Abstract
The flavone apigenin has been previously selected as a potent pharmacological activator of the CFTR Cl(-) channel, however, its utility for the activation of CFTR in vivo is expected to be limited because flavonoids are readily metabolized. We therefore investigated the poorly metabolizable methylether of apigenin, 5,7,4'-trimethoxyflavone (TMF) as a CFTR activator using transepithelial short-circuit current measurements, whole cell and single cell patch clamp techniques, and nasal potential difference (PD) measurements. Transepithelial Cl(-) secretion by Calu-3 epithelia was stimulated by TMF with a halfmaximal concentration of 64+/-5 microM to 55+/-15% of maximal currents achieved by subsequent addition of cAMP agonist forskolin (10 microM). In forskolin-prestimulated tissues, TMF showed small effects and stimulated Cl(-) secretion by an additional 6%. Single channel and whole cell patch clamp techniques were used to verify these effects and identify CFTR as the target of TMF. TMF increased the open probability of silent CFTR (to 0.31+/-0.06) but showed small effects once CFTR had been prestimulated with forskolin. In nasal PD measurements in humans, perfusion of TMF onto the nasal mucosa activated nasal PD by -9.5+/-1.1 mV, which was 69% of the effect of TMF+isoproterenol (-13.8+/-3.9 mV). These data show that TMF is an activator of CFTR in both in vitro and in vivo assays that targets mainly the unstimulated CFTR.