Campus Access Only

All rights reserved. This publication is intended for use solely by faculty, students, and staff of University of the Pacific. No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, now known or later developed, including but not limited to photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author or the publisher.

Date of Award


Document Type

Dissertation - Pacific Access Restricted

Degree Name

Doctor of Philosophy (Ph.D.)


Pharmaceutical and Chemical Sciences

First Advisor

Roshanak Rahimian

First Committee Member

James Blankenship

Second Committee Member

Geoff Lin-Cereghino

Third Committee Member

James Uchizono


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.





To access this thesis/dissertation you must have a valid email address and log-in to Scholarly Commons.

Find in PacificSearch Find in ProQuest



If you are the author and would like to grant permission to make your work openly accessible, please email


Rights Statement

Rights Statement

In Copyright. URI:
This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).