- Email: [email protected]
Age-associated decrease of adenylate cyclase activity in rat myocardium
Mechanisms of Ageing and Development, 16 (1981) 91-95
91
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
AGE-ASSOCIATED DECREASE OF ADENYLATE CYCLASE ACTIVITY IN RAT MYOCARDIUM
STEPHEN W. O'CONNOR, PHILIP J. SCARPACE and ITAMAR B. ABRASS* Geriatric Research, Education and Clinical Center, Veterans Administration Medical Center, Sepulveda, California 91343 (U.S.A.), and Department o f Medicine, University of California, Los Angeles, California 90024 (U.S.A.)
(Received October 14, 1980; in revised form December 31, 1980)
SUMMARY Myocardial inotropic and chronotropic responses to beta-adrenergic agonists are diminished with aging. Since myocardial beta-adrenergic receptors are unaltered with age, we tested the hypothesis that this decreased responsiveness is related to a defect in the adenylate cyclase system. Isoproterenol-sfimulated adenylate cyclase activity was assessed in myocardial membranes from Fischer 344 rats of 3, 12, and 24 months of age. Basal, as well as F-, GTP-, and hormone-stimulated adenylate cyclase activity was decreased by 20-30% with age. The Km of the enzyme for ATP was not found to be statistically different for any age group studied. These data support the hypothesis that the diminished responsiveness seen in senescence is a result of an alteration in either the catalytic subunit or the coupling protein (N) of the adenylate cyclase complex.
INTRODUCTION The loss of responsiveness with age has been demonstrated for many agents including isoproterenol, epinephrine, norepinepherine, cortisol, thyroxine, insulin and estradiol [ 1]. Specifically, myocardial inotropic and chronotropic responsiveness to betaadrenergic agonists is diminished with aging. In isolated cardiac muscle preparations the contractile response to isoproterenol is altered in senescent rats, although the response to increased Ca 2÷ is not altered [2, 3]. Likewise, the chronotropic response to isoproterenol is diminished with age in humans [4, 5] and dogs [4, 6]. Heart rate response to electrical pacing is, however, unaltered with aging [4, 6]. We and others [7, 3] have reported that there are no changes in the density or affinity of myocardial beta-adrenergic receptors with aging as measured by [all] dihydro*Reprint requests should be addressed to: Itamar B. Abrass, M. D., GRECC-11E, Veterans Administration Medical Center, Sepulveda,California 91343, U.S.A.
92 alprenenol binding to the membranes of 3-, 12- and 24-month-old rats. These data suggest that the mechanisms of the age difference in response to catecholamines are distal to the receptor site and lie in the sequence of events that mediate the response between the receptor and contractile elements. To test the hypothesis that the age-related defect is in the adenylate cyclase complex, we assessed myocardial adenylate cyclase activity in Fischer 344 rats of 3, 12, and 24 months of ag.e.
METHODS
Animals Female Fischer 344 rats of 3, 12, and 24 months of age were obtained from Charles River Breeding Laboratory (North Wilmington, MA, U.S.A.) under contract with the National Institute on Aging.
Membrane preparation Rats were sacrificed by cervical dislocation; the hearts were excised, placed in 0.9% NaC1 at 4 °C, and cleaned of adipose tissue, large vessels and dots. All subsequent steps were carried out at 4 °C. Hearts were finely minced in 15 ml of 0.25 M sucrose, 1 mM MgC12, 5 mM Tris'HC1 (pH 7.4). Preparations were exposed to two 10-second bursts of a Tekmar Tissuemizer and homogenized with 10 strokes of a motor-driven Teflon-tipped pestle at moderate speed. The homogenate was centrifuged at 12000 g for 15 minutes and the pellet resuspended in 0.036 volumes (w/v) of 18 mM MgCI2, 0.08 M ascorbic acid, 7 5 mM Tris-HC1 (pH 7.4). The homogenate was filtered through four layers of cheese cloth and the filtrate assayed for adenylate cyclase activity.
Adenylate cyclase assay Approximately 50/ag of membrane protein were incubated in a total volume of 150 /al of 75 mM Tris.HCl (pH 7.4), 8 mM KC1, 7.5 mM theophyUine, 3 mM dithiothreitol, 0.4 mg/ml bovine serum albumin, 0.7 mM EGTA, 6 mM MgC12, 1 mM ATP, 2.5 mM creatine phosphate, 0.075 mg/ml creatine phosphokinase, for 20 minutes at 37 °C. In certain assay conditions either 7 mM NaF, 10-s M GTP, or 10-s M GTP plus 10 - 9 to 10-3 M isoproterenol were included. The reaction was terminated by the addition of 700 /al of cold 14.5 mM EDTA, 50 mM sodium acetate (pH 7.2). Assay tubes were centrifuged at 9000 g and 100/al of supernatant were assayed for cAMP. cAMP was quantified by radioimmunoassay (New England Nuclear, Boston, MA, U.S.A.). Proteins were determined by a modification of the Biuret method [8].
RESULTS Isoproterenol-stimulated adenylate cyclase activity in myocardial membranes from a single experiment is shown in Fig. 1. The apparent Kd (2 × 10-7 M) indicated by the
93
140 .~_120 ~lO0.c_oo-
•
T~ t ~. .06"07
•
~ .04. .03
6o! E
40-' 20-
,,~ .o2.
~ o g ~, 5 -log OSOPROTERENOL](M)
Y,
-2o
-)o
~b
2'0
go
4b
~
6o
.
I/[ATP] (raM) -I
Fig. 1. Isoproterenol-stimulated adenylate cyclase activity in myocardial membranes from 3-month-old (m), 12-month-old (×), and 24-month-old (*) rats. Fig. 2. Lineweaver-Burk plot of the reciprocal of cAMP production vs. the reciprocal of ATP concentration in myocardial membranes from 3-month-old (m), 12-month-old (X) and 24-month-old (o) rats. Assays were performed in the presence of 10-3 M isoproterenol and 10-s M GTP. concentration of isoproterenol to achieve half-maximal stimulation of adenylate cyclase is the same for all three age groups (Fig. 1). The maximal cAMP generation (Vm~x) is greatest in 3-month-old rats, less in 12-month-old and least in 24-month-old rats (Fig. 1). When basal, NaF-, GTP-, and isoproterenol-stimulated activities were assessed, there was a 1 0 - 2 0 % decrease in cAMP production from 3 months to 12 months and a 2 0 - 3 0 % decrease in cAMP production from 3 months to 24 months (Table I). Although both GTP- and hormone-stimulated activities showed a steady decline from 3 to 24 months (R = 0.34 and 0.33, respectively; p < 0.05 by regression analysis), the change from 12 to 24 months was not statistically significant (Student's t-test). The kinetics of adenylate cyclase-ATP interaction were analyzed by LineweaverBurk plots. Figure 2 represents data from a single experiment. There was a progressive decrease in Vrn~,,, calculated from the reciprocal of the ordinate intercept, and no change in K m , calculated from the negative reciprocal of the abscissa intercept, with age. Vm= determined from the mean -+S.E. of five determinations was 71.4 + 7.1 pmoles cAMP per mg protein per minute for 3 months, 65.6 + 7.2 for 12 months and 48.6 + 7.7 for 24 months of age. The Kin, 72 #M, was unchanged with age.
DISCUSSION The data demonstrate a decrease in adenylate cyclase activity with age. The decrease is seen in basal and F--, GTP. and isoproterenol-stimulated adenylate cyclase
94 TABLE I MYOCARDIAL ADENYLATE CYCLASE ACTIVITY 1N F1SCttER 344 RATS Adenylate cyclase activity 3 months protein per min (pmoles cAMP per rag)
Basal FGTP GTP plus isoproterenol
17 ± 2 182 ± 29 55 ± 11 112 ± 20
12 months* (% of 3 months)
24 months* (% o f 3 months)
87 ± 2 * * *
66 ± 8**
80 ± 6** 82 ± 5*** 87 ± 6**
79 ± 9** 74 ± 6*** 80 ± 6***
*Activity was determined as a percentage of the 3-month value in each individual experiment with the data representing the mean +- S.E. of 18 separate determinations. **p < 0.05 by Student paired t. ***p < 0.01 by Student paired t.
activity. These changes occur without a change in beta-adrenergic receptor number or affinity [3, 7]. A decrease in agonist-stimulated adenylate cyclase activity with age has been reported for several tissues including dopamine and histamine in rabbit brain [9], dopamine in rat brain [10], and beta-adrenergic agonists in rat adipocytes [11]. In the above studies, receptor changes paralleled agonist-stimulated adenylate cyclase activity, while fluoride- and guanine nucleotide-stimulated activity were unaltered with age. Data from another group of investigators has demonstrated decreased agonist-, fluoride- and guanine nucleotide.stimulated adenylate cyclase activity in adipocytes from aging rats, suggesting a primary decrease in adenylate cyclase activity [12]. However, these latter investigators did not examine associated receptor changes. Our data suggest a defect in the adenylate cyclase complex not accounted for by receptor changes. Therefore, we suggest that the defect exists in either the nucleotidebinding protein (N) or the catalytic unit of the enzyme complex. The ATP kinetic data with a decreased Vmax and unchanged K m with age suggest a decreased number of active or activated catalytic units which are functionally intact. We are extending these observations to assessment of the functional status of the N protein in light of its critical role in the regulation of adenylate cyclase activity [13].
ACKNOWLEDGEMENTS This research was supported by the Medical Research Service of the Veterans Administration.
95 REFERENCES 1 G. S. Roth, Changes in hormone binding and responsiveness in target cells and tissues during aging. Adv. Exp. Med. Biol., 61 (1975) 195-208. 2 E. G. Lakatta, G. Gerstenblith, C. S. Angell, N. W. Shock and M. L. Weisfeldt, Diminished inotropic response of aged myocardium to catecholamines. Circ. Res., 36 (1975) 262-269. 3 T. Guarnieri, C. R. Filburn, G. Zitnik, G. S. Roth and E. G. Lakatta, Contractile and biochemical correlates of ~-adrenergic stimulation of the aged heart. Am. J. Physiol., 239 (1980) HS01-H508. 4 E. G. Lakatta, Perspectives on the aged myocardium. In J. Roberts et al. (eds.), Pharmacological Intervention in the Aging Process, Plenum Press, New York, 1978, pp. 147-169. 5 R. E. Vestal, A. J. J. Wood and D. G. Shand, Reduced beta-adrenoreceptor sensitivity in the elderly. Clin. Pharmacol. Ther., 26 (1979) 181-186. 6 F. C. P. Yin, H. A. Sprugeon, H. C. Greene, E. G. Lakatta and M. L. Weisfeldt, Age-associated decrease in heart rate response to isoproterenol in dogs. Mech. Ageing Dev., 10 (1979) 17-25. 7 P. J. Scarpace and I. B. Abrass, Beta-adrenergic receptors in aged rats. Fed. Proc., 38 (1979) 361. 8 J. L. Bailey, Miscellaneous analytical methods. In J. L. Bailey, (ed.), Techniques in Protein Chemistry, Elsevier Publishing Company, New York, 1967, pp. 341-352. 9 M. H. Makman, H. S. Ahn, L. J. Thal, N. S. Sharpless, B. Dvorkin, S. G. Horowitz and M. Rosenfeld, Evidence for selective loss of brain dopamine and histamine stimulated adenylate cyclase activities in rabbits with aging. Brain Res., 192 (1980) 177-1'83. 10 S. K. Puri and L. Volicer, Effect of aging on cyclic AMP and adenylate cyclase and phosphodiesterase activities in the rat corpus striatum.Mech. AgeingDev., 6 (1977) 53-58. 11 Y. Giudicelli and R. Pequery, Beta-adrenergic receptors and catecholamine sensitive adenylate cyclase in rat fat cell membranes: influence of growth, cell size, and aging. Eur. J. Biochem., 90 (1978)413-419. 12 B. Cooper and R. I. Gregerman, Hormone-sensitive fat cell adenylate cyclase in the rat: influences of growth, cell size and aging. Z Clin. Invest., 57 (1976) 161-168. 13 M. RodbeU, The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature, 284 (1980) 17-22.
91
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
AGE-ASSOCIATED DECREASE OF ADENYLATE CYCLASE ACTIVITY IN RAT MYOCARDIUM
STEPHEN W. O'CONNOR, PHILIP J. SCARPACE and ITAMAR B. ABRASS* Geriatric Research, Education and Clinical Center, Veterans Administration Medical Center, Sepulveda, California 91343 (U.S.A.), and Department o f Medicine, University of California, Los Angeles, California 90024 (U.S.A.)
(Received October 14, 1980; in revised form December 31, 1980)
SUMMARY Myocardial inotropic and chronotropic responses to beta-adrenergic agonists are diminished with aging. Since myocardial beta-adrenergic receptors are unaltered with age, we tested the hypothesis that this decreased responsiveness is related to a defect in the adenylate cyclase system. Isoproterenol-sfimulated adenylate cyclase activity was assessed in myocardial membranes from Fischer 344 rats of 3, 12, and 24 months of age. Basal, as well as F-, GTP-, and hormone-stimulated adenylate cyclase activity was decreased by 20-30% with age. The Km of the enzyme for ATP was not found to be statistically different for any age group studied. These data support the hypothesis that the diminished responsiveness seen in senescence is a result of an alteration in either the catalytic subunit or the coupling protein (N) of the adenylate cyclase complex.
INTRODUCTION The loss of responsiveness with age has been demonstrated for many agents including isoproterenol, epinephrine, norepinepherine, cortisol, thyroxine, insulin and estradiol [ 1]. Specifically, myocardial inotropic and chronotropic responsiveness to betaadrenergic agonists is diminished with aging. In isolated cardiac muscle preparations the contractile response to isoproterenol is altered in senescent rats, although the response to increased Ca 2÷ is not altered [2, 3]. Likewise, the chronotropic response to isoproterenol is diminished with age in humans [4, 5] and dogs [4, 6]. Heart rate response to electrical pacing is, however, unaltered with aging [4, 6]. We and others [7, 3] have reported that there are no changes in the density or affinity of myocardial beta-adrenergic receptors with aging as measured by [all] dihydro*Reprint requests should be addressed to: Itamar B. Abrass, M. D., GRECC-11E, Veterans Administration Medical Center, Sepulveda,California 91343, U.S.A.
92 alprenenol binding to the membranes of 3-, 12- and 24-month-old rats. These data suggest that the mechanisms of the age difference in response to catecholamines are distal to the receptor site and lie in the sequence of events that mediate the response between the receptor and contractile elements. To test the hypothesis that the age-related defect is in the adenylate cyclase complex, we assessed myocardial adenylate cyclase activity in Fischer 344 rats of 3, 12, and 24 months of ag.e.
METHODS
Animals Female Fischer 344 rats of 3, 12, and 24 months of age were obtained from Charles River Breeding Laboratory (North Wilmington, MA, U.S.A.) under contract with the National Institute on Aging.
Membrane preparation Rats were sacrificed by cervical dislocation; the hearts were excised, placed in 0.9% NaC1 at 4 °C, and cleaned of adipose tissue, large vessels and dots. All subsequent steps were carried out at 4 °C. Hearts were finely minced in 15 ml of 0.25 M sucrose, 1 mM MgC12, 5 mM Tris'HC1 (pH 7.4). Preparations were exposed to two 10-second bursts of a Tekmar Tissuemizer and homogenized with 10 strokes of a motor-driven Teflon-tipped pestle at moderate speed. The homogenate was centrifuged at 12000 g for 15 minutes and the pellet resuspended in 0.036 volumes (w/v) of 18 mM MgCI2, 0.08 M ascorbic acid, 7 5 mM Tris-HC1 (pH 7.4). The homogenate was filtered through four layers of cheese cloth and the filtrate assayed for adenylate cyclase activity.
Adenylate cyclase assay Approximately 50/ag of membrane protein were incubated in a total volume of 150 /al of 75 mM Tris.HCl (pH 7.4), 8 mM KC1, 7.5 mM theophyUine, 3 mM dithiothreitol, 0.4 mg/ml bovine serum albumin, 0.7 mM EGTA, 6 mM MgC12, 1 mM ATP, 2.5 mM creatine phosphate, 0.075 mg/ml creatine phosphokinase, for 20 minutes at 37 °C. In certain assay conditions either 7 mM NaF, 10-s M GTP, or 10-s M GTP plus 10 - 9 to 10-3 M isoproterenol were included. The reaction was terminated by the addition of 700 /al of cold 14.5 mM EDTA, 50 mM sodium acetate (pH 7.2). Assay tubes were centrifuged at 9000 g and 100/al of supernatant were assayed for cAMP. cAMP was quantified by radioimmunoassay (New England Nuclear, Boston, MA, U.S.A.). Proteins were determined by a modification of the Biuret method [8].
RESULTS Isoproterenol-stimulated adenylate cyclase activity in myocardial membranes from a single experiment is shown in Fig. 1. The apparent Kd (2 × 10-7 M) indicated by the
93
140 .~_120 ~lO0.c_oo-
•
T~ t ~. .06"07
•
~ .04. .03
6o! E
40-' 20-
,,~ .o2.
~ o g ~, 5 -log OSOPROTERENOL](M)
Y,
-2o
-)o
~b
2'0
go
4b
~
6o
.
I/[ATP] (raM) -I
Fig. 1. Isoproterenol-stimulated adenylate cyclase activity in myocardial membranes from 3-month-old (m), 12-month-old (×), and 24-month-old (*) rats. Fig. 2. Lineweaver-Burk plot of the reciprocal of cAMP production vs. the reciprocal of ATP concentration in myocardial membranes from 3-month-old (m), 12-month-old (X) and 24-month-old (o) rats. Assays were performed in the presence of 10-3 M isoproterenol and 10-s M GTP. concentration of isoproterenol to achieve half-maximal stimulation of adenylate cyclase is the same for all three age groups (Fig. 1). The maximal cAMP generation (Vm~x) is greatest in 3-month-old rats, less in 12-month-old and least in 24-month-old rats (Fig. 1). When basal, NaF-, GTP-, and isoproterenol-stimulated activities were assessed, there was a 1 0 - 2 0 % decrease in cAMP production from 3 months to 12 months and a 2 0 - 3 0 % decrease in cAMP production from 3 months to 24 months (Table I). Although both GTP- and hormone-stimulated activities showed a steady decline from 3 to 24 months (R = 0.34 and 0.33, respectively; p < 0.05 by regression analysis), the change from 12 to 24 months was not statistically significant (Student's t-test). The kinetics of adenylate cyclase-ATP interaction were analyzed by LineweaverBurk plots. Figure 2 represents data from a single experiment. There was a progressive decrease in Vrn~,,, calculated from the reciprocal of the ordinate intercept, and no change in K m , calculated from the negative reciprocal of the abscissa intercept, with age. Vm= determined from the mean -+S.E. of five determinations was 71.4 + 7.1 pmoles cAMP per mg protein per minute for 3 months, 65.6 + 7.2 for 12 months and 48.6 + 7.7 for 24 months of age. The Kin, 72 #M, was unchanged with age.
DISCUSSION The data demonstrate a decrease in adenylate cyclase activity with age. The decrease is seen in basal and F--, GTP. and isoproterenol-stimulated adenylate cyclase
94 TABLE I MYOCARDIAL ADENYLATE CYCLASE ACTIVITY 1N F1SCttER 344 RATS Adenylate cyclase activity 3 months protein per min (pmoles cAMP per rag)
Basal FGTP GTP plus isoproterenol
17 ± 2 182 ± 29 55 ± 11 112 ± 20
12 months* (% of 3 months)
24 months* (% o f 3 months)
87 ± 2 * * *
66 ± 8**
80 ± 6** 82 ± 5*** 87 ± 6**
79 ± 9** 74 ± 6*** 80 ± 6***
*Activity was determined as a percentage of the 3-month value in each individual experiment with the data representing the mean +- S.E. of 18 separate determinations. **p < 0.05 by Student paired t. ***p < 0.01 by Student paired t.
activity. These changes occur without a change in beta-adrenergic receptor number or affinity [3, 7]. A decrease in agonist-stimulated adenylate cyclase activity with age has been reported for several tissues including dopamine and histamine in rabbit brain [9], dopamine in rat brain [10], and beta-adrenergic agonists in rat adipocytes [11]. In the above studies, receptor changes paralleled agonist-stimulated adenylate cyclase activity, while fluoride- and guanine nucleotide-stimulated activity were unaltered with age. Data from another group of investigators has demonstrated decreased agonist-, fluoride- and guanine nucleotide.stimulated adenylate cyclase activity in adipocytes from aging rats, suggesting a primary decrease in adenylate cyclase activity [12]. However, these latter investigators did not examine associated receptor changes. Our data suggest a defect in the adenylate cyclase complex not accounted for by receptor changes. Therefore, we suggest that the defect exists in either the nucleotidebinding protein (N) or the catalytic unit of the enzyme complex. The ATP kinetic data with a decreased Vmax and unchanged K m with age suggest a decreased number of active or activated catalytic units which are functionally intact. We are extending these observations to assessment of the functional status of the N protein in light of its critical role in the regulation of adenylate cyclase activity [13].
ACKNOWLEDGEMENTS This research was supported by the Medical Research Service of the Veterans Administration.
95 REFERENCES 1 G. S. Roth, Changes in hormone binding and responsiveness in target cells and tissues during aging. Adv. Exp. Med. Biol., 61 (1975) 195-208. 2 E. G. Lakatta, G. Gerstenblith, C. S. Angell, N. W. Shock and M. L. Weisfeldt, Diminished inotropic response of aged myocardium to catecholamines. Circ. Res., 36 (1975) 262-269. 3 T. Guarnieri, C. R. Filburn, G. Zitnik, G. S. Roth and E. G. Lakatta, Contractile and biochemical correlates of ~-adrenergic stimulation of the aged heart. Am. J. Physiol., 239 (1980) HS01-H508. 4 E. G. Lakatta, Perspectives on the aged myocardium. In J. Roberts et al. (eds.), Pharmacological Intervention in the Aging Process, Plenum Press, New York, 1978, pp. 147-169. 5 R. E. Vestal, A. J. J. Wood and D. G. Shand, Reduced beta-adrenoreceptor sensitivity in the elderly. Clin. Pharmacol. Ther., 26 (1979) 181-186. 6 F. C. P. Yin, H. A. Sprugeon, H. C. Greene, E. G. Lakatta and M. L. Weisfeldt, Age-associated decrease in heart rate response to isoproterenol in dogs. Mech. Ageing Dev., 10 (1979) 17-25. 7 P. J. Scarpace and I. B. Abrass, Beta-adrenergic receptors in aged rats. Fed. Proc., 38 (1979) 361. 8 J. L. Bailey, Miscellaneous analytical methods. In J. L. Bailey, (ed.), Techniques in Protein Chemistry, Elsevier Publishing Company, New York, 1967, pp. 341-352. 9 M. H. Makman, H. S. Ahn, L. J. Thal, N. S. Sharpless, B. Dvorkin, S. G. Horowitz and M. Rosenfeld, Evidence for selective loss of brain dopamine and histamine stimulated adenylate cyclase activities in rabbits with aging. Brain Res., 192 (1980) 177-1'83. 10 S. K. Puri and L. Volicer, Effect of aging on cyclic AMP and adenylate cyclase and phosphodiesterase activities in the rat corpus striatum.Mech. AgeingDev., 6 (1977) 53-58. 11 Y. Giudicelli and R. Pequery, Beta-adrenergic receptors and catecholamine sensitive adenylate cyclase in rat fat cell membranes: influence of growth, cell size, and aging. Eur. J. Biochem., 90 (1978)413-419. 12 B. Cooper and R. I. Gregerman, Hormone-sensitive fat cell adenylate cyclase in the rat: influences of growth, cell size and aging. Z Clin. Invest., 57 (1976) 161-168. 13 M. RodbeU, The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature, 284 (1980) 17-22.