Relaxin stimulates NO production via PI3K/Akt. (A) Human CD34⁺ BMDECs were isolated from a healthy volunteer and pretreated with the PI3K inhibitor LY294002 (10μM) for 30 minutes as indicated before the addition of DAF-FM and continued incubation with LY294002 (LY). After 30 minutes of monitoring to confirm that bioavailable NO was stable, either vehicle (Control) or 100 ng/mL of rhRLX was added to the CD34⁺ BMDECs, and NO was measured again after 30 minutes. Fluorescence was determined for each condition in at least 30 cells within 2 separate wells. Data are mean ± SD. *P < .001 versus all other conditions. (B) CD34⁺ BMDECs were isolated from a healthy volunteer and pretreated with the Akt inhibitor MK2206 (20nM) for 30 minutes as indicated before the addition of DAF-FM and continued incubation with MK2206. After 30 minutes of monitoring, to confirm that bioavailable NO was stable, either vehicle (Control) or 50 ng/mL of rhRLX was added to the CD34⁺ BMDECs, and NO was measured again after 30 minutes. Fluorescence was determined for each condition in 2 wells for at least 30 cells per well. Data are mean ± SD. *P < .001 versus control. There is a significant difference between control and MK2206 (P = .025) but no difference between MK2206 and MK2206 + rhRLX-treated cells.