Chronic Hypoxia Induces Peroxynitrite‐Mediated Post‐Translational Modification of cGMP Dependent Protein Kinase (PKG) in Ovine Fetal Pulmonary Arteries.

Abstract

We have reported that chronic hypoxia in utero attenuates PKG-mediated relaxation in fetal pulmonary arteries, partly due to inhibition of PKG activity and partly due to enhanced ROCK activity (AJP, 2006). To determine the mechanisms of decreased PKG activity, we investigated the role of reactive oxygen and nitrogen species (ROS/RNS) in regulating PKG kinase activity via post-translational modification. Pregnant ewes were at 3,801m altitude from ~35–140 days gestation (Chronic Hypoxia CH) and fetuses were delivered near term. Matched sea-level controls were also used. Intrapulmonary arteries from CH and control fetuses were isolated and PKG immunoprecipitated from tissue extracts. PKG immuno-complex was sedimented, washed and fractionated by SDS-PAGE and probed for nitrotyrosine or visualized with silver stain. We found increased PKG protein but also increased PKG nitration in CH arteries. We also found that PKG binding partners were significantly altered in CH arteries compared to controls. Among the binding partners, there was increased binding with calmodulin. We speculate that PKG nitration is involved in the decreased PKG activity as well as the altered interaction with its binding partners. We wish to identify the sites of PKG nitration, confirm that PKG nitration alters PKG activity, identify critical binding partners and determine if and how they modify PKG protein function. HL059435 & 075187 to JUR and HD-31226 to LDL.