Pharmacological Modulation of Akt‐dependent Activation of eNOS/NO in Airway Cells

Rationale Ca 2+ /calmodulin activates endothelial nitric oxide synthase (eNOS). However, other pathway(s) by which eNOS is activated is less studied in the airway cells. We hypothesized protein kinase B/Akt plays a role in the activation of eNOS and subsequent nitric oxide (NO) production. Methods To test if Akt activator SC79 activates Akt, A549 a type II alveolar epithelial cell‐line was transfected with a FRET‐based AktAR sensor and were treated with SC79 (10μg/ml). Cells were treated with SC79 ± phosphoinositide‐3‐kinase (PI3K) inhibitor LY294002(10μg/ml) and were harvested for Western blotting to detect phospho(p)‐eNOS. To test the production of NO, airway cells were loaded with DAF‐FM (5μM) ± eNOS inhibitor L‐NAME (10μM) and were stimulated with SC79. To test if SC79 might be protective against cadmium‐induced barrier functions and inflammation, cells were treated with cadmium( 100μM) or TNF‐α (0.1μg/ml) ± SC79 followed by Western blotting to measure tight junction protein (TJ) Zonula occluden (ZO‐1) and qPCR to measure IL‐8. Results Live cell imaging data show a significant increase in Akt activity (p<0.001) with SC79. AKT‐activation increased p‐eNOS via PI3K. Further, NO was induced with SC79 and was blocked by L‐NAME. Cadmium‐induced reduction in ZO‐1 protein levels and TNF‐α induced IL‐8 were both reversed by SC79. Conclusion Our preliminary data suggest that Akt plays a role in phosphorylating eNOS and NO production in airway cells. Further, Akt activators such as SC79 reversed IL‐8 and induced TJ proteins like ZO‐1. Future work will be directed towards testing the downstream mechanisms of eNOS/NO with Akt activators in patient‐derived primary airway cells. Support or Funding Information R01DC016309, R21AI137484