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CELLULAR AND MOLECULAR

Multiple Differences in Agonist and Antagonist Pharmacology between Human and Guinea Pig Histamine H₁-Receptor

Department of Pharmacology and Toxicology, the University of Kansas, Lawrence, Kansas (R.S., K.W.-S.); Department of Molecular Biosciences, the University of Kansas, Lawrence, Kansas (T.B.); Department of Chemistry and Biochemistry, Free University of Berlin, Berlin, Germany (T.B.); Institute of Pharmacy, Free University of Berlin, Berlin, Germany (H.H.P., W.S.); Institute of Pharmacy, University of Regensburg, Regensburg, Germany (S.D., A.B., S.E.)

Received January 28, 2003; accepted February 21, 2003.

	Abstract

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Species isoforms of histamine H₂-, H₃-, and H₄-receptors differ in their pharmacological properties. The study aim was to dissect differences between the human H₁R (hH₁R) and guinea pig H₁R (ghH₁R). We coexpressed hH₁R and gpH₁R with regulators of G-protein signaling in Sf9 insect cells and analyzed the GTPase activity of G_q-proteins. Small H₁R agonists showed similar effects at hH₁R and gpH₁R, whereas bulkier 2-phenylhistamines and histaprodifens were up to 10-fold more potent at gpH₁R than at hH₁R. Most 2-phenylhistamines and histaprodifens were more efficacious at gpH₁R than at hH₁R. Several first-generation H₁R antagonists were 2-fold, and arpromidine-type H₁R antagonists up to 10-fold more potent at gpH₁R than at hH₁R. [³H]Mepyramine competition binding studies confirmed the potency differences of the GTPase studies. Phe-153Leu-153 or Ile-433Val-433 exchange in hH₁R (hH₁RgpH₁R) resulted in poor receptor expression, low [³H]mepyramine affinity, and functional inactivity. The Phe-153Leu-153/Ile-433Val-433 double mutant expressed excellently but only partially changed the pharmacological properties of hH₁R. Small H₁R agonists and 2-phenylhistamines interacted differentially with human and guinea pig H₂R in terms of potency and efficacy, respectively. Our data show the following: 1) there are differences in agonist- and antagonist-pharmacology of hH₁R and gpH₁R encompassing diverse classes of bulky ligands. These differences may be explained by higher conformational flexibility of gpH₁R relative to hH₁R; 2) Phe-153 and Ile-433 are critical for proper folding and expression of hH₁R; and 3) H₂R species isoforms distinguish between H₁R agonists.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Structures of H1R agonists. 1, histamine; 2 to 4, small histamine-derived agonists; 5 to 17, 2-phenylhistamine derivatives; 18, thioether compound related to 5 to 17; 18 to 23, histaprodifens. For agonist names, see Table 2.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Structures of H1R antagonists. 24 to 32, first-generation H1R antagonists; 33 to 39, arpromidine (35)-derived dual H2R agonists/H1R antagonists; 40, H1R antagonist derived from 2-phenylhistamines; 41 to 45, second-generation H1R antagonists. For antagonist names, see Table 3.

The species differences in pharmacological properties of H₂R, H₃R, and H₄R raise the question whether this is a general characteristic of H_xRs. In fact, the K_d values of [³H]mepyramine for H₁Rs from various species differ by 2 to 6-fold (Chang et al., 1979). Moreover, histaprodifens exhibit different potencies and efficacies in the guinea pig ileum and rat aorta (Elz et al., 2000). Furthermore, 2-(3-chlorophenyl)histamine (12) is a potent H₁R agonist in the guinea pig ileum but failed to exhibit agonistic activity in H₁R-expressing dibutyryl cAMP-differentiated human HL-60 leukemia cells (Seifert et al., 1994). A snake plot of hH₁R depicts the relative positions and topology of amino acid residues in the TM domains, putative agonist and antagonist binding sites, and differences with respect to the gpH₁R (Fig. 3). Mutagenesis data (Leurs et al., 1994, 1995; Ohta et al., 1994; Nonaka et al., 1998) and modeling approaches (Elz et al., 2000) indicated that histamine and histaprodifens interact with amino acid residues in TMs III, IV, V, and VII. Considering the alignment of H₁Rs with bovine rhodopsin (Palczewski et al., 2000) and results of the substituted-cysteine accessibility method with the dopamine D₂-receptor (Ballesteros et al., 2001), there are no amino acid differences in the ligand binding pocket of gpH₁R and hH₁R. The two lipid-directed residues, Phe-153 in TM IV of hH₁R versus Leu in gpH₁R and Ile-433 in TM VI of hH₁R versus Val in gpH₁R, represent the only differences near the binding site. Although these amino acid exchanges are conservative, the amino acids in hH₁R are bulkier than those in gpH₁R, and such differences could have an impact on the ligand-binding pocket.

View larger version (46K): [in this window] [in a new window]   Fig. 3. Snake plot of hH1R showing the relative positions and topology of amino acid residues in the TM domains, putative agonist and antagonist binding sites, and differences with respect to the gpH1R. Figure 3 considers results of the substituted cysteine accessibility method by which the dopamine D2-receptor binding site was mapped (Ballesteros et al., 2001). Red circles, amino acids predicted to face the interior of the TM helix bundle based on corresponding positions in bovine rhodopsin (Palczewski et al., 2000). Blue circles, amino acids predicted to face lipid. Red letters, the corresponding amino acids in the dopamine D2-receptor are water-accessible and protected by ligand binding in substituted-cysteine accessibility experiments (Ballesteros et al., 2001). Magenta letters, amino acids that are water accessible but not protected by ligand binding in substituted cysteine accessibility experiments. Red shading, histamine binding site residues according to site-directed mutagenesis data (Leurs et al., 1994, 1995; Ohta et al., 1994; Nonaka et al., 1998). Green shading, amino acid of the H1R antagonist binding site based on mutagenesis studies (Ohta et al., 1994; Nonaka et al., 1998; Wieland et al., 1999). Yellow shading, amino acids that are essential for both histamine and H1R antagonist binding (Wieland et al., 1999). Orange shading, additional amino acid residues of the histaprodifen binding site based on molecular modeling approaches (Elz et al., 2000). Note that Lys-191 does not interact with histaprodifens. Blue shading, amino acids targeted by site-directed mutagenesis in the present article. Gray shading, other amino acid residues different between hH1R and gpH1R. Brown line, conserved disulfide bridge between Cys-100 and Cys-180. Numbers designate the first and last amino acids, respectively, of each TM domain.

The aim of the present study was to compare recombinant hH₁R and gpH₁R expressed in Sf9 insect cells under identical experimental conditions. We also examined the roles of Phe-153 and Ile-433 in hH₁R function. As read-out, we focused on the determination of the GTPase activity of insect cell G_q-proteins in the presence of the RGS proteins RGS4 and GAIP. This coexpression system provides a sensitive model for studying H₁R at the G-protein level (Houston et al., 2002). The GTPase assay is a steady-state method and eliminates the impact of effector availability/compartmentation and pharmacokinetic barriers on the properties of agonists (Buschauer, 1989; Ostrom et al., 2000). Moreover, we conducted [³H]mepyramine binding studies and analyzed the effects of H₁R agonists on recombinant H₂R-G_s fusion proteins, recently verified as sensitive systems for the analysis of H₂Rs (Kelley et al., 2001).

	Materials and Methods

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Materials. Construction of the cDNAs for hH₁R-F153L, hH₁R-I433V, and hH₁R-F153L/I433V was performed by overlap-extension polymerase chain reaction following recently described procedures (Houston et al., 2002). Dimethindene enantiomers were a kind gift of Dr. G. Lambrecht (Department of Pharmacology, University of Frankfurt/Main, Germany). Ketotifen was a gift from Novartis (Basel, Switzerland), azelastine a gift from Asta Medica (Frankfurt/Main, Germany), fexofenadine a gift from Janssen-Cilag (Neuss, Germany), and terfenadine a gift from Aventis (Frankfurt/Main, Germany). Guanidines 33 to 38 were synthesized as described (Buschauer, 1989). Guanidine 39 was prepared by analogy to the procedures described for guanidines 33 to 38. 2-Methylhistamine (2) and 2-(2-thiazolyl)ethanamine (3) were synthesized using standard procedures. Compounds 5 to 18 were prepared according to published procedures (Zingel et al., 1990; Leschke et al., 1995). Compounds 22, 23, and 40 were available by synthetic pathways reported for the synthesis of 19 to 21 (Elz et al., 2000). Structures of synthesized compounds were confirmed by elemental analysis (C, H, and N), ¹H NMR spectroscopy, and mass spectrometry. The purity of the compounds was >98% as determined by high-performance liquid chromatography or capillary electrophoresis. Tunicamycin, histamine, betahistine, promazine, chlorpromazine, mianserin, cyproheptadine, diphenhydramine, mepyramine, triprolidine, and (+)-chlorpheniramine were from Sigma-Aldrich (St. Louis, MO). Sources of other materials are described elsewhere (Kelley et al., 2001; Houston et al., 2002).

Cell Culture and Membrane Preparation. Recombinant baculoviruses encoding hH₁R-F153L, hH₁R-I433V, and hH₁R-F153L/I433V were generated in Sf9 cells using the BaculoGOLD transfection kit (BD Pharmingen, San Diego, CA), according to the manufacturer's instructions. Infection and culture of Sf9 cells and membrane preparation were performed as described (Kelley et al., 2001; Houston et al., 2002). In some cultures, we added tunicamycin (10 µg/ml) to cultures to inhibit N-glycosylation of H₁Rs (Seifert and Wenzel-Seifert, 2001).

[³H]Mepyramine Binding Assay. Membranes expressing various H₁R constructs plus RGS proteins were thawed and sedimented by a 15-min centrifugation at 4°C and 15,000g. Membranes were resuspended in binding buffer (12.5 mM MgCl₂, 1 mM EDTA, and 75 mM Tris/HCl, pH 7.4). Tubes (total volume 500 µl) contained 20–25 µg of membrane protein. Incubations were conducted for 90 min at 25°C and shaking at 250 rpm. For H₁R saturation binding experiments, tubes contained 0.2 to 20 nM [³H]mepyramine (hH₁R, gpH₁R, and hH₁R-F153L/I433V) or 2 to 100 nM [³H]mepyramine (hH₁R-F153L and hH₁R-I433V). Nonspecific binding was routinely determined in the presence of 10 µM mepyramine (30). Nonspecific binding in the presence of saturating concentrations of compounds 1, 3, 12, 14, 15, 19, 20, 31, 35, and 36 was virtually identical to nonspecific binding in the presence of compound 30 (data not shown). Competition binding experiments were carried out in the presence of 2 nM [³H]mepyramine and unlabeled ligands at various concentrations. Bound [³H]mepyramine was separated from free [³H]mepyramine by filtration through GF/C filters, followed by three washes with 2 ml of binding buffer (4°C). Filter-bound radioactivity was determined by liquid scintillation counting.

Steady-State GTPase Activity Assay. Membranes expressing various H₁R constructs plus RGS proteins or H₂R-G_s fusion proteins were thawed, sedimented, and resuspended in 10 mM Tris/HCl, pH 7.4. Assay tubes contained Sf9 membranes (10 µg of protein/tube), 1.0 mM MgCl₂, 0.1 mM EDTA, 0.1 mM ATP, 100 nM GTP, 1 mM adenylyl imidodiphosphate, 5 mM creatine phosphate, 40 µg of creatine kinase, and 0.2% (w/v) bovine serum albumin in 50 mM Tris/HCl, pH 7.4, and H_xR ligands at various concentrations. Reaction mixtures (80 µl) were incubated for 3 min at 25°C before the addition of 20 µl of [-³²P]GTP (0.2–0.5 µCi/tube). Reactions were conducted for 20 min at 25°C. Reactions were terminated by the addition of 900 µl of slurry consisting of 5% (w/v) activated charcoal and 50 mM NaH₂PO₄, pH 2.0. Charcoal-quenched reaction mixtures were centrifuged for 15 min at room temperature at 15,000g. Seven hundred microliters of the supernatant fluid of reaction mixtures were removed, and ³²P_i was determined by liquid scintillation counting.

SDS-PAGE and Immunoblot Analysis. Membrane proteins were separated on SDS polyacrylamide gels containing 10% (w/v) acrylamide. Proteins were then transferred onto Immobilon-P transfer membranes (Millipore, Bedford, MA). Membranes were reacted with M1 antibody (1:1000). Immunoreactive bands were visualized by sheep anti-mouse IgG (1:1000) coupled to peroxidase, using o-dianisidine and H₂O₂ as substrates. Expression of RGS proteins was verified by immunoblot analysis with specific anti-RGS4 IgG and anti-GAIP IgG, as described (Houston et al., 2002).

Miscellaneous. Protein concentrations were determined using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). All analyses of experimental data were performed with the Prism 3.02 software (GraphPad-Prism, San Diego, CA). K_i and K_B values were calculated according to Cheng and Prusoff (1973). Statistical comparisons were performed with Student's t test.

	Results

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Immunological Detection of H₁R Constructs. The H₁R constructs analyzed in this study were all N-terminally tagged with the FLAG epitope, allowing immunological detection with the M1 monoclonal antibody (Houston et al., 2002). The predicted molecular mass of nonglycosylated hH₁R and gpH₁R is 56 kDa (Fukui et al., 1994; Traiffort et al., 1994). The FLAG epitope-tagged hH₁R expressed in Sf9 membranes migrated as diffuse 75-kDa doublet in SDS-PAGE (Figs. 4, A and B). Treatment of Sf9 cells with the inhibitor of N-glycosylation, tunicamycin (Seifert and Wenzel-Seifert, 2001), shifted the majority of the protein toward 70 kDa and rendered the lower band crisper. Migration of FLAG epitope-tagged gpH₁R in SDS-PAGE differed considerably from the migration of hH₁R. In membranes expressing gpH₁R, faint and diffuse bands in the 36- and 50-kDa regions were detected, and tunicamycin treatment had little effect on migration of gpH₁R in SDS-PAGE (Fig. 4A). Additionally, we detected intense and crisp bands of 16 and 30 kDa. Both hH₁R-F153L and hH₁R-I433V showed a broad ladder of diffuse bands ranging from 30 to 80 kDa, and there was a more intense doublet at 28 to 29 kDa (Fig. 4B). The hH₁R-F153L/I433V double mutant showed the predicted migration in SDS-PAGE, i.e., this mutant migrated as a 56 kDa band.

View larger version (45K): [in this window] [in a new window]   Fig. 4. Analysis of the expression of H1R constructs in Sf9 cell membranes. A, Sf9 cells were cultured in the absence (-) of tunicamycin (Tuna.) or in the presence (+) of tunicamycin (10 µg/ml). Sf9 cell membranes expressing hH1R or gpH1R were separated by SDS-PAGE on a gel that contained 10% (w/v) acrylamide. Membranes were probed with the anti-FLAG Ig (M1 antibody). Each membrane preparation was analyzed in two different amounts (25 and 50 µg of protein/lane, respectively). B, Sf9 cell membranes expressing hH1R-F153L, hH1R-I433V, hH1R-F153L/I433V, and hH1R were separated by SDS-PAGE on a gel that contained 10% (w/v) acrylamide. Membranes (75 µg of protein/lane each) were probed with the anti-FLAG Ig. Numbers on the left of the immunoblots in A and B indicate molecular masses of marker proteins. The horseradish peroxidase-reacted Immobilon P membranes of representative gels are shown. Similar results were obtained with three to six other membrane preparations of hH1R constructs. Expression levels of H1R constructs in terms of [3H]mepyramine saturation binding are given in Table 1.

Potencies and Efficacies of H₁R and H₂R Agonists at H₁R Constructs in the GTPase Assay. We studied three classes of H₁R agonists in the GTPase assay (Fig. 1). As a control we also studied the H₂R agonists amthamine (46) and dimaprit (47) (Hill et al., 1997). Table 2 and Fig. 5 summarize the data for hH₁R and gpH₁R coexpressed with RGS4 and GAIP since no significant differences were observed between the two RGS proteins (data not shown). Only histamine and the small histamine derivatives 2 and 3 were full hH₁R agonists, whereas all other modifications resulted in reductions of efficacy. Additionally, compounds 2 and 3 were less potent hH₁R agonists than histamine. We identified only two agonists that were more potent at hH₁R than histamine, i.e., the histaprodifens 19 and 20. The moderate increase in potency (1.8–2.7-fold) was accompanied by a significant decrease in efficacy, however. The introduction of a phenyl group (6) or particularly a benzyl group (5) at the position 2 of the imidazole ring substantially reduced agonist potency. Introduction of a halogen in the meta-position of the phenyl ring partially restored agonist potency in the order F < Cl < Br I (compare 6, 9, 12, 14, and 15). Other hydrogen-donating meta-substituents (Oe and CF₃) were also favorable (16 and 17), whereas a methyl group (7) and halogen substitutions in the ortho- or para-position of the phenyl ring (8 and 13) further reduced agonist potency. At hH₁R, histaprodifens 21 to 23 were less potent than histamine. The H₂R agonists 46 and 47 were essentially devoid of agonistic activity at the hH₁R (Table 2).

View larger version (14K): [in this window] [in a new window]   Fig. 5. Relations between the potencies of H1R agonists at hH1R and gpH1R expressed in Sf9 membranes in the GTPase assay. pEC50 values of agonists at hH1R and gpH1R were derived from EC50 values shown in Table 1 and analyzed by linear regression. Solid lines represent the actual correlations obtained. Dashed lines represent the 95% confidence intervals of the correlations. The straight dotted lines represent the theoretical correlations describing pharmacological identity between the H1R species isoforms. The theoretical curves have a slope of 1.00. A, correlation of the potencies of small agonists (1–4) at hH1R versus gpH1R. Slope, 0.74 ± 0.03; r2, 0.99; p < 0.001 (significant). B, correlation of potencies of 2-phenylhistamines (6–18) at hH1R versus gpH1R. Slope, 0.96 ± 0.07; r2, 0.95; p < 0.001 (significant). C, correlation of potencies of histaprodifens (19–23) at hH1R versus gpH1R. Slope, 0.54 ± 0.08; r2, 0.96; p < 0.001 (significant).

We did not observe significant differences in potency and efficacy of the small H₁R agonists 1 to 4 between hH₁R and gpH₁R (Table 2). This similarity between the H₁R isoforms is reflected by a linear correlation of the pEC₅₀ values of the small agonists at hH₁R and gpH₁R that is close to the theoretical correlation describing identity of H₁R species isoforms (Fig. 5A). When the effects of 2-phenylhistamines and histaprodifens were analyzed, however, significant differences between hH₁R and gpH₁R emerged. All compounds of these two classes were significantly more potent (3.2- to 9.9-fold) at gpH₁R than at hH₁R. The different interaction of 2-phenylhistamines and histaprodifens with hH₁R and gpH₁R is reflected by a linear correlation of the potencies of each series that is shifted toward the left relative to the theoretical correlation describing pharmacological identity of the GPCR species isoforms (Figs. 5, B and C). These linear correlations also show that the overall structure/activity relationships of those compounds are similar at both H₁R species isoforms. In addition to the higher potency, most 2-phenylhistamines (6, 8–12, 14–17) and 3 of 5 histaprodifens (20, 21, and 23) were significantly more efficacious at gpH₁R than at hH₁R. Finally, the small H₂R agonist dimaprit (47) showed only minimal agonistic effects at gpH₁R, but another small agonist, amthamine (46), was a weak partial gpH₁R agonist with significantly higher efficacy at gpH₁R than at hH₁R.

We failed to detect GTPase stimulation by histamine and compounds 3 and 12 in Sf9 membranes expressing hH₁R-F153L and hH₁R-I433V plus RGS proteins (data not shown). In contrast, histamine and compound 3 stimulated GTP hydrolysis in membranes expressing hH₁R-F153L/I433V as potently and efficiently as in membranes expressing hH₁R or gpH₁R. 2-Substituted histamines and histaprodifens tended to be more potent and efficacious at hH₁R-F153L/I433V than at hH₁R, but only the potency and efficacy of compound 12 were significantly increased.

Constitutive Activity of H₁Rs. hH₁R is constitutively active, and many first- and second-generation H₁R antagonists possess inverse agonistic activity (Bakker et al., 2001; Weiner et al., 2001). The extent of constitutive activity of hH₁R is dependent on the specific expression system, however. All first-generation H₁R antagonists (24–32), second-generation H₁R antagonists (41–45), and guanidines (33–39) examined exhibited only small inverse agonistic activity at hH₁R expressed in Sf9 membranes, i.e., the inhibitory effects of compounds amounted to 5 to 15% of the stimulatory effect of histamine (data not shown). There were no significant differences in the inverse agonist effects of H₁R antagonists at hH₁R and gpH₁R. These data indicate that the constitutive activity of the two GPCR isoforms is similar.

Potencies of H₁R Antagonists at H₁R Constructs in the GTPase Assay. In agreement with the [³H]mepyramine binding studies (Table 1), mepyramine (30) was about 2-fold less potent at inhibiting histamine-stimulated GTP hydrolysis in membranes expressing hH₁R than in membranes expressing gpH₁R (Table 3). A similar difference in potency was observed for two other first-generation H₁R antagonists, triprolidine (31) and (+)-chlorpheniramine (32), whereas the other first-generation antagonists studied [24–28, dimethindene enantiomers (R)-(-)-29 and (S)-(+)-29] did not exhibit significantly different potencies at hH₁R and gpH₁R. (R)-(-)-Dimethindene was 30- to 40-fold more potent than (S)-(+)-dimethindene. The stereoselectivity of recombinant H₁Rs for dimethindene enantiomers is in accordance with data for the H₁R expressed in the guinea pig ileum (Pfaff et al., 1995). Among the second-generation H₁R antagonists 41 to 45, no significant differences in potency between hH₁R and gpH₁R emerged.

Arpromidine (35) and arpromidine-derived guanidines (33, 34, and 36–38) are not only very potent H₂R agonists but also moderately potent H₁R antagonists (Buschauer, 1989). The H₁R-antagonistic properties of guanidines are explained by the structural similarity of compounds 33 to 38 and 30 to 32 (Fig. 2). Guanidines 33 to 38 inhibited histamine-stimulated GTP hydrolysis in Sf9 membranes expressing gpH₁R, with K_B values of 50 to 150 nM (Table 3). Guanidines 33 to 38 were all significantly more potent antagonists at gpH₁R than at hH₁R and showed greater gpH₁R/hH₁R selectivity than compounds 30 to 32. The difference in potency was most pronounced (9-fold) for compound 36 that is distinguished from the other guanidines by a para-Cl in the phenyl moiety (Fig. 2). In contrast, guanidine 39 that possesses a trichlorinated phenyl ring and a thiazole instead of a pyridyl ring (Fig. 2) did not discriminate between hH₁R and gpH₁R. Modifications of the substituents in guanidines 33–39 had a considerably larger impact on antagonist potency at gpH₁R (7-fold) than at hH₁R (2-fold).

In the 2-phenylhistamine derivative 40, the free amino group of histamine was integrated into a piperidine ring (Fig. 2). This modification is predicted to interfere with the binding of the basic nitrogen to Asp-107 (hH₁R) (Ohta et al., 1994). In fact, compound 40 exhibited 6.5- to 8-fold reduced apparent affinity compared with its parent compound (17) (Fig. 1) at hH₁R and gpH₁R (Tables 2 and 3). Moreover, introduction of the piperidine ring into 17 conferred antagonistic properties to compound 40 (Table 3). This was also confirmed in the guinea pig ileum assay (K_B of compound 40, 400 nM). Compound 40 was a severalfold more potent antagonist at gpH₁R than at hH₁R.

In agreement with the binding data (Table 1), mepyramine (30) was similarly potent at inhibiting histamine-stimulated GTP hydrolysis in Sf9 membranes expressing hH₁R and hH₁R-F153L/I433V (Table 3). The double mutation exhibited inconsistent effects on the potencies of guanidines 33 and 35 to 38 as well as of the 2-phenylhistamine derivative 40. Specifically, the F153L/I433V mutation increased the potency of 36 1.5-fold, had no effect on the potency of 35, and 37 and decreased the potency of compounds 33, 38, and 40 by up to 2-fold.

Affinities of H₁R Agonists and Antagonists at H₁R Constructs in the [³H]Mepyramine Binding Assay. Histamine and 2-(3-chlorophenyl)histamine (12) inhibited [³H]mepyramine binding in Sf9 membranes expressing hH₁R or gpH₁R plus RGS proteins according to a monophasic function that was not shifted to the right by guanosine 5'-O-(3-thiotriphosphate) (10 µM) (data not shown). Thus, we could not detect high-affinity agonist binding. These data were expected since there is a paucity of endogenous G-proteins relative to the expressed mammalian GPCRs in Sf9 membranes (Seifert et al., 1998; Houston et al., 2002). Accordingly, the agonist-affinities determined in the [³H]mepyramine competition binding studies reflect the agonist affinities of H₁Rs in the G-protein-uncoupled state. In fact, the K_i values of agonists 1, 3, 12, 14, 15, 19, and 20 at hH₁R and gpH₁R were all higher than the corresponding EC₅₀ values in the GTPase assay (Tables 2 and 4). The K_i value of histamine at hH₁R was 2.3-fold lower than the K_i value of histamine at gpH₁R. Since the amino acids in the histamine-binding H₁R domains are identical in both isoforms (Fig. 3), this difference could point to a better fit of histamine into the G_q-uncoupled hH₁R compared with G_q-uncoupled gpH₁R.

To account for the difference in histamine affinity of H₁R species isoforms, we focused on the comparison of the relative affinities of synthetic agonists at hH₁R and gpH₁R. The relative affinity of the small agonist 3 was similar at hH₁R and gpH₁R, whereas the relative affinities of the 2-phenylhistamines 12, 14, and 15 and of the histaprodifens 19 and 20 were 3- to 7-fold higher at gpH₁R than at hH₁R. These differences fit to the differences in relative agonist potencies observed in the GTPase assay (Table 2). In agreement with the GTPase studies (Table 3), the H₁R antagonists triprolidine (31), arpromidine (35), and BU-E 47 (36) also all exhibited significantly higher binding affinities at gpH₁R than at hH₁R (Table 4).

We also studied the impact of the F153L/I433V mutation in hH₁R on ligand-affinities. The double mutation significantly decreased the affinity of hH₁R for histamine and 2-(2-thiazolyl)ethanamine (3) (Table 4). Similar data were obtained for the comparison of hH₁R and gpH₁R. Additionally, in membranes expressing hH₁R-F153L/I433V, the relative affinities of 2-phenylhistamines and histaprodifens were increased relative to hH₁R, but with the exception of methyl-histaprodifen (19), those changes were not as marked as for the comparison of hH₁R and gpH₁R. The affinities of triprolidine (31), arpromidine (35), and guanidine 36 at hH₁R and hH₁R-F153L/I433V were similar.

Potencies and Efficacies of H₁R Agonists at hH₂R and gpH₂R in the GTPase Assay. The question arose whether H₁R agonists, originally designed for gpH₁R in comparison to gpH₂R, interact differentially with the corresponding human H_xRs. To address this question, we analyzed the effects of H₁R agonists on GTP hydrolysis in Sf9 membranes expressing H₂R-G_sS fusion proteins. We examined all H₁R agonists shown in Fig. 1 and listed in Table 2 (1–23) but included only those compounds into Table 5 that actually exhibited agonistic activity at H₂Rs. To account for the fact that the potency of histamine in the GTPase assay in membranes expressing H₁Rs and H₂Rs differs by almost 10-fold (Tables 2 and 5) (Kelley et al., 2001), we focused on the comparison of relative potencies of H₁R agonists.

View this table: [in this window] [in a new window]   TABLE 5 Potencies and efficacies of H1R agonists in the GTPase assay in Sf9 membranes expressing hH2R-GsS and gpH2R-GsS Steady-state GTPase activity in Sf9 membranes expressing H2R-Gsa fusion proteins was determined as described under Materials and Methods. Reaction mixtures contained H1R ligands at concentrations from 10 nM to 1 mM as appropriate to generate saturated concentration/response curves. Data were analyzed by nonlinear regression and were best fit to sigmoid concentration/response curves. Typical basal GTPase activities ranged between 1.0 to 2.0 pmol/mg/min, and the maximum stimulatory effect of histamine (100 µM) amounted to 200 to 300% above basal. The efficacy (Emax) of histamine was determined by nonlinear regression and was set 1.00 The Emax values of other H1R agonists were referred to this value. Data shown are the means ± S.D. of three to 4 experiments performed in duplicates each. The relative potency (Rel. Pot.) of histamine was set 100, and the potencies of other agonists were referred to this value. We also calculated the ratio of the relative potencies of H1R agonists at hH1R (taken from Table 2) and hH2R-GsS (Rel. Pot. Rat. hH1R/hH2R) and the ratio of the relative potencies of H1R agonists at gpH1R (taken from Table 2) and gpH2R-GsS (Rel. Pot. Rat. gpH1R/gpH2R). Additionally, we calculated the ratio of the EC50 values of H1R agonists for hH2R-GsS and gpH2R-GsS (Pot. Rat. gpH2R/hH2R). Data for compounds 1 and 4 were taken from Kelley et al. (2001). Compounds 11, 13 to 15, 17 to 21, and 23 at concentrations from 10 µM to 1 mM were devoid of any stimulatory effect on GTPase activity in membranes expressing hH2R-GsS and gpH2R-GsS and, therefore, not shown in the table.

2-Methylhistamine (2) and 2-(2-thiazolyl)ethanamine (3) were strong partial agonists at gpH₂R with moderate (2.3- to 5-fold) gpH₁R/gpH₂R selectivity. The introduction of a (substituted) phenyl group at position 2 of the imidazole ring greatly reduced the efficacy of H₁R agonists at gpH₂R and further increased gpH₁R/gpH₂R selectivity in terms of potency. Several 2-phenylhistamines (11, 13–15, 17, and 18) and histaprodifens 19 to 21, and 23 were devoid of agonistic activity at gpH₂R-G_sS.

The analysis of histaprodifens at gpH₂R-G_sS revealed the existence of a strong partial H₁R agonist/moderate partial H₂R agonist, N-(imidazolylethyl)histaprodifen (22) (Tables 2 and 5). The H₂-agonistic activity of this compound can be explained by its structural similarity with guanidines 33 to 38 (Figs. 1 and 2) that are potent H₂R agonists (Buschauer, 1989; Kelley et al., 2001).

Although histamine was similarly potent at stimulating GTP hydrolysis in Sf9 membranes expressing hH₂R-G_sS and gpH₂R-G_sS, 2-methylhistamine (2) and 2-(2-thiazolyl)ethanamine (3) were significantly less potent agonists at hH₂R-G_sS than at gpH₂R-G_sS and showed greater hH₁R/hH₂R selectivity (8.4- to 11.2-fold) than gpH₁R/gpH₂R selectivity (2.3- to 5-fold). If one considers the absolute EC₅₀ values of compound 3 for GTPase activation in membranes expressing hH₁R and hH₂R-G_sS, the selectivity for hH₁R becomes even more striking (75-versus 23-fold for gpH_xRs). In contrast to compound 3, another small H₁R agonist, betahistine (4), exhibited considerably higher gpH₁R/gpH₂R selectivity (10-fold) than hH₁R/hH₂R selectivity (3.5-fold). Similar to the data obtained for gpH₂R, several 2-phenylhistamines (11, 13–15, 17, and 18) and histaprodifens (19–21 and 23) were devoid of agonistic activity at hH₂R-G_sS. As was the case for gpH_xRs, N-(imidazolylethyl)-histaprodifen (22) was a strong partial hH₁R agonist/moderate partial hH₂R agonist. There were no significant differences in the interaction of histaprodifens at hH₂R-G_sS and gpH₂R-G_sS. Finally, the efficacies of the 2-phenylhistamines 6 to 9 were significantly lower at hH₂R-G_sS than at gpH₂R-G_sS (Table 5) and therefore in the same order as observed for hH₁R and gpH₁R (Table 2).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Pharmacological Differences between hH₁R and gpH₁R. hH₁R is an important drug target for treatment of allergic diseases (second-generation H₁R antagonists) and sedation (first-generation H₁R antagonists) (Hill et al., 1997). Preliminary data indicate that pharmacological differences between H₁R species isoforms exist (Chang et al., 1979; Seifert et al., 1994; Elz et al., 2000), but a systematic analysis of this topic has not yet been conducted. Therefore, we studied recombinant hH₁R and gpH₁R with 23 H₁R agonists (1–23) (Fig. 1), 22 H₁R antagonists (24–45) (Fig. 2), and two H₂R agonists (46 and 47) under identical experimental conditions, using the GTPase assay (Fig. 5, Tables 2 and 3) and [³H]mepyramine binding assay (Tables 1 and 4) as read-out.

There were no significant differences between hH₁R and gpH₁R with respect to the potencies and efficacies of small agonists (1-4) in the GTPase assay (Fig. 5; Table 2). With respect to bulkier ligands, however, we found significant differences between hH₁R and gpH₁R. Specifically, H₁R agonists of the 2-phenylhistamine class (6–17) and histaprodifen class (19–23) were generally more potent and efficacious in the GTPase assay in membranes expressing gpH₁R than in membranes expressing hH₁R (Fig. 5; Table 2). Additionally, in the binding assay, 2-phenylhistamines and histaprodifens exhibited higher relative affinities for gpH₁R than for hH₁R (Table 4). The differential interaction of 2-phenylhistamine derivatives with gpH₁R and hH₁R is independent of the agonist or antagonist properties of compounds (compare 17 and 40; Tables 2 and 3). High constitutive GPCR activity results in high agonist potency and efficacy (Kenakin, 1996; Seifert and Wenzel-Seifert, 2002), but we did not find differences in constitutive activity between hH₁R and gpH₁R studying inverse agonists. Finally, several first-generation H₁R antagonists (30–32) and particularly arpromidine-type H₁R antagonists (33–38) showed higher affinities for gpH₁R than for hH₁R. Our data concerning the affinity of ([³H])mepyramine for hH₁R and gpH₁R (Tables 1 and 3) fit very well to previously published data on H₁R species isoforms expressed in native brain (Chang et al., 1979). Collectively, our data suggest that the ligand-binding site of gpH₁R exhibits a higher conformational flexibility than the ligand-binding site of hH₁R, allowing bulky compounds like 2-phenylhistamines, histaprodifens, mepyramine-type antagonists, and guanidines to dock more efficiently into gpH₁R than into hH₁R.

Most of the previous H₁R antagonist development had been conducted with guinea pig models (Hill et al., 1997). Thus, from a therapeutic standpoint, it is fortunate that there are no or only small differences between hH₁R and gpH₁R with respect to commonly used first-generation H₁R antagonists (e.g., 24–28, 30, and 32) and second-generation antagonists (41–45). Nevertheless, with regard to the design of H₁R agonists and guanidine-type H₁R antagonists, which are currently used only as experimental tools (Zingel et al., 1995; Hill et al., 1997), the H₁R species isoform is of much greater relevance.

Differences in Electrophoretic Mobility between hH₁R and gpH₁R. A previous study showed that H₁R isoforms expressed in brain from various species exhibit different migration in SDS-PAGE (Ruat and Schwartz, 1989). These data prompted us to study the electrophoretic mobility of recombinant FLAG epitope-tagged recombinant hH₁R and gpH₁R (Fig. 4). In agreement with the data concerning native H₁R species isoforms, recombinant H₁R species isoforms showed different migration in SDS-PAGE. hH₁R exhibited a moderately higher molecular mass (76 kDa) than predicted (56 kDa) (Fukui et al., 1994). hH₁R migrated as mixture of N-glycosylated and nonglycosylated protein, as assessed by the effect of the inhibitor of N-glycosylation, tunicamycin (Seifert and Wenzel-Seifert, 2001). Recombinant gpH₁R exhibited very different migration in SDS-PAGE than hH₁R, i.e., we detected faint diffuse 36- and 50-kDa bands and intense crisp 16- and 30-kDa bands in Sf9 membranes expressing gpH₁R. In contrast to the results obtained with hH₁R, tunicamycin had no effect on migration of gpH₁R, pointing to different types of glycosylation in the two H₁R species isoforms. Currently, we do not know the identity of the multiple bands in Sf9 membranes expressing gpH₁R, but atypical migration of GPCRs in SDS-PAGE has been repeatedly observed (Grünewald et al., 1996; Kelley et al., 2001; Seifert and Wenzel-Seifert, 2001). Because even complex supramolecular structures such as GPCR dimers are preserved in SDS-PAGE (Fukushima et al., 1997; Hebert and Bouvier, 1998; Kelley et al., 2001), it is possible that the different electrophoretic mobilities of hH₁R and gpH₁R reflect different GPCR conformations. The different GPCR conformations may be associated with the specific pharmacological properties of H₁R species isoforms.

Molecular Basis for the Pharmacological Differences between hH₁R and gpH₁R. Site-directed mutagenesis was successful at identifying the molecular basis for pharmacological differences between species isoforms of H₂R and H₃R (Ligneau et al., 2000; Kelley et al., 2001). We wished to apply the same strategy to H₁R species isoforms. The pharmacological data discussed above indicate that the ligand-binding pocket of gpH₁R is more flexible than the binding pocket of hH₁R. Thus, gpH₁R may possess smaller amino acid substitutions in the ligand-binding domain than hH₁R so that bulkier structures are accommodated more easily in gpH₁R than in hH₁R. In fact, the amino acid substitutions at positions 153 (TM IV) and 433 in hH₁R (TM VI) are bulkier than the corresponding amino acid substitutions in gpH₁R (PheLeu exchange in TM IV and IleVal exchange in TM VI, respectively). Nevertheless, the PheLeu exchange in TM IV and the IleVal exchange in TM VI only partially explain the differences in agonist-pharmacology between hH₁R and gpH₁R (Tables 2 and 4). Moreover, with respect to the differences in antagonist-pharmacology, the PheLeu- and IleVal exchanges between hH₁R and gpH₁R are irrelevant (Tables 3 and 4). Thus, additional mutagenesis studies targeting the top portions of TM II and TM VII are required to elucidate the molecular basis for the pharmacological differences between hH₁R and gpH₁R (Fig. 3).

Although our mutagenesis studies were disappointing in terms of elucidating the molecular basis for the pharmacological differences between hH₁R and gpH₁R, our studies revealed an unexpected role of Phe-153 and Ile-433 in H₁R expression and folding. Specifically, Phe-153Leu-153- or Ile-433Val-433 exchange in hH₁R (hH₁RgpH₁R) resulted in poor receptor expression, low [³H]mepyramine affinity, and functional inactivity (Table 1). Moreover, the mutations grossly altered the electrophoretic mobility of hH₁R (Fig. 4). The double mutation rescued the single mutants in terms of function (Tables 1, 2, 3, 4), and it also changed electrophoretic mobility (Fig. 4). These data suggest that the couples Phe-153/Ile-433 or Leu-153/Val-433 are required for a functionally active H₁R. Thus, even conservative amino acid substitutions in TM regions can have profound effects on antagonist affinity, expression, and folding of a GPCR.

Comparison of the Effects of H₁R Agonists at Recombinant and Native gpH₁R. Historically, the guinea pig ileum has been the standard system for the design of H₁R ligands (Zingel et al., 1995; Hill et al., 1997). Therefore, it is important to compare the intact organ data with the results regarding recombinant H₁R. Although many highly potent H₂R and H₃R agonists (i.e., ligands 50- to 150-fold more potent than histamine) were developed (Hill et al., 1997), the design of potent H₁R agonists has been a much more difficult task. In fact, the most potent 2-phenylhistamine, 2-(3-trifluoromethylphenyl)histamine (17), is only 1.3-fold more potent, and methylhistaprodifen (20) just 3.5-fold more potent, than histamine in the guinea pig ileum (Leschke et al., 1995; Zingel et al., 1995; Elz et al., 2000) (Table 2).

The expression level of H₁R in the guinea pig ileum is much lower than in the Sf9 cell expression system (Table 1) (Hill et al., 1997). If there had been differences in receptor reserves between the two systems, we would have expected higher agonist efficacies in the recombinant system than in the native system (Hoyer and Boddeke, 1993; Kenakin, 1996). The opposite was the case, however (Table 2) (Leschke et al., 1995; Zingel et al., 1995; Elz et al., 2000). Thus, we can rule out differences in receptor reserves accounting for the pharmacological differences between the two systems.

All agonists studied with the exception of 1, 3, 22, and 23 were more potent at the recombinant gpH₁R than at the native gpH₁R (Table 2). The increase in potency at the recombinant gpH₁R ranged from 2-fold to almost 20-fold and was most pronounced for the 2-phenylhistamines 7, 11, and 13. Several explanations that are not mutually exclusive could account for the potency differences in the two systems. First, there may be substantial penetration barriers for certain agonists to reach the tunica muscularis of the ileum. Second, compounds may accumulate in certain irrelevant cells, i.e., epithelial cells, and/or, third, they may be subject to degradation. These pharmacokinetic factors are very unlikely to be of relevance when assessing the effects of ligands in membrane fragments of insect cells. Fourth, it is possible that differences in gpH₁R glycosylation in insect cells versus native tissue contribute to the pharmacological differences in the two systems. Indeed, changes in glycosylation of H₁R have already been shown to alter the pharmacological properties of the GPCR (Mitsuhashi and Payan, 1989). Fifth, we studied coupling of H₁Rs to insect cell G_q-proteins (Houston et al., 2002), and the specific type of G_q-protein may have an impact on the pharmacological properties of gpH₁R (Wenzel-Seifert and Seifert, 2000). In contrast to the above-discussed data, the high potency of compounds 22 and 23 in the guinea pig ileum does not fit to the results obtained with recombinant gpH₁R. Additional studies with 22, 23, and closely related new compounds must be performed to clarify this discrepancy.

Collectively, previous studies on the guinea pig ileum resulted in considerably lower potencies of most H₁R agonists than in the recombinant system. Although the high potency of H₂R-and H₃R agonists has not yet been achieved for H₁R agonists, our present study shows that gpH₁R agonists with up to 12-fold higher potency than histamine exist, provided that the GPCR is analyzed in the GTPase assay using membranes. Thus, future studies on the design of H₁R agonists should be complemented with the recombinant system described herein.

Species Differences in Pharmacological Properties of H_xRs. H₂R, H₃R, and H₄R all exhibit species differences in their pharmacological properties (Ligneau et al., 2000; Lovenberg et al., 2000; Kelley et al., 2001; Liu et al., 2001). Thus, we were not too surprised to uncover differences in the pharmacological properties of H₁R species isoforms. The species differences in pharmacological properties of H_xRs extend into H_xR subtype-selectivity of compounds. There are numerous efficacious H₁R agonists of the 2-phenylhistamine and histaprodifen class with high gpH₁R/gpH₂R selectivity (Tables 2 and 5) (Leschke et al., 1995; Zingel et al., 1995; Elz et al., 2000). For the analysis of hH₁R, however, one has to consider the fact that 2-phenylhistamines and histaprodifens possess substantially lower efficacies than histamine (Table 2). Unexpectedly, 2-(2-thiazolyl)ethanamine (3), a small agonist with full efficacy at hH₁R, exhibited a larger hH₁R/hH₂R-than gpH₁R/gpH₂R-selectivity (Tables 2 and 5). Thus, for the analysis of hH₁R with a selective hH₁R agonist, compound 3 may be the ligand of choice. These findings emphasize the importance to study hH_xR isoforms for the development of hH_xR ligands. Future studies will have to answer the question whether the species differences in pharmacological properties of H_xRs reflect species-specific adaptations to as yet unidentified endogenous and/or exogenous H_xR ligands.

Although H₁Rs and H₂Rs are structurally quite distinct from each other (only 40% homology) (Traiffort et al., 1994; Hill et al., 1997), there is a common aspect in the pharmacological properties of these GPCRs, i.e., the preferential interaction of bulky agonists with gpH_xRs relative to hH_xRs. Most notably, arpromidine-derived guanidines represent a class of ligands that exhibit higher affinities for gpH₁R and gpH₂R relative to hH_xRs (Tables 3 and 4) (Kelley et al., 2001). Those differences may indicate that gpH_xRs in general possess a higher conformational flexibility than hH_xRs.

	Acknowledgements

 
We express gratitude to Dr. G. Lambrecht (Department of Pharmacology, University of Frankfurt/Main, Germany) for providing dimethindene enantiomers, to Dr. H. Fukui (Department of Pharmacology, University of Tokushima, Japan) for providing cDNAs for hH₁R and gpH₁R, and Dr. E. Ross (Department of Pharmacology, University of Southwestern Medical Center, Dallas, TX) for donating baculoviruses encoding RGS proteins. We also thank Dr. F. Schalkhausser (Institute of Pharmacy, University of Regensburg, Germany) for the synthesis of guanidine 39, Dr. K. Kramer (Institute of Pharmacy, Free University of Berlin, Germany) for the synthesis of compounds 2, 3, 22, 23, and 40, Dr. W. Toegel (Institute of Pharmacy, Free University of Berlin, Germany) for the preparation of compound 18, and C. Houston (Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS) for help with the cell culture. Thanks are also due to the reviewers for their constructive critique.

	Footnotes

 
This work was supported by the National Institutes of Health COBRE Award 1 P20 RR15563 and matching support from the State of Kansas and the University of Kansas (R.S.), a grant from the Army Research Office (DAAD 19-00-1-006) (R.S.), grants of the Deutsche Forschungsgemeinschaft (W.S. and A.B.), Fonds der Chemischen Industrie (T.B., W.S., A.B., and S.E.), the Bundesministerium für Bildung und Forschung (T.B.), and the Deutscher Akademischer Austauschdienst within the International Quality Network "Medicinal Chemistry" (S.E. and A.B.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.049619.

ABBREVIATIONS: H_xR, histamine H₁-, H₂-, H₃-, or H₄-receptor; h, human; gp, guinea pig; GPCR, G-protein-coupled receptor; TM, transmembrane domain; RGS protein, regulator of G-protein signaling; GAIP, G-interacting protein; PAGE, polyacrylamide gel electrophoresis; gpH₂R-G_sS, fusion protein of the guinea pig histamine H₂-receptor and the short splice variant of G_s;hH₁R, human histamine H₁-receptor; hH₂R, human histamine H₂-receptor; hH₂R-G_sS, fusion protein of the human histamine H₂-receptor and the short splice variant of G_s; hH₁R-F153L, human histamine H₁-receptor bearing a PheLeu exchange at position 153; hH₁R-I433V, human histamine H₁-receptor bearing an IleVal exchange at position 433; hH₁R-F153L/I433V, human histamine H₁-receptor bearing a PheLeu exchange at position 153 and an IleVal exchange at position 433.

Address correspondence to: Dr. Roland Seifert, Department of Pharmacology and Toxicology, The University of Kansas, Malott Hall, Room 5064, 1251 Wescoe Hall Drive, Lawrence, KS 66045-7582. E-mail: rseifert{at}ku.edu

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