Introduction

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The G_q/11-coupled histamine H₁ receptor (Gutowski et al., 1991; Leopoldt et al., 1997) is expressed in a variety of cells and is believed to mediate many of the histamine-induced symptoms of allergic reactions by coupling to different signaling pathways. Consequently, during the past 20 years, the H₁-receptor antagonists have become one of the most prescribed drug families in Western countries (Woosley, 1996) to relieve the symptoms of allergic reactions.

NF-B is a ubiquitous transcription factor that can be activated by many stimuli and is considered to play an important role in inflammatory processes (Barnes et al., 1998). The physiological roles, including lymphocyte activation and protection from apoptosis, for the heterogeneous family of NF-B proteins and activators thereof have recently gained much interest (Newton and Decicco, 1999). Elevated levels of activated NF-B are found in persons with asthma (Hart et al., 1998), and NF-B is therefore thought to play a pivotal role in asthma (Barnes and Adcock, 1997). Elucidation of activation mechanisms of NF-B proteins is expected to lead to significant advances in our understanding of a variety of inflammatory disorders, including asthma and allergy, as well as their treatment. Much progress has already been made in unraveling pathways activating NF-B via receptor tyrosine kinases. However, several additional activation pathways have recently been identified, many of which can be activated by G-protein-coupled receptors (GPCRs; Shahrestanifar et al., 1999; Xie et al., 2000; Casarosa et al., 2001). An increasing number of GPCRs, including histamine H₁ receptors (Aoki et al., 1998; Hu et al., 1999), have been shown to activate NF-B. However, little is known about the signal-transduction pathways leading to GPCR-mediated NF-B activation.

We have shown in a previous study constitutive activation of phospholipase C (PLC) by the human histamine H₁ receptor (Bakker et al., 2000). Moreover, we could show that the agonist-independent accumulation of inositol phosphates is selectively inhibited by several therapeutics formerly known as H₁ antagonists, which has led to the reclassification of various H₁ antagonists as inverse H₁ agonists. In this report, we show that H₁ agonists induce NF-B activation via the H₁ receptor through activation of G_q/11-proteins. Furthermore, in H₁ receptor-expressing cells, we observed constitutive activation of NF-B via a pathway that involves the liberation of G-subunits that is independent of PLC activation, indicating that constitutive GPCR signaling may not represent the same signaling mechanisms that are deployed in agonist-induced GPCR signaling. All tested H₁ antagonists, including those that are used clinically to relieve symptoms of allergy, inhibited the constitutive activation of NF-B, which means that these drugs act as inverse H₁ agonists. The observed inhibition of the constitutive NF-B activation by inverse H₁ agonists may therefore represent a new mechanism of action of this important class of antiallergic drugs.

	Experimental Procedures

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Materials. pNF-B-Luc was obtained from Stratagene (La Jolla, CA). Doxepine hydrochloride, mepyramine (pyrilamine maleate), (S)-(+)--fluoromethylhistidine hydrochloride, and tripelennamine hydrochloride were obtained from Sigma/RBI (Natick, MA). ATP disodium salt, bovine serum albumin, chloroquine diphosphate, cholera toxin, DEAE-dextran (chloride form), histamine dihydrochloride, pertussis toxin, phorbol 12-myristate 13-acetate, polyethyleneimine, triprolidine hydrochloride, and Tween-20 were purchased from Sigma Chemical (St. Louis, MO). D-Luciferin was obtained from Duchefa Biochemie BV (Haarlem, The Netherlands), glycerol from Sigma-Aldrich Laborchemikalien (Seelze, Germany), and Triton X-100 from Fluka (Buchs, Switzerland). Cell culture media, penicillin, and streptomycin were obtained from Invitrogen (Merelbeke, Belgium). Fetal calf serum (FCS) was obtained from Integro BV (Dieren, The Netherlands), dialyzed fetal calf serum was obtained from HyClone Laboratories (Logan, UT). [³H]Mepyramine (30 Ci/mmol), myo-[2-³H]inositol (17 Ci/mmol), and the ECL reagents were from Amersham Pharmacia Biotech UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Mouse anti-G_t-1-subunit (bovine) was obtained from Calbiochem (San Diego, CA), goat anti-mouse IgG (H+L)-horseradish peroxidase conjugate and 30% acrylamide mix from Bio-Rad (Hercules, CA), and the protein molecular mass marker [7708S, prestained protein marker, broad range (6-175 kDa); Beverly, MA]. Clobenpropit, 4-methyldiphenhydramine, and 4,4-dimethyldiphenhydramine were taken from our own stock.

Cell Culture and Transfection. COS-7 African green monkey kidney cells were maintained at 37°C in a humidified 5% CO₂/95% air atmosphere in either Dulbecco's modified Eagle's medium containing 2 mM L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 5% (v/v) FCS or in Opti-MEM I medium with GlutaMAX I (both from Invitrogen) containing 50 IU/ml penicillin, 50 µg/ml streptomycin, and 0.5% (v/v) dialyzed FCS. COS-7 cells were transiently transfected using the DEAE-dextran method. The total amount of DNA transfected was maintained constant by addition of either pcDEF₃ or pcDNA₃. In coexpression experiments with G₁₁ or G₁₁Q²⁰⁹L, the H₁ receptor-expression level was diminished by 10, 20, or 40%, respectively, whereas coexpression of G_t did not affect H₁ receptor expression, as monitored by [³H]mepyramine binding experiments.

[³H]Inositol Phosphate Formation. Cells were seeded in 24-well plates and 24 h after transfection labeled overnight in inositol-free culture medium supplemented with 1 µCi/ml myo-[2-³H]inositol. Subsequently, the medium was aspirated and cells were incubated with drugs for 1 h at 37°C in Dulbecco's modified Eagle's medium containing 25 mM HEPES, pH 7.4, and 20 mM LiCl. Incubations were stopped by aspiration of the culture medium and the addition of cold 10 mM formic acid. After 90 min of incubation at 4°C, [³H]inositol phosphates were isolated by anion exchange chromatography and counted by liquid scintillation.

Reporter-Gene Assay. Cells transiently cotransfected with pNFB-Luc (125 µg/1 × 10⁷ cells) and either pcDEF₃ or pcDEF₃hH₁ (25 µg/1 · 10⁷ cells) were seeded in 96-well blackplates (Costar, Cambridge, MA) in serum-free culture medium and incubated with drugs. After 48 h, cells were assayed for luminescence by aspiration of the medium and the addition of 25 µL/well luciferase assay reagent [0.83 mM ATP, 0.83 mM D-luciferin, 18.7 mM MgCl₂, 0.78 µM Na₂H₂P₂O₇, 38.9 mM Tris, pH 7.8, 0.39% (v/v) glycerol, 0.03% (v/v) Triton X-100, and 2.6 µM dithiothreitol]. After 30 min, luminescence was measured for 3 s/well in a TopCount (Packard Instrument Co., Meriden, CT) or a Victor² (PerkinElmer Wallac, Gaithersburg, MD).

Western Blot Analysis. To assay the expression of G_t, transfected COS-7 cells were washed twice with cold phosphate-buffered saline (PBS) and lysed with lysis buffer (PBS containing 1% Nonidet P40, 1 mM phenylmethylsulfonyl fluoride, 0.1% SDS, 0.5% sodiumdeoxycholate, 2 µg/ml aprotinin, and 2 µg/ml leupeptin). Insoluble material was pelleted by centrifugation, and supernatants were removed. Samples were prepared for electrophoresis by mixing 25-µg cell protein extracts with loading buffer [350 mM Tris-HCL, pH 6.8, 30% glycerol (v/v), 10% SDS (w/v), 600 mM dithiothreitol, and 0.01% bromphenol blue] and denatured (5 min, 100°C), followed by centrifugation for 3 min at 15,000g. Proteins were separated by SDS-polyacrylamide (12%) electrophoresis according to Laemmli (1970) and analyzed by Western blotting. Western blotting was carried out as described (Navon and Fung, 1988). Briefly, the gel was blotted onto a nitrocellulose filter, after which the washed blot (1 h at room temperature in PBS-T [PBS containing 0.1% Tween-20, 3% dry milk (w/v)]) was subsequently incubated overnight at 4°C with the mouse anti-G_t-1-subunit in PBS-T (1:2000); after washing the blot with PBS containing 0.1% Tween-20, it was incubated for 1 h with the goat anti-mouse IgG (H+L)-horseradish peroxidase conjugate (1:7500) at room temperature. Protein bands were detected by film exposure using ECL chemoluminescence and subsequently quantified using Image Quant (version 1.1; Molecular Dynamics, Sunnyvale, CA).

Histamine H₁ Receptor Binding Studies. The transfected COS-7 cells used for radioligand binding studies were harvested after 48 h and homogenized in ice-cold H₁-binding buffer. The COS-7 cell₂ homogenates were incubated for 30 min at 25°C in 50 mM Na₂/K-phosphate buffer, pH 7.4, in 400 µL with 1 nM [³H]mepyramine. The nonspecific binding was determined in the presence of 1 µM mianserin. The incubations were stopped by rapid dilution with 3 ml of ice-cold 50 mM Na₂/K-phosphate buffer, pH 7.4. The bound radioactivity was separated by filtration through Whatman GF/C filters that had been treated with 0.3% polyethyleneimine. Filters were washed twice with 3 ml of buffer, and radioactivity retained on the filters was measured by liquid scintillation counting. Binding data were evaluated by a nonlinear, least-squares curve-fitting procedure using GraphPad Prism (GraphPad Software, Inc., San Diego, CA).

Analytical Methods. Proteins concentrations were determined according to Bradford (1976), using bovine serum albumin as a standard. All data shown are expressed as means ± S.E.M., statistical analyses were carried out by Student's t test. p values < 0.05 were considered to indicate a significant difference (*, p < 0.05; **, p < 0.01; and ***, p < 0.001).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

NF-B Activation by the Histamine H₁ Receptor. In COS-7 cells expressing the H₁ receptor at a density of 3.2 ± 0.4 pmol/mg of protein (n = 6), histamine stimulated NF-B activation 3.6 ± 0.3-fold over basal with a pEC₅₀ value of 6.8 ± 0.1 (n = 37; Table 1). Histamine did not increase NF-B activation in mock-transfected COS-7 cells (n = 3). Also 2-[3-(fluoromethyl)phenyl]histamine, an H₁ selective agonist (Leschke et al., 1995) with a relatively high affinity (Table 1), increased InsP₃ accumulation as well as NF-B-driven luciferase-expression in H₁-expressing cells (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 The pKi and pEC50 values of the tested agonists for the human H1 receptor and their intrinsic activities () The values are expressed as means ± S.E.M. of separate experiments, each performed in triplicate.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   A, effects of mepyramine on the basal luciferase activity in mock-transfected COS-7 cells () or in COS-7 cells transiently expressing the human histamine H1 receptor at 1 ± 0.1 () or 4.2 ± 0.2 () pmol/mg of protein. B, effects of mepyramine (), ranitidine (), and clobenpropit () on NF-B activation in COS-7 cells transiently expressing the human histamine H1 receptor (3.2 ± 0.4 pmol/mg of protein). C, inhibition of constitutive H1-receptor activity, as measured by the reporter-gene assay, by (R)-cetirizine () and (S)-cetirizine () in COS-7 cells transiently expressing the human histamine H1 receptor (3.2 ± 0.4 pmol/mg of protein). Representative experiments performed in triplicate are shown, each experiment was repeated at least three times.

View this table: [in this window] [in a new window]   TABLE 2 The pKi and pIC50 values of the tested inverse agonists for the human H1 receptor and their intrinsic activities () The values are expressed as means ± S.E.M. of separate experiments, each performed in triplicate.

Competitive Antagonism of Mepyramine. Mepyramine competitively antagonized both the histamine-induced [³H]InsP₃ accumulation (Fig. 2A) and NF-B activation (Fig. 2C). Schild plot analysis of the competitive antagonism by mepyramine of the histamine-induced [³H]InsP₃ accumulation resulted in a pA₂ value for mepyramine of 8.9 (slope = 0.86 ± 0.1, r² = 0.93; Fig. 2B), whereas analysis of the competitive antagonism by mepyramine of the histamine-induced NF-B activity yielded a pA₂ value for mepyramine of 8.0 (slope = 0.94 ± 0.1, r² = 0.93; Fig. 2D). The obtained pA₂ values for mepyramine differ somewhat, but are in good agreement with the obtained pIC₅₀ values of mepyramine as inverse agonist in the two assays (Table 2).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   A, competitive antagonism of [3H]InsP3 accumulation in response to histamine () in COS-7 cells expressing the human H1 receptor (4.5 ± 0.5 pmol/mg of protein) by increasing concentrations of mepyramine [1 nM (), 3 nM (), 10 nM (), 30 nM (), 100 nM (), 300 nM (), or 1 µM ()]. Shown is a representative example of two independent experiments, each performed in triplicate. B, Schild plot analysis of the combined data set of two independent experiments of the competitive antagonism by mepyramine of the histamine-induced [3H]InsP3 accumulation. C, competitive antagonism of luciferase activity in response to histamine () in the reporter-gene assay in COS-7 cells expressing the human H1 receptor (3.2 ± 0.4 pmol/mg of protein) by increasing concentrations of mepyramine [10 nM (), 30 nM (), 100 nM (), or 300 nM ()]. Shown is a representative example from three independent experiments, each performed in triplicate. D, Schild plot analysis of the competitive antagonism by mepyramine of the histamine-induced luciferase activity.

pK_i Values for H₁ receptor Antagonists. To correlate the inverse agonist potencies with H₁-receptor affinities, all tested H₁ antagonists were evaluated in [³H]mepyramine displacement studies using whole cell homogenates of transfected COS-7 cells expressing the human H₁ receptor. As shown in Table 2, the inverse H₁ agonists display a pharmacological profile that is expected for the H₁ receptor, including the known stereospecificity for the stereoisomers of cetirizine (Moguilevsky et al., 1995). Whereas the potencies as inverse agonists determined in the [³H]InsP₃ assay correlate well with their respective pK_i-values (Fig. 3A; slope = 0.97, r² = 0.97, n = 6), the pK_i values correlate less well with their respective potencies as inverse agonists determined in the NF-B reporter-gene assay (Fig. 3B; slope = 0.74, r² = 0.68, n = 17).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Correlation graphs of the pIC50 values obtained for the inverse agonists in the [3H]InsP3 assay (A) and reporter-gene assay (B) versus the pKi values obtained by [3H]mepyramine displacement in COS-7 cells expressing the human H1 receptor (see Table 2).

Constitutive H₁ Receptor Activity Is Not Caused by Endogenous Histamine. Despite initial concern about the presence of residual agonist (Baxter and Tilford, 1995) the existence of constitutively active GPCRs and inverse agonists has now been widely accepted (Milligan and Bond, 1997). This is largely because of the identification of true antagonists (or neutral antagonists), ligands that do not affect constitutive GPCR signaling and compete with both agonists and inverse agonists for the GPCR binding site [e.g., burimamide for the H₂ receptor (Smit et al., 1996; Alewijnse et al., 1998)]. Because all H₁ antagonists tested so far display (partial) inverse agonistic efficacy, we performed additional experiments to exclude contamination of our assays with endogenous histamine. Culturing COS-7 cells for several weeks in Opti-MEM I medium supplemented with 0.5% dialyzed serum did not affect the inverse agonistic activity of mepyramine after cotransfection of the cells and seeding them in serum-free Opti-MEM I medium. Under these conditions, mepyramine still inhibited constitutive luciferase expression with a pIC₅₀ value of 8.0 ± 0.1 (n = 2) in H₁ receptor-expressing cells.

Mechanism of H₁ Receptor-Mediated NF-B Activation. It is known that the human histamine H₁ receptor is coupled to PTX- and CTX-insensitive G_q/11 proteins leading to the activation of PLC in various cell types (Leurs et al., 1994). As an initial approach to address the coupling specificity of the human histamine H₁ receptor to the NF-B pathway, we coexpressed the H₁ receptor with various G-subunits and examined the effects of the bacterial toxins PTX and CTX, which covalently modify specific G-subunits. Pretreatment of the cells with either PTX or CTX or coexpression of the GTPase-deficient mutants of G₁₂ (G₁₂Q²²⁹L), G₁₃ (G₁₃Q²²⁶L), or G_o (G_oQ²⁰⁵L) did not increase basal signaling or alter responsiveness of the H₁ receptor to histamine or mepyramine (data not shown), indicating that members of G_i/o-, G₁₂-, and G_s-families are not involved in the activation of the NF-B pathway in COS-7 cells by the H₁ receptor. Cotransfection of cDNA encoding the H₁ receptor together with expression vectors carrying cDNA inserts for G_q or G₁₁ resulted in a G expression level-dependent increase in (constitutive) H₁ receptor-mediated NF-B activation (Table 3 and Fig. 4). In control experiments using cells that do not express the human H₁ receptor, transfection with cDNA encoding G₁₁ does not significantly alter basal activation of NF-B (basal activation is 85 ± 5% of control). Whereas an elevated constitutive H₁-receptor activity results in an increased basal signaling, the actual -fold increase upon agonist-stimulation is reduced. Thus, whereas coexpression of G₁₁ results in an 2.3 ± 0.5-fold increase of constitutive H₁-receptor activity (Table 3 and Fig. 4A), the histamine-induced stimulation of NF-B activation is reduced to 1.0 ± 0.5-fold over basal levels (Table 3 and Fig. 4B). Mepyramine inhibited the G₁₁-induced raise of constitutive H₁ receptor-mediated activation of NF-B completely (Fig. 4B). Expression of the constitutively active mutant G₁₁Q²⁰⁹L resulted in an activation of NF-B in the absence or presence of the H₁ receptor. In control experiments using cells that do not express the human H₁ receptor, transfection with cDNA encoding G₁₁Q²⁰⁹L resulted in a 1.9 ± 0.1-fold increase of basal NF-B activation. In cells coexpressing the H₁ receptor and the constitutively active mutant G₁₁Q²⁰⁹L, basal NF-B activation is further increased, and the agonist-induced stimulation is reduced to 1.0 ± 0.5-fold over basal (Table 3). Moreover, the inverse H₁ agonist mepyramine is not capable to inhibit the H₁ receptor-independent activation of NF-B by G₁₁Q²⁰⁹L.

View this table: [in this window] [in a new window]   TABLE 3 The pEC50 values of histamine and the pIC50 values of mepyramine for the human H1 receptor Data of the basal NF-B activation are expressed as a percentage of the basal level of NF-B activation in cells transfected with cDNA coding for the H1 receptor but not with cDNAs coding for G-subunits (level of NF-B activation in mock-transfected cells was 35 ± 4% of that of cells transiently expressing the H1 receptor). The pEC50 and pIC50 values are means ± S.E.M. of separate experiments, each performed in triplicate.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   A, effects of mepyramine on the basal luciferase activity in COS-7 cells transiently expressing the human histamine H1 receptor (3.2 ± 0.4 pmol/mg of protein) that had been cotransfected in the absence () or presence of 1.0 µg (), 5.0 µg (), or 25 µg () of cDNA coding for G11/107 cells. B, effects of histamine (square symbols) and mepyramine (round symbols) on NF-B activation in COS-7 cells transiently expressing the human histamine H1 receptor that had been cotransfected in the absence (filled symbols) or presence of 25 µg of cDNA coding for G11/107 cells (empty symbols).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effects of coexpression of the histamine H1 receptor with G11 or the constitutively active mutant G11Q209L (G11QL) subunits on constitutive and agonist-induced H1 receptor-mediated activation of PLC. COS-7 cells were cotransfected with pcDEF3H1 encoding the human histamine H1 receptor (25 µg/107 cells) together with equal amounts of cDNA coding for G11 or G11Q209L; the total amount of transfected DNA was kept constant using pcDEF3. Forty-eight hours after transfection, cells were incubated with medium to determine basal activity () or with 100 µM histamine () or 100 µM mepyramine (), and inositol phosphates were determined as described under Experimental Procedures. Inset, effects of expression of G11 or constitutively active mutant G11Q209L (G11QL) subunits on the activation of PLC in cells not expressing the human H1 receptor.

Role of G- and G-Subunits. Previous studies (Shahrestanifar et al., 1999; Xie et al., 2000; Casarosa et al., 2001) have indicated a role for G-subunits in the activation of NF-B. To address the role of G-subunits in the H₁ receptor-mediated NF-B activation we coexpressed the H₁ receptor either with various G-scavengers or with G-subunits. When over-expressed, G_t is known to scavenge G-subunits and to inhibit G-mediated signaling (Clapham and Neer, 1997). Coexpression of G_t neutralized constitutive activation of NF-B by the H₁ receptor in an expression level-dependent manner (Fig. 6, A-C). When equal amounts of the cDNAs encoding the H₁ receptor and G_t were used for cotransfection of the cells, G_t neutralized the constitutive activation of NF-B by the H₁ receptor without altering H₁-receptor expression (Table 3 and Fig. 6, A and D). Depletion of free G-subunits did not prevent histamine-induced NF-B activation (Table 3 and Fig. 6D), although the absolute increase in NF-B activation was reduced. Although we cannot exclude the possibility of an incomplete scavenging of released G-subunits, these data suggest that signaling pathways other than those using free G-subunits are involved in the agonist-induced NF-B activation. Similar results have been obtained in coexpression experiments using either GRK2 or the kinase deficient mutant GRK2K²²⁰R (Fig. 6A).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effects of coexpression of the histamine H1 receptor with Gt-subunits on constitutive H1 receptor-mediated NF-B activation. A, COS-7 cells were cotransfected with either pcDEF3 (mock, ) or pcDEF3H1 encoding the human histamine H1 receptor (25 µg/107 cells) together with various amounts cDNA coding for Gt (0-25 µg/107 cells; ). The total amount of transfected DNA was kept constant using either pcDEF3 or pcDNA3. Inset, the effect of coexpression of the H1 receptor together with either GRK2 or the kinase deficient GRK2K220R on the constitutive H1-mediated NF-B activation. The asterisks indicate significant differences from the H1 receptor-mediated NF-B activation in the absence of Gt-subunits (Gt = 0 µg). B, Western blotting of COS-7 cells that had been cotransfected with various amounts of cDNA (0, 1, 3, 5, 15, or 25 µg/107 cells) coding for Gt with a Gt-specific antibody resulted in the detection of immunoreactive bands corresponding to Gt-subunits. C, quantification of the Western blot indicates a linear relationship between the amount of transfected cDNA coding for Gt and the expression of Gt. D, effects of histamine (squares) and mepyramine (circles) on NF-B activation in COS-7 cells transiently expressing the human histamine H1 receptor that had been cotransfected in the absence (filled symbols) or presence of 25 µg of cDNA coding for Gt/107 cells (open symbols).

View this table: [in this window] [in a new window]   TABLE 4 Effect of co-expression of various G-combinations on the constitutive H1 receptor-mediated NF-B activation NF-B-driven luciferase expression was measured 48 h after COS-7 cells were transiently transfected with cDNA coding for the human H1 receptor and various G-combinations of G1, 2, and 5 with G1 and 2 in a ratio of 1:1:1. Data are expressed as a percentage of the basal level of NF-B activation in cells transfected with cDNA coding for the H1 receptor but not with cDNAs coding for G-subunits. The values are means ± S.E.M. of separate experiments, each performed in triplicate.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The actual therapeutic importance of constitutive GPCR activity has not yet been fully clarified. However, for a proper evaluation of drug action this new aspect in GPCR pharmacology cannot be ignored. In view of the widespread therapeutic use of H₁ antagonists in allergic conditions (Woosley, 1996; Zhang et al., 1997; Handley et al., 1998), we investigated the constitutive H₁-receptor activity and inverse agonistic activity of several H₁ antagonists. In this study, we show that the H₁ receptor activates the important pro-inflammatory transcription factor NF-B in both a constitutive and agonist-dependent manner. Moreover, a developed NF-B reporter-gene assay proved to be a very sensitive discriminator between positive and negative ligand efficacy for the H₁ receptor. The observed pharmacological differences for mepyramine in the two assays might be due to phenomena such as receptor trafficking (Berg et al., 1998) and is part of our ongoing investigations.

As observed for other GPCRs (Samama et al., 1993; Burstein et al., 1995; MacEwan and Milligan, 1996; Smit et al., 1996), constitutive H₁-receptor activity increases as the receptor or G-protein expression level increases; the inverse agonist mepyramine reduces this constitutive activity with unchanged potency. The inhibition of constitutive H₁ receptor-mediated NF-B activation is specific for H₁ antagonists, because an inverse H₂ agonist or an H₃ antagonist has no effect. Furthermore, in accordance with the known H₁-receptor stereospecificity (Moguilevsky et al., 1995), the enantiomers of cetirizine display stereospecific inhibition of constitutive H₁-receptor NF-B activation. The constitutive H₁ receptor-mediated NF-B activation is inhibited by all tested, clinically used H₁ antagonistsincluding cetirizine (Zyrtec), ebastine (Kestine), epinastine (Flurinol), loratadine (Claritin), mizolastine (Mizolen), and fexofenadine (Allegra)indicating that these drugs in fact all act as inverse H₁ agonists. Previously there has been some debate about whether the presence of endogenous agonists may account for the observed constitutive GPCR signaling (Baxter and Tilford, 1995). Yet we show that in experiments with cells cultured in histamine-free medium or after treatment with FMH, a suicide inhibitor of HDC (Watanabe et al., 1990), constitutive H₁-receptor activity and inverse agonism of H₁-receptor antagonists is still observed. Our observations therefore show that the negative intrinsic activity of the tested H₁ antagonists is a true property of the various drugs and might contribute to their proven successful therapeutic application (Zhang et al., 1997).

The discovery of inverse agonism at GPCRs has also raised the general question of potential differences in therapeutic outcome upon treatment with inverse agonists or neutral antagonists (Milligan et al., 1995; Smit et al., 1996; Milligan and Bond, 1997). An important issue that has been considered in this respect is the modulation of GPCR expression levels (Milligan and Bond, 1997; Leurs et al., 1998). Inverse agonists, but not neutral antagonists, can up-regulate constitutively active GPCRs, an effect that might not be beneficial under all circumstances. Yet, because all of the tested H₁ antagonists are inverse agonists, we cannot currently address this issue experimentally. Moreover, the availability of a neutral antagonist would offer the opportunity to investigate whether inverse agonism at the H₁ receptor is truly beneficial in allergic diseases. The developed NF-B reporter-gene assay will be a useful tool to identify future neutral H₁ antagonists.

The observed constitutive H₁-receptor activation of NF-B gene transcription is another very interesting feature, because both histamine acting at H₁ receptors and NF-B are known to be involved in inflammatory conditions, such as atherosclerosis (Takagishi et al., 1995; Bourcier et al., 1997) and allergy (Zhang et al., 1997; Barnes et al., 1998; Handley et al., 1998). It is attractive to speculate, therefore, that the observed (constitutive) coupling of the H₁ receptor to the NF-B pathway in transfected COS-7 cells can also be of physiological importance. By activating NF-B, H₁ receptors may participate in the regulation of gene expression under physiological and pathological conditions. In the nasal mucosa of patients suffering from allergic rhinitis (Iriyoshi et al., 1996; Hamano et al., 1998), H₁-receptor mRNA is significantly up-regulated. It is tempting to consider that in these conditions the constitutive H₁ receptor signaling is also increased and is at least partly responsible for some of the symptoms. Thus, constitutively active H₁ receptors may regulate basal NF-B-mediated transcriptional activity.

Because a detailed characterization of the histamine-stimulated NF-B-mediated gene transcription will extend our understanding of the molecular actions of histamine and the H₁ antagonists, we investigated the mechanisms of NF-B activation by the H₁ receptor to some extent. In agreement with previous findings that the H₁ receptor is a G_q/11-coupled GPCR (Gutowski et al., 1991; Leopoldt et al., 1997), coexpression of either wild-type G_q or G₁₁ increases H₁ receptor-mediated signaling, confirming the involvement of G_q/11 heterotrimers in H₁ agonist-mediated responses (Fig. 7). Furthermore, coexpression of the constitutively active mutant of G₁₁ (G₁₁Q²⁰⁹L) results in an H₁ receptor-independent activation of NF-B and PLC (Table 3 and Fig. 5), which cannot be inhibited by an inverse H₁ agonist (Table 3). These data show that active G₁₁-subunits can stimulate cellular signaling partners leading to NF-B activation, possibly via protein kinase C (PKC; Shahrestanifar et al., 1999).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Proposed mechanism of NF-B activation via G and G-subunits in COS-7 cells transiently transfected with the human histamine H1 receptor. Activation of the H1 receptor induces GDP/GTP exchange on the G-subunit, upon which the activated Gq/11G heterotrimer dissociates into its corresponding G11-subunit and G heterodimer, which can both activate cellular effectors. Activated G11 (GTP-G11) activates PLC to induce the formation of InsP3 and release diacyl glycerol to activate PKC, which in turn activates the NF-B/I-B complex (Rothwarf and Karin, 1999) and NF-B is translocated to the nucleus to induce gene transcription of a wide variety of genes. Also, free G-subunits activate the NF-B/I-B complex to induce gene transcription by the released NF-B via an as-yet-unidentified mechanism. Our data suggest that the signal transduction pathway of agonist-mediated activation is via the G-subunit, whereas the constitutive H1 receptor-mediated activation is via the free G-subunits (see text).

However, several studies have indicated that GPCR-linked signal transduction pathways other than G-mediated PLC activation can also result in NF-B activation, possibly via G-subunits (Xie et al., 2000; Casarosa et al., 2001). Specific G-heterodimer combinations (G₂₁ and G₂₂) coexpressed together with the H₁ receptor indeed elevate NF-B-activation, indicating that signal transduction pathways other than the G-mediated PLC activation may mediate NF-B activation. The lack of any effect of G-dimers containing G-subunits other than G₂ is not attributable to differences in their expression levels, because previous studies in our laboratory have shown all tested G-subunits to be expressed to a similar extent upon transfection of COS-7 cells (Casarosa et al., 2001).

Likewise, by competing for free G-subunits, G-scavengers completely neutralize constitutive H₁ receptor-mediated NF-B activation, confirming the involvement of G-subunits in the activation of NF-B by GPCRs (Xie et al., 2000; Casarosa et al., 2001). Whereas the studies with coexpressed G-scavengers suggest that G-subunits may be responsible for the H₁-mediated constitutive activation of NF-B, G-scavengers do not affect the (constitutive) H₁ receptor-mediated activation of PLC or the agonist-induced activation of NF-B. The difference in basal signaling that is observed upon expression of G-scavengers may arise from a varying sensitivity of activation of the two pathways. Possibly, the pathway of activation of NF-B activation is much less sensitive to the G_q/11 subunits and highly sensitive to G-subunits, whereas activation of PLC is mostly sensitive to G_q/11 subunits. Activation of PLC by these G_q/11 subunits could thus mask possible effects of G-subunits; therefore, no effects on PLC activation are detected upon G-subunit scavenging. A low sensitivity of PLC for G-subunits may be explained by the lack of expression of the -sensitive PLC-₂ isoenzyme in COS-7 cells (Wu et al., 1993). The observation that G-scavengers completely neutralize basal signaling to NF-B but do not totally reduce the agonist-induced response suggests that signaling pathways other than those using free G-subunits are primarily involved in the agonist-induced NF-B activation. Although we cannot exclude the possibility that not all free G-subunits are scavenged after agonist-induced G-protein activation, these data suggest that G_q/11-subunits are likely to be involved in this response. This suggestion is corroborated by the observations that expression of constitutively active G_q/11Q²⁰⁹L results in NF-B activation and that coexpression of the H₁ receptor with G₁₁ strongly enhances the agonist-induced NF-B activation. The complete reduction of basal H₁-receptor activity by G-scavengers is surprising in view of the fact that some level of activated G_q/11-subunits will be present under these conditions as well. At the moment, we have no explanation for these observations. One can speculate that for activation of the NF-B pathway, the level of basal H₁ receptor-mediated PKC stimulation via G_q/11-dependent PLC activation is insufficient. Only at higher levels of diacylglycerol production (e.g., after agonist stimulation) would the activation of PKC become important and result in the activation of NF-B. Previously, mechanistic differences have also been reported for basal and agonist-induced activation for the phosphatidylinositide-3-OH-kinase-dependent activation of p42/p44 mitogen-activated protein kinase in a variety of different cell types (Versteeg et al., 2000).

In conclusion, we have shown that the histamine H₁ receptor constitutively activates NF-B-mediated transcription in COS-7 cells. The various tested, clinically used H₁ antagonists act as inverse agonists, suggesting that inverse agonism might be part of their mechanism of action. Moreover, we have identified the initial signaling events in the H₁ receptor-mediated NF-B activation in COS-7 cells. Our data indicate that both G_q/11- and G-subunits play a role in the H₁ receptor-mediated NF-B activation similar to NF-B activation mediated via the bradykinin B₂ receptor or US28 (Xie et al., 2000; Casarosa et al., 2001). We suggest that the constitutive H₁ receptor-mediated activation of NF-B is mediated via free G-subunits from G_q/11-proteins, whereas histamine-mediated NF-B activation is most likely regulated via G_q/11-subunits. This study shows for the first time a role for G-subunits in the signal transduction of the histamine H₁ receptor. Although PKC is a likely candidate for the _q/11-mediated NF-B activation (Shahrestanifar et al., 1999) and both phosphatidylinositol 3-kinase and Akt have been shown to be involved in the G-mediated activation (Xie et al., 2000), the actual target(s) of the G- and G-subunits in the H₁ receptor-mediated responses is at present not known and is currently under investigation.