Biochimica et Biophysica Acta, l 120 (1992) 17-23


© 1992 ElsevierScience Publishers B.V. All rights reserved 0167-4838/92/$05,00

BBAPRO 34137

Generation of polyclonal antibodies against the mineralocorticoid receptor and analysis of mineralocortin in rat myocardium by immunophotochemistry M, Mirshahi


M. Pagano b, A. Mirshahi


and M.K. Agarwal c

Department of Ophthalmology. Centre Unirersitaire des Cordeliers, Paris (France), h Department of Biochemistry. Centre Unicersiry de Cordeliers, Paris (France) and c Hormone Laboratory, Centre Unit,ersitaire des Cordeliers, Paris (France)

(Received I0 June 1991)

Key words: Mineralocorticoid;Receptor: Antibody:lmmunohistochemistry;Photoaffinity;Spirolactone:(Heart) Fawn, Burgundy rabbits were immunized with the mineralocorticoid receptor (MCR) purified biochemically from rat kidney by a simple, two step procedure. High anti-MCR titers were observed in radioimmunoassays just 3 weeks after the initial injection and increased further with time. Western blot analysis revealed a single band of 94-98 kDa in renal and cardiac cytosol from the rat, like the antigen prepared biochemically. The two atria from beef heart exhibited far greater MCR-positivity compared to the two ventricles, suggesting physiological relevance. The receptor was also photolabelled for the first time with promegestone in this very 94-98 kDa region which could be displaced by the antagonist RU 26752 specific to MCR. The immune IgG precipitated 3H-aldosterone or 3H-RU 26752-MCR complexes from rat heart, and displaced the MCR-antagonist complex to high molecular weight regions during gel permeation chromatography on Sephacryl columns, lmmunofluorescent labelling showed that MCR was widely distributed in the cytoplasm in rat myocardium with limited staining in what appeared to be the nuclear compartment. These open up the possibility of large scale purification of the endogenous mineralocorticoid binding protein, mineralocortin, for detailed physieochemical characterization. The technique of photoaffinity labelling presented here should also help delineate the nature of the steroid binding domain in the MCR.

Introduction Steroid hormones are believed to bind to high affinity, low capacity receptors in a superfamily of proteins that act as transactivating signals to modulate gene expression specific to the appropriate cell type [1-3]. Mineralocorticoids regulate the hydrosodic balance in higher organisms whose dysfunction can lead to hypertension and related syndromes [4,5]. Analysis of receptor mediated mineralocorticoid action is therefore important not only to understand the organisation and expression of the complex mammalian genuine, but also to alleviate a human disease. The availability of radioligands specific to several receptor classes permitted biochemical purification of

Abbreviations: MCR, mineralucorticoidreceptor; SBD, steroid binding domain; GCR, glucocorticoid; PR, progestin; PC, phosphocellulose; ELISA, enzyme-linked immunoadsorbent assay: PBS, phosphate-bufferedsaline.

Correspondence: M.K. Aganval, Hormone Laboratory,Centre Universitaire de Cordeliers, 15 rue de I'Ecole de M~decine,75270 Paris Cedex 06, France.

the respective proteins that were then used for the generation of monocional a n d / o r polyclonal antibodi,~s [6-12]. Furthermore, the steroid binding domain (SBD) of a number of receptor classes could also be photolabelled [13-14]. Progress with the mineralocorticoid receptor (MCR) has been slow on all these counts due primarily to the unavailability of suitable ligands [15]. Of late, synthesis of MCR-specific spirolactones [16,17] permitted not only the delineation of receptor kinetics [18,19] this protein, mineralocortin, was at long last purified recently in our laboratory [20,21]. We present here the characterstics of a polyclonal antiserum engendered in rabbit against the endogenous, native MCR from rat. We have also succeeded in the photoaffinity labelling of the SBD of mineralocortin for the first time.

Experimental procedures R e c e p t o r purification

The activated form of the mineralocorticoid receptor (MCR), or mineralocortin, was purified from rat kidney by the original procedure developed in our

18 laboratory [20,21]. The organ cytosol from adrenalectomized rats, equilibrated with the MCR specific ligand 3H-RU 26752 in the presence of RU 38486 to block the glucocorticoid (GCR) and the progestin (PR) receptors, was passed through a phosphocellulose (PC) column. The MCR in the PC flowthrough was activated at room temperature, adsorbed onto the DNAcellulose, extracted by high salt buffer, dialyzed to isotonic pH and finally lyophilized. Immunization The lyophilized MCR (150 #g protein) was reconstituted in 1 ml of complete Freund's adjuvant for 30 min at room temperature and injected intradermally at four different foci into fawn, Burgundy rabbits (Cegav, France) maintained on pellet food and water at all times in a climate controlled animal room. A booster shot (140 /.tg) of the same preparation was given 3 weeks after the initial injection. Rabbits were bled periodically from the ear vein and anti-MCR titers were analyzed by ELISA.

Enzyme-linked immunoabsorbent assay (ELISA) Polystyrene microplates (Dynatech, U.K.) were coated overnight at 37°C with 100/.d MCR (3 # g / m l ) in pht~sphate-buffered saline (PBS) containing (per !) 0.25 g potassium dihydrogen phosphate, 1.38 g disodium phosphate, 0.25 g KCI, 9 g NaCI and 0.01% sodium azide. They were washed thrice with 0.2 ml PBS-0.1% Tween 20 prior to incubation (2 h, 37°C) with 100 /.tl serial dilutions of the rabbit MCR antiserum. After three washes with PBS-Tween, the wells were saturated with 100 #1 sheep anti-rabbit biotinylated antibody (1:1000 in PBS-Tween-0.1% bovine serum albumin), Three washes with PBS-Tween followed and 100/.tl of 1 : 1000 streptavidin biotinylated horseradish peroxidase was then added to the wells. The peroxidase activity was finally developed by the addition of 100 ~1 of orthophenylene diamine (0,04%)-hydrogen peroxide (0.01%). The reaction was stopped after 3 min with 50 #! of 2.5 M sulphuric acid, and the intensity of the colour quantitated at 492 nm against a control preimmune serum, as described before [22].

Western blots Organ cytosols were denatured in 220 mM Tris-HCI (pH 6.8), 2% SDS, 15% glycerol and electrophoresed for 3-4 h on 15% acrylamide gels in Tris-glycine-SDS (13 g + 14.4 g + 1 g/l water). The gels were coloured with Coomassie brilliant blue reagent for 10 min and finally destained with 10% each of acetic acid and methanol for the desired period of time. Standards (Bio-Rad) of known molecular weight were run simultaneously [20-22].

The proteins separated by SDS-PAGE as above were electrotransferred to nitrocellulose membranes (Millipore) for 1 h at 5 mA in Tris-glycine-methanol (1.5 g + 7 . 2 g + 1 0 0 ml/l). The membranes were blocked for 1 h with 15% non fat dry milk at 37°C in PBS, incubated with rabbit anti-MCR (1 : 250 in PBSalbumin) for 2 h at 4°C, and the antigen-antibody complexes were saturated with sheep anti-rabbit biotinylated antibody (1:500) for 90 min, followed by 90 min in presence of streptavidin biotinylated horseradish peroxidase complex (1:500), all diluted in PBS-albumin. The blots were washed 3 × 7-10 min between each of the successive steps mentioned above. Finally, the nitrocellulose papers were developed in the dark for 10-20 min at room temperature with a mixture of 4-chloro-l-naphthol (0.05%) hydrogen peroxide (0.01%) methanol (10%), dried, and stored at -20°C, as described before [22].

Photochemistry The organ cytosol in PBS was incubated with 50 nM of 3H-R 5020 alone or in the presence of a 100-fold excess of RU 26752 that is specific to MCR and then irradiated at a distance of < 1 cm for 3 min at 4°C with an ultraviolet lamp (Jelight). The samples were electrophoresed as above along with radiolabelled, but non-irradiated, cytosol, or free steroid, as controls. The electrophoresed gels were fixed for 30 rain with water: acetic acid : isopropanol (65 : 10: 25), wiped dry with soft tissue paper and then flooded with Amplify (Amersham) for 30 rain, all at room temperature and dried overnight at 37°C in a slab drier. The dry gels were exposed to Hyperfilm MP in a film holder equipped with fluorographic screens (Amersham). After varying lengths of time at -80°C, the films were developed with the Kodak processor, fixed, washed with tap water and dried. Autofluorographic patterns were carefully aligned with the original gel calibrated systematically with molecular weight markers (Bio-Rad).

Immune precipitation Whole serum (10 ml) was fractionated on a DEAETrisacryl M column (1.6x 15 cm), equilibrated and eluted with 25 mM Tris-HC! (pH 8.8) containing 35 mM NaCi and 5 mM sodium azide, lgG excluded from the column were quantitated at 280 nm (23), dialyzed against PBS overnight at 4°C and stored frozen in small aliquots. Organ cytosol (0.5 ml) in PBS was incubated 2 h with 20 nM of the desired tritiated steroid alone or in presence of 1000 fold excess of the corresponding radioinert molecule. Free steroids were removed by the addition of 0.5 ml charcoal-dextran (Sigma: 2.5% + 0.25%) for 10 min, followed by eentrifugation (6000 x g) for 10 min. The samples were brought to 0.3 M KC!

19 to dissociate the larger MCR complexes and desired quantities of immune or normal lgG, prepared as above, were added to the assay tubes with additional incubation for 3 h. Finally, 100 #1 of goat anti-rat)bit IgG-Agarose (Sigma), containing 0.6 mg IgG/ml packed resin, was added to each sample and the incubation was continued for an additional 3 h. The incubation tubes were centrifuged for 10 min at 6000 x g and the precipitate was washed twice with 2 ml PBS containing 0.5 M NaCl + 0.01% sodium azide. Rabbit IgG-rat MCR complexes bound to the goat anti-iabbit Agarose resin were eluted once with I ml of 0.1 M glycine-0.15 M NaCI (pH 2.4) and counted in 10 ml Picofluor (Packard). All manipulations were carried out in triplicate in a melting ice bath.

Immune displacement Preswollen Sephacryl S-300-HR (Sigma)was washed repeatedly with PBS and packed in a 1.5 × 80 cm column which was thereafter calibrated with markers of known molecular weight and Stoke's radii (Pharmacia). Organ cytosol in PBS was equilibrated with 20 nM 3H-RU 26752 for 2 h at 4°C, charcoal treated (50 mg/ml) to remove free steroids, brought to 0.3 M KCI and divided into two lots. One lot of 3 ml was incubated with 500 #g anti-MCR lgG for 3 h whereas the other one received an equivalent amount of nonimmune IgG. Each sample was fractionated on the Sephacryl column not to exceed 3% of the bed volume. Deionized and degassed water was used throughout. Fractions of 2 ml were collected at a constant flow rate of 40 m l / h at 4°C and l ml was counted in l0 ml Picofluor.

lmmunofluorescence Rat heart ventricles were frozen in liquid nitrogen and fixed in OCT compound at -30°C. Sections 7/~m thick were cut by a Cryostat at -30°C and spread on microscopic slides prewashed with ethanol. The slides were dried at room temperature for 10 rain and immersed in a bath of 95% methanol : 5% acetic acid at -20°C, followed by repeated washes with PBS. The sections were flooded with 15% nonfat dry milk (Regilait) for 10 min and exposed for 1 h to anti-MCR antiserum diluted 1:40 in Regilait. After three wshes with PBS the slides were incubated with sheep biotinylated anti-rabbit antibody (Amersham) diluted 1:50 in PBS-I% albumin for 60 rain. Following three washes with PBS, they were incubated for 60 min with fluorescent streptavidin (Amersham) diluted 1:50 in PBS. Finally, the slides were washed 5 times with PBS and mounted with a coverslip in Fluoprep. Sections were photographed with a Kodak black and white 400 ASA film in a fluorescent microscope (Nikon).

Anhnais, reagents, chemicals Male, Wistar, adrenalectomized rats, 4-6 weeks (200-250 g) were purchased from iffa-Credo, France. Fawn, Burgundy rabbits, 2-2.5 kg, were obtained from Cegav Farm, France. Pellet food was a product of UAR, Epinay, France. Beef heart from the local slaughter house was frozen immediately after ablation. Cellulose phosphate (lot 39F0907), DNA-cellulose (lot 109F 8120), activated charcoal (lot 27C-0022), dextran (lot 125F-C254), Sephacryl S-300 HR (lot 19F0459), and goat anti-rabbit IgG-Agarose (lot 77F8985) were purchased from Sigma chemie, Verpilliere, France. Sheep anti-rabbit biotinylated antibody (lot 21), streptavidin biotinylated horseradish peroxidase (lot 82), streptavidin fluorescein (lot 32), Amplify (lot 3026) and Hyperfilm MP (RPN 6) were products of Amersham, U.K. DEAE-Trisacryl M (lot 5208) was purchased from Industrie Biologique Francaise, Paris. Tissue-Tek O.C.T. Compound (lot 0355045) was supplied by Miles, Paris. Fluoprep (lot 601500-A) was obtained from BiomErieux, Paris. Polyacrylamide gels were calibrated by the Molecular weight standards kit from Bio-Rad (CA, U.S.A.) as follows: rabbit muscle phosphorylase b 97 kDa; bovine serum albumin 66 kDa; ovalbumin 43 kDa: soybean trypsin inhibitor 21.5 kDa; lysozyme 14 kDa. The gel filtration calibration kit (lot 8197) from Pharmacia (Uppsala, Sweden) contained: thyroglobulin 669 kDa; ferritin 440 kDa; catalase 232 kDa; aldolase 158 kDa; supplemented with (lot 9082) hen egg ovaibumin 43 kDa; and Ribonuclease A 13.7 kDa. The double bore mercury vapour lamp with power supply was purchased from Jelight (CA, U.S.A.). Freund's complete adjuvant (lot 784041 ) was a product of Difco (MI, U.S.A.). Picofluor 40 and polyethylene vials (Packard) were used for radioactive measurements in a Tricarb scintillation spectrometer. Tritiated (50 Ci/mM, reference X 3025 A) and radioinert RU 26752 (lot 7) and RU 38486 (lot 6B0381) were provided free of charge by Roussel-Uclaf, Romainville, France. 3H-R 5020 (84.7 Ci/mM; lot 2668062) and cold promegestone were purchased from New England Nuclear (Paris). 1,2-3H-aldosterone (45 Ci/mM; batch 34) was a product of Amersham, U.K. The radiochemical purity exceeded 97% in all cases. The trivial names of steroids used in this study are: R 5020, promegestone-17-a-methyl; RU 26752, 7 alpha propyl-3-oxo- 17-alpha-pregn-4-ene-21,17-carbalactone; RU 38486, I 1 beta-(4-dimethylaminophenyl)-17 betahydroxy, 17-alpha-(prop- 1-ynyl)-estra-4,9-dien-3-one; aldosterone, 11-/3,21-dihydroxy-3,20-dioxopregn-4-ene18-al. Results Data in Fig. l show antimineralocortin tilers in the rabbit immunized with MCR purified from rat kidney

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D i l u t i o n x 100 "2

FJ#. |. Radit~immunoa',.,,ay~.~,1"rabhil :mlil~dy 1o mincrak~orlin. llh~t~d~,itmplcsw~:rc analyzed 5 weeks after the inilial injection ~l'

1~,(}tag -~ r~t retail MCR u~ingthe immtm¢( I ) or the control (1~) scream. EI.ISA ~,;t~ ~x~:~:utedtnl .,,criat diltttions. ;is described in Matrrials gll0 Me|h~.~d.~.

in the presence of RU 2f~752 that is specific to tttis receptor (lt,.18-_l}. 5 weeks after the initial immunizatitm the anti-MCR activity was positive by the

ELISA technique at tile ~erum dilutitm of l:160t~). increasing over the l: IOtX}O liters observed 3 weeks after the injection of MCR; a booster shot rcsulled in further increase to > I :32t1(1{1 (not shown), Negative results w e r e o b l a i n c d when preimmunc serum was used under these conditions. This 5 week bleed appeared adequately rich in anli-MCR netivity and was used in subsequent experiments lot fijrlher characterization of mineral

Generation of polyclonal antibodies against the mineralocorticoid receptor and analysis of mineralocortin in rat myocardium by immunophotochemistry.

Fawn, Burgundy rabbits were immunized with the mineralocorticoid receptor (MCR) purified biochemically from rat kidney by a simple, two step procedure...
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