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Downloaded by guest on October 1, 2021 Proc. Nati. Acad. Sci. USA Vol. 88, pp. 2603-2607, March 1991 Neurobiology Evidence for thymopoietin and thymopoietin/a-bungarotoxin/ nicotinic receptors within the brain M. QUIK*t, U. BABUf, T. AUDHYA*, AND G. GOLDSTEINt *Department of Pharmacology, McGill University, McIntyre Medical Building, 3655 Drummond Street, Montreal, Quebec H3G 1Y6, Canada; and timmunobiology Research Institute, Route 22 East, P.O. Box 999, Annandale, NJ 08801-0999 Communicated by Edward A. Boyse, December 20, 1990 ABSTRACT Thymopoietin, a polypeptide hormone of the These observations suggested that thymopoietin may repre- thymus that has pleiotropic actions on the immune, endocrine, sent an endogenous ligand for the nicotinic a-BGT-binding and nervous systems, potently interacts with the neuromuscu- site in brain; however, the identification of receptor-specific lar nicotinic acetylcholine receptor. Thymopoietin binds to the binding of radiolabeled thymopoietin to a site in brain that nicotinic a-bungarotoxin (a-BGT) receptor in muscle and, like also recognized a-BGT and nicotinic ligands would substan- a-BGT, inhibits cholinergic transmission at this site. Evidence tially support the above hypothesis. The present studies show is given that radiolabeled thymopoietin similarly binds to a that in brain 125I-thymopoietin binds in a specific manner to nicotinic a-BGT-binding site within the brain and does so with a receptor site that also interacts with nicotinic ligands and the characteristics of a specific receptor ligand. Thus specific a-BGT and, furthermore, that a constituent in brain extracts binding to neuronal membranes was saturable, of high affinity has the immunological reactivity and molecular mass of (Kd = 8 nM), linear with increased tissue concentration, and thymopoietin. readily reversible; half-time was -5 min for association and 10 min for dissociation. Binding of '2sI-labeled thymopoietin was MATERIALS AND METHODS displaced not only by unlabeled thymopoietin but also by a-BGT and the nicotinic receptor ligands d-tubocurarine and Materials. Thymopoietin was isolated and purified from nicotine; various other receptor ligands (muscarinic, adrener- thymus as described (1, 2) and radioiodinated (7.8 AGCi/,ug; 1 gic, and dopaminergic) did not affect binding of 'SI-labeled Ci = 37 GBq) according to Audhya and coworkers (2, 11). thymopoietin. Thymopoietin was shown by ELISA to be pres- a-BGT, purified from Bungarus multicinctus venom as de- ent in brain extracts, displacement curves ofthymus and brain scribed (12), was obtained from Miami Serpentarium Lab extracts being parallel to the standard thymopoietin curve, and (Salt Lake City, UT). All other chemicals were obtained from Western (immuno) blot identified in brain and thymus extracts standard commercial sources. a thymopoietin-immunoreactive polypeptide of the same mo- Preparation of the Tissue Extracts. Brain, liver, thymus, lecular mass as purified thymopoietin polypeptide. We con- and thyroid were removed from female Sprague-Dawley rats clude that thymopoietin and thymopoietin-binding sites are weighing =280 g. The tissues were suspended at 20%o (wet present within the brain and that the receptor for thymopoietin wt/vol) in chilled 10 mM Tris-HCl, pH 6.5, containing 1 mM is the previously identified nicotinic a-BGT-binding site of phenylmethylsulfonyl fluoride/i mM ethylenediamine tetra- neuronal tissue. acetate (EDTA)/1 AtM bacitracin/1 ,uM 2-mercaptoethanol/l mM ascorbic acid and were homogenized in an Eberbach Thymopoietin is apolypeptide hormone ofthe thymus (1-3) that con-torque homogenizer on ice for 5 min. The homogenates has pleiotropic actions on the immune, endocrine, and nervous were then centrifuged at 45,000 x g for 20 min at 40C, and the systems (4). Studies of the thymus and myasthenia gravis supernatants were aliquoted and stored at -20'C until used revealed an effect ofthymopoietin on neuromuscular transmis- for ELISA or immunoblot analysis. sion (5), and thymopoietin was subsequently shown to interact ELISA. Rat thymopoietin and tissue extracts were assayed with the nicotinic acetylcholine receptor (nAChR) of the Tor- with a high titer of rabbit anti-human thymopoietin antibody pedo electroplax (6) and of the neuromuscular synapse (7). that also reacts with rat thymopoietin. A limiting dilution of Thymopoietin bound to the muscle-type nAChR with a Kd of antibody was incubated with different concentrations of rat 0.5-3 nM (6, 7) and appeared to interact with a similar region of thymopoietin or different dilutions of the tissue extracts for the receptor as the snake toxin a-bungarotoxin (a-BGT) be- 2 hr at 370C on a rocking platform. The incubation mixture cause thymopoietin prevented binding of '2-I-labeled a-BGT was then added to a rat thymopoietin-coated polyvinyl chlo- (125I-a-BGT), and a-BGT blocked binding of II5I-labeled thy- ride microtiter plate and incubated at 370C for 2 hr. After the mopoietin (125I-thymopoietin) in Torpedo electroplax (6). Fur- plate was washed with phosphate-buffered saline/Tween 20, thermore, thymopoietin inhibited cholinergic transmission at goat anti-rabbit antibody conjugated to horseradish peroxi- the nAChR by a mode ofaction similar to a-BGT (7), although dase was added, and the plate was incubated for 1 hr at 370C. an additional mechanism involving Ca2+-dependent accelera- After three further washes with phosphate-buffered saline/ tion and maintenance ofthe desensitized state ofthe nAChR has Tween 20, a substrate/chromogen system consisting ofH202 also been described (8). and 3,3',5,5'-tetramethylbenzidine dihydrochloride was a-BGT receptors are also present within the brain, and we added, and color was allowed to develop at room temperature have previously shown that thymopoietin effectively com- for 30 min. The reaction was terminated with the addition of petes for these neuronal 125I-a-BGT-binding sites (9). Fur- 2.5 M HCI; optical density was recorded at 450 nm. thermore, thymopoietin was detected in brain extracts and Immunoblot Analysis. The protein sample was character- supernatants of developing rat spinal cord cultures and of ized by preparing 10 A.l of the extracts in 0.25 A.l of 10% SDS certain neuronal cultures by using an immunoassay (10). and 1 A.l of bromophenol blue and electrophoresing on a The publication costs of this article were defrayed in part by page charge Abbreviations: a-BGT, a-bungarotoxin; nAChR, nicotinic acetyl- payment. This article must therefore be hereby marked "advertisement" choline receptor. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2603 2604 Neurobiology: Quik et al. Proc. Natl. Acad. Sci. USA 88 (1991) Pharmacia PhastSystem SDS electrophoresis system with a 1.5 Ad 20% SDS homogenous polyacrylamide gel. Filter paper and B, -A 0.2-,um nitrocellulose paper were wetted with transfer buffer a (25 mM Tris/192 mM glycine/20% methanol, pH 8.3). A transfer sandwich was assembled as follows: three pieces of filter paper, nitrocellulose paper, the gel, and three pieces of 1.0 filter paper. The protein was then transferred in a Phast- L CO) Transfer system for 15 min at 25 mA per gel. The membrane z was blocked overnight with 3% bovine serum albumin in phosphate-buffered saline/Tween 20, washed three times, In and allowed to react with a horseradish peroxidase- J 0.5- conjugated monoclonal antibody against thymopoietin (37) at 370C for 2 hr. The membrane was washed three times and incubated with the substrate/chromagen mixture (3,3',5,5'- 0 tetramethylbenzidine) for 15-30 min, washed with water, and U-A air dried. 0.1 1 10 100 1000 Preparation of Membranes for the Binding Assay. Male ITPJ (ng/ml) Sprague-Dawley rats were killed by decapitation, and the brains were rapidly removed and dissected on ice. The brain minus the cerebellum was homogenized in 20 vol of 10 mM 2560 640 160 40 10 Tris-HCl, pH 7.4, with a Polytron homogenizer at setting 7 for EXTRACT TITER 10 s. After centrifugation at 45,000 x g for 15 min, the supernatant was discarded, and the membranes were resus- FIG. 1. Immunoreactivity of rat brain (m), thymus (v), liver (o), pended with a Polytron in the appropriate buffer to be used and thyroid (A) extracts as shown by inhibition of ELISA. Parallel in the binding assay. displacement curves were observed with purified rat thymopoietin 125I-Thymopoietin Binding. Membranes were resuspended at (i) and rat brain and thymus extracts. TP, rat thymopoietin. 75 mg/ml (unless otherwise indicated) in 10 mM Tris HCI, pH 7.4. A 160-sl aliquot ofmembranes was preincubated for 15min the same molecular mass position; liver extract provided a at 22°C with the indicated concentration of drug, followed by a negative control with no band detected on the immunoblot. 15- or 20-min incubation at 22°C with 5 nM 125I-thymopoietin '25I-Thymopoietin-Binding to Rat Brain Membranes. Spe- (unless otherwise indicated),. The assay was terminated by cific binding of 125I-thymopoietin to brain membranes was addition of 1.0 ml of 0.2 M NaCl immediately followed by defined as the difference in binding of 125I-thymopoietin centrifugation (5 min) in an Eppendorf microcentrifuge (8800 x without and with 10-7 M thymopoietin. Specific binding was g). The pellet was washed twice, and the radioactivity in the maximal in a 10 mM Tris-HC1 buffer, pH 7.4; other buffer pellet was determined by using a y counter. Specific binding systems tested (50 mM Tris HCI, pH 7.4; 50 mM phosphate was defined as the difference in binding in the absence and buffer, pH 7.4; 50 mM Tris-HCI, pH 7.4, containing 120 mM presence of 10-v M thymopoietin. Specific binding was =40- NaCl, 5 mM KCl, 2 mM CaC12, and 1 mM MgCl2) resulted in 50% of the total binding; this binding could be enhanced by similar or lower specific binding.