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 radio- activity 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). rium being reached after 120-150 min (Fig. 2). Dissociation 125I-APT (retention time, 8.4 min) was found to be quite occurring in the presence of 10 ,uM tiotidine, a H2 antagonist, completely transformed and 125I-AZPT (retention time, 19.0 followed first-order kinetics with a rate constant (k-,) of min) was collected. Extreme care was taken to work under 0.013 min'. The ratio kL1/k, gave an equilibrium dissocia- dim light during the azidation and HPLC steps. 125I-APT was tion constant (Kd) of0.43 nM. Saturation of specific 125I-APT diluted (1.5 times) in EtOH and stored at -20°C and 12511 binding at equilibrium, defined using 3 AM tiotidine, was AZPT was stored at 4°C in the dark, both without losing their binding properties. 100, Membrane Preparation. Brain regions from male Hartley guinea pigs (200-300 g) were homogenized with a Polytron blender in 60 vol of cold 50 mM Na2HPO4/KH2PO4 buffer, pH 7.5. After centrifugation at 260 x g for 1 min, the resulting 100 supernatant was recentrifuged at 20,000 x g for 30 min, and the final pellet was used immediately or stored at -80°C. In the experiments with protease inhibitors, the buffer was 2 200 supplemented with bacitracin (0.1 mg/ml), leupeptin (10 E Timeeh ,ug/ml), pepstatin A (0.1 ,ug/ml), phenylmethylsulfonyl flu- oride (0.1 mM), and soybean trypsin inhibitor (10 ,g/ml). E 50 100 B Protein concentration was determined by the method of Lowry with bovine serum albumin as a standard. Brain membranes from male Wistar rats were prepared in a similar way. '251-APT Binding. Pellets were resuspended in phosphate buffer. Triplicate assays were performed in polypropylene tubes and gelatin was added (final concentration, 0.1%) to prevent 1251-APT adsorption. Membranes (50-100 ,ug of protein) were usually incubated with 125IjAPF for 120-150 0.25 050 0.75 min at 25°C in a final vol of 400 ,ul. Incubations were stopped FREE '251-APTnM by four additions ofcold buffer (3 ml each), followed by rapid FIG. 2. Reversible labeling of the H2 receptor in membranes of filtration through glass fiber filters (GF/B) treated with 0.3% guinea pig striatum using 1251-APT. The saturation curve at equilib- rium was established from 150-min incubations at 25°C, with non- specific binding being evaluated in the presence of 3 AM tiotidine NH C NH NHC using 54 ,ug of protein per 0.4 ml (or 0.8 ml for 125I-APT < 0.05 nM). 7N-CH2iiLO(CH2)3 (CH2)2 (Left Inset) Scatchard plot analysis of specific binding. (Right Inset) N-CN Association of'251-APT (0.2 nM, 25TC, 100 Ag of protein per 0.4 ml) and its dissociation upon addition of 10 ,tM tiotidine (arrow). B, FIG. 1. Chemical structures of 1251-APT (R = NH2), a reversible bound; F, free; *, total binding; A, nonspecific binding; o, specific probe, and 125I-AZPT (R = N3), a photoaffinity probe. binding (5). Downloaded by guest on September 26, 2021 1660 Neurobiology: Ruat et al. Proc. Natl. Acad. Sci. USA 87 (1990)

monophasic (nH = 0.95 + 0.10) leading to a linear Scatchard -10 plot, and computer analysis (23) of the binding isotherms led to a Kd of 0.34 ± 0.10 nM and a Bmax of 135 ± 9 fmol per mg of protein (Fig. 2 and Table 1). Similar Kd values were z obtained from the analysis of I251-APT binding to membranes 0 a. of cerebral cortex or hippocampus in which the Bmax was CD lower (Table 1). With the latter, specific binding at 0.2 nM w -8 125I-APT typically represented 50-60% of the total, to be compared with 70-80% with striatal membranes. Specific 0 - 125I-APT binding to striatal membranes was inhibited in a 0 z monophasic manner (pseudo-Hill coefficients close to unity) 0 by a series of H2 antagonists. Their Ki values were highly cc -6 correlated (r = 0.94) with their apparent equilibrium disso- ciation constants (KB) regarding inhibition of histamine- induced chronotropic responses as reported by others (5, 22, 24, 25) or measured by ourselves (Fig. 3). The KB of I-APT -J was measured after a 60-min contact as the potency of the compound increased with this contact duration. In contrast, H1- or H3-receptor antagonists and various nonhistaminergic 4h7Li Mk-~MeHA agents were poorly effective. Histamine itself, in phosphate buffer and in the presence of -4 -5 -6 -7 -8 -9 -10 1 mM Mg2+, inhibited the binding of 125I-APT (0.2 nM) to LOG (Ki)125I-APT BINDING striatal and hippocampal membranes with IC50 values of 11 ± 2 and 24 ± 6 ,uM, respectively, in the absence of Gpp(NH)p, FIG. 3. Pharmacological characterization of 1251-APT binding to and of 33 ± 7 and 100 ± 24 ,uM, respectively, in the presence striatal membranes. Ki values are compared with KB (or EC50) values of0.1 mM nucleotide (pooled data from two experiments with of the same compounds regarding the H2-receptor-mediated chro- 12 concentrations in each; data not shown). Under the same notropic response at the isolated guinea pig right atrium. Ki values of experimental conditions, tiotidine-displaceable 125I-APT compounds (used at 7-14 different concentrations in two to five was in striatal, hippocampal, and experiments) were determined from the equation Ki = lC50/(1 + binding (0.2 nM) detected L/Kd), where IC50 is their half-inhibition concentration, L is the cortical membranes of rat brain, with specific binding (-30% concentration of 125I4APT (0.2 nM), and Kd is the equilibrium of total binding) representing 6, 6.5, and 10 fmol per mg of dissociation constant of 251I-APT (0.34 nM). Ki values of non-H2 protein, respectively. histaminergic compounds were as follows: mepyramine, >2 A.&M; Autoradiographic Localization of '251-APT Binding Sites in thioperamide, >2 AM; spiperone, >0.3 ,M; Sch 23390, >2 1uM; Guinea Pig Brain. After incubations with 0.1 nM 125I-APT, scopolamine, >10 AiM; phentolamine, >6 AM; propranolol, >6 AM; high contrast autoradiograms were obtained on sagittal brain naloxone, >10 AM; adenosine, >300 AuM. KB (or EC50) values at the sections (Fig. 4). Nonspecific binding generated on adjacent isolated guinea pig atrium are from refs. 5, 22, 24, 25, and the present sections in the presence of 3 ,M tiotidine was extremely faint study. (R)a-MeHA, (R)-a-methylhistamine. and homogeneous, whereas nearly half inhibition of labeling was obtained in the presence of 0.03 ,uM tiotidine (data not parabrachial nuclei, dorsal cochlear nucleus, or nucleus of shown). No labeling occurred at the level of the corpus the solitary tract. callosum. The highest densities of sites were observed in Photoaffinity Labeling of Histamine H2-Receptor Binding caudate putamen, nucleus accumbens, olfactory tubercles, Peptides Using'I25-AZPT. After 18-hr incubations of striatal superficial layers (I-III) of cerebral cortex, superficial gray layer of the superior colliculus, and inferior olive. A faint to Hi pp Mc moderate labeling occurred in deep layers of the cerebral C:hP II cortex (V, VI), in the hippocampal formation (CA1, CA3, Icx CA4), in thalamic nuclei (anterior and lateral groups) and hypothalamus (medial tuberal nucleus, lateral area). Specific I I ~ ~ ~ ~ labeling was also detectable in choroid plexuses, in the mesencephalic central gray and molecular layer of the cere- w _MPB bellum. It was very low in most brainstem areas but detect- ~~~~~- able in various nuclei of the pons-i.e., lateral and medial Table 1. Comparison of the distribution of 1251-APT binding sites UTAcb' \N 10 and histamine-sensitive adenylate cyclase activity in regions of Pir Tu guinea pig brain 4mm1 1251-APT binding Histamine-sensitive FIG. 4. Autoradiographic H2-receptor localization in a sagittal Region Kd Bmax adenylate cyclase section of guinea pig brain incubated for 3 hr at 220C in the presence Hippocampus 0.4 ± 0.1 69 ± 4 100*t of 0.1 nM 1251-APT. Nonspecific labeling generated on an adjacent was uniform Striatum 0.3 ± 0.1 135 ± 9 64,* 33t section in the presence of 3 ,uM tiotidine faint and (data ± ± 92t not shown). Acb, accumbens nucleus; AD, anterodorsal thalamic Cerebral cortex 0.2 0.1 51 6 89,* nucleus; Ccx, cerebral cortex; ChP, choroid plexus; CPu, caudate Kd (nM) and Bmax (fmol per mg of protein) for 1251-APiT binding are putamen; DC, dorsal cochlear nucleus; Hipp, hippocampal forma- derived from saturation studies at equilibrium, similar to those tion; 10, inferior olive; LH, lateral hypothalamic area; LPB, lateral depicted in Fig. 2 (means + SEM of two to four experiments). Values parabrachial nucleus; MC, molecular layer of cerebellar cortex; for adenylate cyclase stimulation by 0.1 mM histamine over basal MPB, medial parabrachial nucleus; Pir, piriform cortex; PMD, activity are expressed as percent of value in hippocampal mem- premammillary nucleus, dorsal part; SN, substantia nigra; Sol, branes. nucleus of the solitary tract; SuG, superficial gray layer of the *Data from ref. 7. superior colliculus; TM, tuberomammillary nucleus; Tu, olfactory tData from ref. 8. tubercle. Downloaded by guest on September 26, 2021 Neurobiology: Ruat et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1661 sensitive radioassay ofH2 receptors. This probe should allow 200- 200- the detection of the H2 receptor in tissues or cell lines with a low abundance as well as its purification. The overall 929-2-*m _ 92- density of H2 receptors in guinea pig brain appears to be in 69-9- 69- the same range as that of H1 receptors (26), also considered 0 Oslo 00 as postsynaptic receptors relative to histaminergic axons, but 45- significantly higher than that of H3 autoreceptors (21). Inhi- 45- bition of1251-APT binding by histamine was modulated by the 30- guanylnucleotide Gpp(NH)p, indicating the coupling of the 30- H2 receptor with a guanine nucleotide binding regulatory 21- protein (G protein), in agreement with its known association with adenylate cyclase (7, 8, 13). The IC50 of histamine, 1 2 3 4 5 6 7 8 9 evaluated in the presence of the nucleotide, was significantly higher than its EC50 for adenylate cyclase stimulation: this FIG. 5. Photoaffinity labeling pattern of striatal and hippocampal could reflect heterogeneity among H2 receptors (see below) membranes using 1251-AZPT. Hippocampal (lanes 1-4) and striatal to (lanes 5-9) membranes were incubated for 3 hr at 25TC and 17 hr at or the existence of spare receptors, but this remains be 100C, respectively, with 60 pM 1251-AZPT alone (lanes 1 and 5) or in confirmed in parallel experiments conducted under strictly the presence of 0.05 ,uM (lane 6) or 3 /LM (lanes 2 and 7) tiotidine, 0.1 similar experimental conditions. AiM Qane 8), or 10 jLM (lanes 3 and 9) ranitidine, or 100 jLM (lane 4) The high contrast autoradiograms generated by 1251-APT cimetidine. Solubilized membranes were analyzed by SDS/PAGE. over a very low background show the H2 receptors to be Molecular mass is indicated in kDa. Data from two independent distributed in many brain regions although in a highly heter- experiments are shown in lanes 1-4 and 5-9. ogeneous manner. The failure to detect them in several brain areas-e.g., the cerebellum or brainstem-by either [3H]- or hippocampal membranes at 100C in the dark (or 150 min at tiotidine binding (15) or histamine-induced activation of ade- 25TC) in phosphate buffer containing 100 mM NaCl and 0.1 nylate cyclase activity (7) might be due to their relatively low nM 125I-AZPT, specific' binding determined by the filtration abundance therein and the low signal/background ratio in assay (and defined with 3 AM tiotidine) represented -30% of these two tests. the total in the presence or absence of protease inhibitors The heterogeneous distribution of H2 receptors, which (data not shown). After UV irradiation and SDS/PAGE display a clearly laminated pattern in such areas as the analysis of the solubilized membranes, autoradiography of cerebral cortex or hippocampus, suggests their major asso- the dried gel indicated that a fraction of the initial radioac- ciation with neuronal elements, as indicated by previous tivity was covalently incorporated (Fig. 5). Among the main electrophysiological (9) and lesion studies (27). This distri- bands of the autoradiograms, labeling of two of them (87 + bution seems more or less in accordance with that of hista- 2 and 51 ± 1.3 kDa) was not significantly prevented by the H2 minergic axons (12), whereas this is not the case for H1 antagonists tiotidine or ranitidine, and cimetidine partially receptors (18). For instance, the striatum and the external prevented the labeling ofthe 87-kDa peptide. In contrast, the layers of the cerebral cortex, which receive an abundant labeling of two others (59 ± 0.5 and 32 ± 1.3 kDa; means + histaminergic innervation (12), are among the areas the most SEM of five independent experiments) was partially pre- heavily labeled with I251-APT, whereas H1 receptors are very vented by the H2 antagonists'when used at low concentra- scarce therein (18). Hence, these comparisons suggest that tions (around 2-5 times their K, values); at higher concen- histaminergic transmission is more often mediated by H2 than trations, the labeling was totally prevented for the 32-kDa by H1 receptors. On the other hand, the hypothalamus, which peptide and to a larger extent, although not completely, for receives the largest input of histaminergic axons, contains the 59-kDa peptide. A similar pattern was evidenced when only modest densities of H2 (but also H1 and H3) receptors, cortical membranes were used (data not shown). an observation that remains to be clarified. In addition, the colocalization ofH1 and H2 receptors-e.g., in several layers of cerebral and hippocampat cortices-accounts for the DISCUSSION synergism in cAMP formation resulting from simultaneous The present data provide information about the precise activation of the two receptors (28), whereas this situation localization and molecular properties of the H2 receptor in does not apply to other areas-e.g., the striatum. brain by using two original'[125I]iodinated probes.' Starting Interestingly, the distribution of H2 receptors among the from a relatively potent antagonist, potentidine-i.e., N- richest regions did not parallel that reported (7, 8) for (2-benzamidoethyl)-N'-cyano-N'-{3-[3-(1-piperidinylmethyl)- H2-linked adenylate cyclase (Table 1). Since the Kd of 1251 phenoxy]propyl}guanidine (KB = 16 nM)§-we found that the APT and Ki of several antagonists (data not shown) were introduction of a p-amino group, leading to APT, was toler- similar in these areas, regional differences in the coupling ated (K, = 10 nM) and that, unexpectedly, iodination of the efficiency of the H2-receptor complex might largely account latter increased the affinity: with a Kd of 0.3 nM, 125I-APT for this discrepancy. Nevertheless, the slight differences in constitutes one of the most potent H2-receptor antagonists histamine potency for inhibition of 125I-APT binding, as well known so far. Conversion of its amino group into an azido as in the modulatory effect of Gpp(NH)p, between two of group led to 125I-AZPT, a photoaffinity probe with a slightly these areas-e.g., striatum and hippocampus (see Results)- lower although acceptable potency. might reflect some heterogeneity among H2 receptors, pos- In striatal membranes, 125I-APT reversibly and stereose- sibly related to their coupling to different G proteins. lectively labeled a homogeneous population of sites that the In fact, after photoaffinity incorporation of 125I-AZPT, inhibition constants of a large panel of compounds unambig- SDS/PAGE analysis revealed that the same two bands (59 uously identify as H2 receptors. The high affinity of the and 32 kDa), presumably derived from the H2-receptor probe, its high specific radioactivity, together with its low binding peptides, were selectively labeled in hippocampal nonspecific binding are all important features enabling a and striatal membranes, their covalent labeling being consis- tently prevented by H2 antagonists with the expected po- §Hirschfeld, J., Buschauer, A. & Schunack, W. (1989) 18th Meeting tency. Although experiments were performed in the presence of the European Histamine Research Society, Breda, Netherlands, of protease inhibitors, we cannot exclude the fact that the abstr. 91. 32-kDa peptide corresponds to a proteolytic product of the Downloaded by guest on September 26, 2021 1662 Neurobiology: Ruat et al. Proc. Natl. Acad. Sci. USA 87 (1990) 59-kDa peptide, which may, therefore, represent the H2- 10. Ozawa, K., Nomura, Y. & Segawa, T. (1987) J. Neurochem. receptor binding subunit. Also its apparent mass is closer to 48, 1392-1398. that of members of the superfamily of G protein-linked 11. Blandina, P., Knott, P. J., Leung, L. K. H. & Green, J. P. receptors with seven transmembrane to (1989) J. Pharmacol. Exp. Ther. 249, 44-51. domains (29), which 12. Airaksinen, M. S. & Panula, P. (1988) J. Comp. Neurol. 273, the H2 receptor is likely to belong. Nevertheless, this hy- 163-186. pothesis remains to be confirmed-namely, because labeling 13. Johnson, C. L. (1982) in Pharmacology of Histamine Recep- of the 59-kDa peptide was only partially prevented by H2 tors, eds. Ganellin, C. R. & Parsons, M. E. (Wright, Bristol, antagonists: the lower affinity of 1251-AZPT as compared to U.K.), pp. 146-216. 1251-APT was accompanied by a relative increase in its 14. Gajtkowski, A. G., Norris, D. B., Rising, T. J. & Wood, T. P. nonspecific binding, illustrated by the labeling of several (1983) Nature (London) 304, pp. 65-67. 15. Norris, D. B., Gajtkowski, G. A. & Rising, T. J. (1984) Agents peptides not related to the H2 receptor. Actions 14, pp. 543-545. In conclusion, 125I-APT and 125I-AZPT are likely to con- 16. Maayani, S., Hough, L. B., Weinstein, H. & Green, J. P. stitute useful probes for localization and molecular studies of (1982) in Typical and Atypical Antidepressants, eds. Racagni, the H2 receptor. In addition, 125II-APr could be used for G. & Costa, E. (Raven, New York), Vol. 31, pp. 131-147. neuropathological studies since it has allowed us to recently 17. Schwartz, J. C., Garbarg, M., Lebrecht, U., Nowak, J., Pol- characterize the H2 receptor in human brain. lard, H., Rodergas, E., Rose, C., Quach, T. T., Morgat, J. L. & Roy, J. (1982) in Advances in Histamine Research, eds. We thank F. Keller (Berlin) for the enantiomers of PPAT. This Uvnas, B. & Tasaka, K. (Pergamon, Oxford), pp. 71-80. work was 18. Bouthenet, M. L., Ruat, M., Sales, N., Garbarg, M. & supported in part by a grant from the Direction des Schwartz, J. C. 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