G protein-coupled estrogen receptor activation by bisphenol-A disrupts the protection from apoptosis conferred by the estrogen receptors ERα and ERß in pancreatic beta cells.

17ß-estradiol protects pancreatic ß-cells from apoptosis via the estrogen receptors ERα, ERß and GPER. Conversely, the endocrine disruptor bisphenol-A (BPA), which exerts multiple effects in this cell type via the same estrogen receptors, increased basal apoptosis. The molecular-initiated events that trigger these opposite actions have yet to be identified. We demonstrated that combined genetic downregulation and pharmacological blockade of each estrogen receptor increased apoptosis to a different extent. The increase in apoptosis induced by BPA was diminished by the pharmacological blockade or the genetic silencing of GPER, and it was partially reproduced by the GPER agonist G1. BPA and G1-induced apoptosis were abolished upon pharmacological inhibition, silencing of ERα and ERß, or in dispersed islet cells from ERß knockout (BERKO) mice. However, the ERα and ERß agonists PPT and DPN, respectively, had no effect on beta cell viability. To exert their biological actions, ERα and ERß form homodimers and heterodimers. Molecular dynamics simulations together with proximity ligand assays and coimmunoprecipitation experiments indicated that the interaction of BPA with ERα and ERß as well as GPER activation by G1 decreased ERαß heterodimers. We propose that ERαß heterodimers play an antiapoptotic role in beta cells and that BPA- and G1-induced decreases in ERαß heterodimers lead to beta cell apoptosis. Unveiling how different estrogenic chemicals affect the crosstalk among estrogen receptors should help to identify diabetogenic endocrine disruptors.

A novel mitochondrial Kv1.3-caveolin axis controls cell survival and apoptosis.

The voltage-gated potassium channel Kv1.3 plays an apparent dual physiological role by participating in activation and proliferation of leukocytes as well as promoting apoptosis in several types of tumor cells. Therefore, Kv1.3 is considered a potential pharmacological target for immunodeficiency and cancer. Different cellular locations of Kv1.3, at the plasma membrane or the mitochondria, could be responsible for such duality. While plasma membrane Kv1.3 facilitates proliferation, the mitochondrial channel modulates apoptotic signaling. Several molecular determinants of Kv1.3 drive the channel to the cell surface, but no information is available about its mitochondrial targeting. Caveolins, which are able to modulate cell survival, participate in the plasma membrane targeting of Kv1.3. The channel, via a caveolin-binding domain (CDB), associates with caveolin 1 (Cav1), which localizes Kv1.3 to lipid raft membrane microdomains. The aim of our study was to understand the role of such interactions not only for channel targeting but also for cell survival in mammalian cells. By using a caveolin association-deficient channel (Kv1.3 CDBless), we demonstrate here that while the Kv1.3-Cav1 interaction is responsible for the channel localization in the plasma membrane, a lack of such interaction accumulates Kv1.3 in the mitochondria. Kv1.3 CDBless severely affects mitochondrial physiology and cell survival, indicating that a functional link of Kv1.3 with Cav1 within the mitochondria modulates the pro-apoptotic effects of the channel. Therefore, the balance exerted by these two complementary mechanisms fine-tune the physiological role of Kv1.3 during cell survival or apoptosis. Our data highlight an unexpected role for the mitochondrial caveolin-Kv1.3 axis during cell survival and apoptosis.

Physiology of pancreatic ß-cells: Ion channels and molecular mechanisms implicated in stimulus-secretion coupling.

The human and mouse islet of Langerhans is an endocrine organ composed of five different cells types; insulin-secreting ß-cells, glucagon-producing α-cells, somatostatin-producing δ-cells, pancreatic polypeptide-secreting PP cells and ɛ-cells that secretes ghrelin. The most important cells are the pancreatic ß-cells that comprise around 45-50% of human islets and 75-80% in the mouse. Pancreatic ß-cells secrete insulin at high glucose concentration, thereby finely regulating glycaemia by the hypoglycaemic effects of this hormone. Different ion channels are implicated in the stimulus-secretion coupling of insulin. An increase in the intracellular ATP concentration leads to closure KATP channels, depolarizing the cell and opening voltage-gated calcium channels. The increase of intracellular calcium concentration induced by calcium entry through voltage-gated calcium channels promotes insulin secretion. Here, we briefly describe the diversity of ion channels present in pancreatic ß-cells and the different mechanisms that are responsible to induce insulin secretion in human and mouse cells. Moreover, we described the pathophysiology due to alterations in the physiology of the main ion channels present in pancreatic ß-cell and its implication to predispose metabolic disorders as type 2 diabetes mellitus.

Bisphenol-S and Bisphenol-F alter mouse pancreatic ß-cell ion channel expression and activity and insulin release through an estrogen receptor ERß mediated pathway.

Bisphenol-S (BPS) and Bisphenol-F (BPF) are current Bisphenol-A (BPA) substitutes. Here we used pancreatic ß-cells from wild type (WT) and estrogen receptor ß (ERß) knockout (BERKO) mice to investigate the effects of BPS and BPF on insulin secretion, and the expression and activity of ion channels involved in ß-cell function. BPS or BPF rapidly increased insulin release and diminished ATP-sensitive K+ (KATP) channel activity. Similarly, 48 h treatment with BPS or BPF enhanced insulin release and decreased the expression of several ion channel subunits in ß-cells from WT mice, yet no effects were observed in cells from BERKO mice. PaPE-1, a ligand designed to preferentially trigger extranuclear-initiated ER pathways, mimicked the effects of bisphenols, suggesting the involvement of extranuclear-initiated ERß pathways. Molecular dynamics simulations indicated differences in ERß ligand-binding domain dimer stabilization and solvation free energy among different bisphenols and PaPE-1. Our data suggest a mode of action involving ERß whose activation alters three key cellular events in ß-cell, namely ion channel expression and activity, and insulin release. These results may help to improve the hazard identification of bisphenols.

Bisphenol A Regulates Sodium Ramp Currents in Mouse Dorsal Root Ganglion Neurons and Increases Nociception.

17ß-Estradiol mediates the sensitivity to pain and is involved in sex differences in nociception. The widespread environmental disrupting chemical bisphenol A (BPA) has estrogenic activity, but its implications in pain are mostly unknown. Here we show that treatment of male mice with BPA (50 µg/kg/day) during 8 days, decreases the latency to pain behavior in response to heat, suggesting increased pain sensitivity. We demonstrate that incubation of dissociated dorsal root ganglia (DRG) nociceptors with 1 nM BPA increases the frequency of action potential firing. SCN9A encodes the voltage-gated sodium channel Nav1.7, which is present in DRG nociceptors and is essential in pain signaling. Nav1.7 and other voltage-gated sodium channels in mouse DRG are considered threshold channels because they produce ramp currents, amplifying small depolarizations and enhancing electrical activity. BPA increased Nav-mediated ramp currents elicited with slow depolarizations. Experiments using pharmacological tools as well as DRG from ERß-/- mice indicate that this BPA effect involves ERα and phosphoinositide 3-kinase. The mRNA expression and biophysical properties other than ramp currents of Nav channels, were unchanged by BPA. Our data suggest that BPA at environmentally relevant doses affects the ability to detect noxious stimuli and therefore should be considered when studying the etiology of pain conditions.

Oestrogen receptor ß mediates the actions of bisphenol-A on ion channel expression in mouse pancreatic beta cells.

AIMS/HYPOTHESIS: Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical that has been associated with type 2 diabetes development. Low doses of BPA modify pancreatic beta cell function and induce insulin resistance; some of these effects are mediated via activation of oestrogen receptors α (ERα) and ß (ERß). Here we investigated whether low doses of BPA regulate the expression and function of ion channel subunits involved in beta cell function. METHODS: Microarray gene profiling of isolated islets from vehicle- and BPA-treated (100 µg/kg per day for 4 days) mice was performed using Affymetrix GeneChip Mouse Genome 430.2 Array. Expression level analysis was performed using the normalisation method based on the processing algorithm 'robust multi-array average'. Whole islets or dispersed islets from C57BL/6J or oestrogen receptor ß (ERß) knockout (Erß-/-) mice were treated with vehicle or BPA (1 nmol/l) for 48 h. Whole-cell patch-clamp recordings were used to measure Na+ and K+ currents. mRNA expression was evaluated by quantitative real-time PCR. RESULTS: Microarray analysis showed that BPA modulated the expression of 1440 probe sets (1192 upregulated and 248 downregulated genes). Of these, more than 50 genes, including Scn9a, Kcnb2, Kcnma1 and Kcnip1, encoded important Na+ and K+ channel subunits. These findings were confirmed by quantitative RT-PCR in islets from C57BL/6J BPA-treated mice or whole islets treated ex vivo. Electrophysiological measurements showed a decrease in both Na+ and K+ currents in BPA-treated islets. The pharmacological profile indicated that BPA reduced currents mediated by voltage-activated K+ channels (Kv2.1/2.2 channels) and large-conductance Ca2+-activated K+ channels (KCa1.1 channels), which agrees with BPA's effects on gene expression. Beta cells from ERß-/- mice did not present BPA-induced changes, suggesting that ERß mediates BPA's effects in pancreatic islets. Finally, BPA increased burst duration, reduced the amplitude of the action potential and enlarged the action potential half-width, leading to alteration in beta cell electrical activity. CONCLUSIONS/INTERPRETATION: Our data suggest that BPA modulates the expression and function of Na+ and K+ channels via ERß in mouse pancreatic islets. Furthermore, BPA alters beta cell electrical activity. Altogether, these BPA-induced changes in beta cells might play a role in the diabetogenic action of BPA described in animal models.

Extranuclear-initiated estrogenic actions of endocrine disrupting chemicals: Is there toxicology beyond paracelsus?

Endocrine Disrupting Chemicals (EDCs), including bisphenol-A (BPA) do not act as traditional toxic chemicals inducing massive cell damage or death in an unspecific manner. EDCs can work upon binding to hormone receptors, acting as agonists, antagonists or modulators. Bisphenol-A displays estrogenic activity and, for many years it has been classified as a weak estrogen, based on the classic transcriptional action of estrogen receptors serving as transcription factors. However, during the last two decades our knowledge about estrogen signaling has advanced considerably. It is now accepted that estrogen receptors ERα and ERß activate signaling pathways outside the nucleus which may or may not involve transcription. In addition, a new membrane estrogen receptor, GPER, has been proposed. Pharmacological and molecular evidence, along with results obtained in genetically modified mice, demonstrated that BPA, and its substitute BPS, are potent estrogens acting at nanomolar concentrations via extranuclear ERα, ERß, and GPER. The different signaling pathways activated by BPA and BPS explain the well-known estrogenic effects of low doses of EDCs as well as non-monotonic dose-response relationships. These signaling pathways may help to explain the actions of EDCs with estrogenic activity in the etiology of different pathologies, including type-2 diabetes and obesity.

Molecular mechanisms involved in the non-monotonic effect of bisphenol-a on ca2+ entry in mouse pancreatic ß-cells.

In regulatory toxicology, the dose-response relationship is a key element towards fulfilling safety assessments and satisfying regulatory authorities. Conventionally, the larger the dose, the greater the response, following the dogma "the dose makes the poison". Many endocrine disrupting chemicals, including bisphenol-A (BPA), induce non-monotonic dose response (NMDR) relationships, which are unconventional and have tremendous implications in risk assessment. Although several molecular mechanisms have been proposed to explain NMDR relationships, they are largely undemonstrated. Using mouse pancreatic ß-cells from wild-type and oestrogen receptor ERß-/- mice, we found that exposure to increasing doses of BPA affected Ca2+ entry in an NMDR manner. Low doses decreased plasma membrane Ca2+ currents after downregulation of Cav2.3 ion channel expression, in a process involving ERß. High doses decreased Ca2+ currents through an ERß-mediated mechanism and simultaneously increased Ca2+ currents via oestrogen receptor ERα. The outcome of both molecular mechanisms explains the NMDR relationship between BPA and Ca2+ entry in ß-cells.

Direct voltage control of endogenous lysophosphatidic acid G-protein-coupled receptors in Xenopus oocytes.

Lysophosphatidic acid (LPA) G-protein-coupled receptors (GPCRs) play important roles in a variety of physiological and pathophysiological processes, including cell proliferation, angiogenesis, central nervous system development and carcinogenesis. Whilst many ion channels and transporters are recognized to be controlled by a change in cell membrane potential, little is known about the voltage dependence of other proteins involved in cell signalling. Here, we show that the InsP(3)-mediated Ca(2+) response stimulated by the endogenous LPA GPCR in Xenopus oocytes is potentiated by membrane depolarization. Depolarization was able to repetitively stimulate transient [Ca(2+)](i) increases after the initial agonist-evoked response. In addition, the initial rate and amplitude of the LPA-dependent Ca(2+) response were significantly modulated by the steady holding potential over the physiological range, such that the response to LPA was potentiated at depolarized potentials and inhibited at hyperpolarized potentials. Enhancement of LPA receptor-evoked Ca(2+) mobilization by membrane depolarization was observed over a wide range of agonist concentrations. Importantly, the amplitude of the depolarization-evoked intracellular Ca(2+) increase displayed an inverse relationship with agonist concentration such that the greatest effect of voltage was observed at near-threshold levels of agonist. Voltage-dependent Ca(2+) release was not induced by direct elevation of InsP(3) or by activation of heterotrimeric G-proteins in the absence of agonist, indicating that the LPA GPCR itself represents the primary site of action of membrane voltage. This novel modulation of LPA signalling by membrane potential may have important consequences for control of Ca(2+) signals both in excitable and non-excitable tissues.

Structural and functional changes induced in the nicotinic acetylcholine receptor by membrane phospholipids.

Ligand-gated ion channels (LGICs) constitute an important family of complex membrane proteins acting as receptors for neurotransmitters (Barnard, 1992; Ortells and Lunt, 1995). The nicotinic acetylcholine receptor (nAChR) from Torpedo is the most extensively studied member of the LGIC family and consists of a pentameric transmembrane glycoprotein composed of four different polypeptide subunits (alpha, beta, gamma, and delta) in a 2:1:1:1 stoichiometry (Galzi and Changeux, 1995; Hucho et al., 1996) that are arranged pseudosymmetrically around a central cation-selective ion channel. Conformational transitions, from the closed (nonconducting), to agonist-induced open (ion-conducting), to desensitized (nonconducting) states, are critical for functioning of the nAChR (Karlin, 2002). The ability of the nAChR to undergo these transitions is profoundly influenced by the lipid composition of the bilayer (Barrantes, 2004). Despite existing information on lipid dependence of AChR function, no satisfactory explanation has been given on the molecular events by which specific lipids exert such effects on the activity of an integral membrane protein. To date, several hypotheses have been entertained, including (1) indirect effects of lipids through the alteration of properties of the bilayer, such as fluidity (an optimal fluidity hypothesis [Fong and McNamee, 1986]) or membrane curvature and lateral pressure (Cantor, 1997; de Kruijff, 1997), or (2) direct effects through binding of lipids to defined sites on the transmembrane portion of the protein (Jones and McNamee, 1988; Blanton and Wang, 1990; Fernández et al., 1993; Fernández-Ballester et al., 1994), which has led to the postulation of a possible role of certain lipids as peculiar allosteric ligands of the protein. In this paper we have reconstituted purified AChRs from Torpedo into complex multicomponent lipid vesicles in which the phospholipid composition has been systematically altered. Stopped-flow rapid kinetics of cation translocation and Fourier transform-infrared (FT-IR) spectroscopy studies have been used to illustrate the lipid dependence of both AChR function and AChR secondary structure, respectively.

Potentiation of P2Y receptors by physiological elevations of extracellular K+ via a mechanism independent of Ca2+ influx.

Many physiological and pathophysiological situations generate a significant increase in extracellular K+ concentration. This is known to influence a number of membrane conductances and exchangers, whereas direct effects of K+ on the activation of G protein-coupled receptors have not been reported. We now show that Ca2+ release evoked by P2Y1 receptors expressed in 1321-N1 astrocytoma cells is markedly potentiated by small increases in external K+ concentration. This effect was blocked by the phospholipase-C inhibitor U-73122 (1-[6-[[17 beta]-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), but not by its analog U-73343 (1-[6-[[17 beta]-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione), and not by nifedipine, Ni2+, Cd2+, or Gd3+. Thus, K+ enhances d-myo-inositol 1,4,5-trisphosphate-dependent Ca2+ release without a requirement for Ca2+ influx. The cation dependence of this effect displayed the order K+ > Rb+ > N-methyl-D-glucamine+, and Cs+ and choline+ were ineffective. The potentiation by K+ is half-maximal at an increase of 2.6 mM (total K+ of 7.6 mM). K+ caused a reduction in EC50 (2.7-fold for a 29 mM increase) without a change of slope; thus, the greatest effect was observed at near-threshold agonist levels. The response to K+ can be explained in part by depolarization-dependent potentiation of P2Y1 receptors [J Physiol (Lond) 555:61-70, 2004]. However, electrophysiological recordings of 1321-N1 cells and megakaryocytes demonstrated that K+ also amplifies ADP-evoked Ca2+ responses independently of changes in membrane potential. Elevated K+ also amplified endogenous UTP-dependent Ca2+ responses in human embryonic kidney 293 cells, suggesting that other P2Y receptors are K(+)-dependent. P2Y receptors display a widespread tissue distribution; therefore, their modulation by small changes in extracellular K+ may represent a novel means of autocrine and paracrine regulation of cellular activity.

Direct voltage control of signaling via P2Y1 and other Galphaq-coupled receptors.

Emerging evidence suggests that Ca2+ release evoked by certain G-protein-coupled receptors can be voltage-dependent; however, the relative contribution of different components of the signaling cascade to this response remains unclear. Using the electrically inexcitable megakaryocyte as a model system, we demonstrate that inositol 1,4,5-trisphosphate-dependent Ca2+ mobilization stimulated by several agonists acting via Galphaq-coupled receptors is potentiated by depolarization and that this effect is most pronounced for ADP. Voltage-dependent Ca2+ release was not induced by direct elevation of inositol 1,4,5-trisphosphate, by agents mimicking diacylglycerol actions, or by activation of phospholipase Cgamma-coupled receptors. The response to voltage did not require voltage-gated Ca2+ channels as it persisted in the presence of nifedipine and was only weakly affected by the holding potential. Strong predepolarizations failed to affect the voltage-dependent Ca2+ increase; thus, an alteration of G-protein betagamma subunit binding is also not involved. Megakaryocytes from P2Y1(-/-) mice lacked voltage-dependent Ca2+ release during the application of ADP but retained this response after stimulation of other Galphaq-coupled receptors. Although depolarization enhanced Ca2+ mobilization resulting from GTPgammaS dialysis and to a lesser extent during AlF4- or thimerosal, these effects all required the presence of P2Y1 receptors. Taken together, the voltage dependence to Ca2+ release via Galphaq-coupled receptors is not due to control of G-proteins or down-stream signals but, rather, can be explained by a voltage sensitivity at the level of the receptor itself. This effect, which is particularly robust for P2Y1 receptors, has wide-spread implications for cell signaling.