Catecholamine Receptor Binding in Rat Kidney: Effect of Aging

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Kidney International, Vol. 33 (1988), pp. 1073—1077

Catecholamine receptor binding in rat kidney: Effect of aging

MIRIAM GALBUSERA, SILvlo GARATTINI, GIUSEPPE REMUZZI, and TIZIANA MENNINI

Mario Negri Institute for Pharmacological Research, Via Eritrea 62, 20157 Milano and Via Gavazzeni 11, 24100 Bergamo, Italy

Catecholamine receptor binding in rat kidney: Effect of aging. Binding With the present study we wanted to assess the distribution to several receptors was compared in kidneys from 3- and 24-month-old of catecholamine receptors in the kidney and their affinity for rats. In crude membrane preparations of aged rat kidneys, the number of f32-adrenergic receptors was significantly reduced but the number of specific binding in rats at 3 and 24 months of age. Our results total /3-adrenoceptors was unchanged. The high-affinity a-adrenocep- can be relevant to the problem of age-associated changes in tor component was significantly reduced in old rats, whereas the low- renal hemodynamics. affinity component was unchanged. The number of a2-adrenoceptors showed a non-significant decrease. 3H-spiperone binding sites were Methods similar in young and old rats. For each receptor binding the KD values were the same in young and old animals. The D1 dopaniLine receptor was Adult (3 months) and aged (24 months) male Sprague-Dawley significantly reduced in old rats. In our experiments, age-related changes CD-COBS rats (Charles River, Calco, Italy) maintained in of specific binding sites in the kidney were selective for some receptors standard laboratory conditions were used. The animals were studied and did not seem to be due to general aging-induced membrane modifications. Moreover, the renal and central receptors were sensitive killed by decapitation and kidneys were immediately removed to aging differently in the same animal model. and frozen at —80°C until binding experiments. The kidneys were thawed, minced and homogenized in 50 volumes of ice-cold washing buffer (as required for the different binding assays except for DA receptors assays when 100 volumes were Catecholamine receptors in the kidney modulate hemody-used) using an Ultra-Turrax TP-1810 (2 ><30sec; Janke & namics [1] and electrolyte balance [1, 2]. Actually dopamineKunkel GMBH & Co KG, Staufen, FRG). To obtain crude receptors can regulate renal vascular tone and sodium excretionmembrane preparation, the homogenate was centrifuged at [3, 4]. Alpha-adrenergic receptors have been found to mediate50,000 x g for 10 minutes, the supernatant was discarded, the renal vasoconstriction and blood flow redistribution from cor-pellet resuspended, incubated at 37°C for 10 minutes and tical to medullary areas, and their activation induces a decreasecentrifuged at 50,000 x g for 10 minutes. The pellet was then in urinary sodium excretion and a rise in renin secretion rate [51.resuspended and filtered through four layers of sterile gauze to Finally,/3-receptors located on the juxtaglomerular cells in theremove large particulate matter, and centrifuged again. The last kidney mediate renin release [5, 6]. So far, a variety ofpellet was resuspended just before the binding assay in 20 experimental models have been used to study catecholaminevolumes of ice-cold incubation buffer. receptors [1, 6—141 and different experimental approaches have been employed to characterize them. At present, no uniform 3H-spiperone binding information is available within a given animal species to evalu- Washing buffer was 50 mrvi Tris-HC1, pH 7.4, containing 3 ate the influence of pathophysiological changes on receptormM CaCl. Incubation buffer was 50 mrvi Tris-HCI, pH 7.1, number and affinity for specific ligands. containing 10 .tMpargyline,5.7 mrvi ascorbic acid, 120 mrvt In the last few years the influence of aging on renal hemody-NaCl, 2 mrvi CaC12, 5 mtvt KC1 and 1 mrvi MgCI2. For 3H- namics and electrolyte excretion has been extensively studiedspiperone binding, samples of I ml final volume containing 10 [151. The results of these investigations have showed that agingmg of tissue and 10 nrvi 3H-spiperone (S.A. 23.4 Ci/mmol, New of the kidney of experimental animals and humans is associatedEngland Nuclear, Boston, Massachusetts, USA) were incu- with dramatic renal functional changes, mainly characterizedbated for 15 minutes at 37°C in the presence of unlabelled by a reduction in glomerular filtration rate, a decline in renalspiperone Ito 1000 M. Non-specific binding was determined in blood flow and a progressive proteinuria. The cause of suchthe presence of 10 tM (+)-butaclamol and represented 40% of profound changes in renal functional parameters which developtotal binding. with age are not completely understood. It has been suggested that elevated glomerular capillary pressures and flows contrib- 3H-serotonin binding ute to the development of glomerular sclerotic lesion leading to Washing buffer was 50 mrvi Tris-HCI, pH 7.4. Incubation progressive renal insufficiency. buffer consisted of 50 mvt Tris-HCI, pH 7.7, containing 10 /.LM pargyline; 5.7 mrs'i ascorbic acid; and 4 mrvi CaCl2. For 3H- serotonin binding, samples of 1 ml final volume containing 10 Received for publication March 9, 1987 mg of tissue and 4 nivt 3H-serotonin (S.A. 14.9 Ci/mmol, and in revised form October 5, 1987 Amersham International plc, Buckinghamshire, UK) were in- © 1988 by the International Society of Nephrology cubated for 15 minutes at 37°C in the presence of unlabelled

1073 1074 Galbusera et al: Renal catecholamine receptor binding serotonin 0.5 to 5.000 nM or unlabelled spiperone 0.5 to 5000 Calculations flM. Binding parameters for 3H-spiperone, 3H-SCH 23390 and 3H-yohimbine were obtained using increasing concentrations of 3H-Ketanserin binding unlabelled ligand; data were analyzed by non-linear fitting Washing and incubation buffer was 50 mM Tris-HCI, pH 7.4.according to the function: For 3H-Ketanserin binding, samples of I ml final volume [3F1-ligand] containing 10 mg of tissue and 1 nM 3H-Ketanserin (S.A. 95 Bound =Bmax [Inhib]\ Ci/mmol, New England Nuclear) were incubated for 15 minutes [3H-ligand] + K0 + (1 + at 37°C in the presence of unlabelled serotonin 0.5 to 5,000 nM Ki ) or unlabelled spiperone 0.5 to 5,000 nM. + non-specific bound 3H-SCH 23390 binding by using the NL FIT program [16] running on the HP8S desk Washing buffer was 50 mrt Tris-HCI, pH 7.4. Incubationcomputer. Binding parameters for other ligands were calculated buffer was 50 mi Tris-HCI, pH 7.9, containing 1 mi EDTA andby non-linear fitting analysis of data from saturation experi- 4 mM MgSO. For 3H-SCH 23390 binding, samples of 1 ml finalments using six to seven different concentrations of 3H-ligand, volume containing 10 mg tissue and 2.5 nM 3H-SCH 23390 (S.A.each assayed in duplicate or triplicate, using the program 85 Ci/mmol, Amersham, mt. plc) were incubated for 30 minutes"Recept" running on an HP85 desk computer [171. For inhibi- at 25°C in the presence of unlabelled SCH 23390 0.1 to 100,000tion experiments binding parameters were calculated by non- n. Non-specific binding was determined in the presence of 100linear fitting analysis of data using 9 to 10 different concentra- /LM cis-fiupentixol and was about 80 to 85% of total binding. tions of inhibitor, each assayed in duplicate or triplicate, using the "Recept" program [17]. 3H-prazosin binding Proteins were determined by the method of Peterson [18] Washing and incubation buffer was 50 mrvi Tris-HCI, pH 7.4.using bovine serum albumin as standard. For 3H-prazosin binding, samples of 1 ml final volume containing 10 mg of tissue and 0.01 to 6 nM 3H-prazosin (S.A. 80.9 Drugs Ci/mmol, New England Nuclear) were incubated for 30 minutes The following were used: bovine serum albumin (Merck, at 25°C, Non-specific binding was determined in the presence ofDarmstadt, FRG); spiperone and haloperidol (Janssen, Beerse, 100 LM phentolamine, and represented 40 to 45% of totalBelgium); (+)-butaclamol and (—)-butaclamol (Research Bio- binding. chemical Inc., Wayland, Massachusetts, USA); phentolamine (Ciba-Geigy, BasIc, Switzerland); P0160 (Pierrel, Milan, Italy); 3H-yohimbine binding zinterol (Mead Johnson and Company, Evansville, Indiana, Washing and incubation buffer was 50 m'vi Tris-HCI, pH 7.4.USA) and (+/—)-propranolol (ICI, Cheshire, UK). Serotonin For 3H-yohimbine binding, samples of 1 ml final volume con-creatinine sulphate (Fluka AG, Buchs, CH), cis(Z)-flupentixol taining 10 mg of tissue and 5 nM 3H-yohimbine (S.A. 90(H Lundbeck & Co., Copenhagen, Denmark) and SCH 23390 Ci/mmol, Amersham International plc) were incubated for 30(Schering Corp., Bloomfield, NJ, USA) were used. minutes at 25°C in the presence of unlabelled yohimbine 1 to 100,000 n. Non-specific binding was determined in the pres- Results ence of 100 ILM phentolamine and was about 70% of total Kidney weight was significantly (P < 0.001) greater in old rats binding. (2.20 0.20 g for old and 1.490.15 g for young) and protein concentration was significantly lower (P < 0.05) in aged animals 3H-dihydroalpreno/ol (3H-DHA) binding (31.96 5.56mg/gtissue in old and 36.914.87 mg/g tissue in Washing and incubation buffer was 50 m Tris-HCI, pH 7.4. young). For 3F1-DHA binding, samples of 1 ml final volume containing Kinetic parameters of 3H-spiperone binding, which is a 10mg of tissue and 3}{-DHA (S.A. 58 Ci/mmol, Amersham; 0.35marker for D2 dopamine receptors, were similar in young and to 6.7 n for saturation studies, 1 nM for inhibition studies)old rats and indicated a very high density of binding sites (1200 were incubated for 25 minutes at 25°C in the presence ofpmol/mg protein) with low-affinity (Table 1). Similar results different concentrations of l-phenyl-3(2-(3- (2-cyanophenoxy)were obtained when membrane preparations were carried out in -2-hydroxypropyl)-aminoethyl) -hydantoinHCI (P0160) or zin-Tris HCI 50 mM, pH 7.4, without the addition of ions (Bmax terol. Non-specific binding was determined in the presence of 1 1706 228 pmol/mg protein, K0 19.82 2.94 >< l0 M). M (+1—)- propranolol, and corresponded to 60 to 70% of totalInhibition experiments with (+)-butaclamol gave an 1C50 of 8.6 binding. 14.1 x l0— M for young and 1.330.93 x lO M for old For all binding assays incubation was stopped by the additionanimals. Using (—)-butaclamol we obtained an 1C50 of 1.63 of ice-cold incubation buffer followed by rapid filtration under0.29 x iO M for young and 4.11 2.8 x i0 M for old vacuum through Whatman GFIBglassfiber filters, and threeanimals, not significantly different from the values obtained washes with incubation buffer. Filters were counted in 8 ml ofwith the (+) form. Filter-Count scintillator (Packard Instrument Co. Inc., Down- To test whether the renal receptor population bound by ers Grove, Illinois, USA) in a Beckman LS 7500 scintillation3H-spiperone presented a serotoninergic component we mea- counter (Beckman, Irvine, California, USA) at 50% efficiency.sured 3H-serotonin and 3H-Ketanserin binding in the presence Galbusera et a!: Renal catecholamine receptor binding 1075

Table 1. Kinetic characteristicsof different receptors in kidneyof young and old rats Agemonths Receptor subtype 3H-Ligand Binding parameter 3 24 Dopamine D1 3HSCH23390 BmafmolImg protein 1631.00 94.00 816.00 54.OOa K0 (nre) 192.68 17.85 103.93 592b D2 3H-spiperone Bmax pmol/mg protein 1200.00 140.00 1190.00 238.00 K0 /.LM 22.40 3.20 22.00 7.60 Adrenoceptors: alpha-I 3H-prazosin high-affinity BfmolImg protein 23.00 4.00 11.00 2.90t K0 flM 0.03 0.04 0.02 0.01 low-affinity Bmaxfmo//mgprotein 850.00 332.00 913.00 308.00 K0 nM 28.70 14.30 28.50 12.00 alpha-2 3H-yohimbine B,,axfmol/mg protein 70.80 29.20 45.40 26.10 K0 n 15.20 4.50 12.80 17.30 beta-I +beta-2 3H-dihydroalprenolol Bmaxfmollmg protein 36.10 0.41 34.40 4.10 KD nM 1.20 0.20 1.10 1.19 beta-I — Br,,axfmol/mg protein 15.70 0.60 21.50 2.60 beta-2 BaxfmolImg protein 20.50 0.20 12.90 Kinetic characteristics of the different ligands were calculated by non-linear fitting of binding data. Results are means SDof 3 to 4 animals per group. B,ax values for total /3-adrenoceptors were obtained in presence of I M (+/—)-propranolol; for /31 determination I M P0160 was used; /32 adrenoceptor numbers were calculated as the difference between the two values. a P<0.001,Student's t-test b P<0.05,Student's t-test of increasing concentrations of unlabelled serotonin and 30 30 spiperone. No specific binding was detected. C 24 0 24 3H-SCH 23390 binding .018 18 C 12 12 The maximum number of D1 receptors (Bmax) was signifi- 6 6 cantly decreased in aged animals (1,631 94and 816 54 fmol/mg protein in young and in old, respectively, P < 0.001; 0 0 Table 1). K0 was also slightly reduced in aged rats, due to —10 —8.6 —7 —5.4—3.8 —10 —8.6 —7 —5.7—3.8 mathematical correlation between the two parameters. 30 30 c 24 24 3H-prazosin binding 0 Saturation experiments indicated two populations of a-1 18 18 12 adrenoceptors in kidney. The high-affinity component gave the C same KD in the two groups (0.03 n in young and 0.02 flM in 6 6 ' / old) but a significantly lower number of receptors in aged 0 0 animals, with a Bmax of 23 4 fmol/mg protein in young and 11 —10 —8.6 —7 —5.4 —3.8 — —10—8.6 —7—5.4—3.8 2.9 fmol/mg protein in old (P <0.05; Table 1). No changes in Log [MI Log IM] relation to aging were found in the low-affinity component. Fig.1. 3H-DHA binding inhibition of P0160 and zinterol. Inhibition curves of 3H-DHA (1 nM)byP0 160 (top) and zinterol (bottom) were 3H-yohimbine binding analyzed by non-linear fitting of experimental data, according to a A non-significant decrease in a-2 adrenoceptor number wastwo-site model. For each inhibition curve the graphs report the final found in aged rats (Bmax 70.8 fitting of experimental points and the calculated functions for high- and 29.2 fmol/mg protein in young low-affinity sites (Methods). Shown are inhibition curves of one animal, and 45.4 26.1 fmol/mg protein in old) and no changes inrepresentative of its group. Each point is mean value of 3 determina- affinity (Table I). tions, varying less than 10%. y axis % inhibition of total 3H-DHA binding; x axis =logarithmof drugs molar concentrations. Symbols are: 3H-dihydroalprenolol (3H-DHA) binding (A) unbroken line, young; (•) broken line, old. Inhibition curves of I nM 3H-DHA binding (a marker for total /3-adrenoceptors), obtained with the /3-i selective antagonist P0160 (Fig. I) and /3-2 selective agonist zinterol (Fig. 1) are Table 2 shows the percentage inhibition of specific renal reported. Competition curves for both compounds were best3H-DHA binding in young and old rats by zinterol and P0 160. In fitted by a two-site model showing the presence of twothis experiment we found an almost equal density of /3-i and 13-2 adrenoceptor populations with different affinities for the selec-adrenergic receptors in young animals and a lower proportion of tive agents. The proportion of beta population that binds P0 16013-2 subtype in old animals, with either P0 160 (low-affinity) or at high-affinity represents the /3-i receptors, and P0160 low-zinterol (high-affinity). affinity binding represents the 13-2receptors[19, 20]. Zinterol Saturation curves using (+/—)-propranolol to define non- binds with high-affinity /3-2 and with low-affinity /3-ireceptor specific binding showed that the total number of beta receptors subtypes [21]. (/3-i + /3-2)wasnot changed by aging (Table 1). Using P0160 1 1076 Galbusera et a!: Renal catecholamine receptor binding

Table 2.Inhibition of specific renal 3H-DHA binding in young and old rats by P0160 (beta I) and zinterol (beta 2) High-affinity Low-affinity IC50 IC50 %RT flM %RT pi P0160 young 52.00 15.00 17.50 17.30 48.00 20.00 4.60 8.30 P0160 old 64.00 14.00 27.60 19.10 36.00 17.00 12.20 26.00 Zinterolyoung 48.00 25.00 103.00 120.00 52.00 22.60 5.70 8.90 Zinterolold 24.00 20.40 26.90 59.00 76.00 18.30 3.80 3.60 3H-DHA was used at mM final concentration. P0 160 and zinterol was used at concentrations of 0.1 to 100,000 nM Data were calculated as means SD from the inhibition curves (of one animal) presented in Fig. 1, by two-site model fitting of the experimental points. Abbreviations are: % RT, percentage of total receptors; and IC50, drug concentration giving half maximal inhibition of specific binding.

M (concentration that completely blocks 13-i receptors; Fig. 1)ligands for serotoninergic receptors, to bind to kidney mem- to define non-specific binding, we obtained saturation curves ofbrane preparation in our experimental conditions would rule out 13-ireceptors[20]. The amount of /3-2 subtype was obtained asthis possibility. the difference between the two curves (Table 1). These data are Alpha-adrenergic receptors have been studied using 3H- in agreement with those obtained in inhibition experiments andprazosin and 3H-yohimbine. The results indicate the presence showed a lower number of /3-2 receptors in aged rats (Table 1)of both a-I and a-2 receptor subtypes. Alpha-I receptors were with an insignificant increase in the /3-i subtype. resolved into two components, one with low and one with high affinity. As far as the receptor density the low-affinity compo- Discussion nent prevailed, the number of receptor being about 40 times Our present results identify catecholamine receptors in themore than the high-affinity component. These results are in rat kidney and characterize their binding parameters. In agree-agreement with those of McPherson and coworkers [9] who ment with previous studies we have documented the presencefound that 3H-prazosin binds to rat renal cortex with two of dopamine receptors, a-adrenergic receptors and /3-adrener-binding sites, a low and a high-affinity one, the low-affinity gic receptors in whole kidney crude membrane preparations. having the higher density. Alpha-2 receptor appears to consist As far as dopamine receptors we have identified the presenceof a single class of high-affinity binding sites. The affinity and of D1 subtype, while we were not able to characterize high-density of renal a-2 receptor binding we found in the present affinity and stereospecific D2 subtype in our preparation, but onstudy were consistent with previously published work [10, 111. 3H-spiperone binding sites. The identification of D1 receptors Beta-adrenoceptors have been identified by 3H-dihydroal- has been possible by virtue of a specific radioligand, 3H-SCHprenolol binding. The results showed a high-affinity, low den- 23390 [22] which has become available only recently and hassity binding. Since 3H-dihydroalprenolol binds to both f3-and never been used to identify D1 receptor subtypes in the kidney13-2 receptor subtypes we have resolved the two components by binding studies, The results indicate a high-affinity binding tousing a selective /3-I antagonist P0160 [19, 20] and a selective D1 receptors. The direct documentation of the existence of D1 /3-2agonistzinterol. It has been documented that rat kidney receptor in kidney homogenates supports previous work [23] inmembrane preparations possess both receptor subtypes with a which the vasodilatory and natriuretic response was studied incomparable density. These data are in agreement with those of dogs with and without pharmacological blockade of D1 receptorHealy, Munzel and Insel [13] who using autoradiographic with infusion of the specific D1 receptor antagonist SCH 23390.techniques found that both receptor subtypes are present in the Moreover our results are also consistent with the previous datarat kidney with an approximately equal relative percentage ofFelderet at [7] who documented the presence of D1 dopaminedistribution. receptors associated with adenylate cyclase in cortical tubules. When the results obtained in young rats were compared to As to the 3H-spiperone binding sites, our results indicate a low-those of old rats a selective reduction in D1, high-affinity a-1 and affinity high-density binding, which in our hands using (+) and13-2 was documented whereas 3H-spiperone binding sites low- (—)butaclamolwas not stereoselective. Our studies are inaffinity a-1, and /3-i were not changed by the process of aging. A contrast with previous studies which identified high-affinity highpossible explanation for the changes in catecholamine receptor density, stereospecific D2 receptor in the kidney labelled bydensity associated with aging rests on the observation that 3H-haloperidol or 3H-spiperone [7, 24]. These contradictoryplasma catecholamine concentrations are higher in aged than in results can be explained by the use of different experimentalyoung rats [26]. Actually high plasma concentrations of epi- conditions, like the strain of rats, the use of whole kidney andnephrine has been found to down-regulate catecholamine re- the procedure used for membrane preparations and bindingceptors [27, 281. That this can be the case is consistent with the measurements. Moreover, since spiperone can also bind sero-observation that endogenous catecholamines can selectively toninergic receptors of SHT2 subtypes [25],wewondered"down-regulate" a-1, but not a-2 adrenergic receptor in the whether the differences in affinity and density found in therenal cortex [29]. An alternative explanation for the selective present study in respect to previously published studies [7, 24]reduction in D1, high-affinity a-1 and /3-2 in old versus young rats were influenced by the presence of serotoninergic receptors.could be the progressive reduction of parenchimal renal mass Failure of both 3H serotonin and 3H Ketanserine, specificwhich occurs as a consequence of aging in the rat. Whether this Galbusera et a!: Renal catecholamine receptor binding 1077 could account for the selective reduction in some but not in all adrenoceptor modulating gluconeogenesis under physiological con- the catecholamine receptors cannot be established on the basis ditions. Br J Pharmacol 77:177—184, 1982 10. SCHMITZ JM, GRAHAM RM, SAGALOWSKY A, PETTINGER WA: of the results of the experiments presented here. Changes in Renal alpha-I and alpha-2 adrenergic receptors; biochemical and renal catecholamine receptors associated with aging in the rat pharmacological correlations. J Pharmaco! Exp Ther 219:400—406, could not have been predicted from the known changes ob- 1981 served under similar experimental condition in the rat brain. 11. WOODCOCK EA, JOHNSTON CI: Characterization of adenylate Actually, in the brain aging is associated with a selective cyclase-coupled alpha 2-adrenergic receptors in rat renal cortex using 3H-yohimbine. Mo! Pharmacol 22:589—594, 1982 decrease in the density of D2 receptors in the striatum [30] and12. SUMMERS RJ, KUHAR MJ: Autoradiographic localization of beta- of total /3-adrenoceptors [311. This would imply that catechol- adrenoceptors in rat kidney. Eur J Pharmacol 91:305—3 10, 1983 amine receptors in different tissues are differently affected by13. HEALY DP, MUNzEL PA, INSEL PA: Localization of /31- and aging process, thus showing tissue-specific adaptive responses. /32-adrenergic receptors in rat kidney by autoradiography. Circ Res 57:278—284, 1985 At the present stage of the knowledge about the functional14. MCPHERSON GA, SUMMERS RJ: Evidence from binding studies for significance of catecholamine receptors in the kidney, it is f31-adrenoceptors associated with glomeruli isolated from rat kid- difficult to define the pathophysiological meaning of the ob- ney. Life Sci 33:87—94, 1983 served changes in relation to age. Whether the reduction in a- 15. ANDERSON S, BRENNER BM: Effects of aging on the renal glomer- adrenoceptors which have been shown to mediate adrenergic- ulus. Am J Med 80:435—442, 1986 16. SACCHI LANDRIANI G, GUARDABASSO V, ROCCHETTI M: NL-FIT: induced vasoconstriction [1] accounts for the age-related max- A microcomputer program for non-linear fitting. Comp Progr imal vasodilation of residual glomeruli is a possibility which Biomed 16:35—42, 1983 merits investigation. 17. BENFENATI F, GUARDABASSO V: Basic concepts to analyze binding In conclusion, our findings identify and characterize cate- data using personal computer. The "recept" program, in Princip!es and Methods in Receptor Binding, edited by CATTABENI S, cholamine receptors in the rat kidney and show that aging is NICOSIA S, New York, Plenum Press, 1984, p. 41 associated with a lowering of D1 subtypes, a-sand 13-2thus 18. PETERSON GL: A simplification of the protein assay method of creating an imbalance in the relative density of the various Lowry et a!. which is more generally applicable. Anal Biochem catecholamine receptors. The possibility that the disproportion 83:346—356, 1977 19. MENNINI T, GOBBI M, RATTI E: Highly selective interaction of P between tise various receptor subtypes alters renal hemody- 0160 with /31-adrenoceptors in the rat heart: In vitro binding namics, thus accounting for adaptive changes associated with studies. (abstract) J Mo! Cell Cardio! 16 (suppl. 2):28, 1984 aging kidney, is worth investigating. 20. G0BBI M, MENNINI T, RATTI E: Biochemical characterization of a new highly cardioselective /3-adrenoceptor antagonist. J Pharm Acknowledgments Pharmacol (in press) 21. MINNEMAN KP, HEGSTRAND LR, MOLINOFF PB: Simultaneous We thank Fidia Research Laboratories for the kind gift of aged rats; determination of beta-I and beta-2-adrenergic receptors in tissues and Pierrel R. & D. Division for providing P0160. We thank Marco containing both receptor subtypes. Mo! Pharmacol l6:34—46, 1979 Gobbi and Stefano Cavanus for technical assistance. Judy Baggott and 22. BILLARD W, RUPERTO V, CROSBY G, IORLO LC, BARNETT A: Cristina Signorelli helped prepare the manuscript. Characterization of the binding of 3H-SC1-I-23390, a selective Dl receptor antagonist ligand, in rat striatum. Life Sci 35:1885—1893, Reprint requests to Dr. Miriam Galbusera, Mario Negri Institute for 1984 Pharmacological Research, Via Gavazzeni, 11, 24100 Bergamo, Italy. 23. 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