The β-adrenergic transduction system in kidneys from young and senescent rats

European Journal of Pharmacology - Molecular Pharmacology Section, 188 (1990) 129-137

!29

Elsevier EJPMOL 90062

The fl-adrenergie transduction system in kidneys from young and senescent rats Patrick Vanscheeuwijck, Eric Van de Velde and Norbert Fraeyman Hevmans hlstitute of Pharmacology', Unit,ersitv of Ghent Medical School, B-9000 Ghent. Belgium

Received 17 July, revised MS received 20 October 1989, accepted 14 November1989

fl-Adrenoceptor density, ligand affinity, high-affinity agonist binding, basal adenylate cyclase activity and cAMP synthesis upon stimulation with either forskolin. F-, guanine nucleotides (GTP or GppNHp) or isoproterenol in the presence of the nucleotides were studied in membranes prepared from kidneys of young (2-3 month) and old (24-25 month) male Wistar rats. There is a significant (P < 0.01) 62% increase in r-receptor density, a ~ignificant (P < 0.05) 115% decrease in ligand affinity, a significant (P < 0.05) 33% decrease of high-affinity binding sites for (-)-isoproterenol and a significant (P < 0.01) 151% decrease of the affinity of the high-affinity agonist binding site. Basal adenylate cyclase activity and the activity after stimulation with guanine nucleotides and forskolin were significantly higher in old animals as compared to young (P < 0.01). Stimulation of the system with isoproterenol in the presence of GTP was more effective in old animals, although the P < 0.05 level of significance was barely reached. It is suggested that age-dependent changes of the fl-adrenoceptors in rat kidney are similar to those described for lungs: changes at the different levels of the fl-adrenergic transduction chain associated with age are compensatory so as to ensure equal cAMP synthesis for a given agonist stimulation. Aging: fl-Adrenoceptors; Kidney (rat): Adenylate cyclase

i. Introduction We recently reported that in rat lung, aging is accompanied by changes in the fl-adrenergic transduction system, with an increase in receptor density and a decrease in percentage of l:;gh-affinity agonist binding sites; in spite of these ,changes. cAMP synthesis upon agonist stimulatiot~ was not altered (Vanscheeuwijck et at., 1989). It is known that fl-adrenoceptors in rat lung are predominantly of the fl2-subtype (Barnett et al,. 1978), In order to extend and confirm our results, we performed experiments with rat kidney membranes

Correspondence to: Norbert Fraeyman, HeymansInstitute of Pharmacology, University of Ghent Medical School, De Pintelaan 185. B-9000Ghent, Belgium.

because in this tissue, the fl-adrenoceptors are mainly of the ill-subtype (Brodde, 1982; Snavely et al., 1982; Moustafa et al., 1984; Michel et al., 1987). Few data are available on the effect of age upon the properties of the fl-adrenoceptors and the fl-adrenoceptor-coupled signal transduction system in rat kidney. However, a shift in r-receptor subtype distribution towards the fl~-adrenoceptor with increasing age has been reported recently (Galbusera et al., 1988). We have investigated the fl-adrenergic system at each level of the transduction chain in membranes prepared from total kidneys of rats aged either 2-3 or 24-25 months. These experiments include the determination of receptor density, ligand affinity, f l ~ / f l 2 - a d r e n o e e p t o r subtype distribution, coupling between receptor and GTP-binding protein and the effect of various agents on

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130 adenylate cyclase activity. The results are comparable to what we found for rat lung (Vanscheeuwijck et al., 1989): the receptor density increased and the amount of high-affinity agonist binding sites decreased with increasing age. Agonist-induced cAMP synthesis was essentially unchanged in kidneys from old animals as compared to the young, suggesting that the abovementioned changes are essentially compensatory in order to assure equal cAMP production for equal agonist stimulation. A preliminary account of these results has been given (Fraeyman et al., 1989). 2. Materials and methods

2.1. Materials [12sI]Iodocyanopindolol (ICYP, spec. act. 1988 Ci/mmol) and [8- 3H]adenosme-3,5 . . . .-cychc monophosphate ([3H]cAMP, spec. act. 23 Ci/mmol) were obtained from Amersham (Amersharn, U.K.). ( -)-Isoproterenol, (+)-metoprolol bit~_rtrate, forskolin and bovine serum albumin (BSA) were purchased from Sigma (Poole, U.K.). ATP, GTP, guanylylimidodiphosphate (GppNHp), creatine phosphokinase (E.C. 2.7.3.2) and creatine phosphate were obtained from Boehringer (Mannheim, F.R.G.). cAMP was from Janssen Chimica (Beerse, Belgium). (+)-Propranolol hydrochloride and ICI 118,551 hydrochloride (erythro-DL-l-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol) were generous gifts from ICI Pharma (Destelbergen, Belgium). All other chemicals were of the highest purity available and obtained from local suppliers. 2.2. Animals Male SPF Wistar rats, aged 2-3 months (young rats) and 24-25 months (old rats), were obtained from the Proefdierencentrum of the University of Leuven, Belgium. They were housed individually and had free access to food and water. The animals were fasted overnight before the experiment. 2. 3. Membrane preparation The animals were decapitated, exsanguinated and the kidneys were rapidly removed and placed

on chilled petri dishes. All subsequent steps were performed at 4 ° C. Connective tissue, kidney capsule, fat, ureter and large blood vessels were removed and the kidneys were homogenized with an Ultra-turrax homogenizer in buffer A containing (mM): sucrose 250; Tris-HCl 50; EGTA 2; pH 7.4. The first 5 s were at 20% of maximal speed followed by 2 × 15 s at 70% of maximal speed with a 1-min interval between each stroke. The homogenate was centrifuged at 2000 × g for 19 min and the pellet was discarded. The supernatant was then centrifuged at 33000 × g for 30 min and the pellet was washed three times with buffer A. The final pellet was resuspended in 5 ml of buffer B containing (raM): Tris-HCl 50; MgCI 2 20; EGTA 2; pH 7.4. All further deterrrfinations were performed on the same day on freshly prepared membranes. The protein content was determined with the dye-binding method of Macart and Gerbaut (1982) using BSA as standard. 2.4. Binding experiments Association binding experiments were done by incubating 67 /xg of kidney membrane proteins with one concentration (96.2 pM) 1CYP in a final volume of 250 p.l at 37°C in glass test tubes. At different time intervals, the incubation mixtures were rapidly filtered through Whatman G F / C glass fiber filter disks (2.5 cm diameter) and rinsed three times with 5 ml cold 0.9% NaCI. The radioactivity on the filters was then counted as described below. The time needed to reach half-maximum binding was 13.3 + 1.5 rain ( n = 3) and 17.3 + 0.5 min (n = 3) for young and old rats, respectively. All further ligand binding experiments were conducted for 60 min. Saturation binding experiments were performed to determine the total number of r-receptors (Bmax in f m o l / m g protein) and their equilibrium dissociation constant (K o in pM) for ICYP. Membrane proteins (67 #g) were incubated with 8 concentrations of ICYP (up to 250 pM) iN a total volume of 250 /~1 for 60 rain at 37°S. Nonspecific binding was defined as the radioaztivity not displaced by 10/tM (+__)-propranolol. Competition binding experiments were done for the determination of the fl-adrenoceptor sub-

131 type distribution and for the differ,nt affinity configurations of the ,8-adrenoceptor.~ for ( - ) isoproterenol. Kidney membrane prot~ ins (67 ~tg) in buffer B and one ICYP concentration (96.2 pM) were incubated for 60 min at 33°C with either 12 concentrations of the ~3~-selective antagonist metoprolol (0.1 nM to 1 mM) or of the /~z-selective antagonist IC1 118,551 ((,.01 nM to 0.! mM) or of the non-selective a~onist ( - ) isoproterenol (0.I nM to 1 mM). In the experiments with isoproterenol, the incubat on mixture was supplemented with 0.01% ascorbi,: acid; in a parallel experiment, 300 p,M G p p N H p was included. All incubations (except for the association binding experiments) were done at least in :luplicate in microtiter pla'es and were stopped by rr_pid filtration over Gelman A / E g!oss fiber "ilter sheets (3.5 x 25.4 cm). The filters were rinsed with 10 ;< 300 btl of ice-cold 0.9% NaCI using the semi-automatic cell harvester method (Vanscheeuwijck and Fraeyman, 1989). The radioactivity on the filters was counted in 4 ml U!~.ima Gold (Packard) with 70-80% counting efficiency in a Packard Tri-Carb Liquid Scintillation Spectrometer (type 4530) (Van Oosterhout et al., 1988).

2.5. Adenvlate c),clase stimulation ~md cAMP determination Basal adenylate eyclase (E.C. 4.6.1.1) activity was measured according to Scarpace and Abrass (1983) with slight modifications. Membrane suspensions (100 t~l containing 100 #g membrane proteins in buffer B) were added to an incubation mixture (volume 150 pl) containing 1.25 m M ATP, 1 m M ascorbic acid, 0.01 m M BSA, 2.5 m M creatine phosphate, 300 I.U. creatine phosphokinase, 0.5 m M theophylline and either (added in a volume of 50 /~1) 33 p M GTP, 33 /~M G p p N H p , 25 p M forskolin, 10 m M N a F or 33 # M (-)-isoproterenol together with G T P or GppNHp. Incubations were carried out for 15 min at 30 o C. After incubation, samples were diluted with 750 /H 10 m M Tris buffer, pH 7.5, containing 2

m M EDTA, heated for t5 rain at 100°C, cooled to 4 ° C and centrifuged (5000 x g for 10 min). The cAMP content was determined in 100-/tl aliquots of the supernatant by a protein binding assay (Tovey et al.. 1974). using [3HIcAMP and a binding protein purified according to Gilman (1970). The calibration curve for cAMP was constructed by incubating, in parallel, a set of tubes containing cAMP (0-160 p m o l / t u b e ) instead of kidney membranes. The between-assay coefficient of variation, determined on a commercial standard cAMP (Amersham) of 6.72 + 0,19 p m o l / t u b e was 9.1% (n = 10). Adenylate cyclase activity is expressed as pmol cAMP formed per mg protein per rain incubation. In some cases basal activity was subtracted from the total activity (see Results).

2.6. Cak'ulations Saturation binding experiments were evaluated with Scatchard plot analysis using linear regression. Only plots with correlation coefficients of at least 0.85 were accepted for further calculations. Displaccment curves were analyzed with the program G r a p h P A D (Motulsky, 1987); the inhibition constants (Krvalues) were calculated from ECs~j values according to Cheug and Prusoff (1973). The distinction between a one-site or a two-site model was based on the partial F-test (Snedecor and Cochran, 1973). All results are presented as m e a n s + S.E.M. Comparisons between the two age groups were evaluated using a two-tailed Stude~'~t's t-test; for comparisons within one age group, the paired t-test was used. The equality of means of several variables was tested by a multivariat~,~ test (Hotelling's t-test). Statistical analysis was done with the computer program Statgraphics or SPSS (Hotelling's t-test).

3. Results

3.1. Receptor density and ligand affinity In fig. 1, a representative example of a saturation binding curve obtained with membrane pre-

132 TABLE 1

A

Saturation binding and ( - t-isoproterenol competition binding experiments of I C Y P to fl-receptors in total kidney membranes from young (2-3 month) and old (24-25 month) rats. H, high-affinity binding site; L low-affinity binding site; K+, inhibition constant.

o

~'=

3o

~

2o

Type of experiment Saturation binding BmaX(fmol/mg prot) K D (pM) 50

I00

150

200

250

([~25I]CYP. pMI

B 5(

~

2-3 month rats (n =10) 26.6± 3.0 27.3_+ 3.7

24-25month rats (n =10) 43.1_+ 4.2 h 58.8+!2.0 ~

Competition binding (with (-)-isoproterenol) Without G p p N H p K i H(nM) %H K i L (nlVl) %L

5.7_+ 1.2 44.1_+ 5.0 522.9+130.5 55.9_+ 5.0

1 4 , 3 + 2.7 b 2 9 . 9 + 3.8 a 658.2+90.4 70.1_+ 3.8 a

With G p p N H p K i L(nM) %L

3 6 5 . 9 + 68.2 100

607.7+63.2 ~ 100

P < 0.05; h p < 0.01 (Student's t-test).

3o

50

100 150 1[t25I] CYP. pM)

.30I'" ° .25 I ~ .20

200

250

C

~

.t5L\ o

0

tO 20 30 40 50+

"

60

[s2~I]CYP bound {fmollmg prof.) Fig. l. Saturation binding curves with rat kidney membranes. A representative example is shown for a young (2 month) animal (A) and an old (24 month) animal (B). Diamonds, total binding; triangles, nonspecific binding; circles, specific binding. (C) Seatchard plots of the curves shown in A and B. Unfilled symbols, young rat; Bma× = 34.6 fmol/mg protein; Ko = 31.7 pM; r = 0.94. Filled symbols, old rat; Bm~ = 50.2 fmol/mg protein; K o = 45.1 pM; r = 0.94.

p a r a t i o n s from a y o u n g (fig. 1A) a n d an old (fig. 1B) rat is shown. In fig. 1C, the c o r r e s p o n d i n g Scatchard p l o t s are depicted. Nonspecific b i n d i n g w i t h 1 0 / ~ M p r o p r a n o l o l was 27.7 + 2.0% a n d 25.5 + 1.3% of total b i n d i n g at the highest ligand concentration, respectively, for the y o u n g and the old a n i m a l s (P > 0.05). W i t h increasing age, the n u m ber of I C Y P b i n d i n g sites increased b y 62% (P < 0.01) and the K D of the tigand receptor increased b y 11570 (P < 0.05) (table 11. The m e a n c o m p e t i t i o n b i n d i n g curves with ( - ) - i s o p r o t e r e n o l are s h o w n in fig. 2. C o m p u t e r analysis of the shallow curves revealed the presence of b o t h high- a n d low-affinity b i n d i n g sites in both age groups. The n u m b e r of high-affinity b i n d i n g sites was 33.1% lower in the old a n i m a l s than in the y o u n g ( P < 0 . 0 5 1 . The K i - v a l u e for ( - ) - i s o p r o t e r e n o l b i n d i n g at the high-affinity b i n d i n g site was 2.5 times higher in old a n i m a l s (P < 0.01). In the presence of G p p N H p , the isoproterenol d i s p l a c e m e n t curves b e c a m e steep for kidneys from both y o u n g and old rats. U n d e r these conditions, fl-adrenoceptors in kidneys from old a n i m a l s had e+ higher K i-value as c o m p a r e d to the y o u n g (P < 0.0:~, table 11.

133 A

~00 80 80 u

40 PO

-t0

-9

-8 -7 -6 -5 log (isoproterenol. H)

-4

-3

100 ~

-1t

-t0

-9

-8 -7 -6 log (I. ~]

-I!

~~0

-9

-8

-5

-4

-3

B

8t)

~

~o

~

4O

6(] •~'

4(]

2O 0

,

-10

-9

-fl -7 -6 -5 log I~.soproterenol. M)

-4-

-3

-7 -6 -5 -4 -3 II. M) Fig. 3. Mean competition curves for the displacement of 1CYP by metoprolol and ICI 118,551. (A) Young (2-3 month) ra~s (n = 6); (B) old (24-25 month) rats (n = 6). Diamonds, ICI 118,551; squares, metoprolol. 10g

Fig. 2. Mean competition curves for the displacement of ICYP by (-)-isoproterenol in the presence (triangles) and absence (circles) of 300 /tM GppNHp. (A) Young (2-3 month) rats (n = 10); (B) old (24-25 month) rats (n = 10).

TABLE 3

TABLE 2 Competition binding experiments with ICYP as radioactive ligand: characteristics of the binding of metoprolol and ICI 118,551 to fl-adrenoceptors in total kidney membranes from young (2-3 month, n = 6) and old (24-25 month, n = 6) rats. H, high-affinity binding; K i l l ; inhibition constant for the high-affinity binding site; KiL, inhibition constant for the low-affinity binding site. %H

Metoprolol Young rats Old rats

75.3_+1.6 68.4_+3.0

ICI 118,551 Youngrats Oldrats

2;..5_+1.0 34.5+_3.1b

KiH (10 -I° M)

K,L (10 -7 M)

915.0_+189.0 2600.0_+700.i~ a

56.8_+ 15.8 136.0_+64.0

6.2-+ 2.6 43.9-+ 13.9 ~

2.l_+ 0.6 4.6_+ 1.2

P < 0.05; b P < 0.01 (Student's t-test).

Adenylate cyclase activity (pmol cAMP. mg protein - ~. rain- i ) in total kidney membranes from young (2-3 month) and old (24-25 month) rats. Additions Basal activity None

Young rats (n =13)

Old rats (n =13)

11.0_+ 1.3

16.0+ 1.2 b

132.3+10.7 47.3+- 4.4 6.1_+ 0.7 40.7_+ 2.5

179.2_+12.2 b 56.4+_ 4.9 15.7_+ 2.0 b 58.6+_ 4.6 h

Increase over GTP or GppNHp activity Isoproterenol + GTP 2.7_+ 0.4 Isoprotercnol+GppNHp 3.6_+ 1.9

4.3_+ 0.7 ~ Z 8 + 2.4

Increase ever basal activity Forskolin NaF GTP GppNHp

P < 0.05; ~' P < 0.01 (Student's t-test).

134 The results of the competition experiments with the fl~-selective antagonist metoprolol and the fl2selective antagonist ICI 118,551 are shown in fig. 3 and table 2. Both antagonists bind to a highand a low-affinity binding site. High-affinity binding of metoprolol amounts to 75.3% of the total in young animals and to 68.4% of the total in old animals (P > 0.05). For ICI 118,551, high-affinity binding amounts to 21.5% in young and to 34.5% in old animals (difference P < 0.01). The equilibrium inhibition constant for the high-affinity binding site of metoprolol and ICI 118,551, respectively, was 2.8 and 7.1 times higher (P < 0.05) in old animals than in young ones; the affinity for the low-affinity binding site was unchanged with aging.

3.2. Stimulation of adenylate cyclase The results of the experiments involving the determination of the activity of adenylate cyclase are summarized in table 3. Basal adenylate cyclase activity (pmol c A M P . mg protein -~. rain -~) was increased by 31% in kidneys of old animals as compared to young ones (P < 0.01). Stimulation of the enzyme with either GTP, GppNHp, NaF or forskolin, increased the adenylate cyclase activity both in young and old animals above basal values (paired Student's t-test, P < 0.01, not indicated in table 3). The net increase (mean v~h,e obtained with stimulation minus basal value) was higher in the old than in "he young animals (P < 0.01) for GTP, G p p N H p and forskolin; the effe~, of NaF was similar in both groups of animals. Stimulation of adenylate cyclase with isoproterenol in the presence of GTP or of GppNHp, resulted in a small, sigrdficant increase in cAMP synthesis above the value obtained with GTP or with G p p N H p alone in both young and old animals (paired Student's t-test, P < t).01, not indicated in table 3). Stimulation with isoproterenol in the absence of GTP or GppNHp did not affect the adenylate cyclase activity (results r~ot shown). The net increase (mean value obtained with stimulation with (-)-isoproterenol in the presence of GTP minus the value obtained with GTP alone) was significantly different for the kidneys from young and old animals (P < 0.05).

When G p p N H p was used instead of GTP, the net increase obtained with isoproterenol stimulation was not significantly different for young and old animals. Hotelling's t-test, using Bmax, K D, percentage of high-affinity coupled receptors, the inhibition constant at the high-affinity binding site and basal adenylate cyclase activity as variables, showed no difference between the two age groups.

4. Discussion Recently, we described our findings on the effect of aging on some of the properties of the fl-adrenoceptor transduction system in rat lung. which contains a majority of fl2-adrenoceptors. It was found that, in spite of alterations at different levels of the transduction chain, agonist stimulation led to a comparable synthesis of cAMP in young and old rats. We concluded that in rat lung, homeostatic mechanisms remain active throughout aging (Vanscheeuwijck et al., 1989). In this study, we investigated the influence of aging on rat kidney/Ladrenoceptors, which are predominantly of the ill-subtype. The effect of aging on the biochemical properties of the /3-adrenergic transduction system in kidney is not well known. Since the fl-adrenergic transduction system is composed of several units (Neer and Clapham, 1988), the expression of the effect of age could be present at each level of the transduction chain. Hence, our experiments can be subdivided into three groups, according to the level of the transduction chain which is mainly involved. In the first group, experlraents were performed yielding information on the binding of different compounds with the receptor. Our results can be summarized as follows: the values for the Bm~x and the ligand affinity of total kidney membranes from young rats are comparable to what is found by others for young rats (Zini et al., 1983; Michel et al., 1987). We found that in old animals, the receptor density was almost doubled and the affinity of this binding site for the ligand was halved. This finding is in keeping with the data we found for rat lung (Vanscheeuwijck et al., 1989). For other tissues (e.g., rat liver, Katz, 1988; and guinea

135 pig lung, Duncan et al., 1982) an increase in Bmax value upon aging is also found. It is, however, in contrast to the ongoing belief that the aging process is a_.companied by a decrease in the number of receptors (Scarpace, 1986). Our results suggest that aging is not necessarily accompanied by a decrease in receptor density, possibly dependent on the strain of rats or the tissue used. Our results on the age-dependent change in the /31//~2-subtype ratio in rat kidney are in contrast with those of Galbusera et al. (1988), who found a shift ~r, the #t//~2 ratio in favor of the #l-subtype in kidneys from old rats. We found a significant increase in the percentage of binding sites which bind ICI 118,551 with high affinity (/32-subtype), while the percentage of binding sites with high affinity for metoprolol (/3~-subtype) were lower, without reaching statistical significance. This can possibly be explained because of the availability of metoprolol in the ( + ) configuration, while ICI 118,551 is in the pure ( - ) form. The use of a different ligand and of different antagonists could explain the discrepancy with the results of Galbusera. As the selectivity of metoprolol (Abrahamsson et al., 1988) and ICI 118,551 (Bilski et al., 1983) is well documented, and as the percentages of /3~- and B2-adrenoceptors within each age group are independent of which antagonist is used, we believe that in rat kidney, the age-dependent increase in /~-receptor density is accompanied by a change of the B~/~2 ratio in favor of the/32-subtype. This change in the/31//32 ratio could have consequences at the level of the respoia~e to agonist stimulation. Indeed, it has been suggested that the different receptor subtypes are heterogeneously distributed in kidney (131 in glomeruli and /~2 in tubules, Healy et al., 1985) and that the response upon isoproterenol stimulation is much higher in tubules than in glomeruli (Sundaresan et al., 1987). Since we used total kidney in our e;:pe,iments, we are unable to draw any conclusion on this item. Experiments with selective and irreversible blockade of one of the receptor subtypes or with glomeruli and tubules separately could yield further information. In the second group of experi~,aents, the degree of coupling between receptor and GTP-binding protein was investigated. It has been shown on

many occasions that part of the fl-receptors are present as high-affinity isoproterenol binding sites which represent the receptor-GTP protein-coupled fraction. The remaining binding sites are present in a low-affinity configuration. Upon addition of GTP or of its non-hydrolysable analogue GppNHp, all binding sites are of the low-affinity binding type (Birnbaumer, 1987; Graziano and Gilman, 1987: Weiss et aL, 1988). In our preparation, both in young and old animals, high- and low-affinity binding sites were detected in the absence of GppNHp. The fraction of the fladrenoceptors in high-affinity configuration, however, was significantly lower in the older animals as compared to the young. This result is compatible with data for rat lung (Scarpace and Abrass, 1983; Vanscheeuwijck et al., 1989) and human lymphocytes (Feldman et al., 1984). Apparently, the aging process is associated with a decrease in the ability of the receptors to be coupled with the GTP-binding protein. As discussed previously, membrane fluidity could play an important role in this p h e n o m e n o n (Hanski et al., 1979; Vanscheeuwijck et al., 1989). In addition to the increase in KD-value, the competition binding experiments demonstrated that there is an age-dependent decrease in affinity for both the agonist and antagonists. This could mean that the age-dependent perturbations of the membrane structure (e.g., as discussed above: alterations of the membrane composition a n d / o r of its fluidity), could be important parameters in the age-dependent alterations of the receptor-ligand interaction. In the third group of experiments, data were collected related to the enzyme adenylate cyclase and to the effects of stimulations at different levels of the transduction chain on the formation of cAMP. Our results show that the basal (non-stimulated) enzyme activity was significantly higher in old rats. This is in contrast to the results obtained for rat lung (Scarpace and Abrass, 1983; Vanscheeuwijck et al.. 1989). rat brown adipose tissue (Scarpace et al., 1988) and human lymphocytes (Abrass and Scarpace, 1982; Krall et al., 1983). The increase of adenylate cyctase activity induced by direct stimulation of the enzyme with

136 forskolin and stimulation at the level of the GTPbinding protein with guanine nucleotides, is also significantly more pronounced in the older animals as compared to the young. Agonist stimulation at the level of the receptor in the presence of GTP, yielded a significant different value for both age groups, although significance was barely reached (P = 0.0497). There are several reasons for cautious interpretation of the significance. First, it is clear that this P-value is so close to the conventional 0.05 level that firm conclusions are at least doubtful (Feinstein, 1985). Second, when the increase in adenylate cyclase activity by isoproterenol stimulation in the presence of GTP is expressed as a percentage of the activity in the presence of GTP alone, no significant difference between the two age groups was obtained. This could mean that the net increase caused by isoproterenol stimulation is at least partially influenced by the activity under GTP stimulatory conditions. As the GTP stimulation of adenylate cyclase is more pronounced in older rats, this could explain part of the difference. Third, applying the multivariate Hotelling's t-test to the variables which are directly related to the agonist-stimulated cAMP production (receptor density, ligand affinity, percentage high-affinity binding site, the inhibition constant of the high-affinity binding sites and basal adenylate cyclase activity) failed to show any significant difference between the two groups. The use of multivariate statistical methods has been discussed by others (Cupples et al., 1984; Stahle and Wold, 1986). In addition, although less convincing, using G p p N H p instead of GTP in combinatio~ with isoproterenot leads to a comparable stimulation in both age groups. For all these reasons, we suggest that the observed difference for the agoniststimulated cAMP production between the two age groups is probably of limited importance. When comparing the present results with those from our previous study on the rat lung fl-adrenergie transduction, it is clear that in both tissues. the aging process is accompanied by changes at each level of the fl-adrenergic transducticn chain: at the level of the receptor, at the level of the coupling between receptor and GTP-binding protein and at the level of adenylate cyelase. Indeed,

in lung as well as in kidney, the receptor density increased upon aging and the ligand affinity decreased. Furthermore, in both tissues, there was a decrease in percentage of high-affinity receptor configuration. As far as adenylate cyclase is concerned, there was, in lung, a tendency for a decrease in cAMP synthesis with increasing age in all types of stimulation, reaching a significant level in the case of forskolin only. In rat kidney, there is a tendency for an increase in adenylate cyclase activity, reaching a significant level for the basal value and in the case of stimulation with forskolin, GTP and G p p N H p . Finally, in both tissues, the cAMP synthesis upon agonist stimulation is probably unaltered with age, as discussed above. Our results strongly suggest that in rat kidneys, in spite of a number of changes occurring at different levels of the fl-adrenergic transduction system, age does not affect the synthesis of cAMP upon a given stimulation with agonist. The observations that the receptor density increases and that the percentage of receptors coupled to the GTP-binding protein decreases, could mean that in this tissue, which contains mainly flt-adrenoceptors, and in lung, which contains mainly flzadrenoceptors, compensatory mechanisms are present in order to assure comparable cAMP production for equal stimulatory conditions.

Acknowledgements The authors are grateful to Dr. M. Bogaert and Dr. R. Lefebvre for critical reviewof the manuscript. Financial support was given by the Fund for Medical Scientific Research (Belgiuml grant no. 3.9006.87. P.V. is a recipient of a grant from the Belgian I.W.O.N.L Statistical analysis with the programs Statgraphics and SPSS was performed by G. Van Maele of the Department of Medical lnformatics at the Ghent University Hospital.

References Abrahamsson, T.B.. Ek and V. Nerrae. 1988. The ill- and flz-adrenoceptor affinity of atenolol and metoprolol, Biochem. PharmacoL37. 2C3.

t37 Abrass, I.B. and P,J. Scarpace, 1982, Catalytic unit of adenylate cyclase; reduced activity in aged human lymphocytes. J. Clin. Endocrinol. Metab. 55, 1026. Barnett, D.B., E.L. Rugg and S.R. NahorskL 1978, Direct evidence of two types of fl-adrenoceptor binding site in lung tissue, Nature 273, 166. Bilski, A.J.o S.E. Halliday. J.D. Fitzgerald and J.L. Wale. 1983. The pharmacology of a fl2-selective adrenoceptor antagonist (ICI 118,551), J. Cardiovasc. Pharmacol. 5. 430. Birnbaumer, L., 1987. Which G-protein subunits are the active mediators ie signal transduetion? Trends PharmacoL Sci. 8. 209. Brodde, O.-E., 1982, Homogeneous class of beta-1 adrenergic receptors in rat kidney, Biochem. Pharmacol. 31. 1743. Cheng. Y.-C. and W.H. Prusoff, 1973, Relation between the inhibition constant (Ki) and the concentration of inhibitor which causes 50'~ inhibition (t~,,) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099. Cupples, L.A., T. Hecren, A. Schatzkin and T. Colton. 1984, Multiple testing of hypothesis in comparing two groups. Ann. Internal Med. 100. 122. Duncan, ,'.G., C. Brink and J.S. Douglas. 1982. B-receptors during aging in respiratory tissues. European J. Pharmacol. 78, 45. Feinstein. A.R.. 1~35, Tempest in a P-pot? Hypertension 7, 313. Feldman, R.D., L.E, Limbird. J. Nadau, D. Robertson and A.J.J. Wood, 1984. Alterations in leukocyte beta-receptor affinity with aging, New Engl. J. Med. 310. 815. Fraeyman, N., P. Vanseheeuwijck and E. Van de Velde, 1989. Age-related alterations of the beta-adrenergie transdoction system in rat kidney, Arch. Int. Physiol. Biochim. 97. B147. Galbusera, M.. S. Garattini. G. Remuzzi and T. Mennini, 1988. Catecholamine receptor binding in rat kidney: effect of aging, Kidney Int. 33, 1073. Gilman, A.G., 1970, A protein binding assay for adenosine 3",5"-cyclic monophosphate, Proc. Natl. Acad. SCi. U.S.A. 67. 305. Graziano, M.P. and A.G. Gilman, 1987. Guanine nucleotidebinding regulatory proteins: mediators of transmembrane signaling. Trends Pharmacol Sci. 8, 478. Hanski, E., G. Rimon and A. Levitzki. 1979, Adenylate cyelase activation by the beta adrenergic receptor as a diffusioncontrolled process, Biochemistry 18, 846. Healy, D.P., P,A. Munzel and P.A. lnsel. 1985, Localization of ill- and B2-adrenergic receptors in rat kidney by autoradiography. Circ. Res. 57. 278. Katz, J.F., 1988, Food restriction modulates B-adrenergic sensitive adenylate cyclase in rat liver during aging. Am. J. Physiol. 254. E54. Krall, J.F.. M. Conndly-Fittingoff and M.L. Tuck. 1983. Lymphocyte adenylate cyclase and human aging. Proc. Soc. Exp. Biol. Med. 173. 475. Macart, M. and L. Gerbaut. 198L An improvement of the Coomassie blue dye binding method allowing an equal sensitivity to various proteins: application to the cerebrospinal fluid, Clin. Chim. Acta 122, 93. Michel, M.C.. X.L. Wang. E. Schlicker. M. G&he~. JA. Be-

ckeringh and O.-E. Brodde. 1987. Increased fl2-adrenoceptor density in heart, kidney and lung of spontaneottsly hypertensive rats. J. Auton. Pharmacol. 7.41. Motulsky, HJ., 1987, ~GraphPAD': Plot, Analyse and Digitize Graphs. ed. M. DiMaggio, ISI software. San Diego. Mnustafa. E.. M.D. Suavely and P.A, lnsel. 1984, Selective inhibition by organic mercurials of binding to the beta~ population of rat renal cortical beta-adrenergic receptors. Biochem. Pharmacol. 33. 1148 Neer, E.J. and D.E. Clapham, 1988, Roles of G-protein subunits in transmembrane signalling. Nature 333. 129. Scarpace. P.J.. 1986. Decrcasea Deta-adrenergic responsiveness during senescence. Fed. Proc. a5. 51 Scat'pace. P.J. and I.B. Abrass, 1983. Decreased beta-adrenergic agonist affinity and adenylate cyclase activity in sene~ent rat lung. J. Gerordol. 3L, 143. Scarpace, PA., A.D. Mooradian and J.E. Morley. 1988. Age-associated decrease in beta-adrenergic receptors and adenylate cyclase activity in rat brown adipo~ tissue. J. Geroutol. 43. B65. Snavely. M.D.. H.J. Motulsky. E. Moustafa. L.C. Mahan and P.A. lnsel. 1982. fl-adrenergic receptor subtypes in the rat renal cortex, Circ. Res. 5t. 504. Snedecor. G.W. and W.G. Cochran. 1973, Curvilinear regression. in: Statistical Methods. ChaFter 15. eds. G.W. Snedecor and W.G. Cochran I Iowa State University Press. Ames. IAt p. 447. Stahle. L. and S. Wold. 1986. On the use of some multivariate statistical methods in pharmacological research. J. PharmecoL Methods 16. 91. Sundaresan. P.R., T.L. Fortin and SL. Ketvie. 1987, a- and fl-adrenergic receptors in proximal ~,ubules of rat kidney, Am. J. Physiol. 253. F848. To~ey. K.C.. K.G. Oldham and J.A.M. Whelan, 1974, A sin~e direct assay for cyclic AMP in plasma and other biological samples using an improved competitive protein binding technique, Clin. Chim. Acta 56. 221. Van Oosterhout. A.J.M., G, Fotkerts, G.A.M. Ten Have and F.P. Nykamp, 1988. Involvement of the spleen in endotoxin-induced determination of the respirato~ air~'ay and lymphocyte B-adrea~g~e :,ystem in guinea pig, European J, Pharmacol. 147. 421. Van~cheeuwijck. P. and N, Fraeymam 1989. Evatua~,~on of a semi-automatic cell harvester filtration for the de~,erminalion of fl-adrenoceptors ill human mononuctear le~k(~cytes, J. i~harmacoL Methods, 21. Vanscheeuwijck, P., E. Van de Velde and N. Fraeyman. 1989, Effect of aging on propertie ~, and function ~f fl-adrenoceptots in rat lung. Eurovean J. PharmacoL (Mot. Pharmacol. Sect-.I t72. 373. Weiss. E.R.. D.J. Ketteher. C.W. Wean. S, St~parkar, S. Osawa. L.E, Heasley and G_L. Johnson. 1988, Receptor activation of G-proteins. FASEB J. Z 2841. ZinL K.. L Gauh. S_ Ledev,3-n, Ph, D'Athis and J_P, TitJement. t983. Binding of propramqol and ic,docyanopindolol to isolated cells, homogenates and plasma membranes of rat liver, tur~o kidney and heart. Biochem. PharmacoL 32. 3375.