RANKL induces eNOS activation and NO production in endothelial cells.(A-D) HUVECs were stimulated with 5 μg/mL RANKL for the indicated times (A-C) and with various concentrations of RANKL for 30 minutes (D). (A) Levels of NOx was determined in the culture medium by using a chemiluminescent NO analyzer. (B) The eNOS enzymatic activity was measured by the production of [³H]-L-citrulline from [³H]-L-arginine. Three independent experiments were performed in duplicate. Data are means ± SEs. Statistical analysis of the results was carried out using ANOVA. * *P < .01 versus untreated control in 0 hour and each time point. (C-D) The levels of eNOS protein and phosphorylation of Akt and eNOS by RANKL were determined by Western blotting (top). Blots are representative of 3 independent experiments. Densitometric analyses are presented as the relative ratio of P-Akt to Akt and P-eNOS to eNOS. The relative ratio in untreated control is arbitrarily presented as 100 (bottom).