Expression of soluble guanylate cyclase (sGC) and its ability to form a functional heterodimer are crucial for reviving the NO-sGC signaling in PAH.

In order to determine the underpinnings of a dysfunctional NO-sGC signal pathway which occurs in pulmonary arterial hypertension (PAH), we investigated pulmonary arterial smooth muscle cells (PASMCs) derived from PAH patients. We found low expression of sGC, a poor sGCα1β1 heterodimer and this correlated with low expression of its facilitator chaperon, hsp90. Treating PASMCs overnight (16 h) with low micromolar doses of a slow release NO donor DETANONOate, reinstated the sGCα1β1 heterodimer and restored its NO-heme dependent activity. Transwell co-culture of HEK cells stably expressing eNOS with PAH PASMCs also restored the sGC heterodimer and its heme-dependent activity with sGC stimulator, BAY 41-2272. To determine whether the dysfunctionality in the NO-sGC pathway stems from a dysfunctional eNOS producing negligible NO, we did transwell co-cultures of pulmonary arterial endothelial cells (PAECs) with PASMCs. Our results indicated that PAECs from both control and PAH samples when activated for eNOS restored both sGC heterodimer and its heme-dependent sGC activity in the corresponding PASMCs, suggesting that PAECs from PAH can also generate NO. In line with these results expression of eNOS, its support chaperon hsp90, its specific kinase Akt, p-Akt or post-translational modifications (PTMs) like OGlcNAc or phospho-tyrosine were unchanged in PAH relative to controls. Additionally there was uniform expression of Hbα/β and Mb in PASMCs or PAECs in PAH or controls and these globins can effectively scavenge the eNOS generated NO, as there was evidence of strong eNOS-Hb/Mb interactions. Our studies suggest that factors such as globin NO scavenging along with vascular remodeling in PAH can cause hampered vasodilation which in the face of poor NO levels as occurs in PAH are additional impediments for effective vasodilation. However importantly our studies suggests that future therapies can use low doses of NO along with sGC stimulators as a potential drug for PAH subjects.