Figure 5.

Placental lactogen (PL) induces both endothelial nitric oxide synthase (eNOS) serine 1177 phosphorylation and the release of nitric oxide (NO) in rat aortic endothelial cells (RAEC). Endothelial cells at 85% confluence were incubated for 8 min in either the presence or the absence of increasing concentrations of PL. Following the incubation period, the culture medium was collected, and the NO level was determined. (A) eNOS 1177 phosphorylation (p-eNOS Ser 1177) was analyzed via Western blot in RAEC. Cell lysates were separated via electrophoresis and transferred to membranes, followed by immunoblotting with antibodies directed against phospho-eNOS serine 1177, total eNOS and β-actin. B) The quantification of eNOS serine 1177 phosphorylation was performed via densitometry after the compound was normalized to β-actin. (C) Total eNOS served as a control to confirm its constitutive presence in the cells after different treatments. (D) NO production was calculated based on the accumulation of NO₂ and NO₃ in the culture medium after 8 min of exposure in the presence of PL, followed by the measurement of absorbance at 490 nm. The results are representative of five independent experiments. Values are mean±SEM. ^bP<0.05 vs control (ctrl). ^cP<0.01 vs control (ctrl).