Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through Gαi

Estrogen causes rapid endothelial nitric oxide (NO) production because of the activation of plasma membrane-associated estrogen receptors (ER) coupled to endothelial NO synthase (eNOS). In the present study, we determined the role of G proteins in eNOS activation by estrogen. Estradiol-17 beta (E-2, 10(-8) M) and acetylcholine (10(-5) hr) caused comparable increases in NOS activity (15 min) in intact endothelial cells that were fully blocked by pertussis toxin (Ptox). In addition, exogenous guanosine 5'-O-(2- thiodiphosphate) inhibited E-2-mediated eNOS stimulation in isolated endothelial plasma membranes, and Ptox prevented enzyme activation by E-2 in COS-7 cells expressing ER alpha and eNOS. Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ER alpha and specific G alpha proteins demonstrated E-2-stimulated interaction between ER alpha and G alpha (i) but not between ER alpha and either G alpha (q) or G alpha (s); the observed ER alpha -G alpha (i) interaction was blocked by the ER antagonist ICI 182,780 and by Ptox, E-2-stimulated ER alpha -G alpha (i) interaction was also demonstrable in endothelial cell plasma membranes. Cotransfection of G alpha (i) into COS-7 cells expressing ER alpha and eNOS yielded a 3-fold increase in E-2-mediated eNOS stimulation, whereas cotransfection with a protein regulator of G protein signaling, RGS4, inhibited the E-2 response. These findings indicate that eNOS stimulation by E-2 requires plasma membrane ER alpha coupling to G alpha (i) and that activated G alpha (i) mediates the requisite downstream signaling events. Thus, novel G protein coupling enables a subpopulation of ER alpha to initiate signal transduction at the cell surface. Similar mechanisms may underly the nongenomic actions of other steroid hormones.