Leiden/Amsterdam Center for Drug Research, Department of
Pharmacochemistry, Vrije Universiteit Amsterdam, Amsterdam, The
Netherlands
 |
Introduction |
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 |
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.
Gifts of 2-(3-trifluoromethyl)phenylhistamine dihydrogenmaleate [Dr.
Schunack (Leschke et al., 1995
)], acrivastine (The Wellcome Foundation
Ltd., London, UK), (R)- and (S)-cetirizine
hydrochloride (UCB Pharma, Brussels, Belgium), diphenhydramine
hydrochloride (DSM Fine Chemicals, Sittard, The Netherlands),
ebastine (Almirall Prodesfarma, Barcelona, Spain), epinastine
hydrochloride (Roche Molecular Biochemicals, Mannheim, Germany),
KW 4679 (Pharmaceutical Research Laboratories, Shizuoka, Japan),
loratadine (Schering Plough, Kenilworth, NJ), mianserine hydrochloride
(Organon NV, Oss, The Netherlands), mizolastine (Synthelabo Recherche,
Bagneux, France), pcDEF3 (Dr. J. Langer), and
(+)-terfenadine carboxylate and (
)-terfenadine carboxylate (Sepracor,
Marlborough, MA), ranitidine dihydrochloride (GlaxoSmithKline,
Uxbridge, Middlesex, UK), and of the cDNAs encoding mouse
G
11 and
G
11Q209L (Dr. H. Umemori), mouse G
q (Dr. B. Conklin), bovine
G
t (Dr. B. Defize),
G
12Q229L and
G
13Q226L (Dr.
Dhanasekaran), G
oQ205L
(Dr. Iyengar), bovine G
1 and
G
2, human G
1 and
G
5 (Dr. M. Lohse),
G
2, bovine GRK2 and
GRK2K220R (Dr. S. Cotecchia), and the human
histamine H1 receptor [Dr. Fukui (Fukui et al.,
1994
)] are greatly acknowledged.
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
G
11 or
G
11Q209L, 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
pNF
B-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 |
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).
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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.
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|
As found for the accumulation of
[3H]InsP3 (Bakker et al.,
2000
), the basal NF-
B activation was increased upon expression of
the human H1 receptor. As can be seen in Fig.
1A, expression of the
H1 receptor at a density of 1.4 ± 0.1 pmol/mg of protein resulted in a 2.6-fold increase of the basal
luciferase expression compared with mock-transfected cells. This
constitutive H1-receptor activity was inhibited
by the H1 antagonist mepyramine, whereas mepyramine had no effect on NF-
B activation in transfected COS-7 cells that did not express the human H1 receptor
(n = 5; Fig. 1A). Mepyramine inhibited the constitutive
NF-
B activation by 78 ± 1% (
=
0.97 ± 0.01)
with a pIC50 value of 7.9 ± 0.1 (n = 55; Table 2).
Whereas increased H1-receptor expression led to a
further rise in the basal response, the pIC50
value of mepyramine was unaffected by varying the
H1-receptor density from 1 to 4 pmol/mg of
protein (Fig. 1A). Furthermore, the constitutive activity of the human
H1 receptor was unaffected by the inverse
H2 agonist ranitidine and the
H3 receptor antagonist clobenpropit (Fig. 1B).

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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.
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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.
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|
The H1 receptor is known to exhibit
stereospecificity toward the enantiomers of H1
antagonists, such as cetirizine (Moguilevsky et al., 1995
). Indeed, the
inhibition of NF-
B activation by the enantiomers of cetirizine was
found to be stereospecific (Fig. 1C); (R)-cetirizine
inhibits the constitutive H1 receptor activity for 59 ± 3% (
=
0.73 ± 0.04) with a
pIC50 value of 8.2 ± 0.1 (n = 13; Table 2), whereas (S)-cetirizine shows an inhibition of 62 ± 3% (
=
0.77 ± 0.04) with a
pIC50 value of 6.6 ± 0.1 (n = 13; Table 2).
The constitutive activation was also inhibited by a variety of other
H1 ligands, including classical
H1 antagonists such as diphenhydramine,
tripelennamine, and triprolidine and other second-generation antihistamines such as acrivastine, ebastine, epinastine, mizolastine, and terfenadine carboxylate (Table 2), some of which are or have been
in clinical use for the treatment of allergic conditions. The negative
intrinsic activities of the tested compounds varied between
1.09 ± 0.06 for loratadine and
0.71 ± 0.06 for triprolidine (Table
2), indicating partial inverse agonism for some of the compounds in the
reporter-gene assay. In general, the potencies of the inverse agonists
to inhibit constitutive H1 receptor-mediated NF-
B activation are in good agreement with their respective
potencies to inhibit constitutive InsP3
accumulation, although some differences are noted (Table 2).
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).

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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.
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pKi 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 pKi-values (Fig.
3A; slope = 0.97, r2 = 0.97, n = 6), the
pKi 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).

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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).
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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.
To address a putative endogenous synthesis of histamine by histidine
decarboxylase (HDC) in COS-7 cells, we treated the cells with
(S)-(+)-
-fluoromethylhistidine (FMH), a selective
irreversible inhibitor and suicide substrate of HDC (Watanabe et al.,
1990
). Prolonged treatment with FMH did not affect constitutive
activity of the H1 receptor. Treatment of
transfected COS-7 cells expressing the human H1
receptor with concentrations up to 100 µM FMH for 48 h did not
affect constitutive activity of the H1 receptor. Under these conditions mepyramine inhibited luciferase expression with
a pIC50 value of 8.0 ± 0.1 (n = 2). Furthermore, mepyramine also inhibited the
constitutive activity of the H1 receptor in COS-7
cells that had been cultured for 5 days in the presence of 100 µM FMH
(pIC50 = 8.2 ± 0.3, n = 4).
Mechanism of H1 Receptor-Mediated NF-
B
Activation.
It is known that the human histamine
H1 receptor is coupled to PTX- and
CTX-insensitive Gq/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 G
12
(G
12Q229L),
G
13
(G
13Q226L), 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-,
G
12-, 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
G
11 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
G
11 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 G
11 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 G
11-induced raise of constitutive
H1 receptor-mediated activation of NF-
B
completely (Fig. 4B). Expression of the constitutively active mutant
G
11Q209L 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 G
11Q209L 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
G
11Q209L, 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 G
11Q209L.
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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.
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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
G 11/107 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
G 11/107 cells (empty symbols).
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Similarly, transfection of cDNA encoding the human
H1 receptor together with expression vectors
carrying cDNA inserts for G
11 or
G
11Q209L resulted in an
increase in constitutive H1 receptor-mediated InsP3 production (Fig.
5). Cotransfection with cDNA encoding
G
11 induces a 77% increase in basal
InsP3 accumulation in cells expressing the human
H1 receptor compared with a 30% increase in
basal InsP3 accumulation in cells not expressing
the human H1 receptor. Expression of
G11Q209L greatly affects
basal InsP3 accumulation; the basal level of InP3 in
G11Q209L-expressing
cells is 3.6-fold the level of that of human
H1-expressing cells and is similar to cells
expressing both G11Q209L
and the human H1 receptor (Fig. 5). The elevated
constitutive H1-receptor activity results in an
increased basal but not agonist-induced signaling. Mepyramine inhibited
the G
11-induced but not the
G
11Q209L-induced
rise of constitutive H1 receptor-mediated
activation of PLC (Fig. 5). Moreover, expression of the constitutively
active mutant G
11Q209L
resulted in a similar activation of PLC in the absence (Fig. 5, inset)
or presence of the human H1 receptor.

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Fig. 5.
Effects of coexpression of the histamine
H1 receptor with G 11 or the constitutively
active mutant G 11Q209L
(G 11QL) 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 G 11 or
G 11Q209L; 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
G 11 or constitutively active mutant
G 11Q209L (G 11QL) 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).

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Fig. 6.
Effects of coexpression of the histamine
H1 receptor with G t-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 G t (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 G t-subunits
(G t = 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 G t with
a G t-specific antibody resulted in the detection of
immunoreactive bands corresponding to G t-subunits. C,
quantification of the Western blot indicates a linear relationship
between the amount of transfected cDNA coding for G t and
the expression of G t. 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 G t/107 cells
(open symbols).
|
|
In coexpression studies with G
-subunits, cells were cotransfected
with a combination of expression vectors carrying various cDNA inserts
for G-protein
- and
-subunits. NF-
B activation is restricted
to cells in which
- and
-subunits are coexpressed together with
the H1 receptor (data not shown). Therefore, a
complex of free G
-subunits act together to elevate constitutive
H1-mediated NF-
B activation, in which
specificity of the G
-subunits is retained in the G
-subunit
because both G
2
1-and
G
2
2-subunits, but
none of the other tested G
-subunit combinations, elevate constitutive H1-receptor activity (Table
4).
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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 G 1,
2, and 5 with G 1 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.
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|
We also examined the possible role of G
-subunits in the
constitutive H1 receptor-mediated
InsP3 accumulation in transfected COS-7 cells. In
contrast to the observed effects on the constitutive H1 receptor-mediated activation of NF-
B,
coexpression of G
t did not affect the
constitutive H1 receptor-mediated
InsP3 accumulation. The basal
InsP3 level in COS-7 cells cotransfected with
cDNAs encoding the human H1 receptor, and
G
t was 82 ± 13% of the basal InsP3 level in control cells expressing the same
level of the human H1 receptor (100 ± 16%).
 |
Discussion |
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
antagonists
including 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
Gq/11-coupled GPCR (Gutowski et al., 1991
;
Leopoldt et al., 1997
), coexpression of either wild-type
G
q or G
11 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 G
11
(G
11Q209L) 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
G
11-subunits can stimulate cellular signaling
partners leading to NF-
B activation, possibly via protein kinase C
(PKC; Shahrestanifar et al., 1999
).

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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 G q/11G heterotrimer dissociates into its
corresponding G 11-subunit and G heterodimer, which
can both activate cellular effectors. Activated G 11
(GTP-G 11) 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).
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|
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 (G
2
1 and
G
2
2) 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 G
2 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 Gq/11 subunits and highly
sensitive to G
-subunits, whereas activation of PLC is mostly
sensitive to Gq/11 subunits. Activation of PLC by
these Gq/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 G
11
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.
Supported in part by UCB Pharma (Belgium) and the EU BIOMED 2 program Inverse Agonism: Implications for Drug Research and by the
Royal Dutch Academy of Arts and Sciences (M.J.S.).
Dr. R. Leurs, Leiden/Amsterdam
Center for Drug Research, Department of Pharmacochemistry, Vrije
Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The
Netherlands. E-mail: leurs{at}chem.vu.nl