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.

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.

Signaling from the membrane via membrane estrogen receptor-alpha: estrogens, xenoestrogens, and phytoestrogens.

Estrogen mimetics in the environment and in foods can have important consequences for endocrine functions. When previously examined for action via genomic steroid signaling mechanisms, most of these compounds were found to be very weak agonists. We have instead tested their actions via several membrane-initiated signaling mechanisms in GH3/B6 pituitary tumor cells extensively selected for high (responsive) or low (nonresponsive) expression of the membrane version of estrogen receptor-alpha (mERalpha).

Xenoestrogens at picomolar to nanomolar concentrations trigger membrane estrogen receptor-alpha-mediated Ca2+ fluxes and prolactin release in GH3/B6 pituitary tumor cells.

Xenoestrogens (XEs) are widespread in our environment and are known to have deleterious effects in animal (and perhaps human) populations. Acting as inappropriate estrogens, XEs are thought to interfere with endogenous estrogens such as estradiol (E2) to disrupt normal estrogenic signaling. We investigated the effects of E2 versus several XEs representing organochlorine pesticides (dieldrin, endosulfan, o',p'-dichlorodiphenylethylene), plastics manufacturing by-products/detergents (nonylphenol, bisphenol A), a phytoestrogen (coumestrol), and a synthetic estrogen (diethylstilbestrol) on the pituitary tumor cell subline GH3/B6/F10, previously selected for expression of high levels of membrane estrogen receptor-alpha.

Xenoestrogen-induced ERK-1 and ERK-2 activation via multiple membrane-initiated signaling pathways.

Xenoestrogens can mimic or antagonize the activity of physiological estrogens, and the suggested mechanism of xenoestrogen action involves binding to estrogen receptors (ERs). However, the failure of various in vitro or in vivo assays to show strong genomic activity of xenoestrogens compared with estradiol (E2) makes it difficult to explain their ability to cause abnormalities in animal (and perhaps human) reproductive functions via this pathway of steroid action. E2 has also been shown to initiate rapid intracellular signaling, such as changes in levels of intracellular calcium, cAMP, and nitric oxide, and activations of a variety of kinases, via action at the membrane.

Mechanisms of membrane estrogen receptor-alpha-mediated rapid stimulation of Ca2+ levels and prolactin release in a pituitary cell line.

The role of membrane estrogen receptor-alpha (mERalpha) in rapid nongenomic responses to 17beta-estradiol (E(2)) was tested in sublines of GH3/B6 rat prolactinoma cells selected for high (GH3/B6/F10) and low (GH3/B6/D9) mERalpha expression. E(2) elicited rapid, concentration-dependent intracellular Ca(2+) concentration ([Ca(2+)](i)) increases in the F10 subline. Lack of inhibition by thapsigargin depletion of intracellular Ca(2+) pools, together with abrogation of the response in Ca(2+)-free medium, suggested an extracellular source of Ca(2+) for this response.

Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways.

Estradiol (E2) and other steroids have recently been shown to initiate various intracellular signaling cascades from the plasma membrane, including those stimulating mitogen-activated protein kinases (MAPKs), and particularly extracellular-regulated kinases (ERKs). In this study we demonstrated the ability of E2 to activate ERKs in the GH3/B6/F10 pituitary tumor cell line, originally selected for its enhanced expression of membrane estrogen receptor-alpha (mERalpha). We compared E2 to its cell-impermeable analog (E2 conjugated to peroxidase, E2-P), and to the synthetic estrogen diethylstilbestrol (DES).

Cell alkalosis elevates cytosolic Ca2+ in rabbit resident alveolar macrophages.

Disruption of cellular acid-base status alters the host defence functions of alveolar macrophages (m phi). These pH effects might be mediated by pH-sensitive changes in the signalling pathways of the effector functions of m phi. The present study examined the effects of intracellular pH (pH(i)) on the free cytosolic calcium concentration ([Ca(2+)](i)), an important second messenger for cell functions.