Serotonergic 5-HT2A/2C Receptors Are Involved in Prolactin Secretion in Hyperestrogenic Rats

Neuroscience Letters 582 (2014) 71–74

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Serotonergic 5-HT2A/2C receptors are involved in prolactin secretion

in hyperestrogenic rats

a,b a,c c a,c,∗

E.S. Mallmann , L. Paixão , M.F. Ribeiro , P.M. Spritzer

Gynecological Endocrinology Unit, Division of Endocrinology, Hospital de Clínicas de Porto Alegre (HCPA), Rua Ramiro Barcelos, 2350, 90035-003 Porto

Alegre, Brazil

Division of Gynecology and Obstetrics, Hospital Presidente Vargas, Avenida Independência 661, 90035-070 Porto Alegre, Brazil

Department of Physiology, Universidade Federal do Rio Grande do Sul (UFRGS), Rua Ramiro Barcelos, 2350, 90035-003 Porto Alegre, Brazil

h i g h l i g h t s

•

Two animal models were assessed: hyperestrogenic and hypoestrogenic rats.

•

We tested whether estradiol modulates prolactin by acting on serotonergic receptors.

•

5-HT2A/2C receptors are involved in prolactin release through serotonergic pathways.

a r t i c l e i n f o a b s t r a c t

Article history: Serotonin (5-HT) has been shown to participate in prolactin secretion through a complex process resulting

Received 31 July 2014

in both positive and negative effects. Estrogen has also been recognized as being involved in this seroto-

Received in revised form 18 August 2014

nergic effect on prolactin release. Therefore, the aim of the present study was to assess whether estradiol

Accepted 1 September 2014

modulates serotonergic involvement in prolactin secretion though 5-HT1A and/or 5-HT2A/2C receptors.

Available online 8 September 2014

Ovariectomized female Wistar rats, treated for 2 weeks with estrogen to induce a hyperprolactinemic

status (hyperestrogenic rats) or with sunﬂower oil vehicle (hypoestrogenic rats), were injected daily with

Keywords:

normal saline solution or 6-chloro-2-(1-piperazinyl)pyrazine (MK-212), an 5-HT2A/2C receptor agonist,

Hyperprolactinemia

Serotonin for 4 days. Other groups of ovariectomized animals received 8-hydroxy-2-(di-N-propylamino)tetralin

MK212 (8-OH-DPAT) or pindolol, an agonist and antagonist of the 5-HT1A receptor respectively, on the last day

8-OH-DPAT of treatment with estrogen or vehicle. Prolactin levels were compared among groups in each experi-

Pindolol ment under the distinct drug treatments. MK-212 was found to increase prolactin concentrations both in

Hyperestrogenism hyper- and hypoestrogenic females compared to controls (p < 0.05). In contrast, prolactin levels remained

similar to those of controls both in hyperestrogenic animals treated with 8-OH-DPAT and pindolol and in

hypoestrogenic rats administered the same treatments. In conclusion, our ﬁndings indicate the involve-

ment of 5-HT2A/2C receptors on prolactin release through serotonergic pathways in female animals,

especially in hyperestrogenic states.

1. Introduction in humans and in diverse animal models [5,27,28] under different

physiological conditions, such as lactation and pregnancy.

Prolactin secretion is largely regulated by dopamine, but sero- Serotonergic receptors such as 5-HT1A, 5-HT2A and 5HT2C have

tonergic neurons also play a role in neuroendocrine regulation of been described in several studies as modulating prolactin release

prolactin secretion. Serotonin (5-hydroxytryptamine, 5-HT) and 5- [1,9,12,14]. However, controversial results have been reported.

hydroxytryptophan (5-HTP) may increase prolactin secretion both Some evidence in rodents indicates that the 5-HT2 receptor is

involved in prolactin regulation [8,16,19]. In contrast, other stud-

ies report that the 5-HT1A subtype also participates in prolactin

release [9,19,23], a ﬁnding contested by others [4,25].

∗ Regarding studies in humans, while some authors have reported

Corresponding author at: Division of Endocrinology, Hospital de Clínicas de Porto

that 5-HT1A receptors play a role in the control of prolactin secre-

Alegre, Rua Ramiro Barcelos, 2350, CEP: 90035-003 Porto Alegre, RS, Brazil.

tion [18,20] others point out that both 5-HT1A and 5-HT2A/2C

Tel.: +55 51 3359 8027; fax: +55 51 3359 8777.

E-mail address: [email protected] (P.M. Spritzer). receptors are required for prolactin secretion [12,13].

http://dx.doi.org/10.1016/j.neulet.2014.09.005

72 E.S. Mallmann et al. / Neuroscience Letters 582 (2014) 71–74

Estrogen has also been recognized as being involved in the

role of serotonergic pathways in prolactin release [15], and evi-

dence suggests that estradiol exerts an inﬂuence on 5-HT1A mRNA

expression in limbic brain regions [17] and modulates serotonin

levels and receptor numbers [2].

We have previously shown that administering estradiol to

animals treated with p-chlorophenylalanine (pCPA), an inhibitor

of 5-HT synthesis, increases prolactin levels as compared

with those observed in sham/normoestrogenic or ovariec-

tomized/hypoestrogenic pCPA-treated rats [10]. Therefore, the

present study aims to assess whether estradiol may modulate the

serotonergic inﬂuence on prolactin secretion though 5-HT1A and

5-HT2A/2C receptors in rats under different conditions of estrogen Fig. 1. Effect of MK-212 (10 mg/kg/day for 4 days), on prolactin levels. Hyperestro-

treatment. genic females (E2 + saline, n = 9; E2 + MK-212, n = 9) were treated for 2 weeks with

300 ␮g per week of estradiol benzoate. Hypoestrogenic females were administered

saline (OVX + saline, n = 9) or MK-212 (OVX + MK-212, n = 8). Results are expressed

2. Materials and methods

as median and 25–75% interquartile range (lower and upper limit of the box); max-

imum and minimum values are shown by the limits of vertical lines. *p < 0.05;

2.1. Animals **p < 0.01 compared to control group (OVX + saline).

Eighty-four female Wistar rats, 65–80 days old, weighing ®

The other half of the group received pindolol (Visken , Sandoz,

180–220 g, were housed six to a cage under controlled tempera-

◦ São Paulo, Brazil), dissolved in 0.9% NaCl, by subcutaneous injec-

ture conditions (22 ± 4 C) and a 12/12 h light–dark cycle with free

tion, as a single 5 mg/kg dose 60 min before decapitation [21,22].

access to standard certiﬁed rodent chow and tap water. All pro-

Prolactin levels were compared among the groups of hypo- and

cedures performed on the rats were in compliance with the EC

hyperestrogenic animals under distinct drug treatments.

Directive 2010/63/EU on the protection of animals used for scien-

tiﬁc purposes. The use of animals was reviewed and approved by

2.5. Prolactin assay

the Research Ethics Committee, Federal University of Rio Grande

do Sul Basic Health Sciences Institute.

Prolactin levels (ng/mL) were measured by a double-antibody

radioimmunoassay with reagents supplied by the National Hor-

2.2. Experimental procedures

mone and Pituitary Program, U.S. National Institute of Diabetes

and Digestive and Kidney Diseases (NIDDK, Bethesda, MD, USA).

All animals were subjected to bilateral ovariectomy (OVX)

Prolactin was radioiodinated by the chloramine-T method as

under anesthesia. At 5 days post-surgery, animals were randomly

described elsewhere [24]. NIDDK rat prolactin RP-3 was used as the

assigned to receive 300 ␮g per week of estradiol benzoate (Ben-

standard. Assay sensitivity was 4 ng/mL and the intra- and interas-

zoginoestril, Sarsa-São Paulo, Brazil), or sunﬂower oil vehicle

say coefﬁcients of variation were 8.6% and 12%, respectively.

(hyperestrogenic and hypoestrogenic animals, respectively) for 2

weeks, as previously reported [24]. In all experiments, the animals

2.6. Statistical analysis

were killed 2 weeks after the ﬁrst estradiol benzoate or vehicle

injection. Animals were killed between 9 and 11 a.m. by decapi-

Prolactin values for each group are expressed as median and

tation. Trunk blood samples were collected from all animals and

◦ interquartile range. Statistical analysis was performed by the

serum was harvested and stored at −20 C until measurement

Mann–Whitney U test. All analyses were performed in the Graph-

of prolactin levels by a double-antibody radioimmunoassay. Drug

PadPrism 5 software environment (GraphPad Software, Inc., La

administration was carried out during the last days of vehicle or

Jolla, CA, USA). Differences were considered signiﬁcant if the prob-

estrogen treatment, between 8 and 11 a.m., as described below.

ability of error was less than 5%.

2.3. Experiment 1: Effect of serotonin receptor agonist MK-212 on

the 5-HT2A/5-HT2C receptor subtypes 3. Results

In the last 4 days before the end of the 2-week vehicle 3.1. Effect of serotonin receptor agonist MK-212 on the

or estradiol treatment period, animals were also injected once 5-HT2A/5-HT2C receptor subtypes

daily with normal saline solution (0.9% NaCl) or 6-chloro-2-(1-

piperazinyl)pyrazine (MK-212, Merck Institute for Therapeutic Fig. 1 shows that prolactin levels were signiﬁcantly higher

Research, West Point, PA, USA), subcutaneously, dissolved in 0.9% in estradiol-treated ovariectomized rats (n = 9) than in vehicle-

NaCl at a dose of 10 mg/kg/day. Animals were killed by decapitation treated animals (n = 9). MK-212 signiﬁcantly increased prolactin

24 h after the end of the 2-week period. concentrations in both hypoestrogenic (n = 8) (p < 0.05) and hypere-

strogenic (n = 9) (p < 0.01) females as compared with controls. Other

2.4. Experiment 2. Effects of serotonin receptor agonist one animal died at the experimentation period.

8-OH-DPAT and antagonist pindolol on the 5-HT1A receptor

subtype 3.2. Effects of serotonin receptor agonist 8-OH-DPAT and

antagonist pindolol on the 5-HT1A receptor subtype

On the last day of the 2-week period of vehicle or estradiol

administration, part of the animals received 8-hydroxy-2-(di-N- Fig. 2 shows the effects of the administration of 8-OH-DPAT

propylamine)tetralin (8-OH-DPAT, Sigma Chemical Co., St. Louis, to hyperestrogenic (n = 8) and hypoestrogenic animals (n = 8).

MO, USA), dissolved in 0.9% NaCl, by intraperitoneal injection, as Although prolactin concentrations were higher in hyperestrogenic

a single 1 mg/kg dose 30 min before decapitation. The dose was females (n = 8) compared to hypoestrogenic animals (n = 8), treat-

determined on the basis of previous studies [4,19]. ment with 8-OH-DPAT did not alter prolactin levels as compared

E.S. Mallmann et al. / Neuroscience Letters 582 (2014) 71–74 73

Concerning pindolol treatment, no differences in prolactin lev-

els were found either in hyper- or in hypoestrogenic females. Few

studies have assessed the interaction between 5-HT1A receptors,

prolactin levels, and estrogenic status. In turn, using the same dose

as described in the present study, a previous investigation found

that pindolol antagonized the antidepressant effect of indorenate

[11]. Evidence suggests that high-dose administration of pindolol

in humans may augment antidepressant response [3]. Therefore,

the absence of response to DPAT and pindolol treatment observed

in the present study suggests that involvement of 5-HT1A receptors

in estradiol-induced prolactin release is unlikely.

5. Conclusions

The present ﬁndings suggest that 5-HT2A/2C receptors are

involved in prolactin release through serotonergic pathways in

female animals, especially in the setting of a hyperestrogenic state.

Fig. 2. Effect of 8-OH-DPAT (1 mg/kg) or pindolol (5 mg/kg) on prolactin levels.

Further studies are needed to better deﬁne the interaction of the

Hyperestrogenic females (E2 + saline, n = 8; E2 + 8-OH-DPAT, n = 8; and E2 + pindolol,

5-HT2A/2C receptor subtypes, prolactin secretion, and the ovarian

n = 7) were treated for 2 weeks with 300 ␮g per week of estradiol benzoate.

Hypoestrogenic females were administered saline (OVX + saline, n = 8), 8-OH-DPAT cycle during antidepressant treatment.

(OVX + 8-OH-DPAT, n = 8), or pindolol (OVX + pindolol, n = 4). Results expressed as

median and 25–75% interquartile range (lower and upper limit of the box); maxi-

Conﬂict of interest

mum and minimum values are shown by the limits of vertical lines.

The authors declare that they have no conﬂict of interest.

with saline control. Similar results were obtained with the adminis-

tration of pindolol: although prolactin concentrations were higher

Authors contribution

in hyperestrogenic (n = 7) than in hypoestrogenic animals (n = 4),

pindolol treatment did not change prolactin levels as compared

E.S.M. contributed to study design, acquisition of data, analy-

with saline control. Other seven animals could not be used for

sis and interpretation of data and drafting manuscript and ﬁnal

experiments or died at the experimentation period.

review. L.P. contributed to analysis and interpretation of data, draft-

ing manuscript and ﬁnal review. M.F.R. contributed to acquisition

4. Discussion

of data, analysis and interpretation of data and manuscript review.

P.M.S. contributed to conception and study design, analysis and

In the present study, we tested whether estradiol can modu-

interpretation of data, drafting manuscript and ﬁnal review. All

late serotonergic effects on prolactin secretion by acting directly

authors approved the ﬁnal version of the manuscript.

on serotonergic receptors. Thus, the experiments were designed

to identify whether the 5-HT2A/2C or 5-HT1A receptors subtypes

were involved in this mechanism. In this sense, treatment with Acknowledgments

the serotonin receptor agonist MK-212 increased prolactin levels

both in hyper- and hypoestrogenic females. Moreover, estradiol This work was supported by a grant from Conselho Nacional

was found to strongly increase prolactin release when adminis- de Desenvolvimento Cientíﬁco e Tecnológico (CNPq INCT

tered with this 5-HT2A/2C agonist [2]. These results suggest that 573747/2008-3). LP is recipient of PhD grant from CAPES

the 5-HT2A receptor subtype is involved in prolactin secretion, as (1172105). The funding sources were not involved in study

reported in a previous study carried out in turkeys [6]. However, design; in the collection, analysis and interpretation of data; in the

while the present results suggest the 5-HT2C receptor subtype may writing of the report; or in the decision to submit the paper for

also be involved in the regulation of prolactin secretion, a deﬁni- publication.

tive role for this receptor can only be conﬁrmed after testing the

administration of new speciﬁc 5-HT2C antagonists [26] in combi- References

nation with dose-response curves for estrogen administration to

ovariectomized rats. [1] E. Aguiar, A. Ranchal, M. Tena-Sempere, L. Pinilla, Serotoninergic control of

prolactin secretion in prepubertal male rats, Eur. J. Endocrinol. 131 (5) (1994)

Studying a hypoestrogenic animal model, Flügge et al. [7]

547–554.

administrated DPAT, a 5-HT1A receptor agonist, to ovariectomized

[2] A.V. Akhmadeev, L.B. Kalimullina, Sex steroids and monoamines in the system

rats. The authors found that, in estradiol-treated females, both the of neuroendocrine regulation of the functions of the amygdaloid complex of

the brain, Neurosci. Behav. Physiol. 43 (1) (2013) 129–134.

number and afﬁnity of 5-HT1A receptors were increased in the

[3] F. Artigas, A. Adell, P. Celada, Pindolol augmentation of antidepressant response,

ventral medial nucleus in comparison with rats not administered

Curr. Drug Targets 7 (2) (2006) 139–147.

estradiol. In contrast, in the present study, despite the increase [4] M.T. Bluet Pajot, F. Mounier, A. di Sciullo, B. Schmidt, C. Kordon, Differential

sites of action of 8OHDPAT, a 5HT1A agonist, on ACTH and PRL secretion in the

in prolactin levels observed due to estradiol treatment, no effect

rat, Neuroendocrinology 61 (2) (1995) 159–166.

was found on prolactin levels with the administration of DPAT or

[5] L. Caligaris, S. Taleisnik, Involvement of neurons containing 5-

of the 5-HT1A antagonist pindolol. A previous study has reported hydroxytryptamine in the mechanism of prolactin release induced by

that DPAT could promote a brief increase in prolactin levels in oestrogen, J. Endocrinol. 62 (1) (1974) 25–33.

[6] Y. Chaiseha, S.W. Kang, B. Leclerc, S. Kosonsiriluk, N. Sartsoongnoen, M.E.

male rats [4]. Another study [1] showed that treatment with DPAT

Halawani, Serotonin receptor subtypes inﬂuence prolactin secretion in the

was efﬁcient in increasing prolactin levels in prepubertal male rats.

Turkey, Gen. Comp. Endocrinol. 165 (1) (2010) 170–175.

However, the experimental conditions of these studies using male [7] G. Flügge, D. Pfender, S. Rudolph, H. Jarry, E. Fuchs, 5HT1 A-receptor binding in

the brain of cyclic and ovariectomized female rats, J. Neuroendocrinol. 11 (4)

animals, as well the doses of DPAT administered, differ from those

(1999) 243–249.

of the present study; this could explain, at least in part, the con-

[8] G.A. Jahn, R.P. Deis, Effect of serotonin antagonist on prolactin and proges-

trasting results. terone secretion in rats: evidence that the stimulatory and inhibitory actions

74 E.S. Mallmann et al. / Neuroscience Letters 582 (2014) 71–74

of serotonin on prolactin release may be mediated through different receptors, [19] J.T. Pan, I.C. Yang, Central administration of 8-OH-DPAT and mCPP stimulates

J. Endocrinol. 117 (3) (1988) 415–422. prolactin secretion in ovariectomized, estrogen-treated rats: lack of an effect

[9] I.M. Lacau-Mengido, C. Libertun, D. Becú-Villalobos., Different serotonin recep- on tuberoinfundibular dopaminergic neuron activity, Life Sci. 58 (14) (1996)

tors types participate in 5-hydroxytryptophan-induced gonadotropins and 1189–1194.

prolactin release in the female infantile rat, Neuroendocrinology 63 (5) (1996) [20] S.B. Park, P.J. Cowen, Effect of pindolol no the prolactin response to d-

415–421. fenﬂuramine, Psychopharmacology 118 (1995) 471–474.

[10] E.S. Mallmann, M.F. Ribeiro, P.M. Spritzer, Effect of serotonin depletion by [21] K. Rasmussen, A.C. McCreary, E.A. Shanks, Attenuation of the effects of ﬂuoxetin

p-chlorophenylalanine on serum prolactin levels in estrogen-treated ovariec- on serotonergic neural activity by pindolol in rats, Neurosci. Lett. 355 (1–2)

tomized rats: insights concerning the serotoninergic, dopaminergic and opioid (2004) 1–4.

systems, Horm. Metab. Res. 33 (6) (2001) 337–342. [22] V.R. Sela, C. Biesdorf, D.H. Ramos, H. Zangrossi Jr., F.G. Graeff, E.A. Audi,

[11] L. Martinez-Mota, E. Estrada-Camarena, C. López-Rubalcava., Indorenate Serotonin-1A receptors in the dorsal periaqueductal gray matter mediate the

produces antidepressant-like actions in the rat forced swimming test via 5- panicolytic-like effect of pindolol and paroxetine combination in the elevated

HT1A, Psychopharmacology 165 (2002) 60–66. T-maze, Neurosci. Lett. 495 (1) (2011) 63–66.

[12] H.Y. Meltzer, M. Maes, Effect of pindolol on the L-5-HTP-induced increase in [23] C.E. Smith, C.J. Ware, P.J. Cowen, Pindolol decreases prolactin and growth hor-

plasma prolactin and cortisol concentrations in man, Psychopharmacology 114 mones responses to intravenous l-tryptophan, Psychopharmacology 103 (1)

(4) (1994) 635–643. (1991) 140–142.

[13] H.Y. Meltzer, M. Maes, Effect of pindolol pretreatment on MK-212-induced [24] P.M. Spritzer, M.F. Ribeiro, M.C. Oliveira, L.M. Barbosa-Coutinho, I.S. Silva,

plasma cortisol and prolactin responses in normal men, Biol. Psychiatr. 38 N. Dahlem, R. Cericatto, M.A. Pavanato, Effects of tamoxifen on serum pro-

(1995) 310–318. lactin levels, pituitary immunoreactive prolactin cells and uterine growth

[14] H.Y. Meltzer, M. Maes, Pindolol pretreatment blocks stimulation by in estradiol-treated ovariectomized rats, Horm. Metab. Res. 28 (4) (1996)

metachlorophenylpiperazine of prolactin but not cortisol secretion in normal 171–176.

men, Psychiatr. Res. 58 (1995) 89–98. [25] L.D. Van de Kar, S.A. Lorens, J.H. Urban, C.L. Bethea, Effect of selective

[15] V. Michopoulos, S.L. Berga, M.E. Wilson, Estradiol and progesterone modify the serotonin (5-HT) agonists and 5-HT2 antagonist on prolactin secretion, Neuro-

effects of the serotonin reuptake transporter polymorphism on serotonergic pharmacology 28 (3) (1989) 299–305.

responsivity to citalopran, Exp. Clin. Psychopharmacol. 19 (6) (2011) 401–408. [26] P.V. Vimala, P.S. Bhutada, F.R. Patel, Therapeutic potential of agomela-

[16] A. Mistry, J.L. Voogt, Role of serotonin in nocturnal and diurnal surges of pro- tine in epilepsy and epileptic complications, Med. Hypotheses 82 (2014)

lactin in the pregnant rat, Endocrinology 125 (6) (1989) 2875–2880. 105–110.

[17] M.K. Osterlund, Y.L. Hurd, Acute 17beta-estradiol treatment down-regulates [27] L.N. Yatham, M. Steiner, Neuroendocrine probes of serotonergic function: a

serotonin 5-HT1A receptor mRNA expression in limbic system of female rats, critical review, Life Sci. 53 (6) (1993) 447–463.

Brain Res. Mol. 55 (1) (1998) 169–172. [28] P.D. Woolf, L. Lee, Effect of the serotonin precursor, tryptophan, on

[18] E. Palazidou, J. Stephenson, J. Butler, P. Coskeran, S. Chambers, A.M. McGreagor, pituitary hormone secretion, J. Clin. Endocrinol. Metab. 45 (1977)

Evidence for 5-hydroxytryptamine 1A receptor involvement in the control of 123–133.

prolactin secretion in man, Psychopharmacology 119 (1995) 311–314.