Differential Effects of Chronic Treatment with Estrogen Receptor Ligands on Regulation of Nitric Oxide Synthase in Porcine Aortic Endothelial Cells

Okano, Hiroya MD*; Jayachandran, Muthuvel PhD* †; Yoshikawa, Akiko MD*; Miller, Virginia M. PhD* †

Journal of Cardiovascular Pharmacology:
doi: 10.1097/01.fjc.0000211749.24196.98
Original Articles

In cultured endothelial cells, estrogen increases expression and activity of endothelial nitric oxide synthase (eNOS). This study was designed to determine whether estrogenic treatments increase eNOS similarly in vivo. Aortic endothelial cells were collected from adult ovariectomized pigs which were untreated (8wk-OVX) or treated with oral 17β-estradiol (E2, 2 mg/day), conjugated equine estrogen (CEE, 0.625 mg/day), or raloxifene (60 mg/day) for 4 weeks. Plasma NOx, estrogen receptors (ERα and ERβ), eNOS, eNOS regulatory proteins, and eNOS mRNA in endothelial cells were determined by Griess reaction, Western blot, and real-time polymerase chain reaction, respectively. Ovariectomy decreased, whereas all treatments restored plasma NOx to pre-OVX levels. On the contrary, eNOS protein and mRNA increased with ovariectomy; E2 and CEE but not raloxifene reduced mRNA; eNOS protein was reduced by CEE and raloxifene treatments. Tyrosine phosphorylation of eNOS and expression of calmodulin increased, but Hsp90 decreased with all treatments and only raloxifene treatment increased caveolin-1 compared with OVX. Expression of ERα/ERβ increased with ovariectomy and was reversed by treatments such that raloxifene>CEE>E2. Three clinically relevant estrogen treatments restore plasma NO after ovariectomy, but do not affect eNOS mRNA, posttranslational regulation of eNOS or expression of estrogen receptors in the same way.

Author Information

*Departments of Surgery

Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN

Supported in part by National Institutes of Health grant HL51736, the Ministry of Health, Labour and Welfare (Japan), and a grant from the Pfizer Health Research Foundation. At the time of the study, Drs. Okano and Yoshikawa were Visiting Scientists at the college.

Reprints: Virginia M. Miller, PhD, Department of Surgery, Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905 (e-mail: miller.virginia@mayo.edu)

Received for publication November 16, 2005; accepted March 14, 2006

© 2006 Lippincott Williams & Wilkins, Inc.