Exercise-induced increases in myocardial adenosine 3′,5′-cyclic monophosphate and phosphodiesterase activity

114

Biochimica et Biophysica Acta, 672 (1981) 114--122

© Elsevier/North-Holland Biomedical Press

BBA 29480 EXERCISE-INDUCED INCREASES IN MYOCARDIAL ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE AND PHOSPHODIESTERASE ACTIVITY

WARREN K. PALMER, THERESE A. STUDNEY and SYLVIA DOUKAS College of Health and Physical Education, University of Illinois, Chicago, IL 60680 (U.S.A.)

(Received May 22nd, 1980) (Revised manuscript received September 16th, 1980) Key words: Exercise; cyclic AMP; Phosphodiesterase; Circadian Rhythm; (Rat heart)

Summary Numerous cellular biochemical events caused by hormones are mediated through cyclic AMP. Although many changes occur in the cell during exercise that could be attributed to this nucleotide, little evidence is available implicating it as an important regulator of exercise metabolism. In this investigation it was found that a 60 min bout of treadmill exercise caused a 2.4-fold increase in myocardial cyclic AMP immediately following the work. Rather than the immediate nucleotide hydrolysis that was expected, it was found that the elevated cyclic AMP level remained for approx. 24 h before returning to control levels. Cardiac glycogen fell to 30% of control after work but supercompensated 60% above control within 1 h following exercise. Therefore, cardiac cyclic AMP was elevated at a time when glycogen was being synthesized. Study of the temporal relationship between the exercise-induced increase in cyclic AMP and cyclic nucleotide phosphodiesterase indicated that the work caused an increase in the hearts' capacity to hydrolyze cyclic AMP. Measurement of heart phosphodiesterase at substrate concentrations of 1.0 and 100 pM produced significant increases in enzyme activity immediately following exercise which remained elevated for 48 h and was back to control activity 96 h following work. These data present a potentially fascinating model for the study of the dissociation between cyclic AMP, glycogenesis and elevations in phosphodiesterase activity in the heart.

Introduction Adenosine 3',5'-cyclic monophosphate (cyclic AMP) mediates a number of intracellular events caused by hormones that do n o t enter the cell. Glycogenol-

115 ysis [1], lipolysis [2] and protein synthesis [3] are events stimulated by this nucleotide. During exercise these biochemical events occur at an elevated rate [4--6]. Circulating plasma hormone concentrations of catecholamines [7], glucagon [8] and glucocorticoids [9], all of which promote an increase in tissue cyclic AMP, are elevated in response to exercise. However, in only one paper [10] has an exercise-induced increase in tissue cyclic AMP been reported. The rapid hydrolysis of the cyclic nucleotide provides technical difficulties that require rapid tissue fixation for the accurate approximation of tissue content. Using intravenous administration of anesthetic, Winder and associates [10] reported that exercise increased hepatic cyclic AMP content only when tissue glycogen reached a critically low level. In this study it was our purpose to determine the influence of an acute bout of exercise upon the concentration of cyclic AMP in the heart. In addition, an a t t e m p t was made to determine if a temporal relationship exists between myocardial cyclic AMP content, heart glycogen c o n t e n t and the activity of the cyclic nucleotide phosphodiesterase(s). Materials and Methods A nima l care and exercise program. Male rats of the Wistar strain obtained from Carworth Farms (Wilmington, MA), weighing between 200 and 300 g, were used t h r o u g h o u t these experiments. All animals were housed in individual cages and received water and Purina rat chow ad libitum for at least 2 weeks prior to killing. This was to let the animals become accustomed to the 12 h (0700 to 1900 h light) light-dark cycle. Animal quarters were maintained at 25 ± 2°C. In the week prior to use, all animals were run for 5 consecutive days on a motor-driven rodent treadmill (Quinton model 42-15) to familiarize the rats with treadmill running. Each practice run was 10 min duration with the b o u t on the fifth day being at a speed of 1.0 mile/h up an 8% gradient. Following a 60 h rest period with no treadmill running, animals were weighed and then put on the treadmill for a 60 min continuous exercise bout. Animals ran for 10 min at 16 m/min, 10 min at 21 m/min, and the remaining 40 min at 26.8 m/min. The entire run was up an 8% incline. After 60-min of running, animals were removed from the treadmill and either killed immediately or returned to their cages, where they had unrestricted access to food and water until time of death. Control rats participated in the indoctrination run-program but n o t the 60 min run. Analytical procedures. Animals were killed by decapitation. Hearts were removed immediately and quick-frozen with Wollenberger tongs prechilled in liquid N2. Tissue was stored at --80°C until analyzed for heart glycogen and cyclic AMP c o n t e n t and the activities of cyclic AMP phosphodiesterase. Comparison of fresh and frozen tissue showed no alteration in these parameters with storage. Preparation of cardiac samples for the determination of cyclic AMP content was carried out on solid CO2. Frozen tissue (80--100 mg) was weighed and immediately homogenized in 10% (w/v) cold 0.1 M HC1 using a Dual groundglass tissue grinder. Nucleotide extraction was carried out by using the method of Weller et al. [11 ]. Extracted samples were concentrated by lyophilization. The resultant material was resuspended in an appropriate volume of 0.12 M

116 sodium acetate (pH 4.5). Myocardial cyclic AMP was assayed using the protein kinase binding assay of Brostrom and Kon [12] using hydroxyapatite to bind the nucleotide-binding protein complex. Samples were counted in aqueous counting scintillant from Amersham Corp. Cyclic AMP recovery using this m e t h o d was greater than 85%. To measure cardiac cyclic AMP phosphodiesterase activities, heart tissue was homogenized in 30 vol. of 50 mM Tris-HC1 (pH 7.4), 5 mM MgCl:. The homogenate was centrifuged at 16 000 X g for 15 min. The supernatant was frozen at --80°C with no loss of activity. High and low K m phosphodiesterase activities were measured by using the m e t h o d of Huang and Kemp [ 13]. Assays of the hypothetical low K m activity were performed at a final cyclic AMP concentration of 1 #M (349 Ci/mol), while the high K m activity was assayed at a final concentration of 100 pM (15.43 Ci/mol). Enzyme incubation was performed for 10 min using between 150 and 200 pg of extract protein. This length of time and quantity of protein were within the linear range of the assay. This applies for both the low and high K m measurements whether the hearts were from exercised or control rats. The 5'-AMP formed was converted to adenosine by adding 100 pg snake venom (Crotalus adamanteus) in 50 pl of 0.12 M EDTA. A carrier of 100 pl of 5 mM adenosine was added to the assay mixture just prior to nucleotide separation on a 4.5 X 0.5 cm column of DEAESephadex. The [3H]adenosine was eluted into scintillation vials and counted. All radioactivity determinations were performed using a Searle Mark III liquid scintillation spectrometer. Appropriate blanks were subtracted from enzymecontaining assays. The a m o u n t of glycogen in the tissue was determined by using the phenol technique of Lo et al. [14]. Recovery of glycogen using this technique was found to be greater than 90% in all experiments. All analyses for glycogen, cyclic AMP and phosphodiesterases were made in duplicate on two separate occasions. Extract protein was determined using the Bio-Rad protein assay kit using bovine serum albumin as standard. Statistical methods. Results are expressed as means ±S.E. The number of animals per group is designated in the legends to the figures and tables. Mean concentrations or activities for various times following exercise were compared using one-way analysis of variance. When an F ratio of P < 0.05 was calculated, a Tukey post-hoc test was performed. Materials. Cyclic [3H]AMP (43 Ci/pmol) was purchased from New England Nuclear Corp. The labelled nucleotide was purified by thin-layer chromatography according to the m e t h o d of Jungas [15] for use in the phosphodiesterase assay to remove contaminants that cause high enzyme blanks. Hydroxypatite was purchased from Bio-Rad Laboratories (Richmond, CA). DEAE A-25 Sephadex was obtained from Pharmacia Fine Chemicals (Piscataway, NJ). Cyclic AMP, cyclic AMP binding protein (Type II from bovine heart) and snake venom were obtained from Sigma Chemical Co. Results Immediately following a submaximal bout of treadmill exercise, the a m o u n t of cyclic AMP measured in the heart is almost 3-fold higher than control levels

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(Fig. 1). When the rate of loss of cardiac cyclic AMP was studied, it was found that the nucleotide remained elevated for more than 24 h following muscle work. This elevation was a result of the work and n o t circadian rhythmicity. No circadian rhythm in heart cyclic AMP was evident when measured at 4-h intervals over a 24 h period {Table I). In Fig. 2, the influence of a b o u t of exercise upon cardiac glycogen can be seen. Immediately following the treadmill run, heart glycogen c o n t e n t was less than 35% of control levels. However, 1 h of recovery was associated with an approx. 65% 'supercompensation'. The significant elevation above control of heart glycogen lasted for at least 4 h following exercise, but returned to control concentration within 8 h of recovery. ..c

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To determine if a temporal relationship existed between the level of myocardial cyclic AMP and the activity of the enzyme(s) that hydrolyze cyclic AMP, phosphodiesterase activity was measured in the 1 6 0 0 0 × g supernatant of hearts at two substrate concentrations. In Fig. 3A and B it is quite apparent that the bout of exercise has caused an elevation in the activity of the enzyme(s) measured at both substrate concentrations. When activity was measured at cyclic AMP concentration of 1.0 #M there was no significant increase in the rate of cyclic nucleotide catabolism until 30 min following exercise. However, when phosphodiesterase activity was measured at a cyclic AMP concentration of 100 #M, activity was significantly elevated above control immediately following exercise and remained elevated for at least 30 h following work. Control activities denoted by the solid line are 0 8 0 0 h activities. It was found that a myocardial circadian rhythm did exist in phosphodiesterase activ-

119 TABLE I CIRCADIAN ACTIVITIES

RHYTHM

IN

RAT

HEART

CYCLIC

AMP

CONTENT

AND

PHOSPHODIESTERASE

A l l v a l u e s are m e a n s -+ S . E . f o r h e a r t s f r o m f i v e r a t s p e r t i m e . H i g h K i n , a c t i v i t y m e a s u r e d a t a s u b s t r a t e c o n c e n t r a t i o n o f 1 0 0 #aM. L o w Kin, a c t i v i t y m e a s u r e d a t a s u b s t r a t e c o n c e n t r a t i o n o f 1 p M . Time of Death 0800 h

1200 h

1600 h

2000 h

2400 h

0400 h

Cyclic AMP (nmol/g)

0.281 +- 0 . 0 2 2

0.262 ± 0.021

0.273 + 0.024

0.277 + 0.028

0.244 +- 0 . 0 1 7

0.233 ± 0.025

High K m phosphodiesterase activity (pmol/mg per rain)

982 ± 94

1109 + 105

1124 -+ 1 3 6

1314 * -+ 7 6

1247 -+ 8 6

1254 ± 127

Low K m phosphodiesterase activity (pmol/mg per min)

69 +- 3 . 7

71 +- 4 . 4

76 -+ 7 . 0

84 * +- 1 . 5

81 ± 4.5

77 ± 6.3

* Significantly higher than 0800 h value; P < 0.05.

ities (Table I), regardless of whether measurements were made at cyclic AMP concentration of 1.0 or 100 #M. With a 12 h light-dark cycle (0700--1900 h light), peak phosphodiesterase activity occurred at 2000 h while the nadir was at 0800 h. Exercise caused phosphodiesterase activity to be significantly higher than control heart activities at all times of the day except 0400 h which was equivalent to 16 h following work. To determine if the increased activity measured in hearts post-exercise was caused by an excess of an activator resulting from exercise, a series of mixing experiments was performed. It can be seen in Table II that when equal amounts of extract from control and exercised (time following exercise indicated) hearts were mixed prior to analysis, the predicted activities and actual activities measured were very close. Discussion

Numerous hormones, of which the actions are mediated through cyclic AMP, are increased in the plasma as a result of exercise. An estimate of the a m o u n t T A B L E II EXTRACT

MIXING EXPERIMENTS

A l l v a l u e s are m e a n s + S . E . f o r h e a r t s f r o m f o u r c o n t r o l s a n d 4 r u n n e r s . R e s u l t s are e x p r e s s e d as p m o l / 1 5 r a i n . O b t a i n e d r e s u l t s ; a c t i v i t i e s a s s a y e d a t 1 #aM c y c l i c A M P i n m i x e d h e a r t extracts. Equal volumes of heart extract from control rats and designated exercised rats were mixed immediately prior to enzyme analysis. Predicted values were determined from heart extract activities obtained prior to mixing.

Control Control Control Control

+ + + +

0 2 4 8

h h h h

runner runner runner runner

Predicted

Obtained

157 143 153 139

149 143 145 134

+ + ++

5.3 1.3 3.2 8.3

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120 of the nucleotide present in tissue following an experimental treatment of the whole animal is difficult because cyclic AMP is rapidly hydrolyzed by the enzyme, cyclic nucleotide phosphodiesterase. Brooker [16] has demonstrated significant changes in cyclic AMP during the cardiac cycle. Dobson et al. [17] have found that with hormonal stimulation of the heart, cyclic AMP levels return to basal activity within 10 s. Keely and co-workers [18] report that 2 min following epinephrine stimulation of isolated hearts, cyclic AMP returns to control levels. In the light of these previous reports, it is quite surprising that a single 60 min submaximal bout of treadmill running should be accompanied by a prolonged elevation in myocardial cyclic AMP. At the same time that this nucleotide was elevated more than 2-fold, glycogen was being synthesized. The cardiac pattern of glycogen supercompensation reported in this study is similar to that reported by Segal et al. [19]. Despite the overwhelming a m o u n t of evidence linking cyclic AMP to glycogenolysis, these data suggests a specific dissociation between the two biochemical events. A possible explanation for this phenomenon may be related to the findings of Keely et al. [18] who found that ~-adrenergic receptor stimulation of perfused rat heart with constant amounts of either epinephrine or glucagon caused prolonged elevations in cyclic AMP and activated cyclic AMPdependent protein kinase with only a transient increase in phosphorylase activity; an activation that decreased to control levels within 15 min. This decline occurred even though both cyclic AMP and the protein kinase activity ratios remained elevated. The role of the prolonged elevation of cyclic nucleotide can only be speculated upon here. Although heart contractile function and glycogen metabolism are both affected by cyclic AMP, these events have long since returned to control levels well within 24 h post-exercise. Two biochemical events that may still be functioning 24 h after physical work are the synthesis of heart protein and/or the metabolism of cardiac lipid. It has been reported that significant cardiac h y p e r t r o p h y in rats results from as little as 2 days of strenuous physical exercise and produces a 30% increase in heart size and total protein within 14 days of work [6]. Cyclic AMP has been implicated indirectly in the stimulation of protein synthesis through the activation of cyclic AMP-dependent protein kinase. This enzyme phosphorylates both ribosomal [20] and nuclear [21] protein in vitro. In addition, Byus and co-workers [22] have reported that growth h o r m o n e injection activated protein kinase in liver and adrenal gland. It is well documented that growth hormone titers increase in response to exercise [23]. Oscai [24] has shown that following an acute bout of exercise, lipid stores in the myocardium do n o t return to control levels for 48 h. These data may indicate a prolonged rate of cyclic nucleotide-stimulated rate of lipid hydrolysis. The temporal relationship between the exercise-induced nucleotide in the heart and the enzyme(s) responsible for its hydrolysis was investigated. However, rather than a reduction in activity, it was found that the single bout of treadmill running was accompanied by significant increases in the activity of cyclic AMP phosphodiesterase. The increase in activity was evident whether measurements were made at 1.0 or 100 pM substrate concentration. The enzyme activity had returned to control within 96 h following work. Although a significant circadian r h y t h m was evident in the enzyme activity of control

121

animals, the r h y t h m i c i t y did n o t seem to be a c o m p o n e n t of the increased enzyme activity measured post-exercise. I n fact, the activity seen at 0400 h in control hearts was n o t significantly different from that seen at the same time (16 h) post-exercise while activities measured at all other times of day were significantly greater in the hearts of exercised animals. Two groups from the National Institutes of Health have reported an induction of phosphodiesterase activity in cultured fibroblasts exposed to dibutyryl cyclic AMP [25,26]. Recently, Ball and co-workers [27] reported that incubation of myoblasts with 1.0 pM dibutyryl cyclic AMP caused an approx. 40% increase in cyclic AMP phosphodiesterase activity after 1 h and a 3-fold increase in enzyme activity with 16 h of incubation with the nucleotide. The increased enzyme activity might well be explained by a nucleotideinduced increase in enzyme protein at some of the longer time points following the prolonged elevation of cyclic AMP. The 75% elevation in activity seen immediately following exercise may be attributed to either enzyme induction or activation. Because our predicted results equalled our obtained results in the mixing experiments shown in Table II, it is assumed that there is no excess activator or inhibitor present in either control or exercised myocardium. These data indicate that exercise induces an increase in myocardial cyclic AMP that does n o t correlate with post-stress changes in glycogen content. The elevated cyclic nucleotide may be responsible for the increased phosphodiesterase activity seen following work. These data suggest a cellular compartmentalization causing the dissociation of these events. The addition of varying amounts of calcium or EGTA to heart extracts was carried out to ascertain if phosphodiesterase activation could be mediated through calmodulin (data not shown). Calcium and EGTA had analagous effects on hearts from control and exercised rats at similar concentrations. These data suggest that the increased phosphodiesterase activity was a result of newly synthesized enzyme protein rather than its activation. It can be concluded from the data presented in this report that a dissociation exists between cyclic AMP, glycogen synthesis and the enzyme(s) cyclic AMP phosphodiesterase. Because of the prolonged elevation in cyclic AMP, the exercised rat heart may provide a potential model for the study of this dissociation. Acknowledgements The authors wish to t h a n k Dr. Lawrence B. Oscai for his reading of this manuscript. Dr. James S. Horgan is acknowledged for his assistance with statistical evaluation of the data. In addition, the assistance of Ms. Mary Ann Johnson in manuscript preparation is greatly appreciated. This project was supported in part by a research grant PHS-RR-07158-04. References 1 Hess, M.E., H o t t e n s t e i n , D., S h a n f e l d , J. a n d H a u g a a r d , N. ( 1 9 6 3 ) J. Pharmaco.1. Exp. Ther. 1 4 1 , 274--279 2 V a u g h a n , M. a n d S t e i n b e r g , D. ( 1 9 6 3 ) J. L i p i d Res. 4, 1 9 3 - - 1 9 9 3 Wicks, W.K. ( 1 9 7 4 ) Adv. Cyclic Nucl. Res. 4, 3 3 5 - - 4 3 8 4 H e r m a n s e n , L., H u l t m a n , E. a n d Saltin, B. ( 1 9 6 7 ) A c t a Physiol. Scand. 71, 1 2 9 - - 1 3 9

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