Abstract

We previously demonstrated that 1-ethyl-2-benzimidazolone (1-EBIO) directly activates basolateral membrane calcium-activated K⁺ channels (K_Ca), thereby stimulating Cl⁻ secretion across several epithelia. In our pursuit to identify potent modulators of Cl⁻ secretion that may be useful to overcome the Cl⁻ secretory defect in cystic fibrosis (CF), we have identified chlorzoxazone [5-chloro-2(3H)-benzoxazolone], a clinically used centrally acting muscle relaxant, as a stimulator of Cl⁻ secretion in several epithelial cell types, including T84, Calu-3, and human bronchial epithelium. The Cl⁻ secretory response induced by chlorzoxazone was blocked by charybdotoxin (CTX), a known blocker of K_Ca. In nystatin-permeabilized monolayers, chlorzoxazone stimulated a basolateral membrane I_K, which was inhibited by CTX and also stimulated an apicalI_Cl that was inhibited by glibenclamide, indicating that the G_Cl responsible for thisI_Cl may be cystic fibrosis transmembrane conductance regulator (CFTR). In membrane vesicles prepared from T84 cells, chlorzoxazone stimulated ⁸⁶Rb⁺ uptake in a CTX-sensitive manner. In excised, inside-out patches, chlorzoxazone activated an inwardly-rectifying K⁺ channel, which was inhibited by CTX. 6-Hydroxychlorzoxazone, the major metabolite of chlorzoxazone, did not activate K_Ca, whereas zoxazolamine (2-amino-5-chlorzoxazole) showed a similar response profile as chlorzoxazone. In normal human nasal epithelium, chlorzoxazone elicited hyperpolarization of the potential difference that was similar in magnitude to isoproterenol. However, in the nasal epithelium of CF patients with the ΔF508 mutation of CFTR, there was no detectable Cl⁻ secretory response to chlorzoxazone. These studies demonstrate that chlorzoxazone stimulates transepithelial Cl⁻ secretion in normal airway epithelium in vitro and in vivo, and suggest that stimulation requires functional CFTR in the epithelia.