| Pulmonary vasodilation is mediated through the activation of protein kinase G (PKG) via a signaling pathway involving nitric oxide (NO), natriuretic peptides (NP), and cyclic guanosine monophosphate (cGMP). In pulmonary hypertension secondary to congenital heart disease, this pathway is endogenously activated by an early vascular upregulation of NO and increased myocardial B-type NP expression and release. In the treatment of pulmonary hypertension, this pathway is exogenously activated using inhaled NO or other pharmacological agents. Despite this activation of cGMP, vascular dysfunction is present, suggesting that NO-cGMP independent mechanisms are involved and were the focus of this study. Exposure of pulmonary artery endothelial or smooth muscle cells (PASMC) to the NO donor, Spermine NONOate (SpNONOate), increased peroxynitrite (ONOO-) generation and PKG-1alpha nitration, while PKG-1alpha activity was decreased. These changes were prevented by superoxide dismutase (SOD) or manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) and were mimicked by the ONOO- donor, 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). Peripheral lung extracts from 4-week old lambs with increased pulmonary blood flow and pulmonary hypertension (Shunt lambs with endogenous activation of cGMP) or juvenile lambs treated with inhaled NO for 24h (with exogenous activation of cGMP) revealed increased ONOO- levels, elevated PKG-1alpha nitration, and decreased kinase activity without changes in PKG-1alpha protein levels. However, in Shunt lambs treated with L-arginine or lambs administered polyethylene glycol conjugated-SOD (PEG-SOD) during inhaled NO exposure, ONOO- and PKG-1alpha nitration levels were diminished, and kinase activity was preserved. Utilizing mass spectroscopy, we found that SIN-1 nitrates PKG-Ialpha at a tyrosine located in the cGMP binding site and at a tyrosine located in the ATP binding site of the enzyme. The tyrosine-phenylalanine mutants were both less susceptible to nitration than wild-type (WT) PKG-Ialpha. However, only the tyrosine located in the cGMP binding site was important for the nitration mediated decrease in the catalytic activity. Utilizing the crystal structure of protein kinase A, we developed a homology molecular model of PKG-Ialpha. Computer simulation of this model predicted that the nitration of PKG-Ialpha decreases the hydrogen bonding energy between the kinase and cGMP. A [³H]-cGMP binding assay confirmed that the nitration of PKG-Ialpha increased the K d and the Km of WT- but not the mutant PKG-Ialpha, for cGMP. AlamarBlue and cell counting assays demonstrated that PASMC transfected with either WT- or mutant PKG-Ialpha were less proliferative compared to the GFP transfected control cells. However, upon SIN-1 challenge, the PASMC expressing the WT-PKG-Ialpha exhibited a proliferative response comparable to that of the control cells, while the cells transfected with the mutant PKG-Ialpha did not display a change in their response. In addition, immunoblot and immunocytochemistry analyses demonstrated that PASMC transfected with WT- and mutant PKG-Ialpha exhibited a contractile phenotype, as illustrated by the increased levels of the contractile markers: MYH, calponin-1, SMA, and SM22-alpha and the decreased levels of the proliferative markers: vimentin and PCNA. However, when these cells were treated with SIN-1, the WT- expressing PASMC acquired a more proliferative phenotype compared to the cells transfected with the mutant. Together our data reveal that vascular dysfunction can occur, despite elevated levels of cGMP, due to a nitration dependent decrease in cGMP binding to PKG-Ialpha and the subsequent attenuation of the kinase activity. |
