title: Hypoxic regulation of pulmonary vascular smooth muscle cyclic guanosine monophosphate-dependent kinase by the ubiquitin conjugating system.
abstract: We previously reported that hypoxia attenuates nitric oxide-cyclic guanosine monophosphate (NO-cGMP)-mediated fetal pulmonary vessel relaxation by inhibiting cGMP-dependent protein kinase 1 (PKG1) activity, but not all the mechanisms by which acute hypoxia inhibits PKG1 activity have been delineated. Here we demonstrate for the first time, to the best of our knowledge, that acute hypoxia induces an accumulation of ubiquitinated PKG1 in ovine fetal and newborn pulmonary artery smooth muscle cells. Such a modification was not evident in ovine fetal systemic (cerebral) artery smooth muscle cells. The accumulation of polyubiquitinated PKG1 observed after 4 hours of hypoxia was affected neither by the activation of PKG1 kinase activity with the cell-permeable cGMP analogue 8-bromo-cGMP, nor by its inhibition with DT-3 in fetal pulmonary artery smooth muscle cells. Ubiquitinated PKG1α was unable to bind the cGMP analogue 8-(2-aminoethyl)thioguanosine-3',5' (AET)-cGMP, a ligand for the unmodified protein. Inhibition of the proteasomal complex with MG132 led to the accumulation of polyubiquitinated PKG1 in normoxia, indicating the involvement of the ubiquitin-26S proteasomal system in degradation and clearance of this protein under normoxic conditions. The ubiquitinated PKG1 under hypoxic conditions, however, was not predominantly targeted for proteasomal degradation. Importantly, reoxygenation reversed the acute hypoxia-induced accumulation of ubiquitinated PKG1. Our results suggest that the PKG1 ubiquitination induced by acute hypoxia plays a unique role in the regulation of the pulmonary vascular smooth muscle cell vasoreactivity and relaxation mediated by the NO-cGMP-PKG1 pathway.
authors: Department of Pediatrics, College of Medicine, University of Illinois at Chicago, 840 S. Wood St., M/C 856, Chicago, IL 60612, USA. ramchan@uic.edu
authors: Ramaswamy
authors: R
authors: Ramchandran
authors: Evgeny
authors: E
authors: Pilipenko
authors: Laura
authors: L
authors: Bach
authors: Aarti
authors: A
authors: Raghavan
authors: Sekhar P
authors: SP
authors: Reddy
authors: J Usha
authors: JU
authors: Raj
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015687:Cell Hypoxia; D002478:Cells, Cultured; D002536:Cerebral Arteries / Q000201:enzymology; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D008024:Ligands; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D010100:Oxygen / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011651:Pulmonary Artery / Q000201:enzymology; D017382:Reactive Oxygen Species / Q000378:metabolism; D012756:Sheep; D015398:Signal Transduction; D013997:Time Factors; D054875:Ubiquitination; D015854:Up-Regulation
chemical_list: D020536:Enzyme Activators; D016166:Free Radical Scavengers; D008024:Ligands; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP; D010100:Oxygen