Neuroscience Letters 582 (2014) 71–74
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Serotonergic 5-HT2A/2C receptors are involved in prolactin secretion in hyperestrogenic rats E.S. Mallmann a,b , L. Paixão a,c , M.F. Ribeiro c , P.M. Spritzer a,c,∗ a Gynecological Endocrinology Unit, Division of Endocrinology, Hospital de Clínicas de Porto Alegre (HCPA), Rua Ramiro Barcelos, 2350, 90035-003 Porto Alegre, Brazil b Division of Gynecology and Obstetrics, Hospital Presidente Vargas, Avenida Independência 661, 90035-070 Porto Alegre, Brazil c 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
Article history: Received 31 July 2014 Received in revised form 18 August 2014 Accepted 1 September 2014 Available online 8 September 2014 Keywords: Hyperprolactinemia Serotonin MK212 8-OH-DPAT Pindolol Hyperestrogenism
a b s t r a c t Serotonin (5-HT) has been shown to participate in prolactin secretion through a complex process resulting in both positive and negative effects. Estrogen has also been recognized as being involved in this serotonergic effect on prolactin release. Therefore, the aim of the present study was to assess whether estradiol modulates serotonergic involvement in prolactin secretion though 5-HT1A and/or 5-HT2A/2C receptors. 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 normal saline solution or 6-chloro-2-(1-piperazinyl)pyrazine (MK-212), an 5-HT2A/2C receptor agonist, for 4 days. Other groups of ovariectomized animals received 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) or pindolol, an agonist and antagonist of the 5-HT1A receptor respectively, on the last day of treatment with estrogen or vehicle. Prolactin levels were compared among groups in each experiment under the distinct drug treatments. MK-212 was found to increase prolactin concentrations both in 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 involvement of 5-HT2A/2C receptors on prolactin release through serotonergic pathways in female animals, especially in hyperestrogenic states. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Prolactin secretion is largely regulated by dopamine, but serotonergic neurons also play a role in neuroendocrine regulation of prolactin secretion. Serotonin (5-hydroxytryptamine, 5-HT) and 5hydroxytryptophan (5-HTP) may increase prolactin secretion both
∗ Corresponding author at: Division of Endocrinology, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350, CEP: 90035-003 Porto Alegre, RS, Brazil. Tel.: +55 51 3359 8027; fax: +55 51 3359 8777. E-mail address: [email protected] (P.M. Spritzer). http://dx.doi.org/10.1016/j.neulet.2014.09.005 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
in humans and in diverse animal models [5,27,28] under different physiological conditions, such as lactation and pregnancy. Serotonergic receptors such as 5-HT1A, 5-HT2A and 5HT2C have been described in several studies as modulating prolactin release [1,9,12,14]. However, controversial results have been reported. Some evidence in rodents indicates that the 5-HT2 receptor is involved in prolactin regulation [8,16,19]. In contrast, other studies 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 that 5-HT1A receptors play a role in the control of prolactin secretion [18,20] others point out that both 5-HT1A and 5-HT2A/2C receptors are required for prolactin secretion [12,13].
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Estrogen has also been recognized as being involved in the role of serotonergic pathways in prolactin release [15], and evidence 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 ovariectomized/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 treatment. 2. Materials and methods 2.1. Animals Eighty-four female Wistar rats, 65–80 days old, weighing 180–220 g, were housed six to a cage under controlled temperature conditions (22 ± 4 ◦ C) and a 12/12 h light–dark cycle with free access to standard certified rodent chow and tap water. All procedures performed on the rats were in compliance with the EC Directive 2010/63/EU on the protection of animals used for scientific purposes. The use of animals was reviewed and approved by the Research Ethics Committee, Federal University of Rio Grande do Sul Basic Health Sciences Institute. 2.2. Experimental procedures
Fig. 1. Effect of MK-212 (10 mg/kg/day for 4 days), on prolactin levels. Hyperestrogenic 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 as median and 25–75% interquartile range (lower and upper limit of the box); maximum and minimum values are shown by the limits of vertical lines. *p < 0.05; **p < 0.01 compared to control group (OVX + saline).
The other half of the group received pindolol (Visken® , Sandoz, São Paulo, Brazil), dissolved in 0.9% NaCl, by subcutaneous injection, as a single 5 mg/kg dose 60 min before decapitation [21,22]. Prolactin levels were compared among the groups of hypo- and hyperestrogenic animals under distinct drug treatments. 2.5. Prolactin assay Prolactin levels (ng/mL) were measured by a double-antibody radioimmunoassay with reagents supplied by the National Hormone and Pituitary Program, U.S. National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, Bethesda, MD, USA). Prolactin was radioiodinated by the chloramine-T method as described elsewhere [24]. NIDDK rat prolactin RP-3 was used as the standard. Assay sensitivity was 4 ng/mL and the intra- and interassay coefficients of variation were 8.6% and 12%, respectively.
All animals were subjected to bilateral ovariectomy (OVX) under anesthesia. At 5 days post-surgery, animals were randomly assigned to receive 300 g per week of estradiol benzoate (Benzoginoestril, Sarsa-São Paulo, Brazil), or sunflower oil vehicle (hyperestrogenic and hypoestrogenic animals, respectively) for 2 weeks, as previously reported [24]. In all experiments, the animals were killed 2 weeks after the first estradiol benzoate or vehicle injection. Animals were killed between 9 and 11 a.m. by decapitation. Trunk blood samples were collected from all animals and serum was harvested and stored at −20 ◦ C until measurement of prolactin levels by a double-antibody radioimmunoassay. Drug administration was carried out during the last days of vehicle or estrogen treatment, between 8 and 11 a.m., as described below.
2.6. Statistical analysis
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 or estradiol treatment period, animals were also injected once daily with normal saline solution (0.9% NaCl) or 6-chloro-2-(1piperazinyl)pyrazine (MK-212, Merck Institute for Therapeutic Research, West Point, PA, USA), subcutaneously, dissolved in 0.9% NaCl at a dose of 10 mg/kg/day. Animals were killed by decapitation 24 h after the end of the 2-week period. 2.4. Experiment 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-Npropylamine)tetralin (8-OH-DPAT, Sigma Chemical Co., St. Louis, MO, USA), dissolved in 0.9% NaCl, by intraperitoneal injection, as a single 1 mg/kg dose 30 min before decapitation. The dose was determined on the basis of previous studies [4,19].
Prolactin values for each group are expressed as median and interquartile range. Statistical analysis was performed by the Mann–Whitney U test. All analyses were performed in the GraphPadPrism 5 software environment (GraphPad Software, Inc., La Jolla, CA, USA). Differences were considered significant if the probability of error was less than 5%.
3.1. Effect of serotonin receptor agonist MK-212 on the 5-HT2A/5-HT2C receptor subtypes Fig. 1 shows that prolactin levels were significantly higher in estradiol-treated ovariectomized rats (n = 9) than in vehicletreated animals (n = 9). MK-212 significantly increased prolactin concentrations in both hypoestrogenic (n = 8) (p < 0.05) and hyperestrogenic (n = 9) (p < 0.01) females as compared with controls. Other one animal died at the experimentation period. 3.2. Effects of serotonin receptor agonist 8-OH-DPAT and antagonist pindolol on the 5-HT1A receptor subtype Fig. 2 shows the effects of the administration of 8-OH-DPAT to hyperestrogenic (n = 8) and hypoestrogenic animals (n = 8). Although prolactin concentrations were higher in hyperestrogenic females (n = 8) compared to hypoestrogenic animals (n = 8), treatment with 8-OH-DPAT did not alter prolactin levels as compared
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Concerning pindolol treatment, no differences in prolactin levels 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
Fig. 2. Effect of 8-OH-DPAT (1 mg/kg) or pindolol (5 mg/kg) on prolactin levels. Hyperestrogenic females (E2 + saline, n = 8; E2 + 8-OH-DPAT, n = 8; and E2 + pindolol, 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 (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); maximum and minimum values are shown by the limits of vertical lines.
with saline control. Similar results were obtained with the administration of pindolol: although prolactin concentrations were higher in hyperestrogenic (n = 7) than in hypoestrogenic animals (n = 4), pindolol treatment did not change prolactin levels as compared with saline control. Other seven animals could not be used for experiments or died at the experimentation period. 4. Discussion In the present study, we tested whether estradiol can modulate serotonergic effects on prolactin secretion by acting directly 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 the serotonin receptor agonist MK-212 increased prolactin levels both in hyper- and hypoestrogenic females. Moreover, estradiol was found to strongly increase prolactin release when administered with this 5-HT2A/2C agonist [2]. These results suggest that the 5-HT2A receptor subtype is involved in prolactin secretion, as reported in a previous study carried out in turkeys [6]. However, while the present results suggest the 5-HT2C receptor subtype may also be involved in the regulation of prolactin secretion, a definitive role for this receptor can only be confirmed after testing the administration of new specific 5-HT2C antagonists [26] in combination with dose-response curves for estrogen administration to ovariectomized rats. Studying a hypoestrogenic animal model, Flügge et al. [7] administrated DPAT, a 5-HT1A receptor agonist, to ovariectomized rats. The authors found that, in estradiol-treated females, both the number and affinity of 5-HT1A receptors were increased in the ventral medial nucleus in comparison with rats not administered estradiol. In contrast, in the present study, despite the increase in prolactin levels observed due to estradiol treatment, no effect was found on prolactin levels with the administration of DPAT or of the 5-HT1A antagonist pindolol. A previous study has reported that DPAT could promote a brief increase in prolactin levels in male rats [4]. Another study [1] showed that treatment with DPAT was efficient in increasing prolactin levels in prepubertal male rats. However, the experimental conditions of these studies using male animals, as well the doses of DPAT administered, differ from those of the present study; this could explain, at least in part, the contrasting results.
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. Further studies are needed to better define the interaction of the 5-HT2A/2C receptor subtypes, prolactin secretion, and the ovarian cycle during antidepressant treatment. Conflict of interest The authors declare that they have no conflict of interest. Authors contribution E.S.M. contributed to study design, acquisition of data, analysis and interpretation of data and drafting manuscript and final review. L.P. contributed to analysis and interpretation of data, drafting manuscript and final review. M.F.R. contributed to acquisition of data, analysis and interpretation of data and manuscript review. P.M.S. contributed to conception and study design, analysis and interpretation of data, drafting manuscript and final review. All authors approved the final version of the manuscript. Acknowledgments This work was supported by a grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq INCT 573747/2008-3). LP is recipient of PhD grant from CAPES (1172105). The funding sources were not involved in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. References [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) 547–554. [2] A.V. Akhmadeev, L.B. Kalimullina, Sex steroids and monoamines in the system of neuroendocrine regulation of the functions of the amygdaloid complex of the brain, Neurosci. Behav. Physiol. 43 (1) (2013) 129–134. [3] F. Artigas, A. Adell, P. Celada, Pindolol augmentation of antidepressant response, Curr. Drug Targets 7 (2) (2006) 139–147. [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 rat, Neuroendocrinology 61 (2) (1995) 159–166. [5] L. Caligaris, S. Taleisnik, Involvement of neurons containing 5hydroxytryptamine in the mechanism of prolactin release induced by oestrogen, J. Endocrinol. 62 (1) (1974) 25–33. [6] Y. Chaiseha, S.W. Kang, B. Leclerc, S. Kosonsiriluk, N. Sartsoongnoen, M.E. Halawani, Serotonin receptor subtypes influence prolactin secretion in the Turkey, Gen. Comp. Endocrinol. 165 (1) (2010) 170–175. [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) (1999) 243–249. [8] G.A. Jahn, R.P. Deis, Effect of serotonin antagonist on prolactin and progesterone secretion in rats: evidence that the stimulatory and inhibitory actions
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[19] J.T. Pan, I.C. Yang, Central administration of 8-OH-DPAT and mCPP stimulates prolactin secretion in ovariectomized, estrogen-treated rats: lack of an effect on tuberoinfundibular dopaminergic neuron activity, Life Sci. 58 (14) (1996) 1189–1194. [20] S.B. Park, P.J. Cowen, Effect of pindolol no the prolactin response to dfenfluramine, Psychopharmacology 118 (1995) 471–474. [21] K. Rasmussen, A.C. McCreary, E.A. Shanks, Attenuation of the effects of fluoxetin on serotonergic neural activity by pindolol in rats, Neurosci. Lett. 355 (1–2) (2004) 1–4. [22] V.R. Sela, C. Biesdorf, D.H. Ramos, H. Zangrossi Jr., F.G. Graeff, E.A. Audi, Serotonin-1A receptors in the dorsal periaqueductal gray matter mediate the panicolytic-like effect of pindolol and paroxetine combination in the elevated T-maze, Neurosci. Lett. 495 (1) (2011) 63–66. [23] C.E. Smith, C.J. Ware, P.J. Cowen, Pindolol decreases prolactin and growth hormones responses to intravenous l-tryptophan, Psychopharmacology 103 (1) (1991) 140–142. [24] P.M. Spritzer, M.F. Ribeiro, M.C. Oliveira, L.M. Barbosa-Coutinho, I.S. Silva, N. Dahlem, R. Cericatto, M.A. Pavanato, Effects of tamoxifen on serum prolactin levels, pituitary immunoreactive prolactin cells and uterine growth in estradiol-treated ovariectomized rats, Horm. Metab. Res. 28 (4) (1996) 171–176. [25] L.D. Van de Kar, S.A. Lorens, J.H. Urban, C.L. Bethea, Effect of selective serotonin (5-HT) agonists and 5-HT2 antagonist on prolactin secretion, Neuropharmacology 28 (3) (1989) 299–305. [26] P.V. Vimala, P.S. Bhutada, F.R. Patel, Therapeutic potential of agomelatine in epilepsy and epileptic complications, Med. Hypotheses 82 (2014) 105–110. [27] L.N. Yatham, M. Steiner, Neuroendocrine probes of serotonergic function: a critical review, Life Sci. 53 (6) (1993) 447–463. [28] P.D. Woolf, L. Lee, Effect of the serotonin precursor, tryptophan, on pituitary hormone secretion, J. Clin. Endocrinol. Metab. 45 (1977) 123–133.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Serotonergic 5-HT2A/2C receptors are involved in prolactin secretion in hyperestrogenic rats E.S. Mallmann a,b , L. Paixão a,c , M.F. Ribeiro c , P.M. Spritzer a,c,∗ a Gynecological Endocrinology Unit, Division of Endocrinology, Hospital de Clínicas de Porto Alegre (HCPA), Rua Ramiro Barcelos, 2350, 90035-003 Porto Alegre, Brazil b Division of Gynecology and Obstetrics, Hospital Presidente Vargas, Avenida Independência 661, 90035-070 Porto Alegre, Brazil c 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
Article history: Received 31 July 2014 Received in revised form 18 August 2014 Accepted 1 September 2014 Available online 8 September 2014 Keywords: Hyperprolactinemia Serotonin MK212 8-OH-DPAT Pindolol Hyperestrogenism
a b s t r a c t Serotonin (5-HT) has been shown to participate in prolactin secretion through a complex process resulting in both positive and negative effects. Estrogen has also been recognized as being involved in this serotonergic effect on prolactin release. Therefore, the aim of the present study was to assess whether estradiol modulates serotonergic involvement in prolactin secretion though 5-HT1A and/or 5-HT2A/2C receptors. 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 normal saline solution or 6-chloro-2-(1-piperazinyl)pyrazine (MK-212), an 5-HT2A/2C receptor agonist, for 4 days. Other groups of ovariectomized animals received 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) or pindolol, an agonist and antagonist of the 5-HT1A receptor respectively, on the last day of treatment with estrogen or vehicle. Prolactin levels were compared among groups in each experiment under the distinct drug treatments. MK-212 was found to increase prolactin concentrations both in 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 involvement of 5-HT2A/2C receptors on prolactin release through serotonergic pathways in female animals, especially in hyperestrogenic states. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Prolactin secretion is largely regulated by dopamine, but serotonergic neurons also play a role in neuroendocrine regulation of prolactin secretion. Serotonin (5-hydroxytryptamine, 5-HT) and 5hydroxytryptophan (5-HTP) may increase prolactin secretion both
∗ Corresponding author at: Division of Endocrinology, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350, CEP: 90035-003 Porto Alegre, RS, Brazil. Tel.: +55 51 3359 8027; fax: +55 51 3359 8777. E-mail address: [email protected] (P.M. Spritzer). http://dx.doi.org/10.1016/j.neulet.2014.09.005 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
in humans and in diverse animal models [5,27,28] under different physiological conditions, such as lactation and pregnancy. Serotonergic receptors such as 5-HT1A, 5-HT2A and 5HT2C have been described in several studies as modulating prolactin release [1,9,12,14]. However, controversial results have been reported. Some evidence in rodents indicates that the 5-HT2 receptor is involved in prolactin regulation [8,16,19]. In contrast, other studies 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 that 5-HT1A receptors play a role in the control of prolactin secretion [18,20] others point out that both 5-HT1A and 5-HT2A/2C receptors are required for prolactin secretion [12,13].
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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 evidence 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 ovariectomized/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 treatment. 2. Materials and methods 2.1. Animals Eighty-four female Wistar rats, 65–80 days old, weighing 180–220 g, were housed six to a cage under controlled temperature conditions (22 ± 4 ◦ C) and a 12/12 h light–dark cycle with free access to standard certified rodent chow and tap water. All procedures performed on the rats were in compliance with the EC Directive 2010/63/EU on the protection of animals used for scientific purposes. The use of animals was reviewed and approved by the Research Ethics Committee, Federal University of Rio Grande do Sul Basic Health Sciences Institute. 2.2. Experimental procedures
Fig. 1. Effect of MK-212 (10 mg/kg/day for 4 days), on prolactin levels. Hyperestrogenic 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 as median and 25–75% interquartile range (lower and upper limit of the box); maximum and minimum values are shown by the limits of vertical lines. *p < 0.05; **p < 0.01 compared to control group (OVX + saline).
The other half of the group received pindolol (Visken® , Sandoz, São Paulo, Brazil), dissolved in 0.9% NaCl, by subcutaneous injection, as a single 5 mg/kg dose 60 min before decapitation [21,22]. Prolactin levels were compared among the groups of hypo- and hyperestrogenic animals under distinct drug treatments. 2.5. Prolactin assay Prolactin levels (ng/mL) were measured by a double-antibody radioimmunoassay with reagents supplied by the National Hormone and Pituitary Program, U.S. National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, Bethesda, MD, USA). Prolactin was radioiodinated by the chloramine-T method as described elsewhere [24]. NIDDK rat prolactin RP-3 was used as the standard. Assay sensitivity was 4 ng/mL and the intra- and interassay coefficients of variation were 8.6% and 12%, respectively.
All animals were subjected to bilateral ovariectomy (OVX) under anesthesia. At 5 days post-surgery, animals were randomly assigned to receive 300 g per week of estradiol benzoate (Benzoginoestril, Sarsa-São Paulo, Brazil), or sunflower oil vehicle (hyperestrogenic and hypoestrogenic animals, respectively) for 2 weeks, as previously reported [24]. In all experiments, the animals were killed 2 weeks after the first estradiol benzoate or vehicle injection. Animals were killed between 9 and 11 a.m. by decapitation. Trunk blood samples were collected from all animals and serum was harvested and stored at −20 ◦ C until measurement of prolactin levels by a double-antibody radioimmunoassay. Drug administration was carried out during the last days of vehicle or estrogen treatment, between 8 and 11 a.m., as described below.
2.6. Statistical analysis
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 or estradiol treatment period, animals were also injected once daily with normal saline solution (0.9% NaCl) or 6-chloro-2-(1piperazinyl)pyrazine (MK-212, Merck Institute for Therapeutic Research, West Point, PA, USA), subcutaneously, dissolved in 0.9% NaCl at a dose of 10 mg/kg/day. Animals were killed by decapitation 24 h after the end of the 2-week period. 2.4. Experiment 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-Npropylamine)tetralin (8-OH-DPAT, Sigma Chemical Co., St. Louis, MO, USA), dissolved in 0.9% NaCl, by intraperitoneal injection, as a single 1 mg/kg dose 30 min before decapitation. The dose was determined on the basis of previous studies [4,19].
Prolactin values for each group are expressed as median and interquartile range. Statistical analysis was performed by the Mann–Whitney U test. All analyses were performed in the GraphPadPrism 5 software environment (GraphPad Software, Inc., La Jolla, CA, USA). Differences were considered significant if the probability of error was less than 5%.
3.1. Effect of serotonin receptor agonist MK-212 on the 5-HT2A/5-HT2C receptor subtypes Fig. 1 shows that prolactin levels were significantly higher in estradiol-treated ovariectomized rats (n = 9) than in vehicletreated animals (n = 9). MK-212 significantly increased prolactin concentrations in both hypoestrogenic (n = 8) (p < 0.05) and hyperestrogenic (n = 9) (p < 0.01) females as compared with controls. Other one animal died at the experimentation period. 3.2. Effects of serotonin receptor agonist 8-OH-DPAT and antagonist pindolol on the 5-HT1A receptor subtype Fig. 2 shows the effects of the administration of 8-OH-DPAT to hyperestrogenic (n = 8) and hypoestrogenic animals (n = 8). Although prolactin concentrations were higher in hyperestrogenic females (n = 8) compared to hypoestrogenic animals (n = 8), treatment with 8-OH-DPAT did not alter prolactin levels as compared
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Concerning pindolol treatment, no differences in prolactin levels 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
Fig. 2. Effect of 8-OH-DPAT (1 mg/kg) or pindolol (5 mg/kg) on prolactin levels. Hyperestrogenic females (E2 + saline, n = 8; E2 + 8-OH-DPAT, n = 8; and E2 + pindolol, 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 (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); maximum and minimum values are shown by the limits of vertical lines.
with saline control. Similar results were obtained with the administration of pindolol: although prolactin concentrations were higher in hyperestrogenic (n = 7) than in hypoestrogenic animals (n = 4), pindolol treatment did not change prolactin levels as compared with saline control. Other seven animals could not be used for experiments or died at the experimentation period. 4. Discussion In the present study, we tested whether estradiol can modulate serotonergic effects on prolactin secretion by acting directly 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 the serotonin receptor agonist MK-212 increased prolactin levels both in hyper- and hypoestrogenic females. Moreover, estradiol was found to strongly increase prolactin release when administered with this 5-HT2A/2C agonist [2]. These results suggest that the 5-HT2A receptor subtype is involved in prolactin secretion, as reported in a previous study carried out in turkeys [6]. However, while the present results suggest the 5-HT2C receptor subtype may also be involved in the regulation of prolactin secretion, a definitive role for this receptor can only be confirmed after testing the administration of new specific 5-HT2C antagonists [26] in combination with dose-response curves for estrogen administration to ovariectomized rats. Studying a hypoestrogenic animal model, Flügge et al. [7] administrated DPAT, a 5-HT1A receptor agonist, to ovariectomized rats. The authors found that, in estradiol-treated females, both the number and affinity of 5-HT1A receptors were increased in the ventral medial nucleus in comparison with rats not administered estradiol. In contrast, in the present study, despite the increase in prolactin levels observed due to estradiol treatment, no effect was found on prolactin levels with the administration of DPAT or of the 5-HT1A antagonist pindolol. A previous study has reported that DPAT could promote a brief increase in prolactin levels in male rats [4]. Another study [1] showed that treatment with DPAT was efficient in increasing prolactin levels in prepubertal male rats. However, the experimental conditions of these studies using male animals, as well the doses of DPAT administered, differ from those of the present study; this could explain, at least in part, the contrasting results.
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. Further studies are needed to better define the interaction of the 5-HT2A/2C receptor subtypes, prolactin secretion, and the ovarian cycle during antidepressant treatment. Conflict of interest The authors declare that they have no conflict of interest. Authors contribution E.S.M. contributed to study design, acquisition of data, analysis and interpretation of data and drafting manuscript and final review. L.P. contributed to analysis and interpretation of data, drafting manuscript and final review. M.F.R. contributed to acquisition of data, analysis and interpretation of data and manuscript review. P.M.S. contributed to conception and study design, analysis and interpretation of data, drafting manuscript and final review. All authors approved the final version of the manuscript. Acknowledgments This work was supported by a grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq INCT 573747/2008-3). LP is recipient of PhD grant from CAPES (1172105). The funding sources were not involved in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. References [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) 547–554. [2] A.V. Akhmadeev, L.B. Kalimullina, Sex steroids and monoamines in the system of neuroendocrine regulation of the functions of the amygdaloid complex of the brain, Neurosci. Behav. Physiol. 43 (1) (2013) 129–134. [3] F. Artigas, A. Adell, P. Celada, Pindolol augmentation of antidepressant response, Curr. Drug Targets 7 (2) (2006) 139–147. [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 rat, Neuroendocrinology 61 (2) (1995) 159–166. [5] L. Caligaris, S. Taleisnik, Involvement of neurons containing 5hydroxytryptamine in the mechanism of prolactin release induced by oestrogen, J. Endocrinol. 62 (1) (1974) 25–33. [6] Y. Chaiseha, S.W. Kang, B. Leclerc, S. Kosonsiriluk, N. Sartsoongnoen, M.E. Halawani, Serotonin receptor subtypes influence prolactin secretion in the Turkey, Gen. Comp. Endocrinol. 165 (1) (2010) 170–175. [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) (1999) 243–249. [8] G.A. Jahn, R.P. Deis, Effect of serotonin antagonist on prolactin and progesterone secretion in rats: evidence that the stimulatory and inhibitory actions
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