Stimulation of Cl- secretion by chlorzoxazone

We previously demonstrated that 1-ethyl-2-benzimidazolone (1-EBIO) directly activates basolateral membrane calcium-activated K+ channels (K-Ca), there by stimulating Cl- secretion across several epithelia. In our pursuit to id entify potent modulators of Cl- secretion that may be useful to overcome th e Cl- secretory defect in cystic fibrosis (CF), we have identified chlorzox azone [5-chloro-2(3H)-benzoxazolone], a clinically used centrally acting mu scle relaxant, as a stimulator of Cl- secretion in several epithelial cell types, including T84, Calu-3, and human bronchial epithelium. The Cl- secre tory response induced by chlorzoxazone was blocked by charybdotoxin (CTX), a known blocker of K-Ca. In nystatin-permeabilized monolayers, chlorzoxazon e stimulated a basolateral membrane I-K, which was inhibited by CTX and als o stimulated an apical I-Cl that was inhibited by glibenclamide, indicating that the G(Cl) responsible for this I-Cl may be cystic fibrosis transmembr ane conductance regulator (CFTR). In membrane vesicles prepared from T84 ce lls, chlorzoxazone stimulated Rb-86(+) uptake in a CTX-sensitive manner. In excised, inside-out patches, chlorzoxazone activated an inwardly-rectifyin g 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 hyperpolarizatio n of the potential difference that was similar in magnitude to isoprotereno l. However, in the nasal epithelium of CF patients with the Delta F508 muta tion of CFTR, there was no detectable Cl- secretory response to chlorzoxazo ne. These studies demonstrate that chlorzoxazone stimulates transepithelial Cl- secretion in normal airway epithelium in vitro and in vivo, and sugges t that stimulation requires functional CFTR in the epithelia.