No abstract

Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca 2؉ signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the net-

The novel G protein‐coupled estrogen receptor 1 (GPER) has been demonstrated to mediate numerous beneficial cardiovascular effects. We have begun to investigate the effects of GPER activation on intracellular Ca2+ stores in the vascular endothelium. We observed that GPER is present in primary endothelial cells in a glycosylated form. Immunostaining locates GPER at both the plasma membrane and in the perinuclear region. In nominally Ca2+‐free condition, treatment with thapsigargin triggers the release of the ER's Ca2+ content. Subsequent addition of the GPER agonist G1 in Ca2+‐free condition surprisingly triggers a slow and sustained Ca2+ rise up to ~ 400 nM, a value comparable to agonist‐induced Ca2+ entry signals. Inhibition of PI3 kinase with wortmannin decreases G1‐triggered Ca2+ signal in Ca2+ free condition, but does not abolish it, suggesting that GPER activation in endothelial cells triggers the release of both IP3‐dependent and IP3‐independent Ca2+ stores. Pretreatment of endothelial cells with brefeldin A, a disruptor of the trans‐Golgi network, abolishes the slow and prolonged phase of the Ca2+ response to G1 treatment. Our data suggest that activation of GPER in primary vascular endothelial cells triggers the release of two different types of intracellular Ca2+ stores, namely the endoplasmic reticulum and the Golgi. The data also suggest that the Golgi represents a substantial intracellular Ca2+ store in the vascular endothelium.

Interactions between vascular endothelial cells and the underlying smooth muscle cells (VSMCs) are of paramount importance in maintaining vascular functions. Calmodulin (CaM) is involved in a wide variety of cellular functions and is a limiting factor in both cell types, with free cytoplasmic CaM constituting only a small fraction of the total cellular CaM. Currently nothing is known about potential interactions between ECs and VSMCs as it involves CaM. We have begun to investigate the possibility that vascular endothelial cells impact VSMC functions via CaM‐dependent activities. Using a co‐culture model of primary culture vascular endothelial cells and smooth muscle cells isolated from the same vessels, we have found that VSMCs in co‐culture with proliferating ECs express on average 90% more CaM than monocultured VSMCs. Media fractionation and eNOS inhibition experiments indicate that the CaM‐elevating effect was exerted by a soluble factor that is not nitric oxide. The CaM‐elevating effect exerted by ECs is strongly dependent on endothelial density, such that at a starting 50% confluency of VSMCs, a starting 20% endothelial confluency triggers the greatest CaM increase in VSMCs, whereas a starting 70% endothelial confluency yields no visible effect on VSMC CaM expression after 48 hrs. Pharmacological inhibition of cyclooxygenase‐1, vascular endothelial cell growth factor (VEGF), and endothelin‐1 receptors (ETA and ETB) does not affect the observed increase in CaM. The data suggest that proliferating endothelial cells produce a soluble factor that can regulate CaM‐dependent signaling in VSMCs via alterations in total cellular CaM expression.

The G protein‐coupled estrogen receptor 1 (GPER) has been demonstrated to have a vast array of cardiovascular effects. Ca2+ extrusion via the plasma membrane Ca2+‐ATPase (PMCA) plays important roles in Ca2+ homeostasis. We tested the hypothesis that GPER is involved in the regulation of calcium efflux in the vasculature. In primary vascular endothelial cells, the specific GPER agonist G‐1 dose‐dependently inhibits PMCA activity. Knockdown of GPER in endothelial cells using antisense directed against GPER is associated with a ~ 40% enhancement of PMCA activity. Heterologous expression of human GPER in fusion with the fluorescent protein DsRed2 in HEK 293 cells results in a 38% reduction of PMCA activity. Co‐IP experiments show that PMCA and GPER form a complex during Ca2+ signals triggered by the SERCA pump inhibitor thapsigargin in endothelial cells. In addition, the fusion proteins PMCA‐DsRed2 colocalizes with GPER‐ECFP heterologously expressed in HEK 293 cells. These data suggest that GPER regulates Ca2+ efflux in endothelial cells by interacting with PMCA. Grant Funding Source: National Institutes of Health