S-nitrosoglutathione supplementation to ovalbumin-sensitized and -challenged mice ameliorates methacholine-induced bronchoconstriction

Abstract

S-nitrosoglutathione (GSNO) is an endogenous bronchodilator present in micromolar concentrations in airway lining fluid. Airway GSNO levels decrease in severe respiratory failure and asthma, which is attributable to increased metabolism by GSNO reductase (GSNOR). Indeed, we have found that GSNOR expression and activity correlate inversely with lung S-nitrosothiol (SNO) content and airway hyperresponsiveness (AHR) to methacholine (MCh) challenge in humans with asthmatic phenotypes (Que LG, Yang Z, Stamler JS, Lugogo NL, Kraft M. Am J Respir Crit Care Med 180: 226-231, 2009). Accordingly, we hypothesized that local aerosol delivery of GSNO could ameliorate AHR and inflammation in the ovalbumin-sensitized and -challenged (OVA) mouse model of allergic asthma. Anesthetized, paralyzed, and tracheotomized 6-wk-old male control and OVA C57BL/6 mice were administered a single 15-s treatment of 0-100 mM GSNO. Five minutes later, airway resistance to MCh was measured and SNOs were quantified in bronchoalveolar lavage (BAL). Duration of protection was evaluated following nose-only exposure to 10 mM GSNO for 10 min followed by measurements of airway resistance, inflammatory cells, and cytokines and chemokines at up to 4 h later. Acute delivery of GSNO aerosol protected OVA mice from MCh-induced AHR, with no benefit seen above 20 mM GSNO. The antibronchoconstrictive effects of GSNO aerosol delivered via nose cone were sustained for at least 4 h. However, administration of GSNO did not alter total BAL cell counts or cell differentials and had modest effects on cytokine and chemokine levels. In conclusion, in the OVA mouse model of allergic asthma, aerosolized GSNO has rapid and sustained antibronchoconstrictive effects but does not substantially alter airway inflammation.

Fig. 1.

Acute S-nitrosoglutathione (GSNO) repletion alters airway hyperresponsivity in the ovalbumin-sensitized and challenged (OVA) mouse. A: schematic showing experimental protocol. B and C: average (Ave) total pulmonary resistance (R_T) in alum control or OVA, tracheotomized, and mechanically ventilated mice was measured by flexiVent immediately after a 15-s dose of aerosolized PBS or 1–100 mM GSNO via ultrasonic nebulizer followed by methacholine (MCh) challenge. Data in C are area under the curve calculated using mean R_T at 0 mM MCh as a baseline within each treatment group. D: total S-nitrosothiols (SNOs) in bronchoalveolar lavage (BAL) from PBS- or GSNO-treated mice were quantified by mercury-coupled photolysis chemiluminescence. i.p., Intraperitoneal. Data in B and C are means ± SE (n = 5–11 per group) and in D are means ± SE (n = 5 per group). *P < 0.02, **P < 0.001.

Fig. 2.

Ameliorative effects of GSNO are sustained. A: schematic showing experimental protocol. B and C: airway resistance in alum control or OVA mice was measured by flexiVent 2 min to 4 h after nose-only inhalation of aerosolized PBS or 10 mM GSNO. Data in C are area under the curve (AUC) of Fig. 1B. D: total S-nitrosothiols in BAL from PBS- or GSNO-treated mice were quantified by mercury-coupled photolysis chemiluminescence. Data in B and C are means ± SE (n = 8–11 per group), *P < 0.001. Data in D are means + SE (n = 3–4 per group).

Fig. 3.

Aerosolized GSNO does not alter markers of allergic inflammation. Mac, macrophages; Eos, eosinophils. Data A, B, and C are means ± SE (n = 4–8 per group), *P < 0.05.

Fig. 4.

GSNO therapy reduces levels of proinflammatory cytokines and chemokines. Levels of IL-4, IL-5, IL-6, IL-13, MCP-1, MIP-1B, MIP-2, and KC were measured from BAL by multiplexed immunoassay. Control data are mean ± range (n = 2) or mean ± SE (n = 3). OVA data are means ± SE (n = 4–6). *P < 0.005 and **P < 0.0001 (2-tailed t-test).