Development of substituted benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel

Chloride channels play an important role in the physiology and pathophysiol ogy of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) an d 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB 07), which we show to be po tent and selective activators of the cystic fibrosis transmembrane conducta nce regulator (CFTR) chloride channel. We examined the effect of MPB compou nds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovar y (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Sin gle and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reve al that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native res piratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MP B compounds was glibenclamide-sensitive and 4,4'-diisothiocyanostilbene-2,2 '-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, RIPE drugs activate CFTR channels and stimulate the secretion of the antiba cterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secreti on without changing intracellular cAMP and ATP levels. Similarly, in CHO ce lls MPB compounds have no effect on the intracellular levels of cAMP and AT P or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or a lkaline phosphatase). Our results provide evidence that substituted benzo[c ]quinolizinium compounds are a novel family of activators of CFTR and of CF TR-mediated protein secretion and therefore represent a new tool to study C FTR-mediated chloride and secretory functions in epithelial tissues.