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Forskolin activation of adenylate cyclase in rat myocardium with age: Effects of guanine nucleotide analogs
Mechanisms of Ageing and Development, 52 (1990) 169-178
169
Elsevier ScientificPublishers Ireland Ltd.
FORSKOLIN ACTIVATION OF ADENYLATE CYCLASE IN RAT MYOCARDIUM WITH AGE: EFFECTS OF GUANINE NUCLEOTIDE ANALOGS
PHILIP J. SCARPACE Geriatric Research, Education and Clinical Center, Veterans Administration Medical Center, Gainesville, FL 32602, and Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610 (U.S.A.)
(Received May 27th, 1989) (Revision receivedJuly 11th, 1989)
SUMMARY Beta-adrenergic and post-receptor activation of adenylate cyclase decreases with age in the rat myocardium. Forskolin-stimulated adenylate cyclase activity decreases with age to the same extent as NaF or isoproterenol stimulation, suggesting that a loss of catalytic unit activity accounts for the loss of activity with age. However, recent evidence suggests that there are both a guanine nucleotidedependent and independent component of forskolin activation. We assessed the possible role of each of these components with age by measuring forskolin doseresponse stimulation of adenylate cyclase activity in the presence and absence of guanosine 5'-O-(2-thiodiphosphate) (GDP-13S) and 13,~-imidoguanine 5'-triphosphate (Gpp(NH)p) in myocardial membranes from F-344 rats of 3, 12, and 24 months of age. Maximal forskolin stimulation of adenylate cyclase was least in the 24- (121 - 21 pmol cAMP/min/mg) as compared with either the 12- (212 - 29) or 3- (190 - 19) month-old rats. The presence of GDP-13S had no effect on either the ECs0 or maximum activity, and the age-related decline persisted. Gpp(NH)p plus isoproterenol enhanced ~ c t i , : a t i ~ but the effect was additive and not synergistic. There was no effect on the ECso~andthe maximum activity was least in the 24-month-old rats. These data reconfirm the~loss of catalytic unit activity with age and indicate that the reduced activity with age is independent of the presence or absence of activated G-protein. K e y words: Aging; Catalytic unit; Rat Address all correspondence and reprint requests to: Philip J. Scarpace, Ph.D., GRECC (182), VA
Medical Center, Gainesville. FL 32602, U.S.A. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990Elsevier ScientificPublishers Ireland Ltd.
170 INTRODUCTION
Adenylate cyclase is part of a receptor-enzyme complex that consists of three components [1]. Located at the outer membrane surface are both stimulatory and inhibitory receptors, each containing a specific binding site for hormones or neurotransmitters. The second component of the complex is a group of guanine nucleotide-binding regulatory proteins (G-proteinS~). These proteins, termed G, and Gi, mediate the transmission of the hormone signal from the receptor to the catalyst and are involved in both the stimulation and inhibition of adenylate cyclase activity, respectively [2]. They are thought to exist as heterotrimers, with G~ and Gi apparently having similar 13 and ~/subunits and a unique a subunit [2]. The Gproteins appear to oscillate between two activity states, an inactive GDP-bound form and an active GTP-bound form. Stimulatory hormones activate the G-proteins by promoting the replacement of bound GDP by GTP, in the G-protein subunit [2]. The GTP-a subunit interacts with the third component of the enzyme complex, the catalytic unit, to form an active enzyme that is the site of the conversion of ATP to cAMP. Beta-adrenergic and post-receptor activation of adenylate cyclase activity decreases with age in the rat myocardium [3]. This loss of activity may contribute to the diminished capacity of the myocardium to respond to catecholamines in senescence [4]. Isoproterenol activates adenylate cyclase through the beta-adrenergic receptor by the mechanism described above. NaF, GTP and the GTP analog, 13,~imidoguanine 5'-triphosphate (Gpp(NH)p), increase adenylate cyclase activity by a post-receptor activation of G-protein. Forskolin, a cardioactive diterpene from the roots of Coleus forskohlii also activates adenylate cyclase by a post-receptor mechanism. It was previously believed that forskolin directly activated the catalytic unit and did not require the presence of a functional G-protein [5]. This laboratory previously reported that forskolin-stimulated adenylate cyclase activity decreases with age to the same extent as NaF or isoproterenol stimulation and based on the tenet that forskolin solely activates the catalytic unit concluded that a loss of catalytic unit activity fully accounts for the loss of adenylate cyclase activity with age [6]. However, recent evidence indicates that forskolin activation of adenylate cyclase can be enhanced by guanine nucleotides [5]. It has been proposed that when the catalytic unit is associated with the activated Gs, the extent of forskolin activation is greater than with the catalytic unit alone [5]. Furthermore, in some tissues guanine nucleotide plus stimulatory hormone reduced the EC.~ofor forskolin activation, suggesting that forskolin interacts with the catalytic unit with high affinity in the presence of activated Gs and with low affinity in the absence of G~ [7]. These effects can be blocked by GDP or its analog, guanosine 5'-0(2-thiodiphosphate) (GDP-13S) . . . . Thus the extent of forskolin activation with age wo~ld be influenced by the presence of any endogenous guanine nucleotides and/or ai~ activated G~. G-protein activity, when assessed by complementation with the G-protein-deficient $49 cyc cell line, is reduced in rat myocardium from older rats [6].\ If forskolin-stimulated adenylate cyclase partially reflects activation through a G~-eatalytic unit complex,
171
then the decreased adenylate cyclase activity with age might possibly be due to the reduced G-protein function with age. A second possibility is that the ability of the activated Gs-catalytic unit complex to enhance forskolin stimulation is diminished with age. In consideration of the above discussion, the age-related activation of myocardial adenylate cyclase by forskolin has been reinvestigated in greater detail. Forskolin activation curves were determined in the presence of the inhibitory and stimulatory guanine nucleotide analogs, GDP-13S and Gpp(NH)p, respectively, in myocardial membrane from rats of 3, 12, and 24 months of age. METHODS
Animals Female F-344 NNia rats of 3, 12, and 24 months of age (received Jan. and June 1988) were obtained from Harlan Industries (Indianapolis, IN) under contract with the National Institute on Aging. Upon arrival, rats were examined and housed individually in 7"x 10" stainless steel cages. All animals were maintained on Purina rat chow ad libitum with a 12-h light/dark cycle: Rats were used within 4 weeks after their arrival. No ill animals were noted or used. No serological or bacteriological tests were performed.
Membrane preparation At sacrifice, the circulatory system was perfused with 20-ml cold saline and the hearts excised. Tissues were finely minced, disrupted in a tissuemizer for 30 s, and homogenized with 10 strokes of a motor-driven pestle. Homogenates were passed through four layers of cheesecloth and centrifuged at 48000g for 15 min at 4°C. Pellets were resuspended in 18 mM MgCI2, 0.08 mM ascorbic acid, and 50 mM 4-(2-hydroxethyl)-I piperazineethanesulfonic acid (HEPES) (pH 7.4.)
Adenylate cyclase assay Adenylate cyclase activity was assayed by the conversion of ATP into cAMP in the presence of 1 mM isobutylmethylxanthine and an ATP regenerating system as described by Scarpace and Abrass [8]. Approximately 75 I~g of membrane protein plus stimulators as indicated were incubated for 15 min at 37°C in a total volume of 150 ~I. The reaction was terminated by addition of 700 I~1 of cold 5.3 mM CaCI2 and immersion into a salt-ice bath. Assay tubes were centrifuged at 9000 g and the supernatant assayed for cAMP. cAMP was quantified by radioimmunoassay. Protein was determined by the method of Bradford [9]. RESULTS Figure 1 shows that GDP-13S partially blocks the activation of adenylate cyclase by forskolin in heart membranes from Sprague-Dawley rats. In the presence of GDP-13S there is a 69% decrease in maximum stimulation by forskolin and a 76% increase in the ECso for forskolin activation.
172
200.
i
100. f
0..
o
~
+b
"
ab
I+o
Fig. 1. Forskolin dose-response curves for adenylate cyclase activation in h©art membranes from Sprague-Dawley rats of 3 months of age. ECso values are 17.1 and 30.5 I~M, respectively, in the absence ([]) and presence (A) of 500-1~M GDP-13S. Data represents the mean - SE of 3 animals. Lines were determined by non-linear least squares analysis.
These observations prompted a reinvestigation of the age-related decrease in forskolin activation of adenylate cyclase in heart membranes from F-344 rats. However, as opposed to the heart membranes from Sprague-Dawley rats, GDP13S had no effect on forskolin activation of adenylate cyclase in membranes from F-344 rats at any age (Fig. 2 and Table I). In both the absence and presence of GDP-I3S there were no differences between the 3- and 12-month-old animals, and there was a 40% decrease in maximal activity in the 24- as compared with either the 3- or 12-month-old rats (Table I). There was no change in the ECso for forskolin activation with age in the absence or presence of GDP-I3S (Fig. 2, Table I). The ability of Gpp(NH)p to activate a d ~ y l a t e cyclase and to potentiate forskolin activation with age was investigated in F-344 rats. Gpp(NH)p stimulated adenylate cyclase to the same extent as forskolin and with a similar age-related decrease in the 24-month as compared with either the 3- or 12-month-old rats (Table II). The activation by Gpp(NH)p was mostly blocked by GDP-I~S, and there were no longer any age-related differences (Table II). In the presence ~of GDP-I3S there remained approximately a twofold stimulation over basal activity (given in Table I). Gpp(NH)p enhanced the activation of adenylate cyclase by forskolin. In the presence of both Gpp(NH)p and forskolin, adenylate cyclase activity was approximately twofold greater than either Gpp(NH)p or forskolin stimulation
173 A
300.
|
t 100'
0~.
o
IrORSKOUN (.M)
Fig. 2. Forskolin dose-response curves for adenylate cyclase activation in heart membranes from F344 rats. Data shown is from 3-month-old animals in the absence (El) and presence (A) of 500-~M GDP-IBS. See Table I for ECso, maximum activity, and data for other ages. Data represent the mean ± S.E. of 5-6 animals. Line was determined by nonlinear least squares analysis for the data in the presence of GDP-13S. a l o n e at e a c h a g e . T h e i n c r e a s e in a c t i v i t y , h o w e v e r , w a s a d d i t i v e a n d n o t s y n e r gistic. B e c a u s e t h e a c t i v i t y w a s a d d i t i v e a n d t h e s t i m u l a t i o n b y G P P ( N H ) p o r b y f o r s k o l i n a l o n e is r e d u c e d in t h e 2 4 - m o n t h - o l d g r o u p c o m p a r e d
alone
to either
y o u n g e r g r o u p , as e x p e c t e d , t h e m a x i m u m a c t i v i t y b y t h e c o m b i n a t i o n o f s t i m u l a t o r s w a s l e a s t in t h e 2 4 - m o n t h - o l d g r o u p ( T a b l e I I I ) . I n t h e p r e s e n c e o f GDP-13S TABLE I EFFECT OF GDP-~8S ON FORSKOLIN ACTIVATION OF ADENYLATE CYCLASE IN HEARTS FROM F-344 RATS OF THREE AGES
Age (months)
(500 IxM)
GDP-~S
EC.~o (IzM)
Maximum activity (pmol cAMP/min/mg protein)
3 3 12 12 24 24
+ + +
7.8 ± 5.0 ± 4.9 ± 9.5 4.4 ~ 3.7 ±
190 ± 19 197 -- 19 212 ± 29 221 ± 34 121 - 21" 21"*
7.7 1.9 5.9 9.7 1.0 2.9
Maximum activity represents the mean ±S.E. of 6 animals for forskolin- (75p, M) stimulated activity minus basal activity, which was 24.6 ± 2.7, 18.3 ± 4.5, and 19.3 ± 3.2 pmol cAMP/min/mg, respectively, for 3-, 12- and 24-month-old rats. *F (2,15)= 4.8, P<0.05 by one-way ANOVA. **F (2,15)= 3.7, P < 0.05 by one-way ANOVA.
174 TABLE II Gpp(NH)p AND FORSKOLIN ACTIVATION OF ADENYLATE CYCLASE ACTIVITY IN PRESENCE AND ABSENCE OF GDP-/3S
Age (months)
3 3 12 12 24 24
GDP-~S (500 #M)
+ + +
Adenylate cyclase activity (pmol cAMPIminlmg protein) Forskolin plus Gpp(NH)p Gpp(NH)p (20 g,M)
(75 p,M)
217 ± 39 55 - 10 96 +- 35 66 ± 8 96 - 20* 45 ± 8
431 - 53 157 - 7 346 -,- 69 193 ± 26 190 ± 50** 119 - 17"**
(20 p,M)
Activity represents stimulated activity above basal activity given in the legend to Table I. Data represents the mean -+S.E. of 5-6 animals except for forskolin plus Gpp(NH)p in the absence of GDPffS, where it represents 3 animals. *F (2,13) = 4.49, P < 0.05 by one-way ANOVA. **F (2,6) = 6.62, P < 0.05 by one-way ANOVA. ***F (2,15) = 5.00, P<0.05 by one-way ANOVA.
the enhancement by Gpp(NH)p of forskolin activation was blocked and the extent of activity was similar to that observed in the presence of forskolin alone. Again, the activity was least in the 24-month-old group (Tables I and II). In a separate group of animals, we investigated the effects of Gpp(NH)p plus isoproterenol on maximum forskolin stimulation and on forskolin dose-response activation curves of adenylate cyclase. Gpp(NH)p plus isoproterenol-stimulated activity was greater in the 3- and 12-month-old rats as compared with the 24month-old rats (Table III). In each age group, adenylate cyclase activity in the presence of all three stimulators was greater than with Gpp(NH)p plus isoproterenol alone or with forskolin alone (Table III). Again, the effect was additive and not synergistic. As with all other combinations of stimulators, the maximal increase in activity was least in the 24-month-old group (Table III). Forskolin dose-response activation curves were determined in the presence and absence of Gpp(NH)p plus isoproterenol. The extent of the forskolin component of the stimulation was the same under both conditions. That is, when the increase in forskolin-stimulated activity minus basal activity is plotted v e r s u s the increase in forskolin activity minus Gpp(NH)p plus isoproterenol activity, the graphs are similar within each age group (Fig. 3). However, the absolute increase in activity at each forskolin concentration was reduced in the older as compared with either younger group (Table IV). The ECso for forskolin activation was unchanged with either age or the presence of Gpp(NH)p plus isoproterenol (Table IV). This suggests the presence of Gpp(NH)p plus agonist increases the enzyme velocity, apparently without affecting the ECso. However, Eadie-Hofstee plots of this same data are curvilinear, indicating two types of activation in both the absence and
175 TABLE III EFFECT OF Gpp(NH)p PLUS ISOPROTERENOL ON FORSKOLIN ACTIVATION OF ADENYLATE CYCLASE Age months
Adenylate cyclase (pmol cAMP/min/mg protein) Basal Gpp(NH)p Forskolin plus ISO
Forskolin plus Gpp(NH)p plus ISO
3 12 24
18.8---4.6 13.7 -+3.6 10.7-+2.0
231-+15 197 -+32 122-+ 11"**
73.1-+13.9 63.3 -+ 4.6 42.1-+ 1.1"
174-+8 145 -+17 64--- 5**
Stimulator concentrations were as follows: ISO (isoproterenol), 100/xM; Gpp(NH)p, 20/~M; and forskolin, 75/~M. Data represents the mean ±S.E. of 3--4 rats. *F (2,9) = 4.7, P < 0.05 by one-wayANOVA. **F (2,8) = 29.4, P < 0.00l by one-way ANOVA. ***F (2,8) = 4.6, P< 0.05 by one-way ANOVA.
presence of Gpp(NH)p plus isoproterenol (Fig. 3). In addition, the Hill slope factor is consistently less in the presence of Gpp(NH)p and isoproterenol, suggesting an alteration in one or both of the two types of activation by forskolin of adenylate cyclase (Table IV). DISCUSSION In the present study we have investigated the age-related forskolin activation of adenylate cyclase in the presence and absence of the guanine nucleotide analogs GDP-13S and Gpp(NH)p. The mechanism of forskolin activation of adenylate cyclase is complex [10]. In some tissues the combination of forskolin plus Gpp(NH)p or forskolin plus Gpp(NH)p plus isoproterenol is synergetic with respect to activation of adenylate cyclase [11]. This synergism of forskolin stimulated adenylate cyclase by activated Gs is blocked by GDP and its analog GDP-13S [5]. Thus, forskolin appears to have two types of interactions with adenylate cyclase; one that requires Gs and one that does not. Quantitation of changes in catalytic unit activity with age must assess both adenylate cyclase activity in the absence and in the presence of activated G~. Preparations of freshly isolated membranes may contain GTP, and thus catalytic unit plus activated G~. Forskolin activation in these preparations may reflect both types of interactions of forskolin with adenylate cyclase. This is the case with myocardial membrane preparations from Sprague-Dawley rats. There was inhibition of forskolin activation of adenylate cyclase in the presence of GDP-13S. However, in F-344 rats of 3, 12, and 24 months of age, forskolin stimulation of adenylate cyclase was unaffected by the presence of GDP-13S. Maximal forskolin activation was unchanged between 3 and 12 months of age, and there was a 36--43% decrease in activity in the 24-month-old rats as compared with either younger group. There was no change in the ECso for forsko-
176 200.
5
~'~
,
1
t
r
,
.,
°'o'
+o
FOm3KOUN (uM)
Fig. 3. Forskolin dose-response curves for adenylate cyclase activation in the absence (l-q) and presence (A) of 20-1~M Gpp(NH)p plus 100-1~M isoproterenol from 3-month-old animals. Only the forskolin component of the activation is shown. That is, data in the presence of Gpp(NH)p plus isoproterenol represent activity above that of stimulation by Gpp(NH)p plus isoproterenol alone. See Table IV for ECs0, maximum activity and data for other age groups. Data represent the mean - S.E. of 4 animals. Lines were determined by nonlinear least squares analysis. Inset. Eadie-HoIstee plots of the same data for activation of adenylate cyclase in the absence (I-q) and presence (A) of Gpp(NH)p plus isoproterenol. AC: adenylate cyclase activity. F: forskolin.
lin activation either with age or with the presence of GDP-I~S. These findings are similar to our previous report; that is, there was a decrease in catalytic unit activity with age in myocardial membranes [6]. The previous study, however, did not consider the stimulation of forskolin in the presence of activated G~. In the current study, forskolin activation was determined in the presence of Gpp(NH)p and forskolin activation curves were determined in the presence of Gpp(NH)p plus isoproterenol. The maximal activation of adenylate cyclase was greater with either combination of stimulators as compared with forskolin activation alone; however, the pattern of activation with age was nearly identical to that with forskolin alone. There was no change between 3 and 12 months of age and there was a 57--64% decrease in adenylate cyclase activity in the 24- as compared with either the 12or 3-month-old rats. When the data is expressed only as the forskolin component of activation, that is, the Gpp(NH)p or the Gpp(NH)p plus isoproterenoi activation is removed, the extent of activity is identical to that of forskolin activation alone at each age group.
177 TABLE IV EFFECT OF Gpp(NH)p PLUS ISOPROTERENOL ON FORSKOLIN DOSE-RESPONSE ACTIVATION OF ADENYLATE CYCLASE
Age (month)
Gpp(NH)p plus isoproterenol
Hill slope
EC.so (IzM)
Increase in maximum activity (pmol cAMPImin/mg protein
3 3 12 12 24 24
+ + +
0.81 0.76 0.81 0.~3 0.90 0.57
3.2 -+ 0.4 2.8 -+ 1.3 3.9 -+ 0.5 4.8 - 5.2 3.8 - 0.9 6.7 +- 8.8
184 -+ 7 165 -+ 24 154 +- 53 223 -+ 46 65 -+ 5 83 -'- 27
Only the forskolin component of the activation is given. That is, the increase in maximum activity represents the increase in activity over basal activity in the absence of Gpp(NH)p plus isoproterenol and represents the increase in activity over Gpp(NH)p plus isoproterenol activity in the presence of Gpp(NH)p plus isoproterenoi. The ECso, Hill slope, and maximum activity were determined from non-linear least squares analysis of forskolin activation curves (see Fig. 3) of the mean of 4 animals.
Thus, the combinations of forskolin plus Gpp(NH)p or forskolin plus Gpp(NH)p plus isoproterenol were additive and not synergistic. Furthermore, the ECsj for activation by forskolin was unaffected by either age or the presence of Gpp(NH)p plus isoproterenol. However, at each age and with every combination of stimulators, Eadie-Hofstee plots for forskolin activation were curvilinear, indicating two sites of forskolin activation. These data are in direct contrast to findings with other preparations. In adipocyte membranes the EC~ for forskolin activation decreases from 15 ~M for forskolin alone to 4 p,M for forskolin in the presence of the stimulatory agonist and Gpp(NH)p [7]. These data indicated that forskolin interacts with high affinity in the presence of Gs and with low affinity in the absence of Gs and that there is synergism between these stimulators, especially at lower concentrations of forskolin. This is further supported by studies in $49 cyc cells that are missing functional G~. In these cells, Eadie-Hofstee plots of forskolin activation are linear, which is indicative of a single site of action with ECso of 244 ~M [10]. In contrast, in wild-type $49 cells, Eadie-Hofstee plots are curvilinear, indicating two sites of action with an ECs0 of 0.35 I~M and 22 IzM, respectively, for the high- and low-affinity interaction [12]. In heart membranes, however, this does not appear to be the case. There is no synergism between forskolin and Gpp(NH)p plus isoproterenol nor is there a shift in ECso to one that represents a higher affinity. Gpp(NH)p plus isoproterenol appears to increase the velocity of the enzyme without an effect on the EC,so. However, in each age group treated with Gpp(NH)p plus isoproterenol, the Hill slope decreased, which is suggestive of a change in the relative potency of the two types of forskolin activation. Catecholamine responsiveness deteriorates with age. This is best demonstrated
178
as the diminished myocardial chronotropic response to beta-adrenergic agonists in older rats [13] and elderly humans [14]. A number of biochemical changes in the beta-adrenergic pathway with senescence have been identified in the rat myocardium. There are no changes in the density of beta-adrenergic receptors [13]; however, there are age-related decreases in receptor-agonist affinity [15], in Gs function [6], and in catalytic unit activity [6]. The present data reconfirms the loss of catalytic unit activity with age and indicates that the reduced activity with age is independent of the presence or absence of activated Gs.
ACKNOWLEDGEMENT
The author appreciates the excellent technical assistance of M. Knight. Supported by the Medical Research Service of the Department of Veterans Affairs. REFERENCES 1 D.R. Sibley and R.J. Letkowitz, Molecular mechanism of receptor desensitization using the betaadrenergic receptor coupled adenylate cyclase system as a model. Nature, 317 (1985) 124-129. 2 E.R. Weiss, D.J. Kelleher, C.W. Woon, S. Soparkar, S. Osawa, L.E. Heasley and O.L. Johnson, Receptor activation of G proteins. FASEB J., 2 (1988) 2841-2848. 3 S.W. O'Connor, P.J. Scarpace and I.B. Abrass, Age-associated decrease of adenylate cyclase activityin rat myocardium. Mech. Ageing Dev., 16 (1981) 91-95. 4 P.J. Scarpace, Decreased beta-adrenergic responsiveness during senescence. Fed. Proc., 45 (1986)51-54. 5 K.B. Seamon, and J.W. Daly, Forskofin: its biological and chemical properties. In P. Greengard and G.A. Robison (eds.), Advances in Cyclic Nucleotide and Protein Phosphorylation Research, Raven Press, New York, 1986, pp. 1-150. 6 S.W. O'Connor, P.J. Scarpace and I.B. Abrass, Age-associated decrease in the catalytic unit activity of rat myocardial adenylate cyclase. Mech. Ageing Dev., 21 (1983) 357-363. 7 Q.-H. Shi, J.A. Ruiz and R.J. Ho, Forms of adenylate cyclase, activation and/or potentiation by forskolin. Arch. Biochem. Biophys., 251 (1986) 156-165. 8 P.J. Scarpace, L.A. Baresi, D.A. Sanford and I.B. Abrass, Desensitization and resensitization of beta-adrenergic receptors in a smooth muscle cell line. Mol. Pharmacol., 28 (1985) 495-501. 9 M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem:, 72 (1976) 248-254. 10 F.J. Darfler, L.C. Mnhan, A.M. Konchman and P.A. Insel, Stimulation by forskolin of intact $49 lymphoma cells involves the nucleotide regulatory protein of adenylate cyclase. J. Biol. Chem., 257 (1982) 11901-11907. 11 J.W. Daly, W. Padgett and K.B. Scamon, Activation of cyclic AMP generating systems in brain membranes and slices by the diterpene forskolin-augmentation of receptor-mediated responses. J. Neurochem., 38 (1982) 532-544. 12 R.B. Clark, T.J. Goka, D.A. Green, R. Barber and R.W. Butcher, Differences in the forskolin activation of adenylate cyclases in wild type and variant lymphoma cells. Mol. Pharmacol., 22 (1982) 609-613. 13 I.B. Abrass, J.L. Davis and P.J. Scarpace, lsoproterenol responsiveness and myocardial betaadrenergic receptors in young and aging rats. J. Gerontol., 37(1982) 156-160. 14 R.E. Vestal, A.J.J. Wood and D.G. Shand, Reduced beta-adrenoceptor sensitivity in the elderly. Clin. Pharmacol. Ther., 26 (1979) 181-185. 15 P.J. Scarpace and I.B. Abrass, Beta-adrenergic agonist mediated desensitization in senescent rats. Mech. Ageing Dev., 35 (1986) 255-264.
169
Elsevier ScientificPublishers Ireland Ltd.
FORSKOLIN ACTIVATION OF ADENYLATE CYCLASE IN RAT MYOCARDIUM WITH AGE: EFFECTS OF GUANINE NUCLEOTIDE ANALOGS
PHILIP J. SCARPACE Geriatric Research, Education and Clinical Center, Veterans Administration Medical Center, Gainesville, FL 32602, and Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610 (U.S.A.)
(Received May 27th, 1989) (Revision receivedJuly 11th, 1989)
SUMMARY Beta-adrenergic and post-receptor activation of adenylate cyclase decreases with age in the rat myocardium. Forskolin-stimulated adenylate cyclase activity decreases with age to the same extent as NaF or isoproterenol stimulation, suggesting that a loss of catalytic unit activity accounts for the loss of activity with age. However, recent evidence suggests that there are both a guanine nucleotidedependent and independent component of forskolin activation. We assessed the possible role of each of these components with age by measuring forskolin doseresponse stimulation of adenylate cyclase activity in the presence and absence of guanosine 5'-O-(2-thiodiphosphate) (GDP-13S) and 13,~-imidoguanine 5'-triphosphate (Gpp(NH)p) in myocardial membranes from F-344 rats of 3, 12, and 24 months of age. Maximal forskolin stimulation of adenylate cyclase was least in the 24- (121 - 21 pmol cAMP/min/mg) as compared with either the 12- (212 - 29) or 3- (190 - 19) month-old rats. The presence of GDP-13S had no effect on either the ECs0 or maximum activity, and the age-related decline persisted. Gpp(NH)p plus isoproterenol enhanced ~ c t i , : a t i ~ but the effect was additive and not synergistic. There was no effect on the ECso~andthe maximum activity was least in the 24-month-old rats. These data reconfirm the~loss of catalytic unit activity with age and indicate that the reduced activity with age is independent of the presence or absence of activated G-protein. K e y words: Aging; Catalytic unit; Rat Address all correspondence and reprint requests to: Philip J. Scarpace, Ph.D., GRECC (182), VA
Medical Center, Gainesville. FL 32602, U.S.A. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990Elsevier ScientificPublishers Ireland Ltd.
170 INTRODUCTION
Adenylate cyclase is part of a receptor-enzyme complex that consists of three components [1]. Located at the outer membrane surface are both stimulatory and inhibitory receptors, each containing a specific binding site for hormones or neurotransmitters. The second component of the complex is a group of guanine nucleotide-binding regulatory proteins (G-proteinS~). These proteins, termed G, and Gi, mediate the transmission of the hormone signal from the receptor to the catalyst and are involved in both the stimulation and inhibition of adenylate cyclase activity, respectively [2]. They are thought to exist as heterotrimers, with G~ and Gi apparently having similar 13 and ~/subunits and a unique a subunit [2]. The Gproteins appear to oscillate between two activity states, an inactive GDP-bound form and an active GTP-bound form. Stimulatory hormones activate the G-proteins by promoting the replacement of bound GDP by GTP, in the G-protein subunit [2]. The GTP-a subunit interacts with the third component of the enzyme complex, the catalytic unit, to form an active enzyme that is the site of the conversion of ATP to cAMP. Beta-adrenergic and post-receptor activation of adenylate cyclase activity decreases with age in the rat myocardium [3]. This loss of activity may contribute to the diminished capacity of the myocardium to respond to catecholamines in senescence [4]. Isoproterenol activates adenylate cyclase through the beta-adrenergic receptor by the mechanism described above. NaF, GTP and the GTP analog, 13,~imidoguanine 5'-triphosphate (Gpp(NH)p), increase adenylate cyclase activity by a post-receptor activation of G-protein. Forskolin, a cardioactive diterpene from the roots of Coleus forskohlii also activates adenylate cyclase by a post-receptor mechanism. It was previously believed that forskolin directly activated the catalytic unit and did not require the presence of a functional G-protein [5]. This laboratory previously reported that forskolin-stimulated adenylate cyclase activity decreases with age to the same extent as NaF or isoproterenol stimulation and based on the tenet that forskolin solely activates the catalytic unit concluded that a loss of catalytic unit activity fully accounts for the loss of adenylate cyclase activity with age [6]. However, recent evidence indicates that forskolin activation of adenylate cyclase can be enhanced by guanine nucleotides [5]. It has been proposed that when the catalytic unit is associated with the activated Gs, the extent of forskolin activation is greater than with the catalytic unit alone [5]. Furthermore, in some tissues guanine nucleotide plus stimulatory hormone reduced the EC.~ofor forskolin activation, suggesting that forskolin interacts with the catalytic unit with high affinity in the presence of activated Gs and with low affinity in the absence of G~ [7]. These effects can be blocked by GDP or its analog, guanosine 5'-0(2-thiodiphosphate) (GDP-13S) . . . . Thus the extent of forskolin activation with age wo~ld be influenced by the presence of any endogenous guanine nucleotides and/or ai~ activated G~. G-protein activity, when assessed by complementation with the G-protein-deficient $49 cyc cell line, is reduced in rat myocardium from older rats [6].\ If forskolin-stimulated adenylate cyclase partially reflects activation through a G~-eatalytic unit complex,
171
then the decreased adenylate cyclase activity with age might possibly be due to the reduced G-protein function with age. A second possibility is that the ability of the activated Gs-catalytic unit complex to enhance forskolin stimulation is diminished with age. In consideration of the above discussion, the age-related activation of myocardial adenylate cyclase by forskolin has been reinvestigated in greater detail. Forskolin activation curves were determined in the presence of the inhibitory and stimulatory guanine nucleotide analogs, GDP-13S and Gpp(NH)p, respectively, in myocardial membrane from rats of 3, 12, and 24 months of age. METHODS
Animals Female F-344 NNia rats of 3, 12, and 24 months of age (received Jan. and June 1988) were obtained from Harlan Industries (Indianapolis, IN) under contract with the National Institute on Aging. Upon arrival, rats were examined and housed individually in 7"x 10" stainless steel cages. All animals were maintained on Purina rat chow ad libitum with a 12-h light/dark cycle: Rats were used within 4 weeks after their arrival. No ill animals were noted or used. No serological or bacteriological tests were performed.
Membrane preparation At sacrifice, the circulatory system was perfused with 20-ml cold saline and the hearts excised. Tissues were finely minced, disrupted in a tissuemizer for 30 s, and homogenized with 10 strokes of a motor-driven pestle. Homogenates were passed through four layers of cheesecloth and centrifuged at 48000g for 15 min at 4°C. Pellets were resuspended in 18 mM MgCI2, 0.08 mM ascorbic acid, and 50 mM 4-(2-hydroxethyl)-I piperazineethanesulfonic acid (HEPES) (pH 7.4.)
Adenylate cyclase assay Adenylate cyclase activity was assayed by the conversion of ATP into cAMP in the presence of 1 mM isobutylmethylxanthine and an ATP regenerating system as described by Scarpace and Abrass [8]. Approximately 75 I~g of membrane protein plus stimulators as indicated were incubated for 15 min at 37°C in a total volume of 150 ~I. The reaction was terminated by addition of 700 I~1 of cold 5.3 mM CaCI2 and immersion into a salt-ice bath. Assay tubes were centrifuged at 9000 g and the supernatant assayed for cAMP. cAMP was quantified by radioimmunoassay. Protein was determined by the method of Bradford [9]. RESULTS Figure 1 shows that GDP-13S partially blocks the activation of adenylate cyclase by forskolin in heart membranes from Sprague-Dawley rats. In the presence of GDP-13S there is a 69% decrease in maximum stimulation by forskolin and a 76% increase in the ECso for forskolin activation.
172
200.
i
100. f
0..
o
~
+b
"
ab
I+o
Fig. 1. Forskolin dose-response curves for adenylate cyclase activation in h©art membranes from Sprague-Dawley rats of 3 months of age. ECso values are 17.1 and 30.5 I~M, respectively, in the absence ([]) and presence (A) of 500-1~M GDP-13S. Data represents the mean - SE of 3 animals. Lines were determined by non-linear least squares analysis.
These observations prompted a reinvestigation of the age-related decrease in forskolin activation of adenylate cyclase in heart membranes from F-344 rats. However, as opposed to the heart membranes from Sprague-Dawley rats, GDP13S had no effect on forskolin activation of adenylate cyclase in membranes from F-344 rats at any age (Fig. 2 and Table I). In both the absence and presence of GDP-I3S there were no differences between the 3- and 12-month-old animals, and there was a 40% decrease in maximal activity in the 24- as compared with either the 3- or 12-month-old rats (Table I). There was no change in the ECso for forskolin activation with age in the absence or presence of GDP-I3S (Fig. 2, Table I). The ability of Gpp(NH)p to activate a d ~ y l a t e cyclase and to potentiate forskolin activation with age was investigated in F-344 rats. Gpp(NH)p stimulated adenylate cyclase to the same extent as forskolin and with a similar age-related decrease in the 24-month as compared with either the 3- or 12-month-old rats (Table II). The activation by Gpp(NH)p was mostly blocked by GDP-I~S, and there were no longer any age-related differences (Table II). In the presence ~of GDP-I3S there remained approximately a twofold stimulation over basal activity (given in Table I). Gpp(NH)p enhanced the activation of adenylate cyclase by forskolin. In the presence of both Gpp(NH)p and forskolin, adenylate cyclase activity was approximately twofold greater than either Gpp(NH)p or forskolin stimulation
173 A
300.
|
t 100'
0~.
o
IrORSKOUN (.M)
Fig. 2. Forskolin dose-response curves for adenylate cyclase activation in heart membranes from F344 rats. Data shown is from 3-month-old animals in the absence (El) and presence (A) of 500-~M GDP-IBS. See Table I for ECso, maximum activity, and data for other ages. Data represent the mean ± S.E. of 5-6 animals. Line was determined by nonlinear least squares analysis for the data in the presence of GDP-13S. a l o n e at e a c h a g e . T h e i n c r e a s e in a c t i v i t y , h o w e v e r , w a s a d d i t i v e a n d n o t s y n e r gistic. B e c a u s e t h e a c t i v i t y w a s a d d i t i v e a n d t h e s t i m u l a t i o n b y G P P ( N H ) p o r b y f o r s k o l i n a l o n e is r e d u c e d in t h e 2 4 - m o n t h - o l d g r o u p c o m p a r e d
alone
to either
y o u n g e r g r o u p , as e x p e c t e d , t h e m a x i m u m a c t i v i t y b y t h e c o m b i n a t i o n o f s t i m u l a t o r s w a s l e a s t in t h e 2 4 - m o n t h - o l d g r o u p ( T a b l e I I I ) . I n t h e p r e s e n c e o f GDP-13S TABLE I EFFECT OF GDP-~8S ON FORSKOLIN ACTIVATION OF ADENYLATE CYCLASE IN HEARTS FROM F-344 RATS OF THREE AGES
Age (months)
(500 IxM)
GDP-~S
EC.~o (IzM)
Maximum activity (pmol cAMP/min/mg protein)
3 3 12 12 24 24
+ + +
7.8 ± 5.0 ± 4.9 ± 9.5 4.4 ~ 3.7 ±
190 ± 19 197 -- 19 212 ± 29 221 ± 34 121 - 21" 21"*
7.7 1.9 5.9 9.7 1.0 2.9
Maximum activity represents the mean ±S.E. of 6 animals for forskolin- (75p, M) stimulated activity minus basal activity, which was 24.6 ± 2.7, 18.3 ± 4.5, and 19.3 ± 3.2 pmol cAMP/min/mg, respectively, for 3-, 12- and 24-month-old rats. *F (2,15)= 4.8, P<0.05 by one-way ANOVA. **F (2,15)= 3.7, P < 0.05 by one-way ANOVA.
174 TABLE II Gpp(NH)p AND FORSKOLIN ACTIVATION OF ADENYLATE CYCLASE ACTIVITY IN PRESENCE AND ABSENCE OF GDP-/3S
Age (months)
3 3 12 12 24 24
GDP-~S (500 #M)
+ + +
Adenylate cyclase activity (pmol cAMPIminlmg protein) Forskolin plus Gpp(NH)p Gpp(NH)p (20 g,M)
(75 p,M)
217 ± 39 55 - 10 96 +- 35 66 ± 8 96 - 20* 45 ± 8
431 - 53 157 - 7 346 -,- 69 193 ± 26 190 ± 50** 119 - 17"**
(20 p,M)
Activity represents stimulated activity above basal activity given in the legend to Table I. Data represents the mean -+S.E. of 5-6 animals except for forskolin plus Gpp(NH)p in the absence of GDPffS, where it represents 3 animals. *F (2,13) = 4.49, P < 0.05 by one-way ANOVA. **F (2,6) = 6.62, P < 0.05 by one-way ANOVA. ***F (2,15) = 5.00, P<0.05 by one-way ANOVA.
the enhancement by Gpp(NH)p of forskolin activation was blocked and the extent of activity was similar to that observed in the presence of forskolin alone. Again, the activity was least in the 24-month-old group (Tables I and II). In a separate group of animals, we investigated the effects of Gpp(NH)p plus isoproterenol on maximum forskolin stimulation and on forskolin dose-response activation curves of adenylate cyclase. Gpp(NH)p plus isoproterenol-stimulated activity was greater in the 3- and 12-month-old rats as compared with the 24month-old rats (Table III). In each age group, adenylate cyclase activity in the presence of all three stimulators was greater than with Gpp(NH)p plus isoproterenol alone or with forskolin alone (Table III). Again, the effect was additive and not synergistic. As with all other combinations of stimulators, the maximal increase in activity was least in the 24-month-old group (Table III). Forskolin dose-response activation curves were determined in the presence and absence of Gpp(NH)p plus isoproterenol. The extent of the forskolin component of the stimulation was the same under both conditions. That is, when the increase in forskolin-stimulated activity minus basal activity is plotted v e r s u s the increase in forskolin activity minus Gpp(NH)p plus isoproterenol activity, the graphs are similar within each age group (Fig. 3). However, the absolute increase in activity at each forskolin concentration was reduced in the older as compared with either younger group (Table IV). The ECso for forskolin activation was unchanged with either age or the presence of Gpp(NH)p plus isoproterenol (Table IV). This suggests the presence of Gpp(NH)p plus agonist increases the enzyme velocity, apparently without affecting the ECso. However, Eadie-Hofstee plots of this same data are curvilinear, indicating two types of activation in both the absence and
175 TABLE III EFFECT OF Gpp(NH)p PLUS ISOPROTERENOL ON FORSKOLIN ACTIVATION OF ADENYLATE CYCLASE Age months
Adenylate cyclase (pmol cAMP/min/mg protein) Basal Gpp(NH)p Forskolin plus ISO
Forskolin plus Gpp(NH)p plus ISO
3 12 24
18.8---4.6 13.7 -+3.6 10.7-+2.0
231-+15 197 -+32 122-+ 11"**
73.1-+13.9 63.3 -+ 4.6 42.1-+ 1.1"
174-+8 145 -+17 64--- 5**
Stimulator concentrations were as follows: ISO (isoproterenol), 100/xM; Gpp(NH)p, 20/~M; and forskolin, 75/~M. Data represents the mean ±S.E. of 3--4 rats. *F (2,9) = 4.7, P < 0.05 by one-wayANOVA. **F (2,8) = 29.4, P < 0.00l by one-way ANOVA. ***F (2,8) = 4.6, P< 0.05 by one-way ANOVA.
presence of Gpp(NH)p plus isoproterenol (Fig. 3). In addition, the Hill slope factor is consistently less in the presence of Gpp(NH)p and isoproterenol, suggesting an alteration in one or both of the two types of activation by forskolin of adenylate cyclase (Table IV). DISCUSSION In the present study we have investigated the age-related forskolin activation of adenylate cyclase in the presence and absence of the guanine nucleotide analogs GDP-13S and Gpp(NH)p. The mechanism of forskolin activation of adenylate cyclase is complex [10]. In some tissues the combination of forskolin plus Gpp(NH)p or forskolin plus Gpp(NH)p plus isoproterenol is synergetic with respect to activation of adenylate cyclase [11]. This synergism of forskolin stimulated adenylate cyclase by activated Gs is blocked by GDP and its analog GDP-13S [5]. Thus, forskolin appears to have two types of interactions with adenylate cyclase; one that requires Gs and one that does not. Quantitation of changes in catalytic unit activity with age must assess both adenylate cyclase activity in the absence and in the presence of activated G~. Preparations of freshly isolated membranes may contain GTP, and thus catalytic unit plus activated G~. Forskolin activation in these preparations may reflect both types of interactions of forskolin with adenylate cyclase. This is the case with myocardial membrane preparations from Sprague-Dawley rats. There was inhibition of forskolin activation of adenylate cyclase in the presence of GDP-13S. However, in F-344 rats of 3, 12, and 24 months of age, forskolin stimulation of adenylate cyclase was unaffected by the presence of GDP-13S. Maximal forskolin activation was unchanged between 3 and 12 months of age, and there was a 36--43% decrease in activity in the 24-month-old rats as compared with either younger group. There was no change in the ECso for forsko-
176 200.
5
~'~
,
1
t
r
,
.,
°'o'
+o
FOm3KOUN (uM)
Fig. 3. Forskolin dose-response curves for adenylate cyclase activation in the absence (l-q) and presence (A) of 20-1~M Gpp(NH)p plus 100-1~M isoproterenol from 3-month-old animals. Only the forskolin component of the activation is shown. That is, data in the presence of Gpp(NH)p plus isoproterenol represent activity above that of stimulation by Gpp(NH)p plus isoproterenol alone. See Table IV for ECs0, maximum activity and data for other age groups. Data represent the mean - S.E. of 4 animals. Lines were determined by nonlinear least squares analysis. Inset. Eadie-HoIstee plots of the same data for activation of adenylate cyclase in the absence (I-q) and presence (A) of Gpp(NH)p plus isoproterenol. AC: adenylate cyclase activity. F: forskolin.
lin activation either with age or with the presence of GDP-I~S. These findings are similar to our previous report; that is, there was a decrease in catalytic unit activity with age in myocardial membranes [6]. The previous study, however, did not consider the stimulation of forskolin in the presence of activated G~. In the current study, forskolin activation was determined in the presence of Gpp(NH)p and forskolin activation curves were determined in the presence of Gpp(NH)p plus isoproterenol. The maximal activation of adenylate cyclase was greater with either combination of stimulators as compared with forskolin activation alone; however, the pattern of activation with age was nearly identical to that with forskolin alone. There was no change between 3 and 12 months of age and there was a 57--64% decrease in adenylate cyclase activity in the 24- as compared with either the 12or 3-month-old rats. When the data is expressed only as the forskolin component of activation, that is, the Gpp(NH)p or the Gpp(NH)p plus isoproterenoi activation is removed, the extent of activity is identical to that of forskolin activation alone at each age group.
177 TABLE IV EFFECT OF Gpp(NH)p PLUS ISOPROTERENOL ON FORSKOLIN DOSE-RESPONSE ACTIVATION OF ADENYLATE CYCLASE
Age (month)
Gpp(NH)p plus isoproterenol
Hill slope
EC.so (IzM)
Increase in maximum activity (pmol cAMPImin/mg protein
3 3 12 12 24 24
+ + +
0.81 0.76 0.81 0.~3 0.90 0.57
3.2 -+ 0.4 2.8 -+ 1.3 3.9 -+ 0.5 4.8 - 5.2 3.8 - 0.9 6.7 +- 8.8
184 -+ 7 165 -+ 24 154 +- 53 223 -+ 46 65 -+ 5 83 -'- 27
Only the forskolin component of the activation is given. That is, the increase in maximum activity represents the increase in activity over basal activity in the absence of Gpp(NH)p plus isoproterenol and represents the increase in activity over Gpp(NH)p plus isoproterenol activity in the presence of Gpp(NH)p plus isoproterenoi. The ECso, Hill slope, and maximum activity were determined from non-linear least squares analysis of forskolin activation curves (see Fig. 3) of the mean of 4 animals.
Thus, the combinations of forskolin plus Gpp(NH)p or forskolin plus Gpp(NH)p plus isoproterenol were additive and not synergistic. Furthermore, the ECsj for activation by forskolin was unaffected by either age or the presence of Gpp(NH)p plus isoproterenol. However, at each age and with every combination of stimulators, Eadie-Hofstee plots for forskolin activation were curvilinear, indicating two sites of forskolin activation. These data are in direct contrast to findings with other preparations. In adipocyte membranes the EC~ for forskolin activation decreases from 15 ~M for forskolin alone to 4 p,M for forskolin in the presence of the stimulatory agonist and Gpp(NH)p [7]. These data indicated that forskolin interacts with high affinity in the presence of Gs and with low affinity in the absence of Gs and that there is synergism between these stimulators, especially at lower concentrations of forskolin. This is further supported by studies in $49 cyc cells that are missing functional G~. In these cells, Eadie-Hofstee plots of forskolin activation are linear, which is indicative of a single site of action with ECso of 244 ~M [10]. In contrast, in wild-type $49 cells, Eadie-Hofstee plots are curvilinear, indicating two sites of action with an ECs0 of 0.35 I~M and 22 IzM, respectively, for the high- and low-affinity interaction [12]. In heart membranes, however, this does not appear to be the case. There is no synergism between forskolin and Gpp(NH)p plus isoproterenol nor is there a shift in ECso to one that represents a higher affinity. Gpp(NH)p plus isoproterenol appears to increase the velocity of the enzyme without an effect on the EC,so. However, in each age group treated with Gpp(NH)p plus isoproterenol, the Hill slope decreased, which is suggestive of a change in the relative potency of the two types of forskolin activation. Catecholamine responsiveness deteriorates with age. This is best demonstrated
178
as the diminished myocardial chronotropic response to beta-adrenergic agonists in older rats [13] and elderly humans [14]. A number of biochemical changes in the beta-adrenergic pathway with senescence have been identified in the rat myocardium. There are no changes in the density of beta-adrenergic receptors [13]; however, there are age-related decreases in receptor-agonist affinity [15], in Gs function [6], and in catalytic unit activity [6]. The present data reconfirms the loss of catalytic unit activity with age and indicates that the reduced activity with age is independent of the presence or absence of activated Gs.
ACKNOWLEDGEMENT
The author appreciates the excellent technical assistance of M. Knight. Supported by the Medical Research Service of the Department of Veterans Affairs. REFERENCES 1 D.R. Sibley and R.J. Letkowitz, Molecular mechanism of receptor desensitization using the betaadrenergic receptor coupled adenylate cyclase system as a model. Nature, 317 (1985) 124-129. 2 E.R. Weiss, D.J. Kelleher, C.W. Woon, S. Soparkar, S. Osawa, L.E. Heasley and O.L. Johnson, Receptor activation of G proteins. FASEB J., 2 (1988) 2841-2848. 3 S.W. O'Connor, P.J. Scarpace and I.B. Abrass, Age-associated decrease of adenylate cyclase activityin rat myocardium. Mech. Ageing Dev., 16 (1981) 91-95. 4 P.J. Scarpace, Decreased beta-adrenergic responsiveness during senescence. Fed. Proc., 45 (1986)51-54. 5 K.B. Seamon, and J.W. Daly, Forskofin: its biological and chemical properties. In P. Greengard and G.A. Robison (eds.), Advances in Cyclic Nucleotide and Protein Phosphorylation Research, Raven Press, New York, 1986, pp. 1-150. 6 S.W. O'Connor, P.J. Scarpace and I.B. Abrass, Age-associated decrease in the catalytic unit activity of rat myocardial adenylate cyclase. Mech. Ageing Dev., 21 (1983) 357-363. 7 Q.-H. Shi, J.A. Ruiz and R.J. Ho, Forms of adenylate cyclase, activation and/or potentiation by forskolin. Arch. Biochem. Biophys., 251 (1986) 156-165. 8 P.J. Scarpace, L.A. Baresi, D.A. Sanford and I.B. Abrass, Desensitization and resensitization of beta-adrenergic receptors in a smooth muscle cell line. Mol. Pharmacol., 28 (1985) 495-501. 9 M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem:, 72 (1976) 248-254. 10 F.J. Darfler, L.C. Mnhan, A.M. Konchman and P.A. Insel, Stimulation by forskolin of intact $49 lymphoma cells involves the nucleotide regulatory protein of adenylate cyclase. J. Biol. Chem., 257 (1982) 11901-11907. 11 J.W. Daly, W. Padgett and K.B. Scamon, Activation of cyclic AMP generating systems in brain membranes and slices by the diterpene forskolin-augmentation of receptor-mediated responses. J. Neurochem., 38 (1982) 532-544. 12 R.B. Clark, T.J. Goka, D.A. Green, R. Barber and R.W. Butcher, Differences in the forskolin activation of adenylate cyclases in wild type and variant lymphoma cells. Mol. Pharmacol., 22 (1982) 609-613. 13 I.B. Abrass, J.L. Davis and P.J. Scarpace, lsoproterenol responsiveness and myocardial betaadrenergic receptors in young and aging rats. J. Gerontol., 37(1982) 156-160. 14 R.E. Vestal, A.J.J. Wood and D.G. Shand, Reduced beta-adrenoceptor sensitivity in the elderly. Clin. Pharmacol. Ther., 26 (1979) 181-185. 15 P.J. Scarpace and I.B. Abrass, Beta-adrenergic agonist mediated desensitization in senescent rats. Mech. Ageing Dev., 35 (1986) 255-264.