Regulation of cGMP-dependent protein kinase-mediated vasodilation by hypoxia-induced reactive species in ovine fetal pulmonary veins

Abstract

We previously reported that hypoxia attenuates cGMP-dependent protein kinase (PKG)-mediated relaxation in pulmonary vessels ( Am J Physiol Lung Cell Mol Physiol 279: L611–L618, 2003). To determine whether hypoxia-induced reactive oxygen and nitrogen species (ROS and RNS, respectively) may be involved in the downregulation of PKG-mediated relaxation, ovine fetal intrapulmonary veins were exposed to 4 h of normoxia or hypoxia, with or without scavengers of ROS [ N-acetylcysteine (NAC)] or peroxynitrite (quercetin and Trolox) and preconstricted with endothelin-1. Hypoxia decreased the relaxation response to 8-bromo-cGMP, PKG protein expression, and kinase activity and increased tyrosine nitration in PKG. However, ROS and RNS scavengers prevented these changes. To determine whether increased PKG nitration diminishes PKG activity, pulmonary vein smooth muscle cells (PVSMC) were exposed to shorter-term (30 min) hypoxia, which increased PKG nitration and decreased PKG activity but did not alter PKG protein expression. Increased dihydro-2,7-dichlorofluorescein diacetate (DCFH2-DA) fluorescence in PVSMC after 4 h or 30 min of hypoxia was not observed in the presence of NAC, quercetin, or Trolox, suggesting increased ROS and RNS production. Increased PKG nitration and the associated decrease in PKG activity in PVSMC after 30 min of hypoxia were also reversed on reoxygenation. The consequences of PKG nitration were assessed by exposure of purified PKG-Iα to peroxynitrite, which caused increased 3-nitrotyrosine immunoreactivity and inhibition of kinase activity. Our data suggest that, after 30 min of hypoxia, reversible covalent modification of PKG by hypoxia-induced reactive species may be an important mechanism by which the relaxation response to cGMP is regulated. However, after 4 h of hypoxia, PKG nitration and decreased PKG expression are involved.