The role of nitric oxide synthase-derived reactive oxygen species in the altered relaxation of pulmonary arteries from lambs with increased pulmonary blood flow

Abstract

Congenital cardiac defects associated with increased pulmonary blood flow (Q(p)) produce pulmonary hypertension. We have previously reported attenuated endothelium-dependent relaxations in pulmonary arteries (PA) isolated from lambs with increased Q(p) and pulmonary hypertension. To better characterize the vascular alterations in the nitric oxide-superoxide system, 12 fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). Twin lambs served as controls. PA were isolated from these lambs at 4-6 wk of age. Electron paramagnetic resonance spectroscopy on fourth-generation PA showed significantly increased superoxide anion generation in shunt PA that were decreased to control levels following inhibition of nitric oxide synthase (NOS) with 2-ethyl-2-thiopseudourea. Preconstricted fifth-generation PA rings were relaxed with a NOS agonist (A-23187), a nitric oxide donor [S-nitrosyl amino penicillamine (SNAP)], polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), or H(2)O(2). A-23187-, PEG-SOD-, and H(2)O(2)-mediated relaxations were impaired in shunt PA compared with controls. Pretreatment with PEG-SOD significantly enhanced the relaxation response to A-23187 and SNAP in shunt but not control PA. Inhibition of NOS with nitro-L-arginine or scavenging superoxide anions with tiron enhanced relaxation to SNAP and inhibited relaxation to PEG-SOD in shunt PA. Pretreatment with catalase inhibited relaxation of shunt PA to A-23187, SOD, and H(2)O(2). We conclude that NOS catalyzes the production of superoxide anions in shunt PA. PEG-SOD relaxes shunt PA by converting these anions to H(2)O(2), a pulmonary vasodilator. The redox environment, influenced by the balance between production and scavenging of ROS, may have important consequences on pulmonary vascular reactivity in the setting of increased Q(p).

Figures

Fig. 1

Electron paramagnetic resonance (EPR) spectroscopy measurement of superoxide anions in pulmonary arteries (PA) isolated from control lambs (control) and lambs with fetal placement of an aortopulmonary shunt (shunt) and the effect of pretreatment with polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) (A) and NOS inhibitor 2-ethyl-2-thiopseudourea (ETU) (B). The decrease in waveform amplitude in control PA following pretreatment with PEG-SOD or ETU is normalized to 1.0. Data are presented as mean change in the amplitude of the EPR waveform of CM · product ± SD, following inhibition with PEG-SOD or ETU, from n = 6 control and n = 6 shunt lambs. Representative waveforms from each group are included. *P < 0.05 compared with control.

Fig. 2

Concentration-response curves to endothelial nitric oxide synthase stimulant and calcium ionophore A-23187 (A) and nitric oxide donor S-nitrosyl amino penicillamine (SNAP; B) in PA isolated from 4- to 6-wk-old control lambs an shunt lambs. All PA were pretreated with indomethacin (10−5 M) and propranolol (10−6 M) and were contracted with norepinephrine (NE; 3 × 10−7 M). Some PA were pretreated with 37.5 units/ml of PEG-SOD. There was a tendency toward enhanced relaxations in control PA following pretreatment with PEG-SOD (P = 0.06). *P < 0.05 compared with control and †P < 0.05 compared with shunt. Data were obtained from 13 control and 12 shunt lambs.

Fig. 3

Concentration-response curves to A-23187 (A), SNAP (B), and PEG-SOD (C) in control and shunt PA with or without pretreatment with superoxide scavenger tiron (10−5 M). Data were obtained from 13 control lambs and 8 shunt lambs. *P < 0.05 and **P < 0.01 compared with control, and †P < 0.05 compared with shunt PA.

Fig. 4

Concentration-response curves to A-23187 (A), SNAP (B), and PEG-SOD (C) in control and shunt PA with or without pretreatment with nitric oxide synthase inhibitor nitro-L-arginine (L-NNA; 10−3 M). Data were obtained from 13 control lambs and 10 shunt lambs. *P < 0.05 and **P < 0.01 compared with control, and †P < 0.05 and ††P < 0.001 compared with shunt PA.

Fig. 5

Concentration-response curves to A-23187 (A), SNAP (B), PEG-SOD (C), and hydrogen peroxide (D) in control and shunt PA with or without pretreatment with PEG-catalase (1,200 units/ml). *P < 0.05 and **P < 0.005 compared with control, and †P < 0.05 compared with shunt PA.