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Laboratorio de Radioisótopos, Facultad de Farmacia y Bioquímica (F.M., N.F., B.L.L., M.E.R., C.D.) and Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales (C.S.), Universidad de Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina (F.M., N.F., A.B., C.S., C.D.); and Instituto de Biología y Medicina Experimental, Buenos Aires, Argentina (N.F., A.B., C.S.)
Received January 27, 2003; accepted May 13, 2003
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionAppendix 1Appendix 2References |
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s-protein (
2-adrenoreceptor) was impeded
by tiotidine. This interference clearly implies that tiotidine biases the
system to Gs-coupled form of the H2 receptor and turns
Gs-protein less available to interact with
2-adrenoreceptor. These findings not only show that tiotidine
is an H2 inverse agonist in U-937 cells but also provide experimental support
for the CTC model.
) in which the receptor
possesses two binding sites, one for the ligand on the extracellular side and
other for the G-protein in the intracellular side. This model is a
generalization of former models, in which only the free receptor and the
receptor ligand complex existed, allowing receptors to interact with ligands
as well as G-proteins. An important advancement in the understanding of GPCRs
was the recognition that they could couple to and functionally activate
G-proteins even in the absence of an agonist. This receptor feature was
suggested by the identification of constitutively activated mutant forms of
several receptors (CAM receptors), such as 1B adrenoreceptor
(Kjelsberg et al., 1992),
thyrotropin receptor (Parma et al.,
1993), and luteinizing hormone receptor
(Shenker et al., 1993). These
experimental results led to the extended ternary complex model (ETC)
(Samama et al., 1993), which
includes two distinct conformational states of the receptor, an active
(R*) and an inactive (R) state, that exist in equilibrium even in
the absence of drugs. This spontaneous equilibrium determines basal activity,
and in CAM receptors, it is driven toward R*, increasing the
response in the absence of drugs. A further modification of the ETC model is
the cubic ternary complex model (CTC) (Weiss et al.,
1996a,b,c),
which completes the ETC model by allowing G-proteins to interact with
receptors in both their active and inactive states (R*G,
LR*G, and RG, LRG). These two models account for the effects of different types of ligands on receptor signaling. Agonists increase receptor activity, neutral antagonists have no effect on it, and inverse agonists are able to reduce the activity of receptor systems that are active in the absence of agonists.
Although initially drugs were classified as agonists and neutral
antagonist, now it is considered that ligands have an efficacy ranging from
agonism through neutral antagonism to inverse agonism. In fact, many drugs
with important therapeutic actions that were originally described as neutral
antagonists are actually inverse agonists. Examples of this are cimetidine
[selective histamine H2 receptor (H2r) ligand]
(Smit et al., 1996),
haloperidol (which acts on dopamine D2 receptors)
(Hall and Strange, 1997),
prazosin (1-adrenoreceptor ligand)
(Rossier et al., 1999),
timolol (which acts on 2-adrenoreceptor)
(Chidiac et al., 1994), and
clozapine (which acts on D2 and 5-HT2C receptors)
(Westphal and Sanders-Bush,
1994; Hall and Strange,
1997).
H2r have the regulatory functions of histamine during cell proliferation,
gastric acid secretion, airway and vascular smooth muscle relaxation and
immune responses (Del Valle and Gantz,
1997). Furthermore, some histamine H2 antagonists are effectively
used as therapeutical agents, such as ranitidine, which is effective in the
modulation of gastric acid secretion. Taking into account that treatment with
antagonists would not be effective for several pathologies associated with
point mutations in GPCRs that lead to increased basal receptor activity and,
on the other hand, that long-term administration of some inverse agonists
leads to the development of tolerance and increased sensitivity after
withdrawal, it is highly important to differentiate between neutral
antagonists and inverse agonists.
The presence of H2r showing typical pharmacological profile and
G-protein-mediated adenylyl cyclase stimulation has been described in U-937
promonocytic cell line, which make it an adequate model to study H2r signaling
(Davio et al., 1995a). However,
in these cells, tiotidine, previously reported as an H2 specific antagonist,
showed inverse agonist characteristics
(Monczor et al., 1998). The
suitability of tiotidine has been a controversial issue
(van der Goot and Timmerman,
2000) because of its complex binding since studies carried out in
gastric mucosal cells (Batzri and Harmon,
1986) and kidney membranes
(Rising and Norris, 1985)
revealed the presence of several binding sites for [3H]tiotidine.
On these bases, we aimed to study the atypical behavior of tiotidine in U-937
promonocytic cell line.
We found a two-site binding unusually sensitive to GTP
S and an
inverse agonist behavior that can be explained only in terms of the cubic
ternary complex model. These observations suggest the existence of an inactive
receptor species coupled to G-protein. These observations shed new light into
the pharmacological classification of H2 antagonists and may offer a plausible
explanation for the results previously reported by other authors and us.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionAppendix 1Appendix 2References |
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Plasmid
H2r was previously cloned into the eukaryotic expression vector pCEFL
(Shayo et al., 2001). Plasmid
purification was performed using reagents from QIAGEN (Valencia, CA) according
to the manufacturer's instructions.
Cell Culture
U-937 and COS-7 cells were cultured at 37°C in a humidified atmosphere
with 5% CO2 in RPMI 1640 medium and Dulbecco's modified Eagle's
medium, respectively, supplemented with 10% fetal calf serum and 50 µg/ml
gentamicin.
Transient Transfection of COS-7 Cells
Transient transfection was performed by the DEAE-dextran technique
(Shayo et al., 2001). COS-7
cells plated in 35-mm dishes were transfected at 80% confluence using 1 µg
of plasmid. Assays were performed 48 h after transfection.
Stable Transfection of U-937 Cells
Cells were transfected as reported previously
(Fernández et al.,
2002). Briefly, cells were harvested by centrifugation from
cultures in exponential growth phase, washed once in phosphate-buffered
saline, and resuspended at 106 cells/ml in fresh RPMI 1640 medium
on ice. Linearized pCEFL-rH2 (10 µg) with SalI was added to cell
suspension (400 µl) and kept on ice for 5 min. The cells and DNA were then
subjected to a pulse of 150 V at a capacitance of 250 µF using a Bio-Rad
"Gene Pulser". Cells were returned to ice for 5 min and
nonselective medium was added overnight. The next day, cells were plated on a
96-well culture tray, switching the medium to RPMI 1640 containing 0.8 mg/ml
G-418. After 2 to 3 weeks, the surviving clones were amplified.
cAMP Assay
Suspension Cells. Cells in Hanks' solution were supplemented with 1
mM IBMX, at a density of 106 cells/ml, preincubated for 3 min at
37°C, and exposed for 9 min to different ligand concentrations. The
reaction was stopped by a 3-min centrifugation at 3,000g. For cAMP
extraction, ethanol was added to the pellet and further centrifuged 5 min at
3000g.
COS-7 Cells. Cells were incubated 3 min in Dulbecco's modified Eagle's medium supplemented with 1 mM IBMX at 37°C, and exposed for 9 min to the ligand at the indicated concentrations. Cells were then washed with phosphate-buffered saline and subjected to ethanol extraction followed by a 5-min centrifugation at 3,000g.
The ethanol phase was dried and resuspended in 50 mM Tris-HCl, pH 7.4, and
0.1% bovine serum albumin. cAMP content was determined by competition of
[3H]cAMP for protein kinase A, as described previously
(Davio et al., 1995b).
Radioligand Binding Assay
Suspension Cells. Triplicate assays were performed in polyethylene
tubes in 50 mM Tris-HCl, pH 7.4. For saturation studies, increasing
concentrations of [3H]tiotidine were incubated with 106
cells/tube in the absence or presence of 1 mM histamine in a total volume of
200 µl. After 40 min at 4°C, incubation was stopped by dilution with 3
ml of ice-cold 50 mM Tris-HCl, pH 7.4; rapid filtration onto Whatman GF/B
glass-fibers filters was performed under reduced pressure, followed by three
washes with 3 ml of ice-cold buffer.
COS-7 Cells. Triplicate assays were performed in COS-7 transfected cells in 24 multiwell plates. For saturation studies, increasing concentrations of [3H]tiotidine were incubated in the absence or presence of 1 µM tiotidine in a total volume of 200 µl of 50 mM Tris-HCl, pH 7.4. After 40 min at 4°C, incubation was stopped by dilution with 3 ml of ice-cold 50 mM Tris-HCl, pH 7.4, followed by washes with ice-cold buffer.
Experiments on intact cells were carried out at 4°C to avoid internalization of the ligand. Kinetic studies showed that equilibrium was reached after 30 min and persisted for 4 h (data not shown).
Membrane-purified fraction was obtained by cell sonication in 50 mM Tris-HCl buffer, pH 7.4, and centrifuged for 15 min at 8,500g; the supernatant was further centrifuged for 15 min at 30,000g. The pellet obtained was resuspended in an adequate volume.
The membrane fraction was pretreated at 37°C with 10 µM GTPS
for 2 h in Hanks' medium.
Analytical Methods
Binding data were analyzed using a weighted, nonlinear, least-squares
program that determines binding to multiple sites using the law of mass action
(LIGAND program) as detailed previously
(Munson and Rodbard, 1980).
Sigmoidal dose-response fittings were done using Prism version 3.00 for
Windows (GraphPad Software, San Diego, CA).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionAppendix 1Appendix 2References |
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Binding assays using [3H]tiotidine, a specific H2r ligand (described previouslyas antagonist), revealed the presence of two binding sites, one with higher affinity and lower capacity (Kd1 = 2.2 ± 0.8 nM; Q1 = 2,000 ± 430 sites/cell) and the other with lower affinity but higher capacity (Kd2 = 20 ± 3 nM; Q2 = 20,000 ± 1800 sites/cell) (Fig. 1). Similar results were obtained when membrane fractions were used (Fig. 2A).
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Because tiotidine specifically binds H2rs, these results can be explained in terms of tiotidine binding to two distinct receptor states. It is worth noting that in competitive binding experiments performed with [3H]tiotidine and various concentration of unlabeled tiotidine, famotidine, and BU-E-75, the maximal inhibition of [3H]tiotidine binding by each compound did not differ significantly from that obtained in the presence of 10 µM tiotidine (data not shown). These results verify that tiotidine-labeled sites are histamine H2 sites.
In assays performed on membrane fractions of cells previously incubated
with 10 µM GTPS, the Scatchard plot showed only one binding site
corresponding to the low affinity site. As it can be observed, the number of
sites with low affinity is equal to the total amount of sites without
GTPS showed in Fig. 1
(Kd = 20 ± 3 nM, Q = 22,000 ± 1,900
sites/cell 
Q1 + Q2) (Fig.
2A).
These data suggest that the high-affinity site corresponds to a subset of
receptors coupled to G-protein, because it disappeared in the presence of
GTPS. This is supported by the fact that binding assays in cells
pretreated for 2 h with H2 agonist, which induces H2r desensitization or
uncoupling from G-protein, only showed the site with low affinity
(Fig. 2B). Based on classic
ligand-receptor occupancy models (the ETC model of
Samama et al., 1993) in which
a ligand that has more affinity for the receptor coupled to G-protein
(R*G) instead of receptor alone (R or R*) necessarily
bias the system to a response-evoking receptor state (LR*G), we can
consider the tiotidine binding profile to be expected for an agonist. This
conception is in opposition to the well established idea of tiotidine as the
reference H2 antagonist. Therefore, we attempted to asses the ability of
tiotidine to induce a second messenger signal and to block an H2
agonist-induced response.
Tiotidine Dose-Response Assays. In dose-response experiments carried out with BU-E-75, a specific H2 agonist, tiotidine not only produced a right shift on the EC50 (700 ± 120 nM versus 1,900 ± 300 nM) but also diminished the maximal response achieved with the agonist from 212.8 ± 7.7 pmol/106 cells to 112.8 ± 6.7 pmol/106 cells, suggesting that tiotidine behavior is more complex than expected for a neutral antagonist. We next evaluated the effect of tiotidine on cAMP accumulation through H2r by dose-response assays. Tiotidine is able to reduce cAMP basal levels in a dose-dependent manner, from 7.0 ± 0.4 pmol/106 cells to 1.3 ± 0.2 pmol/106 cells with an EC50 = 11.5 ± 4.5 nM (Fig. 3, A and B).
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To improve the sensitivity to this reduction in basal levels, different
strategies that allow increasing spontaneous activity so that the negative
effect of inverse agonists becomes clearly evident have been described. To
achieve this, there are at least two experimental approaches; the first one
involves the addition of a chemical agent that increases second messenger
levels and the second one consists of receptor transfection in an
overexpression system (Kenakin et al.,
1995).
The first approach is based on evidence showing that inactivation of
Gs subunits decreases forskolin-induced cAMP levels. Leurs and
coworkers (2002) proposed the
modulation of the forskolin response by H2rs as a quite sensitive and useful
tool to study inverse agonism at H2rs. The last approach is based on evidence
showing that when the receptor number is increased, there is a concomitant
increase in the likelihood of spontaneous receptor coupling to G-protein,
which in turns leads to an increase in basal levels
(Milligan, 1996).
We carried out tiotidine dose-response experiments in forskolin pretreated U-937 cells and in the heterologous system of H2r-transfected COS-7 cells. In both assays, we observed an evident decrease in cAMP levels when cells were treated with increasing tiotidine concentrations (Fig. 4, A and B). Similar results were obtained with U-937 cells overexpressing H2rs, indicating that this effect was not dependent on an intrinsic property of the COS-7 heterologous system (Fig. 4C). It is worth noting that in both COS-7 and U-937 cells overexpressing H2r, [3H]tiotidine binding profile is the same that in U-937—naive cells (data not shown).
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Up to this point, the results obtained in dose-response experiments provide enough evidence to consider tiotidine as an inverse agonist. However, we can associate its binding profile with the characteristic for agonists. As can be observed, the results from binding assays disagreed with what we expected based on the classic models for inverse agonists and challenged us to analyze them using another model capable of explaining them.
Cubic Ternary Complex Receptor-Occupancy Model: a Possible Explanation
for Tiotidine Behavior. The only described model capable of predicting a
higher affinity for the receptor G-protein complex even for an inverse agonist
is the CTC model (Fig. 5). In
this way, if we consider that the species able to generate physiological
responses are R*G and LR*G, we can define a normalized
factor called f* as the addition of that species over the
total receptor amount:
 | (1) |
 | (2) |
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If we represent f* as a function of log [L],
we can simulate a theoretical dose-response assay for a given set of
parameters. Among all parameters, 
describes the extent to which the
joint effect of any two receptor modifications (i.e., receptor activation,
G-protein coupling, and ligand binding) varies in relation to the third. In
other words, it gives an idea of the interaction or synergism between two
modifications in the receptor state over the third one. Considering that the
ligand facilitates receptor activation ( > 1), that the active
receptor has more affinity for G protein than the inactive form ( >
1), and that the ligand improves G protein coupling ( > 1), we are
in the presence of an agonist for the ETC model.
However, by varying value in the CTC model, the ligand can behave
as an agonist, an antagonist, or even as an inverse agonist
(Fig. 6). In the same way, we
can deduce another set of equations that describe the variation of each
possible receptor state concentration (R, R*, RG, R*G,
LR, LR*, LRG, LR*G) as a function of ligand dose.
Keeping constant the parameters mentioned above and giving a value
that makes the ligand work as an inverse agonist, we can observe in
Fig. 7, A and B, that there is
a significant increase in receptor species bound to ligand but unable to evoke
a biological response ([LRG] and [LR]).
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If tiotidine biases the system to a G-protein—coupled but inactive
H2r state, it can be inferred that it may interfere with the signal
transduction pathway of an unrelated GPCR that also signals through a
Gs-protein. We tested this hypothesis by stimulating U-937
cells with a 2-adrenoreceptor agonist (isoproterenol) and
variable doses of tiotidine. At isoproterenol EC50 (50 nM),
tiotidine significantly diminishes isoproterenol-induced cAMP accumulation in
a dose-dependent manner in more than a 60%
(Fig. 8). These results agree
with our prediction that tiotidine should interfere with the transduction
cascade of an unrelated GPCR signaling through a Gs-protein,
such as the 2-adrenoreceptor.
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We next evaluated the binding profile for a ligand with the parameters established above. To do that, we had to consider first that the affinity constant detected in a binding assay is an apparent constant, because we can measure only the fraction of receptors bound to ligands that also involves species uncoupled to G-protein ([LR], [LR*]) and coupled to G-protein ([LRG], [LR*G]). Therefore, even in this case, in which we can differentiate between receptor states coupled and uncoupled to G-protein, we have to measure the ligand affinity for more than one receptor species.
If [R]B is the receptor bound to ligand and
[R]U the free receptor concentrations, we can define an
affinity constant:
 |
 |
Using this equation, the apparent affinity of the ligand for the receptor
species coupled (KappG) and uncoupled
(KappN) to G-protein can be calculated
as follows:
 |
value for which the theoretical relation
KappG/KappN
(8.4) is quite similar to that experimentally obtained for
[3H]tiotidine binding (9.1). This implies that a radiolabeled
inverse agonist can label the uncoupled states with lower affinity and the
coupled states with higher affinity, although it fails to signal. |
Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionAppendix 1Appendix 2References |
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S or in desensitized cells and 2) tiotidine leads to a
dose-dependent decrease in cAMP basal levels in U-937 cells, an effect that is
clearly evident in both homologous and heterologous overexpression systems, as
well as in forskolin pretreated cells. Each of these points is discussed, and
our results are then interpreted with the aid of the CTC model.
First of all, tiotidine was initially described as a potent H2 antagonist
with reported pA2 values of 7.3 to 7.8 (guinea pig right atrium)
(van der Goot et al., 1991).
It was considered a selective tool to establish interactions with the H2r
because of its low affinity for both the H1 and the H3 receptors
(van der Goot and Timmerman,
2000). On the other hand, binding of [3H]tiotidine to
gastric mucosal cells revealed the presence of several binding sites
(Batzri and Harmon, 1986).
Therefore, the use of tiotidine for selectively labeling peripheral H2rs was
considered rather limited. However, these findings can be explained with our
first observation that tiotidine recognizes with different affinities the
G-protein—coupled and -uncoupled forms of the H2r, as can be inferred
from the vanishing of the high-affinity binding site in the presence of
GTPS. This result was confirmed by studies in desensitized cells in
which pretreatment with an H2 agonist that promotes a rapid receptor
desensitization or uncoupling from heterotrimeric G-protein
(Lemos et al., 2000) also
leads to a single binding site for [3H]tiotidine.
GPCRs are affected by various receptor-specific ligands that modulate the GPCR activity in distinct ways. Originally, antagonists were thought to compete with the agonist for the same binding site but not to affect the GPCR activity directly. With the identification of CAM receptors, it has become clear that antagonists should be reclassified as neutral antagonists and inverse agonists.
Considering as an inverse agonist every ligand that is able to reduce the
activity of receptor systems that are active in the absence of agonist, and
taking into account our second observation, that tiotidine reduces cAMP basal
levels in naive and surrogate systems, we believe that this ligand has to be
reclassified as an H2 inverse agonist. Knowing that tiotidine may be regarded
as an analog of cimetidine, a previously described H2 antagonist that was
reclassified as an H2 inverse agonist
(Smit et al., 1996), it is not
surprising that this compound, which shares most of its structure with
cimetidine, has a similar behavior.
It is widely assumed that inverse agonists stabilize certain states of the receptor with different functional activities. Based on the ETC model, they may act by binding to the R state of the receptor in preference to the R* state. Alternatively, they could bind to uncoupled states of the receptor (R and R*) in preference to the coupled state (R*G). A third possibility is that inverse agonists bias the receptor to an inactive conformation that can exist in G-protein coupled and uncoupled forms. The CTC model contains an inactive receptor conformation that can nevertheless couple to G-protein and makes it a suitable model to explain our findings about both tiotidine binding and its negative efficacy. To better explain what we discussed above, it is worth considering that for the ETC model, a ligand with high affinity for receptor species coupled to G-protein necessarily elicits a response. In contrast, the CTC model implies the existence of a receptor state coupled to G-protein, which is unable to evoke a response (RG), allowing a ligand with high affinity for the receptor form coupled to G-protein to behave as an antagonist or even as an inverse agonist (Fig. 6). This point is a distinctive feature of the CTC model.
Although the development of the ETC model was made necessary by experimental observation, the CTC model was originally proposed in a attempt to explore the mathematical and pharmacological implications that can be derived from permitting G-proteins to interact with receptors in their inactive and active forms. Thus, the CTC model was the culmination of a trend in increasing model complexity and statistical and thermodynamic completeness. However, our experimental findings can be explained solely in terms of the CTC model, justifying the use of this heuristic model despite its complexity. The main implication of the present study is that it provides experimental support for the CTC model, showing that it has a practical use in addition to its theoretical use.
It is worth considering that another group obtained similar results for the
CB1 cannabinoid receptor, which, after stimulation with a specific ligand, can
couple to Gi/o-protein without evoking a response
(Bouaboula et al., 1997).
Moreover, Brown and Pasternak
(1998) showed that the binding
of a specific µ-opioid receptor ligand, functionally classified as an
antagonist, labels a G-protein—coupled state of the receptor with high
affinity. These observations could also be explained using the CTC model.
In terms of the CTC model, the fact that tiotidine interferes with the
signal transduction pathway of the 2-adrenoreceptor, which also involves
Gs-protein, implicates that tiotidine is biasing the system to a
G-protein—coupled form of the H2r and making the G-protein less
available to other GPCRs. This result supports the speculations based on our
theoretical simulations considering a limiting G-protein concentration.
Alewijnse et al. (2000)
showed that, as seen for CAM receptors, the affinity of histamine for both
R116A and R116N arginine H2r mutants was significantly increased over that of
the wild-type receptor. However, this increase in agonist affinity was not
accompanied by an increase in constitutive receptor activity. In contrast,
basal cAMP levels of both arginine mutant H2rs were significantly decreased
compared with the wild-type receptors. These mutant receptors seemed to be in
an active conformation, but the ability to couple to or activate G-protein was
decreased. We speculate that the form of the receptor obtained in the presence
of tiotidine resembles the conformation of these mutated H2rs, which has a
high affinity for G-protein but fails to evoke a response.
Taking into account that the ligand-receptor interaction is very complex, a
further consideration is to analyze our results in terms of the proposed
existence of receptor protein as collections of microconformational states
that might be pharmacologically relevant (ensemble theory;
Hilser et al., 1998). In this
context, it is tempting to speculate that tiotidine behaves as a ligand that
preferentially binds to a microconformation coupled to G-protein but inactive,
biasing the system to a non—response-evoking state. This possibility
could be partly a restatement of the CTC model, because it might provide a
conformational explanation for the receptor species present in it.
These results, which enlarge the knowledge about inverse agonists, not only lead to the reclassification of tiotidine as an H2 inverse agonist with an unusual behavior but also provide evidence to consider the CTC an experimentally supported model.
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Appendix 1 |
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TopAbstractMaterials and MethodsResultsDiscussionAppendix 1Appendix 2References |
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Taking into account Eq. 1 (see Results) and considering the
model-assumed equilibrium, its related constants,
 |
 |
 |
 |
 |
 | (3) |
 | (4) |
 | (5) |
 | (6) |
 | (7) |
 | (8) |
 | (9) |
 | (10) |
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Appendix 2 |
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TopAbstractMaterials and MethodsResultsDiscussionAppendix 1Appendix 2References |
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: GPCR, G-protein coupled receptor; CAM, constitutively
activated mutant; ETC, extended ternary complex model; CTC, cubic ternary
complex model; H2r, histamine H2 receptor; GTPS, guanosine
5'-O-(3-thio)triphosphate; RPMI, Roswell Park Memorial
Institute; IBMX, 3-isobutyl-1-methylxanthine; BU-E-75,
(±)-N1-[3-(3,4-difluorophenyl)-3-(pyrid-2-yl)propyl]-N2-[3-(1H-imidazol-4-yl)propyl]guanidine.
Address correspondence to: Dr. Davio Carlos, Laboratorio de Radioisótopos, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires. Junin 956 PB, 1113, Capital Federal, Argentina. E-mail: cardavio{at}ffyb.uba.ar
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) or not (
) with 10 µM GTP
