Nitric oxide- and nitric oxide donors-induced relaxation and its modulation by oxidative stress in piglet pulmonary arteries

Abstract

Inhaled nitric oxide (iNO) is widely used in the treatment of pulmonary hypertension while inhaled NO donors have been suggested as an alternative therapy. The differential susceptibility to inactivation by oxidative stress and oxyhaemoglobin of NO and two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) were analysed in isolated endothelium-denuded pulmonary arteries from 2-week-old piglets stimulated with U46619. NO, SNAP and SNP relaxed the arteries (pIC(30)=7.73+/-0.12, 7.26+/-0.17 and 6.43+/-0.13, respectively) but NO was not detected electrochemically in the bath after the addition of SNP and only at concentrations at which SNAP produced more than 50% relaxation. The sGC inhibitor ODQ (10(-6) M) or the sarcoplasmic Ca(2+)-ATPase thapsigargin (2x10(-6) M) markedly inhibited the relaxation induced by NO, SNAP and SNP. Addition of oxyhaemoglobin (3x10(-7) M) or diethyldithiocarbamate (1 mM) markedly inhibited NO- (pIC(30)=6.88+/-0.07 and 6.92+/-0.18, respectively), weakly inhibited SNAP- and had no effect on SNP-induced relaxation. Xanthine oxidase (5 mu ml(-1)) plus hypoxanthine (10(-4) M) markedly inhibited NO- (pIC(30)=6.96+/-0.12) but not SNAP- or SNP-induced relaxation. Superoxide dismutase (SOD), MnCl(2), diphenileneiodonium and exposing the luminal surface of the rings outwards (inversion) potentiated the relaxant responses of NO (pIC(30)=8.52+/-0.16, 8.23+/-0.11, 8.01+/-0.11 and 8.20+/-0.10, respectively). However, SOD did not modify the NO detected by the electrode and had no effect on SNAP- or SNP-induced relaxation. Therefore, the kinetics and local distribution of NO release of NO donors influence the susceptibility to the scavenging effects of oxyhaemoglobin and superoxide.

Figure 1

Relaxant responses to NO, SNAP and SNP in endothelium-denuded piglet pulmonary arteries and electrochemically detected NO in the organ bath. (A) Original simultaneous recordings of the relaxant effects of NO and the NO detected in the organ bath. NO was added as indicated by the arrows (concentrations are expressed in nM). A small artefact (downward deflection) can be observed upon addition of the NO solution mostly due to the extremely high sensitivity of the electrode to changes in temperature. (B) Representative traces of the time-course of the effects of a single concentration of NO (5×10⁻⁸ M), SNAP (3×10⁻⁷ M) and SNP (3×10⁻⁶ M). (C) The averaged results of relaxation (solid symbols) induced by NO (n=9, circles), SNAP (n=4, diamonds) and SNP (n=3, squares) and the NO detected electrochemically (open symbols). Results (mean±s.e.mean) of relaxation are expressed as a percentage of control tone induced by 10⁻ M U46619. The dashed line indicates the theoretical NO added (note that detected NO is lower than NO added indicating NO loss during pipetting and diffusion in the bath). ND not detectable.

Figure 2

Effects of the sGC inhibitor ODQ (10⁻⁶ M) and the SERCA inhibitor thapsigargin (2×10⁻⁶ M) on (A) NO-, (B) SNAP- and (C) SNP-induced relaxation in endothelium denuded pulmonary arteries stimulated with 10⁻⁷ M U46619. Results are expressed mean±s.e.mean (n=5–7) and ^** denote P<0.05 and P<0.01 vs control.

Figure 3

Effects of the oxyhaemoglobin (3×10⁻⁷ M) on (A) NO-, (B) SNAP- and (C) SNP-induced relaxation in endothelium denuded pulmonary arteries stimulated with 10⁻⁷ M U46619. Results are expressed mean±s.e.mean (n=5–6). ^* and ^** denote P<0.05 and P<0.01 vs control.

Figure 4

Effects of increased levels of superoxide on (A) NO-, (B) SNAP- and (C) SNP-induced relaxation in endothelium denuded pulmonary arteries stimulated with 10⁻⁷ M U46619. Superoxide was increased by adding XO (5 mu ml⁻¹) plus HX (10⁻⁴ M) or by inhibiting SOD with DETCA (1 mM). Results are expressed mean±s.e.mean (n=5–8). ^* and ^** denote P<0.05 and P<0.01 vs control.

Figure 5

Effects of the superoxide scavengers superoxide dismutase (SOD, 100 u ml⁻¹) and MnCl₂ (10⁻⁴ M) on (A) NO-, (B) SNAP- and (C) SNP-induced relaxation in endothelium denuded pulmonary arteries stimulated with 10⁻⁷ M U46619. Results are expressed mean±s.e.mean (n=4–8). ^* and ^** denote P<0.05 and P<0.01 vs control.

Figure 6

Effects of SOD (100 u ml⁻¹), XO (5 mu ml⁻¹) plus HX (10⁻⁴ M) and DETCA (1 mM) on the NO concentrations detected electrochemically in the organ bath after the addition of NO. Results are expressed mean±s.e.mean (n=5–9). ^* and ^** denote P<0.05 and P<0.01 vs control.

Figure 7

(A) Effects of inhibitors of the main enzymatic systems producing superoxide, rotenone (5×10⁻⁵ M), DPI (10⁻⁵ M), L-NAME (10⁻⁴ M), indomethacin (10⁻⁵ M), AA861 (10⁻⁵ M), SKF 525A (10⁻⁵ M) and oxypurinol (10⁻⁴ M) on NO-induced relaxation in endothelium denuded pulmonary arteries stimulated with 10⁻⁷ M U46619 (n=5–6). (B) Effects of the inversion procedure. Control are normal non inverted arteries, inverted indicates arteries in which the adventitia faces inward and inverted ×2 indicates arteries inverted twice in which (as control arteries) the adventitia faces outward. Results are expressed mean±s.e.mean (n=4–6). ^* and ^** denote P<0.05 and P<0.01 vs control.

Figure 8

A model showing the hypothesized role of the adventitial oxidant barrier and the different susceptibility of NO, SNAP and SNP. See text for explanation.