112 - NO and 100% O2 Can Be a Vasoconstrictive Combination in PPHN Pulmonary Artery

Monday, May 1, 2023

9:30 AM – 11:30 AM ET

Poster Number: 112
Publication Number: 112.426

Martha Hinton, University of Manitoba, Winnipeg, MB, Canada; VIKRAM BHATIA, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, WINNIPEG, MB, Canada; Saeid Maghsoudi, University of Manitoba, Winnipeg, MB, Canada; Prashen Chelikani, University of Manitoba, Winnipeg, MB, Canada; Shyamala Dakshinamurti, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada

Presenting Author(s)

MH

Martha Hinton, MSc (she/her/hers)

Technician
University of Manitoba
Winnipeg, Manitoba, Canada

Background:

Persistent pulmonary hypertension of the newborn (PPHN) features hypoxia-induced pulmonary constriction, which is treated with 100% O2 and nitric oxide (NO). Hypoxia is known to impair antioxidant enzymes, therefore we proposed that treatment of PPHN with O2 and NO may increase reactive oxygen (ROS) and nitrogen species (RNS), with uncertain downstream effects.

Objective: We first reported 72 hr exposure to hypoxia sensitizes pulmonary artery smooth muscle (PASMC) to thromboxane and inhibits adenylyl cyclase (AC) activity. We now examine effects of NO (as sodium nitroprusside, SNP) on thromboxane-mediated contraction and NO-independent relaxation pathways; on ROS/RNS accumulation; and additive effects of 2 hr exposure to 100% O2.

Design/Methods:

Newborn porcine PASMC were cultured in normoxia (21% O2) or hypoxia (10% O2) for 72 hr, with or without daily addition of SNP, or peroxynitrite scavenger FeTTPs, or exposure to 100% O2 for 2hr prior to study. ROS was measured by DCF and DHE staining; RNS by nitrate measurement and biotin switch labelling; Ca2+ mobilization to thromboxane studied using fura-2; AC activity determined by terbium norfloxacin assay.

Results:

Hypoxia increased total ROS compared to normoxic PASMC. SNP mitigates this increase, while addition of hyperoxia further increased ROS. Total protein nitrosylation and nitrate accumulation was increased in hypoxic PASMC, the same was achieved by SNP treatment. Cell viability was unchanged by hypoxia, SNP or hyperoxia. Hypoxic PASMC had higher basal Ca2+, and higher peak Ca2+ response to thromboxane challenge. SNP normalized baseline Ca2+ in hypoxic PASMC, however it further increased Ca2+ response to thromboxane. 100% O2 exposure of hypoxic PASMC treated with SNP triggered a markedly elevated Ca2+ response to thromboxane compared to all other groups. AC activity was impaired by hypoxia; addition of SNP almost fully inhibited AC in both normoxic and hypoxic PASMC. Increased Ca2+ mobilization and impaired cAMP generation in SNP-treated hypoxic PASMC were normalized by treatment with FeTTPs.

Conclusion(s): ROS and RNS levels are altered by SNP, hypoxia and hyperoxia. Hypoxia enhances contraction and inhibits relaxation pathways in PASMC; NO exacerbates these changes, while scavenging of RNS reverses them. Even transient exposure to 100% O2 sensitizes the contractile pathway in hypoxic PASMC treated with NO. We conclude that concurrent use of NO with 100% O2 in PPHN carries risk of unchecked vasoconstriction.