Hypothalamic GnRH expression and pulsatility depends on a balance of prolactin receptors in the plains vizcacha, Lagostomus maximus.

In mammals, gestation is considered a physiological hyperprolactinemia status. Prolactin (PRL) is one of the modulators of gonadotropin-releasing hormone (GnRH) neurons function. The South American plains vizcacha (Lagostomus maximus) is a unique model to study the regulation of hypothalamic GnRH neurons by direct and indirect steroid-dependent pathways. The aim was to characterize the hypothalamic expression of endocrine markers in vizcacha during gestation as well as their response to experimental induced hyperprolactinemia. The possible involvement of PRL regulatory pathways on GnRH in the context of hypothalamic and pituitary reactivation in mid-gestating vizcachas was discussed. Using two in vivo approaches, we determined changes in the hypothalamic expression and distribution of prolactin receptor (PRLR), tyrosine hydroxylase (TH), and dopamine type 2 receptor. A significant increment in the number of tuberoinfundibular dopaminergic (TIDA) neurons was determined in the arcuate nucleus from early to term pregnancy. On the other hand, at preoptic area, the number of both TH+PRLR+ and GnRH+PRLR+ double-labeled neurons significantly decreased at mid-pregnancy probably allowing the recovery of GnRH expression indicating that both types of neurons may represent the key points of PRL indirect and direct pathways modulating GnRH. Moreover, in a model of induced hyperprolactinemic vizcachas, the inhibitory effect of PRL on GnRH at both expression and delivery levels were confirmed. These results suggest the concomitant participation of both PRL regulatory pathways on GnRH modulation and pinpoint the key role of PRL on GnRH expression enabling the recovery of the hypothalamic activity during the gestation in this species.

Bu-Shen-Zhu-Yun decoction induces PRLR deubiquitination and JAK2/STAT5 activation via CSN5 in vitro.

PURPOSE: To determine the effect of Bu-Shen-Zhu-Yun Decoction (BSZY-D) on the kisspeptin through JAK2/STAT5 signaling pathway in hyperprolactinemia (HPRL) infertility. METHOD: SD rats were treated with BSZY-D for cerebrospinal fluid (CSF) extraction. GT1-7 cells were subjected to different treatments. The phosphorylation levels of JAK2 and STAT5, and the expressions of PRLR and kisspeptin of GT1-7 cells in different groups were detected by western blot, RT-qPCR and immunofluorescence. The expressions of CSN5 and GATA1 and other molecular features were checked by western blot, RT-PCR, co-immunoprecipitation and renilla luciferase activity. RESULTS: The phosphorylation levels of JAK2 and STAT5, and the expressions of PRLR and kisspeptin in the HPRL group were significantly decreased, and these changes could be reversed after BSZY-D treatment. In addition, the presence of PRLR deubiquitination was detected in the HPRL group, which could be reversed by shRNA-CSN5, suggesting that BSZY-D played a role through targeting CSN5. The binding level of GATA1 and CSN5 promoter in the HPRL group was significantly decreased, but elevated in the HPRL (BSZY-D/CSF) group (P < 0.05). CONCLUSION: BSZY-D improved the transcription activity of GATA1 and increased the binding of GATA1 and CSN5. BSZY-D was involved in the deubiquitination of PRLR, which contributes to alleviating the symptoms of HPRL infertility.

Paeoniflorin ameliorates antipsychotic-induced hyperprolactinemia in rats by attenuating impairment of the dopamine D2 receptor and TGF-ß1 signaling pathways in the hypothalamus and pituitary.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin, a prominent component in some Chinese formulas for hyperprolactinemia-associated disorders, has been found to inhibit prolactin secretion in prolactinoma cells. AIM: To examine the efficacy of paeoniflorin on hyperprolactinemia and the underlying mechanisms of action. MATERIALS AND METHODS: Hyperprolactinemia in female rats was generated by administration of olanzapine (5 mg/kg, by a gavage method, once daily, × 13 weeks). The rats were co-treated with paeoniflorin (10 and 50 mg/kg). Prolactin and TGF-ß1 concentrations were detected by ELISA. Protein expression was determined by Western blot. The effect in MMQ cells was also examined. RESULTS: Paeoniflorin inhibited olanzapine-induced increases in plasma prolactin concentration and prolactin protein overexpression in the pituitary and hypothalamus of rats. Further, paeoniflorin restored olanzapine-induced downregulation of pituitary and hypothalamic dopamine D2 receptor (D2R) protein expression. More importantly, paeoniflorin attenuated olanzapine-suppressed protein expression of transforming growth factor (TGF)-ß1 and its downstream genes, type II TGF-ß receptor, type I TGF-ß receptor and phosphorylated SMAD3 in the tissues. However, paeoniflorin did not affect plasma TGF-ß1 concentration and hepatic TGF-ß1 protein expression. In accord, olanzapine-induced increase in prolactin concentration, upregulation of prolactin protein expression, and downregulation of protein expression of the D2R and TGF-ß1 signals in MMQ cells were attenuated. CONCLUSIONS: This study demonstrates that paeoniflorin ameliorates olanzapine-induced hyperprolactinemia in rats by attenuating impairment of the D2R and TGF-ß1 signaling pathways in the hypothalamus and pituitary. Our findings may provide evidence to support the use of paeoniflorin-contained Chinese herbs and formulas for hyperprolactinemia and its associated disorders.

Chronic estrogen affects TIDA neurons through IL-1ß and NO: effects of aging.

Women are chronically exposed to estrogens through oral contraceptives, hormone replacement therapy or environmental estrogens. We hypothesized that chronic exposure to low levels of estradiol-17ß (E2) can induce inflammatory and degenerative changes in the tuberoinfundibular dopaminergic (TIDA) system leading to reduced dopamine synthesis and hyperprolactinemia. Young (Y; 3­4 months) and middle-aged (MA; 10­12 months) Sprague-Dawley rats that were intact or ovariectomized (OVX) were either sham-implanted or implanted with a slow-release E2 pellet (20 ng E2/day for 90 days). To get mechanistic insight, adult 3- to 4-month-old WT, inducible nitric oxide synthase (iNOS) and IL-1 receptor (IL-1R) knockout (KO) mice were subjected to a similar treatment. Hypothalamic areas corresponding to the TIDA system were analyzed. E2 treatment increased IL-1ß protein and nitrate levels in the arcuate nucleus of intact animals (Y and MA). Nitration of tyrosine hydroxylase in the median eminence increased with E2 treatment in both intact and OVX animals. There was no additional effect of age. This was accompanied by a reduction in dopamine levels and an increase in prolactin in intact animals. E2 treatment increased nitrate and reduced dopamine levels in the hypothalamus and increased serum prolactin in WT mice. In contrast, the effect of E2 on nitrate levels was blocked in IL-1R KO mice and the effect on dopamine and prolactin were blocked in iNOS KO animals. Taken together, these results show that chronic exposure to low levels of E2 decreases TIDA activity through a cytokine-nitric oxide-mediated pathway leading to hyperprolactinemia and that aging could promote these degenerative changes.

Expression of prolactin receptors in the duodenum, kidneys and skeletal system during physiological and sulpiride-induced hyperprolactinaemia.

INTRODUCTION AND AIM: Hyperprolactinaemia in pregnancy leads to mild and reversible changes in the maternal skeletal system, and medicamentous hyperprolactinemia causes more detrimental effects. We conducted an experimental study to evaluate differences between Prlr gene expression in the duodenum, vertebrae and kidneys during physiological and medicamentous hyperprolactinaemia, which could influence calcium homeostasis. METHODS: Experimental animals (18 weeks old, Wistar female rats) were divided as follows: group P (nine rats that were 3 weeks pregnant), group M (ten rats that were intramuscularly administrated sulpiride (10 mg/kg) twice daily for 3 weeks), and the control group (C, ten age-matched nulliparous rats, 18-week-old). Laboratory investigations included measurements of serum ionized calcium, phosphorus, urinary calcium and phosphorus excretion, osteocalcin (OC), serum procollagen type 1 N-terminal propeptide (P1NP), vitamin D, parathyroid hormone (PTH) and prolactin (PRL). Relative quantification of gene expression for prolactin receptors in the duodenum, vertebrae and kidneys was determined using real-time PCR. RESULTS: Expression of the Prlr gene was significantly higher in the duodenum (p < 0.001) and lower in vertebrae (p < 0.001) and kidneys (p < 0.01) in rats with physiological hyperprolactinaemia (PHP) than in the control group. Significantly lower Prlr expression in the duodenum was verified (p < 0.001), along with increased Prlr gene expression in vertebrae (p < 0.001) and kidneys (p < 0.01), in rats with medicamentous hyperprolactinaemia (MHP) than in the C group. CONCLUSIONS: Downregulation of Prlr gene expression in the duodenum may explain the diminished intestinal calcium absorption in medicamentous hyperprolactinaemia. Prolactin takes calcium from the skeletal system following increased Prlr gene expression in the vertebrae to maintain calcium homeostasis, which increases the harmful effect on bone metabolism compared to that of physiological hyperprolactinaemia.

Mucuna pruriens (L.) DC chemo sensitize human breast cancer cells via downregulation of prolactin-mediated JAK2/STAT5A signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Mucuna pruriens (L.) DC (MP) is an ancient Indian medicinal plant traditionally used to treat Parkinson's disease. L-Dopa (LD), precursor of dopamine is abundantly found in the seeds of MP. L-dopa is a natural inhibitor of prolactin (PRL) hormone which is required to maintain lactation in women but it's over production (hyperprolactinemia) plays critical role in advancement of breast cancer. AIM OF THE STUDY: We aim to examine the pharmacological effect of LD and MP on this hyperprolactinemia associated breast cancer and related signaling for effective management of the disease. We also investigated chemo-sensitizing effect of MP on hyperprolactinemia-mediated cisplatin resistance. MATERIALS AND METHODS: Methanolic seed extract of MP were prepared and analysed using HPLC. Effect of LD and MP on the cellular viability of breast cancer cells (T47D, MCF-7, MDA-MB-468 and MDA-MB-231) were evaluated using MTT assay. Further, effect of LD and MP on colony forming potential, DNA damage, cell cycle distribution and apoptosis was determined using agar/agarose method, comet assay and annexin and PI method followed by FACS analysis. To reveal the molecular mechanism involved in the anti-cancer activity of MP, transcriptional and translational level analysis of the key proteins involved in the PRL-mediated signaling, was performed using RT-PCR and western blot analysis. The effect of MP extract on PRL-mediated signaling was validated using dopaminergic agonist bromocriptine. MP extract and cisplatin was given in different combination with appropriate controls to check their effect on chemo-resistivity of breast cancer cells. RESULTS: Our results demonstrated that MP seed extract has the potential to inhibit cellular proliferation of PRL expressing T47D and MCF-7 breast cancer cells via induction of DNA damage, G1 phase of cell cycle arrest and apoptosis more effectively as compare to LD. Further, MP-mediated anti-cancerous effect was associated with the downregulation of PRL expression, further suppressing the JAK2/STAT5A/Cyclin D1 signaling pathway which has been validated using dopaminergic agonist bromocriptine. Cancer-related hyperprolactinemia confers cisplatin resistance, we observed that MP via PRL inhibition, enhances cisplatin efficacy after their combinatorial treatment in breast cancer cells. CONCLUSIONS: Collectively, our study suggests that MP could be recommended as dietary supplement along with the chemotherapeutic agents against breast cancer.

The mechanism of selfheal extract in treating hyperprolactinemia.

OBJECTIVE: Selfheal has been used for many years in hyperprolactinemia induced galactorrhea, menstrual disorders, and dysgenesis with satisfactory curative effect. However, its mechanism is still unclear. This study intended to investigate the effect of selfheal extract on hyperprolactinemia in vivo and in vitro, in order to elucidate its mechanism of anti-hyperprolactinemia. PATIENTS AND METHODS: Hyperprolactinemia rat model was established. High dose (28.8 g/(kg⋅d)), middle dose (14.4 g/ (kg⋅d)), and low dose (7.2 g/(kg⋅d)) of selfheal extract were used to treat the model to observe impact on serum estradiol (E2), progesterone (P), prolactin (PRL), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels. Three cell lines MMQ, GH3, and PC12 were applied to investigate selfheal extract effect on PRL secretion, dopamine D2 receptor, and dopamine transporter (DAT). RESULTS: High and middle dose of selfheal extract significantly reduced PRL level in hyperprolactinemia rat compared with model group (P< 0.01). Compared with normal control, 5 mg/ml and 10 mg/ml selfheal extract obviously inhibited PRL secretion in MMQ cells that high expressed D2 receptor after 24 hours (P< 0.01), but did not affect PRL secretion in GH3 cells lack of D2 receptor. 8 mg/ml selfheal extract markedly suppressed D2 receptor and DAT expression in PC12 cells that strongly expressed D2 receptor and DAT (P< 0.01). CONCLUSIONS: Selfheal extract treated hyperprolactinemia through dopamine D2 receptor with significant effect.

Paeoniflorin and liquiritin, two major constituents in Chinese herbal formulas used to treat hyperprolactinemia-associated disorders, inhibits prolactin secretion in prolactinoma cells by different mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin and liquiritin are major constituents in some Chinese herbal formulas, such as Yiru Tiaojing (YRTJ) Granule (a hospitalized preparation) and Peony-Glycyrrhiza Decoction, used for hyperprolactinemia-associated disorders. AIM OF THE STUDY: To investigate the effect of paeoniflorin and liquiritin on prolactin secretion. MATERIALS AND METHODS: The effect of YRTJ Granule on metoclopramide-induced hyperprolactinemia was tested in rats. Paeoniflorin and liquiritin in the YRTJ Granule extract were identified and quantified by HPLC. The effects of paeoniflorin and liquiritin on prolactin secretion were examined in prolactinoma cells that were identified morphologically and by Western blot. The concentration of prolactin was determined by ELISA. The gene expression was analyzed by Western blot. RESULTS: YRTJ Granule ameliorated metoclopramide-induced hyperprolactinemia in rats. The contents of paeoniflorin and liquiritin in YRTJ Granule were 7.43 and 2.05mg/g extract, respectively. Paeoniflorin, liquiritin and bromocriptine (a dopamine D2 receptor (D2R) agonist) decreased prolactin concentration in MMQ cells expressing D2R. However, the effect of liquiritin and bromocriptine was abolished in GH3 cells lacking D2R expression. Interestingly, paeoniflorin still decreased prolactin concentration in GH3 cells in the same manner. Furthermore, paeoniflorin suppressed prolactin protein expression, and was without effect on D2R protein expression in both MMQ and GH3 cells. CONCLUSIONS: The present results suggest that paeoniflorin and liquiritin play a role in YRTJ Granule-elicited improvement of hyperprolactinemia. While the effect of liquiritin is D2R-dependent, paeoniflorin D2R-independently inhibits prolactin secretion in prolactinoma cells that may especially benefit the hyperprolactinemic patients who are refractory to dopaminergic therapies.

Maternal flaxseed oil intake during lactation changes body fat, inflammatory markers and glucose homeostasis in the adult progeny: role of gender dimorphism.

We evaluated maternal flaxseed oil intake during lactation on body composition, lipid profile, glucose homeostasis and adipose tissue inflammation in male and female progeny at adulthood. Lactating rats were divided into the following: control 7% soybean oil (C), hyper 19% soybean oil (HS) and hyper 17% flaxseed oil+2% soybean oil (HF). Weaned pups received a standard diet. Offspring were killed in PN180. Male HF presented higher visceral adipose tissue (VAT) and triacylglycerol, and female HF showed insulin resistance. Both male and female HF had hyperleptinemia, and only male HF had hyperprolactinemia. In VAT, male HF presented lower PPAR-γ expressions and higher TNF-α, IL-6, IL-1ß and IL-10 expressions; in subcutaneous adipose tissue (SAT), they presented lower PPAR-γ and TNF-α expressions. Female HF presented higher leptin, as well as lower adiponectin, TNF-α, IL-6 and IL-1ß expressions in VAT and lower TNF-α in SAT. Flaxseed oil during lactation leads to gender-specific effects with more adiposity and dyslipidemia in male and insulin resistance in female. Higher prolactin and inflammatory cytokines in male could play a role in these gender differences. We suggest that the use of flaxseed oil during lactation increases metabolic syndrome risk in the adult progeny.

Studies on the regulatory effect of Peony-Glycyrrhiza Decoction on prolactin hyperactivity and underlying mechanism in hyperprolactinemia rat model.

Clinical trials have demonstrated the beneficial effects of Peony-Glycyrrhiza Decoction (PGD) in alleviating antipsychotic-induced hyperprolactinemia (hyperPRL) in schizophrenic patients. In previous experiment, PGD suppressed prolactin (PRL) level in MMQ cells, involving modulating the expression of D2 receptor (DRD2) and dopamine transporter (DAT). In the present study, hyperPRL female rat model induced by dopamine blocker metoclopramide (MCP) was applied to further confirm the anti-hyperpPRL activity of PGD and underlying mechanism. In MCP-induced hyperPRL rats, the elevated serum PRL level was significantly suppressed by either PGD (2.5-10 g/kg) or bromocriptine (BMT) (0.6 mg/kg) administration for 14 days. However, in MCP-induced rats, only PGD restored the under-expressed serum progesterone (P) to control level. Both PGD and BMT administration restore the under-expression of DRD2, DAT and TH resulted from MCP in pituitary gland and hypothalamus. Compared to untreated group, hyperPRL animals had a marked reduction on DRD2 and DAT expression in the arcuate nucleus. PGD (10 g/kg) and BMT (0.6 mg/kg) treatment significant reversed the expression of DRD2 and DAT. Collectively, the anti-hyperPRL activity of PGD associates with the modulation of dopaminergic neuronal system and the restoration of serum progesterone level. Our finding supports PGD as an effective agent against hyperPRL.

Efficacy and Mechanism of Action of Yiru Tiaojing Granule Against Hyperprolactinemia In Vitro and In Vivo.

Yiru Tiaojing Granule, a traditional Chinese medicine formula, is used to treat hyperprolactinemia. This study was conducted to evaluate the mechanism of action and pharmacological activity of Yiru Tiaojing Granule on prolactin secretion. The animal model of hyperprolactinemia was induced by metoclopramide. The dopamine D2 receptor in hyperprolactinemia rat models was analyzed by immunohistochemistry. The biochemical parameters, including a follicle-stimulating hormone, luteinizing hormone, estradiol, progesterone, testosterone, and prolactin, were measured by an enzyme-linked immunosorbent assay. Furthermore, the expression of prolactin and the dopamine D2 receptor was analyzed by Western blotting. The components in the Yiru Tiaojing Granule-medicated serum were assayed by liquid chromatography-tandem mass spectrometry. The Yiru Tiaojing Granule significantly decreased the prolactin level in the hyperprolactinemia rat model, and increased the estradiol, luteinizing hormone, and progesterone levels. The high and medium doses of Yiru Tiaojing Granule reduced dopamine D2 receptor expression in the brain (p < 0.001) and produced a similar effect on bromocriptine (p < 0.001). Yiru Tiaojing Granule-medicated serum reduced (p < 0.001) prolactin expression in MMQ cells in a concentration-dependent manner, but had no effects on GH3 cells. The level of the dopamine D2 receptor in MMQ cells was also increased dose-dependently (p < 0.05). In addition, the protein kinase A and cyclic adenosine monophosphate in MMQ cells were significantly attenuated dose-dependently by treatment with a high and medium dose of Yiru Tiaojing Granule-medicated serum (p < 0.05) and bromocriptine-medicated serum (p < 0.01). The results suggested that Yiru Tiaojing Granule was effective against hyperprolactinemia, and the activation of the dopamine D2 receptor, which was related to the second messenger cyclic adenosine monophosphate and protein kinase A, might be the potential mechanism.

Hypothalamic Prolactin Regulation of Luteinizing Hormone Secretion in the Female Rat.

Prolactin (PRL) levels increase in response to long-term antipsychotic treatment that disrupts reproductive function. Recent evidence suggests that activation of central PRL receptors (PRLR) inhibits LH secretion and in ovariectomized rats. However, the mechanisms involved, the mode of LH secretion affected and relevance to hyperprolactinemia remain unknown. We therefore investigated the contribution of central PRL/PRLR signaling to the control of estradiol-induced surges of LH and PRL and pulsatile LH secretion under basal and hyperprolactinemic conditions. First, by subjecting ovariectomized estradiol-primed rats intracerebroventricularly administered with PRL to frequent blood sampling, we demonstrated that acute activation of hypothalamic PRLR disrupts pulsatile LH secretion. Pretreatment (intracerebroventricularly) with the pure PRLR antagonist, Δ1-9-G129R-hPRL, or the γ-aminobutyric acid receptor type A antagonist, bicuculline, blocked this effect. Next, we revealed that sustained blockade of hypothalamic PRLR using Δ1-9-G129R-hPRL augmented the magnitude of LH surges induced by estradiol benzoate and progesterone treatment and suppressed the concomitant surges of PRL. Finally, we determined that acute antagonism of central PRLR is insufficient to normalize the duration of the LH pulse interval prolonged as a result of hyperprolactinemia induced by chronic exposure to the atypical antipsychotic sulpiride. These data serve as the first evidence to suggest that PRL signaling through hypothalamic PRLR inhibits pulsatile secretion of LH in a γ-aminobutyric acid receptor type A-dependent fashion and tonically restrains the magnitude of the LH surge. Furthermore, our results indicate that transient blockade of hypothalamic PRL/PRLR signaling is not an effective strategy for restoring LH pulsatility perturbed by chronic hyperprolactinemia.

Chronic estradiol exposure induces oxidative stress in the hypothalamus to decrease hypothalamic dopamine and cause hyperprolactinemia.

Estrogens are known to cause hyperprolactinemia, most probably by acting on the tuberoinfundibular dopaminergic (TIDA) system of the hypothalamus. Dopamine (DA) produced by TIDA neurons directly inhibits prolactin secretion and, therefore, to stimulate prolactin secretion, estrogens inhibit TIDA neurons to decrease DA production. However, the mechanism by which estrogen produces this effect is not clear. In the present study, we used a paradigm involving chronic exposure to low levels of estradiol-17ß (E(2)) to mimic prolonged exposures to environmental and endogenous estrogens. We hypothesized that chronic exposure to low levels of E(2) induces oxidative stress in the arcuate nucleus (AN) of the hypothalamus that contains TIDA neurons and causes nitration of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA. This results in a significant decrease in DA and consequently, hyperprolactinemia. To investigate this, adult, intact female cycling rats were implanted with slow-release E(2) pellets (20 ng/day) for 30, 60, or 90 days and were compared with old (16-18 mo old) constant estrous (OCE) rats. Chronic E(2) exposure significantly increased the expression of glial fibrillary acidic protein and the concentrations of interleukin-1ß (IL-1ß) and nitrate in the AN that contains perikarya of TIDA neurons and increased nitration of TH in the median eminence (ME) that contains the terminals. These levels were comparable to those seen in OCE rats. We observed a significant decrease in DA concentrations in the ME and hyperprolactinemia in an exposure-dependent manner similar to that seen in OCE rats. It was concluded that chronic exposure to low levels of E(2) evokes oxidative stress in the AN to inhibit TIDA neuronal function, most probably leading to hyperprolactinemia.

Antipsychotics and hyperprolactinaemia: clinical recommendations.

A group of international experts in psychiatry, medicine, toxicology and pharmacy assembled to undertake a critical examination of the currently available clinical guidance on hyperprolactinaemia. This paper summarises the group's collective views and provides a summary of the recommendations agreed by the consensus group to assist clinicians in the recognition, clinical assessment, investigation and management of elevated plasma prolactin levels in patients being treated for severe mental illness. It also deals with the special problems of particular populations, gives advice about information that should be provided to patients, and suggests a strategy for routine monitoring of prolactin. The recommendations are based upon the evidence contained in the supplement 'Hyperprolactinaemia in schizophrenia and bipolar disorder: Clinical Implications' (2008). The guidance contained in this article is not intended to replace national guidance (such as that of the National Institute of Clinical Excellence), however, it does provide additional detail that is unlikely to be covered in existing guidelines, and focuses on areas of uncertainty and disagreement. We hope it will add to the debate about this topic.

Prolactin directly enhances bone turnover by raising osteoblast-expressed receptor activator of nuclear factor kappaB ligand/osteoprotegerin ratio.

Hyperprolactinemia leads to high bone turnover as a result of enhanced bone formation and resorption. Although its osteopenic effect has long been explained as hyperprolactinemia-induced hypogonadism, identified prolactin (PRL) receptors in osteoblasts suggested a possible direct action of PRL on bone. In the present study, we found that hyperprolactinemia induced by anterior pituitary transplantation (AP), with or without ovariectomy (Ovx), had no detectable effect on bone mineral density and content measured by dual-energy X-ray absorptiometry (DXA). However, histomorphometric studies revealed increases in the osteoblast and osteoclast surfaces in the AP rats, but a decrease in the osteoblast surface in the AP+Ovx rats. The resorptive activity was predominant since bone volume and trabecular number were decreased, and the trabecular separation was increased in both groups. Estrogen supplement (E2) fully reversed the effect of estrogen depletion in the Ovx but not in the AP+Ovx rats. In contrast to the typical Ovx rats, bone formation and resorption became uncoupled in the AP+Ovx rats. Therefore, hyperprolactinemia was likely to have some estrogen-independent and/or direct actions on bone turnover. Osteoblast-expressed PRL receptor transcripts and proteins shown in the present study confirmed our hypothesis. Furthermore, we demonstrated that the osteoblast-like cells, MG-63, directly exposed to PRL exhibited lower expression of alkaline phosphatase and osteocalcin mRNA, and a decrease in alkaline phosphatase activity. The ratios of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) proteins were increased, indicating an increase in the osteoclastic bone resorption. The present data thus demonstrated that hyperprolactinemia could act directly on bone to stimulate bone turnover, with more influence on bone resorption than formation. PRL enhanced bone resorption in part by increasing RANKL and decreasing OPG expressions by osteoblasts.

Hypothalamic prolactin receptor messenger ribonucleic acid levels, prolactin signaling, and hyperprolactinemic inhibition of pulsatile luteinizing hormone secretion are dependent on estradiol.

Hyperprolactinemia can reduce fertility and libido. Although central prolactin actions are thought to contribute to this, the mechanisms are poorly understood. We first tested whether chronic hyperprolactinemia inhibited two neuroendocrine parameters necessary for female fertility: pulsatile LH secretion and the estrogen-induced LH surge. Chronic hyperprolactinemia induced by the dopamine antagonist sulpiride caused a 40% reduction LH pulse frequency in ovariectomized rats, but only in the presence of chronic low levels of estradiol. Sulpiride did not affect the magnitude of a steroid-induced LH surge or the percentage of GnRH neurons activated during the surge. Estradiol is known to influence expression of the long form of prolactin receptors (PRL-R) and components of prolactin's signaling pathway. To test the hypothesis that estrogen increases PRL-R expression and sensitivity to prolactin, we next demonstrated that estradiol greatly augments prolactin-induced STAT5 activation. Lastly, we measured PRL-R and suppressor of cytokine signaling (SOCS-1 and -3 and CIS, which reflect the level of prolactin signaling) mRNAs in response to sulpiride and estradiol. Sulpiride induced only SOCS-1 in the medial preoptic area, where GnRH neurons are regulated, but in the arcuate nucleus and choroid plexus, PRL-R, SOCS-3, and CIS mRNA levels were also induced. Estradiol enhanced these effects on SOCS-3 and CIS. Interestingly, estradiol also induced PRL-R, SOCS-3, and CIS mRNA levels independently. These data show that GnRH pulse frequency is inhibited by chronic hyperprolactinemia in a steroid-dependent manner. They also provide evidence for estradiol-dependent and brain region-specific regulation of PRL-R expression and signaling responses by prolactin.

Hypothalamic levels of NPY, MCH, and prepro-orexin mRNA during pregnancy and lactation in the rat: role of prolactin.

Pregnancy and lactation provide excellent models of physiological hyperphagia and hyperprolactinemia. To identify possible factors associated with the increased feeding in these situations, we measured hypothalamic mRNA levels of three orexigenic neuropeptides--NPY, MCH, and orexins--in nonpregnant, pregnant, and lactating rats by in situ hybridization. NPY mRNA content in the arcuate nucleus was significantly increased during pregnancy and lactation. However, MCH and prepro-orexin expression was decreased in both states. 48 or 72 h of fasting in pregnant and lactating rats further elevated NPY mRNA levels and increased the low MCH mRNA content. Surprisingly, no effect was observed in prepro-orexin mRNA levels. Finally, we investigated the possible effect of high PRL levels on these orexigenic signals using a model of hyperprolactinemia induced by pituitary graft. NPY mRNA content was unchanged, but MCH and prepro-orexin mRNA levels were significantly decreased. Our results suggest that the increased NPY expression might be partly responsible for the hyperphagia observed during pregnancy and lactation. MCH and prepro-orexin may be involved in the adaptation of other homeostatic mechanisms and their decreased levels in these physiological settings could be mediated by the elevated circulating PRL levels.

Superior cervical ganglionectomy effects on median eminence and anterior and mediobasal hypothalamic taurine content in male rats: effects of hyperprolactinemia.

The neuroendocrine sequelae of acute or chronic superior cervical ganglionectomy in control or pituitary-grafted rats were studied by analyzing both plasma prolactin, growth hormone (GH) and ACTH levels, and taurine (TAU) content in the hypophysiotropic area of the hypothalamus or the median eminence. As expected, after either acute or chronic ganglionectomy, norepinephrine (NE) content decreased in the brain areas studied, although the values remained higher in hyperprolactinemic rats. TAU content was differentially modified by acute vs. chronic surgeries, thus indicating the possible existence of hypothalamic interactions between TAU and NE to regulate pituitary hormone secretion. Indeed, associated differential changes in plasma prolactin, GH and ACTH levels may be due to the observed TAU changes. As expected, pituitary grafting increased plasma prolactin, GH and ACTH levels, so that the presence of a pituitary graft differentially interferes with the effects of either surgery not only on TAU content but also on the plasma levels of the hormone studied. Globally, ongoing studies confirm the differential effects of acute and chronic superior cervical ganglionectomy on plasma prolactin, GH and ACTH levels, and provide new evidence about its effects on TAU content in the hypophysiotropic area of the hypothalamus and the median eminence that may partially explain the changes observed in the pituitary hormones studied.

Daily changes of GABA and taurine concentrations in various hypothalamic areas are affected by chronic hyperprolactinemia.

This study was designed to characterize, in anterior, mediobasal, and posterior hypothalamic and median eminence, the 24h changes of gamma aminobutyric acid (GABA) and taurine (TAU) contents in adult male rats and to analyze whether chronic hyperprolactinemia may affect these patterns. Rats were turned hyperprolactinemic by a pituitary graft. Plasma prolactin (PRL) levels increased after pituitary grafting at all time points examined. A disruption of the circadian rhythm was observed in pituitary-grafted rats, whereas GABA and TAU content followed daily rhythms in all areas studied in controls. In the mediobasal hypothalamus, two peaks for each amino acid were found at midnight and midday. In the anterior hypothalamus, GABA and TAU showed only one peak of concentration at midnight. In the posterior hypothalamus, the values of both GABA and TAU were higher during the light as compared to the dark phase of the photoperiod. In the median eminence GABA content peaked at 20:00h, the time when TAU exhibited the lowest values. Hyperprolactinemia abolished the 24h changes of GABA in the mediobasal hypothalamus and reduced its content as compared to controls. Hyperprolactinemia advanced the diurnal peak of TAU to 12:00h in the mediobasal hypothalamus and did not modify the 24:00h peak. In the anterior hypothalamus, hyperprolactinemia increased GABA and TAU contents during the light phase while it decreased them during the dark phase of the photoperiod. In the posterior hypothalamus hyperprolactinemia did not modify GABA or TAU patterns as compared to controls. In the median eminence hyperprolactinemia increased the 20:00h peak of GABA and shift advanced the decrease in TAU content at 20:00h and its maximum at 24:00h as compared to controls. These data show that GABA and TAU content exhibit specific daily patterns in each hypothalamic region studied. PRL differentially affects the daily pattern of these amino acids in each hypothalamic region analyzed.

Changes in substance P content at the hypothalamic-pituitary axis during the Wallerian degeneration of peripheral sympathetic neurons after superior cervical ganglionectomy in male rats: effect of hyperprolactinemia.

The effects of Wallerian degeneration of the peripheral sympathetic neurons projecting to the hypothalamus on the mechanism of interaction between prolactin and substance P (SP) were examined. The effects of superior cervical ganglionectomy (SCGx) on SP content in various hypothalamic regions and in the hypophysis were evaluated in control and hyperprolactinemic rats. Male rats that received pituitary transplants at the age of 5 days and age-matched sham-operated controls were used. Pituitary grafting significantly increased circulating values of prolactin, as did SCGx. In hyperprolactinemic rats, SCGx partially decreased plasma prolactin levels. Neonatal hyperprolactinemia decreased SP content in the anterior (AH) and posterior (PH) hypothalamus and in the median eminence (ME), but increased it in the mediobasal hypothalamus (MBH). Acute SCGx significantly increased SP in the MBH, PH, and ME. SCGx in hyperprolactinemic animals further increased SP content in MBH. In the ME and Ah, SCGx in pituitary grafted rats decreased SP content as compared with the controls. In the pituitary gland (PG), SCGx only decreased SP content in hyperprolactinemic, but not in control rats. An interaction between peripheral nor-adrenergic neurons and prolactin to regulate SP within the hypothalamus was positive in the MBH, AH, ME, and PG, but not in the PH. These data indicate the existence of interactive mechanisms between prolactin and the peripheral sympathetic neurons to regulate SP content at the hypothalamic-pituitary axis. Interrelationships between prolactin and SP were also observed.