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Endocrinology, doi:10.1210/en.2006-1198
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Endocrinology Vol. 148, No. 3 1384-1395
Copyright © 2007 by The Endocrine Society

Estradiol Induces Expression of 5-Hydroxytryptamine (5-HT) 4, 5-HT5, and 5-HT6 Receptor Messenger Ribonucleic Acid in Rat Anterior Pituitary Cell Aggregates and Allows Prolactin Release via the 5-HT4 Receptor

A. Papageorgiou and C. Denef

Laboratory of Cell Pharmacology, University of Leuven, Medical School, Campus Gasthuisberg, B3000 Leuven, Belgium

Address all correspondence and requests for reprints to: Prof. Carl Denef, Laboratory of Cell Pharmacology, University of Leuven, Medical School, Campus Gasthuisberg (O & N), B-3000 Leuven, Belgium. E-mail: Carl.Denef{at}med.kuleuven.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Serotonin [5-hydroxytryptamine (5-HT)] is known to control prolactin (PRL) release at a hypothalamic level, but a pituitary site of action remains poorly studied. The present study explores the acute effect of 5-HT on PRL release in rat anterior pituitary aggregate cell cultures, the influence of steroid and thyroid hormones, and the 5-HT receptor (5-HTR) subtype(s) involved. 5-HT elicited a prompt increase in basal PRL release, an effect strongly potentiated by estradiol (E2) in the culture medium (dose response 1–100 nM). In E2 condition, the PRL response was not affected by the nonselective 5-HTR antagonists methysergide and methiothepin nor by 5-HTR1, 5-HTR2, 5-HTR3, 5-HTR6, and 5-HTR7/5 antagonists, but was fully blocked by the 5-HTR4 antagonist GR 113808. Among various agonist analogs, only the 5-HTR4 agonist cisapride and the 5-HTR2 agonist {alpha}-methyl-5-HT evoked PRL release. The effect of {alpha}-methyl-5-HT also required E2 during culture and was abolished by GR 113808 but not by combined 5-HTR2A, B, and C blockade. In E2-treated aggregates, 5-HT caused a 5-fold increase in cAMP levels. The intact anterior pituitary expressed mRNA of all known members of the 5-HTR family. In aggregates, 5-HTR4, 5-HTR5, and 5-HTR6 mRNA expression required E2 during culture. The effect of 5-HT on PRL release was not affected by blocking the serotonin transporter or the vesicular monoamine transporter. The present data suggest a widespread expression of 5-HTRs in the rat anterior pituitary, several of which are up-regulated by estrogen, and that, in the presence of estrogen, one of these, the 5-HTR4, mediates acute PRL release.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEROTONIN [5-HYDROXYTRYPTAMINE (5-HT)] is a widespread neurotransmitter and neuromodulator in the central and peripheral nervous systems, with numerous functions in sensory-motor, autonomic, and behavioral systems (1, 2). Yet it is also a hormone and paracrine factor in nonneural tissues such as enterochromaffin cells, immune cells, platelets, skin, adrenal, mammary gland, and others, exerting functions related to vasomotor activity and hemostasis, intestinal motility, and secretion, T-cell-mediated immune responses, and reproduction and development (1, 3, 4). Signaling is through a large family of 5-HT receptors (5-HTR), comprising seven groups and different subtypes (5-HTR1A, B, D, E and F, 2A, B and C, 3, 4, 5A, 5B, 6, and 7) (5), although recently Walther et al. (6) demonstrated an additional receptor-independent mode of action in 5-HT-stimulated {alpha}-granule exocytosis from platelets.

5-HT indirectly controls pituitary function through neuronal projections from the medial and dorsal raphe nuclei of the brain stem to specific nuclei of the basal hypothalamus (7, 8), which alters hypophysiotropic hormone delivery into the portal blood vessels reaching the endocrine cells of the anterior pituitary. In vivo, 5-HT stimulates prolactin (PRL), GH, and ACTH release (9, 10, 11), whereas both stimulatory and inhibitory actions on LH release have been reported (12).

5-HT may also have direct effects at the level of the pituitary gland via receptors located on the endocrine cells. More than two decades ago it was shown that the anterior pituitary contains 5-HT (13, 14) and converts tryptophan into 5-HT, presumably by the enzyme tryptophan hydroxylase (15). Subsequently, 5-HT has been demonstrated in nerve fibers originating in the raphe magnus and making synaptoid contact with endocrine cells of the intermediate lobe (16). In the anterior pituitary of bats and mice, 5-HT has been found in secretory granules of the gonadotrophs (14, 17) and in nerve fibers from unknown origin entering the rostral zone with blood vessels (18). Anterior pituitary glandular cells of the rat display a specific and fluoxetine-sensitive uptake system of 5-HT (19) and in bats these uptake cells may be gonadotrophs (20). The fluoxetine sensitivity suggests the carrier is the specific 5-HT transporter [serotonin transporter (SERT)]. However, its mRNA has not been detected in the postnatal anterior pituitary but appears to be transiently expressed during fetal life in the anterior and intermediate lobe of the rat (21).

Little is known about the precise function of 5-HT and the regulation of its action at the pituitary level. A few studies have shown a stimulatory effect on ACTH/ß-endorphin release (22, 23, 24) and an increase in intracellular free Ca2+ in AtT20 cells (25). 5-HT inhibits basal and GnRH-stimulated LH release in intact pituitary (26, 27) but potentiates this response in the LßT2 cell line (28), and in dispersed pituitary cells, 5-HT increases basal LH release (29). 5-HT may also affect PRL release, but the data are controversial. Some investigators showed stimulation of basal and potentiation of TRH-stimulated PRL release in transplanted rat pituitaries and rat hemipituitaries or cultures (30, 31, 32), but another group did not find any effect (33). Still others showed a PRL response in monolayer cultures (34), provided intermediate lobe cells were present, a phenomenon also seen in vivo (35).

Data concerning 5-HTRs expressed in the pituitary and their functions are also very limited. Release of ß-endorphin from pituitary cell cultures is stimulated by 5-HTR2 agonists (23, 36), but also the 5-HTR3 appears to mediate 5-HT-stimulated ACTH release from cultures (24). In only one study, the receptor subtype involved in 5-HT-stimulated PRL release was suggested to be a 5-HTR2 (32), but to our knowledge, no data are available on the implication of other 5-HTR receptors. The 5-HTR6 is expressed at the mRNA level in the normal pituitary (lobe not specified) but no function is known (37). A strong in situ hybridization signal of 5-HTR3 has been reported in the anterior and intermediate lobe of the rat at embryonic d 14.5 (38) but the biological significance of this early developmental expression remains unknown.

The present investigation intended to study the direct effect of 5-HT on basal PRL secretion in reaggregated anterior pituitary cells and to characterize the receptor mechanism behind any intrinsic activity. Extensive research in our laboratory already demonstrated the unique characteristics of the latter in vitro test system in terms of secretory responses to monoamines and peptides (39, 40, 41). The present study also examined potential nonreceptor mechanisms recently reported for certain actions of 5-HT, i.e. serotonylation of small GTPases by intracellular transglutaminase, for which 5-HT first needs to be taken up in the cell (6). We demonstrate that there is indeed an acute stimulatory effect of 5-HT on PRL release and that it is strongly up-regulated in magnitude and sensitivity by estradiol (E2) in culture. Furthermore, we show that there is widespread expression of 5-HTR subtypes in both intact pituitary and culture, but that the in vitro PRL response to 5-HT is mediated mainly, if not exclusively, through the 5-HTR4, the expression of which is also estrogen-induced. Uptake of 5-HT into the cell via SERT or the vesicular monoamine transporter (VMAT) is not obligatory in the effect.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and drugs
5-HT (5-HT), SB-269970, ketanserin, SB-206553, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), cisapride, WAY-100635, BRL-15572, GR113808, Ro 04–6790, pertussis toxin (PTX), isobutylmethylxantine (IBMX), SB-224289, and citalopram were purchased from Sigma Chemical Co. (St Louis, MO). Ondansetron and sumatriptan were purchased from GlaxoSmithKline (Middlesex, UK). Domperidone was obtained from Janssen Pharmaceutica (Beerse, Belgium). Methiothepin, methysergide, 5-carboxamidotryptamine, {alpha}-methyl-5-HT, m-chlorophenyl piperazine, RS-102221, and (+/–)-8-hydroxy-dipropylaminotetralin (8-OHDPAT) were purchased from Tocris Biosciences (Avonmouth, UK). T3, dexamethasone (Dex), and BSA (Cohn fraction V) were from Serva (Heidelberg, Germany). E2 and ascorbic acid were from Merck (Darmstadt, Germany). 5-HT was dissolved in a 1% ascorbic acid solution and was made freshly. Domperidone, E2, Dex, and ketanserin were dissolved in ethanol and stored at 4 C. PTX was dissolved in water and stored at 4 C. IBMX was made freshly and dissolved in DMSO as was GR113808 and cisapride (stock concentration of 10–2 M). All other chemicals (analytical grade) were dissolved in water and stored at –20 C. Stock concentrations were 10–3 M, except for Dex and E2 (stock concentration 10–5 M), and T3 (10–7 M stock concentration).

Animals
Adult male Wistar rats (3 months old) were obtained from Elevage Janvier (Schaijk, The Netherlands) and were housed in an environment with constant temperature, humidity, and day-night cycles, with food and drink ad libitum. All experiments were conducted in accordance with the Guidelines for Care and Use of Experimental Animals and approved by the University Ethical Committee.

Anterior pituitary aggregate cell culture
Rats were decapitated after CO2 anesthesia, and the anterior pituitary was dissected free from the neurointermediate lobe under sterile conditions and dispersed into single cells as described previously (39). Dispersed cells were allowed to aggregate at 2 x 106 cells in nontreated 35-mm petri dishes (Iwaki, Japan) with 2 ml serum-free defined culture medium. For aggregation, the dishes were placed on a gyratory shaker (Applitek, Nazareth, Belgium) at 64 rpm in a 1.5% CO2 humidified incubator at 37 C. Culture medium was serum-free defined medium as previously described (39, 42). It consists of HEPES/TES-buffered DMEM/F12 1/1 with selenite and ethanolamine (prepared as powder by Invitrogen, Grand Island, NY), supplemented with 0.5% BSA, 5 mg/liter insulin-Zn, 5 mg/liter transferrin, 50 mg/liter streptomycin, 35 mg/liter penicillin, 10 mM ethanol, 1 mg/liter catalase, 8 mg/liter phenol red, and 1 g/liter NaHCO3. Final concentration of iron (Fe2+ and Fe3+) was 0.6 µM and of glucose 1.4 g/liter (7.8 mM). Medium was replaced after 2 d, and aggregates were tested on d 5 of culture. Depending on the experimental design, the medium was either hormone-free or supplemented with 1 nM E2, 1 nM T3, or 10 nM Dex (Serva). In certain experiments, PTX (100 ng/ml) (Sigma) was added in the 35-mm petri dishes containing the aggregates for a period of 24 h before the perifusion.

Perifusion of aggregates
The perifusion system was designed to study the continuous release of hormones from aggregates over a period of time, with the possibility of adding substances and observing their effect on hormone secretion at intervals of 1–2 min. A more elaborate description of its design and set-up can be found elsewhere (39). We have validated the system thoroughly and used it in numerous studies on secretory responses to GnRH and TRH, adrenergic agonists, GHRH and vasoactive intestinal peptide, cholinomimetics, dopamine, angiotensin II (39), bombesin (43), and PRL-releasing peptide (41). Briefly, aggregates (2 x 106 cells) are placed on a nylon mesh, in a chamber that is kept at 37 C, and immersed in medium [DMEM with 4.5 g/liter glucose; Invitrogen, supplemented with 15 mM HEPES, 110 mg/liter Na-pyruvate, 1 g/liter NaHCO3, and 0.5 g/liter NaCl (pH 7.5)], which is being pumped at a set rate through the chamber and collected in 2-min fractions. Aggregates are perifused for a period of 1.5 h to stabilize hormone release, before any test substances are applied to the cells. A two-way valve system enables a smooth uninterrupted switch between a "rest-stage," where only medium (with vehicle if appropriate) is passed through the chamber, and an "exposure stage," where test substances (dissolved in the same perifusion medium) perifuse the aggregates. Fractions (0.5 ml/2 min) are collected in 100 µl 2% BSA (Serva) in 0.01 M phosphosaline, vortexed, and stored at –20 C before analysis. To avoid any time delay in the onset of action of receptor antagonists, the antagonists were added to the perifusion medium 30 min before the start of fraction collections and were also present during the perifusion of the agonist to be blocked. In the figures, exposure time to agonists is indicated by a horizontal bar, taking into account the dead time in the system (4 min).

RIA
Hormone levels were measured from duplicate samples by specific competitive RIA using the rat PRL RIA kit obtained from Dr A. F. Parlow (National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, Harbor-UCLA Medical Center, Torrance, CA) with rabbit antirat PRL (S-9) antiserum and rat PRL-RP-3 as reference preparation. The reliability of the assays has been shown in previous work (39, 41). Both intraassay and interassay variation were less than 10%. Labeling of PRL with 125I was done with the Iodogen method according to the manufacturers’ instructions (Pierce, Perbio Sciences, Erembodegem-Aalst, Belgium). Values were expressed in nanogram equivalents of National Institute of Diabetes and Digestive and Kidney Diseases rat PRL-RP-3 standard. The average basal PRL release was around 20 ng/2 min for E2-treated aggregates, 5 ng/2 min for T3-treated aggregates, 4 ng/2 min for Dex-treated aggregates, and 3 ng/2 min for aggregates with no hormone supplement.

cAMP measurements
Aggregates (cultured as mentioned above) from different dishes were combined and subsequently distributed in the same medium with 0.5 mM IBMX in 35-mm diameter petri dishes at a density of roughly 106 cells per dish (based on the cell number counted at the beginning of culture). The cells were then incubated in culture medium with IBMX and 5-HTR agonists or vehicle for 30 min at 37 C in a CO2 humidified incubator, after which aggregates were collected and cell lysis was obtained through the addition of 0.1 M HCl and sonication. cAMP content was quantified through enzymatic immunoassay kit from Assay Designs (Ann Arbor, MI) (44).

RNA extraction and RT-PCR
RNA from whole anterior pituitary or from aggregates was isolated using Tripure RNA isolation reagent (Roche Diagnostics, Mannheim, Germany) following the procedure recommended by the manufacturer. RNA was dissolved in diethyl pyrocarbonate-treated RNase-free water and treated with 1 U (1 µl) of DNase I (amplification grade), in 20 mM Tris-HCl (pH 8.4), 2 mM MgCl2, 50 mM KCl (Invitrogen), followed by the addition of 0.125 mM EDTA (1 µl). RNA quantification was done with the Ribogreen RNA quantification kit (Invitrogen). Subsequent detection of mRNA was done by RT-PCR with defined sets of primers (Table 1Go), flanking one intron in each gene where applicable. Ribosomal L19 gene mRNA detection was carried to ensure RT-PCR performance. RT was performed as follows. The RT mixture contained 4 µl MgCl2 (25 mM), 2 µl 10x PCR buffer III, 1 µl RNase inhibitor (RNasin 40 U/µl; Promega Benelux, Leiden, The Netherlands), 8 µl of dNTP mixture (2.5 mM of each; Applied Biosystems, Applera, Belgium), 1 µl Moloney murine leukemia virus reverse transcriptase (200 U/µl; Invitrogen), 1 µl random hexamers (50 µM, Applied Biosystems), 1 µl of water, and RNA (corresponding to 60 ng for aggregates or whole anterior pituitary) in 2 µl, previously heated at 72 C for 10 min and placed on ice. RT was performed using a four-step program; 15 min at 25 C, 50 min at 42 C, 10 min at 95 C, and cooling down to 4 C.


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  		TABLE 1. Primer cDNA sequences used for PCR

 
The PCR mixture included 0.9 µl MgCl2 (25 mM), 1.1 µl 10x PCR buffer III, 0.15 µl Amplitaq gold (5 U/µl; Applied Biosystems), 1 µl of dNTP mixture (2.5 mM of each; Applied Biosystems), 3.85 µl of H2O, 1 µl of each antisense and sense primer (10 µM; Invitrogen), and 1 µl cDNA solution (corresponding to 3 ng RNA for aggregates). The amplification program was as follows: 7 min at 95 C; followed by 45 cycles of 95 C for 10 sec, 20 sec annealing temperature, 30 sec at 72 C; and finally 7 min at 72 C with subsequent cooling to 4 C. The amplified DNA fragment was visualized by means of electrophoresis in a 2% agarose gel containing 0.5 µg/ml ethidium bromide. The authenticity of the amplified products was confirmed by nucleotide sequence analysis (Lark, Essex, UK) after cloning the fragment in the 2.1 TOPO vector (Invitrogen).

Data expression and statistics
All data from the perifusion experiments are expressed as percentage of basal hormone secretion defined as the average secretion before each drug application. Values are expressed as mean ± SEM. Statistical analysis was performed using either a one-way ANOVA with Fisher’s least significant difference (LSD) multiple comparison test on the cumulative secretion for the duration of drug application (area under the curve, AUC) or two-way ANOVA with Fisher’s LSD multiple comparison test using individual values of each 2-min fraction. All data from the cyclic AMP experiments are expressed as percentage of control cAMP production and values are expressed as mean ± SEM. One-way ANOVA with Fisher’s LSD multicomparison test was used for the statistical analysis, after data were log transformed due to heterogeneity of variance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
5-HT stimulates PRL release in cultured pituitary cell aggregates
In a first series of experiments, aggregates were cultured in hormone-free medium and, on d 5 of culture, exposed to three doses of 5-HT, i.e. 10 nM, 100 nM, and 1 µM each, for a period of 20 min, with rest periods of 40 min between pulses. As shown in Fig. 1AGo, 5-HT did not affect PRL release at the lowest dose, and was slightly stimulatory at a 100-nM dose, although not to a statistically significant degree (AUC 13% above basal release). A significant (P < 0.05) but not impressive effect of 5-HT was found at a dose of 1 µM, with a maximal increase (peak PRL value during exposure to 5-HT) to 39 ± 8.1% and AUC of 20% above basal release.


Figure 1
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  		FIG. 1. Effect of 5-HT on PRL release from perifused pituitary cell aggregates cultured either without (A) (n = 6) or with 1 nM E2 in the culture medium (B and C). A and B, Ten nanomolar, 100 nM, and 1 µM 5-HT (n = 8). C, One and 3 nM 5-HT (n = 3). Data are PRL values at the different time points ± SEM expressed as percentage of basal release (measured during the first 20 min or 20 min preceding the next dose). The average basal PRL release was around 3 ng/2 min for aggregates with no hormone supplement and 20 ng/2 min for E2-treated aggregates. Statistics: A and B, One-way ANOVA with Fisher LSD multiple comparison test on log-transformed data comparing basal secretion values to 5-HT-stimulated values (AUC) or comparing the different doses among each other. C, Two-way ANOVA with Fisher LSD multiple comparison test comparing basal secretion values at the different time points. ##, P < 0.001 vs. basal PRL release; *, P < 0.05 for hormone-free vs. E2-supplemented condition.

 
As previously observed (41, 43, 45, 46, 47), the response to various monoamine or peptide agonists may either depend on, be abolished or even reversed by the presence of thyroid, estrogen, glucocorticoids, or combinations of these hormones during culture. In the present study, the PRL response to 5-HT in aggregates cultured in the presence of 10 nM Dex or 1 nM T3 was not different from the modest response seen in aggregates cultured in hormone-free medium (Table 2Go). In contrast, in aggregates cultured in the presence of 1 nM E2, a significant stimulation of PRL release was already seen at 10 nM 5-HT, with a peak response of 48 ± 15% above basal release. At 100 nM and 1 µM 5-HT, the peak response was 77 ± 13% and 98 ± 17% above basal release, respectively (Fig. 1BGo and Table 2Go) (P < 0.001). A dose-dependent increase was observed between the 10 and 100 nM dose (P < 0.005) and between the 10 nM and 1 µM dose (P < 0.001). In another series of experiments, a dose response to smaller concentrations of 5-HT was studied (Fig. 1CGo). A small increase in PRL secretion was noted at 1 nM 5-HT (P < 0.001) and 3 nM 5-HT (P < 0.001) with a dose-dependent difference (P < 0.001).


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  		TABLE 2. PRL response to 5-HT in anterior pituitary cell aggregates cultured in the presence of T3 (1 nM), Dex (10 nM), or E2 (1 nM) during culture, and effect of PTX pretreatment and of domperidone during perifusion

 
To exclude any blunting effect of 5-HT via dopamine receptors, resulting in an inhibitory action on PRL release, 5-HT (10 nM, 100 nM and 1 µM) was added in the perifusion in the presence of the D2 receptor blocker domperidone (10 nM) (aggregates cultured with 1 nM E2). The PRL response to 5-HT was not altered in magnitude in the presence of domperidone (Table 2Go).

5-HT-induced PRL release is blocked by a selective 5-HTR4 antagonist
To dissect out which of the 5-HTRs is responsible for 5-HT stimulation of PRL release, aggregates cultured with 1 nM E2 were exposed to 5-HT in the absence and presence of nonselective and selective 5-HTR antagonists, all being used at a dose at least two orders of magnitude above their Ki for binding at the respective 5-HTRs. As shown in Fig. 2Go, the PRL response to 5-HT (10 nM) was not changed at any time point in the presence of methysergide or methiothepin, which both block the 5-HTR1, 2, 5, 6, and 7 (48, 49, 50, 51). The 5-HTR2A antagonist ketanserin (48, 52, 53, 54), the 5-HTR2B antagonist SB-206553 (55, 56, 57), and the 5-HTR2C antagonist RS-102221 (50, 56, 57), used separately (each 1 µM) (data not shown, n = 3) or in combination (Fig. 3Go), also did not affect the PRL response, nor did the 5-HTR7 selective antagonist SB-269970 (1 µM); the latter also displayed some blocking potency at the 5-HTR5 (58) (Fig. 3Go). The addition of the 5-HTR3 antagonist ondansetron (3 µM) (59) also did not alter the PRL response to 10 nM 5-HT (Fig. 3Go). In contrast, the 5-HTR4 antagonist GR113808 (1 µM) (60, 61) completely blocked the PRL response (P < 0.03) (Fig. 3Go). The 5-HTR6 blocker Ro 04–6790 (1 µM) (62) and selective antagonists of 5-HTR1A (WAY100635, 50 nM) (63), 5-HTR1B (SB-224289, 1 µM) (57, 64), and 5-HTR1D (BRL 15572, 1 µM) (65) also did not affect the PRL response (in at least three independent experiments, data not shown).


Figure 2
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  		FIG. 2. Effect of 10 nM 5-HT alone or in the presence of the nonselective 5-HT receptor antagonists methiothepin (left panel; n = 3) and methysergide (right panel; n = 4) on PRL release from perifused anterior pituitary cell aggregates cultured in E2-supplemented medium. Data are PRL values at the different time points ± SEM expressed as percentage of basal (measured during the first 20 min) release.

 

Figure 3
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  		FIG. 3. Effect of selective 5-HT receptor antagonists on the PRL response to 10 nM 5-HT in perifused anterior pituitary cell aggregates cultured in E2 supplemented medium. Data are PRL values at the different time points ± SEM expressed as percentage of basal release (measured during the first 20 min). A, 5-HT alone and in the presence of 5-HTR2A antagonist ketanserin, 5-HTR2B antagonist SB-206553, and 5-HTR2C antagonist RS-102221 (1 µM) (n = 3); B, 5-HT alone and in the presence of 5-HTR3 antagonist ondansetron (3 µM) (n = 3); C, 5-HT alone and in the presence of 5-HTR4 antagonist GR113808 (1 µM) (n = 3); and D, 5-HT 10 nM alone and in the presence of 5-HTR5/7 antagonist SB-269970 (1 µM) (n = 3). Statistics: One-way ANOVA with Fisher’s LSD multiple comparison test performed on AUC values during 5-HT vs. 5-HT in the presence of the antagonist; {ddagger}, P < 0.03.

 
Experiments with agonists confirm the mediation of the PRL response to 5-HT by the 5-HTR4
A series of 5-HT agonists with different selectivity for the 5-HTRs (aggregates cultured with 1 nM E2) was tested at doses relevant according to their receptor binding affinity. As expected, the 5-HTR4 agonist cisapride (60, 61, 66), caused a significant increase in PRL secretion at both the 100 nM and 1 µM concentrations (P < 0.001) (Fig. 4Go). Curiously, in contrast to 5-HT, the effect at 100 nM was larger in magnitude than that at 1 µM (P < 0.01). There was no PRL response to the 5-HTR1A agonist 8–0H-DPAT (53) at 10 nM, whereas, at a 100 nM dose, a small inhibitory response was seen (P < 0.01; Fig. 4Go). The 5-HTR1B, D, F agonist sumatriptan (100 nM and 1 µM) (48, 64) had also no effect (Fig. 4Go). In contrast, the preferential 5-HTR2 agonist {alpha}-methyl-5-HT (100 nM and 1 µM) (48, 53, 54, 67) significantly stimulated PRL release at 1 µM concentration (P < 0.001) (Fig. 4Go). However, two other 5-HTR2 agonist, m-Cpp (61, 67, 68) (100 nM and 1 µM) and DOI (100 nM or 1 µM) (48, 67, 69) did not affect PRL release (n = 3; data not shown) (Fig. 4Go). 5-Carboxytryptamine (5-CT), a high affinity agonist of 5-HTR1A, B, D (53, 64, 70), 5-HTR5 (48, 49, 71), and 5-HTR7 (72, 73) did not stimulate PRL release at a 10-nM dose and caused a very slight increase at 100 nM [AUC +19% above basal release, data not shown (P < 0.001)].


Figure 4
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  		FIG. 4. Effect of 5-HT receptor agonists on PRL release from perifused anterior pituitary cell aggregates cultured in E2-supplemented medium. Data are PRL values at the different time points ± SEM, expressed as percentage of basal release (measured during the first 20 min or 20 min preceding the second dose). A, 5-HTR1A agonist 8-OHDPAT (10 and 100 nM, n = 4); B, 5-HTR1B/1D/1F agonist sumatriptan (100 nM and 1 µM, n = 3); C, 5-HTR2 agonist {alpha}-methyl-5-HT ({alpha}-me-5-HT, 100 nM and 1 µM, n = 4); D, 5-HTR2 agonist DOI (100 nM and 1 µM, n = 3); E, 5-HTR4 agonist Cisapride (100 nM and 1 µM, n = 4); and F, 5-HT (10 and 100 nM, n = 6). The 5-HT effects were studied simultaneously with at least several of the other agonists, except cisapride, which was tested separately. Statistics: One-way ANOVA with Fisher’s LSD multiple comparison tests performed comparing basal secretion values and 5-HT stimulation values (AUC) except in A, where a two-way ANOVA with Fisher LSD multiple comparison test was performed with the different time points to compare basal PRL values and 100 nM 8-OHDPAT values. Basal release vs. 5-HT, ##, P < 0.001; cisapride 100 nM vs. 1 µM, **, P < 0.01.

 
In this series of experiments, the dose of 100 nM cisapride and 1 µM {alpha}-methyl-5-HT had an effect of approximately the same magnitude as that of 10 nM 5-HT (Fig. 4FGo).

The PRL response to {alpha}-methyl-5-HT (1 µM) was abolished when perifused together with the 5-HTR4 antagonist GR113808 (1 µM) (aggregates cultured with 1 nM E2) (Fig. 5Go). In contrast, a combined blockade of the 5-HTR2A, 5-HTR2B, and 5-HTR2C by perifusion with ketanserin, SB-206553, and RS-102221 (each at 1 µM) in combination, did not change the PRL response to {alpha}-methyl-5-HT (Fig. 5Go).


Figure 5
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  		FIG. 5. Effect of 5-HTR2 and 5-HTR4 antagonists on the PRL response to 1 µM {alpha}-methyl-5-HT in perifused anterior pituitary cell aggregates (n = 4). Data are PRL values at the different time points ± SEM is expressed as percentage of basal release (measured during the first 20 min). A, One micromolar {alpha}-methyl-5-HT alone and in the presence of 5-HTR2A antagonist ketanserin, 5-HTR2B antagonist SB-206553, and 5-HTR2C antagonist RS-102221 (added together; each 1 µM) in E2-treated aggregates (n = 4). B, One micromolar {alpha}-methyl-5-HT alone (redrawn from A) and in the presence of the 5-HTR4 antagonist GR113808 (1 µM) in E2-treated aggregates (n = 4). C, One micromolar {alpha}-methyl-5-HT in aggregates cultured in hormone-free medium (n = 4). Statistics: One-way ANOVA with Fisher’s LSD multiple comparison test performed on AUC values during {alpha}-methyl-5-HT vs. {alpha}-Methyl-5-HT in the presence of the antagonist and comparing basal secretion values with {alpha}-methyl-5-HT stimulation values (AUC) in E2-treated aggregates. Two-way ANOVA with Fisher LSD multiple comparison test was performed with the different time points to compare basal PRL values vs. stimulated values. ##, P < 0.001

 
One previous study reported mediation of PRL release by 5-HT in a ketanserin-blockable manner (32), indicating mediation via the 5-HTR2A. Our agonist and antagonist studies were performed on aggregates cultured in the presence of E2, which may have blunted mediation via the 5-HTR2A. Therefore, we wanted to verify the hypothesis that the small effect of 5-HT on PRL release in hormone-free medium was the result of an effect via the 5-HTR2 that was counteracted by 5-HT acting at other 5-HTRs. In that case, a 5-HTR2 agonist would elicit a clear-cut PRL release. In aggregates cultured in hormone-free medium, selective agonist stimulation of the 5-HTR2 with 1 µM {alpha}-methyl-5-HT did not cause an appreciable increase in PRL release [only 19 ± 8% above basal release, comparable with the effect of 5-HT in hormone-free medium, but statistically significant (P < 0.01)] (Fig. 5Go).

No effect of PTX on 5-HT evoked PRL release
Because there are 5-HTR subtypes (5-HTR1 and 5-HTR5) that are negatively coupled to adenylyl cyclase through Gi/o (74, 75), and because 8-OH-DPAT slightly inhibited basal PRL release, we tested whether PRL secretion in response to 5-HT was modulated by Gi/o coupling. Therefore, aggregates (cultured in medium with 1 nM E2) were pretreated in culture with PTX (100 ng/ml) for 24 h before the perifusion. The PRL response to 10 nM, 100 nM, and 1 µM 5-HT was not significantly affected by PTX (Table 2Go).

Effect of 5-HT uptake inhibition on the PRL response to 5-HT
We wanted to test the hypothesis that an intracellular mode of action might be involved after 5-HT uptake in the cells because such an intracellular action has recently been discovered by Walther et al. (6) in platelet aggregation (a process called serotonylation). These investigators demonstrated that 5-HT can be covalently bonded through intracellular transglutaminase to small GTPases (such as Rab4 and RhoA), which then become constitutively active as intracellular signaling molecules. To test the possible implication of this intracellular mechanism, the PRL response to 5-HT (aggregates cultured with 1 nM E2) was examined in the presence of substances that block uptake of 5-HT, reasoning that, in this way, activation of GTPases cannot occur and the PRL response would weaken or disappear. 5-HT has been shown to be taken up in anterior pituitary cells by a fluoxetine-sensitive mechanism (17, 19, 20) typical for a SERT-mediated uptake system (76). As shown in Fig. 6Go, however, neither citalopram (100 nM), a highly specific SERT inhibitor known to fully block SERT at a 100 nM concentration (77), nor ketanserin (1 µM), a potent blocker of the VMAT-2 (78), affected 5-HT-induced PRL release. Citalopram did not affect basal PRL release when added alone (data not shown).


Figure 6
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  		FIG. 6. Effect of citalopram (100 nM) and ketanserin (1 µM) on the PRL response to 5-HT in perifused anterior pituitary cell aggregates cultured in the presence of 1 nM E2. Both citalopram and ketanserin were added to the perifusion 50 min prior and during 5-HT exposure. Data are PRL values at the different time points ± SEM expressed as percentage of basal release (measured during the first 20 min). Left panel, Ten nanomolar 5-HT alone or in the presence of 100 nM citalopram (n = 3). Right panel, Ten nanomolar 5-HT alone or in the presence of 1 µM ketanserin (n = 3).

 
5-HTR agonists stimulate cAMP production in aggregates
5-HT (100 nM), added for 30 min to aggregates cultured in hormone-free medium, slightly but significantly stimulated cAMP accumulation compared with the control aggregates treated with vehicle only (P < 0.03) (Fig. 7Go). When aggregates were cultured in the presence of E2 (1 nM), 5-HT (100 nM) caused a more than 5-fold rise in cAMP accumulation (P < 0.001). A comparable response was seen with 1 µM {alpha}-methyl-5-HT (P < 0.001) and cisapride (P < 0.01) (Fig. 7Go).


Figure 7
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  		FIG. 7. Effect of 5-HTR agonists on cAMP accumulation in anterior pituitary cell aggregates. Left panel, Effect of 100 nM 5-HT in aggregates cultured without hormone supplement (n = 2). Right panel, Effect of 100 nM 5-HT, 1 µM {alpha}-methyl-5-HT, and 1 µM cisapride in aggregates cultured with 1 nM E2 (n = 3). Data are presented as percentage basal cAMP levels in control dishes. {alpha}-me5-HT, {alpha}-Methyl-5-HT. Statistics: One-way ANOVA with Fisher’s LSD multiple comparison test performed on log transformed values for agonist data vs. control values. ##, P < 0.001; **, P < 0.01; {ddagger}{ddagger}, P < 0.02.

 
Expression of 5-HTRs in intact pituitary and aggregates
Because our functional data strongly suggested that the 5-HTR4 mediates the effect of 5-HT on PRL release, we examined which 5-HTRs are expressed at the mRNA level in the anterior pituitary and the aggregates by means of RT-PCR of a RNA extract (3 ng RNA used per RT-PCR). In RNA from intact anterior pituitary, cDNA amplicons of all 5-HTRs known today (except 5-HTR1E that was not tested) were detected, each with a size corresponding to that of the gene fragment amplified on the basis of the primers used (Fig. 8Go). In aggregates, cultured in hormone-free medium, a cDNA signal for the 5-HTR4, 5-HTR5A, 5-HTR5B, and 5-HTR6 was not detectable. However, in RNA extract from E2-treated aggregates, the 5-HTR4, 5-HTR5A, 5-HTR5B, and 5-HTR6 amplicons were clearly present again, whereas the expression of 5-HTR2C was lost (Fig. 8Go). The authenticity of all amplicons, including those found in samples of aggregates cultured in the presence of E2, was confirmed by nucleotide sequencing (sequence identical to expected).


Figure 8
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  		FIG. 8. Analysis of 5-HTR1A, 1B, 1D, 1F, 2A, 2B, 2C, 3, 4, 5A, 5B, 6, and 7 mRNA expression by RT-PCR in intact anterior pituitary and aggregates cultured without hormone and with E2 (1 nM E2) supplement. Representative samples from three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present investigation clearly demonstrates that 5-HT elicits a direct stimulation of PRL release in anterior pituitary aggregate cell cultures and that this is largely dependent on estrogen but not on glucocorticoid or thyroid hormone. The PRL response initiates within approximately 2 min of exposure to this agonist and at concentrations of 5-HT as low as 1 nM. The latter concentrations are within the range of plasma 5-HT levels reported to be 0.6–0.9 nM in rats and humans under basal conditions but which can rise 50–100 times during treatment with amphetamines, selective serotonin reuptake inhibitors, and 3,4-methylenedioxymethamphetamine (ecstasy) (79, 80), or under certain pathological conditions such as hypoxia (80, 81).

The magnitude of the PRL response to 5-HT was smaller than that to TRH, but it was comparable to the PRL response to vasoactive intestinal peptide, angiotensin II, acetylcholine, PRL-releasing peptide, and bombesin-like peptides seen in previous studies in the same pituitary cell aggregate system (39, 41, 43, 82, 83), indicating that 5-HT is a putative partner with many other compounds in regulation of PRL secretory activity.

The PRL response to 5-HT in estrogen-supplemented culture appears to be mediated by the 5-HTR4 as the response was completely abolished by the 5-HTR4 antagonist GR-113808 and not affected by methiothepin or methysergide that are known to be potent antagonists of most 5-HTR subtypes (48, 49, 50, 51) but not of the 5-HTR4 (84, 85). Selective antagonists of 5-HTR1A, 5-HTR1B, 5-HTR1D, 5-HTR2A, B, and C, 5-HTR3, 5-HTR6, and 5-HTR7/5 did not affect the PRL response, indicating no role of these receptors in the response. In contrast, the 5-HTR4 agonist cisapride significantly increased PRL secretion. The 5-HT agonist, {alpha}-methyl-5-HT, which preferentially activates the 5-HTR2 subtypes (48, 53, 54, 67), had a marked PRL releasing activity at the 1 µM dose, consistent with its affinity for the 5-HTR4 [reported Ki ranging between 250 nM and 1.5 µM (66, 85)]. The effect of {alpha}-methyl-5-HT was via the 5-HTR4 and not via the 5-HTR2, because a combination of 5-HTR2A, B, and C antagonists did not affect the PRL response to {alpha}-methyl-5-HT, whereas the 5-HTR4 blocker GR-113808 completely abolished it. In confirmation of this conclusion, two other potent agonists of 5-HTR2A, B, and C, DOI and m-chlorophenyl piperazine (48, 61, 67, 68, 69), had no PRL releasing activity. The finding that 10 nM 5-HT had approximately the same magnitude of effect as 100 nM cisapride and 1 µM {alpha}-methyl-5-HT is also consistent with the difference in potencies of these agonists at the 5-HTR4. Ki for agonist binding was reported (tested simultaneously) to be 6.3, 25, and 263 nM for 5-HT, cisapride, and {alpha}-methyl-5-HT, respectively (66).

The small PRL releasing action of 5-CT is peculiar. In view of the low binding affinity of 5-CT for the 5-HTR4 (Ki > 1 µM) (86, 87, 88), it is difficult to explain this effect by an action via the latter receptor. 5-CT has high affinity (Ki < 5 nM) for the 5-HTR1A, B, and D (53, 64, 70) and 5-HTR7 subtypes (72, 73) and medium high affinity (Ki ~ 20 nM) for the 5-HTR5 (48, 49, 71), but these receptors are not likely involved either, as the potent 5-HTR1A agonist 8-OH-DPAT and the 5-HTR1B/D/F agonist sumatriptan had no PRL releasing activity and the 5-HTR7 blocker SB 269970 (which also has blocking activity at the 5-HTR5) did not affect the PRL response to 5-HT. Because the effect of 5-CT was small and considering the evidence for the major implication of the 5-HTR4, we decided to not further invest in this issue. Another puzzling observation is that cisapride had a stronger stimulatory effect on PRL release at the 100 nM than at the 1 µM dose, although this was not the case for 5-HT. Perhaps the 5-HTR4 is more rapidly desensitized by cisapride than by 5-HT.

In further support of the action of 5-HT via the 5-HTR4, we investigated whether aggregates express the 5-HTR4 and whether cAMP production is increased by 5-HT, because the 5-HTR4 is positively coupled to adenylyl cyclase (via Gs) (86). The anterior pituitary expressed 5-HTR4 mRNA, but when aggregates were cultured in hormone-free medium, this mRNA was no longer detectable. However, when the cultures were supplemented with E2, a clear-cut expression of this mRNA was found, indicating that the strong up-regulation of the PRL response on E2 is due to induction of 5-HTR4 expression by E2. The involvement of the 5-HTR4 also was confirmed by finding a cAMP response to the 5-HTR4 agonist cisapride. The cAMP response to 5-HT also was enlarged markedly by estrogen and the agonist {alpha}-methyl-5-HT at 1 µM stimulated cAMP accumulation equally well as 100 nM 5-HT, which is consistent with the difference in potency between these compounds at the 5-HTR4 (86, 87, 88).

Certain 5-HTRs, such as 5-HTR1 and 5-HTR5, are negatively coupled to adenylyl cyclase through Gi/o transduction proteins (89). Therefore, we investigated whether the PRL response to 5-HT included an inhibitory component. If this were the case, PTX pretreatment would enhance the response magnitude to 5-HT. The addition of PTX did not have an amplifying effect on 5-HT stimulation of PRL release, pleading against the involvement of an inhibitory component. Previously, a small and transient inhibition of PRL release by the 5-HT1A agonist 8-OH-DPAT was reported in perifused dispersed pituitary cells (90), and a small inhibition was also observed in the present study. It is possible that a small inhibitory action of 5-HT on PRL release indeed exists but was overlooked or masked in the PTX experiment due to an overruling stimulatory action of 5-HT.

It has been shown recently that the action of 5-HT on platelet function is mediated by a signal transduction via the 5-HTR2A but also requires uptake of 5-HT and subsequent functioning as a substrate for transamidation of small GTPases, such as Rab4 and RhoA, by intracellular tissue transglutaminase II (a process termed "serotonylation") (6). This process renders those GTPases constitutively active as intracellular transduction molecules. Therefore, we explored whether 5-HT needs to be taken up in the cell to establish a PRL response. However, the SERT inhibitor citalopram (91) or the VMAT-2 blocker ketanserin (78) did not affect the PRL response to 5-HT, suggesting that the internalization of 5-HT is not essential for a PRL response to occur and, thus, that an intracellular molecule is not a primary target or cotarget of the 5-HT action. However, we cannot exclude that transport of 5-HT may be through another carrier as other membrane solute carriers have recently been identified (92).

To our knowledge, the implication of the 5-HTR4 in PRL secretion at the pituitary level has not been reported previously. The presence of the 5-HTR4 has been reported in human pituitary, although data as to the neural or adenohypophysial sublocalization or its function were not studied (93). Although, it has been reported that 5-HT administration in vivo stimulates PRL release in hypophysectomized pituitary-implanted rats and that estrogen treatment augments the magnitude of the response (30). However, in the same study, 5-HT failed to affect PRL release in perifused intact anterior pituitary (30). Apfelbaum (31) reported a small stimulatory effect of 5-HT on PRL release from hemipituitaries of ovariectomized rats in a small concentration window of 10–30 nM 5-HT, higher doses being less to not effective and the effect being blocked by methysergide, which suggests no mediation by the 5-HTR4. The same author also found a PRL releasing effect of 5-HT, although small (20% above basal release), in pituitary monolayer cell cultures after 1 h of incubation, an effect blocked by ketanserin (32), suggesting mediation by the 5-HTR2A. Others have reported a stimulatory effect of 5-HT on PRL release in pituitary cell cultures via a primary action on neurointermediate lobe cells (34), but no receptor subtype was determined. Still another group did not detect an effect of 5-HT on PRL release in monolayer cultures (33). How can these discrepancies in response and receptor type be explained? We believe that one reason may be sought in the test systems used previously. Transplanted pituitary most likely suffers from the surgical procedures and may be influenced by inflammatory agents from the surrounding tissue where it is transplanted, whereas intact pituitaries rapidly suffer from anoxia, and cells located in deeper layers of the organ may not be reached by added 5-HT. Monolayer cultures do not represent a three-dimensional tissue structure. In contrast, our pituitary cell aggregates are three-dimensional cultures displaying no diffusion barriers inside the tissue (39), and for several tissues, this type of culture has been shown to display much more fidelity in gene expression compared with the intact tissue than monolayer cell cultures (94, 95, 96, 97, 98, 99, 100). Another possible explanation can be found in the fact that Apfelbaum (32) used pituitary cells cultured with steroid-free serum, which may have had differentiation-altering influences. In our system no serum is added, and in an estrogen environment, the response to 5-HT is virtually exclusively via the 5-HTR4, at least with doses of 5-HT up to 10 nM. Still another point of controversy in the Apfelbaum study is that 5-HT was active only at doses 30 nM or greater, and the antagonist action of ketanserin was examined with 100 nM 5-HT, whereas we tested antagonists with 10 nM 5-HT. Therefore, we cannot exclude that, at higher doses of 5-HT, the 5-HTR2 participates in our test system. However, if that is the case, it is a minor contribution. As a matter of fact, Apfelbaum working in steroid-free condition found only an approximately 20% rise of PRL release in response to 100 nM 5-HT, which is comparable to what we find in hormone-free cultures at this dose. In estrogen-supplemented condition we find an approximately 40% rise in PRL release already at 10 nM and an approximately 70% rise at 100 nM 5-HT. We speculate that, in steroid-free milieu, there is a minor effect on PRL release via the 5-HTR2, whereas, in the presence of estrogen, the PRL response is primarily mediated by the 5-HTR4 in a considerably more impressive manner. Because estrogen represses the 5-HTR2C but not the 5-HTR2A and B, a contribution via the 5-HTR2 may indeed exist. Future work will have to further dissect this out. In any event, we found expression of the 5-HTR4 also in the intact pituitary, indicating 5-HTR4 expression is not due to culture conditions per se.

The estrogen dependency of 5-HTR4 expression and the PRL response to 5-HT is of significant importance as it indicates a role of 5-HT in sexual and reproductive functions governed by pituitary PRL. Moreover, the present study revealed that expression of three other 5-HTRs, i.e. 5-HTR5A and B and 5-HTR6, is up-regulated by E2, whereas 5-HTR2C expression is repressed by estrogen. E2 is known to promote PRL and GH secretion in response to certain peptides in aggregates (43) and various elements of the neuronal serotoninergic system (101, 102, 103, 104). In addition, E2 is well known to stimulate PRL gene expression (105). The estrogen-dependency of the PRL response to 5-HT is even more remarkable if one realizes that the basal secretion of PRL itself is enhanced several times by E2 treatment (41), but nevertheless, 5-HT is capable of further increasing the release. The estrogen-induced responses to 5-HT are also most interesting in view of the PRL induction of a paracrine serotoninergic system promoting mammary gland maturation during pregnancy (3). In clinical practice, however, no change in plasma PRL levels were reported in patients chronically treated with cisapride (106).

In conclusion, we report a stimulatory action of low nanomolar doses of 5-HT on PRL release and cAMP accumulation in anterior pituitary aggregate cell cultures that is largely dependent on estrogen. The response is mediated by the 5-HTR4, which is up-regulated by that hormone. A small effect via the 5-HTR2 in steroid-free medium, as previously reported, cannot be excluded. Estrogen also induces expression of the 5-HTR5A and B and 5-HTR6 mRNA and represses the 5-HTR2C, suggesting a role of several 5-HTRs in the regulation of sexual pituitary functions. Our data do not support the requirement of intracellular uptake of 5-HT for an action on secretion, as is the case in platelets.


   Acknowledgments
 
We thank Dr. A. F. Parlow and the National Hormone and Pituitary Program (National Institute of Diabetes and Digestive and Kidney Diseases, Harbor-UCLA Medical Center) for providing antirat PRL RIA kits. Dr. H. Vankelecom is acknowledged for his help with primer and RT-PCR procedures, and Magaly Boussemaere, Kristine Rillaerts, and Yvonne Van Goethem are acknowledged greatly for skillful technical assistance.


   Footnotes
 
This work was supported by grants from the Flemish Ministry of Science Policy (Concerted Research Actions) and the Fund for Scientific Research Flanders (Belgium).

The authors have nothing to declare.

First Published Online November 22, 2006

Abbreviations: AUC, Area under the curve; 5-CT, 5-carboxamidotryptamine; Dex, dexamethasone; DOI, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane; E2, estradiol; 5-HT, 5-hydroxytryptamine; 5-HTR, 5-HT receptor; IBMX, isobutylmethylxanthine; LSD, least significant difference; 8-OHDPAT, (+/–)-8-hydroxy-dipropylaminotetralin; PRL, prolactin; PTX, pertussis toxin; SERT, serotonin transporter; VMAT, vesicular monoamine transporter.

Received August 31, 2006.

Accepted for publication November 10, 2006.


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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