Sensitization of the Histamine H1 Receptor by
Increased Ligand Affinity*
S. Margreet
Bloemers
§,
Sander
Verheule
§,
Maikel P.
Peppelenbosch
§¶
,
Martine J.
Smit**,
Leon G. J.
Tertoolen
, and
Siegfried
de Laat
From the
Hubrecht Laboratory, Netherlands Institute
for Developmental Biology, Uppsalalaan 8, NL-3584 CT Utrecht,
¶ Laboratory for Experimental Internal Medicine, Academic Medical
Centre, G2-130, Meibergdreef 9, NL-1105 AZ Amsterdam, and ** Department
of Pharmacochemistry, Faculty of Chemistry, Vrije Universiteit, de
Boelelaan 1083, NL-1081 HV Amsterdam, The Netherlands
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ABSTRACT |
Histamine regulates a variety of physiological
processes including inflammation, gastric acid secretion, and
neurotransmission. The cellular response to histamine is subject to
dynamic control, and exaggerated histamine reactivity in response to
cysteinyl leukotrienes and other stimuli is important in a variety
of different pathological conditions. The molecular mechanisms
controlling histamine responsiveness are still unresolved. In
investigating histamine responses in embryonic stem (ES5) and F9
embryonic carcinoma cells, we encountered a novel mechanism controlling
the cellular reaction to histamine. Unstimulated cells displayed
neither [3H]pyrilamine binding nor
histamine-induced increases in cytosolic Ca2+ levels.
Pretreatment of these cells, however, with leukotriene D4,
leukotriene E4, serotonin, or fetal calf serum induced an immediate and transient ability of these cells to respond to histamine with an increase in cytosolic Ca2+ levels. This effect
could be inhibited by pertussis toxin and was mimicked by GTP
analogues. Importantly, the latter compounds also provoked immediate
high affinity [3H]pyrilamine binding. We conclude that in
these cells histamine responsiveness is directly controlled by
pertussis toxin-sensitive G protein-coupled receptors, whose activation
enables the H1 receptor to bind its ligand. These findings
define a novel mechanism for regulating histamine H1
receptor activity and provide for the first time molecular insight into
the mechanism by which cysteinyl leukotrienes and other external
stimuli can increase histamine responsiveness.
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INTRODUCTION |
Histamine, a biogenic amine formed by decarboxylation of the amino
acid L-histidine (1), is found in large quantities in most
tissues, mainly in the granules of mast cells, although numerous other
cell types are capable of histamine synthesis as well (2). Histamine
controls a multitude of physiological functions by activating specific
receptors on target cells. Three types of receptors for histamine have
been described, denominated as the H1, H2, and H3 receptor and are distinguished on the basis of their
sensitivity to specific agonists and antagonists (3). In general, the
H3 receptor is implicated in autoinhibition of histamine
synthesis and release and the H2 receptor in gastric acid
secretion, whereas the H1 receptor is involved in
inflammatory responses, mediating for instance blood vessel and
bronchial constriction, vascular permeabilization, and synthesis of
other inflammatory agents (4). Histamine receptors are subject to
dynamic regulation, receptor activity being increased or diminished in
response to various conditions (5-7), and exaggerated histamine
reactivity is associated with a variety of pathological disorders.
Cysteinyl leukotrienes have been implicated in the stimulation of
histamine reactivity. Inhalational challenge with these inflammatory
eicosanoids increases histamine responsiveness of the airways (8-12),
and cysteinyl leukotriene-induced histamine hypersensitivity is
presumed to be important in asthmatic disease (13). Also, other
signaling molecules stimulate histamine responsiveness. Especially
serotonin, platelet-activating factor, and thromboxanes are known to
enhance histamine reactivity (14, 15). The molecular mechanisms,
however, by which such stimuli can provoke increased histamine
responsiveness have remained obscure.
In the present study we describe a molecular mechanism by which
external stimuli can enhance histamine reactivity by directly controlling the affinity of the histamine H1 receptor for
its ligand. We have reported earlier that the P19 embryo carcinoma (EC)1 cell, a pluripotent
cell type resembling the inner cell mass of the embryo, expresses
functional histamine H1 receptors (16), although its
function with respect to embryogenesis is not clear. To obtain more
insight into the function of histamine receptor expression in
uncommitted cells, we decided to investigate the presence of cellular
responses to histamine in other pluripotent cells. We observed that F9
EC cells and embryonic stem (ES5) cells displayed neither high affinity
[3H]pyrilamine binding nor histamine-induced increases in
cytosolic Ca2+ levels. A pretreatment of these cells with
cysteinyl leukotrienes, serotonin, or FCS, however, induced an
immediate and transient ability of these cells to react to histamine.
This effect was inhibited by pertussis toxin and was mimicked by GTP
analogues. Importantly, induction of histamine responses coincided with
the appearance of high affinity [3H]pyrilamine binding
sites on these cells. Apparently pertussis toxin-activating agents can
regulate histamine responses by inducing high-affinity binding sites
for histamine. These findings define for the first time a molecular
mechanism by which cysteinyl leukotrienes and other external stimuli
can increase histamine responsiveness and identify a novel mechanism
for the regulation of G protein-coupled receptors.
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EXPERIMENTAL PROCEDURES |
Chemicals--
Histamine dihydrochloride, pyrilamine (maleate
salt), leukotrienes, serotonin, and valinomycin were obtained from
Sigma. Fura-2 acetoxymethyl ester was from Molecular Probes (Eugene,
OR), streptolysin O was from Wellcome Diagnostics (Dartford, UK),
GTP
S was from Boehringer Mannheim, okadaic acid was from Life
Technologies, Inc., and [pyridinyl-5-3H]pyrilamine
([3H]pyrilamine) was from Amersham (Buckinghamshire, UK).
The enantiomers of cicletanine were kind gifts from the Henri Beaufour
Institute-IPSEN Laboratories, France. SH-FCS was prepared at our
laboratory by DTT treatment of fetal calf serum. DTT hydrolyzes the
protein S-S bridges and thereby inactivates most of the polypeptide
growth factors in FCS. The thus-treated serum is dialyzed to remove
traces of DTT.
Cell Culture--
F9 EC and P19 EC cells were cultured at 7.5%
CO2 and 37 °C in bicarbonate-buffered DF-medium
supplemented with 7.5% FCS. ES5 and D3 ES cells were maintained in
conditioned minimal essential medium (Life Technologies, Inc.)
supplemented with 10
4 M
-mercaptoethanol
and 20% FCS. The cells were passaged three times a week using EDTA
(0.2 mg/ml) for F9 EC and trypsin (0.05%), EDTA (0.2 mg/ml) for P19 EC
and the ES cell lines. Two days before experimentation the cells were
plated to yield subconfluent cultures for experiments.
Ca2+ Determinations--
For Ca2+
measurements, cells were maintained in serum-free medium for 1 h
and subsequently loaded with 10 µM Fura-2
acetoxymethylester for 30-45 min at 33 °C in a Hepes-buffered
saline of the following composition: 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM Hepes, 10 mM glucose, 0.2% bovine serum albumin adjusted to pH 7.3 with NaOH. During experiments, cells were maintained in Hepes-buffered
saline at 33 °C. For experiments with NaF and AlCl3, use
was made of a saline containing 120 mM NaCl, 20 mM NaF, 75 µM AlCl3, 5 mM KCl, 2 mM CaCl2, 10 mM Hepes, and 10 mM glucose, pH 7.3. The
measurements were carried out with a fluorescence microscope focusing
on a group of 8-12 cells with a 50 × water immersion objective
and a SPEX dual-wavelength fluorimeter. Emission fluorescence was
digitally sampled at 340 and 380 nM and corrected for
background fluorescence as determined from unlabeled cells. The
intracellular Ca2+ concentration was calculated according
to Grynkiewicz et al. (17). For digital image analysis,
pictures were taken from a video recording of Fura-loaded cells
(excited at 340/380 nM) and processed with the Crystal
Particle Package Version 1.08 (Quantel).
Electrophysiology--
For whole-cell patch clamp analysis,
cells were measured in a saline solution of the following composition:
140 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 2 mM CaCl2, 10 mM Hepes, 10 mM glucose adjusted to pH 7.3 with
NaOH. During experiments, cells were maintained at 33 °C.The patch
pipette contained 140 mM KCl, 2 mM
MgCl2, 1 mM CaCl2, 10 mM EGTA, 10 mM Hepes adjusted to pH 7.1 with
KOH. Using the fluorimetric method described by Civitelli et
al. (18), we determined a resting membrane potential for the F9 EC
cells of
51 mV, and cells were clamped at this potential. Currents were analyzed as described earlier (19).
[3H]Pyrilamine Binding--
Scatchard analysis on
membrane preparations and intact cells was performed as described
earlier (16). For Scatchard analysis, cells were serum-starved for
1 h, after which they were labeled for 1 h at 4 °C in
Hepes-buffered Dulbecco's modified Eagle's medium containing 4 nM [3H]pyrilamine and different
concentrations of unlabeled pyrilamine, after which total binding
reached plateau phase (not shown). Subsequently, cells were washed
three times with phosphate-buffered saline, and cell protein was
precipitated with 0.2 M NaOH. The bound radioactivity was
determined by liquid scintillation counting. In each experiment, each
condition was tested in triplicate. For experiments with NaF (20 mM) and AlCl3 (75 µM), the same
buffer was used as described for Ca2+ determinations and
applied for 20 min at room temperature, after which the cells were
placed on ice and the [3H]pyrilamine binding assay was
performed. For experiments with GTP
S, cells were first permeabilized
for 5-10 min at 37 °C with 0.5 IU/ml streptolysin O in 100 mM KCl, 5.6 mM glucose, 1 mg/ml bovine serum
albumin, 1.3 mM CaCl2, 2 mM EGTA,
0.1 mM MgCl2, 1 mM ATP, 10 mM Hepes, pH 7.2 (20) and washed once in the absence of
streptolysin O. GTP
S was added to the permeabilized cells and
incubated shortly (1-2 min) at room temperature to allow GTP
S to
enter and sensitize the cells. Thereafter cells were placed on ice and
labeled as described above. For permeabilized cells, the incubation
buffer contained 150 mM KCl, 5 mM NaCl, 5.6 mM glucose, 1 mg/ml bovine serum albumin, 1 mM
ATP, 1.3 mM CaCl2, 0.1 mM
MgCl2, 2 mM EGTA, 10 mM Hepes, pH
7.2.
In general, Scatchard plots made using intact cells show considerable
nonspecific low affinity binding of [3H]pyrilamine (16),
more so in permeabilized cells when compared with nonpermeabilized
cells. Therefore, Scatchard plots were fitted according to a one- or
two-site model, using the formula, bound/free = 0.5 ([Bmax1
bound]/Kd1 + [Bmax2
bound]/Kd2) + 0.5 ([{Bmax1
bound}/Kd1 + {Bmax2
bound}/Kd2]2 + 4 [{Bmax1
Bmax2}/Kd1
Kd2}]), in which Bmax1, Bmax2, Kd1, and
Kd2 are the respective maximal binding capacities
and dissociation constants of the different affinities. The observed
points of the Scatchard plot of unstimulated cells were satisfactorily
fit with a one-site (low affinity) model, whereas two affinity binding
sites could be distinguished in the sensitized cells. To determine best
fit, we calculated the
2 distribution of the estimated
curve relative to the observed values. We accepted the fit if the
2 did not exceed the probability value of 5%.
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RESULTS |
Induction of Histamine Signal Transduction in F9 EC and D3 ES
Cells--
Different EC and ES cell lines showed marked differences in
their reaction toward histamine (1 µM). P19 EC cells and
ES5 cells responded to histamine with a marked increase in cytosolic
Ca2+ levels and transmembrane currents, as assayed with
whole-cell patch clamp electrophysiology and fluorimetric
Ca2+ determinations (Figs. 1
and 2). Such responses, however, were absent in the F9 EC and D3 ES cells (Figs. 1 and 2; Tables
I and II).
Even digital image analysis (which allows detection of small responses
in single cells) of Fura-2-loaded F9 EC and ES5 cells did not reveal
any response to histamine in these cells (Fig.
3). Importantly, we noted that
stimulation of F9 EC and D3 ES cells with 5% (DTT-treated) fetal calf
serum (SH-FCS), 1 µM leukotriene D4, 1 µM leukotriene E4, or 3 µM
serotonin induced an ability in these cells to respond to histamine:
after prestimulation with one of these compounds, both
histamine-induced Ca2+ responses and transmembrane currents
were easily detected (Figs. 1-3; Tables I and II). Control experiments
consistently showed that F9 EC and D3 ES cells spontaneously reacted
toward ATP (50 µM) and bradykinin (1 µM)
but never did show uninduced histamine responses (n = 29). Furthermore, ATP and bradykinin were not able to induce histamine
responsiveness. We were confident, therefore, to have encountered a
novel form of regulation of histamine responsiveness, as the cellular
reaction to histamine in the F9 EC and D3 ES lines requires
sensitization by specific stimuli.

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Fig. 1.
The effect of histamine (HA) on
the intracellular Ca2+ concentration in P19 EC and F9 EC
cells. Representative traces of the intracellular Ca2+
concentration of Fura-2-loaded P19 EC (A) and F9 EC
(B and C) are shown. The additions of the stimuli
are indicated.
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Fig. 2.
The effect of histamine on transmembrane
currents in embryonal carcinoma cells. Representative whole cell
patch clamp tracings (outward currents down) of P19 EC (A)
and F9 EC cells (B and C) are shown. The
additions of the stimuli are indicated (HA = 0.1 mM histamine; SH-FCS = 5% DTT-treated fetal calf
serum).
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Table I
Ca2+ determinations
The number of histamine responses is shown (i.e. an increase
in cytosolic Ca2+ in excess of 25 nM)/total number
of experiments. ND, not determined.
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Table II
Whole patch clamp experiments in F9 EC cells
The number of histamine responses is shown (i.e. an increase
in transmembrane currents in excess of 10 pA)/total number of experiments and average histamine-induced current ± standard
deviation.
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Fig. 3.
The effect of histamine on the intracellular
Ca2+ concentration in F9 EC cells. Pseudo-color images
of video-recorded Fura-2/AM-loaded cells. Upper panel,
absence of a response to 0.1 mM histamine (a1,
30 s before histamine addition; a2, 5 s before
histamine addition; a3, 15 s; a4, 30 s;
a5, 60 s after histamine addition). Lower
panel, induction of histamine responsiveness by 5% SH-FCS
(b1, 30 s before SH-FCS; b2, 20 s after
SH-FCS; b3, 55 s after SH-FCS, 5 s before
histamine addition; b4, 5 s after histamine;
b5, 10 s after histamine addition; b6,
60 s after histamine addition).
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Histamine Responsiveness Induced by SH-FCS Is Transient and Is
Mediated by the H1 Receptor--
To further characterize
the induction of histamine responsiveness, we performed fluorimetric
Ca2+ determinations in the F9 EC cell line using SH-FCS as
a pre-stimulus. The induction of histamine responsiveness by SH-FCS is
fast, as coapplication of SH-FCS and histamine had a supra-additive
effect. The SH-FCS-induced responsiveness, however, is of a highly
transient nature. Already, 10 min after application of serum, the
sensitivity of these cells to histamine was lost (Fig.
4). Pharmacological studies carried out
after induction of histamine responsiveness with 5% SH-FCS showed that
the reaction to histamine in these cells was inhibited by the
H1 receptor antagonists pyrilamine (1 µM) and
(
)cicletanine (15 µM) but not by the
H1-unspecific enantiomer (+)cicletanine (15 µM). Apparently, SH-FCS transiently enables
H1 receptor signaling in D3 ES and F9 EC cells. Subsequent experiments were performed to obtain insight into the mechanisms implicated in the regulation of this transient histamine H1
receptor responsiveness.

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Fig. 4.
Application of SH-FCS to F9 EC cells yields a
transient responsiveness to histamine. A, the kinetics of
the response to 5% SH-FCS followed by 0.1 mM histamine
(HA) with increasing time intervals. B, example
of a LTE4-induced histamine response as assayed with whole
cell patch clamp. C, relation between the response to
histamine and SH-FCS, measured at the peak of the response, with a time
interval of 50-70 s between the subsequent stimuli. D,
effect of okadeic acid (oka) on histamine responsiveness induced by SH-FCS as analyzed with fluorimetric Ca2+
determinations.
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Role of the Ca2+ Response in the Induction of Histamine
Responsiveness--
Although leukotrienes and serotonin activate only
minor Ca2+ fluxes when compared with SH-FCS,
histamine-induced Ca2+ responses were not different after
induction with either leukotrienes, serotonin, or SH-FCS (Fig.
4B; Table II). Also, stimulation with FCS, which tends to
yield bigger Ca2+ responses when compared with SH-FCS (not
shown), did not produce different Ca2+ responses to
histamine. These results suggest that the size of the Ca2+
response produced by the sensitizing stimulus is not indicative of the
amount of subsequently induced histamine responsiveness. To test this
possibility, we initiated a series of experiments in which the size of
the Ca2+ response to SH-FCS was compared with the
Ca2+ response to histamine added 60 s later. As shown
in Fig. 4C, no relationship between the two Ca2+
responses was detected. We concluded that the induction of histamine responsiveness is independent of the size of the prior Ca2+
response provoked by the sensitizing agent.
Induction of Histamine Responsiveness in F9 EC Cells Requires
Pertussis Toxin-sensitive G Proteins--
To further investigate the
signal transduction pathways regulating this transient histamine
responsiveness, we observed that an increase in intracellular
Ca2+, cAMP analogues or forskolin treatment, cGMP
analogues, inhibitors of phospholipase A2, arachidonic
acid, or the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (acute or overnight
treatment) neither induced nor prevented the sensitization of histamine
responses in F9 EC cells (not shown). Because serum, leukotrienes, and
serotonin are potent inducers of membrane hyperpolarization in this
cell type (not shown), we also tested the effect of the K+
ionophore valinomycin. Although this compound provoked strong hyperpolarization, no effect on the induction of histamine
responsiveness was noted in either F9 EC cells or D3 ES cells (not
shown). Treatment with the phosphatase inhibitor okadeic acid prolonged
histamine responsiveness, suggesting an involvement of serine/threonine phosphorylation in the induction of histamine responsiveness (Fig. 4D).
Because in contrast to the H1 receptor (16, 21), serotonin,
leukotriene D4, leukotriene E4, and serum
activate pertussis toxin-sensitive G proteins, we investigated the
effect of a 4-h pretreatment with pertussis toxin (100 ng/ml). It
appeared that the serum-, leukotriene- and serotonin-provoked
inductions of histamine responsiveness were abolished by this procedure
(as determined either by fluorimetric Ca2+ determinations
or patch clamp electrophysiology; Tables I and II). Therefore, the
sensitization of the histamine response appears to be dependent on the
activation of pertussis toxin-sensitive G protein-coupled receptors. To
test whether activation of G proteins is sufficient for induction of
histamine responsiveness, F9 EC cells were injected with 10 µM GTP
S. This procedure was indeed sufficient for
inducing histamine responsiveness, as assayed with whole-cell patch
clamp (Fig. 5B), whereas
GDP
S injection did not produce this effect. The GTP
S-induced
histamine responsiveness was, however, of a highly transient nature
(Fig. 5C), maybe due to GTP
S-dependent
activation of histamine signaling elements, making further stimulation
of these signaling elements by the receptor impossible. In agreement,
impalement of cells with GTP
S-containing pipettes provoked strong
currents, indicating activation of such signaling elements by GTP
S.
GTP
S-induced histamine responsiveness was eliminated by the
H1 receptor antagonist pyrilamine (Fig. 6) but not by cimetidine (a
H2 receptor antagonist), demonstrating that this histamine
responsiveness is mediated by the H1 receptor. Treatment of
cells with 20 mM NaF and 75 µM
AlCl3 (which potently activates G proteins) led within 5 min after application to a slow but sustained increase in intracellular
Ca2+ (Fig. 5A), probably due to activation of
Ca2+-mobilizing G protein-dependent signaling
elements. Importantly, such a treatment also induced
histamine-dependent Ca2+ responses on top of
the aforementioned sustained increase in intracellular Ca2+
levels within 20 min after application of AlF3 (Fig.
5A; Table I). We concluded that activation that activation
of pertussis toxin-sensitive G proteins is implicated in the induction
of histamine responsiveness.

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Fig. 5.
Induction of histamine responsiveness in F9
EC cells by AlF4 and
GTP S. A shows a representative Ca2+ tracing
of the effect of 20 mM NaF and 75 µM
AlCl3 on the responsiveness to histamine (HA)
added 6 min later. Pretreatment with NaF and AlCl3
permitted a Ca2+ response to histamine in 10 out of 20 experiments. B shows the response to 0.1 mM
histamine on transmembrane currents (whole cell patch clamp) when 10 µM GTP S was included in the patch pipette solution.
Histamine was added 40 s after impalement. The relation between
the response to histamine and the time interval after impalement is
plotted in C.
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Fig. 6.
Effect of pyrilamine on histamine-induced
transmembrane currents in GTP S-injected F9 EC cells. Cells were
impaled with 20 µM GTP S containing whole cell
electrodes for 40 s in the presence of several concentrations of
extracellular pyrilamine and subsequently stimulated with 0.1 mM histamine.
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Induction of Histamine Responsiveness Coincides with the Appearance
of High Affinity [3H]Pyrilamine Binding--
To further
explore the processes underlying the induction of histamine
responsiveness, Scatchard analysis was performed. Surprisingly, in
unstimulated F9 EC cells, no high affinity [3H]pyrilamine
binding was observed (n = 7; Fig.
7A), in contrast to P19 EC
cells (which react unconditionally to histamine), which exhibited high
affinity binding of [3H]pyrilamine (kd
7 nM; n = 2). In accordance, whole cell
membrane preparations of P19 EC cells displayed high affinity binding
of [3H]pyrilamine, but no such binding could be detected
in F9 EC cells. These results suggest that the failure of F9 EC cells
to react to histamine under uninduced conditions is due to the absence of high affinity histamine binding activity, and that induction of
histamine responsiveness is caused by a rapid increase of high affinity
histamine binding sites on the plasma membrane. Indeed, introduction of
GTP
S into the cells rapidly induces [3H]pyrilamine
binding with a Kd of 19 ± 4 nM and
a Bmax of 0.15 ± 0.02 pmol/106
cells (± S.E.; n = 3; Fig. 7). Also, treatment of
nonpermeabilized cells with 20 mM NaF and 75 µM AlCl3 induces high affinity
[3H]pyrilamine binding with an apparent
Kd of 24 ± 3 nM and a
Bmax of 0.31 ± 0.12 pmol/106
cells (n = 4; Fig. 7B). As our observations
with regard to the transient nature of the induced histamine
responsiveness predicted a transient induction of
[3H]pyrilamine by external sensitizing stimuli like
leukotrienes and AlF3, we also tested whether such
transient [3H]pyrilamine binding to cells could be
demonstrated. At 37 °C, these experiments yielded rather variable
results, probably because only after 1 h of labeling,
[3H]pyrilamine binding to cells reaches equilibrium,
whereas induction of histamine responsiveness at this temperature by
such stimuli is short-lived. When experiments were performed at lower
temperatures, however, transient induction of [3H]
pyrilamine binding became apparent (Fig.
8). Together our observations strongly
suggest that a pre-stimulus-induced change in receptor conformation,
resulting in a highly increased affinity for histamine, underlies the
observed regulation of H1 receptor action in F9 EC cells.
Therefore, these results define a hitherto undescribed mechanism
controlling H1 receptor function.

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Fig. 7.
Induction of high affinity
[3H]pyrilamine binding sites by GTP S (A)
and AlF4 (B).
A shows a Scatchard plot representing the binding of
[3H]pyrilamine to streptolysin O (0.5 IU/ml in 100 mM KCl, 5.6 mM glucose, 1 mg/ml bovine serum
albumin, 1.3 mM CaCl2, 2 mM EGTA, 0.1 mM MgCl2, 1 mM ATP, 10 mM Hepes, pH 7.2) permeabilized F9 EC cells (open
circles) and permeabilized F9 EC cells treated with 20 µM GTP S (filled circles). The solid
line indicates the induced high affinity binding, whereas the
dotted line indicates the constitutive nonspecific low
affinity binding (16). B, displays a Scatchard plot of
[3H]pyrilamine binding to unpermeabilized cells
(open circles) and to unpermeabilized cells treated with NaF
and AlCl3 (filled circles). The solid
line indicates the induced high affinity binding, whereas the
dotted line indicates the constitutive nonspecific low
affinity binding. Scatchard analysis and fitting was performed as
described earlier (16).
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Fig. 8.
Transient induction of high affinity
[3H]pyrilamine binding in intact F9 EC cells by
sensitizing stimuli. Our observations with regard to the transient
nature of the induced histamine responsiveness predicted a transient
induction of [3H]pyrilamine by external sensitizing
stimuli like leukotrienes and AlF3, without the need for
cell permeabilization. We tested whether such transient
[3H]pyrilamine binding to intact cells could be
demonstrated. Cells incubated with 4 nM
[3H]pyrilamine in the presence (filled circles,
solid line) or absence (open circles, dotted line) of
the sensitizing agent at 4 °C for the time periods indicated. To
determine high affinity [3H]pyrilamine binding, the
amount of label nondisplaceable with 100 µM cold
pyrilamine was subtracted. Under these conditions, transient induction
of high affinity [3H]pyrilamine binding becomes visible.
[3H]Pyrilamine bound at each time point is the total
specifically bound expressed as a percentage of total unbound and bound
at the same time.
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DISCUSSION |
In the present study we show that H1 receptor
signaling in F9 EC cells and ES5 cells required prestimulation of the
cells with either fetal calf serum, serotonin, leukotriene
D4, or leukotriene E4, whereas bradykinin
and ATP did not produce this effect. This sensitizing effect was
inhibited by pertussis toxin, whereas it was mimicked by GTP
S and
AlF4
. Therefore, pertussis
toxin-sensitive G proteins are probably mediating this regulation of H1
receptor action. The molecular basis for the induction H1
receptor responsiveness appears to be the appearance of high affinity
ligand binding sites, as control cells did not display high affinity
[3H]pyrilamine binding, but introduction of GTP
S or
treatment with AlF4
immediately
provoked such high affinity [3H]pyrilamide binding sites.
These findings strongly suggest that histamine responses in these cells
are controlled by agents that induce high affinity binding sites for
histamine and define a molecular mechanism by which external stimuli
can control histamine H1 receptor action.
The molecular details, however, by which external stimuli enable the
H1 receptor to interact with its ligand, remain unclear. An
explanation for the impaired H1 receptor function in
unstimulated cells may be a physical impossibility for histamine to
interact with its receptor. Generally, receptors may be continuously
recycled between plasma membrane and endosomes. Although this process
has not been reported for H1 receptors, it has been found
to occur with several other G protein-coupled receptors
(e.g. Ref. 22), including the H2 receptor (7),
and some of the signaling elements involved have been identified
(e.g. Refs. 23 and 24). It is conceivable that in cell types
that require sensitization for histamine responsiveness, the balance
between endosome and plasma membrane localization is shifted to the
endosomal state, and that sensitization releases this shift. Such a
scheme would imply that in unstimulated F9 EC and ES5 cells the large
majority of histamine receptors has an endosomal location. In other
cells types, however, which show unconditional histamine responses, the
balance between endosomal and plasma membrane-localized receptors
should be shifted in favor of a plasma membrane location. In this
context it is interesting to note that pertussis toxin-sensitive G
proteins have been implicated in the stimulation of vesicle fusion
(e.g. Refs. 25-27) and that rab3-mediated exocytosis from
mast cells is pertussis toxin-sensitive (28). Such a mechanism,
however, can not explain the absence of high affinity binding of
[3H]pyrilamine in whole cell membrane preparations of
uninduced F9 EC cells, prompting alternative explanations for induction of histamine responses in these cells.
Therefore, control of the histamine response by SH-FCS, leukotrienes,
and serotonin may be mediated by the induction of a conformational
change of the histamine H1 receptor, resulting in increased
ligand affinity. Our experiments using the phosphatase inhibitor
okadaic acid in F9 EC cells implicate a serine/threonine phosphorylation event in the stimulus-dependent
sensitization of the histamine response, opening the possibility that a
phosphorylation of the receptor underlies this conformational change.
In agreement, mutational analysis of the H2 receptor has
shown that relatively small changes in the intracellular domain can
have profound influences on ligand affinity (7), and the primary
sequence of the H1 receptor contains a number of serine and
threonine residues that may serve as potential phosphorylation sites
for an affinity controlling kinase (Yamashita et al. (30)).
Such a mechanism would contrast the proposed regulation of rhodopsin
and the a2-adrenergic receptor, where receptor
phosphorylation is associated with deactivation (29). Further
biochemical and mutational characterization of the H1
receptor is required to determine whether specific phosphorylation sites are involved in the regulation of the receptor affinity and
activity.
H1 receptor activation has been implicated in processes
like inflammation and anaphylaxis, and therefore H1
receptor action must be carefully regulated. It is to be expected that
mechanisms have evolved for controlling the signaling by the
H1 receptor. The regulation of histamine responsiveness as
described in the present study provides such a control mechanism
because for stimulation of the H1 receptor in these cells,
both a pre-stimulus as well as histamine are necessary. Interestingly,
in pathological conditions like asthma and allergy, exaggerated
histamine reactivity is associated with the formation of cysteinyl
leukotrienes (13) and serotonin (15), but no molecular details are
known. The findings described in this study for inducing histamine
responsiveness define for the first time a molecular mechanism by which
such control of histamine reactivity may be exerted, but further
studies are required to assess the importance of this mechanism in
these pathological conditions. These studies are currently under
progress.
 |
ACKNOWLEDGEMENTS |
The authors thank Gert Folkers and the other
members of our laboratories for stimulating discussions.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
These authors contributed equally to this work.
Supported by a fellowship from the Koningin Wilhelmina Fonds
(KWF) and to whom correspondence should be addressed: Tel.:
31-20-5665910; Fax: 31-20-6977192.
1
The abbreviations used are: EC, embryo
carcinoma; ES, embryonic stem; FCS, fetal calf serum; GTP
S,
guanosine 5
-O-(3-thiotriphosphate); [3H]pyrilamine, [pyridinyl-5-3H]pyrilamine;
DTT, dithiothreitol; GDP
S,
guanosine-5
-O-(2-thiodiphosphate).
 |
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