CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector 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 contain- ing 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).