Rebecca A. Alyea

Differential regulation of dopamine transporter function and location by low concentrations of environmental estrogens and 17β-estradiol

Background The effects of 17β-estradiol (E2) and xenoestrogens (XEs) on dopamine transport may have important implications for the increased incidence of neurologic disorders, especially in women during life stages characterized by frequent hormonal fluctuations. Objective We examined low concentrations of XEs [dieldrin, endosulfan, o′, p′-dichlorodiphenyl-ethylene (DDE), nonylphenol (NP), and bisphenol A (BPA)] for nongenomic actions via action of membrane estrogen receptors (ERs). Methods We measured activity of the dopamine transporter (DAT) by the efflux of 3H-dopamine in nontransfected nerve growth factor–differentiated PC12 rat pheochromocytoma cells expressing membrane DAT, ER-α, ER-β, and G-protein–coupled receptor 30. We used a plate immunoassay to monitor trafficking of these proteins. Results All compounds at 1 nM either caused efflux or inhibited efflux, or both; each compound evoked a distinct oscillatory pattern. At optimal times for each effect, we examined different concentrations of XEs. All XEs were active at some concentration < 10 nM, and dose responses were all nonmonotonic. For example, 10−14 to 10−11 M DDE caused significant efflux inhibition, whereas NP and BPA enhanced or inhibited efflux at several concentrations. We also measured the effects of E2/XE combinations; DDE potentiated E2-mediated dopamine efflux, whereas BPA inhibited it. In E2-induced efflux, 15% more ER-α trafficked to the membrane, whereas ER-β waned; during BPA-induced efflux, 20% more DAT was trafficked to the plasma membrane. Conclusions Low levels of environmental estrogen contaminants acting as endocrine disruptors via membrane ERs can alter dopamine efflux temporal patterning and the trafficking of DAT and membrane ERs, providing a cellular mechanism that could explain the disruption of physiologic neurotransmitter function.

Xenoestrogens are potent activators of nongenomic estrogenic responses

Studies of the nuclear transcriptional regulatory activities of non-physiological estrogens have not explained their actions in mediating endocrine disruption in animals and humans at the low concentrations widespread in the environment. However, xenoestrogens have rarely been tested for their ability to participate in the plethora of nongenomic steroid signaling pathways elucidated over the last several years. Here we review what is known about such responses in comparison to our recent evidence that xenoestrogens can rapidly and potently elicit signaling through nongenomic pathways culminating in functional endpoints. Both estradiol (E(2)) and compounds representing various classes of xenoestrogens (diethylstilbestrol, coumestrol, bisphenol A, DDE, nonylphenol, endosulfan, and dieldrin) act via a membrane version of the estrogen receptor-alpha on pituitary cells, and can provoke Ca(2+) influx via L-type channels, leading to prolactin (PRL) secretion. These hormones and mimetics can also cause the oscillating activation of extracellular regulated kinases (ERKs). However, individual estrogen mimetics differ in their potency and temporal phasing of these activations compared to each other and to E(2). It is perhaps in these ways that they disrupt some endocrine functions when acting in combination with physiological estrogens. Our quantitative assays allow comparison of these outcomes for each mimetic, and let us build a detailed picture of alternative signaling pathway usage. Such an understanding should allow us to determine the estrogenic or antiestrogenic potential of different types of xenoestrogens, and help us to develop strategies for preventing xenoestrogenic disruption of estrogen action in many tissues.

Nongenomic Actions of Low Concentration Estrogens and Xenoestrogens on Multiple Tissues

Nongenomic estrogenic mechanisms offer an opportunity to explain the conundrum of environmental estrogen and plant estrogen effects on cells and animals at the very low concentrations which are prevalent in our environments and diets. Heretofore the actions of these compounds have not been adequately accounted for by laboratory tests utilizing assays for actions only via the genomic pathway of steroid action and the nuclear forms of estrogen receptor alpha and beta. Membrane versions of these receptors, and the newly described GPR30 (7TMER) receptor protein provide explanations for the more potent actions of xenoestrogens. The effects of estrogens on many tissues demand a comprehensive assessment of the receptors, receptor levels, and mechanisms that might be involved, to determine which of these estrogen mimetic compounds are harmful and which might even be used therapeutically, depending upon the life stage at which we are exposed to them.

The roles of membrane estrogen receptor subtypes in modulating dopamine transporters in PC-12 cells

The effects of 17β‐estradiol (E2) on dopamine (DA) transport could explain gender and life‐stage differences in the incidence of some neurological disorders. We tested the effects of E2 at physiological concentrations on DA efflux in nerve growth factor‐differentiated rat pheochromocytoma cells that express estrogen receptors (ER) α, ERβ, and G‐protein coupled receptor 30 (GPR30), and DA transporter (DAT). DAT efflux was determined as the transporter‐specific loss of 3H‐DA from pre‐loaded cells; a 9–15 min 10−9 M E2 treatment caused maximal DA efflux. Such rapid estrogenic action suggests a non‐genomic response, and an E2‐dendrimer conjugate (limited to non‐nuclear actions) caused DA efflux within 5 min. Efflux dose–responses for E2 were non‐monotonic, also characteristic of non‐genomic estrogenic actions. ERα siRNA knockdown abolished E2‐mediated DA efflux, while ERβ knockdown did not, and GPR30 knockdown increased E2‐mediated DA efflux (suggesting GPR30 is inhibitory). Use of ER‐selective agonists/antagonists demonstrated that ERα is the predominant mediator of E2‐mediated DA efflux, with inhibitory contributions from GPR30 and ERβ. E2 also caused trafficking of ERα to the plasma membrane, trafficking of ERβ away from the plasma membrane, and unchanged membrane GPR30 levels. Therefore, ERα is largely responsible for non‐genomic estrogenic effects on DAT activity.

Surface-Expressed Enolase Contributes to the Pathogenesis of Clinical Isolate SSU of Aeromonas hydrophila

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU of Aeromonas hydrophila bound to human plasminogen and facilitated the latters tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease alpha(2)-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT) A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability of A. hydrophila SSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied in trans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase in A. hydrophila SSU; the mutated forms of enolase were hyperexpressed in Escherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues ((252)FYDAEKKEY(260)) in the A. hydrophila SSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis of A. hydrophila SSU infections and of any gram-negative bacteria in general.

Conserved estrogen binding and signaling functions of the G protein-coupled estrogen receptor 1 (GPER) in mammals and fish

Recent studies by several research groups have shown that G protein estrogen receptor-1 (GPER) formerly known as GPR30, mediates 17beta-estradiol (E2) activation of signal transduction pathways in a variety of human cancer cells and displays E2 binding typical of a membrane estrogen receptor. However, the importance of GPER as an estrogen receptor has been questioned by Otto and co-workers. Some of the pitfalls in investigating the functions of recombinant steroid membrane receptors that may explain the negative results of these investigators are discussed. The characteristics of GPER have also been investigated in a teleost fish, Atlantic croaker, where it has been shown to mediate E2 inhibition of oocyte maturation. Investigations on newly discovered homologous proteins from distantly related vertebrate groups are valuable for determining their fundamental, evolutionarily conserved functions. Therefore, the functions of croaker and human GPERs were compared. The comparisons show that croaker and human GPER have very similar estrogen binding characteristics, typical of estrogen membrane receptors, and activate the same estrogen signaling pathways via stimulatory G proteins (Gs) resulting in increased cAMP production. These results suggest that the estrogen binding and estrogen signaling functions of GPER arose early in vertebrate evolution, prior to the divergence of the teleosts from the tetrapods, more than 200 million years ago. The finding that estrogen membrane signaling through GPER has been conserved for such a long period in two distantly related vertebrate groups, mammals and fish, suggests that this is a fundamental function of GPER in vertebrates, and likely its major physiological role.

Estradiol effects on the dopamine transporter – protein levels, subcellular location, and function

Background The effects of estrogens on dopamine (DA) transport may have important implications for the increased incidence of neurological disorders in women during life stages when hormonal fluctuations are prevalent, e.g. during menarche, reproductive cycling, pregnancy, and peri-menopause. Results The activity of the DA transporter (DAT) was measured by the specific uptake of 3H-DA. We found that low concentrations (10-14 to 10-8 M) of 17β-estradiol (E2) inhibit uptake via the DAT in PC12 cells over 30 minutes, with significant inhibition taking place due to E2 exposure during only the last five minutes of the uptake period. Such rapid action suggests a non-genomic, membrane-initiated estrogenic response mechanism. DAT and estrogen receptor-α (ERα) were elevated in cell extracts by a 20 ng/ml 2 day NGFβ treatment, while ERβ was not. DAT, ERα and ERβ were also detectable on the plasma membrane of unpermeabilized cells by immunocytochemical staining and by a fixed cell, quantitative antibody (Ab)-based plate assay. In addition, PC12 cells contained RNA coding for the alternative membrane ER GPR30; therefore, all 3 ER subtypes are candidates for mediating the rapid nongenomic actions of E2. At cell densities above 15,000 cells per well, the E2-induced inhibition of transport was reversed. Uptake activity oscillated with time after a 10 nM E2 treatment; in a slower room temperature assay, inhibition peaked at 9 min, while uptake activity increased at 3 and 20–30 min. Using an Ab recognizing the second extracellular loop of DAT (accessible only on the outside of unpermeabilized cells), our immunoassay measured membrane vs. intracellular/nonvesicular DAT; both were found to decline over a 5–60 min E2 treatment, though immunoblot analyses demonstrated no total cellular loss of protein. Conclusion Our results suggest that physiological levels of E2 may act to sequester DAT in intracellular compartments where the transporters second extramembrane loop is inaccessible (inside vesicles) and that rapid estrogenic actions on this differentiated neuronal cell type may be regulated via membrane ERs of several types.

Nongenomic mechanisms of physiological estrogen-mediated dopamine efflux

BackgroundNeurological diseases and neuropsychiatric disorders that vary depending on female life stages suggest that sex hormones may influence the function of neurotransmitter regulatory machinery such as the dopamine transporter (DAT).ResultsIn this study we tested the rapid nongenomic effects of several physiological estrogens [estradiol (E2), estrone (E1), and estriol (E3)] on dopamine efflux via the DAT in a non-transfected, NGF-differentiated, rat pheochromocytoma (PC12) cell model that expresses membrane estrogen receptors (ERs) α, β, and GPR30. We examined kinase, ionic, and physical interaction mechanisms involved in estrogenic regulation of the DAT function. E2-mediated dopamine efflux is DAT-specific and not dependent on extracellular Ca2+-mediated exocytotic release from vesicular monoamine transporter vesicles (VMATs). Using kinase inhibitors we also showed that E2-mediated dopamine efflux is dependent on protein kinase C and MEK activation, but not on PI3K or protein kinase A. In plasma membrane there are ligand-independent associations of ERα and ERβ (but not GPR30) with DAT. Conditions which cause efflux (a 9 min 10-9 M E2 treatment) cause trafficking of ERα (stimulatory) to the plasma membrane and trafficking of ERβ (inhibitory) away from the plasma membrane. In contrast, E1 and E3 can inhibit efflux with a nonmonotonic dose pattern, and cause DAT to leave the plasma membrane.ConclusionSuch mechanisms explain how gender biases in some DAT-dependent diseases can occur.

Estrogens of multiple classes and their role in mental health disease mechanisms

Gender and sex hormones can influence a variety of mental health states, including mood, cognitive development and function, and vulnerability to neurodegenerative diseases and brain damage. Functions of neuronal cells may be altered by estrogens depending upon the availability of different physiological estrogenic ligands; these ligands and their effects vary with life stages, the genetic or postgenetic regulation of receptor levels in specific tissues, or the intercession of competing nonphysiological ligands (either intentional or unintentional, beneficial to health or not). Here we review evidence for how different estrogens (physiological and environmental/dietary), acting via different estrogen receptor subtypes residing in alternative subcellular locations, influence brain functions and behavior. We also discuss the families of receptors and transporters for monoamine neurotransmitters and how they may interact with the estrogenic signaling pathways.

Multi-well plate immunoassays for measuring signaling protein activations/deactivations and membrane vs. intracellular receptor levels.

We developed fixed-cell multi-well plate immunoassays that increase the throughput and ease of quantification for questions formerly assessed by immunoblot scanning. The assays make use of the now abundant antibodies designed to recognize receptor subtypes and posttranslationally modified signaling proteins. By optimizing permeabilization and fixation conditions, mainly based on specific cell types, the assay can be adapted to the study of many different antigens of importance to hormonal and neurotransmitter signaling scenarios.