Psoralens: Novel modulators of Cl- secretion

We evaluated effects of psoralens on Cl- secretion (short-circuit current, I-sc) across T84 monolayers. Methoxsalen failed to increase I-sc. Several observations suggest that psoralens open cystic fibrosis transmembrane conductance regulator Cl- channels. I) After activation of the Ca2+- dependent basolateral membrane K+ channel (K-Ca) by 1-ethyl-2-benzimidazolinone or thapsigargin, methoxsalen (10 mu M) further increased I-sc. 2) When added before carbachol (CCh), methoxsalen potentiated the I-sc response to CCh, as predicted, if it increased apical Cl- conductance. 3) After establishment of a mucosal-to-serosal Cl- gradient and permeabilization of basolateral membrane with nystatin, psoralens increased Cl- current, which was inhibited by glibenclamide. In contrast, neither TS-TM calix[4]arene nor Cd2+, inhibitors of outwardly rectifying Cl- channels and the ClC-2 Cl- channel, respectively, inhibited psoralen-induced Cl- current. In contrast to their effects on Cl- conductance, psoralens failed to significantly affect basolateral membrane K+ conductance; subsequent addition of 1-ethyl-2-benzimidazolinone induced a large increase in K+ conductance. Also, in excised patches, methoxsalen failed to activate K-Ca. In addition to potentiating the peak response to CCh, psoralens induced a secondary, sustained response. Indeed, when added up to 60 min after return of CCh-induced I-sc to baseline, psoralens induced a sustained I-sc. This sustained response was inhibited by atropine, demonstrating the requirement for continuous muscarinic receptor activation by CCh. This sustained response was inhibited also by verapamil, removal of bath Ca2+, and charybdotoxin. These results suggest that return of I-sc to baseline after CCh stimulation is not due to downregulation of Ca2+ influx or K-Ca. Finally, we obtained similar results with psoralens in rat colon and primary cultures of murine tracheal epithelium. On the basis of these observations, we conclude that psoralens represent a novel class of Cl- channel openers that can be used to probe mechanisms underlying Ca2+-mediated Cl- secretion.