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Vol. 61, Issue 1, 85-96, January 2002
s- and Gi/o-Proteins
Department of Receptor Pharmacology, Janssen Research Foundation, B-2340 Beerse, Belgium
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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This study documents differences in ligand binding and signal
transduction properties between the human (h) 5-hydroxytryptamine (5-HT)4a and h5-HT4b receptor splice variants
stably expressed in human embryonic kidney 293 cells. The fraction of
the [3H]5-HT high-affinity site relative to the whole
receptor population measured with [3H]GR113808 was higher
for the h5-HT4a isoform (around 0.4) than for the
5-HT4b isoform (around 0.2) and was independent of the level of expression. The potency and efficacy of reference compounds tested for the cAMP response differed slightly but significantly between both variants. Most remarkably, 5-methoxytryptamine and prucalopride were found more potent on the 5-HT4b variant,
whereas SDZ-HTF 919 and SB204070 were more potent on the
5-HT4a variant. Guanosine-5'-O-(3-[35S]thio)triphosphate
binding on membranes and cAMP assays in whole cells revealed that only
the h5-HT4b isoform coupled to G
i/o-proteins in addition
to its well-documented Gs coupling. In contrast, the
h5-HT4a receptor coupled only to Gs-proteins, however,
was able to trigger an increase in the intracellular calcium
concentration ([Ca2+]i). The observed
[Ca2+]i increase did not occur through
inositol phosphate formation and was not sensitive to Bordatella
pertussis toxin, forskolin, or 3-isobutyl-1-methylxanthine
(pre)treatment but was due to Ca2+ influx from the
extracellular environment. Interestingly, the Ca2+ pathway
was dependent on high receptor expression levels and was
compound-specific, because benzamide-like compounds triggered two to
three times higher responses than indoleamines. Taken together, these
data provide the first evidence for fine functional differences between
C-terminal splice variants of the h5-HT4 receptor, which may contribute to a better understanding of the functional diversity of
this receptor class.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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The
ubiquitous neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) has
so far been shown to interact with seven receptor classes, classified
as 5-HT1 to 5-HT7
receptors. Three of them were defined as Gs-protein-coupled
receptors: the 5-HT4,
5-HT6, and 5-HT7 receptors.
In this study, we focus on the functional properties of the
5-HT4 receptor class, in which a new level of structural diversity has been highlighted in recent years.
The wide distribution of 5-HT4 receptors, from
the central nervous system to the peripheral tissues, suggests a wide
range of functional roles. In the central nervous system,
5-HT4 receptors have been localized in rat basal
ganglia, hippocampus, and olfactory tubercule (Compan et al., 1996
;
Vilaro et al., 1996). In human brain, radioligand binding (Domenech et
al., 1994; Reynolds et al., 1995) and in situ hybridization studies
(Bonaventure et al., 2000) have shown predominant expression of
5-HT4 receptors in basal ganglia and limbic
structures. Functionally, central 5-HT4 receptors
have been implicated in diverse processes such as anxiety, memory, and
cognition (Silvestre et al., 1996; Fontana et al., 1997).
5-HT4 receptors have also been shown to be widely
distributed in peripheral organs such as the gastrointestinal tract,
the myocardium, the urinary bladder, and the adrenal glands (for
review, see Ford and Clarke, 1993). Furthermore, pharmacological
differences have been identified in various tissues and species,
suggesting a heterogeneity of 5-HT4 receptors
(Ford and Clarke, 1993; Leung et al., 1996). Indeed, Gerald et al.
(1995) have initially cloned two alternative splice variants coding for
short (5-HT4S) and long
(5-HT4L) isoforms of the receptor, which could be
the structural basis for functional diversity. So far, seven receptor
variants that differ in their C termini have been identified:
5-HT4a (previously named
5-HT4S), 5-HT4b (previously
named 5-HT4L), 5-HT4c,
5-HT4d, 5-HT4e,
5-HT4f, and 5-HT4g
(Claeysen et al., 1997; Blondel et al., 1998; Bender et al., 2000).
Moreover, by cloning the human 5-HT4 receptor
gene, our group has recently shown the existence of an additional site of alternative splicing (5-HT4h), leading to an
extended second extracellular loop that could theoretically combine
with each C-terminal variant (Bender et al., 2000). Interestingly, the
only experimentally isolated h5-HT4hb-receptor
variant showed an altered functional pharmacology because the
prototypic 5-HT4 receptor antagonist GR113808
exhibited partial agonistic properties. This finding supported the
hypothesis of the existence of a functional diversity among
5-HT4 receptor splice variants. In addition, we have recently shown a differential tissue distribution of the h5-HT4 receptor isoforms (Bender et al., 2000),
which may also contribute to tissue-specific functional differences. To
date, all isoforms have been shown to activate adenylyl cyclase (AC) in
vitro, and no difference in signal transduction between C-terminal 5-HT4 receptor variants has been demonstrated.
Although the functional significance for the existence of
5-HT4 receptor splice variants is currently not
well understood, studies on other receptors suggest that different C
termini may discriminate between signal transduction pathways.
G-protein selectivity was shown to be different for the C-terminal
splice variants of the prostaglandin E receptor EP3 (Satoh et al.,
1999), whereas somatostatin receptor isoforms have been found to differ
in the coupling efficiency and desensitization events (Vanetti et al.,
1993). To better understand the functional significance of
5-HT4 receptor isoforms, we investigated the
pharmacological profile and signal transduction of
h5-HT4a and h5-HT4b
receptors, two initially cloned C-terminal splice variants.
We have found major differences between the two variants, not only in the pharmacology of the cAMP response and the fractions of agonist high-affinity sites, but also in the selection of the G-proteins activated by the receptors and the second messengers triggered by agonists. Interestingly, this differential signaling was receptor variant and compound specific. These data provide further evidence for functional differences between 5-HT4 receptor isoforms, underscoring a physiological relevance for the existence of 5-HT4 receptor splice variants.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials.
[3H]GR113808 (specific
activity of 3.7 TBq/mmol),
5-hydroxy[3H]tryptamine triacetate
([3H]5-HT, specific activity of 4.44 TBq/mmol),
and [35S]GTP
S (specific activity of 37 MBq/ml) were obtained from Amersham Biosciences (Little Chalfont,
Buckinghamshire, UK). The mammalian expression vector pcDNA3 was
obtained from Invitrogen (Carlsbad, CA). Dulbecco's modified Eagle's
medium (DMEM), phosphate-buffered saline (PBS), sodium butyrate,
Geneticin (G-418), and calf serum were from Invitrogen. The Bradford
protein assay was performed with the reagent supplied by Bio-Rad
(Nazareth Eke, Belgium). The Adenylyl Cyclase Activation FlashPlate kit
was supplied by PerkinElmer Life Sciences (Brussels, Belgium).
The Bordatella pertussis toxin (PTX) was from Calbiochem (La
Jolla, CA). The liquid scintillation spectrometer, the scintillation
fluids Ultima Gold MV and Ultimaflo AF, and the Unifilter-96 GF/B
plates were from Packard (Meriden, CT). GDP and dilithium salt were
from Hoffman-La Roche (Basel, Switzerland). Fluo3-acetoxymethyl ester
was purchased from Molecular Probes (Leiden, The Netherlands).
Forskolin and probenecid were from Sigma (St Louis, MO). Cisapride and
prucalopride are proprietary compounds from Janssen Pharmaceutica
(Antwerp, Belgium). Renzapride, SB204070, GR113808, SDZ205070, and SDZ
HTF919 were synthesized by Janssen Pharmaceutica for its own
purposes. 5-HT and 5-MeOT were from Acros Organics (Geel, Belgium). For pharmacological testing, compounds were dissolved and diluted in
dimethyl sulfoxide except for indoleamines, which were dissolved in
water and protected from light throughout the experiment. The final
dimethyl sulfoxide concentration never exceeded 0.5% (v/v). The
GraphPad Prism program was from GraphPad Software, Inc. (San Diego, CA).
Stable Transfections and Selection of Monoclonal Cell Lines.
The h5-HT4a and h5-HT4b
receptors were stably expressed in HEK 293 cells. Expression was under
the control of the constitutive cytomegalovirus promoter
provided by the pcDNA3 vector. The calcium phosphate transfection
method was used with modifications as described previously (Lesage et
al., 1998). Stable monoclonal cell lines were created by limited
dilution and selected by [3H]GR113808
radioligand binding after culture under Geneticin (G-418) selection
(800 µg/ml). Receptor expression levels of a range of isolated
monoclonal cell lines were investigated with the antagonist [3H]GR113808 in radioligand binding
experiments; nontransfected HEK 293 cells did not show specific
[3H]GR113808 binding (data not shown). Of the
39 monoclonal cell lines obtained after transfection with the
h5-HT4a receptor, six had expression levels of
about 2000 fmol/mg of protein after treatment with sodium butyrate (see
below). Clone 8 (maximal expression levels ~3100 fmol/mg) was used
for further studies and characterization. Another clone (clone 1) with
a lower expression level (maximal ~1500 fmol/mg) was used to
investigate the contribution of different receptor expression levels in
the functional tests performed in this study. Of the 33 monoclonal cell
lines expressing the h5-HT4b receptor, cell clone
9 had an expression level of about 7000 fmol/mg of protein. Clone 9 was
used for further study and characterization, and as for the other
receptor splice variant, a clone of lower expression level (clone 4, maximal ~2000 fmol/mg) was used to investigate the effect of
different receptor expression levels in the functional tests performed
in this study.
Cell Culture and Treatments. Cells were grown in DMEM containing 10% dialyzed calf serum, 105 IU/l penicillin G, 0.1 g/l streptomycin, 0.1 g/l pyruvate, and 0.292 g/l L-glutamine, under 5% CO2 at 37°C. Cells were grown under selection (800 µg/ml G418) for 48 h every 2 weeks. Culture plates were coated with PBS containing 10 µg/ml poly(L-lysine) for 45 min at 37°C and washed once with PBS before the cells were seeded.
In certain experiments, receptor expression levels were also manipulated using sodium butyrate, which is a histone deacetylase inhibitor found to arrest growth and to induce differentiation in various cell types by modulating gene expression (Archer et al., 1998).
Therefore, sodium butyrate is a "differentiating agent" able to
change expression levels of various proteins. The observed and
described differences between both isoforms have been obtained using
the same conditions in sodium butyrate-treated or nontreated cells. In
addition, we provide data from clones exhibiting lower receptor levels
whenever appropriate to understand the effects. In our experience,
short-term overnight sodium butyrate treatments can be used to boost
receptor expression without altering the pharmacological properties of
the receptor. For sodium butyrate treatment, cells were incubated in
medium containing 5 mM sodium butyrate for 16 to 18 h before
experiments to boost receptor expression before membrane preparation
for radioligand saturation binding, [35S]GTPS binding, and calcium measurements.
PTX was used to prevent the coupling of Gi/o-proteins to the
receptor and was dissolved in 10 mM sodium phosphate, pH 7.0, and 50 mM
NaCl and added to the cell medium overnight before the experiment at a
concentration of 100 ng/ml.
Membrane Preparations and Radioligand Binding.
Cells were
cultured on 150-mm Petri dishes and washed twice with ice-cold PBS. The
cells were then scraped from the plates with a cell scraper, suspended
in 50 mM Tris-HCl buffer, pH 7.4, and harvested by centrifugation at
16,000g for 10 min. The pellet was re-suspended in 5 mM
Tris-HCl, pH 7.4, and homogenized with an Ultra Turrax homogenizer (IKA
Labortecnik, Staufen, Germany). The resulting membranes were collected
by centrifugation at 25,000g for 20 min and stored at
70°C in 50 mM Tris-HCl buffer, pH 7.4, at a protein concentration
of approximately 1 mg/ml. The Bradford protein assay was used for
protein determination with bovine serum albumin as a standard.
). The maximal number of binding sites
(Bmax) and equilibrium dissociation
constant (KD) of the radioligand were
derived from the curve fitting. Graphs were prepared with the GraphPad
Prism program.
Measurements of cAMP Formation. After 2 days of growth, cells were detached from the Petri dishes with 3 ml of EDTA (0.04% w/v in PBS) and resuspended in PBS without Ca2+ and Mg2+. The cells were centrifuged at 500g for 5 min. The pellet was resuspended in the "stimulation buffer" provided with the PerkinElmer kit and diluted to a concentration of 106 cells/ml, and 50 µl was added per well of the Flashplate (50,000 cells/well). Compounds were diluted in PBS without Ca2+ and Mg2+ containing 1 µM pargyline and 1 µM paroxetine. Fifty microliters of the compound solution was added per well, followed by an incubation for 20 min at 37°C. After the incubation period, a direct radioimmunoassay using 125I-cAMP was performed by the addition of 100 µl of detection mix per well according to the supplier's instructions. After incubation for 18 h at room temperature, counting was performed in a Topcount HTS9912V counter (Packard).
[35S]GTPS Binding Assays.
For further
functional testing, the [35S]GTPS binding
assay was performed on membrane preparations. Membranes were incubated (105 µg/ml) in 20 mM HEPES-NaOH, pH 7.4, containing 10 mM
MgCl2, 1 µM GDP, 1 µM pargyline, and 1 µM
paroxetine, in the presence of 0.25 nM
[35S]GTPS and compounds. The final volume
was 250 µl and the incubation was performed in polypropylene tubes
(PPN-tube-96; Micronic bv, Lelystad, The Netherlands) at 37°C
for 20 min. The incubation was stopped by filtration on Unifilter-96
GF/B plates with ice-cold phosphate buffer, 10 mM
Na2HPO4, adjusted to pH 7.4 with a solution of 10 mM
NaH2PO4. The filter plates
were dried at room temperature, 30 µl of Microscint was added per
well, and plates were counted in a Topcount HTS9912V counter (Packard).
Measurements of Intracellular Calcium Concentration. The intracellular [Ca2+]i was measured with a fluorometric imaging plate reader (FLIPR; Molecular Devices, Crawley, England). Cells were seeded at 40,000 cells/well in 96-well plates (Cluster plates, black with clear bottom; Costar; Merck, Overijse, Belgium) for 2 days until they reached confluence. Cells were loaded with 2 µM Fluo3-acetoxymethyl ester in DMEM supplemented with 20 mM HEPES, pH 7.4, and 2.5 mM probenecid, for 1 h at 37°C under 5% CO2. The plates were washed three times with 5 mM HEPES, pH 7.4, 1.25 mM CaCl2, 140 mM NaCl, 1 mM MgCl2, 5 mM KCl, and 10 mM glucose, containing 2.5 mM probenecid (to prevent extrusion of Fluo-3), 1 µM pargyline, and 1 µM paroxetine. [Ca2+]i was measured by FLIPR, and the peak of the calcium transient was considered as the relevant signal. Treatment of the cells with 10 µM ionomycin (Ca2+-inophore) was performed as a control for dye loading and cell viability.
To investigate the source of the [Ca2+]i increase, we performed the same assay as described above, using several modifications. We performed the assay in the absence of extracellular calcium, where the Fluo3-loaded cells were washed two times with the above buffer without calcium. The final washing step and the measurements were performed in Ca2+-free buffer containing 2 mM EGTA. Ionomycin (10 µM) and 1% Triton X-100 were used as controls to estimate the maximal signals from extracellular and intracellular compartments, respectively. [Ca2+]i measurements were also performed on cells pretreated with 100 ng/ml PTX (overnight), 200 µM bepridil (15 min), 1 mM 3-isobutyl-1-methylxanthine (IBMX; 30 min), and 1 µM forskolin (15 min).| |
Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Characterization of Cell Lines in Ligand Concentration
Binding.
The two human 5-HT4 receptor
isoforms investigated in this study, h5-HT4a and
h5-HT4b, were stably transfected into HEK 293 cells. In this study, we have primarily used a high-level expressing clone of each splice variant, the h5-HT4a clone 8 and the h5-HT4b clone 9. In addition, clones with
lower expression levels, h5-HT4a clone 1 and
h5-HT4b clone 4, were employed in certain
experiments to investigate the effect of receptor expression on the
functional properties of each receptor variant. The isolated monoclonal
cell lines were characterized by saturation binding, using two
radioligands, an antagonist, [3H]GR113808, and
the natural agonist, [3H]5-HT. The experiments
were performed on membrane preparations from cells treated with sodium
butyrate and from untreated cells. Independent of expression levels,
the concentration binding isotherms of the antagonist
[3H]GR113808 to h5-HT4b
and h5-HT4a receptors expressed in different clones showed rectangular hyperbolae. The resulting linear Scatchard plots revealed a single high-affinity binding site
(KD values ranging between 0.05 and 0.1 nM
for both 5-HT4 receptor isoforms). Furthermore,
whereas treatment with sodium butyrate increased all
Bmax values by almost 70%, it did not
alter mean KD values. The calculated
KD and Bmax
values are presented in Table 1. For comparison, the Bmax values of the two
lower level expressing clones, treated with sodium butyrate, were 1500 (±160) and 2087 (±513) fmol/mg of protein for the
h5-HT4a clone 1 and the
h5-HT4b clone 4, respectively.
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Adenylyl Cyclase Activation.
The activity of AC after
h5-HT4 receptor stimulation with various
reference ligands was estimated on living cells, which were not treated
with sodium butyrate to elevate receptor expression levels. An increase
of basal cAMP levels, found in the high expressing clones compared with
wild-type HEK 293 cells, revealed the constitutive activity of both
5-HT4 receptors, which is consistent with
previous reports (Claeysen et al., 1999, 2000). Compared with the basal values measured in the wild-type HEK 293 cells, the measured
constitutive activity was 482% (±135) and 881% (±156) (mean ± S.D., n = 3) in 5-HT4a and
5-HT4b receptor-expressing cells, respectively.
). The reference antagonists
used were GR113808, an indole carboxylate analog, SDZ205,557, a
benzoate analog, and SB204070, an imidazole amide. All compounds were
tested for agonistic efficacy, and the results were expressed as a
percentage of maximal 5-HT response (5-HTmax);
i.e., 100% of stimulation was the maximum of the fitted plots obtained
with 5-HT. The derived pEC50 and
5-HTmax values are presented in Table
2 and the concentration-response curves are shown in Fig. 2. All tested agonists
were found to be full agonists for both h5-HT4a
and h5-HT4b receptors. The antagonist GR113808
did not induce cAMP formation via h5-HT4a or via
h5-HT4b receptors.
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Changes in Intracellular Calcium Concentration.
To investigate
other potential signal transduction pathways, we measured the
modulations of [Ca2+]i
after h5-HT4a and h5-HT4b
receptor activation. The same reference compounds as described for the
cAMP experiments were employed. The h5-HT4a
receptor showed fast increase in
[Ca2+]i after exposure to
all tested agonists, independent of the expression level found in both
investigated clones. Interestingly, the Ca2+
response induced by the h5-HT4a receptor was
dependent on the chemical nature of the agonists. For the two agonistic
benzamide-like compounds, cisapride and prucalopride, the peaks of the
calcium transients were two and three times higher than for the natural agonist 5-HT and showed a different time course (Fig.
3). Both cisapride- and
prucalopride-stimulation needed a longer time (>100 s) to reach
maximal [Ca2+]i than was
observed with indoleamines (<50 s).
|
), we believe that its enhancing effect on the
Ca2+-signaling is rather linked to its
up-regulating effect of receptor levels, because the treatment is not a
prerequisite to measure a response (Fig. 4, D versus C). Taken together
this results indicate that the calcium pathway is relatively
h5-HT4a receptor-specific, dependent on receptor
expression level, and more responsive to benzamide-like compounds in
comparison to indoleamines.
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Inositol Phosphates or Extracellular Calcium Influx. Mobilization of calcium from intracellular stores would classically involve the activation of phospholipase C and was investigated by means of IP measurements. Assays were performed after h5-HT4a receptors had been stimulated with the natural agonist 5-HT and the benzofuran prucalopride. No increase of IP concentrations could be detected (data not shown) in contrast to a parallel control performed with CHO cells expressing h5-HT2A receptors. Our results, therefore, indicate that the h5-HT4a-induced calcium mobilization does not involve the phospholipase C/IP3 pathway.
When calcium was removed from the extracellular environment before h5-HT4a receptor stimulation, no response could be triggered with 1 µM 5-HT or 1 µM prucalopride (Fig. 5A). These results indicate that the h5-HT4a receptor-induced increase in [Ca2+]i was caused by opening of calcium channels located in the plasma membrane. This hypothesis could be confirmed by pretreating the cells with 200 µM bepridil, which resulted in a complete blockade of the Ca2+-signal (Fig. 5B). In addition, the response was not dependent on the contribution of Gi-proteins, because it was
insensitive to PTX pretreatment (Fig. 5A). IBMX treatment (not shown)
and forskolin addition (Fig. 5B) did not modify or induce the
Ca2+ transients, which speaks against a direct
contribution of the cAMP pathway.
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[35S]GTPS Binding.
G-protein coupling to
receptors was investigated by measuring
[35S]GTPS binding to membrane preparations
from cells expressing either h5-HT4 receptor
isoform. Results were expressed as percentage increase in
[35S]GTPS binding over basal levels (Fig.
6). The levels of stimulation were found
to differ between h5-HT4a and
h5-HT4b receptors, and were in the range of 50 and 100% of stimulation, respectively. To investigate potential
differences in pharmacology, the same six agonists and three putative
antagonists were used as described above. The
pEC50 values derived from fitted
concentration-response curves are presented in Table
4. We were not able to detect any stimulatory effects on [35S]GTPS binding
with the two antagonists GR113808 and SDZ205,557. However, SB204070
triggered a weak response via h5-HT4b receptors. Despite the higher levels of stimulation triggered via the
h5-HT4b receptor, no significant differences in
the potency of the agonistic compounds were detected between the both
receptor variants.
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i/o-proteins, we performed
similar [35S]GTPS binding experiments on
membranes from PTX-pretreated cells (Fig. 6; Table 4). We observed, for
all agonists used, a decrease in the maximal levels of
[35S]GTPS binding in the
h5-HT4b receptor preparation treated with PTX.
The percentages of inhibition induced by PTX on the
h5-HT4b-receptor are presented in Table
5. Therefore, the uncoupling of the
receptor from the Gi/o-proteins by PTX pretreatment results in lower
levels of [35S]GTPS binding stimulation via
the h5-HT4b receptor. Interestingly, the
pEC50 values were not significantly modified,
although they tended to be higher on PTX-treated membranes (Table 4).
In contrast, no significant modulation of
[35S]GTPS binding was observed on the
h5-HT4a receptor preparation. Because the
h5-HT4b cell line used for these experiments had
higher receptor expression levels than the
h5-HT4a clone, we investigated whether the
coupling to Gi/o-proteins was not a consequence of the high receptor
levels. To this end, we performed the same experiments on the
lower-expressing h5-HT4b clone (i.e., clone 4, expressing approximately one-third of receptors). We observed the same
decrease in [35S]GTPS binding efficacy after
PTX pretreatment (data not shown). Taken together, these results
indicate that in addition to Gs, the h5-HT4b
receptor splice variant is also coupled to Gi/o-proteins and that
this is independent of the expression levels. In contrast, the lack of
sensitivity to PTX of [35S]GTPS binding
indicates that h5-HT4a receptors do not couple to
Gi/o-proteins. We also investigated the contribution of
Gi/o-proteins to the other h5-HT4b
receptor-mediated signal transduction pathways.
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Adenylyl Cyclase Activation after PTX Pretreatment.
AC
stimulation was investigated on h5-HT4b- and, as
a control, on h5-HT4a receptors on cells
pretreated with PTX and on untreated cells. Because the basal levels of
cAMP were increased after PTX pretreatment the results were
expressed as a percentage of the maximal stimulation obtained on
nontreated cells. Consistent with the findings in
[35S]GTPS binding experiments, cAMP
formation induced by 5-HT or prucalopride was found to be increased by
PTX pretreatment in the h5-HT4b-variant
indicating a des-inhibition of the cAMP-pathway (Fig.
7). The increase in maximally accumulated
cAMP was 27% (±5) for 5-HT and 29% (±6) for prucalopride
stimulation. In contrast, no significant modulation of the AC response
by PTX treatment was found with the h5-HT4a
receptor variant (Fig. 7).
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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This study uses the initially cloned and widely distributed
5-HT4a and 5-HT4b receptors
to investigate the pharmacological and functional properties of these
receptor splice variants in stably transfected HEK 293 cells. Analysis
of saturation binding performed with
[3H]GR113808 revealed
KD values that were consistent with
previous findings on transiently transfected cells (Ansanay et al.,
1996; Van den Wyngaert et al., 1997) and different brain tissues
(Grossman et al., 1993; Waeber et al., 1994; Arranz et al., 1998). We
confirm that C-terminal variations of h5-HT4a and
h5-HT4b receptors did not modify binding
KD values, consistent with the findings on EP3 and somatostatin receptors (Tsunehisa et al., 1993; Vanetti et al.,
1993) and very recently also 5-HT4 receptors
(Bach et al., 2001). Detailed saturation binding performed with
[3H]5-HT, revealed the presence of two affinity
sites for both h5-HT4a and
h5-HT4b receptors. Previous studies could only
show a single affinity site for [3H]5-HT of 20 (±7) nM for the rat 5-HT4b receptor expressed in COS7 cells, although the shallow competition binding curves and partial
GTPS sensitivity indicated the existence of two affinity sites
(Adham et al., 1996). The high concentrations of
[3H]5-HT used in that study (5-100 nM) may
have precluded the detection of the high-affinity site described in
this report (KD values ranging from 1.3 to
3.2 nM).
Comparison of saturation binding Bmax data
using [3H]GR113808 and
[3H]5-HT allowed us to estimate the fraction of
receptors present in the agonist high-affinity state. This fraction of
high-affinity receptors was not dependent on expression levels, either
modified by sodium butyrate pretreatment of cells or using different
monoclonal cell lines, but characteristic for a given splice variant.
The h5-HT4b receptors showed a consistently
higher proportion of non-G-protein-coupled or inactive receptors than
the h5-HT4a splice variant, based on the
assumption that the natural agonist [3H]5-HT
labels only active receptors in R*- and R*G-state with high
affinity. The independence of the fraction of
[3H]5-HT high-affinity sites from expression
levels is analogous to the equilibrium J constant calculated
in the absence of ligand as recently shown by Claeysen and colleagues
(2000). They noticed that in COS-7 cells transfected with human or
mouse 5-HT4a receptors, the expression levels had
no effect on the equilibrium J constant.
The pharmacological profile obtained for cAMP formation was partially
divergent compared with previously reported results. We have shown that
benzamide-like compounds in our system were full agonists on both
variants, in contrast to reports classifying renzapride and cisapride
as partial agonists (Ouadid et al., 1992; Blondel et al., 1998).
Moreover, SB204070 and SDZ205,557, described as antagonists, were found
to be partial agonists on both h5-HT4a and
h5-HT4b receptors. In addition, small but
statistically significant differences in efficacy and/or potency of
agonists were found between the two splice variants. Direct
extrapolation of these results to tissue-based studies should bear in
mind that functional responses found in tissues may reflect not only
different expression levels but also be the result of the stimulation
of several 5-HT4 receptor splice variants
expressed in a given tissue.
A very recent study of Bach et al. (2001) investigating the cAMP
response in h5-HT4a and
h5-HT4b receptors could also not match the
pharmacological pattern found on cells to measurement on right atrium
membranes, although the 5-HT4a isoform is thought to be predominant in that tissue (Blondel et al., 1997). In contrast to
our study, they have found partial agonism of renzapride and cisapride.
However, their pharmacological comparison is focused on antagonists,
and the study uses [-32P]ATP-loaded
membranes for measurements of the cAMP, whereas our study is performed
on intact cells. At similar expression levels, our
EC50 for 5-HT is approximately 10-fold lower than
the ones reported by Bach et al. (2001). Therefore, the membrane based assay used by Bach et al., which allows a direct comparison to measurements performed on tissue samples, may suffer from impaired coupling and may not be able to reveal the subtle differences seen in
this study on intact cells.
In addition to AC activation, 5-HT4 receptors
have also been shown to trigger other signal transduction pathways,
such as reduction of potassium currents in mouse colliculi neurons
(Fagni et al., 1992), calcium influx in adrenocortical cells (Contesse et al., 1996), and atrial myocytes (Ouadid et al., 1992). Our results
revealed a clear functional difference between the two h5-HT4 receptor isoforms in their ability and
pharmacology to increase
[Ca2+]i. The
h5-HT4a receptor was able to trigger strong
[Ca2+]i increase, in
contrast to h5-HT4b receptors, which responded with a weaker signal and only to indoleamines. This signal transduction pathway was dependent on receptor expression levels, however, also
detected the lowest expression level of the
h5-HT4a receptor [i.e., clone 1 expressing 1500 (±160) fmol/mg]. Interestingly, the calcium response was not only
variant-specific but also dependent on compound structure, because
benzamide-like compounds triggered higher responses than indoleamines.
The absence of IP pathway activation, the elimination of the response
after removal of extracellular calcium, and the inhibition of the
response by bepridil suggest that h5-HT4a
receptors were able to activate calcium channels. Nevertheless, the
question of the cascade events triggering the calcium channel
activation has yet to be assessed. The phosphodiesterase inhibitor IBMX
and forskolin, a direct AC activator did not enhance the increase of
[Ca2+]i (data not shown),
suggesting that, in contrast to the
h5-HT4-induced [Ca2+]i response in
adrenocortical cells (Contesse et al., 1996), the h5-HT4a calcium response in HEK 293 cells was not
dependent on the cAMP pathway. Moreover, PTX treatment did not modify
the calcium response, which excluded the hypothesis of Gi/o-protein
involvement in the mechanism. However, because it has been shown that
after activation of G-protein heterotrimers, the 
-subunits are
able to activate calcium channels (for review, see Holler et al.,
1999), such a pathway has to be considered for the
5-HT4a receptor. In addition, opening of
Ca2+-channels subsequent to a
-subunit-mediated activation of potassium channels could be
another mechanism.
In regard to the G-protein coupling to h5-HT4a
and h5-HT4b receptors, we have found a difference
between the level of G-protein stimulation induced by
h5-HT4a and h5-HT4b
receptors. PTX pretreatment induced a reduction in
h5-HT4b receptor-mediated
[35S]GTPS binding to the level found for the
h5-HT4a receptor, suggesting the existence of an
additional Gi/o component in the h5-HT4b receptor G-protein coupling. This was confirmed by an enhanced increase
of cAMP formation induced via the h5-HT4b variant
after a PTX pretreatment, which was not found for the
h5-HT4a receptor. Taken together, these results
indicate that the h5-HT4b receptor is able to
activate both Gi/o- and Gs-proteins, in contrast to the
h5-HT4a-receptor, which couples only to
Gs-proteins. The pEC50 values of both
PTX-pretreated and nontreated cells in
[35S]GTPS binding and cAMP formation were
similar, suggesting that unlike for the D2
receptor, 5-HT4b receptor dual coupling was not
dependent on the agonist concentration (Chang et al., 1997). Until now,
dual G-protein coupling is hypothesized to have two main functions. On
the one hand, it has been shown for other receptors that these were
sequential events contributing to desensitization mechanisms
(Lefkowitz, 1998). On the other hand, Gs- and Gi/o-proteins have
been shown to couple simultaneously to the 2
adrenergic receptor (Eason et al., 1992), the functional net result of
cAMP production representing the integration of the two
G-protein-mediated effects.
Taken together, these results show that the
h5-HT4a and
h5-HT4b-receptor variants do activate partially
different intracellular processes (Fig.
8, A and B). The characteristic sum of
these differences may result in pharmacological differences of
compounds as described in different tissues (Dumuis et al., 1988;
Bockaert et al., 1997). Although possible differences in the
localization of 5-HT4a and 5-HT4b receptors in brain tissue are still
controversial (Gerald et al., 1995; Bender et al., 2000), saturation
binding experiments performed on membranes from different human brain
regions have revealed a higher fraction of agonist binding sites in the
substantia nigra (0.54) than in other brain regions such as the frontal
cortex (0.27), the striatum (0.25), or the hippocampus (0.16)
(Bonaventure et al., 2000). These values parallel very closely the
findings in the present cellular study. Although further studies on
cell- and tissue-specific 5-HT4 receptor
expression patterns are required; the above findings together with the
reported tissue-specific differences in agonist efficacy provide a
physiological context of our study. We have evaluated the influence of
receptor levels on the observed responses, however, for the
Ca2+-response, promiscuous coupling cannot be
completely ruled out. However, we have shown that it is not
cAMP-dependent in HEK cells and has different thresholds and
pharmacology on both receptor isoforms. It is important to state that a
direct comparison of tissue expression levels (guinea pig striatum
~200 fmol/mg; Van den Wyngaert et al., 1997) to data derived on
recombinant cells is problematic, because the physiological
concentration of neurotransmitter receptors in the synaptic cleft is
certainly higher than the tissue average observed in binding studies.
|
On the receptor level the differences in the functional profile of the investigated reference compounds in cAMP and Ca2+ measurements favor different forms of R* for each receptor isoform and signaling pathway, which can be stabilized to a different degree by the compounds investigated. In general, further understanding of the fine-tuning of G-protein-coupled receptor isoform-associated signaling and its compound specificity could, in the future, lead to G-protein-coupled receptor isoform-specific (i.e., "super") drugs with potentially broader safety margins.
| |
Footnotes |
|---|
Received March 28, 2001; Accepted September 25, 2001
1 Present address: CNS Discovery Research, Janssen Research Foundation, B-2340 Beerse, Belgium.
Dr. Mirek Jurzak, Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium. E-mail: mjurzak{at}janbe.jnj.com
| |
Abbreviations |
|---|
5-HT, 5-hydroxytryptamine, serotonin;
h5-HT4 receptor, human 5-hydroxytryptamine4
receptor;
AC, adenylyl cyclase;
DMEM, Dulbecco's modified Eagle's
medium;
GTPS, guanosine-5'-O-(3-thio)triphosphate;
PBS, phosphate-buffered saline;
PTX, Bordatella
pertussis toxin;
HEK, human embryonic kidney;
FLIPR, fluorometric imaging plate reader;
IBMX, 3-isobutyl-1-methylxanthine;
5-MeOT, 5-methoxytryptamine;
IP, inositol phosphate;
RFU, relative
fluorescence units;
SB204070, 1,4-benzodioxin-5-carboxylic acid,
8-amino-7-chloro-2,3-dihydro-(1-butyl-4-piperidinyl)methyl ester;
GR113808, 1H-indole-3-carboxylic acid,
1-methyl-(1-(2-((methylsulfonyl)-amino)ethyl)-4-piperidinyl)methyl
ester;
SDZ205,557, benzoic acid,
4-amino-5-chloro-2-methoxy-2-diethylamino)ethyl ester;
SDZ-HTF919 hydrazinecarboximidamide, 2-((5-methoxy-1H-indol-3-yl)methylene)-N-pentyl-(Z)-2-butenedioate.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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) or nontreated conditions (
). The parabolic Scatchard plots
show the two affinity sites for both h5-HT4 receptor
isoforms. The data shown represent a typical example out of three
independent experiments performed in duplicate.
), cisapride
(
), SDZ-HTF919 (
), renzapride (
), 5-MeOT (
), and SDZ205,557 (
). The maximal response for each compound was
normalized to the maximal stimulation obtained with 5-HT (% of
5-HTmax). Results are expressed as fitted curves to the
mean (±S.D.) of independent experiments performed in duplicate, as
summarized in Table 
6 M 5-HT (A) or 10
) or vehicle (
). The
ordinate represents the mean RFU measured at the peak of the
Ca2+ transient normalized to the mean RFU obtained before
the stimulation (% of basal). Data are expressed as the mean (±S.D.)
of three independent experiments performed in triplicate. B, the
experiments were performed in standard buffer containing 1.25 mM
extracellular calcium after a 15-min preincubation with 200 µM
bepridil (
and 