Iodinated Probes (Photoaffinity Labeling/Guinea Pig/['25I]Iodoaminopotentidine/[1"I]Iodoazidopotentidine) M

Proc. Natd. Acad. Sci. USA Vol. 87, pp. 1658-1662, March 1990 Neurobiology Reversible and irreversible labeling and autoradiographic localization of the cerebral histamine H2 receptor using [1251]iodinated probes (photoaffinity labeling/guinea pig/['25I]iodoaminopotentidine/[1"I]iodoazidopotentidine) M. RUAT*t, E. TRAIFFORT*, M. L. BOUTHENET*, J. C. SCHWARTZ*, J. HIRSCHFELD$, A. BUSCHAUERf, AND W. SCHUNACKt *Unitd de Neurobiologie et Pharmacologie (U. 109) de l'Institut National de la Sante et de la Recherche, Centre Paul Broca, 2ter rue d'Aldsia, 75014 Paris, France; and fFreie Universitat Berlin, Institut fur Pharmazie, Konigin-Luise-Strasse 2+4, 1000 Berlin, Federal Republic of Germany Communicated by Jean-Pierre Changeux, December 11, 1989 ABSTRACT Iodoaminopotentidine (I-APT)-i.e., N-[2- neurons to almost the whole mammalian central nervous (4-amino-3-iodobenzamido)ethyl]-N'-cyano-N"-{3-[3-(1-piperi- system (12), these responses-e.g., adenylate cyclase acti- dinylmethyl)phenoxylpropyl}guanidine-represents one of the vation-could be demonstrated in only a small number of most potent H2-receptor antagonists known so far. In mem- brain areas of a few animal species (13). branes of guinea pig brain 12sI-APT bound reversibly, selec- Various attempts at labeling the H2 receptor with radioac- tively, and with high affinity (Kd = 0.3 nM) to a homogeneous tive probes, a prerequisite for starting localization, regula- population ofsites unambiguously identified as H2 receptors by tion, purification, or molecular cloning studies, have so far inhibition studies conducted with a large panel of antagonists. met with limited success. Whereas [3H]cimetidine, [3H]rani- I2sI.-APT binding was also inhibited by histamine, and the effect tidine, and [3H]impromidine were found to be totally unsuit- was modulated by a guanyl nucleotide, which is consistent with able as ligands (2), [3H]tiotidine was shown to label the H2 the association of the H2 receptor with a guanine nucleotide receptor in membranes of three areas of the guinea pig brain binding regulatory protein. The low nonspecific binding of (14, 15). However, this could not be confirmed in other 125I-APT generated high contrast autoradiographic pictures in laboratories (16, 17); the nonspecific binding was high, and brain sections and established the precise distribution of H2 H2 receptors were undetectable with [3H]tiotidine in many receptors. Their highly heterogeneous distribution and lami- brain areas known to receive histaminergic innervation and in nated pattern in some areas-e.g., cerebral and hippocampal the brain of species other than the guinea pig (14, 15). In cortices-suggest their major association with neuronal ele- addition, and in contrast with H1 and H3 receptors (18-21), ments. These localizations were more consistent than those of no information is available regarding the tissue distribution or H1 receptors with the distribution ofhistaminergic projections, physicochemical properties of the H2 receptor. indicating that H2 receptors mediate a larger number of Here we report the design of the antagonist [125Ij] postsynaptic actions of histamine-e.g., in striatum. Colocal- iodoaminopotentidine (125I-APT), a high-affinity reversible izations of H1 and H2 receptors in some areas account for their probe for H2 receptors, which enables their extremely sen- known synergistic interactions in cAMP formation induced by sitive detection over a low background in membranes as well histamine. The distribution of '2sI-APT binding sites did not as, autoradiographically, in brain sections. In addition, strictly parallel that ofthe H2-receptor-linked adenylate cyclase [125I]iodoazidopotentidine (125I-AZPT), a photoaffinity probe activity, which may reflect heterogeneity among H2 receptors. derived from 125I-APT, was shown to be covalently incorpo- After UV irradiation and SDS/PAGE analysis, ['2sI]iodo- rated into the H2 receptor after UV irradiation, leading to the azidopotentidine (125I-AZPT), a photoaffinity probe derived initial physicochemical characterization ofthe ligand binding from "2I-APT, was covalently incorporated in several pep- peptides of this receptor. tides, among which the labeling of two peptides of 59 and 32 kDa was prevented by H2 antagonists, suggesting that they correspond to H2-receptor binding peptides or proteolysis MATERIALS AND METHODS products of the latter. These probes should be useful for Materials. Na125I (usually 2000 Ci/mmol; 1 Ci = 37 GBq) sensitive radioassays, localization, purification, and molecular was from Amersham. The drugs and their sources were as studies of the H2 receptor, which were previously impractica- follows: cimetidine, zolantidine, burimamide, metiamide, ble. dimaprit, impromidine (Smith Kline & French), mepyramine (Specia), famotidine (Merck Sharp & Dohme), tiotidine (ICI). Histamine is a messenger molecule mainly released by neu- Analytical grade reagents were from Sigma. (R)-a-Meth- rons and mast cells that affects a large variety of target cells ylhistamine and PPAT stereoisomers-i.e., 5-amino-2-(3-{3- by interacting with three pharmacologically distinct sub- [1-(1-pyrrolidinyl)ethyl]phenoxy}propyl)amino-1,3,4-thiadia- classes of receptors termed H1, H2, and H3 (1-5). In brain, zole (22)-were from the Institute of Pharmacy (Berlin, where this amine acts as a neurotransmitter, the presence of F.R.G.). H2 receptors was indirectly evidenced by the histamine- Synthesis of APT. 1,1'-Carbonyldiimidazole (3.43 mmol) induced stimulation of cAMP accumulation in slices (6), was added to a stirred solution of 4-aminobenzoic acid (3.43 activation of adenylate cyclase in membranes (7, 8), changes mmol) in dry tetrahydrofuran (5 ml). The mixture was al- in neuronal firing (9), activation of phospholipid methylation lowed to react for 1 hr at room temperature. The solution was (10), and release of endogenous norepinephrine (11). How- added to the amine N-(2-aminoethyl)-N'-cyano-N'-{3-[3-(1- ever, contrasting the widespread projections ofhistaminergic Abbreviations: 1251I-APT, [125I]iodoaminopotentidine; 251I-AZPIT, The publication costs of this article were defrayed in part by page charge [1251]iodoazidopotentidine; APT, aminopotentidine; G protein, gua- payment. This article must therefore be hereby marked "advertisement" nine nucleotide binding regulatory protein. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 1658 Downloaded by guest on September 26, 2021 Neurobiology: Ruat et al. Proc. Nati. Acad. Sci. USA 87 (1990) 1659 piperidinylmethyl)phenoxy]propyl}guanidine (3.43 mmol) polyethyleneimine. Radioactivity trapped was measured and stirred overnight at room temperature. The mixture was with a LKB y-counter (82% efficiency). Specific binding was poured into water and extracted with CH2Cl2. The organic defined as that inhibited by 3 A.M tiotidine. layer was dried by Na2SO4, filtered, and evaporated under Photoaffinity Labeling and SDS/PAGE. Membranes (0.15- reduced pressure affording the title compound as an oil (1.58 0.30 mg/ml) were incubated in the dark with 50-100 pM g, 3.31 mmol). The product was chromatographically purified 1251I-AZPT for 16-20 hr at 10'C (or 150 min at 250C) in (Chromatotron 7924T, Harrison Research, Muttenz, Swit- phosphate buffer containing 100 mM NaCi. A sample was zerland) by using 4-mm layers of silica gel 60 PF254 (Merck) retained for a I251-AZPT binding assay performed as de- containing gypsum, eluted by CHCl3/MeOH (97:3, vol/vol) scribed above for 125I-APT binding. For photoaffinity label- (ammonia atmosphere). An analytical sample of the purified, ing, another sample was centrifuged at 32,000 x g for 20 min oily base was converted into a salt with oxalic acid and and the pellets were resuspended in phosphate buffer and recrystallized from Et2O/EtOH, C26H35N702-C2H204 0.5 irradiated for 3 min as described (19). The irradiated mem- H20; mp, 920C-950C (dec.). The structure of APT (N- branes were collected by centrifugation and solubilized in the [2-(4-aminobenzamido)ethyl]-N'-cyano-N'-{3-[3-(1-piperidi- presence of 5% 2-mercaptoethanol, and the mixture was nylmethyl)phenoxy]propyl}guanidine was confirmed by 1H analyzed by SDS/PAGE on 11% polyacrylamide gels as NMR, IR, mass spectroscopy, and elemental analysis. described (19). Protease inhibitors were present throughout Synthesis of '25I-APT and 12sI-AZPT. The chemical struc- the experiments. tures of 1251I-APT and 125I-AZPT are shown in Fig. 1. APT (9.5 Autoradiographic Localization of 12sI-APT Binding Sites. gg/2 ,gl of EtOH; 20 nmol) was mixed in a polypropylene tube Sections (10 Am) of guinea pig brains were prepared as with 404ul of 1 M NaOAc buffer, pH 5.6/Na1251 (20 ,ul; 2 mCi). described (18). Slide-mounted sections were incubated for 3 Then chloramine T (44 nmol/10,ul) was added. After 1 min, hr at 220C in 50 mM sodium/potassium phosphate buffer (pH the reaction was stopped with Na2S205 (88 nmol/10,l) and 7.5) containing 0.1 nM 125I-APT at 220C. Nonspecific binding the mixture was analyzed by HPLC (C18 jLBondapak, Wa- was defined by using 3 ,uM tiotidine. After five rinses (4 min ters). The mobile phase was MeCN/10 mM NH4OAc, pH 4.2 each) at 4°C in phosphate buffer, the sections were apposed (32:68, vol/vol), and the flow rate was 1 mI/min. 125I-APT on Ultrofilm (LKB) for a 2-day period and autoradiograms (retention time, 15.2 min) was well separated from APT were developed (18). (retention time, 7.4 min). Usually, >70% of the initial radioactivity was recovered in this peak, a 300-,ul sample of which RESULTS was mixed with 17 M AcOH (30 p1) and NaNO2 (22 ,umol/30 ,ul) for 4 min at 0°C. NaN3 (27 ,mol/30 ,l) was added for 8 Reversible Binding of 12SI-A5r to H2 Receptors. At 25°C min at 20°C and the reaction mixture was immediately 1251I-APT binding to striatal membranes occurred with an submitted to HPLC as described above, the mobile phase association rate constant (kj) of 0.03 min/nM, with equilib- being MeCN/10 mM NH4OAc, pH 4.2 (40:60, vol/vol).

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