S-nitrosothiol repletion by an inhaled gas regulates pulmonary function

Abstract

NO synthases are widely distributed in the lung and are extensively involved in the control of airway and vascular homeostasis. It is recognized, however, that the O(2)-rich environment of the lung may predispose NO toward toxicity. These Janus faces of NO are manifest in recent clinical trials with inhaled NO gas, which has shown therapeutic benefit in some patient populations but increased morbidity in others. In the airways and circulation of humans, most NO bioactivity is packaged in the form of S-nitrosothiols (SNOs), which are relatively resistant to toxic reactions with O(2)/O(2)(-). This finding has led to the proposition that channeling of NO into SNOs may provide a natural defense against lung toxicity. The means to selectively manipulate the SNO pool, however, has not been previously possible. Here we report on a gas, O-nitrosoethanol (ENO), which does not react with O(2) or release NO and which markedly increases the concentration of indigenous species of SNO within airway lining fluid. Inhalation of ENO provided immediate relief from hypoxic pulmonary vasoconstriction without affecting systemic hemodynamics. Further, in a porcine model of lung injury, there was no rebound in cardiopulmonary hemodynamics or fall in oxygenation on stopping the drug (as seen with NO gas), and additionally ENO protected against a decline in cardiac output. Our data suggest that SNOs within the lung serve in matching ventilation to perfusion, and can be manipulated for therapeutic gain. Thus, ENO may be of particular benefit to patients with pulmonary hypertension, hypoxemia, and/or right heart failure, and may offer a new therapeutic approach in disorders such as asthma and cystic fibrosis, where the airways may be depleted of SNOs.

Figures

Figure 1

ENO is a gaseous nitrosant. (A) Nitrogen gas (0.6 liters/min) was passed through a Fisher Milligan gas washer containing selected concentrations of ENO (dissolved in ethanol) and then blended with a room air gas flow (6 liters/min) in a pediatric ventilator. Samples were collected and analyzed by GCMS for ENO content. Data are the mean ± SD of three separate experiments. (B) Mass spectrometry trace of gas chromatography eluate. A gas sample from the ventilator was loaded onto a GS-Q gas chromatography column at 60°C, and column effluent was monitored by negative ion mass spectrometry. ENO elution can be followed at 30 and 46 atomic mass units (amu; the O-nitroso bond is cleaved in the ionization process). The 30-amu trace is shown. The large peak at 3.2 min represents ENO and the very small peak at 1.7 min represents NO. Examination of the column effluent at other atomic masses revealed the complete absence of NO2. (C) The ability of ENO and NO to nitrosate 0.5 mM glutathione (equivalent to airway concentrations) was assessed in vitro. The SNO yield is 0.5 SNO per NO added—in keeping with the nitrosation via a N2O3 intermediate (27)—and ≈1.0 SNO per ENO added, indicating direct transnitrosation of glutathione.

Figure 2

ENO mitigates hypoxia-induced pulmonary hypertension. Neonatal pigs were instrumented and ventilated as previously described (26). Pulmonary hypertension was induced by lowering the FiO2 to 14% (Hypoxia). (A) PAP; (B) MnBP; (C) PVR; (D) SVR; (E) PaO2; and (F) CO. Changes in PAP, PVR, and PaO2 are significant, whereas systemic effects are not evident. ●, 0.0025% ENO; ○, 0.025% ENO; and ▾, 0.125% ENO. *, Significantly different from hypoxia (P < 0.05).

Figure 3

ENO increases the SNO concentration in the lung. ENO or NO was added to the inhaled gas of neonatal pigs breathing room air in three randomized doses. Lung aspirates were collected and assayed immediately for SNO and for protein content. SNO concentrations normalized to protein content are expressed as fold-increase over endogenous levels (0.145 ± 0.03 nM/μg for NO group, 0.22 ± 0.04 nM/μg for ENO group). *, P < 0.05, significantly different from precompound.

Figure 4

Effect of discontinuation of ENO and NO in a piglet model of lung injury. Pulmonary hypertension was induced in intubated neonatal pigs breathing 100% oxygen by repeated saline lavage. Either NO (20 ppm followed by 5 ppm), or ENO (≈5–10 ppm), or nothing (Control, i.e., values after lung injury) was then added to the inhaled gas for 2 hr. (Dosing was designed to achieve comparable reductions in PVR.) Control animals showed a progressive rise in PVR (61%) and fall in CO (40%) over the 2-hr period that followed injury; and accordingly, PAP remained essentially unchanged (±8%). (A) A-a O2 ratio; the mean PaO2 is also shown; (B) PVR; (C) PAP; (D) fall in CO (compared with baseline, i.e., values before injury). Hemodynamics were measured every 5 min for 20 min after abrupt discontinuation of inhaled gases (post). *, P < 0.05 and #, P = 0.06, compared with control.