Effect of Ozone Treatment on Airway Reactivity and Epithelium-Derived Relaxing Factor in Guinea Pigs 1 JEFFREY S. FEDAN, LYNDELL L. MILLECCHIA, RICHARD A. JOHNSTON, APPAVOO RENGASAMY, ANN HUBBS, RICHARD D. DEY, LONG-XING YUAN, DAVID WATSON, W. TRAVIS GOLDSMITH, JEFFREY S. REYNOLDS, LARRY ORSINI, JUANITA DORTCH-CARNES, DEBORAH CUTLER, and DAVID G. FRAZER Pathology and Physiology Research Branch (J.S.F., L.L.M., R.A.J., A.R., A.H., L.-X.Y., D.W., L.O., J.D.-C., D.C.) and Engineering and Controls Technology Branch (W.T.G., J.S.R., D.G.F.), Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia; and Departments of Pharmacology and Toxicology (R.A.J.) and Anatomy (R.D.D.), West Virginia University School of Medicine, Robert C. Byrd Health Sciences Center, Morgantown, West Virginia Accepted for publication February 18, 2000 This paper is available online at http://www.jpet.org ABSTRACT Ozone (O 3 ) is toxic to respiratory epithelium and causes airway inflammation and hyperreactivity. To evaluate the role of the epithelium in the development of hyperreactivity, we examined in guinea pigs the effects of inhaled O 3 (3 ppm for 1 h; 0–24 h after exposure) on 1) reactivity to inhaled methacholine (MCh), 2) reactivity of the isolated, perfused trachea (IPT) to MCh, 3) epithelium-derived relaxing factor (EpDRF)-mediated relax- ations of IPT induced by mucosal hyperosmolar solutions, 4) neurogenic contraction and relaxation responses, 5) transepi- thelial potential difference, and 6) microscopic analysis of ni- trotyrosine immunofluorescence, substance P fiber density, and tracheal morphology. At 0 h, O 3 caused hyperreactivity to inhaled MCh and mucosally but not serosally applied MCh in IPT (only in the presence of the epithelium) and a decrease in transepithelial potential difference. Inhibition of EpDRF-induced relaxation responses occurred at 2 h. All of these changes returned to control by 12 to 18 h. O 3 had no effect on neuro- genic responses. Nitrotyrosine immunofluorescence appeared in the trachea at 0 h in detached epithelial cell ghosts and in intrapulmonary airways by 6 h. Substance P fiber density was elevated in smooth muscle at 0 and 18 h but not in epithelium or lamina propria of intrapulmonary and extrapulmonary bron- chi. Loss of cilia and mucosubstances in the mucosa occurred at 0 h; the epithelium became markedly attenuated over 12 to 24 h. A reversible increase in epithelial permeability and a decrease in EpDRF production may contribute to O 3 -induced hyperreactivity to MCh. Inhalation exposure to ozone (O 3 ) causes several adverse effects in the lung. Typically, the forced expiratory volume in 1 s is reduced, airway reactivity to bronchoconstrictors such as methacholine (MCh) is increased, and neutrophilic inflam- mation occurs in the airways (American Thoracic Society, 1996). Responses of the airways to O 3 occur in three phases: immediate (0 –2 h), early (2–24 h), and late (12–24 h) (Leikauf et al., 1995; American Thoracic Society, 1996). An accumulation of polymorphonuclear leukocytes (PMNs) in the mucosa and submucosa and transit of the cells into the air space occur during the early phase (Schultheis and Bas- sett, 1994; Pendino et al., 1995) and may be transient after acute exposure. O 3 causes morphological damage to respiratory epithe- lium; this effect occurs before the inflammatory response, and it could contribute to the development of airway hyper- reactivity (Murlas and Roum, 1985). Murlas et al. (1990) observed that hyperresponsiveness of tracheal rings in vitro to contractile agonists occurred only in the presence of the epithelium. O 3 causes an increase in epithelial permeability, which, depending on the exposure protocol, may recover within 1 day (Kleeberger and Hudak, 1992; Young and Bhalla, 1992). Guinea pigs became hyperreactive to inhaled but not intravenously administered histamine or MCh (Yea- don et al., 1992; Matsubara et al., 1995), suggesting that the epithelial diffusion barrier to air-borne agents had been com- promised. Inhibition of neutral endopeptidase by O 3 may contribute to hyperreactivity to neuropeptides such as sub- stance P, as well as to histamine and cholinergic agonists (Murlas et al., 1990; Yeadon et al., 1992; Loenders et al., 1996). The airway epithelium has multiple roles in addition to serving as a diffusion barrier (Farmer and Hay, 1991a). For example, it regulates airway surface liquid and is a source of inflammatory mediators and prostanoids. In addition, the epithelium modulates the reactivity of airway smooth muscle Received for publication September 27, 1999. 1 Mention of a brand name does not constitute product endorsement. ABBREVIATIONS: MCh, methacholine; EpDRF, epithelium-derived relaxing factor; MKH, modified Krebs-Henseleit; PMN, polymorphonuclear leukocyte; IL, intraluminal; EL, extraluminal; V T , transepithelial potential difference; EFS, electric field stimulation. 0022-3565/00/2933-0724 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 293, No. 3 U.S. Government work not protected by U.S. copyright Printed in U.S.A. JPET 293:724–734, 2000 /2149/826963 724 at ASPET Journals on October 24, 2016 jpet.aspetjournals.org Downloaded from