Rapid activation of endothelial NO synthase by estrogen: Evidence for a steroid receptor fast-action complex (SRFC) in caveolae

Estrogen has important atheroprotective and vasoactive properties related to its capacity to stimulate nitric oxide (NO) production by endothelial NO synthase. Previous work has shown that these effects are mediated by estrogen receptor (ER) a functioning in a nongenomic manner via calcium-dependent, MAP kinase-dependent mechanisms. Recent studies have demonstrated that estradiol (E-2) activates eNOS in isolated endothelial plasma membranes in the absence of added calcium, calmodulin or eNOS cofactors. Studies of blockade by ICI 182,780 and by ERalpha antibody, and also immunoidentification-experiments indicate that the process is mediated by a subpopulation of plasma membrane-associated ERalpha. Fractionation of endothelial cell plasma membranes has further revealed that ERalpha protein is localized to caveolae, and that E-2 causes stimulation of eNOS in isolated caveolae which is ER-dependent and calcium-dependent, whereas noncaveolae membranes are insensitive. Furthermore, in intact endothelial cells the activation of eNOS by E-2 is prevented by pertussis toxin, and exogenous GDPbetaS inhibits the response in isolated plasma membranes. Coimmunoprecipitation studies have shown that E-2 exposure causes interaction between ERalpha and G(alphai) on the plasma membrane, and eNOS activation by E-2 is enhanced by overexpression of G(alphai) and attenuated by expression of a protein regulator of G protein signaling (RGS), RGS4. Thus, a subpopulation of ERalpha is localized to caveolae in endothelial cells, where they are coupled via G(alphai) to eNOS in a functional signaling module. Emphasizing the dependence on cell surface-associated receptors, these observations provide evidence for the existence of asteroid receptor fast-action complex, or SRFC, in caveolae. (C) 2002 Elsevier Science Inc. All rights reserved.