Rapid activation of eNOS in endothelial cells. (a) Effect of E₂ on eNOS activity in intact PAEC. [³H]l-arginine conversion to [³H]l-citrulline was measured over 5–15 min in the presence of 10^–8 M E₂. (b) Effect of actinomycin D (Act D) on the rapid activation of eNOS. After 120 min preincubation in the absence or presence of 25 μg/ml Act D, 15 min incubations were done with or without continued Act D and either 10^–8 M E₂ or the calcium ionophore A23187 (10^–5 M). (c) Effect of tamoxifen on E₂-stimulated eNOS activity. Fifteen-minute incubations were performed in the absence or presence of 10^–8 M E₂, with or without 10^–6 M tamoxifen (Tam) added simultaneously. Partial inhibition (50%–70%) was also noted with 10^–8 M Tam (13). (d) Effect of ICI 182,780 on E₂-stimulated eNOS activity. Fifteen-minute incubations were performed in the absence or presence of 10^–8 M E₂, with or without 10^–5 M ICI 182,780 added simultaneously. Full inhibition was also observed with 10^–6 M ICI 182,780 (13). Values are mean ± SEM; n = 4–6. *P < 0.05 vs. basal. E₂, estradiol-17β; eNOS, endothelial nitric oxide; PAEC, pulmonary artery endothelial cells.