Lipopolysaccharide-induced alterations in airway reactivity and epithelial bioelectric responses to methacholine and hyperosmolarity

We investigated the in vivo and in vitro effects of lipopolysaccharide(LPS)-treatment (4 mg/kg, i.p.) on guinea-pig airway smooth muscle reactivity and epithelial bioelectric responses to methacholine (MCh) and hyperosmolarity. Hyperosmolar challenge of the epithelium releases epithelium-derived relaxing factor (EpDRF). Utilizing a two-chamber, whole body plethysmograph 18 h post-treatment, animals treated with LPS were hyporeactive to inhaled, MCh aerosol. This could involve an increase in the release and/or actions of EpDRF, since LPS-treatment enhanced EpDRF-induced smooth muscle relaxation in vitro in the isolated, perfused trachea apparatus. In isolated, perfused tracheas the basal transepithelial potential difference (Vt) was increased after LPS-treatment. The increase in V t was inhibited by amiloride and indomethacin. Concentration-response curves for changes in V t in response to serosallyand mucosally-applied MCh were biphasic (hyperpolarization, <3×10 -7 M; depolarization, >3×10 -7 M); MCh was more potent when applied serosally. The hyperpolarization response to MCh, but not the depolarization response, was potentiated after LPS treatment. In both treatment groups, mucosally-applied hyperosmolar solution (using added NaCl) depolarized the epithelium; this response was greater in tracheas from LPS-treated animals. The results of this study indicate that airway hyporeactivity in vivo following LPStreatment is accompanied by an increase in the release and/or actions of EpDRF in vitro. These changes may involve LPS-induced bioelectric alterations in the epithelium. This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on October 17, 2003 as DOI: 10.1124/jpet.103.051672 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from JPET #051672 and 051680 combined 5 Asthma is a chronic disease which is characterized by reversible airway obstruction, airway inflammation, and airway hyperresponsiveness. Lipopolysaccharide (LPS) or endotoxin, the major component of the outer membrane of gram-negative bacteria, poses problems for asthmatic patients. For example, inhaled endotoxin causes a slight decrease in the forced expiratory volume in 1 sec and an increase in histamine responsiveness in asthmatic patients, but not in normal subjects (Michel et al., 1989, 1996). Several reports have demonstrated that LPS administration leads to the development of airway hyperreactivity and inflammation in mice (Held and Uhlig, 2000), rats (Pauwels et al., 1990), and guinea pigs (Toward and Broadley, 2000). Airway reactivity is under the control of numerous physiological mechanisms, including epithelial-derived excitatory and inhibitory substances (Fedan et al., 1988; Goldie and Hay, 1997; Folkerts et al., 1998). One such inhibitory substance which has been shown to modulate airway reactivity is the non-nitric oxide, non-prostanoid epithelium-derived relaxing factor (EpDRF), which is released in response to hyperosmolarity 2 at the mucosal or serosal surface of airway epithelial cells (Munakata et al., 1988, 1989, 1990; Fedan et al., 1999, 2003a). EpDRF release during hyperosmolar challenge occurs in response to the incremental increase in osmolarity (Fedan et al., 2003a), and is associated with epithelial bioelectric events (DortchCarnes et al., 1999; Wu et al., 2003). Evidence has been obtained to suggest that hyperosmolar challenge of the epithelium releases carbon monoxide (Fedan et al., 2003b). Once released, EpDRF initiates airway smooth muscle relaxation via an unknown mechanism. There have been several reports indicating the importance of EpDRF in modulating airway reactivity. For example, in the guinea-pig, isolated perfused trachea, reactivity to methacholine (MCh) is decreased substantially when the release of EpDRF is stimulated by This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on October 17, 2003 as DOI: 10.1124/jpet.103.051672 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from JPET #051672 and 051680 combined 6 challenging the epithelium with hyperosmolar solution (Fedan et al., 1999). In addition, EpDRF-induced smooth muscle relaxation is significantly attenuated in isolated, perfused tracheas from guinea pigs exposed to ozone, which is accompanied by in vitro and in vivo hyperreactivity to MCh (Fedan et al., 2000). Finally, preliminary evidence indicates that sensitization and challenge of guinea pigs with ovalbumin enhances the release of EpDRF and causes in vitro airway hyporeactivity to MCh, at a time when the animals exhibit hyperreactivity to inhaled MCh (Warner et al., 1996). In addition, there is evidence which indicates that the synthesis, release, and/or effects of EpDRF are functionally linked to the electrical activity of epithelial cells. Dortch-Carnes et al. (1999) demonstrated that a decrease in the transepithelial potential difference (V t) preceded EpDRF-induced smooth muscle relaxation elicited by elevating serosal or mucosal osmolarity with either ionic or nonionic osmolytes. Fedan et al. (1999) demonstrated that amiloridesensitive Na channels and 4-4’ diisothiocyanatostilbene-2-2’ disulfonic acid (DIDS)-sensitive Cl channels are involved in EpDRF-induced smooth muscle relaxation, and Tamaoki et al. (1997) published evidence for involvement of Ca -activated K channels in the synthesis and/or release of EpDRF in human bronchial strips. LPS has been shown to alter airway reactivity in human and animal subjects. Therefore, the purpose of this study was to examine the possible relationship between alterations in airway reactivity induced by LPS and alterations in the smooth muscle effects of EpDRF and the epithelial bioelectric events associated with its release. This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on October 17, 2003 as DOI: 10.1124/jpet.103.051672 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from JPET #051672 and 051680 combined 7