Plasma Membrane Estrogen Receptors Are Coupled to Endothelial Nitric-oxide Synthase through Gα_i

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-17β (E₂, 10^-8 M) and acetylcholine (10^-5 M) 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 E₂-mediated eNOS stimulation in isolated endothelial plasma membranes, and Ptox prevented enzyme activation by E₂ in COS-7 cells expressing ERα and eNOS. Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ERα and specific Gα proteins demonstrated E ₂-stimulated interaction between ERα and Gα_i but not between ERα and either Gα_q or Gα _s; the observed ERα-Gα_i interaction was blocked by the ER antagonist ICI 182,780 and by Ptox. E₂-stimulated ERα-Gα_i interaction was also demonstrable in endothelial cell plasma membranes. Cotransfection of Gα_i into COS-7 cells expressing ERα 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 E₂ response. These findings indicate that eNOS stimulation by E₂ requires plasma membrane ERα coupling to Gα_i and that activated Gα_i mediates the requisite downstream signaling events. Thus, novel G protein coupling enables a subpopulation of ERα to initiate signal transduction at the cell surface. Similar mechanisms may underly the nongenomic actions of other steroid hormones.