Oral or Poster Presentation
Concurrent Session 4B - Neo Cardiovascular Science & Hemodynamics
Introduction: Persistent pulmonary hypertension of the newborn (PPHN) features hypoxemia and pulmonary vasoconstriction. The adenylyl cyclase (AC) pathway (upon stimulation of Gαs-coupled receptors, AC converts ATP to cAMP) is crucial for pulmonary vasodilation and cardiac contraction. We previously reported inhibited AC enzyme activity due to cysteine nitrosylation in pulmonary artery myocytes exposed to hypoxia. Globally AC activity is low in PPHN tissues, and in hypoxic arterial myocytes; inhibition of AC activity in PPHN persists in vitro, is worsened by exposure to hypoxia in vitro, and by nitric oxide in vitro. We sought to determine which AC isoforms were susceptible to inhibition.
Methods: AC content, isoform expression and catalytic activity, in presence or absence of forskolin and/or trans-nitrosylating agent s-nitrosocysteine, were measured in neonatal porcine pulmonary artery myocytes (PASMC). AC isoforms were stably overexpressed in HEK293T cells and exposed to 72hr hypoxia (10%O2) or normoxia (21%O2), or to s-nitrosocysteine. Computational models of human AC isoforms were built using homology modeling to investigate their catalytic and allosteric binding sites.
Results: PASMC express mainly ACs 3,6,7 and 9. Only AC6 is inhibited by hypoxia or by nitrosylation. All AC isoforms have a conserved ATP binding site. The AC6 forskolin binding site (allosteric activation site) is defined by V514, N518 and S942 amino acids. Site directed mutational analysis of these residues was pursued and results will be discussed. Substitution of cysteine residues results in conformational change suggesting AC6 structure may be stabilized by disulfide bonds that could be sensitive to nitrosylation.
Conclusion: AC inhibition by hypoxia or nitric oxide exposure is a critical element of PPHN. AC6 is the only AC isoform thus inhibited. Structural detail on AC6 reveals mechanisms by which it can be inhibited. Potential for selective reactivation of AC6 as treatment for PPHN depends on the uniqueness of the AC6 forskolin binding site.