Cell Type‐Specific Differential Effects of Nitric Oxide on PDE3 Expression in Pulmonary Vasculature

Abstract

Persistent pulmonary hypertension of the newborn (PPHN) is an important cause of morbidity and mortality in neonates. Often used in combination with hyperoxia, the pulmonary vasodilator, inhaled nitric oxide (iNO), is the only FDA‐approved therapy for PPHN, although up to 40% of patients are non‐responders. In the pulmonary vasculature, NO increases cGMP levels, promoting vasodilation. cGMP competitively inhibits phosphodiesterase 3 (PDE3), thereby decreasing cAMP hydrolysis and further enhancing vasodilation. However, in animal models, NO treatment has been shown to increase PDE3 expression and/or activity and has been postulated to be the reason for rebound PH upon iNO withdrawal. Furthermore, it has been reported that that addition of a PDE3 inhibitor to iNO enhances pulmonary vasorelaxation. AMP‐activated protein kinase (AMPK), a critical regulator of energy homeostasis with downstream effects on NO synthase (NOS), has been shown to be regulated by PDE3 isoforms in other cell types and implicated in PH pathogenesis. We hypothesize that NO regulates the AMPK pathway via modulating PDE3 activity in a cell‐type specific manner in the pulmonary vasculature. Human PASMC (hPASMC) were treated with the NO donor DETA NONOate (1 mM) or DMSO and incubated in 21% or 85% O2 for 48 h. PDE3A, PDE3B, phosphorylated (p) and total (T)‐AMPK, p‐ and T‐ACC, and β‐actin protein expression were quantified by Western blot analyses. A similar experiment was performed using hPMVEC. Additionally, p‐ and T‐endothelial NOS (eNOS) protein expression were quantified. Our results show that hPASMC PDE3A protein expression was increased following Deta NONOate treatment in normoxia and hyperoxia (5.5±0.4 ‐fold, p=0.0004; 7.5±0.8 ‐fold, p<0.0001) relative to 21% O2 vehicle. Additionally, p/T‐AMPK protein expression was increased by 3‐fold after Deta NONOate treatment (p<0.0001). Consistent with these results, p/T‐ACC (a downstream target of AMPK), was also increased with the addition of NO donor in both conditions. In hPMVEC, the addition of NO donor increased PDE3A protein expression (2.4±0.1 ‐fold, p<0.0001). The combination of hyperoxia + NO donor, however, blunted the increase in PDE3A to a level similar to that in 21% O2, vehicle‐treated cells. Similarly, NO donor alone increased p/T‐AMPK (4.1±0.1 ‐fold, p<0.0001), whereas the combination of hyperoxia + NO blunted this increase. Moreover, p‐eNOS/β‐actin and T‐eNOS/β‐actin were both increased with the NO donor (1.8±0.03 ‐fold, p<0.0001; 2.9‐fold±0.20 SEM, p=0.0068), whereas the combination of hyperoxia + NO blunted the increase in p‐eNOS levels. In conclusion, exogenous NO donor increased PDE3A expression and AMPK activation in hPASMCs in both normoxia and hyperoxia. Alternatively, in hPMVEC, the increase in PDE3A expression, AMPK and eNOS activation after the addition of NO donor was blunted by hyperoxia. We speculate that these cell‐type specific differences in regulation of PDE3 and AMPK may be the reason for the variability in response of the pulmonary vasculature to PDE3 inhibition and AMPK modulators in PH.Support or Funding InformationNIH, NHLBI, R01, 1R01HL136963‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.