Figure 4. PKA pathway mediates stretch-induced phosphorylation of eNOS and NO production in the early phase.

(A) Upper: Western blots of phospho-Akt (Ser⁴⁷³), phospho-eNOS (Ser¹¹⁷⁷) and PKA substrates in HUVECs under continuous stretch (15 min), pretreated with H89 (10–100 µM). Lower: quantitative analysis of Ser¹¹⁷⁷ phosphorylation of eNOS. (B) Upper: Western blots of phospho-Akt (Ser⁴⁷³), phospho-eNOS (Ser¹¹⁷⁷) and PKA substrates in HUVECs expressing scrambled (Scr) or PKA-targeting (PKA KD) shRNAs under continuous stretch. Lower: quantitative analysis of Ser¹¹⁷⁷ phosphorylation of eNOS. (C) Upper: DAF-FM staining of HUVECs expressing scrambled (Scr) or PKA-targeting (PKA KD) shRNAs with or without 50 µM H89 pretreatment, under continuous stretch. Lower: quantitative analysis of NO release. (D) Upper: Western blots of phospho-Akt (Ser⁴⁷³) and phospho-eNOS (Ser¹¹⁷⁷) in HUVECs pretreated with or without 50 µM H89 under continuous stretch for the indicated time periods. Lower: quantitative analysis of Ser¹¹⁷⁷ phosphorylation of eNOS. Results are representative of 3 individual experiments and expressed as mean ± S.D. (n = 4). *p<0.05; **p<0.01.