Karen A. Gregerson

Mammary gland serotonin regulates parathyroid hormone-related protein and other bone-related signals.

Breast cells drive bone demineralization during lactation and metastatic cancers. A shared mechanism among these physiological and pathological states is endocrine secretion of parathyroid hormone-related protein (PTHrP), which acts through osteoblasts to stimulate osteoclastic bone demineralization. The regulation of PTHrP has not been accounted for fully by any conventional mammotropic stimuli or tumor growth factors. Serotonin (5-HT) synthesis within breast epithelial cells is induced during lactation and in advancing breast cancer. Here we report that serotonin deficiency (knockout of tryptophan hydroxylase-1) results in a reduction of mammary PTHrP expression during lactation, which is rescued by restoring 5-HT synthesis. 5-HT induced PTHrP expression in lactogen-primed mammary epithelial cells from either mouse or cow. In human breast cancer cells 5-HT induced both PTHrP and the metastasis-associated transcription factor Runx2/Cbfa1. Based on receptor expression and pharmacological evidence, the 5-HT2 receptor type was implicated as being critical for induction of PTHrP and Runx2. These results connect 5-HT synthesis to the induction of bone-regulating factors in the normal mammary gland and in breast cancer cells.

Serotonin Transport and Metabolism in the Mammary Gland Modulates Secretory Activation and Involution

CONTEXT Serotonin [5-hydroxytryptamine (5-HT)] is an important local regulator of lactation homeostasis; however, the roles for the serotonin reuptake transporter and monoamine oxidase have not been known. OBJECTIVE The aim of the study was to determine whether drugs that impact 5-HT affect human lactation physiology. DESIGN AND SETTING We conducted laboratory studies of human and animal models and an observational study of the onset of copious milk secretion in postpartum women at a university medical center. PARTICIPANTS We studied women expecting their first live-born infant; exclusion criteria were: referred to the medical center for another medical condition, known contraindication to breastfeed, and less than 19 yr of age and unable to obtain parental consent. INTERVENTION(S) The mothers were interviewed. The cell and animal studies consisted of a variety of biochemical, pharmacological, and genetic interventions. MAIN OUTCOME MEASURE(S) The human subjects outcome was prevalence of delayed onset of copious milk secretion. The cell and animal outcomes were physiological and morphological. RESULTS Inhibiting serotonin reuptake in mammary epithelial cells altered barrier function, and the effects were amplified by coadministering a monoamine oxidase inhibitor. Direct delivery of fluoxetine by slow-release pellets caused localized involution. TPH1 knockout mice displayed precocious secretory activation. Among a cohort of 431 women, those taking SSRI were more likely (P = 0.02) to experience delayed secretory activation. CONCLUSIONS Medications that perturb serotonin balance dysregulate lactation, and the effects are consistent with those predicted by the physiological effects of intramammary 5-HT bioactivity. Mothers taking serotonergic drugs may need additional support to achieve their breastfeeding goals.

Estrogen regulation of the dopamine-activated GIRK channel in pituitary lactotrophs: implications for regulation of prolactin release during the estrous cycle

Prolactin (PRL), synthesized and secreted from lactotrophs of the anterior pituitary gland, is tonically inhibited by hypothalamic dopamine (DA) throughout the female reproductive (estrous) cycle. Our laboratory has shown that DA hyperpolarizes these cells by activating G protein-coupled inwardly rectifying K(+) (GIRK) channels; however, this response is only observed on proestrus. While the cellular mechanisms that allow for functional expression of this unique DA-signaling pathway are unclear, we hypothesized that activation of the DA-GIRK effector pathway is due to the rise in circulating estrogen (E₂) during the preceding day of diestrus. Thus, we examined the effects of E₂ on primary lactotrophs isolated from female rats. Treatment with a physiological concentration of E₂ (40-80 pg/ml, in vivo or in vitro) induced a proestrous phenotype in diestrous lactotrophs. These cells exhibited a DA-induced membrane hyperpolarization, as well as a secretory rebound of PRL following DA withdrawal (characteristic of proestrous cells). Internal dialysis of GTPγS demonstrated that E₂ exposure enabled functional expression of GIRK channels, and this regulation by E₂ did not involve the D₂R. The effect of E₂ was blocked by the receptor antagonist, ICI 182,780, and by the protein synthesis inhibitor, cycloheximide. Single-cell analysis revealed increased mRNA expression of GIRK channel subunits in E₂-treated lactotrophs. While E₂ is known to have multiple actions on the lactotroph, the present findings illuminate a novel action of E₂ in lactotrophs-regulation of the expression of a DA effector, the GIRK channel.

Mice treated with a benzodiazepine had an improved survival rate following Pseudomonas aeruginosa infection.

Psychological stress has a high incidence after burn injury, therefore, anxiolytic drugs are often prescribed. Unfortunately, to date, no burn study has investigated the effects of anxiolytic drugs on the ability to fight infection. This study was undertaken to determine if psychological stress, anxiety-modulating drugs, or both, alter survival following an infection. On day 0, 7-week-old male C57Bl/6 mice either received a 15% full-thickness flame burn or were sham treated (anesthesia and shaved), whereas controls received no treatment. Mice received midazolam (1 mg/kg intraperitoneally) or saline daily and were stressed by exposure to rat in a guinea pig cage or placed in an empty cage for 1 hour a day, beginning on postburn day 1. For the survival experiments, mice either received bacteria after 2 or 8 consecutive days of predator exposure and drug treatment, which continued daily for 7 days after inoculation. In a separate set of experiments, after eight daily injections of midazolam, mice were given lipopolysaccharide, bacteria, or saline and were killed 12 hours later. Mice that received midazolam had improved survival rates when compared with their saline-treated counterparts, and the protective effect was more significant the more days they received the drug. For most of the cytokines, the bacteria-induced increase was significantly attenuated by midazolam as was the amount of bacteria in the liver. The protective effect seems to be independent of the drugs anxiolytic activity as there were no significant differences in survival between the predator-stressed and the nonstressed mice. The mechanisms responsible for the protective effect remain to be elucidated.

Functional expression of the dopamine-activated K(+) current in lactotrophs during the estrous cycle in female rats: correlation with prolactin secretory responses.

It is well established that hypothalamic dopamine (DA) is the major physiologic regulator of prolactin (PRL) secretion, exerting a tonic inhibition throughout most of the estrous cycle. A dramatic drop in the amount of DA perfusing the anterior pituitary occurs in the afternoon of proestrus and is critical for the production of the surge of PRL that occurs at that time. In my laboratory, we have identified and characterized a DA-activated K+ channel (KDA) in lactotrophs derived from proestrous rats that underlies DA-induced membrane hyperpolarization of lactotrophs. We have also demonstrated that this hyperpolarization plays a critical role in both the inhibition of PRL release from proestrous cells and the PRL secretory rebound that occurs following DA withdrawal. We now report that the ability of DA to activate the KDA channel and elicit hyperpolarization in primary lactotrophs changes dramatically during the estrous cycle. Lactotrophs isolated from cycling female rats were studied using whole-cell voltage clamp. DA (1 µM) elicited a robust membrane K+ current in the majority of proestrous lactotrophs (86%; 24.0 ± 2.9 pA). By contrast, DA activated a considerably smaller membrane current (3.3 pA) in very few lactotrophs isolated from rats on either diestrus or estrus (8 and 0%, respectively). Using a perifusion system to examine temporal patterns of PRL release, we found that following application and withdrawal of DA, proestrous cells produced a robust secretory rebound, but diestrous and estrous cells did not. However, DA inhibited PRL release to the same extent regardless the stage of the cycle from which the cells were derived. These data are consistent with the presence of multiple DA effectors in lactotrophs and demonstrate that their relative importance shifts dramatically with changes in the endocrine status of the animal. We propose that the DA-activated K+ channel (KDA) is a critical effector governing the unique secretory profile of PRL observed in proestrous animals.

Enhanced Responsiveness to Selective Serotonin Reuptake Inhibitors during Lactation

The physiology of mood regulation in the postpartum is poorly understood despite the fact that postpartum depression (PPD) is a common pathology. Serotonergic mechanisms and their dysfunction are widely presumed to be involved, which has led us to investigate whether lactation induces changes in central or peripheral serotonin (5-HT) systems and related affective behaviors. Brain sections from lactating (day 10 postpartum) and age-matched nulliparous (non-pregnant) C57BL/6J mice were processed for 5-HT immunohistochemistry. The total number of 5-HT immunostained cells and optical density were measured. Lactating mice exhibited lower immunoreactive 5-HT and intensity in the dorsal raphe nucleus when compared with nulliparous controls. Serum 5-HT was quantified from lactating and nulliparous mice using radioimmunoassay. Serum 5-HT concentrations were higher in lactating mice than in nulliparous controls. Affective behavior was assessed in lactating and non-lactating females ten days postpartum, as well as in nulliparous controls using the forced swim test (FST) and marble burying task (MBT). Animals were treated for the preceding five days with a selective serotonin reuptake inhibitor (SSRI, citalopram, 5mg/kg/day) or vehicle. Lactating mice exhibited a lower baseline immobility time during the FST and buried fewer marbles during the MBT as compared to nulliparous controls. Citalopram treatment changed these behaviors in lactating mice with further reductions in immobility during the FST and decreased marble burying. In contrast, the same regimen of citalopram treatment had no effect on these behaviors in either non-lactating postpartum or nulliparous females. Our findings demonstrate changes in both central and peripheral 5-HT systems associated with lactation, independent of pregnancy. They also demonstrate a significant interaction of lactation and responsiveness to SSRI treatment, which has important implications in the treatment of PPD. Although recent evidence has cast doubt on the effectiveness of SSRIs, these results support their therapeutic use in the treatment of PPD.

Stress and prolactin effects on bone marrow myeloid cells, serum chemokine and serum glucocorticoid levels in mice.

Objective: Current evidence supports the conclusion that prolactin (PRL) is not an obligate immunoregulatory hormone and influences the immune system predominantly during stress conditions. In this study, we examined the impact of PRL on the psychogenic stress-induced responses of myeloid cells. Methods: Seven-week-old PRL+/– (normal) and PRL–/– (deficient) mice were exposed to a predator for 1 h/day on 3 consecutive days. Another group of PRL-deficient mice received either 1 pituitary graft (hyperprolactinemic) or sham surgery at 5 weeks of age, while PRL-normal mice only received sham surgery. Two weeks later, these mice were also subjected to predator exposure. One day after the last predator exposure session, all mice were killed and the bone marrow and blood harvested. Results: Significant differences in the myeloid cells between PRL-normal and PRL-deficient mice only occurred in stressed conditions. The median serum corticosterone levels were consistently higher in PRL-deficient mice. The implantation of a pituitary graft lowered the corticosterone levels to those observed in PRL-normal mice. The absolute number of immature neutrophils as well as the numbers of granulocyte macrophage, monocyte/macrophage and granulocyte colonies were significantly higher in the stressed PRL-deficient mice; however, only the increased number of immature neutrophils was reversed by pituitary grafting. Conclusions: Our findings support previous observations that PRL influences myeloid cells of the bone marrow most profoundly in stressed conditions. However, the mechanism by which PRL influences bone marrow myeloid cells during stress cannot be explained solely by its effect on serum corticosterone.

CHAPTER 32 – Prolactin: Structure, Function, and Regulation of Secretion

This chapter discusses the structure, functions, and regulation of secretion of prolactin (PRL). Prolactin (PRL)—a protein hormone secreted from the anterior pituitary gland—has been identified in all vertebrate classes. It has numerous and diverse biological functions, including water and electrolyte balance, growth and development, metabolism and endocrine regulation, behavior, immune function, and reproduction. The amino acid sequence and tertiary structure of PRL is closely related to that of growth hormone (GH), also secreted from the anterior pituitary gland. PRL secretion is regulated in a complex manner by a variety of hormones and neurotransmitters and is influenced by photoperiod, sleep patterns, and stress. In nonmammalian vertebrates, the principal form of regulatory input is stimulatory with specific releasing factors (PRL-releasing factors [PRFs]) required for PRL secretion. In addition, there exists an extensive list of factors that demonstrate a stimulatory action on acute PRL release, and the episodic release of pituitary PRL. However, none of these releasing factors has emerged as a required input for normal PRL secretion.

Characterization of mammary-specific disruptions for Tph1 and Lrp5 during murine lactation

Serotonin is a homeostatic regulator of the mammary gland during lactation. The contribution of mammary-derived serotonin to circulating serum serotonin concentrations was previously unknown. We have developed mice with mammary-specific disruptions of tryptophan hydroxylase 1 (Tph1) or low-density lipoprotein receptor-related protein 5 (Lrp5) that are induced during late pregnancy and lactation via use of the whey acidic protein (WAP)-Cre cre-lox system. Our objective was to characterize dams with a lactation- and mammary-specific disruption of Lrp5 (WAP-Cre × Lrp5FL/FL) or Tph1 (WAP-Cre × Tph1FL/FL). Milk yield and pup weights were recorded throughout lactation. Dams were euthanized on d10 postpartum and mammary glands and duodenal tissue were harvested. WAP-Cre × Lrp5FL/FL dams had elevated serotonin concentrations in both the mammary gland and circulation compared to controls. In contrast, WAP-Cre × Tph1FL/FL dams had decreased mammary gland and serum serotonin concentrations compared to controls. Alveolar morphology, milk yield, and pup weights were similar. Mammary-derived serotonin makes a significant contribution to circulating serotonin concentrations during lactation, with no effect on milk yield or alveolar morphology. These transgenic models can and should be confidently used in future lactation studies to further elucidate the contribution of serotonin to the maintenance of lactation.

The voltage sensor of ion channels revealed

Voltage-gated ion channels control the excitability of nerve, muscle, endocrine and other cell types, thereby regulating a broad spectrum of cellular processes. How these channels sense changes in the potential across the membrane has remained a mystery for decades. The long-awaited crystal structure of a voltage-gated ion channel was recently published by Jiang et al., revealing an unexpected and surprisingly simple mechanism of voltage sensing and gating in these channels.