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Date of Award
Dissertation - Pacific Access Restricted
Doctor of Philosophy (Ph.D.)
Pharmaceutical and Chemical Sciences
First Committee Member
Second Committee Member
Third Committee Member
The effect of estrogen on the vasculature is mediated in part by influences on NO bioavailability. Nitric oxide (NO) is a potent vasodilator which is synthesized in endothelial cell by endothelial nitric oxide synthase (eNOS) catalyzed conversion of L-arginine to L-citrulline. Although estrogen has been shown to increase eNOS expression and/or activity, the mechanism of estrogen-mediated increased eNOS activity in endothelial cells remains elusive. The Ca 2+ /calmodulin complex is known to aid in eNOS activation by dissociating eNOS from the membrane bound protein, caveolin-1. We investigated the role of estrogen on the Ca 2+ homeostasis of the human endothelial cell line, EA.hy926, using thapsigargin (TG), a sarco(endo)plasmic reticulum Ca 2+ -ATPase, or ATP, a purinergic receptor agonist, to evoke increased intracellular calcium concentration ([Ca 2+ ] i ). [Ca 2+ ] i in Fura 2-AM-loaded EA.hy926 cell populations were measured by fluorescence spectrophotometry. Treatment of cells with 17 β-estradiol (E 2 , 1 μM, 24 hours) showed an increased agonist-evoked [Ca 2+ ] i increase due to both higher Ca 2+ release and Ca 2+ influx, which accompanied an increased eNOS protein expression. Both increased [Ca 2+ ] i and eNOS expression were attenuated with the nonselective estrogen receptor (ER) inhibitor, ICI 182,780. We further analyzed the role of ER in E 2 - mediated effects using ERα-knockdown cells. ERα-knockdown was achieved by transfecting the cells with ERα-specific siRNA. E 2 did not influence agonist-evoked [Ca 2+ ] i increase in the ERα-knockdown cells, indicating that the E 2 -mediated effects were ERα-dependent. In the vasculature, both the genomic and nongenomic effects of estrogen are mediated via ERα. In the current study, the effect of E 2 on agonist-evoked [Ca 2+ ] i increase was only observed in chronically treated (1 μM, 24 hours) cells and not acutely treated (1 μM, 5 minutes) cells, suggesting a genomic action of E 2 . The genomic action was verified by treating cells with E 2 in the presence of actinomycin D, a transcription inhibitor. Actinomycin D attenuated the effect of E 2 on agonist-evoked [Ca 2+ ] i increase. The present work revealed a transcription-dependent and ERα-mediated modulation of Ca 2+ homeostasis in human endothelial cells treated with estrogen for the long-term. This data suggests a novel mechanism by which estrogen-mediated NO release may occur in endothelial cells.
Thor, Der. (2009). The effect of estrogen on intracellular calcium homeostasis in human endothelial cells. University of the Pacific, Dissertation - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/2397
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