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-
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.
Plasma estrogen is strongly linked to cardiovascular health. Calmodulin (CaM) is required for the activities of numerous proteins but is insufficiently expressed for all its targets, leading to competition among CaM targets. It is completely unknown if estrogen modulates cardiovascular functions via the CaM network. We observed in endothelial cells that chronic estrogen treatment (CE2T) increases total and free CaM. Chronic treatment with G‐1, an agonist of the estrogen‐sensitive receptor GPER/GPR30 mimics this effect, while agonists of ERa or ERb do not. ICI182780, an ERa/ERb antagonist and GPER/GPR30 agonist, also increases CaM level. CE2T increases CaM binding to different categories of CaM targets, including the plasma membrane Ca2+‐ATPase (PMCA), eNOS, ERa, and GPER/GPR30 itself. For GPER/GPR30, CaM antagonism or CaM binding‐negating mutations in the receptor's multiple CaM‐binding domains prevent GPER/GPR30‐mediated ERK1/2 phosphorylation. For PMCA, CE2T‐induced stimulation of activity through enhanced CaM binding is masked by Src‐dependent phosphorylation. These effects sustain cytoplasmic Ca2+ for enhanced interactions between CaM and other targets. For eNOS, CE2T doubles CaM binding. Kinetic modeling using in‐cell and in vitro data allowed comparison of CE2T's promotion of eNOS point activity and NO accumulation via effects on determinants of eNOS function, including Ca2+, CaM, and phosphorylation. Our data indicate that CE2T improves endothelial functions via a feed‐forward mechanism in which CaM is upregulated through GPER/GPR30 activation, leading to enhanced CaM binding and functional linkage in the network of CaM‐binding proteins.
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