Post-translational regulation of endothelial nitric oxide synthase (eNOS) by estrogens in the rat vagina.
BACKGROUND
Estrogens control vaginal blood flow during female sexual arousal mostly through nitric oxide (NO). Although vascular effects of estrogens are attributed to an increase in endothelial NO production, the mechanisms of endothelial NO synthase (eNOS) regulation by estrogens in the vagina are largely unknown.


OBJECTIVE
Our hypothesis was that estrogens regulate eNOS post-translationally in the vagina, providing a mechanism to affect NO bioavailability without changes in eNOS protein expression.


METHODS
We measured eNOS phosphorylation and eNOS interaction with caveolin-1 and heat shock protein 90 (HSP90) in the distal and proximal vagina of female rats at diestrus, 7 days after ovariectomy and 2 days after replacement of ovariectomized rats with estradiol-17beta (15 microg).


METHODS
Molecular mechanisms of eNOS regulation by estrogen in the rat vagina.


RESULTS
We localized phospho-eNOS (Ser-1177) immunohistochemically to the endothelium lining blood vessels and vaginal sinusoids. Estrogen withdrawal decreased phosphorylation of eNOS on its positive regulatory site (Ser-1177) and increased eNOS binding to its negative regulator caveolin-1 (without affecting eNOS/HSP90 interaction), and they were both normalized by estradiol replacement. Protein expressions of phosphorylated Akt (protein kinase B) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) were not affected by estrogen status, suggesting that the effect of estrogens on eNOS (Ser-1177) phosphorylation was not mediated by activated AKT or ERK1/2. eNOS phosphorylation on its negative regulatory site (Ser-114) was increased in the vagina by estrogen withdrawal and normalized by estradiol replacement, implying that the maintenance of low phosphorylation of eNOS on this site by estradiol may limit eNOS interaction with caveolin-1 and preserve the enzyme's activity. Total eNOS, inducible NOS, caveolin-1, and HSP90 protein expressions were not affected by ovariectomy or estradiol replacement in the distal or proximal vagina.


CONCLUSIONS
These results define novel estrogen signaling mechanisms in the vagina which involve eNOS phosphorylation and eNOS-caveolin-1 interaction.
Department of Urology, The Johns Hopkins University, Baltimore, MD 21287, USA. bmusicki@jhmi.edu
Biljana
B
Musicki
Tongyun
T
Liu
Travis D
TD
Strong
Gwen A
GA
Lagoda
Trinity J
TJ
Bivalacqua
Arthur L
AL
Burnett
D000818:Animals; D015153:Blotting, Western; D051242:Caveolin 1 / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology; D004967:Estrogens / Q000502:physiology*; D005260:Female; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D011916:Rats, Inbred F344; D014599:Uterus / Q000187:drug effects / Q000378:metabolism; D014621:Vagina / Q000187:drug effects / Q000378:metabolism*
D051242:Caveolin 1; D004967:Estrogens; D018841:HSP90 Heat-Shock Proteins; D004958:Estradiol; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat