Seafinding revisited: how hatchling marine turtles respond to natural lighting at a nesting beach.

Hatchling marine turtles emerge at night from underground nests on oceanic beaches and then use visual cues to crawl from the nest site to the sea ("seafinding"). However, the light wavelengths (λ's) used to accomplish this orientation have not been thoroughly documented, nor do we understand why some λ's are favored over others. We measured nocturnal radiance on the horizon at 20 nm intervals between 340 and 600 nm at two nesting beach sites and then, under laboratory conditions, determined the lowest intensities of those λ's that induced green turtle and loggerhead hatchlings to crawl toward each light source (a low positive "phototaxis threshold"). Both species were similarly sensitive and were attracted to all λ's. Radiance measures at all λ's were greater toward the seaward horizon than toward the landward horizon, providing an important orientation cue regardless of variation in lunar illumination. Previous studies document that both species detect λ's longer than those that are most attractive. We hypothesize that seafinding is a specialized response mediated by cones that are sensitive to the shorter λ's (to minimize the effects of dark noise) but such as rods, are especially sensitive to low levels of nocturnal illumination.

A Selective Ligand for Estrogen Receptor Proteins Discriminates Rapid and Genomic Signaling.

Estrogen exerts extensive and diverse effects throughout the body of women. In addition to the classical nuclear estrogen receptors (ERα and ERß), the G protein-coupled estrogen receptor GPER is an important mediator of estrogen action. Existing ER-targeted therapeutic agents act as GPER agonists. Here, we report the identification of a small molecule, named AB-1, with the previously unidentified activity of high selectivity for binding classical ERs over GPER. AB-1 also possesses a unique functional activity profile as an agonist of transcriptional activity but an antagonist of rapid signaling through ERα. Our results define a class of small molecules that discriminate between the classical ERs and GPER, as well as between modes of signaling within the classical ERs. Such an activity profile, if developed into an ER antagonist, could represent an opportunity for the development of first-in-class nuclear hormone receptor-targeted therapeutics for breast cancer exhibiting reduced acquired and de novo resistance.

GPER regulates endothelin-dependent vascular tone and intracellular calcium.

AIMS: An increase in intracellular vascular smooth muscle cell calcium concentration (VSMC [Ca(2+)](i)) is essential for endothelin-1 (ET-1)-induced vasoconstriction. Based on previous findings that activation of the G protein-coupled estrogen receptor (GPER) inhibits vasoconstriction in response to ET-1 and regulates [Ca(2+)](i) in cultured VSMC, we investigated whether endogenous GPER regulates ET-1-induced changes in VSMC [Ca(2+)](i) and constriction of intact arteries. MAIN METHODS: Pressurized carotid arteries of GPER-deficient (GPER(0)) and wildtype (WT) mice were loaded with the calcium indicator fura 2-AM. Arteries were stimulated with the GPER-selective agonist G-1 or solvent followed by exposure to ET-1. Changes in arterial diameter and VSMC [Ca(2+)](i) were recorded simultaneously. Vascular gene expression levels of ET(A) and ET(B) receptors were determined by qPCR. KEY FINDINGS: ET-1-dependent vasoconstriction was increased in arteries from GPER(0) compared to arteries from WT mice. Despite the more potent vasoconstriction to ET-1, GPER deficiency was associated with a marked reduction in the ET-1-stimulated VSMC [Ca(2+)](i) increase, suggesting an increase in myofilament force sensitivity to [Ca(2+)](i). Activation of GPER by G-1 had no effect on vasoconstriction or VSMC [Ca(2+)](i) responses to ET-1, and expression levels of ET(A) or ET(B) receptor were unaffected by GPER deficiency. SIGNIFICANCE: These results demonstrate that endogenous GPER inhibits ET-1-induced vasoconstriction, an effect that may be associated with reduced VSMC Ca(2+) sensitivity. This represents a potential mechanism through which GPER could contribute to protective effects of endogenous estrogen in the cardiovascular system.

Deletion of G protein-coupled estrogen receptor increases endothelial vasoconstriction.

Endogenous estrogens mediate protective effects in the cardiovascular system, affecting both endothelium-dependent and endothelium-independent mechanisms. Previous studies have suggested that nonselective estrogen receptor agonists such as endogenous estrogens inhibit endothelium-dependent vasoconstriction; however, the role of estrogen receptors in this response has not yet been clarified. This study investigated whether the intracellular transmembrane G protein-coupled estrogen receptor (GPER) regulates vascular reactivity in mice. Effects of chronic deficiency (using mice lacking the GPER gene) and acute inhibition (using the GPER-selective antagonist G15) on endothelium-dependent and endothelium-independent vascular reactivity, and the effects of GPER deficiency on vascular gene expression and structure were investigated. We found that chronic GPER deficiency is associated with increased endothelial prostanoid-mediated vasoconstriction but has no effect on endothelial nitric oxide bioactivity, gene expression of endothelial nitric oxide synthase and thromboxane prostanoid (TP) receptor, or vascular structure. GPER deletion also increases TP receptor-mediated contraction. Acute GPER blockade enhances endothelium-dependent contractions and reduces endothelial nitric oxide bioactivity. Contractions in response to TP receptor activation are unaffected by G15. In conclusion, this study identifies GPER as the first estrogen receptor with inhibitory activity on endothelium-dependent contractility. These findings may be important for understanding and treating diseases associated with increased endothelial vasoconstrictor prostanoid activity such as hypertension and obesity.

Identification of a GPER/GPR30 antagonist with improved estrogen receptor counterselectivity.

GPER/GPR30 is a seven-transmembrane G protein-coupled estrogen receptor that regulates many aspects of mammalian biology and physiology. We have previously described both a GPER-selective agonist G-1 and antagonist G15 based on a tetrahydro-3H-cyclopenta[c]quinoline scaffold. The antagonist lacks an ethanone moiety that likely forms important hydrogen bonds involved in receptor activation. Computational docking studies suggested that the lack of the ethanone substituent in G15 could minimize key steric conflicts, present in G-1, that limit binding within the ERα ligand binding pocket. In this report, we identify low-affinity cross-reactivity of the GPER antagonist G15 to the classical estrogen receptor ERα. To generate an antagonist with enhanced selectivity, we therefore synthesized an isosteric G-1 derivative, G36, containing an isopropyl moiety in place of the ethanone moiety. We demonstrate that G36 shows decreased binding and activation of ERα, while maintaining its antagonist profile towards GPER. G36 selectively inhibits estrogen-mediated activation of PI3K by GPER but not ERα. It also inhibits estrogen- and G-1-mediated calcium mobilization as well as ERK1/2 activation, with no effect on EGF-mediated ERK1/2 activation. Similar to G15, G36 inhibits estrogen- and G-1-stimulated proliferation of uterine epithelial cells in vivo. The identification of G36 as a GPER antagonist with improved ER counterselectivity represents a significant step towards the development of new highly selective therapeutics for cancer and other diseases.

Synthetic estrogen derivatives demonstrate the functionality of intracellular GPR30.

Estrogen mediates its effects through multiple cellular receptors. In addition to the classical nuclear estrogen receptors (ERalpha and ERbeta), estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPCR) GPR30. Although estrogen is a cell-permeable ligand, it is often assumed that all GPCRs function solely as cell surface receptors. Our previous results showed that GPR30 appeared to be expressed predominantly in the endoplasmic reticulum. A critical question that arises is whether this localization represents the site of functional receptor. To address this question, we synthesized a collection of cell-permeable and cell-impermeable estrogen derivatives. We hypothesized that if functional GPR30 were expressed at the cell surface, both permeable and impermeable derivatives would show activity. However, if functional GPR30 were predominantly intracellular, like ERalpha, only the permeable ligands should show activity. Cell permeability was assessed using cells expressing ERalpha as a model intracellular estrogen-binding receptor. Our results reveal that despite exhibiting similar binding affinities for GPR30, only the cell-permeable ligands are capable of stimulating rapid calcium mobilization and phosphoinositide 3-kinase (PI3K) activation. We conclude that GPR30 expressed intracellularly is capable of initiating cellular signaling and that there is insufficient GPR30 expressed on the cell surface to initiate signaling in response to impermeable ligands in the cell lines examined. To our knowledge, this is the first definitive demonstration of a functional intracellular transmembrane estrogen receptor.