Proc. Natd. Acad. Sci. USAVol. 87, pp. 1658-1662, March 1990Neurobiology

Reversible and irreversible labeling and autoradiographiclocalization 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-(4-amino-3-iodobenzamido)ethyl]-N'-cyano-N"-{3-[3-(1-piperi-dinylmethyl)phenoxylpropyl}guanidine-represents one of themost potent H2-receptor antagonists known so far. In mem-branes of guinea pig brain 12sI-APT bound reversibly, selec-tively, and with high affinity (Kd = 0.3 nM) to a homogeneouspopulation of sites unambiguously identified as H2 receptors byinhibition studies conducted with a large panel of antagonists.I2sI.-APT binding was also inhibited by histamine, and the effectwas modulated by a guanyl nucleotide, which is consistent withthe association of the H2 receptor with a guanine nucleotidebinding regulatory protein. The low nonspecific binding of125I-APT generated high contrast autoradiographic pictures inbrain sections and established the precise distribution of H2receptors. Their highly heterogeneous distribution and lami-nated pattern in some areas-e.g., cerebral and hippocampalcortices-suggest their major association with neuronal ele-ments. These localizations were more consistent than those ofH1 receptors with the distribution of histaminergic projections,indicating that H2 receptors mediate a larger number ofpostsynaptic actions of histamine-e.g., in striatum. Colocal-izations of H1 and H2 receptors in some areas account for theirknown synergistic interactions in cAMP formation induced byhistamine. The distribution of '2sI-APT binding sites did notstrictly parallel that of the H2-receptor-linked adenylate cyclaseactivity, which may reflect heterogeneity among H2 receptors.After UV irradiation and SDS/PAGE analysis, ['2sI]iodo-azidopotentidine (125I-AZPT), a photoaffinity probe derivedfrom "2I-APT, was covalently incorporated in several pep-tides, among which the labeling of two peptides of 59 and 32kDa was prevented by H2 antagonists, suggesting that theycorrespond to H2-receptor binding peptides or proteolysisproducts of the latter. These probes should be useful forsensitive radioassays, localization, purification, and molecularstudies of the H2 receptor, which were previously impractica-ble.

Histamine is a messenger molecule mainly released by neu-rons and mast cells that affects a large variety of target cellsby interacting with three pharmacologically distinct sub-classes of receptors termed H1, H2, and H3 (1-5). In brain,where this amine acts as a neurotransmitter, the presence ofH2 receptors was indirectly evidenced by the histamine-induced stimulation of cAMP accumulation in slices (6),activation of adenylate cyclase in membranes (7, 8), changesin neuronal firing (9), activation of phospholipid methylation(10), and release of endogenous norepinephrine (11). How-ever, contrasting the widespread projections of histaminergic

neurons to almost the whole mammalian central nervoussystem (12), these responses-e.g., adenylate cyclase acti-vation-could be demonstrated in only a small number ofbrain areas of a few animal species (13).

Various attempts at labeling the H2 receptor with radioac-tive probes, a prerequisite for starting localization, regula-tion, purification, or molecular cloning studies, have so farmet with limited success. Whereas [3H]cimetidine, [3H]rani-tidine, and [3H]impromidine were found to be totally unsuit-able as ligands (2), [3H]tiotidine was shown to label the H2receptor in membranes of three areas of the guinea pig brain(14, 15). However, this could not be confirmed in otherlaboratories (16, 17); the nonspecific binding was high, andH2 receptors were undetectable with [3H]tiotidine in manybrain areas known to receive histaminergic innervation and inthe brain of species other than the guinea pig (14, 15). Inaddition, and in contrast with H1 and H3 receptors (18-21),no information is available regarding the tissue distribution orphysicochemical properties of the H2 receptor.Here we report the design of the antagonist [125Ij]

iodoaminopotentidine (125I-APT), a high-affinity reversibleprobe for H2 receptors, which enables their extremely sen-sitive detection over a low background in membranes as wellas, autoradiographically, in brain sections. In addition,[125I]iodoazidopotentidine (125I-AZPT), a photoaffinity probederived from 125I-APT, was shown to be covalently incorpo-rated into the H2 receptor after UV irradiation, leading to theinitial physicochemical characterization of the ligand bindingpeptides of this receptor.

MATERIALS AND METHODSMaterials. Na125I (usually 2000 Ci/mmol; 1 Ci = 37 GBq)

was from Amersham. The drugs and their sources were asfollows: cimetidine, zolantidine, burimamide, metiamide,dimaprit, impromidine (Smith Kline & French), mepyramine(Specia), famotidine (Merck Sharp & Dohme), tiotidine (ICI).Analytical grade reagents were from Sigma. (R)-a-Meth-ylhistamine and PPAT stereoisomers-i.e., 5-amino-2-(3-{3-[1-(1-pyrrolidinyl)ethyl]phenoxy}propyl)amino-1,3,4-thiadia-zole (22)-were from the Institute of Pharmacy (Berlin,F.R.G.).

Synthesis of APT. 1,1'-Carbonyldiimidazole (3.43 mmol)was added to a stirred solution of 4-aminobenzoic acid (3.43mmol) in dry tetrahydrofuran (5 ml). The mixture was al-lowed to react for 1 hr at room temperature. The solution wasadded to the amine N-(2-aminoethyl)-N'-cyano-N'-{3-[3-(1-

Abbreviations: 1251I-APT, [125I]iodoaminopotentidine; 251I-AZPIT,[1251]iodoazidopotentidine; APT, aminopotentidine; G protein, gua-nine nucleotide binding regulatory protein.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Nati. Acad. Sci. USA 87 (1990) 1659

piperidinylmethyl)phenoxy]propyl}guanidine (3.43 mmol)and stirred overnight at room temperature. The mixture waspoured into water and extracted with CH2Cl2. The organiclayer was dried by Na2SO4, filtered, and evaporated underreduced pressure affording the title compound as an oil (1.58g, 3.31 mmol). The product was chromatographically purified(Chromatotron 7924T, Harrison Research, Muttenz, Swit-zerland) by using 4-mm layers of silica gel 60 PF254 (Merck)containing gypsum, eluted by CHCl3/MeOH (97:3, vol/vol)(ammonia atmosphere). An analytical sample of the purified,oily base was converted into a salt with oxalic acid andrecrystallized from Et2O/EtOH, C26H35N702-C2H204 0.5H20; mp, 920C-950C (dec.). The structure of APT (N-[2-(4-aminobenzamido)ethyl]-N'-cyano-N'-{3-[3-(1-piperidi-nylmethyl)phenoxy]propyl}guanidine was confirmed by 1HNMR, IR, mass spectroscopy, and elemental analysis.

Synthesis of '25I-APT and 12sI-AZPT. The chemical struc-tures of 1251I-APT and 125I-AZPT are shown in Fig. 1. APT (9.5gg/2 ,gl of EtOH; 20 nmol) was mixed in a polypropylene tubewith 404ul of 1 M NaOAc buffer, pH 5.6/Na1251 (20 ,ul; 2 mCi).Then chloramine T (44 nmol/10,ul) was added. After 1 min,the reaction was stopped with Na2S205 (88 nmol/10 ,l) andthe mixture was analyzed by HPLC (C18 jLBondapak, Wa-ters). The mobile phase was MeCN/10mM NH4OAc, pH 4.2(32:68, vol/vol), and the flow rate was 1 mI/min. 125I-APT(retention time, 15.2 min) was well separated from APT(retention time, 7.4 min). Usually, >70% of the initial radio-activity was recovered in this peak, a 300-,ul sample of whichwas 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 8min at 20°C and the reaction mixture was immediatelysubmitted to HPLC as described above, the mobile phasebeing MeCN/10 mM NH4OAc, pH 4.2 (40:60, vol/vol).125I-APT (retention time, 8.4 min) was found to be quitecompletely transformed and 125I-AZPT (retention time, 19.0min) was collected. Extreme care was taken to work underdim light during the azidation and HPLC steps. 125I-APT wasdiluted (1.5 times) in EtOH and stored at -20°C and 12511AZPT was stored at 4°C in the dark, both without losing theirbinding properties.Membrane Preparation. Brain regions from male Hartley

guinea pigs (200-300 g) were homogenized with a Polytronblender in 60 vol of cold 50 mM Na2HPO4/KH2PO4 buffer,pH 7.5. After centrifugation at 260 x g for 1 min, the resultingsupernatant was recentrifuged at 20,000 x g for 30 min, andthe final pellet was used immediately or stored at -80°C. Inthe experiments with protease inhibitors, the buffer wassupplemented with bacitracin (0.1 mg/ml), leupeptin (10,ug/ml), pepstatin A (0.1 ,ug/ml), phenylmethylsulfonyl flu-oride (0.1 mM), and soybean trypsin inhibitor (10 ,g/ml).Protein concentration was determined by the method ofLowry with bovine serum albumin as a standard. Brainmembranes from male Wistar rats were prepared in a similarway.

'251-APT Binding. Pellets were resuspended in phosphatebuffer. Triplicate assays were performed in polypropylenetubes and gelatin was added (final concentration, 0.1%) toprevent 1251-APT adsorption. Membranes (50-100 ,ug ofprotein) were usually incubated with 125IjAPF for 120-150min at 25°C in a final vol of 400 ,ul. Incubations were stoppedby four additions of cold buffer (3 ml each), followed by rapidfiltration through glass fiber filters (GF/B) treated with 0.3%

7N-CH2iiLO(CH2)3NH C NH (CH2)2NHCN-CN

FIG. 1. Chemical structures of 1251-APT (R = NH2), a reversibleprobe, and 125I-AZPT (R = N3), a photoaffinity probe.

polyethyleneimine. Radioactivity trapped was measuredwith a LKB y-counter (82% efficiency). Specific binding wasdefined as that inhibited by 3 A.M tiotidine.

Photoaffinity Labeling and SDS/PAGE. Membranes (0.15-0.30 mg/ml) were incubated in the dark with 50-100 pM1251I-AZPT for 16-20 hr at 10'C (or 150 min at 250C) inphosphate buffer containing 100 mM NaCi. A sample wasretained for a I251-AZPT binding assay performed as de-scribed above for 125I-APT binding. For photoaffinity label-ing, another sample was centrifuged at 32,000 x g for 20 minand the pellets were resuspended in phosphate buffer andirradiated for 3 min as described (19). The irradiated mem-branes were collected by centrifugation and solubilized in thepresence of 5% 2-mercaptoethanol, and the mixture wasanalyzed by SDS/PAGE on 11% polyacrylamide gels asdescribed (19). Protease inhibitors were present throughoutthe experiments.

Autoradiographic Localization of 12sI-APT Binding Sites.Sections (10 Am) of guinea pig brains were prepared asdescribed (18). Slide-mounted sections were incubated for 3hr at 220C in 50 mM sodium/potassium phosphate buffer (pH7.5) containing 0.1 nM 125I-APT at 220C. Nonspecific bindingwas defined by using 3 ,uM tiotidine. After five rinses (4 mineach) at 4°C in phosphate buffer, the sections were apposedon Ultrofilm (LKB) for a 2-day period and autoradiogramswere developed (18).

RESULTSReversible Binding of 12SI-A5r to H2 Receptors. At 25°C

1251I-APT binding to striatal membranes occurred with anassociation rate constant (kj) of 0.03 min/nM, with equilib-rium being reached after 120-150 min (Fig. 2). Dissociationoccurring in the presence of 10 ,uM tiotidine, a H2 antagonist,followed first-order kinetics with a rate constant (k-,) of0.013 min'. The ratio kL1/k, gave an equilibrium dissocia-tion constant (Kd) of 0.43 nM. Saturation of specific 125I-APTbinding at equilibrium, defined using 3 AM tiotidine, was

100,

100

2 200E Timeeh

E 50 100 B

0.25 050 0.75FREE '251-APTnM

FIG. 2. Reversible labeling of the H2 receptor in membranes ofguinea 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 tiotidineusing 54 ,ug of protein per 0.4 ml (or 0.8 ml for 125I-APT < 0.05 nM).(Left Inset) Scatchard plot analysis of specific binding. (Right Inset)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,bound; F, free; *, total binding; A, nonspecific binding; o, specificbinding (5).

Neurobiology: Ruat et al.

1660 Neurobiology: Ruat et al.

monophasic (nH = 0.95 + 0.10) leading to a linear Scatchardplot, and computer analysis (23) of the binding isotherms ledto a Kd of 0.34 ± 0.10 nM and a Bmax of 135 ± 9 fmol per mgof protein (Fig. 2 and Table 1). Similar Kd values wereobtained from the analysis of I251-APT binding to membranesof cerebral cortex or hippocampus in which the Bmax waslower (Table 1). With the latter, specific binding at 0.2 nM125I-APT typically represented 50-60% of the total, to becompared with 70-80% with striatal membranes. Specific125I-APT binding to striatal membranes was inhibited in amonophasic manner (pseudo-Hill coefficients close to unity)by a series of H2 antagonists. Their Ki values were highlycorrelated (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-APTwas measured after a 60-min contact as the potency of thecompound increased with this contact duration. In contrast,H1- or H3-receptor antagonists and various nonhistaminergicagents were poorly effective.

Histamine itself, in phosphate buffer and in the presence of1 mM Mg2+, inhibited the binding of 125I-APT (0.2 nM) tostriatal and hippocampal membranes with IC50 values of 11 ±2 and 24 ± 6 ,uM, respectively, in the absence of Gpp(NH)p,and of 33 ± 7 and 100 ± 24 ,uM, respectively, in the presenceof 0.1 mM nucleotide (pooled data from two experiments with12 concentrations in each; data not shown). Under the sameexperimental conditions, tiotidine-displaceable 125I-APTbinding (0.2 nM) was detected in striatal, hippocampal, andcortical membranes of rat brain, with specific binding (-30%of total binding) representing 6, 6.5, and 10 fmol per mg ofprotein, respectively.

Autoradiographic Localization of '251-APT Binding Sites inGuinea Pig Brain. After incubations with 0.1 nM 125I-APT,high contrast autoradiograms were obtained on sagittal brainsections (Fig. 4). Nonspecific binding generated on adjacentsections in the presence of 3 ,M tiotidine was extremely faintand homogeneous, whereas nearly half inhibition of labelingwas obtained in the presence of 0.03 ,uM tiotidine (data notshown). No labeling occurred at the level of the corpuscallosum. The highest densities of sites were observed incaudate putamen, nucleus accumbens, olfactory tubercles,superficial layers (I-III) of cerebral cortex, superficial graylayer of the superior colliculus, and inferior olive. A faint tomoderate labeling occurred in deep layers of the cerebralcortex (V, VI), in the hippocampal formation (CA1, CA3,CA4), in thalamic nuclei (anterior and lateral groups) andhypothalamus (medial tuberal nucleus, lateral area). Specificlabeling was also detectable in choroid plexuses, in themesencephalic central gray and molecular layer of the cere-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 sitesand histamine-sensitive adenylate cyclase activity in regions ofguinea pig brain

1251-APT binding Histamine-sensitiveRegion Kd Bmax adenylate cyclase

Hippocampus 0.4 ± 0.1 69 ± 4 100*tStriatum 0.3 ± 0.1 135 ± 9 64,* 33tCerebral cortex 0.2 ± 0.1 51 ± 6 89,* 92tKd (nM) and Bmax (fmol per mg of protein) for 1251-APiT binding are

derived from saturation studies at equilibrium, similar to thosedepicted in Fig. 2 (means + SEM of two to four experiments). Valuesfor adenylate cyclase stimulation by 0.1 mM histamine over basalactivity are expressed as percent of value in hippocampal mem-branes.*Data from ref. 7.tData from ref. 8.

Proc. Natl. Acad. Sci. USA 87 (1990)

-10

z0a.CDw -8

0-

0z0cc -6

-J

4h7Li Mk-~MeHA-4 -5 -6 -7 -8 -9 -10

LOG (Ki)125I-APT BINDING

FIG. 3. Pharmacological characterization of 1251-APT binding tostriatal membranes. Ki values are compared with KB (or EC50) valuesof the same compounds regarding the H2-receptor-mediated chro-notropic response at the isolated guinea pig right atrium. Ki values ofcompounds (used at 7-14 different concentrations in two to fiveexperiments) were determined from the equation Ki = lC50/(1 +L/Kd), where IC50 is their half-inhibition concentration, L is theconcentration of 125I4APT (0.2 nM), and Kd is the equilibriumdissociation constant of 251I-APT (0.34 nM). Ki values of non-H2histaminergic compounds were as follows: mepyramine, >2 A.&M;thioperamide, >2 AM; spiperone, >0.3 ,M; Sch 23390, >2 1uM;scopolamine, >10 AiM; phentolamine, >6 AM; propranolol, >6 AM;naloxone, >10 AM; adenosine, >300 AuM. KB (or EC50) values at theisolated guinea pig atrium are from refs. 5, 22, 24, 25, and the presentstudy. (R)a-MeHA, (R)-a-methylhistamine.

parabrachial nuclei, dorsal cochlear nucleus, or nucleus ofthe solitary tract.

Photoaffinity Labeling of Histamine H2-Receptor BindingPeptides Using'I25-AZPT. After 18-hr incubations of striatal

Hi pp Mc

C:hP II

I I ~ ~ ~ ~

Icx

w ~~~~~-_MPB

UTAcb' \NPir Tu

10

4mm1

FIG. 4. Autoradiographic H2-receptor localization in a sagittalsection of guinea pig brain incubated for 3 hr at 220C in the presenceof 0.1 nM 1251-APT. Nonspecific labeling generated on an adjacentsection in the presence of 3 ,uM tiotidine was faint and uniform (datanot shown). Acb, accumbens nucleus; AD, anterodorsal thalamicnucleus; Ccx, cerebral cortex; ChP, choroid plexus; CPu, caudateputamen; DC, dorsal cochlear nucleus; Hipp, hippocampal forma-tion; 10, inferior olive; LH, lateral hypothalamic area; LPB, lateralparabrachial nucleus; MC, molecular layer of cerebellar cortex;MPB, medial parabrachial nucleus; Pir, piriform cortex; PMD,premammillary nucleus, dorsal part; SN, substantia nigra; Sol,nucleus of the solitary tract; SuG, superficial gray layer of thesuperior colliculus; TM, tuberomammillary nucleus; Tu, olfactorytubercle.

Proc. Natl. Acad. Sci. USA 87 (1990) 1661

92-69-

45-

30-

5 6 7 8 9

FIG. 5. Photoaffinity labeling pattern of striatal and hippocampalmembranes using 1251-AZPT. Hippocampal (lanes 1-4) and striatal(lanes 5-9) membranes were incubated for 3 hr at 25TC and 17 hr at100C, respectively, with 60 pM 1251-AZPT alone (lanes 1 and 5) or inthe presence of 0.05 ,uM (lane 6) or 3 /LM (lanes 2 and 7) tiotidine, 0.1AiM Qane 8), or 10 jLM (lanes 3 and 9) ranitidine, or 100 jLM (lane 4)cimetidine. Solubilized membranes were analyzed by SDS/PAGE.Molecular mass is indicated in kDa. Data from two independentexperiments are shown in lanes 1-4 and 5-9.

or hippocampal membranes at 100C in the dark (or 150 min at25TC) in phosphate buffer containing 100 mM NaCl and 0.1nM 125I-AZPT, specific' binding determined by the filtrationassay (and defined with 3 AM tiotidine) represented -30% ofthe total in the presence or absence of protease inhibitors(data not shown). After UV irradiation and SDS/PAGEanalysis of the solubilized membranes, autoradiography ofthe dried gel indicated that a fraction of the initial radioac-tivity was covalently incorporated (Fig. 5). Among the mainbands of the autoradiograms, labeling of two of them (87 +2 and 51 ± 1.3 kDa) was not significantly prevented by the H2antagonists tiotidine or ranitidine, and cimetidine partiallyprevented the labeling of the 87-kDa peptide. In contrast, thelabeling of two others (59 ± 0.5 and 32 ± 1.3 kDa; means +SEM of five independent experiments) was partially pre-vented by the H2 antagonists'when used at low concentra-tions (around 2-5 times their K, values); at higher concen-trations, the labeling was totally prevented for the 32-kDapeptide and to a larger extent, although not completely, forthe 59-kDa peptide. A similar pattern was evidenced whencortical membranes were used (data not shown).

DISCUSSIONThe present data provide information about the preciselocalization and molecular properties of the H2 receptor inbrain by using two original'[125I]iodinated probes.' Startingfrom a relatively potent antagonist, potentidine-i.e., N-(2-benzamidoethyl)-N'-cyano-N'-{3-[3-(1-piperidinylmethyl)-phenoxy]propyl}guanidine (KB = 16 nM)§-we found that theintroduction of a p-amino group, leading to APT, was toler-ated (K, = 10 nM) and that, unexpectedly, iodination of thelatter increased the affinity: with a Kd of 0.3 nM, 125I-APTconstitutes one of the most potent H2-receptor antagonistsknown so far. Conversion of its amino group into an azidogroup led to 125I-AZPT, a photoaffinity probe with a slightlylower although acceptable potency.

In striatal membranes, 125I-APT reversibly and stereose-lectively labeled a homogeneous population of sites that theinhibition constants of a large panel of compounds unambig-uously identify as H2 receptors. The high affinity of theprobe, its high specific radioactivity, together with its lownonspecific binding are all important features enabling a

sensitive radioassay ofH2 receptors. This probe should allowthe detection of the H2 receptor in tissues or cell lines witha low abundance as well as its purification. The overalldensity of H2 receptors in guinea pig brain appears to be inthe same range as that of H1 receptors (26), also consideredas postsynaptic receptors relative to histaminergic axons, butsignificantly higher than that of H3 autoreceptors (21). Inhi-bition of 1251-APT binding by histamine was modulated by theguanylnucleotide Gpp(NH)p, indicating the coupling of theH2 receptor with a guanine nucleotide binding regulatoryprotein (G protein), in agreement with its known associationwith adenylate cyclase (7, 8, 13). The IC50 of histamine,evaluated in the presence of the nucleotide, was significantlyhigher than its EC50 for adenylate cyclase stimulation: thiscould reflect heterogeneity among H2 receptors (see below)or the existence of spare receptors, but this remains to beconfirmed in parallel experiments conducted under strictlysimilar experimental conditions.The high contrast autoradiograms generated by 1251-APT

over a very low background show the H2 receptors to bedistributed in many brain regions although in a highly heter-ogeneous manner. The failure to detect them in several brainareas-e.g., the cerebellum or brainstem-by either [3H]-tiotidine binding (15) or histamine-induced activation of ade-nylate cyclase activity (7) might be due to their relatively lowabundance therein and the low signal/background ratio inthese two tests.The heterogeneous distribution of H2 receptors, which

display a clearly laminated pattern in such areas as thecerebral cortex or hippocampus, suggests their major asso-ciation with neuronal elements, as indicated by previouselectrophysiological (9) and lesion studies (27). This distri-bution seems more or less in accordance with that of hista-minergic axons (12), whereas this is not the case for H1receptors (18). For instance, the striatum and the externallayers of the cerebral cortex, which receive an abundanthistaminergic innervation (12), are among the areas the mostheavily labeled with I251-APT, whereas H1 receptors are veryscarce therein (18). Hence, these comparisons suggest thathistaminergic transmission is more often mediated by H2 thanby H1 receptors. On the other hand, the hypothalamus, whichreceives the largest input of histaminergic axons, containsonly modest densities of H2 (but also H1 and H3) receptors,an observation that remains to be clarified. In addition, thecolocalization ofH1 and H2 receptors-e.g., in several layersof cerebral and hippocampat cortices-accounts for thesynergism in cAMP formation resulting from simultaneousactivation of the two receptors (28), whereas this situationdoes not apply to other areas-e.g., the striatum.

Interestingly, the distribution of H2 receptors among therichest regions did not parallel that reported (7, 8) forH2-linked adenylate cyclase (Table 1). Since the Kd of 1251APT and Ki of several antagonists (data not shown) weresimilar in these areas, regional differences in the couplingefficiency of the H2-receptor complex might largely accountfor this discrepancy. Nevertheless, the slight differences inhistamine potency for inhibition of 125I-APT binding, as wellas in the modulatory effect of Gpp(NH)p, between two ofthese areas-e.g., striatum and hippocampus (see Results)-might reflect some heterogeneity among H2 receptors, pos-sibly related to their coupling to different G proteins.

In fact, after photoaffinity incorporation of 125I-AZPT,SDS/PAGE analysis revealed that the same two bands (59and 32 kDa), presumably derived from the H2-receptorbinding peptides, were selectively labeled in hippocampaland striatal membranes, their covalent labeling being consis-tently prevented by H2 antagonists with the expected po-tency. Although experiments were performed in the presenceof protease inhibitors, we cannot exclude the fact that the32-kDa peptide corresponds to a proteolytic product of the

200- 200-

929-2-*m _69-9-45-

30-21-

1 2 3 4

§Hirschfeld, J., Buschauer, A. & Schunack, W. (1989) 18th Meetingof the European Histamine Research Society, Breda, Netherlands,abstr. 91.

Neurobiology: Ruat et al.

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Proc. Natl. Acad. Sci. USA 87 (1990)

59-kDa peptide, which may, therefore, represent the H2-receptor binding subunit. Also its apparent mass is closer tothat of members of the superfamily of G protein-linkedreceptors with seven transmembrane domains (29), to whichthe H2 receptor is likely to belong. Nevertheless, this hy-pothesis remains to be confirmed-namely, because labelingof the 59-kDa peptide was only partially prevented by H2antagonists: the lower affinity of 1251-AZPT as compared to1251-APT was accompanied by a relative increase in itsnonspecific binding, illustrated by the labeling of severalpeptides not related to the H2 receptor.

In conclusion, 125I-APT and 125I-AZPT are likely to con-stitute useful probes for localization and molecular studies ofthe H2 receptor. In addition, 125II-APr could be used forneuropathological studies since it has allowed us to recentlycharacterize the H2 receptor in human brain.

We thank F. Keller (Berlin) for the enantiomers of PPAT. Thiswork was supported in part by a grant from the Direction desRecherches et Etudes Techniques.

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