Functional Antagonism of Gonadal Steroids at the 5-Hydroxytryptamine Type 3 Receptor
Christian H. R. Wetzel1,2,
Bettina Hermann1,
Christian Behl,
Elmar Pestel,
Gerhard Rammes,
Walter Zieglgänsberger,
Florian Holsboer and
Rainer Rupprecht
Max Planck Institute of Psychiatry 80804 Munich, Germany
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ABSTRACT
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Steroid hormone action involves binding to cognate
intracellular receptors that, in turn, bind to respective response
elements and thus modulate gene expression. The present study shows
that the gonadal steroids, 17ß-estradiol and progesterone, may also
act as functional antagonists at the 5-hydroxytryptamine
type 3 (5-HT3) receptor in whole-cell
voltage-clamp recordings of HEK 293 cells stably expressing the
5-HT3 receptor. Functional antagonistic
properties at this ligand-gated ion channel could also be shown for
17
-estradiol, 17
-ethinyl-17ß-estradiol, mestranol, R 5020,
testosterone, and allopregnanolone but not for pregnenolone sulfate and
cholesterol. An antagonism at the 5-HT3
receptor could further be observed with the aromatic alcohol
4-dodecylphenol but not with phenol or ethanol. Thus, the modulation of
5-HT3 receptor function by steroids or alcohols
is dependent on their respective molecule structure. The antagonistic
action of steroids at the 5-HT3 receptor is not
mediated via the serotonin binding site because the steroids did not
alter the binding affinity of [3H]GR65630 to
the 5-HT3 receptor, and kinetic experiments
revealed a quite different response pattern to 17ß-estradiol when
compared with the competitive antagonist metoclopramide. BSA-conjugated
gonadal steroids labeled with fluorescein isothiocyanate bound to
membranes of HEK 293 cells expressing the 5-HT3
receptor in contrast to native HEK 293 cells. However, there was no
dose-dependent displacement of the binding of gonadal steroids to
membranes of cells expressing the 5-HT3
receptor in binding experiments or fluorescence studies. Thus, gonadal
steroids probably interact allosterically with the
5-HT3 receptor at the receptor-membrane
interface. The functional antagonism of gonadal steroids at the
5-HT3 receptor may play a role for the
development and course of nausea during pregnancy and of psychiatric
disorders.
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INTRODUCTION
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Serotonin (5-hydroxytryptamine, 5-HT) is a major
neurotransmitter within the central nervous system that acts
through multiple receptor subtypes (1). The majority of these receptors
are coupled to G proteins and mediate slow modulatory responses via
second messenger signaling pathways (1). Only the 5-HT3
receptor constitutes a ligand-gated ion channel (2) that shares
structural features with
-aminobutyric acid type A
(GABAA), glycine, and nicotinic acetylcholine receptors
(2). Within the central nervous system, the 5-HT3 receptor
is predominantly expressed in neurons in the area postrema and the
mesolimbic system (3, 4), which receives a major dopaminergic input
from the ventral tegmental area. Thus, 5-HT3 receptors
located at this interface between limbic and motor structures (5)
appear to be a potential target for the development of drugs for the
treatment of nausea and of behavioral disorders (6). 5-HT3
receptor antagonists prevent emesis induced by cytostatic drugs that
are commonly employed in cancer therapy (7). Moreover, based on animal
models and preliminary clinical studies, it has been suggested that
5-HT3 receptor antagonists display anxiolytic (8) and
atypical antipsychotic properties (9, 10). Fluctuations in gonadal
steroid hormone concentrations may be involved in the development and
course of the respective underlying disorders. Nausea is observed
frequently during the first trimester of pregnancy (11), whereas
psychiatric disturbances are more common during the postpartum period
(12). Moreover, there is considerable evidence that the function of the
GABAA receptor may be affected by a variety of naturally
occurring neuroactive steroids (13, 14). Therefore, we investigated
whether the function of the 5-HT3 receptor is also
sensitive to a modulation by endogenous steroids. In the present study
we show that the gonadal steroids, 17ß-estradiol and progesterone,
may act as functional noncompetitive antagonists at the
5-HT3 receptor.
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RESULTS
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Human embryonic kidney cells (HEK 293 cells) that had been stably
transfected with an expression vector for the 5-HT3
receptor (5-HT3R-A) (2, 15) were recorded in the whole-cell
voltage-clamp configuration. The application of 10 µM
serotonin elicited a rapidly developing, inward cation current (Fig. 1A
). The simultaneous application of the
competitive 5-HT3 receptor antagonist metoclopramide
markedly reduced the serotonin-evoked cation current (Fig. 1A
). This
effect was even more pronounced when metoclopramide was already present
before the serotonin pulse (Fig. 1C
). However, the antagonistic
properties of metoclopramide could be reversed by subsequent challenge
with serotonin in the absence of metoclopramide (Fig. 1B
).
17ß-Estradiol was also very effective as an inhibitor of the
serotonin-evoked cation current when the steroid was present before and
during the serotonin pulse (Fig. 1F
). However, the different modes of
application of 17ß-estradiol elicited response patterns to serotonin
that differed considerably from those observed with metoclopramide. The
simultaneous application of 17ß-estradiol did not markedly reduce the
peak amplitude of the serotonin-evoked cation current. Instead, there
was an apparent acceleration of the desensitization kinetics in the
presence of 17ß-estradiol (Fig. 1D
). In contrast, the application of
17ß-estradiol in the form of a prepulse and subsequent challenge with
serotonin in the absence of the steroid resulted in a reduced peak
amplitude and a delayed response to serotonin. The reexposure to
17ß-estradiol induced an apparent acceleration of receptor
inactivation (Fig. 1E
). Thus, the mode of action of 17ß-estradiol
differed considerably from that of metoclopramide in that a prolonged
exposure to the steroid was required for a pronounced antagonistic
effect at this ligand-gated ion channel. The response of the
5-HT3 receptor to 10 µM serotonin could be
fully blocked by 17ß-estradiol with an IC50 of 5.3
± 0.6 µM. Progesterone also displayed inhibitory
properties at the 5-HT3 receptor. However, this steroid was
much less effective as a functional antagonist (Fig. 2
). The effects of 17ß-estradiol
(Fig. 3
) and progesterone (data not
shown) on the serotonin-evoked cation current were voltage
independent.

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Figure 1. Effect of Metoclopramide and 17ß-Estradiol on the
Serotonin-Evoked Cation Current
HEK 293 cells expressing the 5-HT3 receptor were recorded
in the whole-cell voltage-clamp configuration. All records in panels A,
B, and C and in panels D, E, and F were obtained from the same cell.
Results are shown as a representative experiment of at least four
independent experiments. A, Simultaneous application of 10
µM serotonin and 1 µM metoclopramide
without preexposure to metoclopramide. The upper bar
indicates the application of 10 µM serotonin; the
lower bar indicates the presence of 1 µM
metoclopramide. B, Preexposure to 1 µM metoclopramide and
application of 10 µM serotonin without simultaneous
application of metoclopramide. A second application of 1
µM metoclopramide followed the application of 10
µM serotonin. C, Preexposure to 1 µM
metoclopramide and simultaneous application of 10 µM
serotonin and 1 µM metoclopramide. The upper
bar indicates the application of 10 µM serotonin;
the lower bar indicates the presence of 1
µM metoclopramide. D, Simultaneous application of 10
µM serotonin and 10 µM 17ß-estradiol
without preexposure to 17ß-estradiol. The upper bar
indicates the application of 10 µM serotonin; the
lower bar indicates the presence of 10 µM
17ß-estradiol. E, Preexposure to 10 µM 17ß-estradiol
and application of 10 µM serotonin without simultaneous
application of 17ß-estradiol. A second application of 10
µM 17ß-estradiol followed the application of 10
µM serotonin. F, Preexposure to 10 µM
17ß-estradiol and simultaneous application of 10 µM
serotonin and 10 µM 17ß-estradiol. The upper
bar indicates the application of 10 µM serotonin;
the lower bar indicates the presence of 10
µM 17ß-estradiol. Control experiments without
metoclopramide or 17ß-estradiol are represented by tracing a;
experiments with metoclopramide or 17ß-estradiol are represented by
tracing b.
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Figure 2. Dose-Response Relationship of the Functional
Antagonism of 17ß-Estradiol and Progesterone at the 5-HT3
Receptor Expressed in HEK 293 Cells
The steroids were applied via the superfusion at the indicated
concentrations. The amplitude of the current evoked by 10
µM serotonin without addition of steroids was set at
100%. Results are expressed in percent of the amplitude obtained
without steroid and represent the mean ± SEM of four
to eight independent experiments. The antagonism became significant at
300 nM for 17ß-estradiol and at 1 µM for
progesterone (P < 0.05, Wilcoxon signed-ranks
test). Solid circles, 17ß-Estradiol; open
circles, progesterone.
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Figure 3. Voltage Dependence of the Effect of 17ß-Estradiol
on the Serotonin-Evoked Cation Current
HEK 293 cells expressing the 5-HT3 receptor were recorded
in the whole-cell voltage-clamp configuration. All records were
obtained from the same cell. A, Voltage dependence of the
serotonin-evoked cation current. The bar indicates the
application of 10 µM serotonin. B, Voltage dependence of
the antagonism of 17ß-estradiol at the 5-HT3 receptor.
The upper bar indicates the application of 10
µM serotonin; the lower bar indicates the
presence of 10 µM 17ß-estradiol. C, Amplitude/voltage
(I/V) curve of the experiment shown in panels A and B for the
control experiment without addition of 17ß-estradiol (solid
circles) and after application of 10 µM
17ß-estradiol (open circles). D, Voltage dependence of
the effect of 17ß-estradiol at the 5HT3 receptor. The
data are derived from the I/V curve shown in panel C. The amplitude of
the current evoked by 10 µM serotonin in the presence of
17ß-estradiol at the different holding potentials was divided by the
amplitude of the respective control experiment without addition of
17ß-estradiol. Results are shown as a representative experiment of
four independent experiments.
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An antagonistic effect at the serotonin-evoked cation current could
further be demonstrated for 10 µM 17
-estradiol,
testosterone, allopregnanolone (3
-hydroxy-5
-pregnan-20-one),
a major metabolite of progesterone, and for 10 µM R 5020,
a synthetic progestin. Moreover, 17ß-estradiol and progesterone
continued to display an antagonistic activity when they were coupled to
BSA. However, this antagonistic effect was not shared by all steroid
molecules, as 10 µM pregnenolone sulfate and cholesterol
were devoid of any significant modulatory activity at the
5-HT3 receptor (Fig. 4
).

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Figure 4. Antagonistic Properties of Steroids and
Metoclopramide at the 5-HT3 Receptor
The complete inhibition of the current evoked by 10 µM
serotonin was set at 100%. Results are expressed as percent inhibition
of the amplitude of the response evoked by 10 µM
serotonin and are presented as the mean ± SEM of four
to eight independent experiments. The inhibition was significant
(P < 0.05, Wilcoxon signed-ranks test) for
metoclopramide, 17ß-estradiol (17ßE2), R 5020,
17 -estradiol (17 E2), testosterone, progesterone,
allopregnanolone, and the steroid-BSA complexes. Pregnenolone sulfate,
cholesterol, and BSA alone (data not shown) did not significantly
affect the serotonin-evoked cation current.
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To address the question of whether the gonadal steroids act as
competitive antagonists at the 5-HT3 receptor, we
investigated whether they may alter the binding properties of
[3H]GR65630 both in HEK 293 cells stably expressing the
5-HT3 receptor, and after reconstitution of the receptor in
COS-1 cells. As previously shown (2), the selective 5-HT3
receptor antagonist GR65630 bound with high affinity to the respective
membrane fraction of transfected COS-1 cells [dissociation constant
(Kd) = 1.3 ± 0.2 nM] and HEK 293 cells
stably expressing the 5-HT3 receptor (Kd =
1.4 ± 0.3 nM) (Fig. 5A
). In contrast to metoclopramide and
MDL 72222, both of which are antagonists at the serotonin-binding site
of the 5-HT3 receptor, the steroids did not displace
[3H]GR65630 from HEK 293 cells stably expressing the
5-HT3 receptor (Fig. 5B
). Moreover, no displacement of
[3H]GR65630 was achieved by either steroid after
reconstitution of the 5-HT3 receptor in COS-1 cells (data
not shown). The binding affinity of [3H]GR65630 to the
5-HT3 receptor was not changed in the presence of either 10
µM 17ß-estradiol or progesterone (Table 1
). In addition, 10 µM
17ß-estradiol or progesterone did not reduce the apparent affinity of
the 5-HT3 receptor to serotonin (EC50) in the
electrophysiological recordings (Table 1
). Thus, the antagonism of
progesterone is mediated by a mechanism different from that achieved
with typical 5-HT3 receptor antagonists.

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Figure 5. Binding Properties of [3H]GR65630 to
Membrane Fractions of HEK 293 Cells Stably Expressing the
5-HT3 Receptor
A, Binding of [3H]GR65630 to membrane fractions of HEK
293 cells stably expressing the 5-HT3 receptor. The results
are shown as the mean ± SEM of four independent
experiments. The inset shows the Scatchard
transformation of the binding data yielding a Kd value
(mean ± SD) of 1.4 ± 0.3 nM and a
Bmax value of 1268 ± 219 fmol/mg protein. B,
Competition of metoclopramide (solid squares); MDL 72222
(open squares); progesterone (solid
circles), 17ß-estradiol (open circles) and
allopregnanolone (open diamonds) for
[3H]GR65630 binding to membrane fractions of HEK 293
cells stably expressing the 5-HT3 receptor. Membranes were
incubated in the presence of 2 nM [3H]GR65630
and increasing concentrations of unlabeled competitor. Results are
expressed as the mean ± SEM of three independent
experiments. The Ki values (mean ± SD)
were 196 ± 49 nM for metoclopramide and 45 ± 17
nM for MDL 72222. The steroids did not displace
[3H]GR65630.
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Table 1. Effects of 10 µM 17ß-Estradiol
and 10 µM Progesterone on the Dose Response of the
Serotonin-Evoked Cation Current (EC50) and the Binding
Affinity of [3H]GR65630 (Kd) in HEK 293 Cells
Stably Expressing the 5-HT3
Receptor
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Therefore, we questioned whether gonadal steroids exert their
antagonistic effects via an allosteric interaction with the
5-HT3 receptor. To address this question, we applied either
1 µM 17ß-estradiol or progesterone linked to
fluorescein isothiocyanate (FITC)-labeled BSA to HEK 293 cells stably
expressing the 5-HT3 receptor and to native HEK 293 cells.
While fluorescence labeling was rare in native HEK 293 cells, the
membranes of HEK 293 cells stably expressing the 5-HT3
receptor were clearly labeled both by the estradiol-BSA-FITC complex
(Fig. 6
) and the progesterone-BSA-FITC
complex (Fig. 7
). In addition, COS-1
cells that had been transiently transfected with an expression vector
for the 5-HT3 receptor were also labeled by the
steroid-BSA-FITC complexes in contrast to untransfected cells (data not
shown). However, the labeling of cells by the steroid-BSA-FITC
complexes is not a general phenomenon dependent on the presence of
either membrane receptor because neither Chinese hamster ovary (CHO)
cells stably expressing the G protein-coupled dopamine D4.4 receptor
subtype (16) (Fig. 8
) nor COS-1 cells
that had been transiently transfected with an expressison vector for
the D4.4 receptor (16) (data not shown) could be labeled with either
steroid-BSA-FITC complex. The labeling of cells expressing the
5-HT3 receptor appears to occur at the receptor-membrane
interface or an extracellular site, as the steroid-BSA-FITC complexes
do not cross the cell membrane. However, this fluorescence labeling
could not be prevented by pretreating the cells with unlabeled steroids
at a concentration of 10 µM. Moreover, there was
no dose-dependent displacement of the binding of
[3H]17ß-estradiol and [3H]progesterone to
membrane fractions of cells expressing the 5-HT3 receptor
by increasing concentrations of unlabeled steroids in binding assays
(data not shown). In addition, photoaffinity labeling experiments did
not reveal a displacable peak for [3H]17ß-estradiol and
[3H]progesterone corresponding to the respective signal
of the 5-HT3 receptor in the Western blot (data not
shown).

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Figure 6. Fluorescence Labeling of HEK 293 Cells Stably
Expressing the 5-HT3 Receptor with a 17ß-Estradiol
6-(O-carboxymethyl)oxime-BSA-FITC Conjugate
Fluorescence studies were performed as separate blind experiments.
Fluorescence pictures are shown together with their respective phase
contrast photographs. A, Fluorescence labeling of native HEK 293 cells.
B, Bright field view of panel A. C, Fluorescence labeling of HEK 293
cells expressing the 5-HT3 receptor. D, Bright field view
of panel C.
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Figure 7. Fluorescence Labeling of HEK 293 Cells Stably
Expressing the 5-HT3 Receptor with a Progesterone
3-(O-carboxymethyl)oxime-BSA-FITC Conjugate
Fluorescence studies were performed as four separate experiments.
Fluorescence pictures are shown together with their respective phase
contrast photographs. A, Fluorescence labeling of native HEK 293 cells.
B, Bright field view of panel A. C, Fluorescence labeling of HEK 293
cells expressing the 5-HT3 receptor. D, Bright field view
of panel C.
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Figure 8. Fluorescence Labeling of CHO cells Stably
Expressing the Human D4.4 Receptor with a 17ß-Estradiol
6-(O-carboxymethyl)oxime-BSA-FITC Conjugate or a
Progesterone 3-(O-carboxymethyl)oxime-BSA-FITC Conjugate
Fluorescence studies were performed as four separate experiments.
Fluorescence pictures are shown together with their respective phase
contrast photographs. A, Fluorescence labeling with a 17ß-estradiol
6-(O-carboxymethyl)oxime-BSA-FITC conjugate. B, Bright
field view of panel A. C, Fluorescence labeling with a progesterone
3-(O-carboxymethyl)oxime-BSA-FITC conjugate. D, Bright
field view of panel C.
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To identify the structural components of the estradiol molecule that
are necessary to exert an antagonistic effects at the 5-HT3
receptor, we studied the effects of various aromatic alcohols and
synthetic derivatives of estradiol (Fig. 9
) on the serotonin-evoked cation
current. While ethanol and the aromatic alcohol phenol did not affect
the serotonin response at a 10 µM concentration,
4-dodecylphenol, a nonsteroidal derivative of phenol with an incomplete
B, C, and D ring, displayed a pronounced antagonistic effect at the
5-HT3 receptor. Moreover, also
17
-ethinyl-17ß-estradiol and mestranol, two synthetic
derivatives of 17ß-estradiol, markedly reduced the serotonin response
(Fig. 10
). Thus, while the A
ring of the steroid molecule alone is not sufficient for the
antagonistic effects of estrogens at the serotonin-evoked cation
current, structural components of the B, C, or D ring appear to be
required for an antagonistic effect at the 5-HT3
receptor.

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Figure 10. Antagonistic Properties of Aromatic and
Nonaromatic Alcohols and of Synthetic Estrogen Derivatives at the
5-HT3 Receptor
The complete inhibition of the current evoked by 10 µM
serotonin was set at 100%. Results are expressed as percent inhibition
of the amplitude of the response evoked by 10 µM
serotonin and are presented as the mean ± SEM of four
to eight independent experiments. The inhibition was significant
(P < 0.05, Wilcoxon signed-ranks test) for
4-dodecylphenol, 17ß-estradiol (17ßE2),
17 -ethinyl-17ß-estradiol (EthinylE2), and mestranol.
Ethanol and phenol did not significantly affect the serotonin-evoked
cation current.
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DISCUSSION
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Our data show that the gondal steroids, 17ß-estradiol and
progesterone, may act as functional antagonists at the
5-HT3 receptor. Unlike metoclopramide, the steroids do not
act as competitive antagonists because they did not displace the
binding of [3H]GR65630 to the serotonin binding site.
Moreover, neither the binding affinity of [3H]GR65630 nor
the apparent affinity (EC50) to serotonin in the
electrophysiological recordings was decreased in the presence of either
17ß-estradiol or progesterone. In addition, 17ß-estradiol markedly
reduced the peak amplitude of the serotonin-evoked cation current only
when applied in the form of a prepulse before challenge with serotonin,
whereas the simultaneous application of metoclopramide together with
serotonin nearly abolished the serotonin response. These kinetic
experiments suggest that a competitive antagonist like metoclopramide
acts immediately, while the antagonistic effect observed with a steroid
requires a longer exposure time. Thus, the apparent effects of
17ß-estradiol on the desensitization or inactivation of the
serotonin-evoked cation current most likely reflect the delayed onset
of the steroid effect.
Another possibility might be that the antagonistic properties of
gonadal steroids at the 5-HT3 receptor are conferred by an
action of these steroids as open channel blockers. Although the
antagonistic effect of 17ß-estradiol and progesterone on the
serotonin-evoked cation current was voltage-independent, this does not
completely exclude this possibility because steroids are uncharged.
However, the observation that a preexposure to 17ß-estradiol was
sufficient to reduce the peak amplitude of serotonin-evoked cation
current when serotonin was applied in the absence of the steroid argues
against this explanation.
Thus, the inhibitory properties of steroids appear to be conferred via
an allosteric interaction with the 5-HT3 receptor. The
observation that the presence of gonadal steroids affects neither the
EC50 value for the response to serotonin nor the binding
affinity of [3H]GR65630 and the results of the kinetic
experiments are compatible with this assumption, because comparable
results have been obtained regarding the effects of progesterone and
some of its neuroactive metabolites on the function and binding
properties of the nicotinic acetylcholine receptor (17, 18) or the
kainate receptor (19). However, different findings have been obtained
with the GABAA receptor, which is also an important target
for nongenomic effects of progesterone metabolites, e.g.
allopregnanolone (13, 14, 20). At this ligand-gated ion channel,
neuroactive steroids such as allopregnanolone, which potentiate
the GABA-evoked chloride current, enhance the binding of muscimol and
of benzodiazepines (13, 14, 21).
Moreover, the structure of the steroids also plays an important role
for the modulation of the 5-HT3 and the GABAA
receptor. At the GABAA receptor, allopregnanolone is a
positive allosteric modulator, whereas progesterone and 17ß-estradiol
are devoid of modulatory properties (13, 14). Pregnenolone sulfate,
however, acts as a functional antagonist at this neurotransmitter
receptor (13, 14). At the N-methyl-D-aspartate
(NMDA) receptor, 17ß-estradiol displays antagonistic properties
whereas pregnenolone sulfate is a positive allosteric modulator (22).
This contrasts to our findings at the 5-HT3 receptor. At
this ligand-gated ion channel, 17ß-estradiol, progesterone, and
allopregnanolone are functional antagonists, whereas pregnenolone
sulfate is inactive. Thus, the structure-activity requirements for
modulation of ligand-gated ion channels by steroids also appear to be
quite different between various members of this neurotransmitter
receptor family.
The modulation of the 5-HT3 receptors by steroids cannot be
attributed only to their lipophilic properties. This is supported by
the observation that not all steroids display an antagonistic effect at
this ligand-gated ion channel and that the alcohols, ethanol and
phenol, are devoid of antagonistic properties. However, in contrast to
phenol, 4-dodecylphenol is an antagonist at the 5-HT3
receptor. These findings show that apparently structural components of
the B, C, or D ring of the estradiol molecule are required for an
antagonism of the serotonin response while the A ring alone is not
sufficient. Moreover, an alcoholic group at position 3 within the A
ring of the steroid molecule, which has been shown to be essential for
a modulation of the GABAA receptor by neuroactive steroids
(14), is not necessary for an antagonsim at the 5-HT3
receptor because mestranol is also a potent functional antagonist at
this neurotransmitter receptor.
However, ligand-gated ion channels are not generally sensitive to the
modulation by steroids because the Drosophila
GABAA receptor displays only a marginal response to
allopregnanolone when compared with vertebrate GABAA
receptors in the same Xenopus laevis oocyte expression
system (23). Moreover, in contrast to cells transfected with the
5-HT3 receptor, cells expressing the G protein-coupled
human dopamine D4.4 receptor could not be labeled by the
steroid-BSA-FITC complexes. Although these data do not exclude the
possibility that certain G protein-coupled receptors may be a
target for an allosteric modulation by steroids, they clearly show that
distinct interactions of steroids with membrane receptors are not
necessarily observed with all membrane receptors but are determined
also by the amino acid composition of ligand-gated ion channels or G
protein-coupled receptors.
The allosteric interaction of steroids with the 5-HT3
receptor probably occurs within the receptor-membrane interface. This
is supported by the fluorescence studies showing that the membranes of
cells expressing the 5-HT3 receptor could be labeled with
steroid-BSA complexes that cannot cross the cell membrane and also
displayed a functional antagonistic activity in the
electrophysiological recordings. The fluorescence labeling of cells
expressing the 5-HT3 receptor with BSA-conjugated steroids
could not be prevented by the addition of an excess of unlabeled
steroid, and there was no dose-dependent displacement of the binding of
[3H]17ß-estradiol and [3H]progesterone to
membrane fractions of cells expressing the 5-HT3 receptor
by increasing concentrations of unlabeled steroids. Moreover,
photoaffinity labeling experiments did not reveal a specific
displaceable steroid binding corresponding to the respective signal of
the 5-HT3 receptor in the Western blot. These observations
argue against a specific saturable steroid-binding site within the
extracellular domain of this neurotransmitter receptor. Nevertheless,
the presence of the 5-HT3 receptor appears to favor the
binding of steroids to cell membranes as shown by the fluorescence
studies. Thus, a more likely explanation would be that the steroids
enter the membrane at the receptor-membrane interface and thereby
allosterically modulate the function of the ligand-gated ion channel in
a structure-specific manner. The effects of the steroid-BSA complexes
are not contradictory to this assumption because parts of the steroid
molecules may enter into the extracellular lipid component of the cell
membrane.
It has been demonstrated that the membranous cholesterol concentration
appears to be a critical determinant for the insertion of steroids such
as 17ß-estradiol in the cell membrane (24). Moreover, recent
experiments revealed that cholesterol (25), progesterone (26), and
anesthetic steroids that modulate the GABAA receptor (27)
may interfere with membrane fluidity in HEK 293 cells, spermatozoa, or
artificial membranes. However, the most potent steroids in this context
are not necessarily the most effective in modulating the
5-HT3 receptor. For example, 17ß-estradiol was much less
potent in the pertubation of membranes when compared with progesterone
(26), although it is a much stronger functional antagonist than
progesterone at the 5-HT3 receptor. Thus, changes in
membrane fluidity appear not to be the only determinants for the
allosteric modulation of ligand-gated ion channels by steroids in view
of the structure-activity relationship of the effects of steroids at
the 5-HT3 receptor, the known stereoselectivity of the
effects of steroids at the GABAA receptor (14), and the
lack of efficacy of cholesterol in the modulation of ligand gated
ion-channels. It appears that the allosteric modulation of
neurotransmitter receptors by steroids is a highly complex phenomenon
that is dependent on the molecule structure of the steroid, the amino
acid composition of the receptor, and the physicochemical properties of
the cell membrane.
Although considerable electrophysiological work from the last decade
suggested a putative specific steroid-binding site at the
GABAA receptor, up to now there is no biochemical evidence
for a direct interaction of steroids with this neurotransmitter
receptor, and attempts to identify such a binding-site have not yet
been successful (13, 14, 21). In addition, the molecular mechanism
underlying the modulation of
-receptors by steroids has not yet
been elucidated (28, 29, 30). On the other hand, previous investigations
reporting progesterone binding to membranes (31, 32) did not identify
the respective receptors. Current theories even postulate the existence
of membrane receptors for gonadal steroids in the brain (33).
However, it cannot be excluded that putative membrane receptors
that mediate the rapid nongenomic effects of gonadal steroids,
e.g. progesterone or estradiol (34), are not unique yet
unidentified proteins, but rather represent allosteric interactions of
steroids with already known proteins such as distinct neurotransmitter
receptors.
While steroid concentrations in the nanomolar range are sufficient to
activate intracellular steroid receptors (35, 36), the antagonistic
effects of 17ß-estradiol, progesterone, or pregnane steroids at the
N-methyl-D-aspartate receptor (22, 37), the
glycine receptor (38), or the nicotinic acetylcholine receptor (17, 18)
require micromolar concentrations, which is consistent with our
findings at the 5-HT3 receptor. The plasma concentrations
of gonadal steroids usually are in the nanomolar range. However, it
cannot be excluded that considerably higher steroid concentrations may
occur locally in the brain (39) and that there may be an enrichment of
steroids at the receptor-membrane interface due to their lipophilic
properties.
In conclusion, our data show that the gonadal steroids, 17ß-estradiol
and progesterone, may act as functional antagonists at the
5-HT3 receptor via an allosteric mechanism. Such a
functional antagonism of gonadal steroids at the 5-HT3
receptor might play a role in the development and course of nausea
during pregnancy and of psychiatric disorders such as postpartum
psychosis.
 |
MATERIALS AND METHODS
|
---|
Electrophysiological Recordings
Human embryonic kidney cells (HEK 293 cells) stably expressing
the 5-HT3 receptor (15) were grown in MEM supplemented with
10% FCS. Transfected cells were recorded in the whole-cell
voltage-clamp configuration of the giga-seal technique (40) under
visual control using an inverted microscope (Zeiss, Jena, Germany). The
cells were kept in a bath solution containing 140 mM NaCl,
2.8 mM KCl, and 10 mM HEPES, pH 7.2. Patch
electrodes were pulled from borosilicate glass (Hilgenberg, Malsfeld,
Germany) using a horizontal pipette puller (Zeitz-Instruments,
Augsburg, Germany) to yield pipettes with a resistance of 36 M
.
Pipettes were filled with a solution containing 130 mM
CsCl, 2 mM MgCl2, 2 mM
CaCl2, 2 mM ATP, 0.2 mM Tris-GTP,
10 mM glucose, 10 mM HEPES, and 10
mM EGTA, pH 7.2. After the whole-cell configuration was
established, the cells were lifted from the glass substrate, and
serotonin, metoclopramide, aromatic and nonaromatic alcohols, or
steroids were applied at the indicated concentrations using a fast
superfusion device. A piezo translator-driven double-barreled
application pipette was used to expose the lifted cell either to
serotonin-free or serotonin-containing solution for control
experiments. A 2-sec serotonin pulse was delivered every 60 sec. The
steroids were dissolved in ethanol and diluted with bath solution to
the desired concentration. To control for any possible confounding
solvent effects (41), the ethanol concentration was 0.1% in all
serotonin-free or serotonin-containing solutions with the exception of
the experiment using 100 µM 17ß-estradiol or
progesterone, where the respective serotonin-free or
serotonin-containing solutions contained 1% ethanol. Current signals
were recorded at a holding potential of -50 mV with an EPC-9 amplifier
(Heka, Lamprecht, Germany), and they were analyzed using the PulseFit
(Heka) and IgorPro (Wavemetrics, Lake Oswego, Oregon) software on a
Macintosh II computer.
Binding of [3H]GR65630 to Membrane
Fractions of Cells Expressing the 5-HT3
Receptor
HEK 293 cells stably expressing the 5-HT3 receptor
(15) were grown as described above. COS-1 cells were grown in DMEM
supplemented with 10% FCS and transfected by electroporation with 10
µg p5-HT3R-A and 5 µg of carrier DNA (pGEM4) (Promega
Corp., Madison WI) after determination of the optimal electric field
strength as described previously (36). After culture for 24 h,
transfected cells were harvested, washed with PBS, and homogenized in 5
volumes of 0.32 M sucrose, 50 mM Tris-HCl, 1
mM EDTA, pH 7.5, containing the protease inhibitors,
aprotinin (10 µg/ml), leupeptin (0.5 µg/ml), pepstatin (0.75
µg/ml), benzamidine (0.1 mM), phenylmethylsulfonyl
fluoride (0.5 mM), and
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane
(1 µM) as described previously (2). After centrifugation
at 750 x g for 10 min, the supernatant fraction was
recentrifugated at 100,000 x g for 45 min. The
resulting pellet was resuspended in 50 mM Tris-HCl, 1
mM EDTA, pH 7.5, containing the same protease inhibitors
described above. For ligand-binding experiments membrane fractions were
incubated in microtiter plates in a total volume of 250 µl at 37 C
for 30 min with the indicated concentrations of
[3H]GR65630 (New England Nuclear, 64 Ci/mmol).
Bound ligand was separated from free ligand by washing with ice-cold
assay buffer and rapid filtration through Whatman GF/B filters with a
Titertek cell harvester (Nunc, Wiesbaden, Germany).
Radioactivity was determined by liquid scintillation spectroscopy.
Nonspecific binding was determined in the presence of 10
µM MDL 72222. Specific binding represented 65% to 80%
of the total binding. Binding data were analyzed with the EBDA and
LIGAND programs, which provide a nonlinear, least-square regression
analysis.
Fluorescence Labeling of Cells Expressing
5-HT3 Receptor and the D4.4 Receptor
HEK 293 cells stably expressing the 5-HT3 receptor
(15) and native HEK 293 cells were grown as described above. A Chinese
hamster ovary (CHO-K1) cell line stably expressing the human dopamine
D4.4 receptor subtype (16) was grown in Dulbeccos
-MEM
supplemented with 2.5% FCS and 2.5% horse serum in the presence of
geneticine (G418, 400 µg/ml) as described previously (16). COS-1
cells were grown and transfected as described above. After culture for
24 h, the cells were incubated with 1 µM
17ß-estradiol 6-(O-carboxymethyl)oxime-BSA-FITC conjugate
or 1 µM progesterone
3-(O-carboxymethyl)oxime-BSA-FITC conjugate (Sigma,
Deisenhofen, Germany) for 45 min at 37 C. Cells were washed with PBS
and viewed with an Olympus fluorescence microscope (Olympus Corp. of
America, New Hyde Park, NY) using fluorescence optics.
Fluorescence studies were performed as four independent
experiments.
Photoaffinity Labeling Experiments
Membrane fractions of HEK 293 cells stably expressing the
5-HT3 receptor were obtained as previously described (2),
and 0.8 mg protein was incubated for 90 min at 37 C in a total volume
of 1 ml of 10 mM Tris-HCL, 2 mM EDTA, pH 7.5,
containing 100 µM PMSF, 2.5 µg/ml chymostatin, 20
µg/ml aprotinin, and 2 µg/ml leupeptin with 100 nM of
[3H]estradiol (Amersham, Braunschweig, Germany; 54
Ci/mmol) or [3H]progesterone (Amersham, 51 Ci/mmol) for
45 min at 37 C. Photoaffinity labeling was performed at 4 C by
illumination for 60 min at 365 nm with a distance of 5 cm between the
suspension and the lamp (Philips TLD 36W/08, Eindhoven, The
Netherlands). Proteins were washed with buffer containing 10
µM unlabeled estradiol or progesterone and were
solubilized as described previously (42). After electrophoresis, the
respective lanes were either stained by Coomassie blue or cut into
1.5-mm slices to determine the retained radioactivity by scintillation
counting. Western blots of solubilized proteins were incubated with a
polyclonal antibody against the 5-HT3 receptor (43) at a
titer of 1:4000 for 1 h. The detection of signals was performed
with a peroxidase-labeled antibody against rabbit IgG
(Boehringer, Mannheim, Germany) at a titer of 1:4000 for 20
min.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Drs. A. Maricq and D. Julius for the generous
gift of the p5-HT3-RA plasmid and the HEK cell line stably
expressing the 5-HT3 receptor; Drs. M. Morales and F. Bloom
for providing the antibody against the 5-HT3 receptor; Dr.
H. H. M. van Tol for providing the CHO cell line stably
expressing the D4.4 receptor and the expression vector for the D4.4
receptor; and Dr. P. H. Seeburg for valuable comments on the
manuscript. We also appreciate the expert technical assistance of Mrs.
I. Vollmer and B. Burkart-Lauer.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. R. Rupprecht, Department of Psychiatry, Ludwig Maximilian University, Nußbaum-strasse 7, 80336 Munich, Germany. E-mail: Rainer.Rupprecht{at}psy.med.uni-muenchen.de
This work was supported by the Gerhard-Heß-Programm of the Deutsche
Forschungsgemeinschaft (to R.R.).
1 These authors contributed equally to this work. 
2 Present address: Department of Cell Physiology, Ruhr-University of
Bochum, 44780 Bochum, Germany. 
Received for publication December 19, 1997.
Revision received April 14, 1998.
Accepted for publication May 18, 1998.
 |
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