Adenylyl Cyclase Isoform 6 in the Pulmonary Artery Is Inhibited by Hypoxia via Cysteine Nitrosylation

Abstract

Persistent pulmonary hypertension of the newborn (PPHN) is a hypoxic disorder of pulmonary vascular relaxation, mediated in part by adenylyl cyclase (AC). Neonatal pulmonary arteries (PAs) express mainly the AC6 isoform, followed by AC3, AC7, and AC9. AC6 expression is upregulated in hypoxia. We reported AC enzyme inhibition due to S-nitrosylation in PPHN PAs and in PA myocytes exposed to hypoxia. We hypothesized that hypoxia promotes cysteine thiol nitrosylation of AC6, impairing cAMP production. HEK293T cells stably expressing AC isoforms (AC3, 5, 6, 7, 9) or cysteine-to-alanine mutants AC6_C1004A, AC6_C1145A, or AC6_C447A were cultured in normoxia (21% O₂) or hypoxia (10% O₂) for 72 hours or challenged with nitroso donor S-nitrosocysteine (CysNO). AC activity was determined by real-time live-cell cAMP measurement (cADDis assay) or terbium-norfloxacin AC catalytic assay, with or without challenge by allosteric agonist forskolin; protein S-nitrosylation was detected by the biotin switch method and quantified by affinity precipitation. Only AC6 catalytic activity is inhibited in hypoxia or by S-nitrosylating agent in the presence or absence of forskolin; impaired cAMP production in hypoxia correlates with increased cysteine nitrosylation of AC6. Selective AC6 inhibition in pulmonary artery myocytes extinguishes AC sensitivity to inhibition by hypoxia. Alanine substitution of C1004, but not of other cysteines, decreases S-nitrosylation of AC6. AC activity is diminished in AC6_C1004A compared with AC6 wild type. Substitution of C1004 also extinguishes the inhibition of AC6 by hypoxia. We conclude AC6 is uniquely S-nitrosylated in hypoxia, inhibiting its activity and cAMP generation. We speculate that S-nitrosylation at C1004 may inhibit AC6 interaction with G_αs, playing a role in PPHN pathophysiology.

Keywords: ADCY6; S-nitrosylation; adenylyl cyclase; hypoxia; persistent pulmonary hypertension of the newborn.