Effect of isoproterenol on myocardial mechanical function and cyclic AMP content in the fetal rabbit

j Mol Cell Cardiol 19, 151-I57 (1987)

Effect of lsoproterenol on Myocardial Mechanical Function and Cyclic AMP Content in the Fetal Rabbit H i r o f u m i O k u d a , T o s h i o N a k a n l s h i , * M a k o t o Noktt=awa a n d A t s u y o s h i T a k a o

Pediatric Cardiology, Heart Institute of Japan, Tokyo Women's Medical College, 10 Kawadacho, Shinjuku, Tokyo, Japan (Received 7 May 1986, acceptedin revisedform 14 October 1986) H. OKUDA,T. NAKANISHI,M. NAKAZAWAANDA. TAKAO.Effect of Isoproterenol on Myocardial Mechanical Functions and Cyclic AMP Content in the Fetal Rabbit. ffoumal of Molecularand CellularCardiology(1987) 19, 151-157. The effect ofisoproterenol on mechanical function was studied in the isolated arterially perfused heart of the fetal (21st and 28th day of gestation) and newborn rabbits. The inotropic effect of isoproterenol in the fetus was less than in the newborn. In contrast, myocardial cyclic AMP levels after isoproterenol infusion in the fetus were greater than in the newborn. The inotropic effects ofdibutyryl cyclic AMP and of calcium in the fetus were less than in the newborn. These data suggest that the process from ~-receptor to cyclic AMP in the fetus was equally or even more responsive to isoproterenol than in the newborn. The diminished inotropic effect of isoproterenol in the fetus may be due, at least in part, to the decreased inotropic effect of calcium.

KEYWORDS: Isoproterenol ; Fetus; Newborn ; Inotropic agent.

Introduction Several investigators have shown in in vivo studies t h a t the inotropic effect of isoproterenol in n e w b o r n animals is less than in the a d u l t [1, 2], b u t since c a t e c h o l a m i n e changes pretoad, afterload, a n d h e a r t rate, it is difficult to e x a m i n e the effect of isoproterenol on m y o c a r d i a l contractility p e r se in an in vivo study. Nishioka et al. [3] used the isolated h e a r t p r e p a r a t i o n a n d showed t h a t the inotropic effect of isoproterenol in the n e w b o r n r a b b i t was indeed greater t h a n in the adult. T h e effect of isoproterenol on the fetal m y o c a r d i u m has not been studied extensively. F r i e d m a n [4] utilized a p a p i l l a r y muscle p r e p a r a t i o n a n d showed t h a t sensitivity to isoproterenol in the fetal sheep was similar to that in the adult. C h e n g et al. [5] used the ventricular strip o f the fetal sheep a n d their d a t a were similar to those o f F r i e d m a n [4]. These authors, however, did not show w h e t h e r there is any age-related difference in the inotropic effect ofisoproterenol. Isoproterenol a c t i v a t e s / L a d r e n e r g i c receptors a n d the a d e n y l a t e cyclase system. This * To whom all correspondence should be addressed. 0022-2828/87/020151 + 07 $03.00/0

increases m y o c a r d i a l cyclic A M P content a n d cytosolic calcium c o n c e n t r a t i o n a n d causes inotropic effect [6]. Previous investigators have shown that fl-receptor density a n d a d e n y l a t e cyclase activity increase with develo p m e n t in the fetal r a b b i t [7, 8, 9]. T h e relationship between the inotropism of isoproterenol a n d d e v e l o p m e n t a l changes in the/~r e c e p t o r - a d e n y l a t e cyclase system in the fetal m y o c a r d i u m remains unclear. Therefore, this study was designed to investigate the effect of isoproterenol on (t) m e c h a n i c a l function; a n d (2) m y o c a r d i a l cyclic A M P content in the isolated fetal h e a r t of the rabbit.

Methods T h e experiments utilized the fetus at the 91st and 98th d a y of gestation (term: 31 days), a n d 3 to 5 days old n e w b o r n New Z e a l a n d W h i t e rabbits. After the doe was killed by a sharp blow to the h e a d the fetuses were delivered by cesarian section a n d used within 1 min after delivery. T h e fetal a n d n e w b o r n r a b b i t s were killed b y a s h a r p blow to the

9 1987 Academic Press Inc. (London) Limited

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head. The heart was then excised from the ( n = 7 in e a c h age group). The duration of chest cavity and used for mechanical function perfusion was 10 min at each isoproterenol concentration. Mechanical function reached a study. new steady state 2 min after switching to a new isoproterenol concentration, and all meaPerfusion solution surements were made at 5 min. The control Krebs-Henseleit solution contained in m M : NaC1, 118; KC1, 6.0; CaC12, (2) Response to dibutyryl adenosine Y, .Y-cyclic 1.5; glucose, 6.0; MgC12, 1.0; N a H C O 3 , monophosphate (dibutyryl cyclic AMP) 24.0; N a H 2 P O 3 , 0 . 4 3 6 . The control solution Muscles were perfused with solutions containwas equilibrated with 95% O2 and 5% CO2 ing dibutyryl cyclic A M P (10 -4 M to 10 -3 M) yielding a final p H of 7.35 to 7.42. for 20 rain. In all experiments the maximal inotropic effect was observed within 20 min.

Experimental preparation Experiments were performed in the isolated, arterially perfused ventricular preparation as described previously [10, 12-15]. The aorta was canulated with PE-50 polyethylene canula and then perfused with oxygenated perfusate at a constant perfusion rate of 2.5 ml/g tissue per minute using a Harvard pump. The base of the right and left ventricle was fixed between two H a r m o n forceps and the apex was attached to the Statham (UC 3) force transducer using a silk suture. The muscle was stimulated at 40 bpm, and its temperature was maintained at 2 7 _ 0.5~ The following parameters of mechanical function were monitored continuously: developed tension (DT), resting tension (RT), maximum rate of tension development (+dT/dtmax) , and half time to relaxation (1/2 RT).

Experimental protocol Initially the muscles were perfused with a control solution containing 1.5 mM calcium for 60 min to allow for stabilization of the mechanical function. During the initial 40 min of each experiment, the length of the muscle preparation was adjusted so that the tension was equal to 90% of the maximal tension. After this initial period, both resting tension and the length-tension relationship remained unchanged under control conditions. The following studies were then performed.

(1) Response to isoproterenol After stabilization of mechanical function, muscles were perfused with solutions containi n g l x 10 - s , 5 x 10 - s , 1 x 10 -7 , 5 x 10 -7 , 1 x 10 -6 , and 1 x 10 -5 M isoproterenol

(3) Response to calcium After stabilization of mechanical function, the perfusate was switched to solutions containing 3, 7.5, 15, and 30 mM calcium. The duration of perfusion was 5 min at each calcium concentration. Measurements were made 4 min after reaching a steady state. Parameters describing mechanical function were expressed as a percentage of control values and a g/g tissue wet weight.

Myocardial cyclic A M P content After stabilization of mechanical function, muscles were perfused with solutions containing 10 - s M isoproterenol for 10 min and then frozen with metal clamps in liquid nitrogen. Control muscles were perfused with solutions which did not contain isoproterenol and frozen in liquid nitrogen. Muscles were then homogenized in ten volumes of 6% tricholoroacetic acid using a ground-glass homogenizer and centrifuged at 0~ The supernatant was neutralized by adding C a C O 3 [16] and centrifuged again. Cyclic A M P concentration in the neutralized supernatant was measured by radioimmunoassay methods [17] using cAMP [12sI] R I A Kit (New England Nuclear, MA, USA).

Statistical analysis Results were expressed as mean -t-s.c. One way analysis of variance was used for multigroup components. Statistical significance of the difference between group means was determined using the modified t test [18]. Percentage changes were compared using nonparametric methods (Wilcoxon's rank sum test) [18]. Statistical significance of response

Isoproterenol and c A M P i n the Fetus

153

Fetus

to isoproterenol and calcium was analyzed using a paired t test and repeated measurements of analysis of variance. T h e probability was considered to be significant if the P value was less than 0.05.

Isoproterenol g

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IO-~M

,

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Results

-I0

Mechanical function data under control conditions in the fetus were not significantly different from those in the newborn except that +dT/dt(max) in the 21-day-fetus was less than that in the other groups (Table 1).

Newborn

f Control Isoproterenol I 0 - 5 M '~

13n v

.=tn_o

J f i

Responseto isoproterenol Typical data of the experiments are shown in Figure 1. T h e responses of D T and + d T/dt (max) to isoproterenol were similar and therefore only the D T data will be shown. Significant inotropic effects were observed and D T reached m a x i m u m at a concentration of 10 -6 M of isoproterenol in the three age groups (Fig. 2). Significant difference in D T between the newborn and fetus were observed at concentrations from 10 - s M to 10 -5 M. At 10-5 M isoproterenol D T increased to 197 __+6% of the control in the newborn and only to 1 3 0 _ 10% of the control in the 28-day fetus (significant difference between the newborn and fetus: P < 0.001). Inotropic effect of isoproterenol in the 21-day-fetus (138 + 11%) was not significantly different from that in the 28-day-fetus. T h e sensitivity to isoproterenol was expressed as a percentage of the maximal value and the data showed that the sensitivity was similar in the three age groups (Fig. 3). A double reciprocal plot of D T as a function of

o~

-~

4O 2oi o

"u

-20

-40 F I G U R E 1. Typical experiments that show the effect of isoproterenol on D T and d T/dt. The inotropic effect of isoproterenol in the fetus (28th day of gestation) was less than in the newborn.

isoproterenol concentration showed that D T reached one-half maximal value at 3.4 x 10 -8 M in the newborn, 3.6 x 10 -8 in the 28-day fetus, and 4.5 x 10 -8 in the 21-day fetus (no significant difference between the three age groups). Isoproterenol (10 -5 M) decreased 1/2 R T and the decrease in the 21-day fetus was significantly less than that in the newborn (no significant difference between the newborn and 28-day fetus; Fig. 4).

TABLE 1. Base*line data of mechanical function n

RT (g/g tissue)

DT (g/g tissue)

+dT/dt max

Age

(g/s/g tissue)

1/2RT (ins)

Fetus (21 days) Fetus (28 days) Newborn

15

5.4 _ 0.5

4.6 4- 0.9

13.8 Jr 2.3"

175 4- 17

27

3.3 4- 0.5

6.1 Jr 0.8

28.4 4- 5.3

189 4- 9

27

3.9 4- 0.5

6.8 4- 0.5

29.8 Jr 2.8

201 + 14

Values are means + s.~. RT, resting tension; DT, developed tension; + maximal rate of tension development; I/2RT, half time to relaxation. a = significantly less than in the 28-day fetus and newborn.

dT/dt max,

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15

•

s=

c

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_ 5 I ...... I_I ........... i

f5...... t-- . . . . i

0

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-~

i

/ /

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I0 -8 I0 -7 I0 -s Isoproterenol ( M )

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FIGURE 2. Effect of isoproterenol on developed tension (DT). * = significantly (P < 0.05) different from the value in the fetus. (O: Newborn; A: Fetus (28 day); A: Fetus (21 day)). I00

" "

Newborn

Fetus (28 day)

Fetus (21 day)

FIGURE 4. Effect of isoproterenol on half relaxation time (1/2RT). Shortening of 1/2RT in the 21-day fetus was significantly less than in the newborn. No significant difference between the 21-day fetus and the 28-day fetus or between 28-day fetus and newborn was observed.

Myocardial cyclic A M P content

2

T h e c o n t r o l v a l u e o f m y o c a r d i a l cyclic A M P c o n t e n t was similar in the three age g r o u p s ( T a b l e 2). I s o p r o t e r e n o l infusion i n c r e a s e d tissue cyclic A M P levels, a n d the v a l u e after i s o p r o t e r e n o l infusion in t h e 2 1 - d a y fetus was significantly g r e a t e r t h a n the values in the o t h e r t w o groups.

E

"5 50

0

,6-s

,6-,

,6-,

+5

Isoproterenol (M)

FIGURE 3. Sensitivity to isoproterenol, expressed as percentage of maximal inotropic effect in each age group. Although the curve in the 21-day fetus was slightly shifted to the right, there were no significant differences in the values. (Q: Newborn; A: Fetus (28 day); A: Fetus (21 day)).

Response to dibutyryl cyclic A M P S i n c e there was no difference in the i n o t r o p i c effect o f i s o p r o t e r e n o l b e t w e e n the 2 1 - d a y fetus a n d the 2 8 - d a y fetus, effect o f d i b u t y r y l cyclic A M P was s t u d i e d o n l y in the n e w b o r n a n d the 2 8 - d a y fetus. I n a p r e l i m i n a r y e x p e r i m e n t , the i n o t r o p i c effect o f this d r u g r e a c h e d

TABLE 2. Effect of isoproterenol on tissue cyclic A M P contents Age

n

1. Fetus (21-day) 2. Fetus (28-day) 3. Newborn

Control After isoproterenol (pmol/mg tissue wet wt) 0.99 + 0.05 [6]

7.51 • 0.45 a [9]

0.92 • 0.06 [6]

2.46 • 0.67

[7]

0.89 • 0.06 [6]

1.30 • 0.09

[16]

Values are means + s.z. Numbers in parenthesis = numbers of measurements. Values after isoproterenol infusion were significantly (P < 0.05) greater than control in all age groups. a = significantly (P < 0.05) greater than the values in the 28-day fetus and newborn.

l s o p r o t e r e n o l and cAMP in the Fetus

155

cyclase activity stimulated by isoproterenol increased in the fetal rabbit from the 21st day to the 28th day of gestation. Schumacher et al. [9] showed that adenylate cyclase activity stimulated by isoproterenol in the 27-day C2~ fetus was similar to that in the newborn <3 rabbit. These investigations suggest that the /]-receptor-adenylate cyclase system in the Newborn Fetus 21-day fetus (expressed as per mg membrane FIGURE 5. The Inotropic effect of dibutyryl cyclic protein) is premature. In contrast to these AMP 10-4 M). DT = the increase in developed tension, expressed as % of control. * = the value in the fetus (28- findings, myocardial cyclic AMP content after day) was significantly (P< 0.001) different from the isoproterenol infusion in the 21-day fetus was valuein the newborn. greater than that in the newborn (Table 2). The reasons for the discrepancy between these a maximal value at 10 -4 M. The effect of data remain unclear. Since the myocardial dibutyryl cyclic AMP (10 -4 M) on D T in the cell in the fetus is small [11, 19, 20], the cell 28-day fetus was significantly (P < 0.001) less surface/volume ratio or the amount of sarcothan that in the newborn (Fig. 5). lemma in unit weight of the muscle may be greater in the fetus and this might result in the Response to calcium greater production of cyclic AMP, despite the The effect of high extracellular calcium low fl-adrenergic receptor density and low ([Ca]) in the newborn and fetus was similar specific activity ofadenylate cyclase. In agreeto previously reported data [10]. The ment with this speculation, Cheng et al. [4] maximal D T obtained at high [-Ca] in the showed that the density of/~-receptor in the 21-day fetus (DT = 144 +_ 8% of control) fetal sheep, expressed as per mg protein, was and the 28-day fetus (161 + 12%) was signifi- less than in the adult but when expressed as cantly less than in the newborn (262 +_ 25% ; per tissue weight /~-receptor density in the no significant difference between the 21-day- fetus was even greater than in the adult. O f course, other factors such as phosphodiesterfetus and 28-day-fetus). ase activity may also be responsible for the age-related difference in the myocardial cyclic Discussion AMP content. This study demonstrates that in the isolated From the present study it is unlikely that heart preparation the increase of D T and the decreased inotropic effect of isoproterenol +dT/dt(max) during isoproterenol infusion in the fetus is due to the decreased response of in the fetus is less than in the newborn. Isopro- myocardial cyclic AMP levels. However, it is terenol interacts with /Lreceptors and acti- still possible that compartmentalization of vates adenylate cyclase, which in turn cyclic AMP [22] occurred and subcellular stimulates a production of cyclic AMP [6]. levels of cyclic AMP which is physiologically Cyclic AMP, by activating protein kinase, important might be less in the fetus. Further enhances calcium release from the sarco- studies regarding compartmentalization of plasmic reticulum. This causes an increase in cyclic AMP and protein kinase will be intracellular calcium and a positive inotropic required. Different inotropic effects of isoproterenol effect. Isoproterenol also accelerates the relaxation process by stimulating calcium uptake in the newborn and fetus may be due to the age-related differences in (1) the process from by the sarcoplasmic reticulum. Hatjis and McLaughlin [8] showed that cyclic AMP to sarcoplasmic reticulum and/or the density of fl-adrenergic receptor (per mg (2) the inotropic effect of calcium per se. In an membrane protein) was less in the 21-day attempt to study the inotropic effect of cyclic fetus than in the 28-day fetus and newborn AMP, dibutyryl cyclic AMP was used. It is rabbit (no difference between the 28-day fetus thought that this drug passes the cell memand the newborn). Hatjis r7] also showed in brane and activates protein kinase directly membrane preparations that adenylate [21]. In the fetal heart the inotropic effect of

156

H. O k u d a et al.

dibutyryl cyclic AMP was less than in the newborn (Fig. 5). This finding supports the hypothesis that the diminished response to isoproterenol in the fetus resulted from the difference in the process after the cyclic AMP production. In the present study, enhancement of the relaxation process by isoproterenol in the 21-day fetus, expressed as a shortening of half relaxation time, was less than in the newborn. This suggests that the effect of isoproterenol on the sarcoplasmic reticulum is depressed in the 21-day fetus and this may partly explain the decreased inotropism of isoproterenol. The mechanism for this is not clear but underdevelopment of the sarcoplasmic reticulum in this age-group may be one of the reasons. Since both isoproterenol and dibutyryl cyclic AMP increase the amount of calcium reaching the myofilament, the inotropic effect of calcium per se was examined. In the present study, the inotropic effect of high [Ca]0 in the fetus was diminished. Therefore, it is likely that the decreased inotropic effect of isoproterenol in the fetus may be due, at least in part, to the age-related difference in the effect of calcium. It may be possible that developmental changes in contractile protein may explain the diminished response to cyclic AMP and calcium. Indeed, the studies of Nakanishi and Jarmakani [10] and Page and Buecker [11] indicate that the amount of myofibrils in the fetal rabbit is less than in the newborn and this may explain the lower contractile force in the fetus before and after administration of various inotropic agents. In the present study, however, not only the absolute value but also the relative change of D T after administration of inotropic agents were diminished in the fetus. In our previous study [10], sensitivity of myofibritlar ATPase to calcium did not change with development. Therefore, it is difficult to explain the diminished inotropic effect (relative change of DT) of calcium in the fetus by the difference in the contractile protein.

The precise mechanisms of the diminished inotropism of calcium in the fetus remained unclear but we previously postulated that premature development of the Casequestering system i.e., sarcoplasmic reticulum, T tubular system, and mitochondria in the fetus resulted in relatively high intracellular calcium concentrations under control conditions [10]. The higher cytosolic calcium in the fetus may enhance the contractility under control conditions but may prevent a further increase in contractile function at high [Ca]0. Nishioka et al. [3] showed that the inotropic effect ofisoproterenol and high [Ca]0 in the newborn rabbit was greater than that in the adult. These authors also showed that the inotropic effect of isoproterenol was dependent on the baseline contractility. These studies suggest that the age-related difference in the inotropic effect of isoproterenol is largely explained by the difference in the baseline contractility in the fetus, newborn, and adult rabbit [10]. Finally, in the present study the sensitivity of mechanical function to isoproterenol was similar in the fetus and newborn (Fig. 3). This finding is in agreement with previous mechanical function and biochemical studies [3-5,

7-9]. In conclusion, the present data indicate that in the isolated heart preparation the inotropic effect of isoproterenol in the fetus is less than that in the newborn. The diminished inotropic effect of isoproterenol in the fetus may be due, at least in part, to the underdevelopment of sarcoplasmic reticulum and the decreased inotropism of cyclic AMP and calcium.

Acknowledgement This study was supported by Research Grants 59480241 and 59570411 from the Japanese Ministry of Education, Science, and Culture and a grant-in-aid from J a p a n Research Promotion Society for Cardiovascular Diseases.

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3 NISHIOKA,K., NAKANISHI,T., GEORGE,B. L., JARMAKANI,J. M. The effect of calcium on the inotropy ofcatecholamine and paired electrical stimulation in the newborn and adult myocardium. J Mol Cell Cardiol 13, 511-520 (1981). 4 FRXEDMAN,W. F. The intrinsic physiologic properties of the developing heart. Prog Cardiovasc Dis 15, 87-111 (1972). 5 CHENG,J. B., GOLDFXSH,A., CORNETT, L. E., ROBERTS,J. M. Identification of~-adrenergic receptors using [3H] dehydroalprenolol in fetal sheep heart: direct evidence of qualitative similarity to the receptors in adult sheep heart. Pediatr Res 15, 1083-1087 (1982). 6 KATZ,A. M., DEPKE, D. Calcium-membrane interaction in the myocardium. A m J Cardio131, 193-201 (1973). 7 HATJXS,C. G. Adenylate cyclasc activity in fetal rabbit myocardium. Pediatr Res 19, 75-78 (1985). 8 HATJIS,C. G., McLAUGHLIN,M. K. Identification and ontogenesis of beta-adrenergic receptors in fetal and neonatal rabbit myocardium. J Dev Physiol 4, 327 338 (1982). 9 SCHUMACHER,W. A., SHEPPARD,J. R., MmKIN, B. L. Biological maturation and beta-adrenergic effectors: preand postnatal development of the adenylate cyclase system in the rabbit heart. J Pharmacol Exp Ther 223, 587-593 (1982). 10 NAKANISHI,T.,JARMAKANI,J. M. Developmental changes in myocardial function and subcellular organelles. A m J Physio1246, H615-625 (1984). 11 PAOE, E., BUECKER, J. L. Development of dyadic junctional complexes between sarcoplasmic reticulum and plasmalemma in rabbit left ventricular myocardial cell. Circ Res 48, 519-522 ( 1981). 12 NAKANISm,T., JARMAKANI,J. M. The effect of acetyl strophanthidin on myocardial function and potassium and calcium exchange in the newborn rabbit. Am J Physio1241, H637-645 (1981). 13 NAKANISHI,T., MATUOKA, S., UEMURA,S., SHIMIZU,T., NISHIOKA, K., NEUFELD,N. D., JARMAKANI,J. M. Myocardial excitation-contraction coupling in the fetus of alloxan-diabetic rabbit. Pediatr Res 18, 1344-1349 (1984). 14 NAKANISHI,T., SmMmU, T., UEMURA, S., JARMAKANX,J. M. Ouabain effect on myocardial mechanical function and sodium pump in the fetus. A m J Physio1246, H213 H221 (1984). 15 NAKANtSHI,T., OKUDA, H., NAXAZAWA,M., TAKAO, A. Effect of acidosis on contractile function in the newborn rabbit heart. Pediatr Res 19, 482-488 (1985). 16 TIHON, C., GOREN, M. B., SPITZ, E., RmKENBERO, H. V. Convenient elimination of TCA prior to radioimmunoassay cyclic nucleotides. Ann Biochem 80, 652-653 (1977). 17 STEINER,A. L., PAGLIARA,A. S., CHASE,L. R., KIPNXS,A. M. Radioimmunoassay for cyclic nucleotides. J Biol Chem 247, 1114~1120 (1972). 18 WALLENSTEIN,S., ZUCKER,C. L., FLEISS,J. L. Some statistical methods useful in circulation research. Circ Res 27, 1 4 (1980). 19 RoLPIL T. P., JonEs, C. T., PI~RRV,D. Ultrastructual and enzymatic development of fetal guinea pig heart. A m J Physio1243, H87 H93 (1982). 20 PAGE,E., EARLEY,J., POWER, B. Normal growth ofuhrastructures in rat left ventricular myocardial cells. Circ Res 34, 35, II12-16 (1974). 21 IMAI,S., OTORn, T., TAKEDA, K., KATANO,Y., HoRn, D. Effect of cyclic AMP and dibutyryl cyclic AMP on the heart and coronary circulation. Jpn j Pharmaco120, 499-510 (1974). 22 BUXTON,I. L., BRUIn'ON, L. L. Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. J Biol Chem 258, 10233-10239 (1983).