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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}-estradiol, 17{alpha}-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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {gamma}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). The simultaneous application of the competitive 5-HT3 receptor antagonist metoclopramide markedly reduced the serotonin-evoked cation current (Fig. 1AGo). This effect was even more pronounced when metoclopramide was already present before the serotonin pulse (Fig. 1CGo). However, the antagonistic properties of metoclopramide could be reversed by subsequent challenge with serotonin in the absence of metoclopramide (Fig. 1BGo). 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. 1FGo). 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. 1DGo). 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. 1EGo). 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. 2Go). The effects of 17ß-estradiol (Fig. 3Go) 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.

 
An antagonistic effect at the serotonin-evoked cation current could further be demonstrated for 10 µM 17{alpha}-estradiol, testosterone, allopregnanolone (3{alpha}-hydroxy-5{alpha}-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. 4Go).



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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{alpha}-estradiol (17{alpha}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.

 
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. 5AGo). 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. 5BGo). 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 1Go). 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 1Go). 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

 
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. 6Go) and the progesterone-BSA-FITC complex (Fig. 7Go). 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. 8Go) 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.

 
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. 9Go) 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{alpha}-ethinyl-17ß-estradiol and mestranol, two synthetic derivatives of 17ß-estradiol, markedly reduced the serotonin response (Fig. 10Go). 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 9. Molecule Structure of Different Aromatic Alcohols and Estrogens

 


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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{alpha}-ethinyl-17ß-estradiol (EthinylE2), and mestranol. Ethanol and phenol did not significantly affect the serotonin-evoked cation current.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {varsigma}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 3–6 M{Omega}. 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 Dulbecco’s {alpha}-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. Back

2 Present address: Department of Cell Physiology, Ruhr-University of Bochum, 44780 Bochum, Germany. Back

Received for publication December 19, 1997. Revision received April 14, 1998. Accepted for publication May 18, 1998.


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 MATERIALS AND METHODS
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