Attenuated vasodilatation in lambs with endogenous and exogenous activation of cGMP signaling: Role of protein kinase G nitration

Abstract

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 to the NO donor, Spermine NONOate (SpNONOate), increased peroxynitrite (ONOO−) generation and PKG-1α nitration, while PKG-1α activity was decreased. These changes were prevented by superoxide dismutase (SOD) or manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) and 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-1α nitration, and decreased kinase activity without changes in PKG-1α 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-1α nitration were diminished and kinase activity was preserved. Together our data reveal that vascular dysfunction can occur, despite elevated levels of cGMP, due to PKG-1α nitration and subsequent attenuation of activity.

Pulmonary Hypertension Is a Probable NO/ONOO−Cycle Disease: A Review

“…is study followed an earlier one showing that nitration of the G kinase lowered its responsiveness to cGMP stimulation and therefore lowered its ability to produce NOstimulated vasodilation and such attenuated vasodilation was also demonstrated in this study [239]. One part of the study showed that inhaled NO produced elevation of ONOO − , increased G kinase nitration, and decreased G kinase activity, providing a mechanism for lowered responsiveness to inhaled NO with time.…”

“…One part of the study showed that inhaled NO produced elevation of ONOO − , increased G kinase nitration, and decreased G kinase activity, providing a mechanism for lowered responsiveness to inhaled NO with time. Interestingly, in one part of the study [239], L-arginine was found to lower both ONOO − following simultaneous NO inhalation and also helped to maintain G kinase activity; this is important because L-arginine increases NO synthesis while lowering superoxide production from uncoupled nitric oxide synthases; the arginine �nding argues, therefore, that superoxide generated on the uncoupled nitric oxide synthase enzyme may have a key role here. e causal role of ONOO − here is demonstrated by its known role in protein nitration, the demonstrated causation of lowered G kinase activity by a compound that breaks down to produce ONOO − and also the demonstrated causal role in this study of both of the ONOO − precursors, NO, and superoxide.…”

“…e Lakshminrusimha et al study [231] showed that inhaled NO produced increases in ONOO − . e Aggarwal et al study [239] showed that inhaled NO not only raised ONOO − , but also was ONOO − dependent nitration of the G kinase enzyme, leading to lowered vasodilation, an important part of the rebound phenomenon. Oishi et al [237] found that both ONOO − and superoxide were elevated following NO inhalation and established a causal role of superoxide in the rebound response by using polyethylene glycol-conjugated SOD to inhibit the rebound response.…”

“…Aggarwal et al [239] showed that addition of an NO donor to pulmonary artery endothelial and smooth muscle cells increased ONOO − and increased nitration of protein kinase G. is response was shown to be blocked by either superoxide dismutase (SOD) or by an SOD mimic drug. is study followed an earlier one showing that nitration of the G kinase lowered its responsiveness to cGMP stimulation and therefore lowered its ability to produce NOstimulated vasodilation and such attenuated vasodilation was also demonstrated in this study [239].…”

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

“…Similarly, peripheral lung tissue from the control lambs and the lambs with pulmonary hypertension secondary to increased pulmonary blood flow (shunt) was prepared as described earlier (1). Cell extracts (25 g), lung tissue, or recombinant PKG-1␣ protein (1) were resolved using 4 -20% Tris-SDS-Hepes PAGE, electrophoretically transferred to Immuno-Blot TM PVDF membrane (Bio-Rad), and then blocked with 5% nonfat dry milk in Tris-buffered saline.…”