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 24 h (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.

Fig. 1. Peroxynitrite (ONOO⁻) increases protein kinase G-1α (PKG-1α) nitration, and decreases PKG activity in pulmonary arterial endothelial cells (PAEC)

ONOO⁻ formation induced by the nitric oxide (NO) and the superoxide donor, 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), was assayed by monitoring the ONOO⁻ dependent oxidation of dihydrorhodamine 123 (DHR 123) to rhodamine 123. The results are expressed as fold DHR oxidation compared to untreated control cultures. SIN-1 increases ONOO⁻ levels in PAEC, and this was attenuated by the addition of polyethylene glycol conjugated superoxide dismutase (PEG-SOD) or manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) (A). In addition, protein extracts (1000μg) were subjected to immunoprecipitation analysis using an antibody raised against PKG-1α. The immunoprecipitates were resolved using 4-20% Tris-SDS-Hepes PAGE and electrophoretically transferred to a PVDF membrane. The level of nitrated PKG-1α was then determined by probing the membranes with an antiserum raised against 3-nitrotyrosine. Blots were then stripped and re-probed for PKG-1α to normalize for the efficiency of the immunoprecipitation. A representative blot is shown. The exposure of PAEC to SIN-1 results in a significant increase in PKG-1α nitration (B). PEG-SOD or MnTMPyP both inhibit PKG-1α nitration (B). Using an ELISA based assay, we also found that SIN-1 attenuated total PKG activity, and this was prevented by pretreatment with PEG-SOD or MnTMPyP (C). Data are mean ± SEM, n=4, *p<0.05 vs. untreated, † p<0.05 vs SIN-1.

Fig. 2. Nitric oxide elevates ONOO⁻ levels, increases PKG-1α nitration, and decreases PKG activity in pulmonary arterial endothelial cells

ONOO⁻ formation induced by the NO donor, Spermine NONOate (SpNONOate), was assayed by monitoring the ONOO⁻ dependent oxidation of DHR 123 to rhodamine 123. The results are expressed as fold DHR oxidation compared to untreated control cultures. SpNONOate increases ONOO⁻ levels in PAEC, and this was attenuated by the addition of PEG-SOD or MnTMPyP (A). In addition, protein extracts (1000μg) were subjected to immunoprecipitation analysis using an antibody raised against PKG-1α. The immunoprecipitates were resolved using 4-20% Tris-SDS-Hepes PAGE and electrophoretically transferred to a PVDF membrane. The level of nitrated PKG-1α was then determined by probing the membranes with an antiserum raised against 3-nitrotyrosine. Blots were then stripped and re-probed for PKG-1α to normalize for the efficiency of the immunoprecipitation. A representative blot is shown. The exposure of PAEC to SpNONOate results in a significant increase in PKG-1α nitration (B). PEG-SOD or MnTMPyP both inhibit PKG-1α nitration (B). Using an ELISA based assay, we also found that SpNONOate attenuated total PKG activity, and this was prevented by pretreatment with PEG-SOD or MnTMPyP (C). Data are mean ± SEM, n=4, *p<0.05 vs. untreated, † p<0.05 vs SpNONOate.

Fig. 3. Peroxynitrite increases PKG-1α nitration, and decreases PKG activity in pulmonary arterial smooth muscle cells (PASMC)

ONOO⁻ formation induced by the NO and the superoxide donor, SIN-1, was assayed by monitoring the ONOO⁻ dependent oxidation of DHR 123 to rhodamine 123. The results are expressed as fold DHR oxidation compared to untreated control cultures. SIN-1 increases ONOO⁻ levels in PASMC, and this was attenuated by the addition of PEG-SOD or MnTMPyP (A). In addition, protein extracts (1000μg) were subjected to immunoprecipitation analysis using an antibody raised against PKG-1α. The immunoprecipitates were resolved using 4-20% Tris-SDS-Hepes PAGE and electrophoretically transferred to a PVDF membrane. The level of nitrated PKG-1α was then determined by probing the membranes with an antiserum raised against 3-nitrotyrosine. Blots were then stripped and re-probed for PKG-1α to normalize for the efficiency of the immunoprecipitation. A representative blot is shown. The exposure of PASMC to SIN-1 results in a significant increase in PKG-1α nitration (B). PEG-SOD or MnTMPyP both inhibit PKG-1α nitration (B). Using an ELISA based assay, we also found that SIN-1 attenuated total PKG activity, and this was prevented by pretreatment with PEG-SOD or MnTMPyP (C). Data are mean ± SEM, n=4, *p<0.05 vs. untreated, † p<0.05 vs SIN-1.

Fig. 4. Nitric oxide elevates ONOO⁻ levels, increases PKG-1α nitration, and decreases PKG activity in pulmonary arterial smooth muscle cells

ONOO⁻ formation induced by the NO donor, SpNONOate, was assayed by monitoring the ONOO⁻ dependent oxidation of DHR 123 to rhodamine 123. The results are expressed as fold DHR oxidation compared to untreated control cultures. SpNONOate increases ONOO⁻ levels in PASMC, and this was attenuated by the addition of PEG-SOD or MnTMPyP (A). In addition, protein extracts (1000μg) were subjected to immunoprecipitation analysis using an antibody raised against PKG-1α. The immunoprecipitates were resolved using 4-20% Tris-SDS-Hepes PAGE and electrophoretically transferred to a PVDF membrane. The level of nitrated PKG-1α was then determined by probing the membranes with an antiserum raised against 3-nitrotyrosine. Blots were then stripped and re-probed for PKG-1α to normalize for the efficiency of the immunoprecipitation. A representative blot is shown. The exposure of PASMC to SpNONOate results in a significant increase in PKG-1α nitration (B). PEG-SOD or MnTMPyP both inhibit PKG-1α nitration (B). Using an ELISA based assay, we also found that SpNONOate attenuated total PKG activity, and this was prevented by pretreatment with PEG-SOD or MnTMPyP (C). Data are mean ± SEM, n=4, *p<0.05 vs. untreated, † p<0.05 vs SpNONOate.

Fig. 5. ONOO⁻ decreases the kinase activity of recombinant bovine PKG1-α protein

The kinase activity of purified recombinant bovine PKG-1α protein was measured by an ELISA in the presence of ONOO⁻. PKG1-α kinase activity is decreased in the presence of ONOO⁻. We also confirmed that the presence of 4.7% NaOH (the solvent for ONOO⁻) did not alter PKG-1α kinase activity. Data are mean ± SEM, n=3; *p<0.05 vs. untreated.

Fig. 6. The SIN-1 mediated attenuation of PKG kinase activity is independent of cellular cGMP levels

PAEC and PASMC were treated with B-type natriuretic peptide (BNP) (10μM) in two doses of 5μM, 30min apart for a total duration of 1h in the presence or absence of SIN-1 (500μM, 30min) pretreatment. Using an ELISA based assay, we demonstrate in PAEC that BNP increases cGMP levels in the presence or absence of SIN-1 (A). Immunoblot analysis shows that SIN-1 decreases the PKG dependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at Serine²³⁹ induced by BNP (B). Similarly, our results show in PASMC that SIN-1 does not alter cGMP levels (C) but attenuates the BNP induced increase in pSer²³⁹VASP levels (D). Data are mean ± SEM, n=3, *p<0.05 vs. untreated, † p<0.05 vs BNP.

Fig. 7. Shunt lambs have elevated ONOO⁻ levels, increased PKG-1α nitration, and decreased PKG activity

ONOO⁻ levels were determined by monitoring the ONOO⁻ dependent oxidation of DHR 123 to rhodamine 123 in the peripheral lung tissue obtained from 4-week old control and Shunt lambs treated with or without L-arginine. The results, expressed as fold DHR oxidation, show that vehicle treated Shunt lambs have elevated ONOO⁻ levels compared to vehicle control lambs. However, in Shunts treated with L-arginine, ONOO⁻ levels are comparable to vehicle control lambs (A). Protein extracts (25μg) prepared from the peripheral lung tissue of Shunt and age-matched control lambs were resolved using 4-20% Tris-SDS-Hepes PAGE, electrophoretically transferred to a PVDF membrane, and then subjected to Western blot analysis using a specific antiserum raised against PKG-1α. A representative blot is shown. Densitometric analysis indicated that the PKG-1α protein levels are unchanged between Shunt and control lambs (B). Protein levels were normalized by re-probing the membrane with an antibody to β-actin. In addition, protein extracts (1000μg) were subjected to immunoprecipitation analysis using an antibody raised against PKG-1α. The immunoprecipitates were resolved using 4-20% Tris-SDS-Hepes PAGE and electrophoretically transferred to a PVDF membrane. The level of nitrated PKG-1α was then determined by probing the membranes with an antiserum raised against 3-nitrotyrosine. Blots were then stripped and re-probed for PKG-1α to normalize for the efficiency of the immunoprecipitation. The immunoblot analysis shows that PKG-1α nitration is increased in vehicle treated Shunt lambs. However, there is a significant reduction in PKG-1α nitration in Shunts treated with L-arginine (C). Using an ELISA based assay, we also found that total PKG activity is attenuated in vehicle treated Shunt lambs. However, in Shunt lambs treated with L-arginine, there is no significant decrease in total PKG kinase activity (D). Data are mean ± SEM, n=4, *p<0.05 vs. vehicle control, † p<0.05 vs. vehicle treated Shunt lambs.

Fig. 8. Inhaled NO elevates ONOO⁻ levels, increases PKG-1α nitration, and decreased PKG activity

ONOO⁻ levels were determined by monitoring the ONOO⁻ dependent oxidation of DHR 123 to rhodamine 123 in the peripheral lung tissue obtained from 1-month old lambs prior to and post exposure to 24h of inhaled NO in the presence of either PEG or PEG-SOD. The results, expressed as fold DHR oxidation, show that in PEG treated lambs inhaled NO increases ONOO⁻ levels. However, in lambs treated with PEG-SOD, ONOO⁻ levels do not increase (A). Protein extracts (25μg) prepared from the peripheral lung tissue of these lambs were resolved using 4-20% Tris-SDS-Hepes PAGE, electrophoretically transferred to a PVDF membrane, and then subjected to Western blot analysis using a specific antiserum raised against PKG-1α. A representative blot is shown. Densitometric analysis indicates that the PKG-1α protein levels are unchanged by inhaled NO exposure (B). Protein levels were normalized by re-probing the membrane with an antibody to β-actin. In addition, protein extracts (1000μg) were subjected to immunoprecipitation analysis using an antibody raised against PKG-1α. The immunoprecipitates were resolved using 4-20% Tris-SDS-Hepes PAGE and electrophoretically transferred to a PVDF membrane. The level of nitrated PKG-1α was then determined by probing the membranes with an antiserum raised against 3-nitrotyrosine. Blots were then stripped and re-probed for PKG-1α to normalize for the efficiency of the immunoprecipitation. The immunoblot analysis shows that PKG-1α nitration is increased by inhaled NO. However, there is a significant reduction in PKG-1α nitration in the lambs treated with PEG-SOD (C). Using an ELISA based assay, we also found that total PKG activity is attenuated in PEG treated lambs exposed to inhaled NO. However, in PEG-SOD treated lambs, there is no significant decrease in total PKG kinase activity (D). Data are mean ± SEM, n=4, *p<0.05 vs. pre+PEG, † p<0.05 vs. post+PEG.