Estradiol induces the calcium-dependent translocation of endothelial nitric oxide synthase

Although estrogen is known to stimulate nitric oxide synthesis in vascular endothelium, the molecular mechanisms responsible for this effect remain to be elucidated. Using quantitative immunofluorescence imaging approaches, w e have investigated the effect of estradiol on the subcellular targeting of endothelial nitric oxide synthase (eNOS) in bovine aortic endothelial cell s. In unstimulated endothelial cells, eNOS is predominantly localized at th e cell membrane. Within 5 min after the addition of estradiol, most of the eNOS translocates from the membrane to intracellular sites close to the nuc leus. On more prolonged exposure to estradiol, most of the eNOS returns to the membrane. This effect of estradiol is evident at a concentration of 1 p M, and a maximal estradiol effect is seen at a concentration of 1 nM. Neith er progesterone nor testosterone has any effect on eNOS distribution. After estradiol addition, a transient rise in intracellular Ca2+ concentration p recedes eNOS translocation, Both the Ca2+-mobilizing and eNOS-translocating effects of estradiol are completely blocked by the estrogen receptor antag onist ICI 182,780, and the intracellular Ca2+ chelator 1,2-bis-(o-aminophen oxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) prevents estradiol-induced eN OS translocation, Use of the nitric oxide-specific dye diaminofluorescein s hows that estradiol treatment increases nitric oxide generation by endothel ial cells; this response is blocked by ICI 182,780 and by the eNOS inhibito r N-omega-nitro-L-arginine. These results show that estradiol induces subce llular translocation of eNOS by a rapid, Ca2+-dependent, receptor-mediated mechanism, and they suggest a nongenomic role for estrogen in the modulatio n of NO-dependent vascular tone.