Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow: a role for oxidative stress

Abstract

Cardiac defects associated with increased pulmonary blood flow result in pulmonary vascular dysfunction that may relate to a decrease in bioavailable nitric oxide (NO). An 8-mm graft (shunt) was placed between the aorta and pulmonary artery in 30 late gestation fetal lambs; 27 fetal lambs underwent a sham procedure. Hemodynamic responses to ACh (1 microg/kg) and inhaled NO (40 ppm) were assessed at 2, 4, and 8 wk of age. Lung tissue nitric oxide synthase (NOS) activity, endothelial NOS (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS), and heat shock protein 90 (HSP90), lung tissue and plasma nitrate and nitrite (NO(x)), and lung tissue superoxide anion and nitrated eNOS levels were determined. In shunted lambs, ACh decreased pulmonary artery pressure at 2 wk (P < 0.05) but not at 4 and 8 wk. Inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). In control lambs, ACh and inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). Total NOS activity did not change from 2 to 8 wk in control lambs but increased in shunted lambs (ANOVA, P < 0.05). Conversely, NO(x) levels relative to NOS activity were lower in shunted lambs than controls at 4 and 8 wk (P < 0.05). eNOS protein levels were greater in shunted lambs than controls at 4 wk of age (P < 0.05). Superoxide levels increased from 2 to 8 wk in control and shunted lambs (ANOVA, P < 0.05) and were greater in shunted lambs than controls at all ages (P < 0.05). Nitrated eNOS levels were greater in shunted lambs than controls at each age (P < 0.05). We conclude that increased pulmonary blood flow results in progressive impairment of basal and agonist-induced NOS function, in part secondary to oxidative stress that decreases bioavailable NO.

Fig. 1.

Changes in pulmonary artery pressure (PAP), expressed as percent change from baseline, in response to ACh (1 μg/kg), an endothelium-dependent agent, and inhaled nitric oxide (NO; 40 ppm), an endothelium-independent agent, in shunted lambs at 2, 4, and 8 wk of age. At 2 wk of age, PAP decreased significantly from baseline in response to ACh and inhaled NO. At 4 and 8 wk of age, PAP decreased significantly in response to inhaled NO but not to ACh. n = 5 for each group. Values are means ± SD. *P < 0.05 compared with baseline.

Fig. 2.

Lung tissue endothelial nitric oxide synthase (eNOS) protein expression in sham-operated control and shunted lambs at 2, 4, and 8 wk of age. Top: representative Western blots are shown for eNOS protein extracts prepared from lung tissue separated on a 7.5% SDS-polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against eNOS and reprobed with β-actin to demonstrate equal loading. Bottom: densitometric values for eNOS protein shown relative to control. *P < 0.05 vs. age-matched control; n = 5 for each group. Values are means ± SD.

Fig. 3.

In vivo expression of inducible NOS (iNOS) in the pulmonary vasculature of sham-operated control and shunted lambs at 2- (A), 4 (B), and 8 (C) wk of age. The expression in the smooth muscle cell layer was identified by colocalization with caldesmon and in the endothelial cell layer by colocalization with eNOS. iNOS expression is in green, and caldesmon and eNOS are in red. For each cell layer, controls are shown at the top and shunted are shown at the bottom. Images were taken as z-stack using a ×40 objective. The merged images are an orthogonal view showing a clear colocalization in x-, y-, and z-planes indicating that iNOS localized to both the smooth muscle cell and endothelial cell layers. The magnified view of the boxed area is also shown adjacent to merged image. Results are representative of 6 different sets of lambs. D represents the mean fluorescent intensity of iNOS for control and shunted lambs at each age group. iNOS quantification by fluorescent intensity did not demonstrate differences between control and shunted lambs at any age.

Fig. 4.

Total NOS activity and calcium-independent NOS activity in lung tissue from sham-operated control and shunted lambs at 2, 4, and 8 wk of age. In sham-operated control lambs, total NOS activity did not change from 2 to 8 wk of age (white bars). In shunted lambs, total NOS activity increased from 2 to 8 wk of age (ANOVA) and was greater at 4 and 8 wk of age than at 2 wk of age (black bars). Total NOS activity was greater than calcium-independent NOS activity in both sham-operated control lambs (gray bars) and shunted lambs (crossed bars) at all ages. n = 5 for each group. Values are means ± SD. *P < 0.05 compared with 2-wk shunt; †P < 0.05 compared with calcium-independent NOS activity within each age group; §P < 0.05 compared with 2-wk sham-operated control.

Fig. 5.

Plasma (top) and lung tissue (bottom) nitrate and nitrite (NO_x) levels at 2, 4, and 8 wk of age in sham-operated control and shunted lambs. Plasma and tissue NO_x levels decreased from 2 to 8 wk of age in sham-operated control and shunted lambs (ANOVA). Plasma and tissue NOx levels were greater in shunted lambs than sham-operated controls at 2 wk. n = 5 for each group. Values are means ± SD. *P < 0.05 compared with age-matched sham-operated control; †P < 0.05 compared with 2-wk sham-operated control; §P < 0.05 compared with 2-wk shunt.

Fig. 6.

NOx levels expressed relative to total NOS activity at 2, 4, and 8 wk of age in sham-operated control and shunted lambs. Relative NOx levels were lower than controls at 4 and 8 wk of age. n = 5 for each group. *P < 0.05 compared with age-matched sham-operated control.

Fig. 7.

Lung tissue heat shock protein 90 (HSP90) expression in sham-operated control and shunted lambs at 2, 4, and 8 wk of age. Top: representative Western blots are shown for HSP90 protein extracts prepared from lung tissue separated on a 7.5% SDS-polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against HSP90 and reprobed with β-actin to demonstrate equal loading. Bottom: densitometric values for HSP90 protein shown relative to control. n = 5 for each group. Values are means ± SD.

Fig. 8.

A: superoxide levels in lung tissue in sham-operated control and shunted lambs at 2, 4, and 8 wk of age estimated by electron paramagnetic resonance (EPR) assay using 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine·HCl (CMH), which forms a stable chemical product with superoxide. Superoxide concentrations increased from 2 to 8 wk of age in both sham-operated control and shunted lambs (ANOVA) and were greater in shunted lambs than sham-operated control lambs at each age. B: S-ethylisothiourea hydrobromide (ETU)-mediated inhibition of NOS resulted in almost no change in EPR amplitude of control lambs, whereas shunted lambs showed up to ∼30% reduction in signal, suggesting that uncoupled eNOS contributed to superoxide levels in shunted but not control lambs. n = 5 for each group. Values are means ± SD. *P < 0.05 compared with age-matched sham-operated control; †P < 0.05 compared with previous sham-operated control; §P < 0.05 compared with previous shunt.

Fig. 9.

Developmental increases in eNOS nitration in shunted lambs. Nitrated eNOS protein levels were determined by immunoprecipitation (IP) using specific antiserum raised against eNOS in tissue extracts prepared from 2- (n = 4), 4- (n = 5), and 8-wk-old (n = 4) shunted and age-matched control lambs. Immunoprecipitated extracts were separated on 4–20% SDS-polyacrylamide gradient gels, electrophoretically transferred to Hybond membranes, and analyzed using antisera against either eNOS or 3-nitrotyrosine residues. Representative Western Blots are shown. Densitometric analysis indicates that the levels of nitrated eNOS protein are increased in shunted lambs and that this nitration increases over time. Values are means ± SE. *P < 0.05 vs. age matched control; †P < 0.05 vs. previous age. IB, immunoblot.