Age-associated decrease in the catalytic unit activitiy of rat myocardial adenylate cyclase

Mechan~ms of Ageing and Development, 21 (1983) 357-363

357

Elsevier Sdentific PubLishersheland Ltd.

AGE-ASSOCIATED DECREASE IN THE CATALYTIC UNIT ACTIVITY OF RAT MYOCARDIAL ADENYLATE CYCLASE

STEPHEN W. O'CONNOR, PHILIP J. SCARPACE and ITAMAR B. ABRASS Geriatric Research, Education and Clinical Center (GRECC), Sepulveda VA Medical Center, Sepulveda, Califo~ia 91343 (U.S.A.) and Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024 (U.S.A.) (Received June 29th, 1982) (Revision received November 2nd, 1982)

SUMMARY Two techniques were employed to define the site of diminished adenylate cyclase activity observed in aged-rat myocardium. We previously reported no change in betaadrenergic receptor number or affinity but decreased NaF- and 5'-guanylylimidodiphosphate (GppNHp)-stimulated adenylate cyclase activity with age in the rat myocardium. These data suggest a defect in either N-protein or the catalytic unit component of adenylate cyclas¢. N-protein activity was assessed by the ability of myocardial membrane extracts from 3-, 12, and 24-month-old Fischer 344 rats to complement the N-protein deficient cyc- $49 mouse lymphoma cell line. Catalytic unit activity was assessed by the ability of forskolin to stimulate adenylate cyclase in myocardial membranes from young and old rats. The results demonstrate that both N-protein activity and catalytic unit activity are diminished with age in the rat myocardium. However, since N-protein is present in excess relative to catalytic unit, the data are consistent with the hypothesis that the loss of catalytic unit activity accounts for the loss of overall enzyme activity. The effect is tissuespecific, since erythrocyte membranes do not show the same age-dependent loss of adenylate cyclase activity.

Key words: Adenylate cyclase; N-protein; Age; Catalytic unit; Rat myocardium

INTRODUCTION Senescence is associated with the loss of target tissue responsiveness to many hormones [1 ]. These changes are often accompanied by alterations in receptor number and/ or adenylate cyclase activity [2-7]. Specifically, in the rat myocardium the inotropic 0047-6374/83/$03.00

Printed and Publishedin Ireland

© 1983 Elsevier ScientificPublishersIreland Ltd.

358

and chronotropic responsiveness to beta-adrenergic agonists is diminished with aging [8--12]. The intrinsic contractile mechanisms are unaltered with age [8-10,12], suggesting that the diminished responsiveness is a consequence of changes along the hormone stimulated cascade. Both beta-adxenergic receptor number and affinity are unchanged in aging rat hearts [9,13] ; however, we have recently shown a 25% loss in isoproterenol-, GTP-, and fluoride-stimulated adenylate cydase activity as rats age from 3 to 24 months [7]. The data suggest a diminution in adenylate cydase activity not accounted for by receptor changes. This loss in activity could, at least in part, account for the physiological changes seen with age. ,~denylate cyclase is an enzyme complex consisting of at least three components. Located at the outer surface is the receptor component, the specific site for the binding of hormones and neurotransmitters. Distal to the receptor at the inner surface of the membrane is the nucleotide-regulatory protein (N-protein). This is the site at which GTP, GppNHp, and NaF activate the enzyme. The third component, also located at the inner surface of the membrane, is the catalytic unit which converts ATP to cyclic AMP (cAMP) [14]. Since either a loss of N-protein or a loss of catalytic unit could account for the overall changes in adenylate cyclase activity, we have assessed both N-protein and catalytic unit activity independently. Catalytic unR activity was assessed by use of the cardioactive diterpene, forskolin, which stimulates adenylate cyclase by acting directly on the catalytic unit [15]. N-protein activity was assessed by the use of the N.protein deficient clone of the $49 mouse lymphoma cell line (cyc-). NaF, GTP, and horraonestimulated adenylate cyclase activity are absent in cyc- membranes, but can be restored by the addition of N-protein from other sources. Since activity is dependent on the amount of N-protein added, this reconstitution assay can be used to quantify N.protein [16]. Using these methods, we have compared myocardia from 3-, 12-and 24-month-old rats and demonstrated a loss of both N-protein and catalytic unit activity in aging. METHODS

Animals Female Fischer 344 rats of 3, 12, and 24 months of age were obtained from Charles River Breeding Laboratory (North Wilmington, IdA, U.S.A.) under a contract with the National Institute of Aging. Membrane preparations Erythrocytes were obtained by cardiac puncture with a heparinized syringe. The cells were washed in saline and the buffy coat w u removed. The red cell pellet was lysed in 50 vols. of 5 mM phosphate buffer (pH 8.0). The red cell ghosts were centrifuged at 22 000 g for 15 rain, washed twice, and the final pellet was suspended in 18 mM ggC12, 0.08 mM ascorbic acid, 50 mM HE[ES, pH 7.4. Rats were sacrificed by cervical d~location; the hearts were perfused with saline and excised. Myocardial membrar~es were prepared as previously described [7]. The 48 000

359 g pellet, suspended in 25 vols. (w/v) of 18 mM MgC12, 0.08 mM ascorbic acid, 50 mM HEPES, pH 7.4, was used in these studies. The cyc- clone of the $49 mouse lymphoma cell line was obtained from the University of San Francisco cell culture facility and grown in suspension in Dulbecco's modified Eagle Medium with 10% horse serum under 5% CO2. Cells were harvested by centrifugation followed by hypotonic lysis in cold 5 mM Tris, pH 7.4. The lysate was diluted 1:1 with 0.25 M sucrose, 1 mM MgC12, 5 mM Tris HCI, pH 7.4, and homogenized with 10 strokes of a Teflon-tipped pestle at moderate speed. The homogenate was centrifuged at 48 000 g and the pellet was suspended in 18 mM MgCI2, 0.08 mM as~orbic acid, 50 mM HEPES, pH 7.4, and 15% glycerol. Membrane preparations were frozen in liquid nitrogen and stored at -70°C.

A denylate cyclase assay Approximately 75 jag of membrane protein were incubated in a total volume of 150 gl of 8 mM KC1, 1 mM 3-isobutyl-l-methyLxanthine (IBMX), 3 mM dithiothreitol, 0.7 mM EGTA, 6 mM MgCI2, 1 mM ATP, 2.5 mM creatine phosphate, 0.075 mg/ml creatine phosphokinase, 0.4 mg/ml bovine serum albumin, 50 mM HEPES, pH 7.4, for 15 min at 37°C. These incubations had either 10 mM NaF or 0.75-33.3/Al forskolin present as a stimulant~ The reaction was terminated by the addition of 700 91 of cold 14.5 mM EDTA, 50 mM sodium acetate, pH 7.4. Assay tubes were centrifuged at 9000 g and 100 gl of supemate were assayed for cAMP. cAMP was quantified by radioimmunoassay (New England Nuclear, Boston, MA, U.S.A.).

Cyc- complementation assay Heart membrane (2.0 mg/ml) was incubated for 1 h at 4°C in 0.2% lubrol to extract N-protein. Following the incul:ation, the mixture was centrifuged at 48 000 g for 20 min and the supernate containing the extracted N-protein was collected. Heart extract (20 /al) was mixed with an excess of cyc- membrane (4 X 106 cells) and assayed for adenylate cyclase activity with 10 mM NaF. The concentration of lubrol was equal in all tubes. Under these conditions the extent of adenylate cyclase activity was interpreted as a measure of N-protein activity [16]. Proteins were determined by a modification of the biuret method [17]. RESULTS Dose-response curves of forskolin-stimulated adenylate cyclase activity were determined in rat myocardial membranes from 3-, 12-, and 24-month-old rats. A single set of animals is shown in Fig. 1. The Kact (3 X 10 -6 M) of the catalytic unit for forskolin, as indicated by the concentration of forskolin to achieve half-maximal stimulation of adenylate cyclase, is the same for all three age groups. However, the maximal cAMP generation (Vmax) is greatest in the 3-month-old rats, less in the 12-month-old, and least in the 24-month-old rat. Hofstee plots of the data indicate that there is a single

360

6o~-

.~ 50

o

|

; 30

D

IO

20 [FORSKOLIN] (pM)

30

Fig. 1. Forskolin-stimulated adenylate cydue activity in myocLtd~l membranes from 3- (o), 12- (~), and 24- (o) -month-old rats. Data are from a ~ animal in each age group and mprelent the mean

of duplicate determinations. class o f binding sites, the Kaet is u n d u m s e d with age, and total enzyme activity is reduced with age. Adenylate cyclase activity, following maximal forskolin stimulation (33 /~M), from 10 animals in each age group is shown in Table I. There is a 30% decrease in adenylate cyclase activity as rats age from 3 to 24 months. Similarly, N a F (10 m M ) s t i m u l a t i o n of the enzyme from the same set o f rats showed a corresponding loss in adenylate cyclase activity (Table I). TABLE I ADENYLATE CYCLASE ACTIVITY IN MYOCARDIAL AND ERYTHROCYTE MEMBRANES Data represent the mean ± S.E. from I0 animals in each age group.

Age

Adenylate cycl~eactivity (pmoles cAMP per rag protein per rain)

(months: Forskolin (33 t~l)

NaF (10 mM)

57±7 47 ± 6 39 ± 6*

47±3 41 ± 4 37 ± 6*

3 12

55±9 50±8

54±8 51 ± 6

24

50 ± 8

49 ± 6

Myocardium 3 12 24

Erythrocyte

*/7 < 0.025 as determined by two-tailed t-te=t for the difference between the means of the 24- and 3-month-old animals.

361

!6 O.

Q. 2"

O

!

!

5 I0 I~ HEART (pl exfrocl)

,

20

Fig. 2. N-protein activity in myocardial membranes from 3- (o), 12- (~), and 24- (o) -month-old rats. NaF-stimulated adenylate cyclase activity was determined following the addition of 0 - 2 0 ~I of myocardial membrane extract to cyc- membranes derived from 4 X 10 -6 ceils. Data represent duplicate determinations from a single animal in each age group.

In contrast, erythrocyte membranes from aging rats did not demonstrate a decrease in forskolin- of NaF-stimulated activity. The apparent Kact (9 X 10 -6 M) of the catalytic unit for forskolin was the same for the three age groups studied. Forskolin- and NaFstimulated adenylate cyclase activity in the three age groups is shown in Table I. There was no significant change in either forskolin- or NaF~timulated adenylate cyclase activity in erythrocyte membranes from aging rats. Myocardial N-protein activity from 3-, 12-, and 24-month-old rats demonstrated an age-associated decrease. Complementation of cyc" membranes with myocardial extracts from a single set of 3-, 12-, and 24-month-old rats is shown in Fig. 2. Adenylate cyclase TABLE 1I MYOCARDIAL N-PROTEIN ACTIVITY WITH AGE NaF-stimulated adenylate cyclase activity was determined following the addition of 20 lal of myocardial membrane extract to cyc- membranes derived from 4 X 106 cells: Data represent the mean + S .E. of 8 animals in each age group.

Age

Adenylate cyclase activity

(months) fpmoles cAMP per 10' cells per mln) . 3 12 24

8.0 + 0.2 7.1 ± 0.3 6.0 ± 0.6*

*p < 0.01 as determined by two-tailed t-test for the differences between the means of the 24- and 3-month-old animals,

362 activity was directly proportional to added heart extract, demonstrating in this assay that there is an excess of catalytic unit and that N-protein is the rate-limiting component. Myocardial membrane extracts from 3-month-old rats have a greater ability to complement cyc- membranes than membranes from 12-month-old rats, while membranes from 24-month-old rats have the least ability to complement. N-protein activities from $ animals in each age group are shown in Table II. There is a significant decrease in N-protein activity as rats age from 3 to 24 months. DISCUSSION A decrease in agonist-stimulated adenylate cyclase activity has been reported for dopamine and histamine in rabbit brain [2], dopamine in rat brain [3], and isoproterenol in rat adipocytes [4]. These studies demonstrated a parallel loss in receptor number and adenylate cyclase activity, while NaF- and guanine-nucleotide-stimulated activity was unaltered. Others have shown a decrease in agonist-, fluoride., and gnanine-nucleotidestimulated adenylate cyclase activity in aged rat adipocytes, indicating a defect beyond the receptor [5]. We and other investigators have shown that there are no changes in beta-adrenergic receptor number or affinity in the rat heart [9,13]; however, there is a decrease in basal, as well as NaF-, GTP-, and hormone-stimulated adenylate cyclase activity [7]. Since GTP and NaF both stimulate adenylate cyclase activity through N-protein, these experiments were unable to diltingnish between a defect in N.protein or catalytic unit activity. Forskolin has been demonstrated to stimulate directly the catalytic unit of adenylate cyclase [ 15 ]. In this report we demonstrate that forskolin stimulation decreases to the same extent as NaF, GTP and isoproterenol stimulation, suggesting that the loss of catalytic unit activity accounts for the loss of adenylate cyclase activity in aging. There is also an equivalent diminution in the ability of N.protein extracts from older animals to complement cyc- membranes. This could result from N.protein being less extractable from the membranes of older animals, or a decrease in hi.protein activity in the membranes of older animals. The loss of N.protein activity could account for some of the changes in adenylate cyclase activity that have been reported [7]. However, in some tissues N-protein has been demonstrated to be present in excess relative to catalytic unit [14] ; if such a state exists in the myocardium, the changes in N-protein observed here would not account for the loss of adenylate cyclase activity. Although these data do not def'me the difference between a quantitative or functional change in active catalytic unit with age, our previous data demonstrating an unaltered Km for ATP [7] and our present data on an unaltered Kact for forskolin suggest that there is not a qualitative change of active catalytic unit in rat myocardium with age. ACKNOWLEDGEMENT This research was supported by the Medical Research Service of the Veterans Administration.

363 REFERENCES 1 S. Goldstein, Senescence, Grune and Stratton, New York, 1979, pp. 2201-2025. 2 M.H. Makman, H.S. Ahn, L.J. Thai, 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) 177-183. 3 S.K. Puff and L. Volicer, Effect of aging on cyclic AMP and adenylate cyclase and phosphodiesterase activities in the rat corpus striat~m. Mech. Ageing Dev., 6 (1977) 53-58. 4 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. Z Biochem., 90 (1978) 413-419. 5 B. Cooper and R.I. Gregerman, Hormone~ensitive fat cell adenylate cyclase in the rat: influences of growth, cell size and aging. J. Clin. Invest., 57 (1976) 161-168. 6 J.F. Krall, M. Connelly and M.L. Tuck, Evidence for reversibility of age-related decrease in human lymphocyte adenylate cyclase activity. Blochem. Biophys. Res. Commun., 99 (1981) 10281034. 7 S.W. O'Connor, P.J. Scat"pace and I.B. Abrass, Age.associated decrease of adenylate cyclase activity in rat myocardium. Mech. AgeingDev., 16 (1981) 91-95. 8 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. 9 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..l. Physiol., 239 (1980) H 5 0 1 H508. 10 E.G. Lakatta, Perspectives on the aged myocardium. In J. Roberts et al. (eds.), Pharmacological Intervention in the Agir~ Process, Plenum Press, New York, 1978, pp. 147-169. 11 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. 12 E.C. Yin, H.A. Sprugeon, H.C. Greene, E.G. Lakatta and M.L. Weisfeldt, Age-associated decrease in heart rate response to iscproterenol in dogs. Mech. AgeingDev., 10 (1979) 17-25. 13 I.B. Abrass, J.L. Davis and P.J. Scarpace, Isoproterenol responsiveness and myocardial beta adrenergic receptors in young and old rats. Z G~'rontol., 37 (1982) 156-160. 14 M. RodbeU, The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature, 284 (1980) 17-22. 15 K. Seamen and J.W. Daly, Activation of adenylate cyclase by the diterpine forskolin does not require the guanine nucleotide regulatory protein. J. Biol. Chem., 256 (1981) 9799-9801. 16 Z. Farfel, A.S. Brickman, H.R. Kaslow, V.M. Brothers and H.R. Bourne, Defect of receptorcyclase coupling protein in pseudohypopamthyroidism. N. Engl. ~ bled., 303 (1980) 237-242. 17 J.L. Bailey, Miscellaneous analytical methods. In J.L. Barley (ed.), Techniques in Protein Chemistry, Elsevier Publishing Company, New York, 1967, pp. 341-352.