Received for publication July 9, 2004.
Revised October 12, 2004.
Accepted for publication October 19, 2004.

Estrogen receptor activation of PI-3 kinase, Akt and nitric oxide signaling in cerebral blood vessels: rapid and chronic effects

Abstract

Estrogen receptor regulation of nitric oxide (NO) production by vascular endothelium may involve rapid, membrane-initiated signaling pathways in addition to classic genomic mechanisms. Here we demonstrate using intact cerebral blood vessels that 17-estradiol acutely activates endothelial NO synthase (eNOS) via the phosphoinositide-3 (PI-3) kinase-Akt kinase pathway. The effect is mediated by estrogen receptors (ER), consistent with co-localization of ER and caveolin-1 immunoreactivity at the plasma membrane of endothelial cells lining cerebral arteries. Treatment with 17-estradiol (10 nM) for 30 min increased NO production, as measured by total nitrite assay, in cerebral vessels isolated from ovariectomized rats. This effect was significantly decreased by membrane cholesterol depletion with -methyl-cyclodextrin, the ER antagonist ICI 182,780, and two inhibitors of PI-3 kinase: wortmannin and LY294002. In parallel with NO production, acute 17-estradiol treatment increased phosphorylation of both eNOS (p-eNOS) and Akt (p-Akt). PI-3 kinase inhibitors also blocked these effects. ER protein (66 and 50 kDa) co-immunoprecipitated with eNOS as well as with the p85 regulatory subunit of PI-3 kinase, further implicating ER in kinase activation of eNOS. Little is known regarding effects of estrogen on cellular kinase pathways in vivo; therefore we compared cerebral blood vessels isolated from ovariectomized rats that were either untreated or given estrogen replacement for 4 weeks. Chronic estrogen exposure increased levels of cerebrovascular p-Akt and p-eNOS, as well as basal NO production. Thus, in addition to rapid activation of the PI3 kinase-pAkt-peNOS pathway, maintenance of estrogen signaling via non-transcriptional mechanisms has long-term consequences for vascular function.

Key words: Nitric oxide, Nitric oxide synthases, Sex hormones, Protein Kinases (other)