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

Abstract

Regulation of cGMP-dependent protein kinase-mediated vasodilation by hypoxia-induced reactive species in ovine fetal pulmonary veins. Am J Physiol Lung Cell Mol Physiol 293: L1012-L1020, 2007. First published July 6, 2007 doi:10.1152/ajplung.00061.2007.-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 PKGmediated 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 (DCFH 2-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-I␣ 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. hypoxic vasoconstriction; vascular smooth muscle; protein kinase G GUANOSINE 3Ј,5Ј-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) is a serine/threonine kinase that plays an important role in the regulation of vascular smooth muscle (SMC) contractility (6,22,36). Activation of soluble guanylyl cyclase by nitric oxide (NO) leads to increased synthesis of the second messenger cGMP and stimulation of PKG (2, 27). PKG, in turn, mediates SMC relaxation by a number of mechanisms, including phosphorylation of myosin light chain phosphatase, lowering of intracellular free Ca 2ϩ levels, and desensitization of the contractile apparatus to Ca 2ϩ (6,22,36). Mammalian PKG exists in two major forms: PKG-I, a soluble enzyme consisting of ␣-and ␤-isoforms derived from alternative splicing from one gene, and PKG-II, a myristoylated, membrane-associated form derived from a second gene (23,36,46). PKG-I␣ is the primary isoform involv...

Caveolin-1 Deficiency Signaling Novel Mechanisms of Pulmonary Hypertension

“…These Cav1 null cells may experience mitochondria dysfunction, leading to nitrative stress as evident by peroxynitrite formation. In contrast to the observation in Cav1 -/-mice, hypoxia-induced PKG nitration is eNOSindependent but iNOS-dependent [45]. Thus, it is possible that both tissue hypoxia and inflammation will result in increased nitrative stress and PKG nitration through iNOS.…”

“…It has been shown that hypoxia impairs PKG activity and PKG-mediated relaxation in ovine fetal intrapulmonary veins through both downregulation of PKG expression and tyrosine nitration of PKG [45]. Other studies also show decreased PKG activity and resultant attenuated vasodilatory responses to exogenous NO and cGMP in rats following chronic hypoxia [44].…”

“…PKG nitration and resultant impairment of PKG activity in the pulmonary vasculature is also involved in the mechanism of PH induced by hypoxia [44,45]. It has been shown that hypoxia impairs PKG activity and PKG-mediated relaxation in ovine fetal intrapulmonary veins through both downregulation of PKG expression and tyrosine nitration of PKG [45].…”

Nitration of Tyrosine 247 Inhibits Protein Kinase G-1α Activity by Attenuating Cyclic Guanosine Monophosphate Binding

“…In addition, the nitration and subsequent attenuation of PKG activity in the right ventricle appears to be responsible for the deterioration of right ventricle function in a mouse model of pulmonary hypertensive induced by chronic hypoxia (43). However, the increase in protein nitration associated with hypoxia reduces PKG activity through changes at the transcriptional and post-translational levels (2). The clinical relevance of PKG nitration has also been shown by the observation that patients with idiopathic pulmonary arterial hypertension have increased PKG nitration in their lungs with no noticeable alteration in PKG protein levels (3).…”

“…Previous in vitro and in vivo studies have demonstrated that the nitration of cGMP-dependent protein kinase G-1␣ (PKG-1␣) 4 is a key post-translational event responsible for the impaired PKG activity in the lungs of acute and chronic pulmonary hypertensive lambs (1), mice with hypoxia-induced pulmonary hypertension (2), and humans with idiopathic pulmonary arterial hypertension (3). However, the tyrosine residues susceptible to this nitration event and the mechanism(s) by which nitration inhibit PKG-1␣ activity are unclear and were the focus of this study.…”