O0697 Oct.3: 3:50pm - Treatment of human pulmonary microvascular endothelial cells with NO leads to increased PDE3A protein expression and AMPK activation

Background: Phosphodiesterase 3 (PDE3), of which there are two isoforms, 3A and 3B, hydrolyzes both cAMP and cGMP to alter vascular tone. In varying cell types, PDE3 inhibition has been shown to increase endothelial nitric oxide synthase (eNOS) expression and activation via AMP-activated protein kinase (AMPK) activation. Additionally, impaired AMPK regulation has been shown to contribute to endothelial dysfunction in models of pulmonary hypertension. Although PDE3 inhibition has been utilized in the treatment of persistent pulmonary hypertension of the newborn (PPHN), the effects of standard of care treatments – inhaled nitric oxide (NO) and supplement oxygen (hyperoxia) – on the PDE3 isoforms have not been elucidated in the pulmonary vasculature. Furthermore, the downstream effects of each PDE3 isoform on AMPK expression are unknown.

Objective: To delineate the effects of NO and hyperoxia on each of the PDE3 isoforms in human pulmonary microvascular endothelial cells (hPMVEC), and evaluate the downstream effects on AMPK and eNOS activation.
Design/

Methods: hPMVEC were treated with the NO donor DETA NONOate (250 µM) or vehicle and incubated in 21% O2 (normoxia) or 85% O2 (hyperoxia) for 48 h. PDE3A, PDE3B, phosphorylated (p) and total (T)-AMPK and eNOS protein expression were quantified by Western blot analyses (n = 5-10).

Results: In normoxia, PDE3A protein expression was increased in hPMVEC treated with DETA NONOate (p<0.001), with no effect on PDE3B. Interestingly, the treatment combination of NO+hyperoxia blunted the NO-induced PDE3A protein expression (p<0.004) in hPMVEC, as well as decreased PDE3B protein expression (p=0.034) compared to NO+normoxia. Similar to PDE3A protein expression, p/T-AMPK was increased by DETA NONOate in hPMVEC (p=0.021) and the combination of NO+hyperoxia prevented the NO-induced AMPK activation (p=0.014). T-eNOS was increased in hPMVEC incubated in hyperoxia alone (p=0.014), which was blunted with the combination of NO+hyperoxia (p<0.05).
Conclusion(s): The combination of NO+hyperoxia resulted in a decrease in PDE3A, PDE3B, and AMPK activation in hPMVEC compared to NO treatment alone. Similarly the combination of NO+hyperoxia blunted the hyperoxia-induced increase in T-eNOS protein expression. We speculate that PDE3 regulates AMPK and that the treatment combination of NO+hyperoxia may have detrimental effects on this pathway in hPMVEC. Isoform-specific PDE3 inhibition may play a role to differentially regulate AMPK activation and eNOS expression in hPMVEC.