17β-Estradiol increases endothelial nitric oxide synthase mRNA copy number in cerebral blood vessels: quantification by real-time polymerase chain reaction

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Abstract

The enzyme endothelial nitric oxide synthase (eNOS) plays a critical role in the maintenance of vascular tone. The mechanism by which estrogen increases eNOS function remains controversial. We demonstrate here using real-time polymerase chain reaction (PCR) and immunoblot analysis that in vivo estrogen treatment leads to a 100% increase in eNOS messenger RNA (mRNA) copy number and increases eNOS protein levels by 47% in mouse cerebral blood vessels. These data suggest that estrogen can modulate eNOS at the transcriptional level in blood vessels in vivo.

Introduction

Maintenance of endothelial nitric oxide synthase (eNOS) protein levels and activity are critical to vascular function, as diminished nitric oxide (NO) availability contributes to systemic hypertension, endothelial dysfunction and atherosclerosis (Shaul, 2002). Effects on eNOS are believed to mediate some of the vascular protective effects associated with estrogen treatment McNeill et al., 1999, Hodgin et al., 2002. However, the mechanisms by which in vivo estrogen treatment modulates eNOS function are not established.

It is now established that chronic in vivo estrogen exposure substantially elevates eNOS protein levels in rat cerebral blood vessels, but the mechanism behind this process is not understood McNeill et al., 1999, Geary et al., 2001. In cultured human endothelial cells, estrogen can increase eNOS mRNA and protein via estrogen receptors Hayashi et al., 1995, Kleinert et al., 1998 without affecting mRNA stability. It is therefore likely that chronic estrogen exposure specifically increases eNOS protein levels in cerebral blood vessels through a receptor-mediated increase in transcription and translation of the eNOS gene. However, data produced in cultured cells remain controversial as signal transduction may not accurately represent in vivo conditions and must therefore be validated in intact vascular tissue. In the present study, we provide evidence that chronic estrogen treatment in vivo increases eNOS mRNA copy number and eNOS protein levels in mice cerebral blood vessels.

Section snippets

In vivo treatments and cerebral vessel preparation

All animal procedures were approved by the U.C. Irvine Institutional Animal Care and Use Committee. Ovariectomized (OVX) C57BL/6 female mice were obtained from Charles River Laboratories and, in some cases, implanted with a 17β-estradiol pellet (OE) as described previously (Geary et al., 2000). Implants were left in place for 4 weeks; all animals were then anesthetized by CO2 and killed by decapitation. We have previously demonstrated by this procedure that serum estrogen levels in

Real-time PCR

In cerebral blood vessels of mice chronically exposed to physiological estrogen levels for 1 month, eNOS mRNA copy number per nanogram of total RNA was significantly increased as compared to cerebral vessels of ovariectomized mice (Fig. 1A). In estrogen-treated animals, the ratio of eNOS/18S rRNA was also higher than that of ovariectomized animals (Fig. 1B). The real-time PCR reactions were linear over six orders of magnitude of starting cDNA standards, with a detection sensitivity of 20

Discussion

In the present study, we have confirmed and extended our previous findings that chronic in vivo estrogen treatment increases eNOS protein levels in cerebral blood vessels. Using the method of real-time PCR to quantify eNOS mRNA levels in cerebral vessels from female mice, we demonstrate that there is a concomitant increase in eNOS mRNA copy number with increases in vessel eNOS protein. Our data indicate that there are approximately twice the number of eNOS mRNA copies in estrogen-treated mice

Acknowledgments

The authors would like to thank Jonnie Stevens for the technical assistance with surgical procedures and Amin Boroujerdi for the help with Western blots.

This work was supported in part by National Heart, Lung and Blood Institute Grant R01-HL-50775 and by a grant from the American Heart Association.

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