Contraction of human airways by oxidative stress

Abstract

We examined the in vitro effects of tert-butylhydroperoxide (tBu-OOH) in human bronchial muscle. tert-Butylhydroperoxide produced concentration-dependent contractions of bronchial rings (maximum effect was 56.5 ± 9.6% of contraction by 1 mM acetylcholine; effective concentration 50% was ∼100 μM). tert-Butylhydroperoxide (0.5 mM)-induced contraction was enhanced by epithelial removal but abolished by indomethacin (cyclooxygenase inhibitor) and zileuton (lipoxygenase inhibitor). tert-Butylhydroperoxide produced a transient rise in intracellular calcium in human cultured airway smooth muscle cells (HCASMC). The bronchial reactivity to acetylcholine and histamine was not altered by tBu-OOH. In HCASMC, tBu-OOH (0.5 mM, 30 min) increased malondialdehyde levels (MDA; from 7.80 ± 0.83 to 26.82 ± 1.49 nmol mg⁻¹ protein), accompanied by a decrease of reduced glutathione (GSH; from 16.7 ± 2.6 to 6.9 ± 1.9 nmol mg⁻¹ protein) and an increase of oxidized glutathione (from 0.09 ± 0.03 to 0.18 ± 0.03 nmol mg⁻¹ protein). N-acetylcysteine (0.3 mM) inhibited by approximately 60% the bronchial contraction resulting from tBu-OOH (0.5 mM) and protected cultured cells exposed to tBu-OOH (MDA was lowered to 19.51 ± 1.19 nmol mg⁻¹ protein, and GSH content was replenished). In summary, tBu-OOH caused contraction of human bronchial muscle mediated by release of cyclo-oxygenase and lipoxygenase products without producing airways hyperreactivity.N-acetylcysteine decreases tBu-OOH-induced contraction and protects human cultured airway smooth muscle cells exposed to tBu-OOH.

Introduction

Reactive oxygen species from external sources (inhalation of airborne oxidants such as cigarette smoke and oxidizing pollutants) or those derived from activated inflammatory cells in the lung may influence airway and pulmonary vascular reactivities [1], [2], [3] and appear to be involved in the pathogenesis of a number of lung diseases including chronic obstructive pulmonary disease and bronchial asthma [4], [5], [6], [7], [8]. Cells defend themselves against reactive oxygen species by means of endogenous antioxidant mechanisms [9], [10]. A known therapeutic approach to the inhibition of oxidant-mediated injury is the use of glutathione-modulating agents like thiol or sulfydryl compounds. Among them, N-acetylcysteine is probably one of the most widely investigated and has shown beneficial effects in disease states in which free radicals have been involved [11].

The general mechanism by which oxidizing agents produce biologic effects is ascribed to their oxidative and reactive capacities resulting in the generation of free radicals that may oxidize amino acids in tissue proteins, react with polyunsaturated fatty acids in cell membranes, or damage DNA [12]. However, the precise mechanisms underlying their pulmonary effects are still under investigation and are likely to involve various pulmonary cell types including airway smooth muscle [13], [14], [15]. Recent studies show that the airway smooth muscle cell, in addition to its contractile function, participates in the lung inflammatory response [16], but the effects of oxidizing agents on the functional and biochemical responses of airway smooth muscle preparations have been scarcely studied, particularly in humans. This is in contrast with detailed studies made in other cell types of the airways such as the epithelial cells [5], [17], [18], [19], [20].

In the present work, we examined the functional response of human isolated bronchus to tert-butylhydroperoxide (tBu-OOH), a stable organic hydroperoxide widely used as a model oxidative agent in biologic systems. The oxidative stress produced in HCASMC by exposure to tBu-OOH was also studied by measuring MDA content and glutathione status. In addition, we assessed the protective action of N-acetylcysteine on the effects produced by tBu-OOH on human airway smooth muscle cells.