.82 k 0.07 (12) 1.17 + 0.18 (6) 3.12 k 0.25 (12)* (P < 0.05)
’ Values are means 2 SEM with the number of observations in parentheses. b Statistically significant difference between trained and untrained.
EFFECT
OF TRAINING
ON
ADENYL
141
CYCLASE
keeping with the differences in function between skeletal muscle and heart. It is well known (12) that athletes with considerable endurance usually have a slow resting heart rate. In addition, endurance training enables an athlete to achieve a certain cardiac output during exercise with a slower heart rate and a larger stroke volume (12). These observations, coupled with the fact that cyclic AMP mediates the positive chromotropic and inotropic effects of the catecholamines (7) may explain why adenyl cyclase is decreased in heart while it is increased in skeletal muscle. The changes in muscle and heart adenyl cyclase do not appear to be due to an increased level of the enzyme since the fluoride-stimulated activity was unchanged. Thus, the changes in basal adenyl cyclase activity of heart and muscle must be caused by either an alteration in the control properties of the enzyme or the presence of an endogenous effector (inhibitor or activator). Epinephrine can probably be ruled out as an endogenous activator of muscle basal adenyl cyclase activity since the stimulation upon epinephrine addition was approximately the same in trained and untrained rats. Yakovlev (13) has reported that epinephrine-stimulated adenyl cyclase activity was increased in muscle, liver, and adipose tissue, but only muscle showed an increase in basal activity. In agreement with our results, he found that fluoride-stimulated adenyl cyclase activity was not changed by training in any of the tissues. Although we did not find liver epinephrine-stimulated adenyl cyclase to be elevated by training, the results of Yakovlev (13) do confirm our findings in muscle. In conclusion, the changes in adenyl cyclase activity in muscle and heart and the alteration of phosphodiesterase in liver indicate that the capacity to respond to hormones may be altered by training. SUMMARY Adaptive changes in the activities of adenyl cyclase and phosphodiesterase in response to exercise training were studied in muscle, heart, and liver. Basal adenyl cyclase activities were increased in muscle and decreased in heart as a result of training. Epinephrine-stimulated adenyl cyclase activity of heart was depressed by training. Fluoride-stimulated adenyl cyclase activity was not altered by training in any of the tissues studied. Phosphodiesterase activity was depressed in liver of trained rats but unaltered in muscle and heart. These changes in adenyl cyclase and phosphodiesterase activities suggest that the capacity to respond to hormones may be altered by training. ACKNOWLEDGMENT This research Association.
was supported
by Grant
No.
1973-74-A-3
from
the North
Carolina
Heart
142
DOHM, PENNINGTON
AND BARAKAT
REFERENCES 1. Askew, E. W., Dohm, G. L., Huston, R. L.. Sneed, T. W., and Dowdy, R. P.. Pro
MEDICINF
Effect
16, 13x-142 (1976,
of Exercise
Training on Adenyl Cyclase Phosphodiesterase in Skeletal Muscle, Heart, and Liver
and
G. LYNIS DOHM, SAM N. PENNINGTON,AND HISHAM BARAKAT Department
of Biochemistry. School of Medicine, Greernille. North Carolina
East Carolina 27834
University.
Received June 24, 1976
Numerous studies have shown that exercise training is accompanied by adaptive changes which increase the capacity of the animal to mobilize (l-3) and oxidize metabolic substrates (4-6). In general these adaptations enhance the capacity for ATP synthesis to cover an increased energy demand in the muscle. The enzymes responsible for mobilization of carbohydrate (phosphorylase) and lipid (triglyceride lipase) are known to exist in active and inactive forms. Since the activation of both phosphorylase and triglyceride lipase is ultimately under the control of cyclic AMP (7), it would seem that adaptive changes in the enzymes which synthesize and degrade cyclic AMP might be as important for the mobilization of fat and carbohydrate during exercise as the levels of phosphorylase and triglyceride lipase. Therefore, we have investigated the effect of exercise training on adenyl cyclase and phosphodiesterase. METHODS Male SpragueDawley rats weighing approximately 220 g at the start of the experiment were housed in individual cages and given water and commercial lab chow ad lib. Rats were divided into two groups: untrained, which remained sedentary in their cages, and trained, which were subjected to running on a locally built treadmill. The training schedule consisted of a daily run, 5 days a week beginning at 15 m/min for 15 min and gradually increasing time and rate to 25 m/min for 25 min, 30 m/min for 40 min, 35 m/min for 50 min, 35 m/min for 60 min, and 35 m/min for 60 min with an 8% grade by the end of weeks 1, 2, 3, 4, and 5, respectively, and maintaining this final rate for weeks 6 and 7 of the experiment. Rats were sacrificed by decapitation, and the heart, gastrocnemius muscle group, and liver were quickly excised and chilled in ice-cold 0.15 M KCl. Heart and liver were homogenized in a medium containing 250 mM sucrose, 10 mM Tris-HCl (pH 7.5). and 1 mM MgC&. The homogeni138 Copyright @ 1576 by Academic Press. Inc. All rights of reproduction in any form reserved.
EFFECT
OF TRAINING
ON
ADENYL
CYCLASE
139
zation medium for muscle consisted of 100 mM KCl, 50 mM Tris (pH 7.5), 5 mM MgSO,, and 1 mM EDTA. Tissues were minced with scissors, homogenized with five strokes in a glass-Teflon homogenizer, followed by a second homogenization in a glass-glass Ten-Broeck homogenizer. Muscle and liver homogenates were filtered through cheesecloth, and the whole homogenates were used for adenyl cyclase and phosphodiesterase assays. For the assay of heart adenyl cyclase, membrane particles were prepared by centrifuging the homogenate at 20008 for 15 min, washing the pellet twice, and suspending in the homogenization medium. Whole heart homogenate was used for the phosphodiesterase assay. Basal adenyl cyclase activity was assayed in a medium consisting of 20 mM Tris-HCl (pH 7.5), 10 mM creatine phosphate (pH 7.5), 10 mM MgCl,, 10 mM theophylline, 1.5 mM [3H]ATP (10&i, Amersham/Searle, Arlington Heights, Illinois) and 10 units of creatine phosphokinase in a total volume of 1.0 ml. Substrate was added to initiate the assay, after which the reaction was incubated at 37” for 10 min and terminated by boiling for 3 min. Cyclic AMP was separated from other labeled nucleotides by the method of Krishna et al. (8), and the 3H-labeled cyclic AMP was counted in a Packard Tri-Carb liquid scintillation counter. Blanks were prepared by adding [3H]ATP after boiling the enzyme and reaction medium. In addition to the blank and basal reactions, adenyl cyclase activity was also determined in the presence of 0.1 mM epinephrine and 10 mM NaF. The phosphodiesterase assay medium contained 35 mM Tris-HCl (pH 7.0), 10 mM MnCl,, and 0.33 mM cyclic AMP in a total volume of 1.0 ml. The reaction was incubated at 37” for 30 min after initiating with enzyme. The assay was terminated by boiling for 2 min, and the disappearance of cyclic AMP was quantitated by high speed liquid chromatography as described by Pennington (9). The conditions listed above were found to give maximal adenyl cyclase and phosphodiesterase activities, and enzyme activities were proportional to time and enzyme concentration. Protein was determined by the biuret procedure of Gornall et al. (10). RESULTS
AND DlSCUSSlON
The effects of exercise training on adenyl cyclase activity in muscle, heart, and liver are shown in Table 1. Training increased the basal adenyl cyclase activity in muscle, depressed basal and epinephrine-stimulated activity in heart, and did not alter adenyl cyclase activity in liver. Epinephrine-stimulated adenyl cyclase activity in muscle tended to be higher in the trained animals but the difference was not statistically significant (0.1 > P > 0.05). Phosphodiesterase was lowered by training in liver but was unchanged in heart and muscle (Table 2). It is now well established that the plasma concentrations of a number of hormones are altered by exercise and physical training (11). Since cyclic
140
DOHM. PENNINGTON
AND BARAKAT
TABLE THE
EFFECT
OF TRAINING IN MUSCLE,
1
ON AL~NYL HEART,
AND
CYCLASE
ACTWIT>
LIVER
Adenyl cyclase activity (pmoles/min/mg
Epinephrine Stimulated
6.42 k 0.66 (9)a 8.91 2 0.72 (9)” P < 0.025
34.72 + 1.31 (9) .34.42 IT 1.67 (9)
12.40 2 0.87 (8) 15.41 2 1.33 (8)
16.9 2 1.7 (8) 12.5 f 0.8 (8)b
60.0 f 4.3 (8) 55.2 f 3.8 (8)
Basal
Heart Untrained Trained
protein)
Fluoride Stimulated
Tissue Muscle Untrained Trained
of
24.7 t 2.7 (8) 18.0 4 1.2 (8)*
P < 0.05
Liver Untrained Trained
P < 0.05
7.06 f 0.47 (9) 7.02 f 0.63 (9)
11.81 k 0.68 (9) 11.37 + 0.79 (7)
30.21 k 1.88 (9) 29.75 t 1.51 (9)
’ Values are means f SEM with the number of observations in parentheses. b Statistically significant difference between untrained and trained.
AMP has come to be recognized as a second messenger mediating a variety of hormonal effects (7), it seems likely that the increased adenyl cyclase activity of muscle and the decreased phosphodiesterase activity of liver might be significant adaptations to endurance training. The decreased adenyl cyclase activity in heart is at variance with the conclusions drawn for muscle and liver. However, this variance may be in TABLE THE
EFFECT
OF TRAINING IN MUSCLE,
2
ON PHOSPHODIESTERASE HEART, AND LIVER
ACTIVITY
Phosphodiesterase activity (nmoles/min/mg of protein) Tissue
Untrained
Muscle Heart Liver
0.83 2 0.07 (12) 1.46 f 0.22 (6) 4.44 f 0.52 (12)
Trained 0.82 k 0.07 (12) 1.17 + 0.18 (6) 3.12 k 0.25 (12)* (P < 0.05)
’ Values are means 2 SEM with the number of observations in parentheses. b Statistically significant difference between trained and untrained.
EFFECT
OF TRAINING
ON
ADENYL
141
CYCLASE
keeping with the differences in function between skeletal muscle and heart. It is well known (12) that athletes with considerable endurance usually have a slow resting heart rate. In addition, endurance training enables an athlete to achieve a certain cardiac output during exercise with a slower heart rate and a larger stroke volume (12). These observations, coupled with the fact that cyclic AMP mediates the positive chromotropic and inotropic effects of the catecholamines (7) may explain why adenyl cyclase is decreased in heart while it is increased in skeletal muscle. The changes in muscle and heart adenyl cyclase do not appear to be due to an increased level of the enzyme since the fluoride-stimulated activity was unchanged. Thus, the changes in basal adenyl cyclase activity of heart and muscle must be caused by either an alteration in the control properties of the enzyme or the presence of an endogenous effector (inhibitor or activator). Epinephrine can probably be ruled out as an endogenous activator of muscle basal adenyl cyclase activity since the stimulation upon epinephrine addition was approximately the same in trained and untrained rats. Yakovlev (13) has reported that epinephrine-stimulated adenyl cyclase activity was increased in muscle, liver, and adipose tissue, but only muscle showed an increase in basal activity. In agreement with our results, he found that fluoride-stimulated adenyl cyclase activity was not changed by training in any of the tissues. Although we did not find liver epinephrine-stimulated adenyl cyclase to be elevated by training, the results of Yakovlev (13) do confirm our findings in muscle. In conclusion, the changes in adenyl cyclase activity in muscle and heart and the alteration of phosphodiesterase in liver indicate that the capacity to respond to hormones may be altered by training. SUMMARY Adaptive changes in the activities of adenyl cyclase and phosphodiesterase in response to exercise training were studied in muscle, heart, and liver. Basal adenyl cyclase activities were increased in muscle and decreased in heart as a result of training. Epinephrine-stimulated adenyl cyclase activity of heart was depressed by training. Fluoride-stimulated adenyl cyclase activity was not altered by training in any of the tissues studied. Phosphodiesterase activity was depressed in liver of trained rats but unaltered in muscle and heart. These changes in adenyl cyclase and phosphodiesterase activities suggest that the capacity to respond to hormones may be altered by training. ACKNOWLEDGMENT This research Association.
was supported
by Grant
No.
1973-74-A-3
from
the North
Carolina
Heart
142
DOHM, PENNINGTON
AND BARAKAT
REFERENCES 1. Askew, E. W., Dohm, G. L., Huston, R. L.. Sneed, T. W., and Dowdy, R. P.. Pro
![[Effects of bufetolol on rat heart and liver adenyl cyclase activities].](/assets/img/hold.png)
