- Email: [email protected]
Decreased response with age of the cardiac catecholamine sensitive adenylate cyclase system
Life Sciences, Vol. 33, pp. 1679-1686 Printed in the U.S.A.
Pergamon Press
DECREASED RESPONSE WITH AGE OF THE CARDIAC CATFCHOLAMINE SENSITIVE ADENYLATE CYCLASE SYSTEM John W. Kusiak and Josef Pitha Macromolecular Chemistry Section, Gerontology Research Center, National Institute on Aging, NIH, Baltimore, Maryland 21224 (Received in final form August 9, 1983)
The cardiac $-adrenergic coupled adenylate cyclase system was examined in young and old male Wistar rats. The concentration of binding sites for (-)3H-DHA in membranes prepared from cardiac ventricles was 21.1 + 2.78 (SD) fmoles/mg protein in 3-4 month old rats (young rats) and 3i.2 + 2.20 fmoles/mg protein in 24 month old rats (old rats). The dissociation constant, I$ was 4.3 + 1.8 nM and 6.7 + 1.7 nM for young and old rats, respectively. Varyous compounds were used to study the characteristics of activation of adenylate cyclase in homogenates from cardiac ventricles. Basal adenylate cyclase was reduced 30% in old animals compared to young (6.1 pmoles/min/mg protein in 24 month vs. 8.6 pmoles/min/mg protein in 3-4 month). (-)Is0proterenol (10m5M) alone stimulated adenylate cyclase greater than two-fold in young rats (10.6 pmoles/min/mg protein above basal) and this stimulation was 34% lower in old animals. GppNHp (100 PM), fluoride (10 mM), and forskolin (100 PM) activation of adenylate cyclase above basal was reduced 38, 37, and 34%, respectively, in the old animals. No significant changes between the two groups were noted in the apparent affinity of GppNHp either alone or in the presence of (-)isoproterenol nor in the affinities of catecholamine agonists for activation of cyclase. These results suggest a reduction in the amount of functional regulatory protein or possibly cyclase in 24 month old rat ventricular tissue compared to 3-4 month old tissue. However, this data does not rule out the possibility of altered molecular interactions of a full complement of regulatory protein(s) with t3adrenergic receptor and/or catalytic adenylate cyclase. One effect of age on cardiac function is the diminished response of cardiac tissue to stimulation by catecholamines (l-3). The molecular mechanism by which catecholamines stimulate cardiac (inotropic and chronotropic) activity involves the $-adrenergic coupled adenylate cyclase system (E.C.4.6.1.1 ATP pyrophosphate-lyase [cyclizingl). Theories explaining the reduced response of cardiac tissue with age include reductions in one or more of the components of this adenylate cyclase system, or alterations/reductions of components involved in the elaboration of the catecholamine effect subsequent to cyclase activation. Lakatta et al. (1) and Guarnieri et al. (2) using Wistar rats proposed that the reduced inotropic responses to catecholamines in aged tissue might involve impaired mobilization of calcium. In a recent report, Abrass et al. (3) using Fisher 344 rats, concluded that the impairment of chronotropic responses to (-)isoproterenol in old rats is at a step subsequent to catecholamine interaction with B-adrenergic receptors.
0024-3205183 $3.00 + .OO
1680
Aging and the Adenylate Cyclase System
Vol. 33, No. 17, 1983
In related studies, Schocken and Roth (4) reported a negative correlation of $-adrenoceptor number in membranes from human mononuclear lymphocytes with age. However, Abrass and Scarpace (5) using intact human lymphocytes, found no differences in receptor number in an elderly (64 yrs. mean) and young (24 yrs. mean) population and Landmann et al. (6) found similar numbers of 8,adrenoceptors in homogenates of human mononuclear leucocytes from populafions of young and old donors. These latter two groups suggested that the reduced hormonal response of tissues in the elderly was not due to impaired 8-receptor function. Furthermore, Era11 et al. (7,8) reported an age related decrease in the response of human lymphocyte adenylate cyclase to (-)isoproterenol and guanine nucleotide. They suggested that an age related decrease in guanine nucleotide regulatory subunit(s) (variously referred to as G or N protein or G/F) or adenylate cyclase catalytic subunits might be responsible for diminished hormonal responses in these cells and by extrapolation to other catecholamine sensitive tissues. Narayanan and Derby (9) recently reported impairments in agonist-receptor interactions and in guanine nucleotide regulation of adenylate cyclase in cardiac membrane preparations of aged Sprague-Dawley rats. Their results and interpretations were in agreement with work on lymphocytes that suggested impairments beyond the level of the B-receptor. However, their membrane preparations showed only 4-S% increase in adenylate cyclase activity in response to 10 l.@l(-)isoproterenol suggesting that some necessary components of the system were absent in their preparation of membranes from both young and old rats. In fact, we (manuscript submitted) and others (lo-121 showed a large loss of (-)isoproterenol sensitive adenylate cyclase activity in cardiac membrane preparations and the necessity for soluble components in maintaining full enzymatic activity in the presence of (-)isoproterenol. Furthermore, Narayanan and Derby (9) did not report apparent affinities of (-jisoproterenol, fluoride, or guanine nucleotides for activation of cyclase. We undertook the present study to examine age-related changes in the cardiac catecholamine-sensitive adenylate cyclase system using membrane preparations for binding studies and homogenates for adenylate cyclase activity. We were interested specifically in examining the effects of agents that were thought to by-pass 8-adrenergic receptors in the stimulation of cyclase and exert their influence either directly on cyclase or through regulatory proteins of this complex. We examined in detail the 8-adrenergic coupled adenylate cyclase system of young (3-4 man) and old (24 man) male Wistar rats and suggest that the diminished adenylate cyclase activity noted in old rats might be due to a decrease in the number of functional regulatory proteins. However, we cannot rule out at this time the possibility of abnormal interactions of these regulatory proteins with either receptors, catalytic units or both. Methods 3 Materials: 3H-cyclic AMP (34.5 Ci/mMol, H-cAMP) and 3H-dihydroalprenolo1-HC1 (49.0 Ci/mMol, 3H-DHA) were from New England Nuclear, Boston, MA. 32P-c-ATP (450 Ci/mMol) was from ICN, Irvine, CA. (-)Isoproterenol(+)bitartrate, (-)epinephrine(+)bitartrate, (-jnorepinephrine-HCl, 5'-guanylylimidodiphosphate (GPPNHP)I ATP, CAMP, and phosphocreatine were from Sigma Chemical Co., St. Louis, MO. Creatine kinase was from Boehringer-Mannheim, Indianapolis, IN, (+)alprenolol-HCl from Hlssle, MiSlndal, Sweden, and forskolin was from CalBiochem-Behring Corp., LaJolla, CA. Rats were male Wistar 3-4 months old and 24 months old from the Animals : Animal Resources Facility of the Gerontology Research Center, which is fully accredited by the American Association for Accreditation of Laboratory Animal Care.
Vol. 33, No. 17, 1983
Aging and the Adenylate Cyclase System
1681
3 H-DHA Binding Assay: Cardiac ventricular membranes were prepared and binding assays were carried out exactly as previously described (13,141. Total 3HDHA binding to these membranes was measured in duplicate at eleven different concentrations of 3H-DHA between 1 and 20 nM. Non-specific binding was measured in the presence of 10 W (+)alprenolol and the specific binding was analyzed by the method of Scatchard-( Adenylate Cyclase Assay: Hat heart homogenates were prepared according to Minneman et al. (10). Adenylate cyclase was measured in duplicate 10 minute assays at 30°C. Approximately 100 pg of rat heart homogenate protein was incubated in a total volume of 100 ul of 50 mM Tris-IiCl,pH 7.5, containing: 0.1 mM 32P-a-ATP(2.23 x 106dpm/assay), 1 mM 3H-cAMP(4-5 x 103cpm/assay), 1 IIM MgCl2, 0.5 mM ethyleneglycol-bis-(B-aminoethylether)-N,N,N',N'-tetraacetic acid, 0.75 ti isobutylmethylxanthine, 1 mM ascorbate, 0.1 mM dithiothreitol, 10 nM phosphocreatine, and 0.1 mg/ml creatine kinase. 32P-cAMP was isolated by the two column method of Salomon et al. (16) using the modification and regeneration procedures of Iyengar et al. (17). Results Scatchard analysis of 3H-DHA binding data in ventricular membranes indicated that the maximal concentration of binding sites (E&ax) in old rats was slightly increased (31.2 + 2.20 S.D. fmcl/mg protein in old rats vs. 21.1 + 2.78 fmol/mg protein in young, n = 3 in each group, p < .Ol student's t test fzr two means). The dissociation constants (I$-,) were 6.7 + 1.7 nM and 4.3 + 1.8 nM in old and young, respectively and were not significantly different f&n each other. The Bmax and Ic,values were calculated from lines derived by the method of least squares and regression coefficients were greater than 0.86. Basal adenylate cyclase in cardiac ventricular homogenates from old rats was reduced 30% from that of young rats (TABLE I). Similarly, maximal (-)isoproterenol and GppNHp stimulated cyclase activity was reduced 34% and 38% in old rats (TABLE I). Furthermore, the activation of cyclase by 10 m&l fluoride ion and 100 uM forskolin was also reduced by 37% and 34%, respectively. The TABLE I Adenylate Cyclase Activity in Young and Old Rat Heart Homogenates Additions
None (Basal) 10 nM (-Jisoproterenol 108 F1MGPPNHP 5 mM Fluoride 10 r&d Fluoride 10 uM Forskolin 100 llMForskolin
pmol/mg/min protein CAMP old young 8.6 10.6 13.1 8.0 10.4 86.0 126.3
+ 1.7 ‘i 4.9 T 3.3 T 3.2 + 3.6 + 18.8 7 24.2 -
6.1 + 1.9* 7.0 7 3.2 8.17 2.7* 4.5 + 2.3 6.6 T 3.1 48.4 T15.8* 82.9 7 2.58*
% decrease in Old
30 34 38 43 37 44 34
Adenylate cyclase was assayed as described in the Methods without (Basal) or with the additions listed above. The results are the means + 1 standard deviation of at least four separate experiments. The absolute values of the cyclase assay to which additions were made represent activity above that with nothing added (i.e., above Basal).
* Statistically significant at p < .05.
1682
Aging and the Adenylate Cyclase System
Vol. 33, No. 17, 1983
shape of the dose-response curves for these last two effecters appeared to be similar with only a reduced response in the old rats. Fluoride ion stimulation at 10 mM was slightly less than the maximal stimulation observed by GppNHp in the respective tissues (TABLE I). The affinity of catecholamine agonists for stimulation of cyclase was reduced only slightly in old animals (Fig. 1). The dose-response curves for
1.33.59x10-’ 1.6%.34x10-~
-log
[AGONIST]
FIG. 1. Dose-response curves for catecholamine sensitive adenylate cyclase. Adenylate cyclase activity was measured as described in the Methods with the addition of increasing amounts of (-)isoproterenol 0 - 0, (-1epinephrine O-0, or (-lnorepinephrine A - A, in either young (left) or old (right) cardiac ventricular homogenates. Apparent Ka values are the concentrations of agonists stimulating cyclase to 50% of the maximal stimulation above basal. Results are the averages of four experiments + S.E.M. (-Jisoproterenol, (-jepinephrine, and (-lnorepinephrine were right-shifted only to a minor extent in the old rats which was statistically insignificant. Each agonist showed the same fold stimulation in the young and the >ld set of homogenates, however, the maximum activity in the old was reduced 34%. Figure 2 shows the apparent affinity of GppNHp alone and in the presence of 10 uM (-)isoproterenol. The apparent Ka's were slightly right-shifted in the old animals (statistically insignificant). Discussion It is well established that with age there is a diminished response of cardiac tissue to catecholamines in both man and rodents. What remains unresolved is the molecular mechanism(s) by which this diminution occurs. Indeed, only recently has broad agreement been reached on the molecular mechanism of
Aging and the Adenylate Cyclase System
Vol. 33, No. 17, 1983
1683
action of the $-adrenergic coupled adenylate cyclase system which is thought to mediate the hormonal/neurotransmitter response. Most of the recent evidence including this report is in agreement that the diminished response of cardiac tissue to catecholamines is not due to a loss of f3-adrenoceptors. We have
GppNHp 1.06-‘0.55 x10-5 GppNHp+k) IS0 1.490.51x 10-0 676543
676543 - IogIGppNHpl
- IogIGppNHpl
FIG. 2. Dose-response curves for guanine nucleotide stimulation of adenylate cyclase activity. Cyclase activity was measured as described in the Methods with &cheaddition of GppNHp alone 0 - 0, or together with 10 ?JM (-)isoproterenol A - A, in either young (left) or old (right) homogenates. Apparent I$,values were calculated as described in the legend to Fig. 1. Results are the averages of four experiments + S.E.M. 3 measured a slight increase in H-DHA binding sites in cardiac membranes of old Wistar rats. Narayanan and Derby (9) and Abrass et al. (3) have also measured slight increases in old Sprague-Dawley and Fisher 344 rats, respectively. In human lymphocytes there also appear to be no changes in receptor number with age (5-7) and it was thought that these cells may reflect receptor numbers in other catecholamine sensitive tissues. A recent review by Roth and Hess (18) catalogues receptor changes with age in the catecholaminergic system and other hormonal/neurotransmitter systems. Consistent with these results are the studies by Lakatta and co-workers (1,2,19) which suggest that the age related defect in cardiac contractile response to catecholamines lies beyond the level of the receptor and may involve differences in the phosphorylation of proteins in the hormonal cascade or altered calcium mobilization both resulting from normal catecholamine-receptor interaction. Our present results clearly demonstrate an age-related defect in the cardiac f3-adrenergic-coupledadenylate cyclase system. Our conclusion that there is diminished efficacy of the regulatory factor(s) is in basic agreement with a recently published report by Narayanan and Derby (9) also done with cardiac tissue but from Sprague-Dawley rats. However, based upon the minimal
1684
Aging and the Adenylate Cyclase System
Vol. 33, No. 17, 1983
changes in our dose-response curves for agonists and nucleotide stimulation of cyclase and the consistent effects of fluoride, forskolin, and GppNHp on activation of cyclase in old animals (approximately 65% of young), we propose that the defect in the aged system may possibly be a reduction in the number of functional regulatory proteins. There were only slight, statistically insignificant right-shifts in the dose-response curves for catecholamine agonists (Fig. 1) and GppNHp (Fig. 2) on adenylate cyclase activity, which suggest that regulatory protein which is present is interacting normally with receptor and cyclase. Since the maximal cyclase activity was reduced 35% in each case, this suggests that there is a reduction in the amount of regulatory protein available for functional interaction with the other components. Furthermore, each of the activators of cyclase which we tested stimulated cyclase activity to 65% of that in young heart homogenates (Table I). There is good evidence and general agreement that GppNHp (20-22) and fluoride ion (23) activate adenylate cyclase via regulatory proteins independently of receptor occupation. Forskolin, a diterpene, was originally thought to act directly on the catalytic unit (24) but recent evidence (25,26) in human platelets and S49 lymphoma cells seems to suggest a requirement for the regulatory protein. Because of the differences in the structures of these activators and because forskolin activates cyclase approximately lo-fold more than either fluoride or GppNHp (both of which activate cyclase about 2-fold) they all probably stimulate cyclase at different sites and/or by different mechanisms involving the same regulatory protein(s). Therefore, it would seem that the reduced activation seen in old rats by each of these compounds, approximately 35% in each case, is due to a reduction in the amount of regulatory protein and not to abnormal interactions with catalytic subunit. It would seem highly coincidental, although possible, that the activation or modification of regulatory protein by different mechanisms would, in each case, reduce cyclase activity by 35% in old rats unless there was a reduction in the amount of regulatory protein interacting with the cyclase. Recent evidence (27) indicates that the number of regulatory proteins may be similar to the number of $-adrenoceptors in cardiac tissue suggesting that a diminution in the number of regulatory components may reduce the response of tissue to catecholamines. This phenomenon probably would not be observed if regulatory proteins were in large excess in this tissue. Furthermore, in the disease state pseudohypoparathyroidism, it has been hypothesized (28,291 that there is a generalized deficiency of regulatory protein in target organs as measured by either complementation assay or by ADP ribosylation of regulatory protein by cholera toxin, substantiating the point that deficiencies of the regulatory protein occur. Alternatively, regulatory protein concentrations may be normal in aged hearts but the cellular location may be changed, i.e., less in association with the membrane and more located within the cell milieu reducing the actual amount associated with the catalytic subunit. The data could also indicate altered interactions of regulatory protein with the cyclase or altered kinetics of nucleotide interaction with the regulatory protein (i.e., rate of off reaction of GDP or rate of GTP hydrolysis). Furthermore, although untestable at this time, catalytic subunits may be deficient in the old rats. One approach to differentiating these seemingly conflicting hypotheses would be to quantitate regulatory subunits and catalytic subunits in young and old tissue by titration with antibody. Also, the interpretation of these results is limited by the different theories of regulation of adenylate cyclase activity by guanine nucleotides and receptors (30-33). The interpretation of the results of Narayanan and Derby (9) must be done with caution, since the membrane preparations they utilized to measure adenylate cyclase activity, were devoid of cyclase activity in the presence of (-)iso-
Vol. 33, No. 17, 1983
Aging and the Adenylate Cyclase System
1685
proterenol. As mentioned previously, we (manuscript submitted) and others (10-12) have noted large decreases in (-)isoproterenol stimulated cyclase activity when extensively washed membrane preparations are used. Therefore, we feel that our results with heart homogenates are more reliably interpreted in terms of a regulatory protein deficiency. The results of Narayanan and Derby (9) may reflect a differential removal of essential soluble or extrinsic membrane components of the cyclase complex which are critical for the regulation of cyclase catalytic subunit. In lieu of our criticisms, we are also skeptical of the significance of the binding data although our experiments (not reported here) confirm their measurements of reduced affinities of agonists for displacing specific 3H-DHA binding. (See also ref. 34 on differences in 8receptor adenylate cyclase systems of fragmented membranes and cells.) We also think that the species difference is insignificant since some of our preliminary results with young and old Sprague-Dawley rat heart homogenates were very similar to our results reported here with Wistar rats. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
E.G. LAKATTA, G. GERSTENBLITH, C.S. ANGELL, N.W. SHOCK, and M.L. WEISFELDT., Circ. Res. 36 262-269 (1975). T. GUARNIER~ C.R. FILBURN, G. ZITNIK, G.S. ROTH, and E.G. LAKATTA, Am. J. Physiol. 239 H501-H508 (1980). 1-B. ABRASS,yL. DAVIS, and P.J. SCARPACE, J. Gerontol. 37 156-160 (1982). D.D. SCHOCKEN and G.S. ROTH, Nature 267 856-858 (1977). I.B. ABRASS and P.J. SCARPACE, J. GeGtol. 36 298-301 (1981). R. LANDMANN, H. BITTIGER, and F.R. BUHLER, L%e Sci. 29 1761-1771 (1981). J.F. KRALL, M. CONNELLY, and M.L. TUCK, Biochem. BiopFs. Res. Commun. -99 1028-1034 (1981). J.F. KRALL, M. CONNELLY, R. WEISBART, and M.L. TUCK, J. Clin. Endocrinol. Metabol. 52 863-867 (1981). N. NARAYG and J. DERBY, Mech. Ageing Dev. 19 127-139 (1982). K.P. MINNEMAN, L.R. HEGSTRAND, and P.B. MOLINZF, Mol. Pharmacol. -16 2133 (1979). G. LE FUR, P.H. SCHMELCK, P. GBYNET, J. HANOUNE, and A. UZAN, Life Sci. 23 1841-1850 (1978). c KRISHNA BHAT, R. IYENGAR, J. ABRAMOWITZ, M.E. BORDELON-RISER, and L. BIRNBAUMER, Proc. Natl. Acad. Sci. USA 77 3836-3840 (1980). J.W. KUSIAK and J. PITHA, Biochem. Pha&col. 31 2071-2076 (1982). J. PITHA, J. MILECKI, T. CZAIKOWSKA, and J.W. ESIAK, J. Ned. Chem. 26 7-11 (1983). G. SCATCHARD, Ann. NY Acad. Sci. 11.660-672 (1949). Y. SALOMCN, C. LONDDS, and M. RODBELL, Anal. Biochem. 58 541-548 (1974). R. IYENGAR, J. ABRAMOWITZ, M. BORDELON-RISER, A.J. BL&, and L. BIRNBAUMER, J. Biol. Chem. 255 10312-10321 (1980). G.S. ROTH and G.D. HESS, Mech. Ageing Dev. 20 175-194 (1982). E.G. LAKATTA, Fed. Proc. 39 3173-3177 (198Or J.K. NORTHUP, P.C. STERNW?%, M.D. SMIGEL, L.S. SCHLEIFER, E.M. ROSS, and A.G. GILMAN, Proc. Natl. Acad. Sci. USA 77 6516-6520 (1980). J.M. STADEL, R.G.L. SHORR, L.E. LIMBIRD,Gd R.J. LEFKOWITZ, J. Biol. Chem. 256 8718-8723 (1981). P.C. STEFNWEIS, J.K. NORTHUP, M.D. SMIGEL, and A.G. GILMAN, J. Biol. Chem. 256 11517-11526 (1981). R.W. DOWNS, JR., A.M. SPIEGEL, M. SINGER, S. REEN, and G.D. AURBACH, J. Biol. Chem. 255 949-954 (1980). K. SEAMON and J.W. DALY, J. Biol. Chem. 256 9799-9801 (1981). P.A. INSEL, D. STENGEL, N. FERRY, and J.?&OUNE, J. Biol. Chem. 257 7485-7490 (1982). F.J. DARFLER, L.C. NAHAN, A.M. KOACHMAN, and P.A. INSEL, J. Biol. Chem. -257 11901-11907 (1982).
1686
27. 28. 29. 30. 31. 32. 33. 34.
Aging and the Adenylate Cyclase System
Vol. 33, No. 17, 1983
S.P. EiAKER and L.T. POTTER, J. Biol. Chem. 256 7925-7931 (1981). M.A. LEVINE, R.W. DOWNS, JR., M. SINGER, S.rklARX, G.D. AURBACH, and A.M. SPIEGEL, Biochem. Biophys. Res. Commun. 94 1319-1324 (1980). 2. FARFEL, V.M. BROTHERS, A.S. BRICKMAN, F. Ck, R. NEER, and H.R. BOURNE, Proc. Natl. Acad. Sci. USA 78 3098-3102 (1981). A.M. TQLKOVSKY and A. LEVITZKI, J. cclic Nucleotide Res. 7 139-150 (1981). L.E. LIMBIRD, Biochem. J. -195 l-13 (1981). A. DE LEAN, J.M. STADEL, and R.J. LEFKOWITZ, J. Biol. Chem. -255 7108-7117 (1980). L. BIRNBAUMER, C.F. BEARER, and R. IYENGAR, J. Biol. Chem. -255 3552-3557 (1980). H. PORZIG, Trends in Pharmacol. Sci. 2 75-78 (1982).
Pergamon Press
DECREASED RESPONSE WITH AGE OF THE CARDIAC CATFCHOLAMINE SENSITIVE ADENYLATE CYCLASE SYSTEM John W. Kusiak and Josef Pitha Macromolecular Chemistry Section, Gerontology Research Center, National Institute on Aging, NIH, Baltimore, Maryland 21224 (Received in final form August 9, 1983)
The cardiac $-adrenergic coupled adenylate cyclase system was examined in young and old male Wistar rats. The concentration of binding sites for (-)3H-DHA in membranes prepared from cardiac ventricles was 21.1 + 2.78 (SD) fmoles/mg protein in 3-4 month old rats (young rats) and 3i.2 + 2.20 fmoles/mg protein in 24 month old rats (old rats). The dissociation constant, I$ was 4.3 + 1.8 nM and 6.7 + 1.7 nM for young and old rats, respectively. Varyous compounds were used to study the characteristics of activation of adenylate cyclase in homogenates from cardiac ventricles. Basal adenylate cyclase was reduced 30% in old animals compared to young (6.1 pmoles/min/mg protein in 24 month vs. 8.6 pmoles/min/mg protein in 3-4 month). (-)Is0proterenol (10m5M) alone stimulated adenylate cyclase greater than two-fold in young rats (10.6 pmoles/min/mg protein above basal) and this stimulation was 34% lower in old animals. GppNHp (100 PM), fluoride (10 mM), and forskolin (100 PM) activation of adenylate cyclase above basal was reduced 38, 37, and 34%, respectively, in the old animals. No significant changes between the two groups were noted in the apparent affinity of GppNHp either alone or in the presence of (-)isoproterenol nor in the affinities of catecholamine agonists for activation of cyclase. These results suggest a reduction in the amount of functional regulatory protein or possibly cyclase in 24 month old rat ventricular tissue compared to 3-4 month old tissue. However, this data does not rule out the possibility of altered molecular interactions of a full complement of regulatory protein(s) with t3adrenergic receptor and/or catalytic adenylate cyclase. One effect of age on cardiac function is the diminished response of cardiac tissue to stimulation by catecholamines (l-3). The molecular mechanism by which catecholamines stimulate cardiac (inotropic and chronotropic) activity involves the $-adrenergic coupled adenylate cyclase system (E.C.4.6.1.1 ATP pyrophosphate-lyase [cyclizingl). Theories explaining the reduced response of cardiac tissue with age include reductions in one or more of the components of this adenylate cyclase system, or alterations/reductions of components involved in the elaboration of the catecholamine effect subsequent to cyclase activation. Lakatta et al. (1) and Guarnieri et al. (2) using Wistar rats proposed that the reduced inotropic responses to catecholamines in aged tissue might involve impaired mobilization of calcium. In a recent report, Abrass et al. (3) using Fisher 344 rats, concluded that the impairment of chronotropic responses to (-)isoproterenol in old rats is at a step subsequent to catecholamine interaction with B-adrenergic receptors.
0024-3205183 $3.00 + .OO
1680
Aging and the Adenylate Cyclase System
Vol. 33, No. 17, 1983
In related studies, Schocken and Roth (4) reported a negative correlation of $-adrenoceptor number in membranes from human mononuclear lymphocytes with age. However, Abrass and Scarpace (5) using intact human lymphocytes, found no differences in receptor number in an elderly (64 yrs. mean) and young (24 yrs. mean) population and Landmann et al. (6) found similar numbers of 8,adrenoceptors in homogenates of human mononuclear leucocytes from populafions of young and old donors. These latter two groups suggested that the reduced hormonal response of tissues in the elderly was not due to impaired 8-receptor function. Furthermore, Era11 et al. (7,8) reported an age related decrease in the response of human lymphocyte adenylate cyclase to (-)isoproterenol and guanine nucleotide. They suggested that an age related decrease in guanine nucleotide regulatory subunit(s) (variously referred to as G or N protein or G/F) or adenylate cyclase catalytic subunits might be responsible for diminished hormonal responses in these cells and by extrapolation to other catecholamine sensitive tissues. Narayanan and Derby (9) recently reported impairments in agonist-receptor interactions and in guanine nucleotide regulation of adenylate cyclase in cardiac membrane preparations of aged Sprague-Dawley rats. Their results and interpretations were in agreement with work on lymphocytes that suggested impairments beyond the level of the B-receptor. However, their membrane preparations showed only 4-S% increase in adenylate cyclase activity in response to 10 l.@l(-)isoproterenol suggesting that some necessary components of the system were absent in their preparation of membranes from both young and old rats. In fact, we (manuscript submitted) and others (lo-121 showed a large loss of (-)isoproterenol sensitive adenylate cyclase activity in cardiac membrane preparations and the necessity for soluble components in maintaining full enzymatic activity in the presence of (-)isoproterenol. Furthermore, Narayanan and Derby (9) did not report apparent affinities of (-jisoproterenol, fluoride, or guanine nucleotides for activation of cyclase. We undertook the present study to examine age-related changes in the cardiac catecholamine-sensitive adenylate cyclase system using membrane preparations for binding studies and homogenates for adenylate cyclase activity. We were interested specifically in examining the effects of agents that were thought to by-pass 8-adrenergic receptors in the stimulation of cyclase and exert their influence either directly on cyclase or through regulatory proteins of this complex. We examined in detail the 8-adrenergic coupled adenylate cyclase system of young (3-4 man) and old (24 man) male Wistar rats and suggest that the diminished adenylate cyclase activity noted in old rats might be due to a decrease in the number of functional regulatory proteins. However, we cannot rule out at this time the possibility of abnormal interactions of these regulatory proteins with either receptors, catalytic units or both. Methods 3 Materials: 3H-cyclic AMP (34.5 Ci/mMol, H-cAMP) and 3H-dihydroalprenolo1-HC1 (49.0 Ci/mMol, 3H-DHA) were from New England Nuclear, Boston, MA. 32P-c-ATP (450 Ci/mMol) was from ICN, Irvine, CA. (-)Isoproterenol(+)bitartrate, (-)epinephrine(+)bitartrate, (-jnorepinephrine-HCl, 5'-guanylylimidodiphosphate (GPPNHP)I ATP, CAMP, and phosphocreatine were from Sigma Chemical Co., St. Louis, MO. Creatine kinase was from Boehringer-Mannheim, Indianapolis, IN, (+)alprenolol-HCl from Hlssle, MiSlndal, Sweden, and forskolin was from CalBiochem-Behring Corp., LaJolla, CA. Rats were male Wistar 3-4 months old and 24 months old from the Animals : Animal Resources Facility of the Gerontology Research Center, which is fully accredited by the American Association for Accreditation of Laboratory Animal Care.
Vol. 33, No. 17, 1983
Aging and the Adenylate Cyclase System
1681
3 H-DHA Binding Assay: Cardiac ventricular membranes were prepared and binding assays were carried out exactly as previously described (13,141. Total 3HDHA binding to these membranes was measured in duplicate at eleven different concentrations of 3H-DHA between 1 and 20 nM. Non-specific binding was measured in the presence of 10 W (+)alprenolol and the specific binding was analyzed by the method of Scatchard-( Adenylate Cyclase Assay: Hat heart homogenates were prepared according to Minneman et al. (10). Adenylate cyclase was measured in duplicate 10 minute assays at 30°C. Approximately 100 pg of rat heart homogenate protein was incubated in a total volume of 100 ul of 50 mM Tris-IiCl,pH 7.5, containing: 0.1 mM 32P-a-ATP(2.23 x 106dpm/assay), 1 mM 3H-cAMP(4-5 x 103cpm/assay), 1 IIM MgCl2, 0.5 mM ethyleneglycol-bis-(B-aminoethylether)-N,N,N',N'-tetraacetic acid, 0.75 ti isobutylmethylxanthine, 1 mM ascorbate, 0.1 mM dithiothreitol, 10 nM phosphocreatine, and 0.1 mg/ml creatine kinase. 32P-cAMP was isolated by the two column method of Salomon et al. (16) using the modification and regeneration procedures of Iyengar et al. (17). Results Scatchard analysis of 3H-DHA binding data in ventricular membranes indicated that the maximal concentration of binding sites (E&ax) in old rats was slightly increased (31.2 + 2.20 S.D. fmcl/mg protein in old rats vs. 21.1 + 2.78 fmol/mg protein in young, n = 3 in each group, p < .Ol student's t test fzr two means). The dissociation constants (I$-,) were 6.7 + 1.7 nM and 4.3 + 1.8 nM in old and young, respectively and were not significantly different f&n each other. The Bmax and Ic,values were calculated from lines derived by the method of least squares and regression coefficients were greater than 0.86. Basal adenylate cyclase in cardiac ventricular homogenates from old rats was reduced 30% from that of young rats (TABLE I). Similarly, maximal (-)isoproterenol and GppNHp stimulated cyclase activity was reduced 34% and 38% in old rats (TABLE I). Furthermore, the activation of cyclase by 10 m&l fluoride ion and 100 uM forskolin was also reduced by 37% and 34%, respectively. The TABLE I Adenylate Cyclase Activity in Young and Old Rat Heart Homogenates Additions
None (Basal) 10 nM (-Jisoproterenol 108 F1MGPPNHP 5 mM Fluoride 10 r&d Fluoride 10 uM Forskolin 100 llMForskolin
pmol/mg/min protein CAMP old young 8.6 10.6 13.1 8.0 10.4 86.0 126.3
+ 1.7 ‘i 4.9 T 3.3 T 3.2 + 3.6 + 18.8 7 24.2 -
6.1 + 1.9* 7.0 7 3.2 8.17 2.7* 4.5 + 2.3 6.6 T 3.1 48.4 T15.8* 82.9 7 2.58*
% decrease in Old
30 34 38 43 37 44 34
Adenylate cyclase was assayed as described in the Methods without (Basal) or with the additions listed above. The results are the means + 1 standard deviation of at least four separate experiments. The absolute values of the cyclase assay to which additions were made represent activity above that with nothing added (i.e., above Basal).
* Statistically significant at p < .05.
1682
Aging and the Adenylate Cyclase System
Vol. 33, No. 17, 1983
shape of the dose-response curves for these last two effecters appeared to be similar with only a reduced response in the old rats. Fluoride ion stimulation at 10 mM was slightly less than the maximal stimulation observed by GppNHp in the respective tissues (TABLE I). The affinity of catecholamine agonists for stimulation of cyclase was reduced only slightly in old animals (Fig. 1). The dose-response curves for
1.33.59x10-’ 1.6%.34x10-~
-log
[AGONIST]
FIG. 1. Dose-response curves for catecholamine sensitive adenylate cyclase. Adenylate cyclase activity was measured as described in the Methods with the addition of increasing amounts of (-)isoproterenol 0 - 0, (-1epinephrine O-0, or (-lnorepinephrine A - A, in either young (left) or old (right) cardiac ventricular homogenates. Apparent Ka values are the concentrations of agonists stimulating cyclase to 50% of the maximal stimulation above basal. Results are the averages of four experiments + S.E.M. (-Jisoproterenol, (-jepinephrine, and (-lnorepinephrine were right-shifted only to a minor extent in the old rats which was statistically insignificant. Each agonist showed the same fold stimulation in the young and the >ld set of homogenates, however, the maximum activity in the old was reduced 34%. Figure 2 shows the apparent affinity of GppNHp alone and in the presence of 10 uM (-)isoproterenol. The apparent Ka's were slightly right-shifted in the old animals (statistically insignificant). Discussion It is well established that with age there is a diminished response of cardiac tissue to catecholamines in both man and rodents. What remains unresolved is the molecular mechanism(s) by which this diminution occurs. Indeed, only recently has broad agreement been reached on the molecular mechanism of
Aging and the Adenylate Cyclase System
Vol. 33, No. 17, 1983
1683
action of the $-adrenergic coupled adenylate cyclase system which is thought to mediate the hormonal/neurotransmitter response. Most of the recent evidence including this report is in agreement that the diminished response of cardiac tissue to catecholamines is not due to a loss of f3-adrenoceptors. We have
GppNHp 1.06-‘0.55 x10-5 GppNHp+k) IS0 1.490.51x 10-0 676543
676543 - IogIGppNHpl
- IogIGppNHpl
FIG. 2. Dose-response curves for guanine nucleotide stimulation of adenylate cyclase activity. Cyclase activity was measured as described in the Methods with &cheaddition of GppNHp alone 0 - 0, or together with 10 ?JM (-)isoproterenol A - A, in either young (left) or old (right) homogenates. Apparent I$,values were calculated as described in the legend to Fig. 1. Results are the averages of four experiments + S.E.M. 3 measured a slight increase in H-DHA binding sites in cardiac membranes of old Wistar rats. Narayanan and Derby (9) and Abrass et al. (3) have also measured slight increases in old Sprague-Dawley and Fisher 344 rats, respectively. In human lymphocytes there also appear to be no changes in receptor number with age (5-7) and it was thought that these cells may reflect receptor numbers in other catecholamine sensitive tissues. A recent review by Roth and Hess (18) catalogues receptor changes with age in the catecholaminergic system and other hormonal/neurotransmitter systems. Consistent with these results are the studies by Lakatta and co-workers (1,2,19) which suggest that the age related defect in cardiac contractile response to catecholamines lies beyond the level of the receptor and may involve differences in the phosphorylation of proteins in the hormonal cascade or altered calcium mobilization both resulting from normal catecholamine-receptor interaction. Our present results clearly demonstrate an age-related defect in the cardiac f3-adrenergic-coupledadenylate cyclase system. Our conclusion that there is diminished efficacy of the regulatory factor(s) is in basic agreement with a recently published report by Narayanan and Derby (9) also done with cardiac tissue but from Sprague-Dawley rats. However, based upon the minimal
1684
Aging and the Adenylate Cyclase System
Vol. 33, No. 17, 1983
changes in our dose-response curves for agonists and nucleotide stimulation of cyclase and the consistent effects of fluoride, forskolin, and GppNHp on activation of cyclase in old animals (approximately 65% of young), we propose that the defect in the aged system may possibly be a reduction in the number of functional regulatory proteins. There were only slight, statistically insignificant right-shifts in the dose-response curves for catecholamine agonists (Fig. 1) and GppNHp (Fig. 2) on adenylate cyclase activity, which suggest that regulatory protein which is present is interacting normally with receptor and cyclase. Since the maximal cyclase activity was reduced 35% in each case, this suggests that there is a reduction in the amount of regulatory protein available for functional interaction with the other components. Furthermore, each of the activators of cyclase which we tested stimulated cyclase activity to 65% of that in young heart homogenates (Table I). There is good evidence and general agreement that GppNHp (20-22) and fluoride ion (23) activate adenylate cyclase via regulatory proteins independently of receptor occupation. Forskolin, a diterpene, was originally thought to act directly on the catalytic unit (24) but recent evidence (25,26) in human platelets and S49 lymphoma cells seems to suggest a requirement for the regulatory protein. Because of the differences in the structures of these activators and because forskolin activates cyclase approximately lo-fold more than either fluoride or GppNHp (both of which activate cyclase about 2-fold) they all probably stimulate cyclase at different sites and/or by different mechanisms involving the same regulatory protein(s). Therefore, it would seem that the reduced activation seen in old rats by each of these compounds, approximately 35% in each case, is due to a reduction in the amount of regulatory protein and not to abnormal interactions with catalytic subunit. It would seem highly coincidental, although possible, that the activation or modification of regulatory protein by different mechanisms would, in each case, reduce cyclase activity by 35% in old rats unless there was a reduction in the amount of regulatory protein interacting with the cyclase. Recent evidence (27) indicates that the number of regulatory proteins may be similar to the number of $-adrenoceptors in cardiac tissue suggesting that a diminution in the number of regulatory components may reduce the response of tissue to catecholamines. This phenomenon probably would not be observed if regulatory proteins were in large excess in this tissue. Furthermore, in the disease state pseudohypoparathyroidism, it has been hypothesized (28,291 that there is a generalized deficiency of regulatory protein in target organs as measured by either complementation assay or by ADP ribosylation of regulatory protein by cholera toxin, substantiating the point that deficiencies of the regulatory protein occur. Alternatively, regulatory protein concentrations may be normal in aged hearts but the cellular location may be changed, i.e., less in association with the membrane and more located within the cell milieu reducing the actual amount associated with the catalytic subunit. The data could also indicate altered interactions of regulatory protein with the cyclase or altered kinetics of nucleotide interaction with the regulatory protein (i.e., rate of off reaction of GDP or rate of GTP hydrolysis). Furthermore, although untestable at this time, catalytic subunits may be deficient in the old rats. One approach to differentiating these seemingly conflicting hypotheses would be to quantitate regulatory subunits and catalytic subunits in young and old tissue by titration with antibody. Also, the interpretation of these results is limited by the different theories of regulation of adenylate cyclase activity by guanine nucleotides and receptors (30-33). The interpretation of the results of Narayanan and Derby (9) must be done with caution, since the membrane preparations they utilized to measure adenylate cyclase activity, were devoid of cyclase activity in the presence of (-)iso-
Vol. 33, No. 17, 1983
Aging and the Adenylate Cyclase System
1685
proterenol. As mentioned previously, we (manuscript submitted) and others (10-12) have noted large decreases in (-)isoproterenol stimulated cyclase activity when extensively washed membrane preparations are used. Therefore, we feel that our results with heart homogenates are more reliably interpreted in terms of a regulatory protein deficiency. The results of Narayanan and Derby (9) may reflect a differential removal of essential soluble or extrinsic membrane components of the cyclase complex which are critical for the regulation of cyclase catalytic subunit. In lieu of our criticisms, we are also skeptical of the significance of the binding data although our experiments (not reported here) confirm their measurements of reduced affinities of agonists for displacing specific 3H-DHA binding. (See also ref. 34 on differences in 8receptor adenylate cyclase systems of fragmented membranes and cells.) We also think that the species difference is insignificant since some of our preliminary results with young and old Sprague-Dawley rat heart homogenates were very similar to our results reported here with Wistar rats. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
E.G. LAKATTA, G. GERSTENBLITH, C.S. ANGELL, N.W. SHOCK, and M.L. WEISFELDT., Circ. Res. 36 262-269 (1975). T. GUARNIER~ C.R. FILBURN, G. ZITNIK, G.S. ROTH, and E.G. LAKATTA, Am. J. Physiol. 239 H501-H508 (1980). 1-B. ABRASS,yL. DAVIS, and P.J. SCARPACE, J. Gerontol. 37 156-160 (1982). D.D. SCHOCKEN and G.S. ROTH, Nature 267 856-858 (1977). I.B. ABRASS and P.J. SCARPACE, J. GeGtol. 36 298-301 (1981). R. LANDMANN, H. BITTIGER, and F.R. BUHLER, L%e Sci. 29 1761-1771 (1981). J.F. KRALL, M. CONNELLY, and M.L. TUCK, Biochem. BiopFs. Res. Commun. -99 1028-1034 (1981). J.F. KRALL, M. CONNELLY, R. WEISBART, and M.L. TUCK, J. Clin. Endocrinol. Metabol. 52 863-867 (1981). N. NARAYG and J. DERBY, Mech. Ageing Dev. 19 127-139 (1982). K.P. MINNEMAN, L.R. HEGSTRAND, and P.B. MOLINZF, Mol. Pharmacol. -16 2133 (1979). G. LE FUR, P.H. SCHMELCK, P. GBYNET, J. HANOUNE, and A. UZAN, Life Sci. 23 1841-1850 (1978). c KRISHNA BHAT, R. IYENGAR, J. ABRAMOWITZ, M.E. BORDELON-RISER, and L. BIRNBAUMER, Proc. Natl. Acad. Sci. USA 77 3836-3840 (1980). J.W. KUSIAK and J. PITHA, Biochem. Pha&col. 31 2071-2076 (1982). J. PITHA, J. MILECKI, T. CZAIKOWSKA, and J.W. ESIAK, J. Ned. Chem. 26 7-11 (1983). G. SCATCHARD, Ann. NY Acad. Sci. 11.660-672 (1949). Y. SALOMCN, C. LONDDS, and M. RODBELL, Anal. Biochem. 58 541-548 (1974). R. IYENGAR, J. ABRAMOWITZ, M. BORDELON-RISER, A.J. BL&, and L. BIRNBAUMER, J. Biol. Chem. 255 10312-10321 (1980). G.S. ROTH and G.D. HESS, Mech. Ageing Dev. 20 175-194 (1982). E.G. LAKATTA, Fed. Proc. 39 3173-3177 (198Or J.K. NORTHUP, P.C. STERNW?%, M.D. SMIGEL, L.S. SCHLEIFER, E.M. ROSS, and A.G. GILMAN, Proc. Natl. Acad. Sci. USA 77 6516-6520 (1980). J.M. STADEL, R.G.L. SHORR, L.E. LIMBIRD,Gd R.J. LEFKOWITZ, J. Biol. Chem. 256 8718-8723 (1981). P.C. STEFNWEIS, J.K. NORTHUP, M.D. SMIGEL, and A.G. GILMAN, J. Biol. Chem. 256 11517-11526 (1981). R.W. DOWNS, JR., A.M. SPIEGEL, M. SINGER, S. REEN, and G.D. AURBACH, J. Biol. Chem. 255 949-954 (1980). K. SEAMON and J.W. DALY, J. Biol. Chem. 256 9799-9801 (1981). P.A. INSEL, D. STENGEL, N. FERRY, and J.?&OUNE, J. Biol. Chem. 257 7485-7490 (1982). F.J. DARFLER, L.C. NAHAN, A.M. KOACHMAN, and P.A. INSEL, J. Biol. Chem. -257 11901-11907 (1982).
1686
27. 28. 29. 30. 31. 32. 33. 34.
Aging and the Adenylate Cyclase System
Vol. 33, No. 17, 1983
S.P. EiAKER and L.T. POTTER, J. Biol. Chem. 256 7925-7931 (1981). M.A. LEVINE, R.W. DOWNS, JR., M. SINGER, S.rklARX, G.D. AURBACH, and A.M. SPIEGEL, Biochem. Biophys. Res. Commun. 94 1319-1324 (1980). 2. FARFEL, V.M. BROTHERS, A.S. BRICKMAN, F. Ck, R. NEER, and H.R. BOURNE, Proc. Natl. Acad. Sci. USA 78 3098-3102 (1981). A.M. TQLKOVSKY and A. LEVITZKI, J. cclic Nucleotide Res. 7 139-150 (1981). L.E. LIMBIRD, Biochem. J. -195 l-13 (1981). A. DE LEAN, J.M. STADEL, and R.J. LEFKOWITZ, J. Biol. Chem. -255 7108-7117 (1980). L. BIRNBAUMER, C.F. BEARER, and R. IYENGAR, J. Biol. Chem. -255 3552-3557 (1980). H. PORZIG, Trends in Pharmacol. Sci. 2 75-78 (1982).