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Cyclic AMP phosphodiesterase activity in fetal and adult muscle of the rhesus monkey
DEVELOPMENTAL
BIOLOGY
48,
382-391
(19’76)
Cyclic AMP Phosphodiesterase Activity in Fetal and Adult Muscle the Rhesus Monkey R. M. Division
of Perinatal Department
BOCEK
C. H.
AND
Physiology, Oregon Regional Primate of Biochemistry, University of Oregon Accepted
September
of
BEATTY
Research Center, Beaverton, Oregon 97005, Medical School, Portland, Oregon 97201
and
25,1975
Total cyclic AMP phosphodiesterase activity of voluntary skeletal muscle of the rhesus monkey was highest in the loo-day fetal series, decreased near term, and was lowest in the adult series. Kinetic data indicated the existence of two cyclic AMP phosphodiesterase enzymes in both the fetal and adult muscle. The apparent K, values for the high-affinity phosphodiesterase were similar in the loo-day fetal and adult skeletal muscle, whereas those for the lowaffinity enzyme were twofold higher in the fetal series. The V,,, of the highl(, enzyme was tenfold higher in the fetal than in the adult series and the low K, V,,, was fourfold higher in the fetal series. Both caffeine and theophylline were competitive inhibitors of the low K, phosphodiesterase activity and noncompetitive inhibitors of the high K, enzyme. No difference was observed in the sensitivity of the fetal and adult enzyme preparations to the methylxanthines or to Ro20-1724. INTRODUCTION
muscle just before birth than in adult muscle, increased rapidly during the first lo20 days after birth, and then decreased with aging to adult levels.On the other hand, the levels of cyclic AMP were higher in fetal rhesus muscle during the last half of gestation than in adult muscle (Bocek et al., 1975). In addition, the sensitivity of the adenylate cyclase enzyme to prostaglandin E, in vitro was highest in 78- to loo-day-old fetal muscle (47-61% of gestation), decreased near term, and was lowest in the adult series (Bocek et al., 1975). Because intracellular levels of cyclic AMP are regulated by degradative as well as synthetic enzymes, we wanted to ascertain whether the higher level of cyclic AMP found in the fetal muscle was due to a lower cyclic AMP phosphodiesterase activity. Changes in the total phosphodiesterase activity during growth had already been reported for rat skeletal muscle, and its developmental pattern appeared to differ from that of adenylate cyclase; fetal and postnatal levels did not vary until after Day 30 and were higher than in adult muscle (Hommes and Beere, 1971; Williams and Thompson, 1973). Multiple forms of cyclic AMP phosphodiesterase ac-
Evidence obtained in the past 5 years indicates that cyclic AMP plays a critical role in the growth, differentiation, and/or specialization of cells. Numerous studies with tissue cultures and bacterial preparations (Voorhees et al., 1974) have led to the formulation of the hypothesis that the cellular level of cyclic AMP is inversely related to cell proliferation (mitosis). Otten et al. (19721, Pardee et al. (19741, and Voorhees et al. (1974) have shown that cells in vitro that have been transformed or stimulated to grow with various agents generally contain lower internal cyclic AMP, and Zalin and Montague (1975) suggested a causal relationship between the increases in adenylate cyclase activity, the peak of intracellular cyclic AMP levels, and the onset of cell fusion in myoblasts. Changes in cyclic AMP levels and adenylate cyclase activities in uiuo associated with development have been reported in several rat tissues, including skeletal muscle (Novak et al., 1972; Hommes and Beere, 1971; Williams and Thompson, 1973) and heart (Novak et al., 1972). Both cyclic AMP levels and adenylate cyclase activities were generally lower in fetal rat 382 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
BOCEK
AND
BEATTY
Cyclic
tivity had been reported in several tissues of adult mammals (Thompson and Appleman, 19711, but no data on such forms in fetal muscle were available. Therefore, we undertook this study and have determined the total phosphodiesterase activity and the kinetics of a high and low K, cyclic AMP phosphodiesterase in muscle from rhesus fetuses and have compared the results with data from adult monkeys. METHODS Rhesus fetuses (Macczca mulatta) from 85 to 103 days gestational age (designated the loo-day fetal series), 155 days gestational age, and adult rhesus monkeys were used in these experiments. The average gestational age in our colony was 165 days. The mothers were anesthetized intramuscularly with 1.0 mg/kg of l-(l-phenylcyclohexyl) piperidine hydrochloride and the fetuses were removed and rapidly exsanguinated. Adult monkeys were anesthesized with Ketaject (dl-2-{O-chlorophenyl}2-{methyl amino} cyclohexanone hydrochloride). Samples of voluntary muscles from the upper arms and thighs of the fetuses and from the sartorius muscle of the adult animals, and of diaphragm and cardiac muscle from both the fetal and adult monkeys were rapidly excised and stripped of fat and connective tissue. When the skeletal and diaphragm muscles were immediately homogenized (unfrozen at 0 to 4°C) or frozen and homogenized later, cyclic AMP phosphodiesterase activity in these samples was similar. Therefore, we chose the more convenient method and froze the samples in a Wollenberger clamp cooled in liquid nitrogen, wrapped them in aluminum foil, and stored them in a liquid nitrogen refrigerator for future analysis. Homogenates (10% w/v) were prepared in 0.25 M sucrose plus 1 mIt4 EDTA. The frozen tissues were weighed and chopped in a conical homogenizing tube containing frozen sucrose-EDTA medium. The tissues were then thawed in a slurry of the medium and homogenized with a glass pes-
AMP-PDE
in Developing
Muscle
383
tle (lo-12 passes) (0-4°C). The homogenate was frozen, thawed, and centrifuged 1OOOg for 15 min at O”C, and the supernatant was assayed. Cyclic AMP phosphodiesterase activity was determined by a modification of the two-stage method of Loten and Sneyd (1970). For total phosphodiesterase activity, the reaction mixture contained 0.02 &i [3H]cyclic AMP (38.15 Ci/mmole), 1 x lop3 M cyclic AMP 40 mM Tris buffer at pH 8.0, 2 mM MgCl, plus supernatant in a final volume of 0.2 ml. Blank values were determined either with boiled supernatant or sucrose-EDTA medium. The activity in the blanks was similar with both methods. The mixture was incubated for 15 min at 30°C with shaking and the assay was terminated by immersing the tubes in a dry ice-acetone freezing mixture and then heating them at 100°C for 45 sec. After the addition of 0.05 ml of a solution of snake venom (ophiaphagus hannah, 1 mg/l ml water), the assay tubes were reincubated for 15 min and then held at 4°C. For kinetic studies, cyclic AMP concentrations were varied from 0.9 x loo-” to 1 x 10m4M and the [3H]cyclic AMP from 0.01 to 0.02 &i/assay tube. The reaction mixture was applied to 2.5 x 0.5 cm columns of Bio-Rad Agl-X2 (200400 mesh) and the labeled adenosine eluted with 40 mIt4 Tris buffer, pH 7.4. An aliquot of the eluate was added to Triton X-114 xylene-omnifluor and counted in a liquid scintillation counter. The amount of hydrolyzed cyclic AMP was calculated from the specific activity of the substrate cyclic AMP and expressed in terms of nanomoles of cyclic AMP hydrolyzed per minute per milligram of protein. At cyclic AMP concentrations of 5 a, the reaction rate was linear for 20 min with approximately 2 mg protein/ml of incubation medium. The rate remained constant up to 21% substrate hydrolysis. Kinetic studies were routinely run for 15 to 20 min and the enzyme concentration used was such that less than 25% of the substrate cyclic AMP was hydrolyzed. K, values were calculated
384
DEVELOPMENTAL
BIOLOGY
by the method of least squares. Protein content of the supernatant was determined by the method of Lowry et al. (1951) standardized with bovine serum albumin. In the assay of cyclic AMP phosphodiesterase activity in tissue extracts the amount of labeled adenosine recovered may not be indicative of the total amount of labeled cyclic AMP hydrolyzed since enzymes in the unpurified extract may catalyze the breakdown of product 5’-AMP to such compounds as adenine, hypoxanthine, and inosine as well as adenosine. However, Rutten et al. (1973) found that elution of the Bio-Rad column with 0.1 M NaHC03 recovered all four products of 5’AMP metabolism. When we ran these four compounds through individual columns and eluted them with 40 mM Tris buffer at pH 7.4, our recoveries on the basis of optical density measurements at 260 nm were essentially similar to those of Rutten et al. (1973) with 0.1 M NaHCO,. Recovery of added adenosine, the major compound, was 98 to 100%. [3H]cyclic AMP was purified on a BioRad AGl-X2 column (2 ml of resin volume in a Pasteur pipette). The [3Hlcyclic AMP was applied and the column washed with 10 to 15 ml of water. The purified cyclic AMP was eluted with 4 to 5 ml of 0.05 N HCl, diluted with ethanol to make a final concentration of 50% ethanol, and stored at -20°C. Radioactivity in the blanks was usually less than 1% of the total. RESULTS
The total cyclic AMP phosphodiesterase activity of voluntary skeletal muscle was sixfold higher in the loo-day fetal than in the adult series, decreased near term, and was lowest in the adult series (Table 1). The data on diaphragm and heart muscle followed the same pattern of enzyme activity during development as skeletal muscle but were too few for statistical evaluation. In contrast to intermittently active voluntary skeletal muscle, diaphragm is a specialized muscle adapted to constant activ-
VOLUME
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1976 TABLE
1
CYCLIC AMP PHOSPHODIESTERASE ACTIVITY 1OOOg SUPERNATANT OF THE HOMOGENATES FETAL
AND
ADULT
MUSCLE MONKEY”
FROM
THE
IN THE OF
RHESUS
Series
W-day
fetal bmoles
Adult
155&y
fetal cyclic AMP hydrolyzed/min/ 100 mg protein)
-
Skeletal muscle Diaphragm
272 2 25
6) 266
(2) Heart -
790 (1)
172 + 16*
(6) 193 (1) 499 (1)
46 k 4’ (10) 49 k 5 (4) 360 T 18 (4)
’ Values are means 2 SE or averages of duplicate assays. Numbers in parentheses are the number of animals in each series. Cyclic AMP concentration 1 x lo-SM.
* 155-day vs loo-day, P < 0.01. ’ Adult vs 155-day series, P < 0.001.
ity and differing in several metabolic parameters from voluntary skeletal muscle (Peterson et al., 1963). However, within each age group, total phosphodiesterase activities were similar in diaphragm and skeletal muscle but were three to eight times lower than in heart muscle. Kinetic data on rhesus skeletal muscle indicated two cyclic AMP phosphodiesterase activities in both the fetal and adult series; typical Lineweaver-Burk plots for skeletal muscle are shown in Figs. 1 and 2. The apparent K, values for the high affnity (low K,) phosphodiesterase activities were similar in the loo-day fetal and adult voluntary skeletal muscle (Table 21, whereas those for the low affinity (high K,) enzyme were higher in the loo-day fetal than in the adult series. V max values for both the low and high K, enzymes decrease with development and were lowest in adult muscle. The V,,, of the high K, enzyme was about sevenfold higher in the loo-day fetal than in the adult series and the low K, V,,, was fourfold higher in the fetal muscle. V,,, values for diaphragm muscle from fetal and adult animals appear to be similar to those of
BOCEK
AND BEATTY
Cyclic
AMP-PDE
in Developing
385
Muscle
Adult Muscle
6Or
FIG. 1. Kinetic plot of cyclic AMP phosphodiesterase activity in a muscle extract of a loo-day rhesus fetus. The extract represents the 1OOOg supernatant of the homogenate prepared as described in the Methods section. TABLE KINETIC
-
DATA
FOR PHOSPHODIESTERASE
2 ACTIVITY
Low -N
-
AND ADULT
affinity
Km (CLM)
MUSCLE”
High V maxb Voluntary
loo-day Adult
fetal
3 4
46 k 4 24 + 3
-N
affinity
Km (40
Skeletal
380 -c 10 58 -+ 13” (’
-.--
V,,Xh
Muscle 5 7
5.8 + 0.6 5.8 2 0.7
1 2
6.0 5.4, 6.3
81 2 12 21 t 6’
Diaphragm
-
-
IN FETAL
-____ Series
-
FIG. 2. Kinetic plot ofcyclic AMP phosphodresterase activity in a muscle extract of an adult rhesus monkey. The extract represents the 1OOOg supernatant of the homogenate prepared as described in the Methods section.
loo-day Adult
fetal
1 2
48 28, 45
340 30, 40
a Values are means 2 SE, duplicate analyses. N = the number of animals ase activity determined as stated in Methods. Concentration of cyclic AMP * Nanomoles of cyclic AMP hydrolyzed per minute per 100 mg protein. “P for difference, adult vs loo-day fetal series,
voluntary skeletal muscle (Table 2). As early as 100 days gestation, both caffeine and theophylline were competitive inhibitors of the low K, phosphodiesterase activity (Fig. 31, and noncompetitive inhibitors of the high K, enzyme assayed at concentrations of 50 to 200 fl cyclic AMP (data not shown). Methylxanthines were reported to be both competitive and noncompetitive inhibitors of the cyclic nucleotide phosphodiesterases in various adult tissues (Appleman et al., 1973). Huang and Kemp (1971) found that the methylxan-
75 12. 16 .in each series. Phosphodiestervaried from 1 to 100 fl.
thines were competitive inhibitors of the low K, cyclic nucleotide phosphodiesterase in adult rabbit muscle. The K, values for the inhibition of both the low and high phosphodiesterases in rhesus muscle by the methylxanthines were similar in the loo-day fetal and adult series. For the low K, enzyme, K, values ranged from 1.3-1.8 mM for caffeine (Fig. 4) and 0.8-1.4 mM for theophylline (Fig. 5). Ki values for 155day fetal muscle were within these ranges. Ki values for the high K, enzyme ranged from 2.3-4.0 mM and 1.2-2.0 mM for caf-
386
DEVELOPMENTAL
5
IOO-Day
BIOLOGY
VOLUME
48, 1976
Fetus
Adult
, 2SmM
1
I/[cAMP]-pM
Theoph.
I/[cAMP]-UM
FIG. 3. Lineweaver-Burk double reciprocal plot showing competitive inhibition of the low K, cyclic AMP phosphodiesterase by theophylline and caffeine of skeletal muscle from loo-day fetal and adult rhesus monkeys. Assay conditions were as described in Fig. 1 and in the Methods section. Concentrations of substrate cyclic AMP were varied from 0.75 - 5 fl. Lines of best fit were obtained by the method of least squares.
IOO-Day
Fetus
Ki -1.8
,
Adult W5PM CAMP
/ .
I.OUM CAMP
2.9uM 5.8PM
I
3.0 [Caffeine]-mM
-2.0
-1.0
1.0 2.0 [Caffeine]-mM
3.0
FIG. 4. Kinetic data for determination of the K, value for caffeine in homogenates of loo-day fetal and adult rhesus muscle. Assays were incubated for 20 min; other conditions were as described in the Methods section. V = nanomoles of cyclic AMP hydrolyzed per minute per milligram of protein.
feine and theophylline respectively. The relative effect of inhibitors on the activity of phosphodiesterase at 5 fl cyclic AMP concentrations is shown in Table 3. The sensitivity of the enzyme preparations to the methylxanthines did not differ in fetal and adult muscle. The inhibitory effect of theophylline appeared to be slightly greater than that of caffeine; this agrees
with the data of Huang and Kemp (1971). Several other types of compounds inhibit cyclic nucleotide phosphodiesterase (Appleman et al., 1973), including the analogues of 4-(3,4-dimethoxybenzyl)-2-imidazolidinone (Sheppard and Wiggen, 1971). The 3butoxy-4-methoxy derivative, Ro20-1724, a more potent inhibitor of erythrocyte phosphodiesterase than the methylxanthines,
BOCEK
AND
IOO-Day
Cyclic
BEATTY
AMP-PDE
in Developing
387
Muscle
Adult
Fetus
80-
Q75vM Ki-
1.4mM
L -20
-1.0
I.0
[Theophyl
21)
-2.0
30
-1.0
I.0 20 [Theophyllinel-mM
line] - mM
FIG. 5. Kinetic data for determination muscle. Conditions were similar to those milligram of protein.
of the Ki value for theophylline in loo-day fetal in Fig. 4. V = nanomoles of cyclic AMP hydrolized
was also a more effective inhibitor of skeletal muscle phosphodiesterase at 0.4 mM concentrations than the methylxanthines at 1 mM levels. Like the methylxanthines, Ro20-1724 inhibited the enzyme preparations from fetal and adult muscle to a similar degree. Recent studies have shown that a specific protein activator of cyclic AMP phosphodiesterase is present in several mammalian tissues other than skeletal muscle (Smoake et al., 1974) whose action is completely dependent on the formation of a Ca’+-activator complex (Teo and Wang, 1973). The low V,,, values for adult muscle may have been due to a deficiency in calcium and/or in activator protein. To test this possibility, we combined samples of homogenate from fetal and adult muscle and assayed them for total phosphodiesterase activity. When fetal and adult samples were combined in ratios of 1:l and 2:l no stimulation of the adult enzyme preparation was apparent, i.e., the activities were proportional to the activity in the original homogenates (Table 4). The addition of 100 pM Ca*+ either to the 1:l fetal-adult combination or to a preparation of adult muscle (substrate level of cyclic AMP 5 $50 did not affect the rate of substrate hydrolysis.
TABLE RELATIVE
AMP
EFFICIENCY
and adult rhesus per minute per
3
OF INHIBITORY
PHOSPHODIESTERASE SKELETAL
3.0
FROM MUSCLE”
OF CYCLIC VOLUNTARY
Inhibitors
Percentage
of Control
Value”
Fetal
Adult
loo-day
15day
-1 mM
caffeine 52 56 56 1 mM theophylline 49 49 48 0.4 mM R020-1724’ 27 32 29 0.7 mM R020-1724 17 13 a Assays were carried out for 20 min at a cyclic AMP concentration of 5 fl and other conditions as described in the Methods section. Values represent the average of duplicate assays, two or three animals in each series. b The uninhibited control values were assumed to be 100%. ’ 14(3 - butoxy - 4 - methoxybenzyl) - 2 - imidazolidinone]. DISCUSSION
Levels of cyclic AMP in tissues are determined by a balance between activities of adenylate cyclase forming and of phosphodiesterase degrading the nucleotide. In a previous paper (Bocek et al., 1975), we reported that cyclic AMP levels in rhesus fetal muscle were highest at 100 days, decreased near term, and were lowest in the adult. Adenylate cyclase activity as well
388
DEVELOPMENTAL
TABLE TOTAL
PHOSPHODIESTERASE
4 ACTIVITY
IN MIXTURES
OF SUPERNATANT FROM HOMOGENATES FETAL AND ADULT SKELETAL MUSCLE RHESUS
BIOLOGY
OF lOO-DAY OF THE
MONKEYS
Nanomoles lyzed/ruin/100 Obeerved values
of CAMP hydromg protein Calculated values
-
Control Fetal 384 Adult 50 Combined Fetal/adult ratio 1:l 214 217 Fetal/adult ratio 2:l 283 213 a 1OOOg supernatant preparations of homogenates were prepared as described in Methods. Fifty microliters of either the original supernatants or the indicated combinations of supernatants from fetal and adult muscle homogenates were assayed as previously described. Concentration of substrate cyclic AMP was 1 mA4, 1 x 105 dpm [3H]cAMP per assay. Protein concentrations of the enzyme preparations were 0.25 and 0.30 mglassay for fetal and adult muscle, respectively. Values are means of triplicate assays.
as its sensitivity to epinephrine (Bocek et al., 1973) and to prostaglandin Ez (Bocek et al., 1975) were also higher in fetal than in adult muscle. In this paper, we report that phosphodiesterase activity parallels both the levels of cyclic AMP and the activity of adenylate cyclase in developing rhesus muscle. That is, all three were highest in loo-day fetal muscle. There are several reasons why the level of cyclic AMP and the activity of the phosphodiesterase degrading it were both high. High levels of cyclic AMP induce phosphodiesterase activity in certain systems (Schwartz and Passonneau, 1974; Manganiello and Vaughn, 19721, and Schwartz and Passonneau (1974) suggested that this type of activation leads to long-term regulation of cyclic AMP levels, an effect that may be operative in developing fetal muscle. On the other hand, an endogenous factor has been isolated from rat adipocytes after their exposure to agents that raise the intracellular levels of cyclic AMP. This fac-
VOLUME
48,
1976
tor inhibits the activity of cyclic AMP and cyclic GMP phosphodiesterase in several tissues of the rat, including the diaphragm and voluntary skeletal muscle (Ho et al., 1975). Low levels of cyclic GMP can also activate the hydrolysis of cyclic AMP by the high K, enzyme of liver and fat cells (Appleman et al., 19731, and the ratio of cyclic AMP to cyclic GMP may be critical to the coordinated regulation of cell biochemistry (Voorhees et al., 1974). Levels of cyclic GMP in fetal tissues are not yet available. Cyclic AMP phosphodiesterase from various sources appears sensitive to several other endogenous factors capable of activating or inhibiting the enzyme (Appleman et al., 1973). Ca*+ is an essential component in the action of a protein activator of cyclic AMP phosphodiesterase from bovine heart (Tea and Wang, 1973). Activation enhanced the V,,, and decreased the K, for cyclic AMP. The concentration of activator protein in crude tissue preparations appears to be in excess of the amount of enzyme (Tea et al., 1973). Smoake et al. (1974) reported that in eight rat tissues other than muscle the ratio of activity of the protein activator to that of the phosphodie&erase varied not only during development but from tissue to tissue. The fact that we were unable to show any stimulation of phosphodiesterase from adult muscle with Ca2+ or by combination with a more active fetal muscle preparation suggests that these factors are not responsible for the lower phosphodiesterase activity in adult muscle. However, the relation between the actual levels of activator protein and phosphodiesterase in developing skeletal muscle needs to be determined. Hommes and Beere (1971) reported that in developing rat muscle adenylate cyclase activity was low before birth and that the activity of total phosphodiesterase did not parallel that of adenylate cyclase. The difference in the developmental pattern of the activities of the enzymes that govern the concentrations of cyclic AMP in the rat
BOCEK AND BEATTY
Cyclic
AMP-PDE
in Developing
Muscle
389
and rhesus monkey may be due to the found with kinetic analysis of crude exvariation in the stage of development of tracts of rabbit skeletal muscle (5 and 20 the muscle between the rat and rhesus @) (Huang and Kemp, 1971) and of purimonkey with gestational age. Our data fied enzyme preparations of rat muscle (2.2 support the hypothesis that ontogenetic and 57 fl) (Thompson and Appleman, changes in cyclic AMP levels are related to 1971). There are two phosphodiesterases in the developmental stages of the muscle fetal as well as in adult rhesus muscle; the fiber rather than to the age of the animal apparent K, values for the high affinity relative to birth (Beatty et al., 1967); Bo- enzyme are similar in the two series. We cek et al., 1975). have no explanation for the higher K, An inverse relation between intracelluvalue of the low-affinity phosphodiesterase lar levels of cyclic AMP and the rate of in the fetal muscle. The intracellular conDNA synthesis has been demonstrated in centration of cyclic AMP is low, probably cultures of several cell lines (Abel1 and below 1 fl (Steiner et al., 1972; Bocek et Monahan, 1973). Mandel and Pearson al., 19751, and unless this compound is (1975) reported that high levels of intracelhighly compartmentalized, the physiologilular cyclic AMP inhibited the differentiacal significance of the low affinity-high tion of myoblasts. Hypothetically, cyclic K, phosphodiesterase activity might be AMP levels in viuo should be low during limited. The actual difference in the K, the period when the rate of proliferation is values was less than twofold. The differhigh (presumptive or premyoblast stage) ence in enzyme activity between the two and high when proliferation is completed series would imply either that the enand the myoblasts differentiate and fuse to zymes are different or that the relative form myotubes. At birth, rat muscle is concentration of a factor which affects the relatively immature with a majority of the affinity of enzyme for its substrate is differfibers still in the myotube stage (Kamienent. The V,,, for both the low and high iecka and Ostenda, 1959; Dubowitz, affinity enzymes is higher in the fetal se1965a). In the rhesus fetus, the myotube ries. This agrees with previous data demstage occurs before the midpoint of gesta- onstrating that most of the metabolic pation; this observation is compatible with rameters we have measured are higher in the decreasing level of cyclic AMP from rhesus fetal than in adult muscle: oxygen 504 of term to maturity (Bocek et al., consumption, CO, production, glucose up1975). Rhesus fetal muscle shows a typical take, and conversion to lactate (Beatty et adult distribution of red and white fibers al., 1968)and palmitate uptake and converby 70% of gestation with only an occa- sion to lipid (Beatty and Bocek, 1970) were sional immature fiber. The developmental all higher in fetal than in adult muscle in stages of human fetal muscle relative to vitro. From the V,,, values, one can estithe percent of total gestation are similar to mate that the ratios of high to low K,, those of the rhesus monkey (Dubowitz, phosphodiesterase activities in the loo-day 1965a,b; Bocek et al., 1975). fetal and adult skeletal muscle are about According to kinetic evidence, there are 3.7 and 1.9, respectively. This suggests a at least two forms of cyclic AMP phosphodi- relatively greater amount of high-affinity e&erase in a variety of adult tissues, in- enzyme in the adult muscle which may be cluding skeletal muscle (Thompson and related to the fourfold lower levels of cyclic Appleman, 1971). Our data indicate that AMP found in adult but not in 78- to lOOthe 1OOOgsupernatant fraction of rhesus day fetal muscle (Bocek et al., 1975). Our muscle homogenate contains two cyclic data, therefore, agree with those of Huang AMP phosphodiesterase activities whose and Kemp (19711, who showed that a apparent K,n values are similar to those greater proportion of the phosphodiester-
390
DEVELOPMENTAL
BIOLOGY
ase activity of adult skeletal muscle is of the low K,-high affinity type. Determining enzyme levels in whole homogenate generates some uncertainties because of possible assay artifacts (Rutten et al., 1973). However, when Huang and Kemp (1971) compared the K, for the highaffinity phosphodiesterase in a crude muscle extract with that obtained with a purified enzyme, the K, values were similar (about 5 @f). Whether an isolated and semipurified enzyme behaves the same way in its natural milieu has always interested enzymologists (Woo, 1973). The kinetics of unpurified enzyme preparations in a more physiological milieu may more nearly approximate their in situ activity. The fact that the K, values for both the high- and low-affinity enzymes isolated and purified from rat muscle were similar to those obtained in our study lends credence to the values obtained by both methods. Publication No. 829 of the Oregon Regional Primate Research Center, supported in part, by Grant RR-00163 from the National Institutes of Health. This investigation was supported by Public Health Service Research Grants HD-06069 and HD-06425 from the National Institutes of Child Health and Human Development, by General Research Support Grant RR-05694 from the National Institutes of Health, and by the Muscular Dystrophy Associations of America, Inc. We thank Paul Herrington for his competent technical assistance. REFERENCES ABELL, C. W., and MONAHAN, T. M. (1973). The role of adenosine 3’S’-cyclic monophosphate in the regulation of mammalian cell division. J. Cell Biol. 59,549-558. APPLEMAN, M. M., THOMPSON, W. J., and RUSSELL, T. R. (1973). Cyclic nucleotide phosphodiesterases. In “Advances in Cyclic Nucleotide Research” (P. Greengard and G. A. Robison, eds.), Vol. 3, pp. 65-98. Raven Press, New York. BEATTY, C. H., BASINGER, G. M., and BOCEK, R. M. (1967). Differentiation of red and white fibers in muscle from fetal, neonatal and infant rhesus monkeys. J. Histochem. Cytochem. 15, 93-103. BEATTY, C. H., BASINGER, G. M., and BOCEK, R. M. (1968). Oxygen consumption and glycolysis in fetal, neonatal and infant muscle of the rhesus monkey. Pediatrics 42, 5-16.
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BEATTY, C. H., and BOCEK, R. M. (1970). Metabolism of palmitate by fetal, neonatal and adult muscle of the rhesus monkey. Amer. J. Physiol. 219, 13111316. BOCEK, R. M., YOUNG, M. K., and BEATTY, C. H. (1973). Effect of insulin and epinephrine on the carbohydrate metabolism and adenylate cyclase activity of rhesus fetal muscle. Ped. Res. 7, 787793. BOCEK, R. M., YOUNG, M. K., and BEATTY, C. H. (1975). Cyclic AMP in developing muscle of the rhesus monkey, effect of prostaglandin E,. Biol. Neonate, in press. DUBOWITZ, V. (1965a). Enzyme histochemistry of skeletal muscle. I. Developing animal muscle. J Neurol. Neurosurg. Psychiat. 28, 516 - 519. DUBOWITZ, V. (1965b). Enzyme histochemistry of skeletal muscle. II. Developing human muscle. J. Neurol. Neurosurg. Psychiat. 28, 519-521. Ho, R. J., RUSSELL, T. R., ASAKAWA, T., and HUCKS, M. W. (1975). Inhibition of cyclic nucleotide phosphodiesterase activity by an endogenous factor. J. Cyclic Nucleotide Res. 1, 81-88. HOMMES, F. A., and BEERE, A. (19711. The development of adenyl cyclase in rat liver, kidney, brain, and skeletal muscle. B&him. Biophys. Acta 237, 296-300. HUANG, Y-C., and KEMP, R. G. (1971). Properties of phosphodiesterase with high affinity for adenosine 3’,5’-cyclic phosphate. Biochemistry 10,22782283. KAMIENIECKA, Z., and OSTENDA, M. (1969). Histochemical investigations in maturation of rat skeletal muscle after whole-body gamma-irradiation at the fourth day of life. J. Neural. Sci. 9,347-359. LOTEN, E. G., and SNEYD, J. G. T. (1970). An effect of insulin on adipose-tissue adenosine 3’:5’-cyclic monophosphate phosphodiesterase. B&hem. J. 120, 187-193. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chern. 193,265275. MANDEL, J. L., and PEARSON, M. (1975). Effect of insulin and cyclic nucleotides on differentiation of a myoblast cell line. In “Advances in Cyclic Nucleotide Research” (P. Greengard, C. A. Robison, and R. Paoletti, eds.1, Vol. 5, abstract. Raven Press, New York. MANGANIELLO, V., and VAUGHAN, M. (1972). Prostaglandin E, effects on adenosine 3’:5’-cyclic monophosphate concentration and phosphodiesterase activity in fibroblasts. Proc. Nat. Acad. Sci. USA 69, 269-273. NOVAK, E., DRUMMOND, G. I., SKALA, J., and HAHN, P. (1972). Developmental changes in cyclic AMP, protein kinase, phosphorylase kinase and phosphorylase in liver, heart and skeletal muscle of
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AND BEATTY
Cyclic
the rat. Arch. Biochem. Biophys. 150.511-518. OTTEN, J., JOHNSON, G. S., and PASTAN, I. (1972). Regulation of cell growth by cyclic adenosine 3’,5’monophosphate. Effect of cell density and agents which alter cell growth on cyclic adenosine 3’,5’monophosphate levels in fibroblasts. J. Biol. Chem. 247, 7082-7087. PARDEE, A. B., JIMENEZ DE ASUA, L., and ROZENCURT, E. (1974). Functional membrane changes and cell growth: Significance and mechanism. In “Control of Proliferation in Animal Cells” (B. Clarkson and R. Baserga, eds.), pp. 547-561. Cold Spring Harbor Laboratory, New York. PETERSON, R. D., BEATTY, C. H., and BOCEK, R. M. (1963). Effects of insulin and glucagon on carbohydrate and protein metabolism of adductor muscle and diaphragm. Endocrinology 72, 71-77. RUTTEN, W. J., &HOOT, B. M., and DE PONT, J. J. H. H. M. (1973). Adenosine 3’,5’-monophosphate phosphodiesterase assay in tissue homognates. Biochim. Biophys. Acta 315, 378 - 383. SCHWARTZ, J. P., and PASSONNEAU, J. V. (1974). Cyclic AMP-mediated induction of the cyclic AMP phosphodiesterase in C-6 glioma cells. Proc. Nat. Acad. Sci. USA 71, 3844-3848. SHEPPARD, H., and WIGGAN, G. (1971). Analogues of 4-l3,4-dimethoxybenzyl)-2-imidazolidinone as potent inhibitors of rat erythrocyte adenosine cyclic 3’,5’-phosphate phosphodiesterase. Mol. PIcarmacol. 7, 111-115. SMOAKE, J. A., SONG, S-Y., and CHEUNG, W. Y. (1974). Cyclic 3’,5’-nucleotide phosphodiesterase. Distribution and developmental changes of the enzyme and its protein activator in mammalian tissues and cells. B&him. Biophys Acta 341,402411. STEINER, A. L., PAGLIARA, A. S., CHASE, L. R., and KIPNIS, D. M. (1972). Radioimmunoassay for cyclic nucleotides. Adenosine 3’,5’-monophosphate and guanosine 3’,5’-monophosphate in
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in Developing
Muscle
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mammalian tissues and body fluids. J. B/o/. Chem. 247, 1114-1120. TEO, T. S., and WANG, J. H. 11973). Mechanism of activation of cyclic adenosine 3’:5’-monophosphate phosphodiesterase from bovine heart by calcium ions. Identification of the protein activator as a Ca’+ binding protein. J. Biol. Chem 248, 5950-5955. TEO, T. S., WANG, T. H., and WANG, J. H. \19731. Purification and properties of the protein activator of bovine heart cyclic adenosine 3’,5’-monophosphate phosphodiesterase. J. Biol. Chom. 24X, 588-595. THOMPSON, W. J., and APPLEMAN, M. M. 119711. Characterization of cyclic nucleotide phosphodiesterases of rat tissues. J. Biol. Chen. 246. 31453150. VOORHEES, J. J., DUELL, E. A., STAWISKI, M.. and HARRELL, E. R. (1974). Cell nucleotide metabolism in normal and proliferating epidermis. In “Advances in Cyclic Nucleotide Research” iP. Greengardand G. A. Robison, eds.), Vol. 4, pp 117-162. Raven Press, New York. WILLIAMS, R. H., and THOMPSON, W. J. (1973). Effect of age upon guanyl cyclase, adenyl cyclase, and cyclic nucleotide phosphodiesterases in rats. Proc. Sot. Exptl. Biol. Med. 143, 382-387. Woo, Y-T., and MANERY, J. F. (1973). Cyclic AMP phosphodiesterase activity at the external surface of intact skeletal muscles and stimulation of the enzyme by insulin. Arch. Biochem. Biophys 154, 510-519. ZALIN, R., and MONTAGUE, W. (19751. Changes in the level of 3’,5’-cyclic adenosine monophosphate and the activities of adenylate cyclase and protein kinase during the differentiation of primary cultures of chick myoblasts. In “Advances In Cyclic Nucleotide Research” (P. Greengard, G. A. Rohison, and R. Paoletti, eds), Vol. 5, p. 832, abstract. Raven Press, New York.
BIOLOGY
48,
382-391
(19’76)
Cyclic AMP Phosphodiesterase Activity in Fetal and Adult Muscle the Rhesus Monkey R. M. Division
of Perinatal Department
BOCEK
C. H.
AND
Physiology, Oregon Regional Primate of Biochemistry, University of Oregon Accepted
September
of
BEATTY
Research Center, Beaverton, Oregon 97005, Medical School, Portland, Oregon 97201
and
25,1975
Total cyclic AMP phosphodiesterase activity of voluntary skeletal muscle of the rhesus monkey was highest in the loo-day fetal series, decreased near term, and was lowest in the adult series. Kinetic data indicated the existence of two cyclic AMP phosphodiesterase enzymes in both the fetal and adult muscle. The apparent K, values for the high-affinity phosphodiesterase were similar in the loo-day fetal and adult skeletal muscle, whereas those for the lowaffinity enzyme were twofold higher in the fetal series. The V,,, of the highl(, enzyme was tenfold higher in the fetal than in the adult series and the low K, V,,, was fourfold higher in the fetal series. Both caffeine and theophylline were competitive inhibitors of the low K, phosphodiesterase activity and noncompetitive inhibitors of the high K, enzyme. No difference was observed in the sensitivity of the fetal and adult enzyme preparations to the methylxanthines or to Ro20-1724. INTRODUCTION
muscle just before birth than in adult muscle, increased rapidly during the first lo20 days after birth, and then decreased with aging to adult levels.On the other hand, the levels of cyclic AMP were higher in fetal rhesus muscle during the last half of gestation than in adult muscle (Bocek et al., 1975). In addition, the sensitivity of the adenylate cyclase enzyme to prostaglandin E, in vitro was highest in 78- to loo-day-old fetal muscle (47-61% of gestation), decreased near term, and was lowest in the adult series (Bocek et al., 1975). Because intracellular levels of cyclic AMP are regulated by degradative as well as synthetic enzymes, we wanted to ascertain whether the higher level of cyclic AMP found in the fetal muscle was due to a lower cyclic AMP phosphodiesterase activity. Changes in the total phosphodiesterase activity during growth had already been reported for rat skeletal muscle, and its developmental pattern appeared to differ from that of adenylate cyclase; fetal and postnatal levels did not vary until after Day 30 and were higher than in adult muscle (Hommes and Beere, 1971; Williams and Thompson, 1973). Multiple forms of cyclic AMP phosphodiesterase ac-
Evidence obtained in the past 5 years indicates that cyclic AMP plays a critical role in the growth, differentiation, and/or specialization of cells. Numerous studies with tissue cultures and bacterial preparations (Voorhees et al., 1974) have led to the formulation of the hypothesis that the cellular level of cyclic AMP is inversely related to cell proliferation (mitosis). Otten et al. (19721, Pardee et al. (19741, and Voorhees et al. (1974) have shown that cells in vitro that have been transformed or stimulated to grow with various agents generally contain lower internal cyclic AMP, and Zalin and Montague (1975) suggested a causal relationship between the increases in adenylate cyclase activity, the peak of intracellular cyclic AMP levels, and the onset of cell fusion in myoblasts. Changes in cyclic AMP levels and adenylate cyclase activities in uiuo associated with development have been reported in several rat tissues, including skeletal muscle (Novak et al., 1972; Hommes and Beere, 1971; Williams and Thompson, 1973) and heart (Novak et al., 1972). Both cyclic AMP levels and adenylate cyclase activities were generally lower in fetal rat 382 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
BOCEK
AND
BEATTY
Cyclic
tivity had been reported in several tissues of adult mammals (Thompson and Appleman, 19711, but no data on such forms in fetal muscle were available. Therefore, we undertook this study and have determined the total phosphodiesterase activity and the kinetics of a high and low K, cyclic AMP phosphodiesterase in muscle from rhesus fetuses and have compared the results with data from adult monkeys. METHODS Rhesus fetuses (Macczca mulatta) from 85 to 103 days gestational age (designated the loo-day fetal series), 155 days gestational age, and adult rhesus monkeys were used in these experiments. The average gestational age in our colony was 165 days. The mothers were anesthetized intramuscularly with 1.0 mg/kg of l-(l-phenylcyclohexyl) piperidine hydrochloride and the fetuses were removed and rapidly exsanguinated. Adult monkeys were anesthesized with Ketaject (dl-2-{O-chlorophenyl}2-{methyl amino} cyclohexanone hydrochloride). Samples of voluntary muscles from the upper arms and thighs of the fetuses and from the sartorius muscle of the adult animals, and of diaphragm and cardiac muscle from both the fetal and adult monkeys were rapidly excised and stripped of fat and connective tissue. When the skeletal and diaphragm muscles were immediately homogenized (unfrozen at 0 to 4°C) or frozen and homogenized later, cyclic AMP phosphodiesterase activity in these samples was similar. Therefore, we chose the more convenient method and froze the samples in a Wollenberger clamp cooled in liquid nitrogen, wrapped them in aluminum foil, and stored them in a liquid nitrogen refrigerator for future analysis. Homogenates (10% w/v) were prepared in 0.25 M sucrose plus 1 mIt4 EDTA. The frozen tissues were weighed and chopped in a conical homogenizing tube containing frozen sucrose-EDTA medium. The tissues were then thawed in a slurry of the medium and homogenized with a glass pes-
AMP-PDE
in Developing
Muscle
383
tle (lo-12 passes) (0-4°C). The homogenate was frozen, thawed, and centrifuged 1OOOg for 15 min at O”C, and the supernatant was assayed. Cyclic AMP phosphodiesterase activity was determined by a modification of the two-stage method of Loten and Sneyd (1970). For total phosphodiesterase activity, the reaction mixture contained 0.02 &i [3H]cyclic AMP (38.15 Ci/mmole), 1 x lop3 M cyclic AMP 40 mM Tris buffer at pH 8.0, 2 mM MgCl, plus supernatant in a final volume of 0.2 ml. Blank values were determined either with boiled supernatant or sucrose-EDTA medium. The activity in the blanks was similar with both methods. The mixture was incubated for 15 min at 30°C with shaking and the assay was terminated by immersing the tubes in a dry ice-acetone freezing mixture and then heating them at 100°C for 45 sec. After the addition of 0.05 ml of a solution of snake venom (ophiaphagus hannah, 1 mg/l ml water), the assay tubes were reincubated for 15 min and then held at 4°C. For kinetic studies, cyclic AMP concentrations were varied from 0.9 x loo-” to 1 x 10m4M and the [3H]cyclic AMP from 0.01 to 0.02 &i/assay tube. The reaction mixture was applied to 2.5 x 0.5 cm columns of Bio-Rad Agl-X2 (200400 mesh) and the labeled adenosine eluted with 40 mIt4 Tris buffer, pH 7.4. An aliquot of the eluate was added to Triton X-114 xylene-omnifluor and counted in a liquid scintillation counter. The amount of hydrolyzed cyclic AMP was calculated from the specific activity of the substrate cyclic AMP and expressed in terms of nanomoles of cyclic AMP hydrolyzed per minute per milligram of protein. At cyclic AMP concentrations of 5 a, the reaction rate was linear for 20 min with approximately 2 mg protein/ml of incubation medium. The rate remained constant up to 21% substrate hydrolysis. Kinetic studies were routinely run for 15 to 20 min and the enzyme concentration used was such that less than 25% of the substrate cyclic AMP was hydrolyzed. K, values were calculated
384
DEVELOPMENTAL
BIOLOGY
by the method of least squares. Protein content of the supernatant was determined by the method of Lowry et al. (1951) standardized with bovine serum albumin. In the assay of cyclic AMP phosphodiesterase activity in tissue extracts the amount of labeled adenosine recovered may not be indicative of the total amount of labeled cyclic AMP hydrolyzed since enzymes in the unpurified extract may catalyze the breakdown of product 5’-AMP to such compounds as adenine, hypoxanthine, and inosine as well as adenosine. However, Rutten et al. (1973) found that elution of the Bio-Rad column with 0.1 M NaHC03 recovered all four products of 5’AMP metabolism. When we ran these four compounds through individual columns and eluted them with 40 mM Tris buffer at pH 7.4, our recoveries on the basis of optical density measurements at 260 nm were essentially similar to those of Rutten et al. (1973) with 0.1 M NaHCO,. Recovery of added adenosine, the major compound, was 98 to 100%. [3H]cyclic AMP was purified on a BioRad AGl-X2 column (2 ml of resin volume in a Pasteur pipette). The [3Hlcyclic AMP was applied and the column washed with 10 to 15 ml of water. The purified cyclic AMP was eluted with 4 to 5 ml of 0.05 N HCl, diluted with ethanol to make a final concentration of 50% ethanol, and stored at -20°C. Radioactivity in the blanks was usually less than 1% of the total. RESULTS
The total cyclic AMP phosphodiesterase activity of voluntary skeletal muscle was sixfold higher in the loo-day fetal than in the adult series, decreased near term, and was lowest in the adult series (Table 1). The data on diaphragm and heart muscle followed the same pattern of enzyme activity during development as skeletal muscle but were too few for statistical evaluation. In contrast to intermittently active voluntary skeletal muscle, diaphragm is a specialized muscle adapted to constant activ-
VOLUME
48,
1976 TABLE
1
CYCLIC AMP PHOSPHODIESTERASE ACTIVITY 1OOOg SUPERNATANT OF THE HOMOGENATES FETAL
AND
ADULT
MUSCLE MONKEY”
FROM
THE
IN THE OF
RHESUS
Series
W-day
fetal bmoles
Adult
155&y
fetal cyclic AMP hydrolyzed/min/ 100 mg protein)
-
Skeletal muscle Diaphragm
272 2 25
6) 266
(2) Heart -
790 (1)
172 + 16*
(6) 193 (1) 499 (1)
46 k 4’ (10) 49 k 5 (4) 360 T 18 (4)
’ Values are means 2 SE or averages of duplicate assays. Numbers in parentheses are the number of animals in each series. Cyclic AMP concentration 1 x lo-SM.
* 155-day vs loo-day, P < 0.01. ’ Adult vs 155-day series, P < 0.001.
ity and differing in several metabolic parameters from voluntary skeletal muscle (Peterson et al., 1963). However, within each age group, total phosphodiesterase activities were similar in diaphragm and skeletal muscle but were three to eight times lower than in heart muscle. Kinetic data on rhesus skeletal muscle indicated two cyclic AMP phosphodiesterase activities in both the fetal and adult series; typical Lineweaver-Burk plots for skeletal muscle are shown in Figs. 1 and 2. The apparent K, values for the high affnity (low K,) phosphodiesterase activities were similar in the loo-day fetal and adult voluntary skeletal muscle (Table 21, whereas those for the low affinity (high K,) enzyme were higher in the loo-day fetal than in the adult series. V max values for both the low and high K, enzymes decrease with development and were lowest in adult muscle. The V,,, of the high K, enzyme was about sevenfold higher in the loo-day fetal than in the adult series and the low K, V,,, was fourfold higher in the fetal muscle. V,,, values for diaphragm muscle from fetal and adult animals appear to be similar to those of
BOCEK
AND BEATTY
Cyclic
AMP-PDE
in Developing
385
Muscle
Adult Muscle
6Or
FIG. 1. Kinetic plot of cyclic AMP phosphodiesterase activity in a muscle extract of a loo-day rhesus fetus. The extract represents the 1OOOg supernatant of the homogenate prepared as described in the Methods section. TABLE KINETIC
-
DATA
FOR PHOSPHODIESTERASE
2 ACTIVITY
Low -N
-
AND ADULT
affinity
Km (CLM)
MUSCLE”
High V maxb Voluntary
loo-day Adult
fetal
3 4
46 k 4 24 + 3
-N
affinity
Km (40
Skeletal
380 -c 10 58 -+ 13” (’
-.--
V,,Xh
Muscle 5 7
5.8 + 0.6 5.8 2 0.7
1 2
6.0 5.4, 6.3
81 2 12 21 t 6’
Diaphragm
-
-
IN FETAL
-____ Series
-
FIG. 2. Kinetic plot ofcyclic AMP phosphodresterase activity in a muscle extract of an adult rhesus monkey. The extract represents the 1OOOg supernatant of the homogenate prepared as described in the Methods section.
loo-day Adult
fetal
1 2
48 28, 45
340 30, 40
a Values are means 2 SE, duplicate analyses. N = the number of animals ase activity determined as stated in Methods. Concentration of cyclic AMP * Nanomoles of cyclic AMP hydrolyzed per minute per 100 mg protein. “P for difference, adult vs loo-day fetal series,
voluntary skeletal muscle (Table 2). As early as 100 days gestation, both caffeine and theophylline were competitive inhibitors of the low K, phosphodiesterase activity (Fig. 31, and noncompetitive inhibitors of the high K, enzyme assayed at concentrations of 50 to 200 fl cyclic AMP (data not shown). Methylxanthines were reported to be both competitive and noncompetitive inhibitors of the cyclic nucleotide phosphodiesterases in various adult tissues (Appleman et al., 1973). Huang and Kemp (1971) found that the methylxan-
75 12. 16 .in each series. Phosphodiestervaried from 1 to 100 fl.
thines were competitive inhibitors of the low K, cyclic nucleotide phosphodiesterase in adult rabbit muscle. The K, values for the inhibition of both the low and high phosphodiesterases in rhesus muscle by the methylxanthines were similar in the loo-day fetal and adult series. For the low K, enzyme, K, values ranged from 1.3-1.8 mM for caffeine (Fig. 4) and 0.8-1.4 mM for theophylline (Fig. 5). Ki values for 155day fetal muscle were within these ranges. Ki values for the high K, enzyme ranged from 2.3-4.0 mM and 1.2-2.0 mM for caf-
386
DEVELOPMENTAL
5
IOO-Day
BIOLOGY
VOLUME
48, 1976
Fetus
Adult
, 2SmM
1
I/[cAMP]-pM
Theoph.
I/[cAMP]-UM
FIG. 3. Lineweaver-Burk double reciprocal plot showing competitive inhibition of the low K, cyclic AMP phosphodiesterase by theophylline and caffeine of skeletal muscle from loo-day fetal and adult rhesus monkeys. Assay conditions were as described in Fig. 1 and in the Methods section. Concentrations of substrate cyclic AMP were varied from 0.75 - 5 fl. Lines of best fit were obtained by the method of least squares.
IOO-Day
Fetus
Ki -1.8
,
Adult W5PM CAMP
/ .
I.OUM CAMP
2.9uM 5.8PM
I
3.0 [Caffeine]-mM
-2.0
-1.0
1.0 2.0 [Caffeine]-mM
3.0
FIG. 4. Kinetic data for determination of the K, value for caffeine in homogenates of loo-day fetal and adult rhesus muscle. Assays were incubated for 20 min; other conditions were as described in the Methods section. V = nanomoles of cyclic AMP hydrolyzed per minute per milligram of protein.
feine and theophylline respectively. The relative effect of inhibitors on the activity of phosphodiesterase at 5 fl cyclic AMP concentrations is shown in Table 3. The sensitivity of the enzyme preparations to the methylxanthines did not differ in fetal and adult muscle. The inhibitory effect of theophylline appeared to be slightly greater than that of caffeine; this agrees
with the data of Huang and Kemp (1971). Several other types of compounds inhibit cyclic nucleotide phosphodiesterase (Appleman et al., 1973), including the analogues of 4-(3,4-dimethoxybenzyl)-2-imidazolidinone (Sheppard and Wiggen, 1971). The 3butoxy-4-methoxy derivative, Ro20-1724, a more potent inhibitor of erythrocyte phosphodiesterase than the methylxanthines,
BOCEK
AND
IOO-Day
Cyclic
BEATTY
AMP-PDE
in Developing
387
Muscle
Adult
Fetus
80-
Q75vM Ki-
1.4mM
L -20
-1.0
I.0
[Theophyl
21)
-2.0
30
-1.0
I.0 20 [Theophyllinel-mM
line] - mM
FIG. 5. Kinetic data for determination muscle. Conditions were similar to those milligram of protein.
of the Ki value for theophylline in loo-day fetal in Fig. 4. V = nanomoles of cyclic AMP hydrolized
was also a more effective inhibitor of skeletal muscle phosphodiesterase at 0.4 mM concentrations than the methylxanthines at 1 mM levels. Like the methylxanthines, Ro20-1724 inhibited the enzyme preparations from fetal and adult muscle to a similar degree. Recent studies have shown that a specific protein activator of cyclic AMP phosphodiesterase is present in several mammalian tissues other than skeletal muscle (Smoake et al., 1974) whose action is completely dependent on the formation of a Ca’+-activator complex (Teo and Wang, 1973). The low V,,, values for adult muscle may have been due to a deficiency in calcium and/or in activator protein. To test this possibility, we combined samples of homogenate from fetal and adult muscle and assayed them for total phosphodiesterase activity. When fetal and adult samples were combined in ratios of 1:l and 2:l no stimulation of the adult enzyme preparation was apparent, i.e., the activities were proportional to the activity in the original homogenates (Table 4). The addition of 100 pM Ca*+ either to the 1:l fetal-adult combination or to a preparation of adult muscle (substrate level of cyclic AMP 5 $50 did not affect the rate of substrate hydrolysis.
TABLE RELATIVE
AMP
EFFICIENCY
and adult rhesus per minute per
3
OF INHIBITORY
PHOSPHODIESTERASE SKELETAL
3.0
FROM MUSCLE”
OF CYCLIC VOLUNTARY
Inhibitors
Percentage
of Control
Value”
Fetal
Adult
loo-day
15day
-1 mM
caffeine 52 56 56 1 mM theophylline 49 49 48 0.4 mM R020-1724’ 27 32 29 0.7 mM R020-1724 17 13 a Assays were carried out for 20 min at a cyclic AMP concentration of 5 fl and other conditions as described in the Methods section. Values represent the average of duplicate assays, two or three animals in each series. b The uninhibited control values were assumed to be 100%. ’ 14(3 - butoxy - 4 - methoxybenzyl) - 2 - imidazolidinone]. DISCUSSION
Levels of cyclic AMP in tissues are determined by a balance between activities of adenylate cyclase forming and of phosphodiesterase degrading the nucleotide. In a previous paper (Bocek et al., 1975), we reported that cyclic AMP levels in rhesus fetal muscle were highest at 100 days, decreased near term, and were lowest in the adult. Adenylate cyclase activity as well
388
DEVELOPMENTAL
TABLE TOTAL
PHOSPHODIESTERASE
4 ACTIVITY
IN MIXTURES
OF SUPERNATANT FROM HOMOGENATES FETAL AND ADULT SKELETAL MUSCLE RHESUS
BIOLOGY
OF lOO-DAY OF THE
MONKEYS
Nanomoles lyzed/ruin/100 Obeerved values
of CAMP hydromg protein Calculated values
-
Control Fetal 384 Adult 50 Combined Fetal/adult ratio 1:l 214 217 Fetal/adult ratio 2:l 283 213 a 1OOOg supernatant preparations of homogenates were prepared as described in Methods. Fifty microliters of either the original supernatants or the indicated combinations of supernatants from fetal and adult muscle homogenates were assayed as previously described. Concentration of substrate cyclic AMP was 1 mA4, 1 x 105 dpm [3H]cAMP per assay. Protein concentrations of the enzyme preparations were 0.25 and 0.30 mglassay for fetal and adult muscle, respectively. Values are means of triplicate assays.
as its sensitivity to epinephrine (Bocek et al., 1973) and to prostaglandin Ez (Bocek et al., 1975) were also higher in fetal than in adult muscle. In this paper, we report that phosphodiesterase activity parallels both the levels of cyclic AMP and the activity of adenylate cyclase in developing rhesus muscle. That is, all three were highest in loo-day fetal muscle. There are several reasons why the level of cyclic AMP and the activity of the phosphodiesterase degrading it were both high. High levels of cyclic AMP induce phosphodiesterase activity in certain systems (Schwartz and Passonneau, 1974; Manganiello and Vaughn, 19721, and Schwartz and Passonneau (1974) suggested that this type of activation leads to long-term regulation of cyclic AMP levels, an effect that may be operative in developing fetal muscle. On the other hand, an endogenous factor has been isolated from rat adipocytes after their exposure to agents that raise the intracellular levels of cyclic AMP. This fac-
VOLUME
48,
1976
tor inhibits the activity of cyclic AMP and cyclic GMP phosphodiesterase in several tissues of the rat, including the diaphragm and voluntary skeletal muscle (Ho et al., 1975). Low levels of cyclic GMP can also activate the hydrolysis of cyclic AMP by the high K, enzyme of liver and fat cells (Appleman et al., 19731, and the ratio of cyclic AMP to cyclic GMP may be critical to the coordinated regulation of cell biochemistry (Voorhees et al., 1974). Levels of cyclic GMP in fetal tissues are not yet available. Cyclic AMP phosphodiesterase from various sources appears sensitive to several other endogenous factors capable of activating or inhibiting the enzyme (Appleman et al., 1973). Ca*+ is an essential component in the action of a protein activator of cyclic AMP phosphodiesterase from bovine heart (Tea and Wang, 1973). Activation enhanced the V,,, and decreased the K, for cyclic AMP. The concentration of activator protein in crude tissue preparations appears to be in excess of the amount of enzyme (Tea et al., 1973). Smoake et al. (1974) reported that in eight rat tissues other than muscle the ratio of activity of the protein activator to that of the phosphodie&erase varied not only during development but from tissue to tissue. The fact that we were unable to show any stimulation of phosphodiesterase from adult muscle with Ca2+ or by combination with a more active fetal muscle preparation suggests that these factors are not responsible for the lower phosphodiesterase activity in adult muscle. However, the relation between the actual levels of activator protein and phosphodiesterase in developing skeletal muscle needs to be determined. Hommes and Beere (1971) reported that in developing rat muscle adenylate cyclase activity was low before birth and that the activity of total phosphodiesterase did not parallel that of adenylate cyclase. The difference in the developmental pattern of the activities of the enzymes that govern the concentrations of cyclic AMP in the rat
BOCEK AND BEATTY
Cyclic
AMP-PDE
in Developing
Muscle
389
and rhesus monkey may be due to the found with kinetic analysis of crude exvariation in the stage of development of tracts of rabbit skeletal muscle (5 and 20 the muscle between the rat and rhesus @) (Huang and Kemp, 1971) and of purimonkey with gestational age. Our data fied enzyme preparations of rat muscle (2.2 support the hypothesis that ontogenetic and 57 fl) (Thompson and Appleman, changes in cyclic AMP levels are related to 1971). There are two phosphodiesterases in the developmental stages of the muscle fetal as well as in adult rhesus muscle; the fiber rather than to the age of the animal apparent K, values for the high affinity relative to birth (Beatty et al., 1967); Bo- enzyme are similar in the two series. We cek et al., 1975). have no explanation for the higher K, An inverse relation between intracelluvalue of the low-affinity phosphodiesterase lar levels of cyclic AMP and the rate of in the fetal muscle. The intracellular conDNA synthesis has been demonstrated in centration of cyclic AMP is low, probably cultures of several cell lines (Abel1 and below 1 fl (Steiner et al., 1972; Bocek et Monahan, 1973). Mandel and Pearson al., 19751, and unless this compound is (1975) reported that high levels of intracelhighly compartmentalized, the physiologilular cyclic AMP inhibited the differentiacal significance of the low affinity-high tion of myoblasts. Hypothetically, cyclic K, phosphodiesterase activity might be AMP levels in viuo should be low during limited. The actual difference in the K, the period when the rate of proliferation is values was less than twofold. The differhigh (presumptive or premyoblast stage) ence in enzyme activity between the two and high when proliferation is completed series would imply either that the enand the myoblasts differentiate and fuse to zymes are different or that the relative form myotubes. At birth, rat muscle is concentration of a factor which affects the relatively immature with a majority of the affinity of enzyme for its substrate is differfibers still in the myotube stage (Kamienent. The V,,, for both the low and high iecka and Ostenda, 1959; Dubowitz, affinity enzymes is higher in the fetal se1965a). In the rhesus fetus, the myotube ries. This agrees with previous data demstage occurs before the midpoint of gesta- onstrating that most of the metabolic pation; this observation is compatible with rameters we have measured are higher in the decreasing level of cyclic AMP from rhesus fetal than in adult muscle: oxygen 504 of term to maturity (Bocek et al., consumption, CO, production, glucose up1975). Rhesus fetal muscle shows a typical take, and conversion to lactate (Beatty et adult distribution of red and white fibers al., 1968)and palmitate uptake and converby 70% of gestation with only an occa- sion to lipid (Beatty and Bocek, 1970) were sional immature fiber. The developmental all higher in fetal than in adult muscle in stages of human fetal muscle relative to vitro. From the V,,, values, one can estithe percent of total gestation are similar to mate that the ratios of high to low K,, those of the rhesus monkey (Dubowitz, phosphodiesterase activities in the loo-day 1965a,b; Bocek et al., 1975). fetal and adult skeletal muscle are about According to kinetic evidence, there are 3.7 and 1.9, respectively. This suggests a at least two forms of cyclic AMP phosphodi- relatively greater amount of high-affinity e&erase in a variety of adult tissues, in- enzyme in the adult muscle which may be cluding skeletal muscle (Thompson and related to the fourfold lower levels of cyclic Appleman, 1971). Our data indicate that AMP found in adult but not in 78- to lOOthe 1OOOgsupernatant fraction of rhesus day fetal muscle (Bocek et al., 1975). Our muscle homogenate contains two cyclic data, therefore, agree with those of Huang AMP phosphodiesterase activities whose and Kemp (19711, who showed that a apparent K,n values are similar to those greater proportion of the phosphodiester-
390
DEVELOPMENTAL
BIOLOGY
ase activity of adult skeletal muscle is of the low K,-high affinity type. Determining enzyme levels in whole homogenate generates some uncertainties because of possible assay artifacts (Rutten et al., 1973). However, when Huang and Kemp (1971) compared the K, for the highaffinity phosphodiesterase in a crude muscle extract with that obtained with a purified enzyme, the K, values were similar (about 5 @f). Whether an isolated and semipurified enzyme behaves the same way in its natural milieu has always interested enzymologists (Woo, 1973). The kinetics of unpurified enzyme preparations in a more physiological milieu may more nearly approximate their in situ activity. The fact that the K, values for both the high- and low-affinity enzymes isolated and purified from rat muscle were similar to those obtained in our study lends credence to the values obtained by both methods. Publication No. 829 of the Oregon Regional Primate Research Center, supported in part, by Grant RR-00163 from the National Institutes of Health. This investigation was supported by Public Health Service Research Grants HD-06069 and HD-06425 from the National Institutes of Child Health and Human Development, by General Research Support Grant RR-05694 from the National Institutes of Health, and by the Muscular Dystrophy Associations of America, Inc. We thank Paul Herrington for his competent technical assistance. REFERENCES ABELL, C. W., and MONAHAN, T. M. (1973). The role of adenosine 3’S’-cyclic monophosphate in the regulation of mammalian cell division. J. Cell Biol. 59,549-558. APPLEMAN, M. M., THOMPSON, W. J., and RUSSELL, T. R. (1973). Cyclic nucleotide phosphodiesterases. In “Advances in Cyclic Nucleotide Research” (P. Greengard and G. A. Robison, eds.), Vol. 3, pp. 65-98. Raven Press, New York. BEATTY, C. H., BASINGER, G. M., and BOCEK, R. M. (1967). Differentiation of red and white fibers in muscle from fetal, neonatal and infant rhesus monkeys. J. Histochem. Cytochem. 15, 93-103. BEATTY, C. H., BASINGER, G. M., and BOCEK, R. M. (1968). Oxygen consumption and glycolysis in fetal, neonatal and infant muscle of the rhesus monkey. Pediatrics 42, 5-16.
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