Downregulation of parathyroid hormone receptors in renal ...

Downregulation of parathyroid hormone in renal membranes from aged rats

receptors

H. HANAI, D. P. BRENNAN, L. CHENG, M. E. GOLDMAN, M. CHOREV, M. A. LEVINE, B. SACKTORT, AND C. T. LIANG Laboratory of Biological Chemistry, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore 21224; Division of Endocrinology and Metabolism, Department of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205; and Department of New Lead Pharmacology, and Parathyroid Hormone Research Laboratory, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486

HANAI, H., D. P. BRENNAN, L. CHENG, M. E. GOLDMAN, M. CHOREV, M. A. LEVINE, B. SACKTOR, AND C. T. LIANG. Downregulation of parathyroid hormone receptors in renal membranes from aged rats. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F444-F450, 1990.-The mechanism of the inhibition or blunting of parathyroid hormone (PTH) -stimulated Na+-Ca2+ exchange activity in renal cortical cells from aged rats was examined. The number of PTH binding sites in basolateral membranes prepared from adult (6 mo) and old (24 mo) rats was quantitated by the binding of the synthetic analogue 1251-labeled [ Nle8y18, Tyr”“]bPTH-( l-34) amide to the membrane. The maximum number of specific PTH binding sites, B,,,, was 92.7 t 9.3 and 36.7 t 6.1 fmol/mg protein, respectively, in membranes prepared from adult and old rats. The affinity of the receptor to PTH was unaffected with age. The level of PTH binding components (68 and 70 kDa) estimated by a ligand affinity blot technique using biotinylated bPTH-( l-34) as the ligand was similarly reduced in membranes isolated from senescent rats. To test the hypothesis that change in the number of PTH binding sites and level of PTH binding components represented an adaptive response to a high serum PTH level, rats were parathyroidectomized (PTX) and the changes were reexamined. Decreases in the number of PTH binding sites and PTH binding components were either partially or completely negated by the surgery. These findings suggest that the blunting of both the PTH-stimulated Na+Ca2+ exchange and adenylate cyclase activities in the kidneys of aged rats was due, in part, to the loss of PTH receptors in basolateral membranes and that this defect could be partially reversed by removal of the parathyroid gland. adenylate cyclase; roidism; aging

desensitization;

secondary

hyperparathy-

EXPOSURE of the cell or tissue to hormone can lead to a subsequent impaired response to this hormone or other stimulants (6, 29). This phenomenon, known as desensitization, has been shown in many hormone-regulated adenylate cyclase systems. There are two discrete mechanisms by which desensitization is believed to occur. In homologous desensitization, downregulation is accompanied by the loss of receptor sites or change of receptor affinity to the specific hormone, resulting in a selective loss in the response of adenylate cyclase to the PROLONGED

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same hormone (3, 18). In heterologous desensitization, changes in levels of G proteins may alter the responsiveness of adenylate cyclase to other hormones (9, 19). Homologous desensitization to parathyroid hormone (PTH) has been shown in many experimental models, including cultured renal cells (19), bone cells (30) and bone tumor cells (11) pretreated with PTH, canine kidney perfused with exogenous PTH (23), and animals maintained on a low-Ca or vitami n D-deficient diet (8). The general consensus from these studies is that the loss of PTH receptor sites occurred in response to prolonged exposure to exogenous PTH or to elevated levels of circulating PTH. In contrast, heterologous desensitization to PTH has not been as widely characterized. In studies of renal membrane adenylate cyclase from vitamin D-deficient rats, desensitization to PTH was also associated with heterologous desensitization, presumably due to the reduction in levels of G proteins noted (24). PTH has been shown to regulate renal Na+-Ca2+ exchanger via both adenosine 3’,5’-cyclic monophosphate (CAMP)-dependent (12) and CAMP-independent (26,27) pathways. Recently, we reported that PTH-stimulated Na’-Ca2’ exchange and adenylate cyclase activities in renal cells were blunted in aged rats (12). Experiments on toxin-catalyzed ADP-ribosylation of renal membranes showed a decrease of both G, and Gi (13). These findings are consistent with the hypothesis that prolonged exposure to elevated circulating PTH levels observed in aged rats (1, 13, 16) could cause heterologous desensitization of the adenylate cyclase complex. In this study, we quantitate the number of PTH binding sites in renal basolateral membranes by use of a radiolabeled PTH analogue and a ligand affinity blot technique (5) to determine whether downregulation of PTH receptors in renal membranes is also involved in the blunting of responsiveness to PTH in senescence. MATERIALS

AND METHODS

Materials. [Nle8y18,Tyr34] bPTH- (l-34) amide was obtained from Bachem (Torrance, CA) and Peninsula Laboratories. bPTH- (l-34) was purchased from Peninsula Laboratories. Collagenase CLSII was from Cooper Biomedical. Hyaluronidase (type IS) was obtained from

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Sigma Chemical (St. Louis, MO). Animals. Male Wistar rats were obtained from the Animal Facility, Gerontology Research Center, National Institute on Aging. Animals of two age groups were used in this study. The 6-mo-old rat represents the sexually mature adult group, and the 24mo-old rat represents the senescent group. The mean 50% mortality age of our rats is -24 mo. Rats were maintained ad libitum on standard National Institutes of Health rat chow consisting of 23.5% protein, 1.2% Ca, and 1.0% P. The photo period consisted of 12-h light and 12-h dark. Because some of our old rats developed severe renal failure they were discarded. The blood urea nitrogen (BUN) values of the rats used for this study are in the range of 17-40 and 2065 mg/dl for young and old rats, respectively. Preparation of renal cortical basolateral membranes. The number of PTH binding sites in basolateral membranes isolated by the Percoll gradient procedure described previously (25) were determined by use of 1251labeled bPTH. The purity of the isolated basolateral membranes was monitored by the marker, Na+-K+-ATPase, which showed a ninefold increase in enzyme activity compared with crude membranes. Adenylate cyclase activity was also enriched in this isolated membrane fraction. Forskolin-stimulated adenylate cyclase activity was 1.08 t 0.18 and 3.76 t 0.24 nmolmg protein-l.20 min-‘, respectively for crude and basolateral membranes, an increase of X&fold in specific activity. The extent of enrichment of Na’-K’-ATPase and adenylate cyclase activities were unchanged in renal basolateral membranes prepared from the two age groups. 1251-labeledbPTH binding to receptors on renal basolatera1 membranes. 1251-labeled [NlesF18,Tyr34] bPTH- (l34) amide (1251-bPTH) was synthesized as described previously (10) and used to examine the number of PTH binding sites in basolateral membranes. Approximately 50 rugof membrane protein was incubated at 22°C for 30 min with 2.5 X lo4 counts/min (cpm) of 1251-bPTH in the presence and absence of 1 x 10s6 M unlabeled hormone analogue in 250 ~1 of medium containing 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris HCl), pH 7.4, 7 mM MgC12, 2.6 mM KCl, and 1% bovine serum albumin. A 200~~1aliquot of the reaction mixture was pipetted into microfuge tubes on ice and centrifuged at 4°C for 3 min at 12,000g. The supernatant, containing unbound radioligand, was removed by aspiration. The tips of the tubes containing the radioligand bound to the membranes were cut off, and 1251activity was measured in a Packard gamma scintillation spectrometer. Total binding of 1251-bPTH was defined as binding in the absence of unlabeled hormone analogue. Binding activity in the presence of unlabeled ligand was defined as nonspecific binding. Background binding (without membranes) was ~200 cpm. In some experiments, the specific binding of ‘251-bPTH, calculated as the difference of total and nonspecific binding, was determined. Identification of PTH binding components in renal membranes. Renal cortical cells were isolated from adult (6 mo) and old (24 mo) rats as described previously (12). Cells were homogenized at a concentration of 5-10 mg of cell protein/ml in a medium containing 20 mM Tris

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HCl, pH 7.4 and centrifuged at 10,000 g for 20 min at 4°C. The crude plasma membranes, obtained as the pellet, were used to measure PTH receptor level using a ligand affinity blotting technique with biotinylated bPTH-( l-34) as the ligand (5). Briefly, the membrane pellet was resuspended in 0.5% (vol/vol) 3-[ (3-cholamidopropyl) dimethylammono] -1-propanesulfonate (CHAPS) and 10 mM Tris . HCl, pH 7.4, to a protein concentration of 7 mg/ml. The suspension was incubated at 23°C for 30 min and centrifuged at 48,000 g for 90 min at 4°C. The supernatant fraction containing the soluble receptor proteins was diluted with 4 vol double-distilled H20 to yield a final concentration of CHAPS 0.1% (vol/ vol). Aliquots of this suspension were incubated at 23°C for 40 min in a mixture containing 17 nM biotinyl-bPTH, and 0.1% CHAPS in a total volume of 110 ,~l. The crosslinking agent disuccinimidyl substrate (DSS) was freshly dissolved in dimethyl sulfoxide and used at a concentration of 2 mM to attach covalently biotinyl-bPTH to the receptor. The cross-linking reaction was continued for 15 min on ice and terminated by addition of 2 vol of icecold phosphate-balanced salt solution (PBS). The sample was then incubated at room temperature for 30 min in electrophoresis sample buffer [ 1% sodium dodecyl sulfate (SDS), 10% sucrose, 62.5 mM Tris HCl, pH 8.6, 100 mM dithiothreitol (DTT), and 0.005% bromophenol blue] and subjected to slab-gel vertical electrophoresis in l

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FIG. 2. Effect of increasing concentrations of bPTH on binding of 1251bPTH to basolateral membrane from adult and old rats. Basolateral membranes were incubated with radioligand (2.5 x lo-l1 M) and various concentrations of unlabeled bPTH, and specific binding of hormone was determined as describedin MATERIALS AND METHODS. Combined concentrations of PTH are shown. For each experiment, membranes were prepared from 2 rats from each age group. Data represent mean t SE of 4 experiments with triplicate determinations in each experiment. Scatchard plots of B/F, ratio of bound-to-free ligands as a function of total ligand added, were prepared and are shown in inset.

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7.5% acrylamide gel with 0.1% SDS. Proteins were electrophoretically transferred from the slab gel to nitrocellulose in 50 mM Tris HCl, pH 7.5, 200 mM glycine, 20% (vol/vol) methanol at 200 mA for 10 h. Blots were then incubated with blocking solution (100 mM Tris HCl, pH 7.5, 100 mM NaCl, 2 mM MgC12, 5% nonfat dry milk, and 0.05% Tween 20) for 2-5 h. To detect biotinylbPTH, blots were incubated for 10 min with streptavidin (2 pg/ml), washed three times, and incubated with biotinylated calf intestinal alkaline phosphatase (1 pg/ml) for 10 min, and then washed three times. PTH binding proteins were visualized by chromogenic development with use of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. The relative concentration of PTH binding components detected by this technique was determined by densitometric scanning of blots. Under these conditions binding of biotinyl-bPTH to renal membrane components is both saturable and specifically displaced by PTH analogues only (5). Infusion of bPTH. [Nle8~18,Tyr34] bPTH- (l-34) was dissolved in 20 mM acetic acid. Each rat received 0.61 pi/h of [Nle’,“, Tyr34]bPTH (1 pg/pl) for 8-10 days via an Alzet miniosmotic pump (model 2002). The control group was infused with 20 mM acetic acid. Renal cortical basolateral membranes were prepared from rats after the infusion, and the number of PTH binding sites was quantitated by ‘?-labeled bPTH binding as described above. Analytical methods. Protein was determined by a standard method (22) with the use of bovine serum albumin as the reference protein. Adenylate cyclase activity was assayed as described previously (21). l

RESULTS

Time course of 12V-bPTH binding in isolated basolateral membranes. Both PTH receptors and hormone-responsive adenylate cyclase are localized in the basolateral membrane of the renal tubular cell. Because binding of

PTH is coupled to increases in CAMP (2, 20), we examined the time course of 1251-bPTH binding to the receptor in basolateral membranes isolated from adult and old rats. The binding of 1251-bPTH was time dependent (Fig. 1). Fifty percent saturation was observed at 5 min and maximum binding after 20 min of incubation. The time course for 1251-bPTH binding was the same in basolateral membranes isolated from either adult or old rats. Nonspecific binding was measured in the presence of 10e6M unlabeled bPTH. Specific binding sites, calculated as the difference of the total and nonspecific binding sites measured at 20 min, were 0.87 t 0.10 and 0.39 t 0.06 fmol/ mg protein, respectively, for membranes prepared from 6- and 24-mo rats. Unless otherwise stated, measurement of 1251-bPTH binding was routinely determined in a 30min incubation. Characteristics of 1251-bPTH binding in isolated basolateral membranes. The effect of increasing concentrations of unlabeled hormone analogue on the binding of 1251-bPTH in basolateral membranes is shown in Fig. 2. In the presence of 2 x lo-l1 M 1251-bPTH, specific binding was 6.2 t 0.5 and 2.8 t 0.3% for membranes isolated from adult and old rats, respectively. Specific inhibition of 1251-bPTH binding was observed when unlabeled PTH analogue was added at concentrations as low as 3 X 10-l’ M. To determine the kinetic parameters of bPTH binding, the data were transformed by the method of Scatchard. In both adult and aged rats binding of 1251-bPTH was characterized by a single class of highaffinity binding sites. The apparent & was 1.36 t 0.12 and 1.25 t 0.24 nM for membranes isolated from adult and senescent rats, respectively (Fig. 2). The maximum number of bPTH binding sites, B,,,, was reduced 60% in membranes from aged rats (92.1 t 9.3 vs. 36.7 t 6.1 fmol/mg protein for adult and aged rats, respectively, P c 0.01).

PTH binding components in renal membranes. PTH binding proteins were identified using biotinyl-bPTH-

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(l-34) as the ligand. Biotinyl-bPTH-( l-34) specifically labeled a main protein band with a molecular mass of 68 kDa and a minor band of 70 kDa (Fig. 3). The levels of PTH receptor, determined by densitometric scanning of the intensity of the bands were higher in membranes prepared from adult compared with old rats (104 f 20 vs. 63 f 15 relative optical density, respectively, P < 0.05). However, the molecular masses of the apparent PTH binding proteins were unaffected by the age of the animal. In separate experiments, membranes prepared from young rats were used to check the specificity of the ligand blotting method. Addition of 13.5 PM underivatized bPTH-(l-34), but not 20 PM oxidized rat PTH(l-34), before the cross-linking with biotinylated bPTH-(1-34) completely abolished the labeling of the 68 and 70-kDa proteins (data not shown). Effect of parathyroidectomy on kinetics of 1251-bPTH binding. We have reported previously that the serum immunoreactive PTH (irPTH) concentration of the adult and senescent rats used in this study was 100 + 4 and 153 + 19 pmol/l, respectively (13). Because of the elevated levels of irPTH in the aged rat, we tested the hypothesis that the increase in serum irPTH contributed to the loss of PTH binding sites. Adult and senescent rats were divided into two groups, 1) parathyroidectom-

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ized (PTX) animals for which the parathyroids were surgically removed and 2) sham-operated animals. The effectiveness of the PTX procedure was monitored by measuring the PTX reduction of serum irPTH and serum calcium, as described (13). Renal basolateral membranes were prepared from rats 48-72 h after surgery to examine the effect of PTX on the binding of lz51-bPTH. Kinetic parameters of the binding were determined from analyses of Scatchard plots (Fig. 4). PTX did not affect & and B maxof bPTH binding in membranes from adult rats. However, in membranes prepared from senescent rats, PTX was associated with a 35% increase in B,,, (39.8 f 2.4 vs. 53.5 + 24 fmol/mg protein for sham-operated and PTX rats, respectively, P < 0.05). The & was unchanged under the same conditions. Effect of PTX on the levels of PTH binding components in renal membranes. The levels of PTH binding proteins were measured after PTX by use of biotinyl-bPTH-( l34) as the ligand (Fig. 5). In sham-operated animals, the level of receptor protein, quantitated from the chromogenic intensity was lower in membranes prepared from old rats (65 + 18 relative optical density) than for young rats (105 + 21 relative optical density). The levels of PTH binding proteins in adult rats was unchanged after PTX. However, the decrement in PTH binding components seen in the old rat was reversed by PTX (65 f 18 vs. 145 f 20 relative optical density, for sham-operated and PTX senescent rats, respectively). Effect of age and PTX on PTH-stimulated adenylate cyclase. Previously, we reported that PTH-stimulated adenylate cyclase in whole cell membrane was blunted in aged rats (13). In this study, we examined PTHstimulated adenylate cyclase in isolated renal basolateral

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